Table of contents

Hormones produced by glands in the endocrine system and neurotransmitters produced by the nervous system control many bodily functions. The concentrations of these molecules in the body are an indication of its state, hence the use of the term biomarker. Excess concentrations of biomarkers, such as cortisol, serotonin, epinephrine, dopamine, are released by the body in response to a variety of conditions - emotional state (euphoria, stress), disease, etc. The development of simple, low-cost modalities for point-of-use (PoU) measurements of biomarkers levels in various bodily fluids (blood, urine, sweat, saliva) as opposed to conventional hospital or lab settings is receiving increasing attention. The presentation starts with a review of the basic properties of primary stress-related biomarkers: origin in the body (i.e. if they are produced as hormones, neurotransmitters or both), chemical composition, molecular weight (small/medium size molecules and polymers, ranging from ~100Da to ~100kDa), hydro- or lipo-philic nature. Next, a review of the published literature is presented regarding the concentration of these biomarkers found in several bodily fluids that can serve as the medium for determination of the condition of the subject: blood, urine, saliva, sweat and, to a lesser degree, interstitial tissue fluid. The concentration of various biomarkers in most fluids covers a range of 5-6 orders of magnitude, from 100s of ng/mL (~1µM) down to a few pg/mL (sub 1pM). Mechanisms and materials for point-of-use biomarker sensors are summarized and key properties are reviewed. Illustrative examples from the literature are discussed for several sensor device categories, including lateral flow (immuno)assay devices and microfluidic devices. Selected methods for detecting these biomarkers are reviewed, including antibody- and aptamer-based colorimetric assays, electrochemical and optical detection. Finally, the presentation outlines key challenges of the field and provides a look ahead to future prospects.

A. J. Steckl and P. Ray, "A Review of Stress Biomarkers Concentrations in Bodily Fluids and Their Point-of-Use Detection", ACS Sensors, 3 (10), 2025-2044, Sept 2018 (10.1021/acssensors.8b00726)

V. Venkatraman and A. J. Steckl, "Quantitative Detection in Lateral Flow Immunoassay Using Integrated Organic Optoelectronics", IEEE Sensors, 17 (24), 8343, Dec 2017 (10.1109/JSEN.2017.2764178 ).

P. Ray and A. J. Steckl, "Label-Free Optical Detection of Multiple Stress Biomarkers in Sweat, Plasma, Urine and Saliva", ACS Sensors4, 2019. (10.1021/acssensors.9b00301)

S. Dalirirad and A. J. Steckl, "Aptamer-Based Lateral Flow Assay for Point of Care Cortisol Detection in Sweat", Sensors & Actuators 283, 79-86, Jan 2019 (https://doi.org/10.1016/j.snb.2018.11.161).

Figure 1

Introduction

Cells intake nutrients and oxygen to produce cellular energy (adenosine triphosphate, ATP) and simultaneously excrete acidic waste and carbon dioxide. Cell activity can be estimated by measuring cellular metabolism-related molecules, such as oxygen, lactate and carbon dioxide. Furthermore, the rate of oxidative phosphorylation (OXPHOS) can be used as a probe to evaluate mitochondira activity and carcinomatosis degree. Normal cells produce most ATP molecules via OXPHOS, but cancer cells mainly produce ATP via cytosol glycolysis, which consumes less oxygen and produces more protons. Therefore, to measure the consuming rate of oxygen and the change in extracellular pH values is meaningful for the estimation of cell activity and carcinomatosis degree. In our previous studies [1-4], Clark and non-Clark oxygen sensing chips were designed and fabricated for estimating the respiratory activity of mammalian cells, such as HeLa cells [3]. The time-lapse monitoring shows that the respiratory activity of HeLa cells increases with the cultivation time. Moreover, an oxygen electrode array integrated with a microfluidic channel and microstructures can be constructed to measure the oxygen consumption rate of single bovine embryo for the estimation of embryo development [2]. It is worth noting that the microfluidic flow of cell-based chip may induce a shear force or a viscous force to move the attached cell away from the detecting electrodes during medium replacement. In our previous study, a cell-based chip consisting of position-raised microchannel and an open chamber slab was easily used for the cell operation such as cell seeding and medium replacement. The position-raised microchannel can reduce the effect of shear force on the cell attachment during perform medium replacement. In this presentation, the different cell-based chips are used to construct a platform to estimate the cellular respiratory activity.

Fast Measuring Cellular Activity

Adipocyte activity determines the metabolism of carbohydrate and fatty acid of human beings, related to the formation of diabetes. Evaluation of adipocyte activity allows the researchers to realize the causes of type II diabetes and therapeutic methods. In the study, a microfluidic chip containing dissolved oxygen (DO) detectors of three-electrode electrochemical system was developed for the measurement of DO around the cultivated cells. Adipocytes were estimated with the stimulation of different glucose concentration, insulin and mitochondria activity-controlled drugs, such as oligomycin, FCCP, rotenone and antimycin A.

Method

The microfluidic chip comprises a polymethylmethacrylate (PMMA) slab containing a position-raised channel-connected container and a glass substrate containing gold ultramicroelectrode to form a three-electrode electrochemical detector. The PMMA slab can be ablated to form a channel with a laser cutter, adhered with a 3M adhesive tape on the channel-side surface, and then cut to form three holes, which were acted as two containers for cell cultivation and reference-electrode solution and one outlet of channel. 50-nm Ti and 200-nm Au thin films were patterned on the glass substrate as the working electrodes, reference electrode and counter electrode. Subsequently, a 10-mm thick negative photoresist, SU8-3010, was employed to define the sensitive area of electrodes. The open container-containing PMMA slab was bond with the electrode-containing glass substrate to form the cell-based microfluidic chip. The cell suspension can be directly dripping in the working electrode container for cell seeding and cultivation. The medium replacement can be performed via the position-raised channel. The DO detector applied by a −0.8 V potential for oxygen reduction was used to estimate the oxygen consumption of cells.

Results and Conclusions

The result of optical images shows the adipocytes after 1-h cultivation were not removed when replacing the culture medium via the position-raised microchannel. Potential-pulse amperometry was used for the measurement of DO concentration at −0.8 V for 120 s in each measurement. The reductive current of DO is proportional to DO concentration. When applying different mitochondria activity-modulated drugs, the cell-based microfluidic chip can obtain good correlation between the oxygen consumption rate and the cellular metabolic response. For example, oligomycin is a ATPase inhibitor, which decreased the oxygen consumption rate (OCR). FCCP is an uncoupling protein, dissipating proton gradient, to increase mitochondria activity, which increases OCR. The microfluidic chip has a great promise to fast estimate the effect of drugs on the cellular metabolic behavior in several tens of minutes cultivation, which is useful for drug screening in a short time.

References

[1] C.-C.Wu, T. Yasukawa, H. Shiku, T. Matsue, Fabrication of miniature Clark oxygen sensor integrated with microstructure, Sensors and Actuators B 110 (2005) 342–349. doi.org/10.1016/j.snb.2005.02.014.

[2] C.-C.Wu, T. Saito, T. Yasukawa, H. Shiku, H. Abe, H. Hoshi, T. Matsue, Microfluidic chip integrated with amperometric detector array for in situ estimating oxygen consumption characteristics of single bovine embryos, Sensors and Actuators B 125 (2007) 680–687. doi.org/10.1016/j.snb.2007.03.017.

[3] C.-C. Wu, H.-N. Luk, Y.-T. Tsai Lin, C.-Y. Yuan, A Clark-type oxygen chip for in situ estimation of the respiratory activity of adhering cells, Talanta 81 (2010) 228–234. doi: 10.1016/j.talanta.2009.11.062

[4] C.-C.Wu, W.-C. Lin, S.-Y. Fu, The open container-used microfluidic chip using IrOx ultramicroelectrodes for the in situ measurement of extracellular acidification, Biosensors and Bioelectronics 26 (2011) 4191–4197. doi.org/10.1016/j.bios.2011.04.034

Measurements of neurotransmitter concentrations in blood and tissues are of great physiological and pathological importance and could be useful for monitoring and understanding the state and progress of a variety of neurological diseases. Electrochemical microbiosensors can provide real-time measurements of neurotransmitters, but sensors that function reliably and maintain accuracy in hypoxic environments are limited. This presentation will describe an enzyme-based electrochemical sensing technology that enables real time monitoring of key analytes associated with neural signaling and function during ischemia and high frequency stimulation. To design our sensors we utilize unique redox, catalytic and oxygen storage/release properties of ceria nanoparticles, originating from their dual oxidation state and the highly mobile lattice oxygen present at their surface. The construction and analytical performance of several enzyme sensors based on these nanoparticles will be discussed, along with results demonstrating their capabilities for monitoring of neurological activity (dopamine, lactate, glutamate) during hypoxia in brain slices and in intact awake animals, with high sensitivity, wide linear range, and low oxygen dependence. The results demonstrate promising characteristics of ceria nanoparticles as a biosensor material for in vivo monitoring of oxidase enzyme substrates, particularly in settings where the availability of oxygen may be limiting. The use of these materials opens new avenues for application of this technology in different fields such as biosensors, biofuel cells, point-of-care testing and implantable diagnostic devices.

Using low noise electronics and micro-electrodes we can now electrochemically detect and count single, nanometer-scale objects in solution.1,2 This talk will cover recent discoveries in the field of single entity electrochemistry showing that electro-inactive species, including proteins, DNA, and even cells, can be detected individually using unmodified electrodes.3–5 Despite these advances, however, limitations persist. The location upon which the individual analyte adsorbs onto the electrode surface influences the magnitude of the electrochemical signal it produces.6,7 Consequently, the electronic output does not reflect the true physical properties of the biological entity, thus inhibiting the analytical resolution desired for applications.

In response, we have shown that coupling the electrochemical reaction to a homogeneous, rate-limiting chemical reaction ameliorates this problem, improving the precision of single-entity electrochemical blocking measurements. Using both finite element simulations and experimental statistical analysis we provide guidance for the sizing of redox-inactive materials with high analytical precision under a range of conditions, including variations in electrode size and analyte concentration. The presented nanoscale electrocatalytic approach could offer a novel means of achieving cost-effective, rapid, real-time detection of nanometer-scale "insulators" in complex media, an advance that could be of significant relevance to point-of-care medical diagnostics.

References:

(1) M. Crooks, R. Concluding Remarks: Single Entity Electrochemistry One Step at a Time. Faraday Discuss. 2016, 193 (0), 533–547. https://doi.org/10.1039/C6FD00203J.

(2) V. Sokolov, S.; Eloul, S.; Kätelhön, E.; Batchelor-McAuley, C.; G. Compton, R. Electrode–Particle Impacts: A Users Guide. Phys. Chem. Chem. Phys. 2017, 19 (1), 28–43. https://doi.org/10.1039/C6CP07788A.

(3) Bard, A. J.; Zhou, H.; Kwon, S. J. Electrochemistry of Single Nanoparticles via Electrocatalytic Amplification. Isr. J. Chem. 2010, 50 (3), 267–276. https://doi.org/10.1002/ijch.201000014.

(4) Quinn, B. M.; van't Hof, P. G.; Lemay, S. G. Time-Resolved Electrochemical Detection of Discrete Adsorption Events. J. Am. Chem. Soc. 2004, 126 (27), 8360–8361. https://doi.org/10.1021/ja0478577.

(5) Deng, Z.; Renault, C. Detection of Individual Insulating Entities by Electrochemical Blocking. Curr. Opin. Electrochem. 2021, 25, 100619. https://doi.org/10.1016/j.coelec.2020.08.001.

(6) Fosdick, S. E.; Anderson, M. J.; Nettleton, E. G.; Crooks, R. M. Correlated Electrochemical and Optical Tracking of Discrete Collision Events. J. Am. Chem. Soc. 2013, 135 (16), 5994–5997. https://doi.org/10.1021/ja401864k.

(7) Deng, Z.; Elattar, R.; Maroun, F.; Renault, C. In Situ Measurement of the Size Distribution and Concentration of Insulating Particles by Electrochemical Collision on Hemispherical Ultramicroelectrodes. Anal. Chem. 2018, 90 (21), 12923–12929. https://doi.org/10.1021/acs.analchem.8b03550.

In the last decade the field of research in biosensors has had a great interest in the development and manufacture of sensors capable of detecting and monitoring analytes in body fluids such as sweat, tears, and saliva. This interest arises since the traditional method for monitoring a large majority of analytes related to diseases or health conditions is carried out through blood analysis. However, analysis of analytes in blood is a painful method, it is invasive sampling, and more expensive compared to sweat analysis. In recent years, a variety of wearables have been developed for the analysis of analytes in sweat, through electrochemical and optical interfaces. However, the problem with the development of these sweat biosensors has been limited to monitoring a small number of biomarkers such as lactate, glucose, electrolytes, and pH. Here, we present a biosensor for the detection and monitoring of Neuropeptide Y in sweat using flexible materials and different types of working electrodes. Neuropeptide Y has an important role in the energy balance and is related to different diseases and conditions such as diabetes, obesity, depression, anxiety, sleep problems, and a relationship of neuropeptide Y and heart attacks has also been found.

Introduction

Real-time bioprocess monitoring and control is needed for the scalable production and deployment of cancer cell therapies at reasonable cost. For example, CAR-T cell therapy shows promise as an effective treatment for cancer with high (83%) remission rates.¹ However, the cost of these treatments is too expensive for their mass adoption.² One reason for the steep price is the difficulty scaling production while ensuring delivery of an efficacious therapy to the patient. One factor preventing scalability is the lack of effective process analytical technology. As a potential solution to this issue, we have developed a fully integrated, wireless, 3D-printed sensor capsule to be used for multiplexed sensing of critical quality attributes (CQAs), namely pH, glucose, and lactate concentrations within cell bioreactors. CQAs are used not only as metrics for cell viability, but also as determinants of a cell's ability to deliver efficacious treatment. Unlike current process monitoring technology, capsule sensors are buoyant and can be propelled using impellers within the bioreactor. This allows for measurements uniformly inside the bioreactor. To our knowledge, this is the first time that 3D-printed, wireless sensor capsules have been developed for use in cell bioreactors for cancer cell therapy.

Methods

The capsule consists of electrochemical sensors, and read-out and wireless transmission electronics integrated into a capsule made of a biocompatible polymer (Figure 1). The different sensors for glucose, lactate, and pH along with an Ag on-chip reference electrodes were fabricated using standard lithographic techniques on silicon substrates. The reference electrode consisted of a thin film of Ag deposited by e-beam evaporation that was later coated with polyvinyl butyral (PVB) to improve stability.³ Glucose and lactate sensing was achieved amperometrically using enzymes, glucose oxidase or lactase oxidase respectively, immobilized on a Pt working electrode biased to 0.7 V. pH sensing was achieved by depositing a pH-sensitive oxide on a gold electrode. A pH-sensitive Al₂O₃ or HfO₂ layer was deposited through atomic layer deposition.

Figure 1 shows the capsule and sensor design. The capsule packaging was 3D printed using a Stratasys Connex 350 and was designed to be fully insulated from solution except for an opening on the top revealing the various sensors. The sensor chip is installed within the cap of the capsule which can be screwed off, but with wired connection to the electronics housed in the body of the capsule. This allows for simple replacement of the multiplexed sensor chip while retaining the capsule electronics, rechargeable battery, and packaging for reuse.

Results and Conclusions

Before testing within the capsule, the sensors were tested using microfluidics. Figure 2 shows the performance of the Ag/PVB on-chip pseudo reference electrode (pseudo-RE). The pseudo-RE shows a stable potential in different pH buffer solutions and shows similar sensitivity results for pH sensing when compared to a commercial reference electrode. By having a stable microfabricated pseudoRE, the sensing components will not be a limiting factor for miniaturization of the capsule. Figure 3 shows the response of the pH, lactate, and glucose sensors at different concentrations. The pH sensor shows high sensitivity (~53 mV/pH) over the range of pH 3 to 7. Glucose and lactate sensing was tested in 1X PBS. The glucose sensor shows a linear response to glucose concentrations from 0 to 25 mM validating the use of the sensor within cell culture media.⁴ The glucose sensor shows the same linear range when tested in human mesenchymal stem cell culture. In this case, the glucose concentration was first tested using a commercial sensor and then diluted to targeted glucose concentrations by dilution with PBS. The lactate sensor shows sensitivity in the concentration range of 1 to 5 mM with saturation occurring around ~6 mM. The capsule's wireless sensing capability was validated using pH sensing in a beaker. The pH sensing results in Figure 4 were obtained wirelessly from the capsule and were nearly identical to the results measured using our microfluidic system and conventional parameter analyzer.

This work has successfully demonstrated wireless sensing using a 3D-printed sensor capsule and functionality of electrochemical sensors for multiple CQAs for cell growth in bioreactors. These sensor capsules can enable scalability of the cell manufacturing process while ensuring delivery of an efficacious treatment. Future work will focus on capsule sensor validation in cell growth media and eventually monitoring of cell growth in bioreactors.

References

[1] NOVARITIS. "Novartis Receives First Ever FDA Approval for a CAR-T Cell Therapy, Kymriah (TM)(CTL019), for Children and Young Adults with B-cell ALL That Is Eefractory or Has Relapsed At least Twice." (2019).

[2] Dolgin, Elie. "Bringing down the cost of cancer treatment." Nature 555.7695 (2018).

[3] Guinovart, Tomàs, et al. "A reference electrode based on polyvinyl butyral (PVB) polymer for decentralized chemical measurements." Analytica chimica acta 821 (2014): 72-80.

[4] "Glucose in Cell Culture." Sigma Aldrich <https://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-expert/glucose.html>

Figure 1

A multi-metric armband system capable of simultaneous measurement of electrocardiogram (ECG) and electrodermal activity (EDA) from left arm is presented for the assessment of sympathetic nervous response. The performance of of EDA module was validated against a BIOPAC MP160 system while a single-lead ECG module was used to capture heart rate variations simultaneously. The presented armband is anticipated to provide reliable data for the detection and prognosis of different physical and neuropsychological disorders such as autism and Alzheimer's disease.

Keywords: Sympathetic Nervous System; EDA; ECG; Left Arm; I. Introduction

Sympathetic nervous response (SNR) has been found to be correlated with numerous bodily and mental health disorders. Frequency analysis of heart rate variability (HRV) and electrodermal activity (EDA) are the only non-invasive methods to assess the dynamics of the autonomic nervous system. However, frequency analysis of the HRV method cannot separate the dynamics of the sympathetic and parasympathetic nervous systems. EDA is a reflection of the autonomic innervation of sweat glands resulting in the reflection of activity within the sympathetic branch of the autonomic nervous system. EDA, however, suffers from motion artifact and movement. Therefore, the simultaneous monitoring of EDA and electrocardiogram (ECG) will provide more reliable and comprehensive indices of sympathetic nerve activities. Time-domain features of EDA along with ECG has been commonly utilized to assess the overall SNR.

The mere relationship between the EDA and SNR has been investigated via different approaches such as the analysis of power spectral density and time-varying analysis of EDA. The combined use of ECG/HRV and EDA has lent itself to assessing mental stress and numerous mental disorders such as schizophrenia, autism, Down syndrome. Also, it was found that EDA and SNR are heavily invested in the volume of white matter in the cingulum and inferior parietal and thus with Alzheimer's disease.

In this paper we have laid out the groundwork required for SNR evaluation, which takes advantage of the simultaneous EDA and ECG data acquisition from the left arm. II. System Design

The wearable armband system, Gen2.0, is constructed with commercial off-the-shelf components (COTS) and equipped with a BLE-enabled Nordic nRF51822 microcontroller unit (MCU) and is considered low-power [1]. We custom designed the filters for the AD8232 ECG analog frontend chip so that the left arm ECG signal is clear and reliable. The analog frontend for EDA was also custom designed using LTC 6081 op-amp to achieve a sufficiently high resolution. The MCU interfaces with an ADC 1114 for ECG measurement and uses an internal ADC for interfacing the EDA signal. Under the control of an internal timer, the ADC chip examples voltage and conductance signals from ECG and EDA frontends at a specific point depending on sampling frequency. Then the signals are converted into digital data and fed into the MCU. The MCU stores it inside buffers (capacity of 64 data samples) for ECG and EDA data separately. Every time the buffer is full, data will be either stored in a flash or transmitted via BLE. Figure 1 shows the system and the optimal ECG electrodes' positions. III. EDA Optimization And Validation

The performance of our armband ECG was validated against the BIOPAC direct ECG1 system. The optimal positions for EDA electrodes were determined through a set of external physical stimuli (pinch) tests. The accuracy of the used ECG module has already been verified in our previous study [2]. Figure 2 shows the system diagram and the candidate EDA electrode positions. Both the BIOPAC and our Gen2.0 modules were assigned an equal sample rate of 10Hz. IV. Results And Discussion B. EDA Validation

EDA curves were collected from a 38YO non-smoker male subject by both the BIOPAC and our proposed systems in an IRB- approved study (12418, North Carolina State University). The subject relaxed for an undisclosed amount of time (about 25s) and was then pinched in the right hand for 1s and relaxed for the rest of the test. The optimal electrodes' positions for our EDA module were found to be positions 10&13 or 1&2 (Figure 2). Figure 3 shows that the EDA obtained by the proposed system was more stable than that of BIOPAC addressing the relaxation and tension periods more distinctively. C. Simultaneous EDA and ECG

A testing protocol including resting, reading Latin, and being pinched (for 1s) and resting each for about 60s was followed. The ECG and EDA measurements were both carried out using the same armband and in real-time (Figures 4).

Considering the EDA results, the nervous response to the physical stimulus (being pinched) was stronger than the cognitive stimulus (reading). HRV analysis was done in time domain by detecting RR intervals in order to capture instant changes in heart rate in the ECG data. V. Conclusions

A wearable and low-power multi bio-metric armband system was proposed and validated for simultaneous monitoring of ECG and EDA. The current research lays out the groundwork for more in-depth characterization of the autonomic sympathetic nervous system and its relationship to both bodily and neurological disorders obtained from the left arm.

References

• Nozariasbmarz et al, "Review of wearable thermoelectric energy harvesting: From body temperature to electronic systems," Applied Energy, vol. 258, pp. 114069, 2020.

• Mohaddes et al, "A Pipeline for Adaptive Filtering and Transformation of Noisy Left-Arm ECG to Its Surrogate Chest Signal," Electronics (Basel), vol. 9, (5), pp. 866, 2020.

Figure 1

Physical confinement have been shown to alter cancer cell migration mechanisms and regulate intracellular signaling events during tumor development and metastasis. However, the effect of confinement on cytokine secretion remains largely unknown. To better understand this, we designed an in-vitro confinement environment with cell vertically confined and sandwiched between two collagen coated glass slides. This method can enable vertical confinement down to 5. Human epithelial cell MCF10A and breast cancer cell MDA-MB-231 were then cultured inside the in-vitro vertical confinement chamber for 48h. Using PCR test, we found that IL-6 expression was largely upregulated in MDA-MB 231 cell line after vertical confinement. We then designed and fabricated a nanopatterned electrochemical immunosensor to determine the IL-6 level in cell culture serum after confinement. The immunosensor was constructed by employing Au nanopatterned surface immobilized with antibody, exhibited a low limit of detection. The obtained results point towards rapid, sensitive, and specific early diagnosis of breast cancer at the point of care and other low-resource settings.

Currently, one of the approaches in neuroscience research is the analysis and understanding of neurochemical behaviors. This is a great challenge since, although neurotransmitter measurements have been possible, these techniques have limitations, among which are the selectivity, sensitivity, and temporal resolution of the measurement. This work seeks to improve the Electrochemical Impedance Spectroscopy (EIS) technique using a Carbon Fiber Microelectrode and a Platinum Microelectrode for measurement with a conductive polymer modification of Pyrrole and Aniline. The EIS technique allows the measurement of the interaction of molecules in a solution and the surface of the electrode. The modification with conductive polymer is carried out since it is biocompatible, and having a load causes the selectivity to increase and, in turn, its sensitivity, in addition to being modifiable according to the application. Our objective is to find the optimal conditions for the modification of the surface of the microelectrode with the conductive polymers for use with EIS by measuring different neurotransmitters to study and understand its release mechanism and then be able to measure different concentrations. Our neuropeptide of interest is Neuropeptide Y (NPY) because it is related with many processes in the human body as regulation of appetite, stress, and diseases as Alzheimer and Parkinson disease. For conductive polymer modification we used 0.5M Pyrrole and Aniline in 0.5M H2SO4. Cyclic Voltammetry (CV) is applied to carry out the electropolimerization with 1 cycle per depositions 3 times. We used two solutions of ACSF (artificial cerebral-spinal fluid) one of them mixed with 10pg/ml of NPY and the other one without NPY to study the current behavior. SEM and EDS were used to study the morphology. As results for ACSF and ACSF-NPY we observed an increased in current at the first electropolymerization and a decreased after the second electropolymerization for CV.

Introduction

A major and largely underestimated [1] concern of today's society is metabolic health (e.g. obesity) that is related to several diseases (e.g., diabetes, cardiovascular illnesses). Various diet strategies (e.g., ketogenic [2], intermittent fasting [3]) as well as exercising are explored but the assessment of treatment effectiveness on an individual level remains difficult. Desired are simple and accurate integrated devices that communicate wirelessly [4] and monitor metabolic changes conveniently at the point-of-care. This is possible through non-invasive acetone detection, a metabolic breath marker of lipolysis [5]. In specific, acetone is formed during hepatic β-oxidation of fatty acids that further divide into acetoacetate [6] that undergoes decarboxylation and enzymatic degradation to acetone [6], which is highly volatile and measureable in exhaled breath [7]. This has led to the development of several acetone sensors in the last decade.

Most, however, suffer from cross-interference to endogenous (e.g., isoprene) and background interferants (e.g., alcohol from disinfectants) [8], and their evaluation in the application has been hardly considered, a general challenge in sensor science [9]. Catalytic filters [10] offer a simple and most effective approach to enhance selectivity and continuously mitigate the interference of confounders. Here, we present a low-cost and compact detector based on a flame-made Pt-loaded Al₂O₃ catalytic filter [11] coupled to a Si/WO₃ sensor [8] for rapid and highly selective acetone detection. We apply it for breath analysis to monitor exercising and fasting.

Method

Nine volunteers attended two separate appointments. In the first, they performed an exhaustive spiroergometry test for determination of the cardiorespiratory fitness, maximum oxygen uptake (VO₂) and second ventilatory threshold (VT₂). During the second appointment, all volunteers performed a cardiorespiratory fitness-adapted submaximal aerobic exercise protocol [12]. The protocol started at 20% of the individual VT₂ (determined in the first appointment) and increased by 10% every 5 min. Breath was sampled every 5 min during exercise, as well as every 30 min during 3 hours of post-exercise rest with an end-tidal breath sampler [13] and analyzed by PTR-ToF-MS and with and without a Pt/Al₂O₃ filter-enhanced Si/WO₃ sensor.

Results and Conclusions

Figure 1 shows the normalized (i.e., to the baseline at t = 0) end-tidal acetone concentrations during exercising (0 ≤ t < 60 min) and fasting (60 < t ≤ 240 min) for all nine volunteers, as determined with bench-top PTR-ToF-MS (circles) and the Pt/Al₂O₃ filter-enhanced Si/WO₃ sensor (triangles). The error bars indicate the standard error of the mean (SEM) for all volunteers. Baseline acetone concentrations (0.4 - 1.7 ppm) increase marginally during the exercise phase (i.e., < 5%). In contrast, a strong increase (up to 124%) takes place during subsequent fasting. This indicates that the exercise stimulated the body fat metabolism.

The impact of the filter is shown best when monitoring breath acetone in situ during exercising. In fact, the sensor with filter follows closely the bench-top PTR-ToF-MS, while the sensor alone deviates (Figure 1a, squares). This might be related to endogenous isoprene (Figure 1b, diamonds), that spikes immediately in breath during muscle activity (up to 656 ppb within the first 5 min [14]) and then declines. While the sensor responds to isoprene [8], this is mitigated by the filter (triangles). Also varying background ethanol (stars) from hand disinfection [15] may affect the sensor. Thus, with filter, this detector is promising for robust and in situ metabolic monitoring to guide personalized dieting and exercising.

References

[1] S. T. Nyberg et al., Lancet Public Heal., 3, (2018), 490–497.

[2] A. T. Güntner, J. F. Kompalla, H. Landis, S. J. Theodore, B. Geidl, N. A. Sievi, M. Kohler, S. E. Pratsinis, and P. A. Gerber, Sensors, 18, (2018), 3655.

[3] S. D. Anton, K. Moehl, W. T. Donahoo, K. Marosi, S. A. Lee, A. G. Mainous, C. Leeuwenburgh, and M. P. Mattson, Obesity, 26, (2018), 254–268.

[4] S. Abegg, L. Magro, J. van den Broek, S. E. Pratsinis, and A. T. Güntner, Nat. Food, 1, (2020), 351–354.

[5] M. P. Kalapos, Biochim. Biophys. Acta, 1621, (2003), 122–139.

[6] M. Evans, K. E. Cogan, and B. Egan, J. Physiol., 595, (2017), 2857–2871.

[7] A. T. Güntner, N. A. Sievi, S. J. Theodore, T. Gulich, M. Kohler, and S. E. Pratsinis, Anal. Chem., 89, (2017), 10578–10584.

[8] I. C. Weber, H. P. Braun, F. Krumeich, A. T. Güntner, and S. E. Pratsinis, Adv. Sci., 7, (2020), 2001503.

[9] A. Lewis and P. Edwards, Nature, 535, (2016), 29–31.

[10] J. Van den Broek, I. C. Weber, A. T. Güntner, and S. E. Pratsinis, Mater. Horizons, (2020), DOI:10.1039/D0MH01453B.

[11] R. Strobel, W. J. Stark, L. Mädler, S. E. Pratsinis, and A. Baiker, J. Catal., 213, (2003), 296–304.

[12] K. Königstein, S. Abegg, A. N. Schorn, I. C. Weber, N. Derron, A. Krebs, P. A. Gerber, A. Schmidt-Trucksäss, and A. T. Güntner, J. Breath Res., 15, (2020), 016006.

[13] S. Schon, S. J. Theodore, and A. T. Güntner, Sensors Actuators, B Chem., 273, (2018), 1780–1785.

[14] J. King, A. Kupferthaler, K. Unterkofler, H. Koc, S. Teschl, G. Teschl, W. Miekisch, J. Schubert, H. Hinterhuber, and A. Amann, J. Breath Res., 3, (2009), 027006.

[15] V. Bessonneau and O. Thomas, Int. J. Environ. Res. Public Heal., 9, (2012), 868–879.

Figure 1

Current approaches towards drug dosing rely on venous draws measurements performed on test patients which are laboratory-analyzed and returned the following days. This practice forces physicians to administer potentially toxic or ineffective concentrations of drugs to patients since dosages are determined based on weight and age of this test group even though pharmacokinetics may differ in each individuals. Having a technology that would in contrast allow, direct, continuous, real-time monitoring of drugs in the living body would revolutionize healthcare and allow personalized drug dosage and adjustment while enabling the development of artificial organs responsible of adjusting these levels.

Motivated by this goal, we have developed a class of electrochemical aptamer-based (E-AB) sensors[1]. These sensors are comprised of a redox-reporter-modified DNA "probe" that is attached by one terminus to a self-assembled monolayer deposited on an interrogating electrode. The binding of an analyte to this probe alters the kinetics with which electrons exchange to/from the redox reporter via binding-induced conformational changes producing an easily measured change in current when the sensor is interrogated using square-wave voltammetry (see Figure) [2]. E-AB sensors are capable of detecting with high specificity their molecular targets in flowing whole blood and directly in the living body. I will present during this presentation some of these sensors deployed in the vein [3] and in the brain of sedated rats to monitor the pharmacokinetics of the antibiotic, vancomycin. The ability of acquiring high frequency measurements of drug plasma levels using these biosensors has also allowed us to develop a technology that improves our ability to deliver them [3]. Due to their small size, these sensors can also be deployed directly in the brain of freely moving animals to monitor molecules with unmatched temporal resolution [4]. All these advancement in developing E-AB sensors are aimed towards developing new analytical tools for personalized medicine while improving our understanding of drug metabolism.

[1]: Dauphin Ducharme, P. and Plaxco, K. W. Anal. Chem. 2016, 88, 11654–11662.

[2]: Li, H., Dauphin Ducharme, P., Ortega, G., Plaxco, K. W. J Am. Chem. Soc. 2017, 139, 11207-11213.

[3]: Dauphin-Ducharme, P., Yang, K., Arroyo-Currás, N., Ploense, K., Zhang, Y., Gerson, J., Kurnik, M., Kippin, T. E., Stojanovic, M., Plaxco, K. W. ACS Sensors 2019, 4, 2832-2837.

[4]: Ploense, K., Dauphin-Ducharme, P., Arroyo-Currás, N., Williams, S.; Schwarz, N., Kippin, T. E., Plaxco, K. W. 2020 Under review.

Figure 1

Here we present our results on the electric field-induced melting of surface-bound DNA (i.e. electrochemical melting). In particular, we examine the effects of the DNA-binding compound cisplatin on the stability of DNA within high electric fields. Cisplatin is an anticancer drug that has been used to successfully treat testicular, ovarian, and bladder cancers, among others (1). It crosslinks DNA between the N7 atoms of purine bases, resulting in changes in structure and stability, ultimately interfering with vital cellular processes in vivo.

Self-assembled monolayers (SAMs) of thiol-modified oligonucleotides on gold electrodes provide selective and sensitive interfaces for the development of chemical and biological sensors (2). While many transduction mechanisms have been utilized (including electrochemical, optical, and gravimetric) and many analytes targeted (e.g. small DNA-binding molecules, heavy metals, and specific DNA sequences), the unique steric and electrostatic environment of the crowded DNA in the electrical double-layer complicates the behavior of these biosensors (3). Electrical potentials applied at the DNA-SAM have been shown to modulate the behavior of these monolayers (4). Here, we utilize the electric field-effect to induce melting of the bound doublestranded DNA, and we monitor the kinetics of the melting process using square wave voltammetry (5).

We find that the effect of cisplatin on the melting behavior depends on the method used to the prepare the monolayer, in particular the melting depends on surface-coverage and heterogeneity of the resulting layers. Under some conditions, cisplatin results in an apparent stabilization of the DNA-SAM, and a destabilization in others. Significant differences in the resulting melting curves suggest that the mode of cisplatin-DNA binding varies depending on the SAM preparation method. The methodology presented here has the potential of (1) providing an electrochemical approach for studying the interaction between DNA and small molecules, and (2) providing a method for indirectly assessing the heterogeneity of DNA-SAMs via electrochemical melting in the presence of cross-linking agents.

1. R. A. Alderden, M. D. Hall and T. W. Hambley, Journal of chemical education, 83, 728 (2006).

2. J. J. Gooding and N. Darwish, The Chemical Record, 12, 92 (2012).

3. P. Gong and R. Levicky, Proceedings of the National Academy of Sciences, 105, 5301 (2008).

4. U. Rant, K. Arinaga, S. Fujita, N. Yokoyama, G. Abstreiter and M. Tornow, Organic & biomolecular chemistry, 4, 3448 (2006).

5. D. Ho, W. Hetrick, N. Le, A. Chin and R. M. West, Journal of The Electrochemical Society, 166, B236 (2019).

Introduction

Abnormal dopamine levels lead to severe diseases and disorders, including Parkinson's disease, Alzheimer's disease, and schizophrenia [1]. There is a need for sensitive and cost-effective dopamine sensors to provide rapid diagnosis at the point of care. Several approaches, such as high-performance liquid chromatography-mass spectrometry, fluorescence, and chemiluminescence based sensors, have been reported for dopamine detection with good sensitivity. However, these techniques generally are tedious, time-consuming, require sophisticated instruments, and thus not suitable for point of care (POC) testing. On the contrary, electrochemical sensors are promising due to their numerous advantages, including simple operation, faster response, low-cost, and accessibility for onsite monitoring. In addition, tracing dopamine, an electroactive molecule, with an electrochemical technique is also favorable. However, dopamine's electrochemical oxidation is often disrupted by the interference from other electroactive molecules (e.g., ascorbic acid, uric acid) due to their overlapping oxidation peak potentials and the higher physiological concentrations comparing to dopamine in biological fluids.

A wide range of chemically modified electrodes is explored to improve the electrocatalytic capability towards dopamine oxidation. In this regard, the modification of electrodes with carbon-based nanomaterials and redox mediators has gained keen interest. For instance, ferrocene (Fc) and its derivatives can catalyze the oxidation or reduction of several biomolecules with advantages of fast response, stable redox states, electrochemical reversibility, and regeneration at low potential [2]. However, immobilization of Fc on electrodes remains challenging due to their weak adsorption and low stability. In this work, we create a new sensor by decorating ferrocene (Fc) functionalized gold nanoparticles (AuNPs) on carbon nanotube (CNT) to form an electroactive composite (Fc-AuNPs-CNT) for electrochemical detection of dopamine. The Fc functionalized AuNPs provide excellent catalytic activity towards dopamine with improved electron conductivity. Furthermore, the larger active surface area provided by CNT also enhances the dopamine reaction on the electrode.

Method

The synthesis of AuNPs decorated multiwall CNT (MWCNT) was obtained through citrate reduction. During the pre-mixture of MWCNT and chloroauric acid (HAuCl₄), HAuCl₄ molecules are absorbed on the MWCNT and initiate the growth of AuNPs upon the addition of sodium citrate at room temperature. The AuNP surfaces were then functionalized with cysteamine (Cys) for the conjugation of ferrocene carboxaldehyde (Fc-CHO), leading to a Fc-AuNPs-CNT nanocomposite. The resulting nanocomposite was drop-casted with chitosan (CS) as a binder onto a screen-printed carbon electrode (SPE). The film morphology and elemental composition were characterized by scanning electron microscopy (SEM) and energy dispersive X-Ray analysis (EDX). Electrochemical responses were obtained with cyclic voltammetry (CV).

Results and discussion

The Fc-AuNPs-CNT composite exhibits a pair of stable and well-defined redox peaks in CV contributed by the ferrocene and ferrocenium (Fc/Fc+) redox couples. The Fc element peak in EDX further confirms the successful conjugation of Fc towards AuNPs. The Fc-AuNPs-CNT composite was demonstrated for dopamine sensing within 10 s incubation. Dopamine oxidation peak appears around +0.3V in anodic sweep, and its two reduction peaks appear around 0.15V and -0.25V. Increasing dopamine concentrations raises both oxidation and reduction currents. Compared with the AuNPs-CNT composite, Fc-AuNPs-CNT composite exhibits ~3 times higher response for the oxidation of 1 mM dopamine. The synergistic effects of Fc-AuNPs-CNT improve electron transfer rate between dopamine and electrode and convey superior electrocatalytic activity toward dopamine oxidation and reduction. The proposed sensor has a dynamic range of detection ranging from 10 µM to 1 mM dopamine and a low detection limit of 3 µM. The sensor also provides selective detection to discriminate ascorbic acid, glucose, hydrogen peroxide. The electrochemical property of Fc remains stable under repetitive potential sweep. No significant change in peak intensity was observed.

Conclusion

The Fc-AuNPs-CNT nanocomposite-based sensor presented here provides simple, cost-effective, portable, and rapid detection of dopamine with good sensitivity and selectivity, making it suitable for POC testing.

References

[1] X. Liu, H. Zhu, X. Yang, An electrochemical sensor for dopamine based on poly (o-phenylenediamine) functionalized with electrochemically reduced graphene oxide, Rsc Advances 4(8) (2014) 3706-3712.

[2] E. Scavetta, R. Mazzoni, F. Mariani, R. Margutta, A. Bonfiglio, M. Demelas, S. Fiorilli, M. Marzocchi, B. Fraboni, Dopamine amperometric detection at a ferrocene clicked PEDOT: PSS coated electrode, Journal of Materials Chemistry B 2(19) (2014) 2861-2867.

Figure 1

Light-addressable potentiometric sensor (LAPS) is a kind of semiconductor sensor based on electrolyte-insulating -semiconductor (EIS) structure that is sensitive to surface potential. Due to the light-addressability, it can record the photocurrent signals point to point where the modulated laser beam focused on. Cells encapsuled in hydrogel scaffold to constructed the 3D cell model can form chemical and oxygen gradient thus causing different metabolism state in different spatial position. In this study, a partial thinned Si₃N₄-LAPS chip integrated with microfluidic channel has been constructed for real-time 3D cell metabolism state monitoring. HepG2 cells are encapsulated in hydrogel to construct 3D liver cancer model. The system consists of sensing component, optical component, precision electronically controlled stage and a homemade software based on LabVIEW. In the bioreactor, cells take in high-glucose medium to secrete acidic metabolites, which leads to a decrease in photocurrent dramatically. With the help of this system, real-time visualization of cell metabolic dynamic process can be achieved. Moreover, medium or anti-cancer drugs can be automatically delivered to the bioreactor with the assistance of microfluidic technology to carry out cell culture and drug efficacy evaluation. This high-resolution photoelectrochemical imaging system has potential to achieve long-term drug screening platform.

Key words Light-addressable potentiometric sensor, 3D cell model, chemical gradient, cell metabolic visualization, drug screening

Introduction

Cellular metabolism is one of the most basic physiological functions of cells to maintain viability. Cells absorb and decompose various nutrients to produce energy necessary for proliferation and differentiation, while secreting acidic metabolites. Glucose, as the main nutrition source of cells, will be decomposed into energy and acidic substance under both aerobic and anaerobic conditions. The acidic products (such as carbon dioxide, lactic acid) will cause the pH decrease in the extracellular microenvironment. Therefore, the extracellular acidification rate (ECAR) can be used to characterized the cellular metabolic level. Nowadays, three-dimensional cell culture model has attracted the attentions of researchers, which can maintain more physiologically relevant microenvironment resemble to in vivo. Among all these models, 3D cell culture integrated with microfluidic chip has the unique advantages to recapitulating the cell microenvironment. Nevertheless, there exists general problems (oxygen concentration, chemical gradient) due to the special property of 3D cell model.Traditional ECIS and MEA system can not detect the spatial distribution of extracellular acidification rate,but laser beam could be focused and irradiated on the designated LAPS area to realize the light-addressable function based on the characteristics of LAPS. So,scanning the light spot along the chip can obtain spatial distribution of current signal to solve this problem.

Method

Si₃N₄-LAPS chip and backside illumination method were adopted with backside-thinned was thinned. The thickness of the silicon substrate is 500 μm, and the central area is thinned to 100 μm (Fig.1B) to reduce the lateral diffusion of carriers in the semiconductor layer and improve the sign-to-noise ratio while providing sufficient mechanical support.

Before seeding cells, the LAPS chip was cleaned by deionized water, sterilized by UV and 75% ethanol, and incubated with 5 μg/mL gelatin at 4 °C overnight to improve the adhesion ability to cells.

HepG2 cells were cultured in DMEM medium with 10% FBS, 100 U/ml Penicillin and 100 mg/ml Streptomycin. Then cells suspended in the medium were mixed in matrigel and cross-linked in a 37 °C humidified incubator with 5% CO₂ for 30 minutes. Drug test is conducted by doxorubicin.

The stage performed grid scanning. In the X-axis direction, the resolution is determined by the stepping distance, and in the Y-axis direction, the motor keeps continuously moving, and the signal is continuously collected. The spatial resolution is determined by the movement rate and the sampling rate.

Results and Conclusions

In order to maintain highly stability of the optical lens group, the optical path is fixed and the sensing chip can move with the stage; the chip is mounted on the stage by an adjustable fixing scaffold, and the laser reflector is fixed on an immovable base. The 680 nm red light emitted by the laser diode is transmitted through a multi-mode fiber and collimated by fiber collimator, then irradiated on the back of the chip from the vertical direction through an aspheric lens (Figure1A).

The horizontal resolution, which was consistent with motor, was step set to 50 μm. The motor speed is 2.5mm/s, the sampling rate is 50k/S, the number of samples per pixel is 2000. The obtained real-time current grayscale image is shown in Figure 1C. The white part (high current value) is the thinning area of the chip, that is, the effective sensitive area; the black part is the non-thinning area, and the gray is the intermediate transition area. The converted pseudo-color image is shown in Figure 1D. The number of pixels of the image is 142×128, and the actual size is 6mm×6mm, indicating that this LAPS scanning imaging system can obtain an imaging resolution of less than 50 μm.

Hanging drop method was conducted to achieve cell 3D culture. The microscope brightfield image of 3D HepG2 cell sphere is shown in figure 1E and green fluorescence image is shown in figure 1F. Then 3D cell sphere were seeded into microfluidic LAPS chip to realize real-time visualization of cell metabolism.

Figure 1

Introduction

Cortisol, as a steroid hormone secreted by the human body, is a typical stress marker. It plays an important role in regulating blood pressure, blood sugar, carbohydrate metabolism and other physiological processes. Among them, measuring saliva cortisol seems to be advantageous because it is a relatively easy and non-invasive method to extract saliva samples, and saliva can be conveniently processed and stored. Under normal circumstances, the body can well control the secretion of cortisol and regulate the content of cortisol in the blood. However, under greater physical and psychological stress, a high level of cortisol would be induced and kept for a long time, which will lead to the blood sugar, increased appetite, increased weight, and extreme fatigue, etc. The traditional methods for cortisol detection have biochemical analysis, radioimmunoassays, which is based on large-scale equipment and difficult to real-time monitoring. Therefore, the development of rapid, on-situ and sensitive cortisol detection products has important national defense strategic significance.

In this study, the Cu-MOF catalyst integrated-antibody was used for cortisol in-situ colorimetric immunoassay based on the smartphone-based portable electric eye (E-eye) (Fig.1). As a metal-organic framework, Cu-MOF can conjugate with antibody without complicated operations, and then specifically recognize cortisol. As a catalase, Cu-MOF can not only catalyze the coloration of TMB, but also amplify the signal, and also protect the antibody from long-term high temperature. Based on the indirect competition method, the cortisol in the samples competes with the cortisol-BSA coated on the microtiter plate to bind the cortisol antibody. And then the conjugation of MOF-antibody (Cu-MOF@IgG) will bind with the antibody, which could catalyze the H₂O₂ and TMB substrates to blue color and amplify the signal, which could be quantitative recognition by portable E-eye. The degree of blue is inversely proportional to the cortisol content. Based on the portable E-eye, the proposed method processes the features of rapid, simple, high sensitivity and high throughout, which could achieve cortisol in-situ detection in practical applications.

Preparation of Cu-MOF

The Cu-MOF was fabricated by mixing 7.8mM CuCl₂ aqueous and 31.2mM 4,4'-bipyridine equally with 2h gentle stirring under room temperature. The blue precipitates were obtained by centrifugation and washed by ultrapure water. Then it was re-dissolved in 500µl pure water for further use. The catalytic activity of Cu-MOF was verified by adding Cu-MOF to the H₂O₂ and TMB mixture. Under the successful preparation of Cu-MOF, the mixed solution will turn blue due to the catalase activity of Cu-MOF. The pH of the system was adjusted by NaAC/HAc buffer. The morphology (Fig.2(a) and (b)) and the catalase activity (Fig.2(c)) of Cu-MOF materials were characterized by transmission electron microscope and microplate reader.

Preparation of Cu-MOF@IgG

The Cu-MOF@IgG materials were formed via the following two steps: (1) 100µl of 20mg/L IgG was mixed with the 2mL of 7.8mM CuCl₂ aqueous at 4 ℃ for 10 min. (2) Then 2mL of 31.2mM 4,4'-bipyridine was added to the above mixture with 12h continuously stirring at 4 ℃. Also, the 4,4'-bipyridine above was prepared by dissolving 0.0487g of 4,4'-bipyridine powder in 10 mL ultrapure and water mixture with equal volume. (3) The product obtained from the above reaction was thoroughly centrifuged and washed thrice with ultrapure water, and then re-dissolved in 500ul pure water. The prepared Cu-MOF@IgG are stored at 4 ℃ for further use. The verification of the combination of Cu-MOF and IgG was as follows: First, it has been verified that the material of Cu-MOF has no absorption to the microtiter plate (Fig. 2(d)). The obtained Cu-MOF@IgG solution was diluted with carbonate buffer, and then coated on a microtiter plate. After incubating for 12 h at 4℃, the microtiter plate was washed by PBST (0.01 M PBS + 0.01% Tween) four times to remove excess free Cu-MOF and IgG. Then the mixture of H₂O₂ and TMB was added to the coated microtiter plate. After incubating of 20 min at 37℃, the color of the solution would be reserved for the successful preparation of Cu-MOF@IgG (Fig.2(e)).

Cortisol detection based on Cu-MOF@IgG

The cortisol-BSA was diluted to 2.7μg/mL with carbonate buffer (pH = 9.6), and then coated on a microtiter plate with a volume of 100μL per well. After incubating overnight at 4°C, the coated plate was washed with PBST four times to remove the excess free primary antigen. Then the free binding sites were sealed with 2% BSA solution for 2h at 37℃ to prevent the nonspecific binding. After washing with PBST four times, the cortisol standard and cortisol MAb was added to the plate and incubate at 37°C for 1 h. Then 100µl of Cu-MOF@IgG was added to binding with the coated cortisol MAb. After incubating at 37℃ for 2h, the unbound Cu-MOF@IgG was removed by washing with PBST four times. Then the mixture of H₂O₂ and TMB was added to wells and incubate for 20 min at 37°C. Finally, the absorbance of wells at 650nm was detected by the microplate reader. Also, the color changes could be detected by the homemade portable E-eye. As shown in Fig. 2(f), the detection limit of cortisol in the saliva is 0.12µg/L with the detection range of 0.88-1000µg/L.

Figure 1

Introduction

Halitosis refers to the offensive or unpleasant gas emitted from the mouth, also known as oral odor. Previous studies have shown that 80-90% of halitosis patients are caused by oral diseases^[1]. The patients with thick tongue coating and periodontal disease usually have obvious halitosis and the concentrations of hydrogen sulfide and methyl mercaptan in the breath of the oral cavity were significantly higher than those of the control group. Currently there are many methods for the diagnosis of halitosis in clinic. The commonly used direct measurement techniques include organoleptic measurement, gas chromatography and portable sulfide monitor^[2]. Organoleptic measurement, which involves using the human nose to score the intensity of odours emanating from the patient's mouth, is considered the gold standard for halitosis measurement^[3]. Gas chromatography is expensive and complex to operate, which requires professionals to operate. Commercial portable gas chromatography such as Oral Chroma™ (Oral Chroma™, Abimedical, Abilit Corp., Osaka, Japan) can detect the concentration of hydrogen sulfide, methyl mercaptan and dimethyl sulfide, but its price is still relatively expensive. Portable sulfide monitor such as the Halimeter (Interscan Corp., Chatsworth, LA, USA) is specially used to measure the total concentration of sulfide. But it takes a long time to measure and is only suitable for clinical application.

Based on the breath of patients with halitosis, this study uses an electrochemical sensor array to construct a small and intelligent electronic nose device for detecting bad breath, which realized the rapid quantitative detection of hydrogen sulfide and methyl mercaptan, and gave the preliminary screening results of halitosis grade and oral diseases.

Method

Halitosis samples were collected in the dental clinic. All patients had not smoked, drank or ate spicy food before the examination. The dentist first checks the patient's oral health, and then uses organoleptic measurement to evaluate the level of Halitosis. This information will be marked on the sampling bags. Finally, 100 ml exhaled air was collected with Tedlar sampling bag.

The volatile sulfur compounds in 81 samples of breath were qualitatively and quantitatively detected using GC-MS(gas chromatography and mass spectrometry) in the laboratory. ROC method was used to establish the threshold model of halitosis grade. In addition, a diagnosis model of orogenic diseases based on linear discriminant analysis, logistic regression and support vector machine was established. On the basis of the measurement result, a small intelligent electronic nose device for halitosis detection was constructed with an electrochemical sensor array, and the calibration was carried out using hydrogen sulfide and methyl mercaptan standard gases. The device can establish a connection with the mobile phone through Bluetooth. The level threshold model of halitosis and the diagnosis model of oral diseases are transplanted into the app on the mobile phone. The diagnosis results can be calculated by substituting the concentration value transmitted by the device into the model.

Results and Conclusions

The halitosis level threshold model and oral disease diagnosis model show that halitosis can be heard when the total concentration of methyl mercaptan and hydrogen sulfide is greater than 0.2ppm. When the total concentration is greater than 0.4ppm, the patient's halitosis is very obvious. The severity of periodontal disease (including periodontitis and gingivitis) is significantly correlated with the concentration of methyl mercaptan. When the concentration ratio of methyl mercaptan to hydrogen sulfide is greater than 1:1, patients usually have obvious periodontitis. The size of the electronic nose device based on the diagnosis model is 50 mm × 50 mm × 25 mm and the detection range of methyl mercaptan and hydrogen sulfide is 0 to 2 ppm with a detection limit of 0.05 ppm. In addition, the response time and recovery time of the sensor array are both within 15s, which greatly shortens the time compared with the common clinical detection methods. The standard gases of hydrogen sulfide and methyl mercaptan are mixed with air to simulate the breath samples with different odor levels. The gas detection process is controlled by smart phones, and the respective concentrations of hydrogen sulfide and methyl mercaptan can be calculated within 15 seconds. Taking threshold model as the evaluation standard, the accuracy of the detection results is more than 90%, which indicates that the device has the potential to become a household or portable electronic nose for halitosis detection.

References

[1] ZALEWSKA A, ZATOŃSKI M, JABŁONKA-STROM A, et al. Halitosis--a common medical and social problem. A review on pathology, diagnosis and treatment [J]. 2012, 75(3): 300-9.

[2] BICAK D A J T O D J. A current approach to halitosis and oral malodor-A mini review [J]. 2018, 12(322.

[3] SETIA S, PANNU P, GAMBHIR R S, et al. Correlation of oral hygiene practices, smoking and oral health conditions with self perceived halitosis amongst undergraduate dental students [J]. 2014, 5(1): 67.

Figure 1

In recent years, a significant amount of research has focused on nanoparticle-based "theranostic" agents for the treatment of a wide array of diseases, including cancer. This new paradigm in personalized medicine intends to exploit nanoplatforms that carry both therapeutic and diagnostic (theranostic) modalities. Compared with delivering drugs or imaging agents separately, theranostic agents can simultaneously deliver them to specific sites, enabling detection and treatment of disease in a single procedure. Many theranostic nanoplatforms are triggered by light, however, the vast majority of these are limited in that the ultraviolet or visible excitation light used has minimal applicability in biological applications. Lanthanide doped nanoparticles, on the other can be excited with biologically friendly near-infrared light (in the biological windows) and can emit in the ultraviolet, visible or near-infrared regions through a multiphoton upconversion process while simultaneously emitting in the near-infrared region through a Stokes or downshifted process. Hence, the upconversion luminescence can be used to trigger the therapeutic application (drug delivery, photodynamic therapy, etc.) while the near-infrared luminescence can be used for the diagnostic modality (bioimaging, nanothermometry, etc).

In this presentation, we will introduce lanthanide doped nanoparticles and demonstrate their usefulness in theranostics. In particular, we will show complex nanoparticle architectures can endow further functionality to these nanoparticles including the ability to decouple the theranostic processes that are conventionally delivered simultaneously.

In this presentation, a series of different plasmonic sensors for the detection of cancer biomarkers, COVID-19, cancer therapeutic drugs and the response of patients to oncological biological drugs will be presented. The plasmonic sensors are based on a portable 4-channel SPR instrument and surface chemistry developed to present fouling of the sensors in crude serum or plasma. Sensors for detecting methotrexate and for the immune response induced by asparaginase in leukemia treatments directly in sera of patients during ongoing therapies will be presented. Our work towards the detection of HER-2 positive breast cancers will also be presented, with a comparison of commercial and point of care SPR instruments. Our initial work on the development of a SPR-electrochemiluminescence instrument will be presented. We recently revealed that performing SPR and electrochemiluminescence on a single instrument provides unprecedented information about the interfacial processes occurring on the electrode. In addition, studying the energy transfer between the ECL and the plasmon revealed that the optically excited plasmon reduced the ECL intensity in the far-field by about 40% due to a lower plasmon mediated luminescence process. Hence, the combination of SPR and ECL is highly advantageous to study electrochemical processes.

Early diagnosis plays an increasingly significant role in current clinical drive. Detection, identification, and quantification of low abundance biomarker proteins form a promising basis for early clinical diagnosis and offer a range of important medical benefits. Amplification of light from NIR fluorophores by coupling to metal nanostructures, i.e. Metal Induced Fluorescence Enhancement (MIFE), represents a promising strategy for dramatically improving the detection and quantification of low abundance biomarker proteins, and potentially increase already sensitive fluorescence-based detection by up to three orders of magnitude. The amplification of the fluorescence system is based on interaction of the excited fluorophores with the surface plasmon resonance in metallic nanostructures. The enhanced fluorescence intensity due to the existence of metal nanostructures makes it possible to detect much lower levers of biomarkers tagged with fluorescence molecules either in sensing format or for tissue imaging. The first part of my talk will focus on some recent developments of plasmonic metal nanostructures by both "top-down" and "bottom up" methods. I will then discuss the prepared plasmonic nanostructures in the applications of biosensing and bioimaging, with the emphasis on plasmonic enhancement towards NIR I and NIR II regions.

Near-infrared (NIR) absorbing and emitting nanomaterials attract significant attention in bioimaging, which shows great potential for disease detection due to its high sensitivity at the subcellular level and low cost of related imaging facilities. However, currently available optical probes are mainly based on visible-emitting materials. The tissue-induced optical extinction and autofluorescence in the visible range result in limited penetration depth and ambiguous photoluminescence signal, which restricts their in vivo use. To address this issue, photoluminescent probes, with both absorption and emission wavelengths operating in the biological windows in the NIR range, in which tissues are optically transparent, are highly desired. Their integration with superparamagnetic nanomaterials to make a multifunctional platform further opens a wide range of promising applications, including bimodal imaging (photoluminescence and magnetic resonance imaging), synergistic hyperthermia (magnetothermal and photothermal), magnetic confinement of trace amounts of biospecies for ultra high-sensitivity biodetection, etc. In this talk, I will present our most recent work on the synthesis of NIR-emitting water soluble, stable core/shell/shell quantum dots (QDs) and multifunctional (NIR photoluminescent and superparamagnetic) nanoparticles and their use in biomedicine. For instance, in one case, multifunctional particles contain single superparamagnetic nanoparticles as cores and NIR-luminescent nanomaterials as shells. In another case, the multifunctional nanoplatform is compose of multiple superparamagnetic nanoparticles and NIR quantum dots in single particles. These different types of multifunctional particles are designed for different biomedical applications.

References: [1] ACS Nano 2019, 13, 408-420; [2] Adv. Funct. Mater. 2018, 1706235 (Inside Back Cover); [3] Chem. Mater. 2019, 31, 3201-3210.

The second near-infrared (NIR-II) biological window operating in the wavelength range of 1000-1700 nm is of considerable current interest for biomedical applications. This is mainly attributed to the high biological tissue penetration depth of the NIR-II light because of the less absorption and low optical scattering. However, previous studies regarding NIR-II optical materials mainly focused on the fluorescent upconversion nanomaterials, carbon nanomaterials, organic molecules and polymers for fluorescent bioimaging, photothermal/photodynamic therapy, and photoacoustic imaging. As is well-known, these optofunctional materials have limitations of low fluorescence efficiency, poor stability and poor multifunctionality. This talk will introduce NIR-II plasmonic materials recently developed in our lab for versatile biomedical applications. We have developed NIR-II plasmonic Au-Ag nanostructures, copper chalcogenides and their hybrids with excellent optical properties tunable by their size, composition and structure. We have demonstrated the excellent plasmonic properties of these materials and systematically elucidated their structure-plasmonic property relationship. We also exemplified the applicability of these NIR-II optofunctional materials for SERS biosensing, photoacoustic imaging, CT, and light-assisted cancer therapy with excellent performance. We believe that these NIR-II plasmonic materials hold considerable potential for multimodality theranostics of cancer.

The development of flexible and textile-based wearable pressure sensors has provided the opportunity of continuous and real time measurement of human physiological and biomechanical signals during daily activities. Pressure sensors are transducers that convert an exerted compression stress into a detectable electrical signal. Different transduction mechanisms have been introduced so far including triboelectricity, transistivity, capacitance, piezoelectricity, and piezoresistivity. Piezoresistive pressure sensors are the most widely used type due to the simplicity of their structure, and the wide range of materials that can be selected along with low-cost fabrication methods, and easy read-out system required for signal extraction.

A vast majority of piezoresistive sensors developed so far are on-skin sensors developed to detect subtle pressures (1 Pa-10 kPa) for touchpads and electronic skin applications. However, to sense physiological signals such as pulse, respiration, and phonation the sensor range of detection must fall within medium range of 10 kPa to 100 kPa. As expected, for larger-scale human motion detection such as sleep posture and footwear evaluation, the sensor must be able to sense compression stresses larger than 100 kPa. This wide range of detection required by the piezoresistive pressure sensors is one of the important challenges in designing these sensors.

Many of the piezoresistive sensors function based on employing the composite of conductive additives in an elastomer as an active layer. The functionality and sensitivity of these sensors are highly limited by the poor bulk mechanical properties of the elastomer in addition to unbreathability and the complications arising from the skin-sensor interface. Textile-based sensors overcome the issues regarding the elastomer sensors to a good extend. These sensors are mainly developed through coating fibers by conductive inks or intrinsically conductive polymers (ICPs). However, these sensors suffer from major drawbacks. First, the high conductivity of the conductive coatings leads to shortening in signals upon the application of a small amount of pressure. These sensors can respond either to static or dynamic pressures and once being pressed by a pressure, completely lose their sensitivity to further pressure exertions which resembles a connection/disconnection mode of performance. Second, the sensors need to be used in tight-fitting clothing to be able to capture signals which makes it quite uncomfortable and hinders the widespread adoption of the device in society.

Here, we introduce an all-fabric piezoionic pressure sensor, called "PressION", that successfully overcomes the mentioned drawbacks. We gained significant wide range of pressure detection (several kPa to larger than 100 kPa) through tunning the conductivity of the active layer by coating the fabric with an ion-conductive polysiloxane polymer. This optimal conductivity of the fabric makes the sensor capbale of simultaneously responding to both static and dynamic pressures. To overcome challenge of tight-fitting clothing, we took advantage of the fact that even with loose-fitting garments, there are still some parts of the garment which are pressed due to the exerted pressure by the body limbs over the torso or against an external surface such as bed/chair or even a subtle pressure exerted by a blanket over the body. The success of PressION was confirmed by data acquisition from different body locations (Fig1a-h). All these characteristics makes PressION an ideal candidate to be embedded in daily garments for future biomedical and psychological studies.

-Figure Caption-

A variety of physiological signals can be extracted by gently placing PressION on different locations of the body: (a) artery pulse from the face; (b) phonation from throat; (c) joint motion at the elbow or knee; (d) heartbeat from the chest while lying face down on a bed; (e) steps from the sensor used as an insole in footwear; and respiration from the sensor placed on the (f) back, (g) side or (h) front of the body.

Figure 1

Escalating rapidity in the technology leads to the exploration of wearable and mobile devices. There is paramount importance of wearable technology for health management as diseases can be timely monitored which in turn circumvent severe medical situations and decrease economic losses. Wearable technology involves the integration of electronic components into textiles or fabrics for wide range of applications including sensors, medical devices and safety instruments [1, 2]. Textiles, owing to their flexible and capillary characteristic, hold imperative position in the fabrication of rapid and inexpensive point-of-care sensing devices [3]. Metal oxides are affirmed to have potential applications in the fabrication of wearable sensors because of their unique electrical, electrochemical and biocompatible properties. With high surface to volume ratio, the sensitivity, selectivity and catalytic activity of metal oxide semiconductors is greatly enhanced. They serve as excellent candidates for the next generation of wireless sensing and online monitoring applications due to the fast response and long lifetime in different environmental conditions. Among the metal oxide semiconductors, SnO₂ is considered as one of the important material due to their number of properties, like biocompatibility, anti-oxidant , anti-bacterial, cytotoxic properties, strong physical and chemical interactions with adsorbed species, high degree of transparency in the visible spectrum, low operating temperature etc. [4]. There is an increased usage of SnO₂ as modifier electrode materials because of its astonishing features of thermal stability, biocompatibility, excellent bandgap, cost effective and abundant availability. The surface of working electrode is modified by nanomaterials of SnO₂ in combination with various metals, semiconductors and carbon derivatives for improved sensing performance. As per the recent reports, tin oxide in find its applications in the field of glucose sensing [5], gas sensing [6], monitoring heavy metals [7], phenol sensing [8] etc. Apart from the reported applications, tin oxide is proved to be a useful candidate in stealth technology [9].

References

1. Y. Yun, W.G. Hong, N. J.Choi, H.B. Kim, Y. Jun, H.K. Lee, Sci. Rep. 5 (2015) 10904.

2. Skrzetuska, M. Puchalski, I. Krucinska, Sensors 14 (2014) 16816.

3. Li, J. Tian, W. Shen, ACS Appl. Mater. Interfaces 2 (2010) 1.

4. Vidhu, D. Philip, Spectrochim. Acta, Part A 134 (2015) 372.

5. Sedighi, M. Montazer, S. Mazinani, Biosens. Bioelectron. 135 (2019) 192.

6. Wang, C. Zhao, T. Han, Y. Zhang, S. Liu, T. Fei, S. Liu, T. Zhang, Sens. Actuators, B 242 (2017) 269.

7. Wei, C. Gao, F.L. Meng, H.H. Li, L. Wang, J.H. Liu, X.J. Huang, J. Phys. Chem. C 116 (2011)1034.

8. Guo, P. Cai, , J. Sun, W. He, X. Wu, T. Zhang, X. Wang, Carbon 99 (2016) 571.

9. M Jeong, J. Ahn, Y. K. Choi, T. Lim, K. Seo, T. Hong, G. H. Choi, H. Kim, B. W. Lee, S.Y. Park, S. Ju, NPG Asia Materials 12 (2020) 32.

Introduction

Cardiovascular diseases (CVD), diabetes, and chronic kidney disease are among the most society concerns with a high correlation to blood pressure (BP) factor. As a result, developing a wearable device with a BP monitoring function is highly demanded health caring and monitoring. In order to measure BP, traditional methods are based on a sphygmomanometer as a gold standard, but it is not suitable for wearable applications. A solution, therefore, employed some sensors placed along artery paths of a human body and estimated BP from time transfer delay or Pulse Transit Time (PTT) of blood volume through these paths [1]. In practice, it is comfortable if all sensors are located centrally in a specific region and the wrist hand is considered the most convenient. For detecting the pulse wave of blood flow, photoplethysmography (PPG) is the king reigning the wearable device market for healthcare because of its small form factor, none-electrode-contact requirement, and multi-wavelength applications in extracting health indexes such as heart rate, SPO2, Glucose, Hydration levels, etc. Hence, a custom PPG sensor aiming to estimate BP in this paper is designed to optimize signal strength collecting and then applied to measure PTT, Reflective PTT (R-PTT) on the radial artery at three wrist's location as Fig. 1.

Method

First, a three-sensor array with four LEDs at different wavelengths (525nm, 630nm, 850nm, and 940nm) is designed. The distances between LEDs and a single photodetector (PD) are considered to find the optimized places to detect blood pulse and the intensity of light by controlling the electric current on LEDs. The PD is a broadband sensitivity sensor permitting it absorbs all reflecting lights from LEDs on blood flow.

Next, because light penetrates the skin depends on wavelength and intensity, we inherited the research work by Paul C.-P. Chao et al. [2] to select the optimal distances for placing LEDs. Next, a wearable device utilizing an ultra-low-energy analog-front-end, high-resolution 24bit-ADC, and small form factor is designed for collecting PPG data. After that, three well-known locations on a radial artery in Traditional Chinese Medicine at wrist, namely Cun, Guan, and Chi are considered to measure PTT for inter-sites and reflective PTT (R-PTT) for each site and compare each other's to evaluate how different in each method, which one is better for estimating BP. The position of these points and the depth of the radial artery had been widely studied in [3].

Finally, BP estimation based on R-PTT with a validation using a 24-hour ambulatory BP meter namely Oscar 2TM from SunTech Medical® permits us to extract heart rate, systolic BP and diastolic BP. Five healthy subjects with an average age of 30 ± 6, height 170 ± 8 cm are recruited for seven days (9am-9pm) experiments.

Results and Conclusions

From our experiment, we discover the in-phase and invert-phase effect which affect the ratio AC/DC for each wavelength. In invert-phase status, the longer wavelength gives the better AC/DC ratio while this rule will change in in-phase condition. In other words, depending on the locations and status of the PPG pulse, the AC/DC ratio of these signals have a different outcome. Next, for measuring PTT between each location pair Cun-Guan, Chi-Guan and Cun-Chi are ambiguous by phase effect and resolution of data sampling while R-PTT takes advantage because of using just a single site. The correlation values of the R-PTT to SBP and DBP are r = 0.8 and 0.65 respectively.

References

[1] Mukkamala, Ramakrishna, Jin-Oh Hahn, Omer T. Inan, Lalit K. Mestha, Chang-Sei Kim, Hakan Töreyin, and Survi Kyal, Toward ubiquitous blood pressure monitoring via pulse transit time: theory and practice, IEEE Trans. on Biomedical Engineering 62, no. 8 (2015): 1879-1901.

[2] Kao, Y.H., Chao, P.C.P. and Wey, C.L., 2018. Design and validation of a new ppg module to acquire high-quality physiological signals for high-accuracy biomedical sensing. IEEE Journal of Selected Topics in Quantum Electronics, 25(1), pp.1-10.

[3] Kim, J.U., Lee, Y.J., Lee, J. and Kim, J.Y., 2015. Differences in the properties of the radial artery between cun, guan, chi, and nearby segments using ultrasonographic imaging: a pilot study on arterial depth, diameter, and blood flow. Evidence-Based Complementary and Alternative Medicine, 2015.

Figure 1

Current technologies rely on bulkier instruments for detecting small molecule concentration in in the bodyfluids. However, they required time consuming preparation of mixture prior to analysis. In this study, we have demonstrated the new sensing method by using conducting polymer combined with our designed anchor molecule. The sensing interface has secured the ultra-high sensitivity to specific adsorption, which reform the electronic structure of polyaniline substrate by altering the delocalized electrons. Moreover, the PANI structure can be easily tuned for any given sensitivity of detection. Computationally, it was confirmed that the interactions between fentanyl and anchor interface are significantly strong (≈ -1.19 eV). The range of interaction energies is found due to the variation of the positions of key functional groups of fentanyl relative to the PAni-Arg structure. Particularly with strong interactions, spontaneous proton transfer is observed from the sensor to the fentanyl molecule. The sensor showed excellent response when used in different human bodily fluids. In addition to these interactions at the interface, we have demonstrated a new sensing technique as the first type of phase angle detection sensor. Using this technique, the changes at the interface have inherently amplified signals compared to changes in resistance, potential, and/or current signals. The new interface of biocompatible and flexible composite layers can be fabricated via electrochemical approach on variety of surface with minimal maintenance. This approach is particularly useful for assays that requires extremely low sample volume. We showed our designed method and device can be used for testing strip/vial for tear and blood samples and testing patch for in-situ monitoring through skin perspiration. Lastly, electrochemical approach is facile to use, it enables the portability of the sensor for on-site field measurements as well as in clinical settings. This work helped to resolve the challenge of monitoring small molecule drugs accurately and rapidly for point-of-care (POC) diagnosis in current clinical settings.

Introduction

Brevetoxins (named using a notation system PbTx 1-n) produced by the algae species Karenia brevis have been increasing in geographical distribution [1]. Humans' exposure to brevetoxins can occur through consumption of contaminated shellfish as well as aerosol exposure, causing a series of adverse symptoms clinically described as neurologic shellfish poisoning (NSP) including cramps, diarrhea, and coma, etc. Current methods for brevetoxin detection such as mouse bioassay (MBA), high-performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA), etc., feature the advantages of high sensitivity and reliability [2]. However, the dependence on bulk volume laboratory equipment and complicated pretreatment limit the widespread application in on-site brevetoxins detection. Therefore, a sensitive and efficient method for on-site determination of brevetoxin in contaminated shellfish is of great necessity.

In this work, an aptamer-based colorimetric assay is designed for sensitive detection of PbTx-2 which is the most predominant form of brevetoxins. The concentration of PbTx-2 can be easily transformed to the absorbance change which can either be directly visualized by a color change or can be quantified using a portable spectrometer. This approach provides an efficient way for PbTx-2 on-site detection combining the low cost and sensitivity of aptamer and the portability of self-designed spectrometer.

Method

The self-designed portable spectrometer (SDPS) is composed of a spectrometer sensor, a broad-band LED module (wavelength range of 470-850 nm), a micro-control module, and a communication module to contact with portable devices e.g., a personal computer (PC) (Fig.1 B, C). Optic fibers were used for guiding light emitted from the LED or reflected from the sample to reduce the volume of SDPS.

Aptamer BT10 was synthesized with high affinity to PbTx-2 according to published literature and it was heat-treatment at 92 ℃ for 10 min, followed by snap-chilling on ice for 5min and bringing it back to the room temperature to get an appropriate secondary structure [3]. Tyrosine-capped AuNPs were synthesized using tyrosine amino acid as a reducing and capping agent. 10 µL BT10 with a concentration of 0.3 µM was incubated with 20 µL different concentrations of PbTx-2 for 30 min to ensure complete binding. Then, 100 µL AuNPs solution was added into the above mixture for 5 min followed by 5 µL positively charged 3,3',5,5'-tetramethylbenzidine (TMB) for 5 min to induce aggregation of AuNPs. Therefore, free aptamers can be adsorbed on the surface of AuNPs and prevent the AuNPs from aggregation while the aptamer binding to PbTx-2 cannot (Fig.1 A). All reactions were performed in a solution chamber and finally the chamber was placed into the SDPS. Through signal processing on a PC, the concentration of PbTx-2 can be determined.

Results and Conclusions

From the spectrum results, the absorbance at 520 nm is decreasing while the absorbance at 610 nm is increasing with the concentration of PbTx-2 increasing (Fig.1 D). The ratio of absorbance at 610 nm and 520 nm is linear to the concentration of PbTx-2 with a linear range of 0.1 ppm to 10 ppm (Fig.1 E). Moreover, tyrosine-capped instead of citrate-capped AuNPs were chosen for colorimetric assay for experiment results show PbTx-2 can interact with citrate-capped not the tyrosine-capped AuNPs. The positively charged TMB was utilized for inducing AuNPs aggregation not the traditional sodium solution, for we find TMB can be more sensitive for aptamer number change on the surface of AuNPs. This approach allowed a limit of detection of 0.05 ppm PbTx-2 within a detection time of 40 min. Compared with other methods for PbTx-2 detection, this approach features portability and cost-effectiveness, which can be used in resource-limited areas.

References

[1] S.M. Watkins, A. Reich, L.E. Fleming, R. Hammond, Neurotoxic shellfish poisoning, Mar Drugs, 6 (2008) 431-455.

[2] J. Naar, A. Bourdelais, C. Tomas, J. Kubanek, P.L. Whitney, L. Flewelling, K. Steidinger, J. Lancaster, D.G. Baden, A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish, and mammalian body fluid, Environ Health Persp, 110 (2002) 179-185.

[3] S. Eissa, M. Siaj, M. Zourob, Aptamer-based competitive electrochemical biosensor for brevetoxin-2, Biosens Bioelectron, 69 (2015) 148-154.

Figure 1

Introduction

Toxic methanol occurs naturally in most distillates. Yet, suitable detectors to check product adherence to legal limits and, most importantly, monitor the methanol concentration in situ during distillation are not available. The legal limits for methanol contamination in the E.U. are 1350 g methanol per hL ethanol for Williams pear spirits, 1200 g/hL for apple and plum spirits and 1000 g/hL for cherry spirits. Significantly lower ones apply for brandy (i.e. 200 g/hL), Vodka from agricultural alcohol (30 g/hL) and London gin (5 g/hL). However, these limits are exceeded frequently, as shown for 183 commercial Williams pear spirits with methanol contents up to 1865 g/hL [1]. Usually, distillers rely on error-prone human olfaction while "gold standard" but off-line, time-consuming and expensive liquid or gas chromatography (GC) is rarely used.

Here, we monitor methanol concentrations during industrial distillation of cherry, apple, plum and herb liquor (196 samples) with a low-cost and handheld detector. Therein, it quantified individual methanol concentrations (0.1 – 1.25 vol% in liquid) with high coefficient of determination (R² = 0.92) by headspace analysis, as confirmed by GC. Typical ethanol contents (5 – 90 vol%) and aromas did not interfere, whereas methanol levels above legal limits were recognized within two minutes. As a result, this device enables distillers to guide distillation and check product quality, to prevent deadly methanol exposure and occupational hazard.

Method

Mash of plum, apple, cherry as well as a mixture of agricultural alcohol and herbs (for herb spirit) were processed in a distillery located in Switzerland (S. Fassbind AG). Distillate samples were drawn every 1 to 10 min (as compatible with the still operation) for sensor and GC analysis. The methanol detector is shown in Figure 1a. In brief, it consists of a needle (Sterican, B. Braun AG) mounted on a 4 mm (inner diameter) Teflon tube, a packed bed column with 150 mg of Tenax TA particles (35 m²/g, Sigma Aldrich), a flame-made chemoresistive gas sensor of 1 mol% Pd-doped SnO₂ [2] nanoparticles and a rotary vane pump (135 FZ 3V, Schwarz Precision). The pump and sensor are controlled by a microcontroller (Raspberry Pi Zero W) mounted on a tailor-made printed circuit board with wireless communication to a laptop or smartphone [3].

For each measurement, the vial containing the sample is shaken for 30 s, the sampling needle is inserted through the septum and the headspace is extracted at 25 ml/min for 10 s. Thereafter, the sampling needle is removed and ambient air is drawn for 6 min to convey the sample through the separation column and to analyze it by the sensor. Finally, the column and sensor are recovered by flushing with ambient. Methanol is quantified from the sensor response at a retention time of 1.5 - 1.7 min [4], based on previous calibrations in ternary mixtures of 0.1, 0.3, 0.5, 0.70, 1, and 1.25 vol% methanol (> 99.9 %, Sigma Aldrich) in 10, 30, 50, 70, 90 vol% ethanol (> 99.8 %, Fisher Scientific) and water (Milli-Q Synthesis A10, Merck).

Results and Conclusions

Figure 1b shows the results of selected samples (for better visibility) for plum (triangles), apple (circles), cherry (squares) and herb (stars), Already low methanol concentrations of 0.1 (cherry), 0.13 (apple) and 0.21 vol% (plum) in the mash (first data points) of the fermented fruits are detected. Highest methanol levels occur in plum distillates (triangles) that vary between 1.2 and 0.85 vol% and only drop significantly after 80 min reaching 0.2 vol% at the end of distillation. Similar methanol dynamics were observed for apple (circles), though at slightly lower concentrations ranging from 1 to 0.7 for t < 80 min. Distinctly lower methanol levels (0.5 – 0.4 vol%) were measured for cherry (squares). Lastly, there is herb spirit (stars) where methanol concentrations stayed constantly below 0.22 vol%. So, the handheld detector is able to follow the individual methanol dynamics of fruit and herb during distillation. Figure 1c shows a scatter plot of methanol concentrations in all (196) samples of herb (stars), cherry (squares), apples (circles) and plum (triangles) distillates, as measured by the handheld device and GC. Close agreement (ideal line, dashed) is observed over the entire concentration range of 0.1 to 1.25 vol%, as quantified with high coefficient of determination (R² = 0.92).

As a result, this handheld device is capable to identify critical methanol levels and could support distillers who mostly rely on olfactory analysis that is error-prone (given the indistinct smell of methanol over ethanol). Since the detector is fully integrated, pocket-sized, battery operated, indicating results on a laptop or smartphone and consists mostly of low-cost components, it is ideal for on-site application and should be affordable even for small distilleries.

References

[1] Adam, L.; Versini, G., A study on the possibilities to lower the content of methyl-acohol in eaux-de-vie de fruits. Office for Official Publications of the European Communities: Luxembourg, 1996.

[2] Pineau, N. J.; Keller, S. D.; Güntner, A. T.; Pratsinis, S. E., Palladium embedded in SnO₂ enhances the sensitivity of flame-made chemoresistive gas sensors. Microchim Acta 187 (2020), 96.

[3] Abegg, S.; Magro, L.; van den Broek, J.; Pratsinis, S. E.; Güntner, A. T., A pocket-sized device enables detection of methanol adulteration in alcoholic beverages. Nat Food 1 (2020), 351-4.

[4] van den Broek, J.; Abegg, S.; Pratsinis, S. E.; Güntner, A. T., Highly selective detection of methanol over ethanol by a handheld gas sensor. Nat Commun 10 (2019), 4220.

Figure 1

Introduction

Over 800 people died this year in Iran alone as a result of methanol poisoning [1]. In fact, such outbreaks are quite common with thousands of victims each year [2]. However, no inexpensive and fast point-of-care method exists for diagnosis of methanol intoxication to rapidly respond to disasters. Currently, methanol poisoning is detected directly through blood analysis by gas chromatography or indirectly through blood gas analysis [3]. Both require trained personnel, are expensive and rarely available in developing countries where most outbreaks occur. Blood methanol levels can also be determined non-invasively in exhaled breath (Figure 1a), analogous to ethanol as widely applied by law enforcement [4]. However, current chemical sensors cannot distinguish methanol from the usually much higher ethanol background in the breath of poisoned victims. Here, we present an inexpensive and handheld detector for rapid and highly selective methanol detection.

Method

The handheld detector is shown in Figure 1b. It consists of a capillary inlet for sampling from Tedlar bags, a separation column consisting of a packed bed of Tenax TA polymer sorbent to separate the breath mixture [5], a chemoresistive Pd-doped SnO₂ microsensor to quantify the methanol and ethanol concentrations, and a rotary vane pump (SP 135 FZ 3 V, Schwarzer Precision, Germany) drawing the sample through the column to the sensor. A microcontroller (Raspberry Pi Zero W, Great Britain) with integrated circuits on a custom-designed printed circuit board (PCB) is used for autonomous sensor heating, film resistance readout, pump flow control as well as wireless communication with a computer or smartphone [6].

The detector was validated with methanol-spiked breath of drunken volunteers (0.1% blood ethanol) [7]. Late expiratory breath was sampled into Tedlar bags and subsequently spiked with 10–1000 ppm methanol on a dynamic gas mixing setup to simulate poisoning without intoxicating volunteers. Samples were then measured by the detector and a high resolution proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS).

Results and Conclusions

The main challenge for chemical sensors is the selective detection of breath methanol over high ethanol concentrations present after consumption of contaminated beverages or during methanol poisoning treatment where ethanol is even used as an antidote. The present detector (Figure 1b) achieves this with a compact separation column, where ethanol absorbs stronger (and thus is retained longer) than methanol, analogous to GC. A downstream chemoresistive microsensor based on Pd-doped SnO₂ nanoparticles quantifies the methanol and ethanol sequentially with high sensitivity. Figure 1c shows the sensor response to breath (green) after consumption of an alcoholic beverage and when spiked with 23 (blue), 66 (purple) and 148 ppm (red) methanol. These methanol levels correspond to endogenous (0–10 ppm), harmless exogenous (10–52 ppm) and toxic concentrations (>52 ppm), respectively. Most importantly, the sensor detects no significant methanol concentration in the original breath (PTR-TOF-MS, <1 ppm) with sensor response below the LOD, as expected from physiological breath methanol concentrations (median 0.26 ppm), while the spiked concentrations are recognized distinctly at 1.8 min.

In total, 105 methanol-spiked breath samples from 20 volunteers after consumption of water, beer, liquor or wine were evaluated and the measured methanol concentrations of the detector and PTR-TOF-MS are shown in Figure 2a. Indicated also are the concentration ranges where antidote (>52 ppm, grey shaded) and hemodialysis (>131 ppm, red shaded) treatments are recommended from the corresponding blood methanol concentrations. The detector shows excellent agreement with PTR-TOF-MS (R² = 0.966) over the entire concentration range (14–1079 ppm) and in the presence of 0–316 ppm ethanol.

As a result, this device is promising to screen methanol poisoning and classify severity. This detector can be equipped with a disposable mouthpiece, as for commercial breath alcohol testers, and readily applied as a point-of-care diagnostic tool for fast screening of methanol poisoning by first responders and clinicians.

References

[1] H. Hassanian-Moghaddam, N. Zamani, A.-A. Kolahi, R. McDonald, K.E. Hovda, Double trouble: methanol outbreak in the wake of the COVID-19 pandemic in Iran—a cross-sectional assessment, Critical Care. 24 (2020) 402.

[2] The American Academy of Clinical Toxicology Ad Hoc Committee on the Treatment Guidelines for Methanol Poisoning:, D.G. Barceloux, G. Randall Bond, E.P. Krenzelok, H. Cooper, J. Allister Vale, American Academy of Clinical Toxicology practice guidelines on the treatment of methanol poisoning, Journal of Toxicology: Clinical Toxicology. 40 (2002) 415-446.

[3] J.A. Kraut, Diagnosis of toxic alcohols: limitations of present methods, Clinical Toxicology. 53 (2015) 589-595.

[4] A.T. Güntner, S. Abegg, K. Königstein, P.A. Gerber, A. Schmidt-Trucksäss, S.E. Pratsinis, Breath Sensors for Health Monitoring, ACS Sensors. 4 (2019) 268-280.

[5] J. van den Broek, S. Abegg, S.E. Pratsinis, A.T. Güntner, Highly selective detection of methanol over ethanol by a handheld gas sensor, Nat. Commun. 10 (2019) 4220.

[6] S. Abegg, L. Magro, J. van den Broek, S.E. Pratsinis, A.T. Güntner, A pocket-sized device enables detection of methanol adulteration in alcoholic beverages, Nature Food. 1 (2020) 351-354.

[7] J. van den Broek, D. Bischof, N. Derron, S. Abegg, P.A. Gerber, A.T. Güntner, S.E. Pratsinis, Screening Methanol Poisoning with a Portable Breath Detector, Analytical Chemistry. (Just Accepted) (2020)

Figure 1

The QS-B220 Explosives Trace Detector (ETD; Implant Sciences Corporation, Wilmington, MA, USA) was tested to determine whether Sample Traps (Impant Sciences Part No. 42200191 REV) contaminated with severe acute respiratory syndrome coronavirus (SARS-CoV-2) can be effectively decontaminated for re-use during normal operation. Sample Traps were inoculated with a known concentration of SARS-CoV-2 (strain USA-WA1/2020), allowed to dry at ambient conditions, and inserted into the QS-8220 ETD's Sample Desorber (i.e., the sample port). During normal operation, heat is generated and directed at the Sample Trap undergoing analysis, and it was the heating process that was evaluated for decontamination efficacy against the virus. Each Sample Trap was inserted into the QS-8220 ETD at least two times (constituted a single exposure) to ensure that it was completely heated. Multiple exposures were tested, but it only took a single exposure inside the QS-8220 ETD to result in no recoverable infectious SARS-CoV-2 (based on the 1.31 × 10¹ median tissue culture infectious dose [TCID₅₀] limit of detection) and expressed as 5.29 log reduction from the Sample Traps.

Introduction

Ascorbic acid (AA), also known as vitamin C, is a water-soluble vitamin that can promote the body's absorption of iron to prevent iron-deficiency anemia. It also has an antioxidant effect, which can inhibit free radicals from oxidative damage to the human body, thereby preventing tumors. Because of its antioxidant properties, AA can also be used as antioxidants in foods and has a wide range of applications in foods such as cured meat products, canned fruits and vegetables, and beer beverages.

Conventional AA detection methods such as fluorescence [1] and electrochemical methods are complicated and time-consuming, and are not suitable for rapid and portable detection. Based on this, this article proposes a high-efficient handheld spectrometer, which is mainly composed of a miniature spectrometer sensor, a broadband LED light source, an Arduino UNO board and a self-designed circuit board (Figure.1C). This device is equipped with a touch screen and some buttons which are designed to facilitate user operation (Figure.1 D). The entire system is powered by a rechargeable lithium battery. In addition, the device is designed by Solidworks software and fabricated by 3D printing (Figure.1 E). As shown in Figure 1C, the detection system is also equipped with 3D printed guide rails and a light-transmitting bracket, combined with commercial microplates to achieve detection of AA. The detected signal can be transmitted to a smartphone or PC via Bluetooth. The handheld spectrometer has high portability and simplicity and shows excellent sensitivity. Combined with the above high-efficient spectrometer, a colorimetric assay based on MnO₂ nanosheet for AA detection was established with great sensitivity and portability.

Method

MnO₂ nanosheets were firstly synthesized acting as a biomimetic oxidase with which TMB can be oxidized to oxide TMB with a color change from pale yellow to blue (Figure.1 A). The hydrochloric acid added can turn the color of the solution to yellow with characteristic absorption at 450 nm. When the solution contains AA, MnO₂ nanosheets can be reduced to Mn²⁺ resulting in the oxidation rate decreasing. That induces an absorption change at 450 nm indicating the concentration of AA (Figure.1 B) [2].

All the above reactions were performed in microplates and the microplates were pushed along the track in the middle of the handheld device and pushed out from the other end. The top of the device is equipped with a broadband LED light source. The emitted light passes through the solution in the microplates and is coupled to the micro spectrometer through a collimator below [3]. The optical signal is converted into an electrical signal and processed by the detection circuit board, and then transmitted to the Arduino microcontroller for analyze. Since the detection process is carried out inside the device, in a relatively dark environment, and the first well of the microplates contains the blank solution to eliminate the weak ambient light interference, the handheld spectrometer can be used on almost all occasions.

Results and Conclusions

Under optimized experimental conditions, different concentrations of AA samples were prepared using multiple dilution from 80 μM to 0.1563 μM. The blank solution without AA was also prepared as a control group. All the AA detection experiments were conducted by both the microplate reader and the handheld spectrometer, and the analytical performance was compared. The comparison results demonstrated the two instruments both have good linearity in the AA concentration range from 0.6250 μM to 40 μM. Furthermore, according to the 3σ/slope rule, the LOD of the handheld spectrometer is 0.4946 μM, which is slightly lower than the microplate reader. The good performance of the handheld spectrometer demonstrates it can be a promising tool for POC applications in food security, disease diagnosis, and environmental monitoring.

References

[1] Q. Jin, Y. Li, J.Z. Huo, X.J. Zhao, The "off-on" phosphorescent switch of Mndoped ZnS quantum dots for detection of glutathione in food, wine, and biological samples, Sens. Actuators B Chem. 227 (2016) 108e116.

[2] Y. Gan, N. Hua, C.J. He, S.Q. Zhou, J.W. Tu, T. Liang, Y.X. Pan, D. Kirsanov, A. Legin, H. Wan, P. Wang, MnO2 nanosheets as the biomimetic oxidase for rapid and sensitive oxalate detection combining with bionic E-eye, Biosens. Bioelectron. 130 (2019) 254e261

[3] Bayram, A., Horzum, N., Metin, A. U., Kilic, V., & Solmaz, M. E. (2018). Colorimetric Bisphenol-A Detection With a Portable Smartphone-Based Spectrometer. IEEE Sensors Journal, 18(14), 5948–5955. doi:10.1109/jsen.2018.2843794.

Figure 1

Quinoxaline heterocyclic aromatic amines (HAAs), are formed during meat and fish cooking, frying, or grilling at high temperatures. HAAs are classified as potent hazardous carcinogens, even though the HAAs are usually generated at very low concentrations (~ng per g of a food sample). This is because the HAA food contaminants effectively damage DNA by intercalation or strand break. Hence, chronic exposure to HAAs, even in low doses, can cause cancers of the lung, stomach, breast, etc. Currently, HPLC is used for the determination of these toxins in food matrices. However, this technique is expensive, tedious, and time-consuming. Therefore, fast, simple, inexpensive, and reliable HAAs determination procedures, without the need for separation of these toxins, in the protein food matrices are in demand. Molecularly imprinted polymers (MIPs) are excellent examples of bio-mimicking recognition materials. Therefore, they have found numerous applications in selective chemosensing. Within the present project, we synthesized a nucleobase-functionalized molecularly imprinted polymer (MIP) as the recognition unit of an electrochemical sensor for selective DPV and capacitive detection and determination of 2-amino-3,7,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (7,8-DiMeIQx) HAA. MIP-(7,8-DiMeIQx) film-coated electrodes were sensitive and selective to 7,8-DiMeIQx. The linear dynamic concentration range of the devised chemosensor extended from 12 µM to 0.4 mM 7,8-DiMeIQx and the imprinting factor was high, IF = 13.

The present work is part of a project that has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 711859. Scientific work was funded from the financial resources for science in the years 2017-2021 awarded by the Polish Ministry of Science and Higher Education for the implementation of an international co-financed project and National Science Centre Poland (grant No. NCN 2014/15/B/NZ7/01011 to W.K.)

Aluminum is one of the most widely distributed elements (7.5%) of the earth crust. The current research interest in the aluminum content in our foods and diets are related to the concerns about the possible association of excessive intake and/or elevated tissue levels of this element and linked with various disorders viz. Alzheimer's, Dialysis encephalopathy/dementia, bone disorder. Aluminum (Al) is present in our daily food naturally, but mostly in elevated level through the various food additives, by the use Al cookware, storage canes and Al wrappings. The migration of Al is known to increase with the acidity (pH of the food), duration of storage, and presence of salts and organic acids. As it is found commonly in our daily food products and it may not be the reach of every common people to have sophisticated laboratory test to understand the Aluminum content in the daily food products. Herein, we have developed a quick portable electrochemical platform to measure the threshold Al content in the readily available food samples mostly canned foods (juices having the pH acidic (pH 3.5-5.5). For that, we used Pd/C (palladium black catalysts) modified Au screen- printed electrode. The activity of the catalyst influenced by the added Al³⁺ content was analyzed using Electrochemical impedance spectroscopy. The calibrated dose rate (CDR) is obtained as a function of the change in Z mod (Ω) value from the Bode plot vs the concentration of added Al³⁺ in buffer of different pH in the concentration ranging 0.5µg/mL to 1mg/mL. The sensors were tested for real canned juice samples. Such method can be an easy and quick qualitative testing procedure for Aluminum poisoning in our everyday food products

Figure 1

Lung cancer is one the most common diseases worldwide with more than 1.6 million dead since 2012 [1]. The survival rate for lung cancer is also very low as the disease is generally diagnosed only at an advanced stage when the treatment is not possible as significant damage has been done [2]. Hence, there is a need for an early and timely non-invasive method for the diagnosis of lung cancer. Previous studies have shown the use of endogenously produced volatile organic compounds (VOCs) as target analytes for the detection of lung cancer. Endogenous VOCs are produced in the bloodstream as result of cellular metabolic pathways involved in maintaining homeostasis[3]. Subsequently, the VOCs diffuse into the alveolar air in the blood-gas interface quickly in the lung because of their low solubility in the blood and they are exhaled out of the lung[4]. Therefore, detection of VOC levels in exhaled breath can be used to study the biochemical pathways that have been altered as a result of tumorigenesis. A breathomics device can be developed and used as a tool for non-invasive, sensitive, and specific detection of lung cancer. Moreover, the presence of lung cancer tumor is not masked by other diseases since every disease has its own volatiles fingerprint[5] , thereby making the detection mechanism specific in nature. Electrochemical sensors are gaining widespread interest as they are robust in nature, have faster response time, and specific and sensitive signal response. Electrochemical gas sensors usually involve a two or three electrode setup that causes change in signal as a function of the output current/potential. Electrochemical sensors require an electrode, a transducer, and an electrolyte. A good electrolyte should have high chemical and electrochemical stability, should be stable at elevated temperatures and have high charge transfer ability. Room temperature ionic liquids (RTIL) are being explored as suitable electrolytes for gas sensing applications as they follow all these parameters. There is an increasing interest in the field of non-invasive disease diagnosis, primarily using breath as a sample for monitoring the presence of various metabolites. Breath alkanes such as heptane can be used as a signature target analyte to differentiate the healthy subjects from lung cancer patients. Levels of heptane in breath for a healthy individual are within 50 ppb-200 ppb range. Concentrations greater than 400 ppb are found in breath as a result of altered metabolic pathways. We demonstrate the proof of concept of an electrochemical VOC sensor specific and sensitive for the detection of heptane up to 400 ppb toward developing a handheld breath analyzer for on-site applicability (Scheme 1). We use RTIL as a transducer and the inherent redox properties of these VOCs such as heptane make them a suitable candidate for electrochemical detection. Moreover, RTILs allow easy removal of VOCs by joule heating and prevent fouling of the system, thereby increasing the shelf life of the sensor.

For this study, RTIL Triethylsulfonium Bis(trifluoromethanesulfonyl)imide with over 98 percent purity and used as received without any further purification. Trace Source™ disposable permeation tube for Heptane was purchased from KINTEK analytical. Chronoamperometry was used as transduction principle for detection and voltage applied was set within the electrochemical window of the RTIL. An electrochemical sensing platform is developed for the detection of heptane in vapor phase. The sensing platform can be used to monitor the presence of heptane in breath upto 400 ppb (LOD). The sensor showed dose dependent response for the concentrations ranging from 400 ppb to 5 ppm.

Scheme 1- Schematic illustration of the sensing strategy used for the development of the electrochemical RTIL-based sensor

Figure 1-Chronoamperometry scan was performed at −0.6 V for 60 s RTIL-modified interdigitated electrode (IDE) for the target analytes. Calibrated dose response chronoamperograms were observed for heptane, which varies with concentration.

References

1. J.-L. Tan, Z.-X. Yong, C.-K. Liam, J Thorac Dis, 8 (2016) 2772–2783.

2. R.L. Siegel, K.D. Miller, A. Jemal, CA Cancer J Clin, 65 (2015) 5–29.

3. W. Cao, Y. Duan, Clin Chem, 52 (2006) 800–811.

4. A.G. Dent, T.G. Sutedja, P. V Zimmerman, J Thorac Dis, 5 Suppl 5 (2013) S540-50.

5. G. Peng, M. Hakim, Y.Y. Broza, S. Billan, R. Abdah-Bortnyak, A. Kuten, U. Tisch, H. Haick, Br J Cancer, 103 (2010) 542–551.

Figure 1

Introduction

Chemical (eg pesticides, veterinary drugs, etc.) and bacteriological contaminants (eg. foodborne pathogens) could contaminate animal and plant derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites, dyes) are banned in foodstuffs of animal origin (eg. milk, meat, eggs, etc.) in European Union because of toxicological risks for the consumer. The European Regulation has set Minimum Required Performance Limits (MRPL) [1] or Reference Point for Action (RPA) for banned substances [2]. Food containing residues of substances at or above the MRPL or RPA are declared non-compliant and consignments are rejected from the consumer's market.

Screening methods are the first stage of food control and so are essential for food safety monitoring. Conventional screening methods are microbiological methods (eg. plate tests, tube tests), immunological methods (eg. ELISA, radioimmunoassays) or physico-chemical methods (Thin Layer Chromatography, High Performance Liquid Chromatography (HPLC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). These methods sometimes lack of sensitivity or specificity; they also could be time and money consuming. There is thus a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach.

Electrochemical immunosensor

An innovative electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [3]. This technique presents major advantages: low cost (eg. disposable electrodes, potentiostat), promising detection limits, portability, and possible automatisation. Our laboratory has evaluated the transferability of the method. An electrochemical immunosensor has been developed for the detection of chloramphenicol residues in honey as a proof of concept. Honey composition and colour varies considerably depending on the botanical origin. Moreover some honey ingredients can interfere with the electrochemical detection, especially substances with antioxidant activities (eg. polyphenols). Therefore a lot of work had to be done to improve sample extraction to reduce matrix effects.

The objective is to develop an electrochemical bead-based immunosensor for the multiplex detection of banned antibiotics (eg. chloramphenicol, nitrofuran metabolites, dyes) in bovine milk.

Method

Antibodies (Abs) against antibiotics are grafted on the surface of magnetic beads (MBs). Milk samples and antibiotic conjugated with Horseradish peroxidase (HRP) are mixed with MBs-Abs. A competition occurs between the HRP conjugates and the antibiotic residues if present in the sample, for the binding to the antibody. The MBs are washed to remove free antibiotics and conjugates. Then, a Screen Printed Carbon Electrode (SPCE) with MBs on its surface (maintained by a magnet) is soaked into a buffer solution containing hydroquinone; when adding hydrogen peroxide (H2O2) to the solution, an electrochemical signal is produced, due to the enzymatic activity of HRP and measured. The electrochemical signal is inversely proportional to the antibiotic concentration in the sample.

Results and Conclusions

Screening methods for the detection of veterinary drugs in food products have to be validated according to the European regulation [1] and to the European guideline for the validation of screening methods [4]. After the development and the optimization of the analytical parameters (eg. sample preparation, HRP concentration, incubation time, applied potential, etc), the methods developed for single compounds will be evaluated and validated according to the European regulations. Then the single compound methods will be merged into one multiplex method if possible. The results will be presented to the conference, discussing the advantages and drawbacks of electrochemical biosensors for the screening of antibiotic residues in food products.

References

1. Commission Decision (EC) N° 2002/657 of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. 2002: Official Journal of European Communities. p. 8-36.

2. Commission Regulation (EC) N° 470/2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin 2009, The European parliement and the Council: Official Journal of the European Union p. 11-22.

3. Conzuelo F, Ruiz-Valdepeñas Montiel V, Campuzano S, Gamella M, Torrente-Rodríguez RM, Reviejo AJ, Pingarrón JM. 2014. Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors. Anal. Chim. Acta. 820:32-38.

4. CRL, Guideline for the validation of screening methods for residues of veterinary medicines (initial validation and transfer). 2010: Available from:<http://ec.europa.eu/food/food/chemicalsafety/residues/lab_analysis_en.htm>: Guideline_Validation_Screening_en.pdf. p. 1-18.

Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all or none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectable with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all-or-none. Here we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39317 ± 1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13310 ± 1127) for each stimulated exocytosis event. Thus, beta cells release only one-third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type-2 diabetes. Finally, these data therefore raise the tantalising possibility that pharmacological intervention can be used to increase the fraction released during beta cell exocytosis and lead to new therapies for type-2 diabetes.

Molecularly imprinted polymers (MIPs) belong to the illustrious examples of bio-mimicking recognizing materials.¹ They have found numerous applications in the fabrication of selective chemosensors.² Their analytical parameters, such as sensitivity, selectivity, and detectability, are almost as high as those of biosensors. Additionally, MIP based chemosensors are superior to biosensors concerning their ease of fabrication, durability, and tolerance to harsh conditions, including elevated or decreased temperature, high ionic strength, extreme pH values, the presence of heavy metal ions and organic solvents in the samples. Conductive MIPs have recently become more frequently applied. That is mainly due to the easy control of MIPs deposition as thin films by electropolymerization.³

For the electrochemical determination of non-electroactive analytes, some external redox probe is usually added to the test solution. It is assumed that target analyte molecules' binding into molecular cavities causes MIP film swelling or shrinking. According to the so-called "gate effect" mechanism, this polymer "breathing" causes changes in the redox probe permeability through an MIP film, thus changing faradaic current corresponding to the redox probe's reduction or oxidation in cyclic voltammetry (CV) and differential pulse voltammetry (DPV) determinations.^4-5 This mechanism is operative for nonconductive MIP films. Another mechanism may be considered for surface imprinted macromolecular compounds, e.g., proteins. A drop in the faradaic current of the redox probe accompanying protein adsorption originates from physical blocking of the electrode surface by their bulky nonconductive molecules.⁶ But both of these mechanisms seem to be invalid in case of electrochemical sensors based on conductive MIP films. In our previous studies, we demonstrated that a drop in the DPV current, caused by the appearance in a solution of an analyte, at conductive MIP film-coated electrodes might originate not from hindering the diffusion of the redox probe through the film but from changes in electrochemical properties of the film itself ⁷. Suppose the redox probe diffusion through the MIP film is not a decisive parameter for the faradaic current involving. Then, in the, e.g., DPV, determinations of electroinactive analytes at conductive MIP film-coated electrodes, this diffusion may be eliminated. For that the redox probe could be immobilized inside the MIP film matrix.

Herein, we propose to deposit a self-reporting MIP film and apply it for fabrication of the selective electrochemical sensor determining the target analyte in the redox probe free test solutions. For that purpose, a ferrocene redox probe was covalently immobilized in a bis-bithiophene polymer molecularly imprinted with the p-synephrine template. Simultaneously, this polymer was deposited on the Pt electrode as a thin film. After the template extraction from the film, the analyte was determined with differential pulse voltammetry (DPV) in a redox probe free solution. That was possible because the internal ferrocene redox probe generated the DPV analytical signal. The thickness and morphology of the film were crucial for the sensor's performance. The mechanism of this redox self-reporting MIP film-based chemosensor was examined with electrochemical methods, simultaneous piezomicrogravimetry and electrochemistry at an electrochemical quartz crystal microbalance, and surface plasmon resonance spectroscopy. The devised chemosensor was applied for selective p-synephrine determination in a concentration range of 2.0 to 75 nM.

References

1. Cieplak, M.; Kutner, W., Artificial biosensors: How can molecular imprinting mimic biorecognition? Trends Biotechnol. 2016,34 (11), 922-941.

2. Uzun, L.; Turner, A. P. F., Molecularly-imprinted polymer sensors: realising their potential. Biosens. Bioelectron. 2016,76, 131-144.

3. Huynh, T.-P.; Sharma, P. S.; Sosnowska, M.; D'Souza, F.; Kutner, W., Functionalized polythiophenes: Recognition materials for chemosensors and biosensors of superior sensitivity, selectivity, and detectability. Prog. Polym. Sci. 2015,47, 1-25.

4. Yoshimi, Y.; Narimatsu, A.; Nakayama, K.; Sekine, S.; Hattori, K.; Sakai, K., Development of an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer. J. Artif. Organs 2009,12 (4), 264-270.

5. Sharma, P. S.; Garcia-Cruz, A.; Cieplak, M.; Noworyta, K. R.; Kutner, W., 'Gate effect' in molecularly imprinted polymers: the current state of understanding. Curr. Opin. Electroche. 2019,16, 50-56.

6. Moreira, F. T. C.; Dutra, R. A. F.; Noronha, J. P. C.; Fernandes, J. C. S.; Sales, M. G. F., Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-screen printed electrodes. Sens. Actuators, B 2013,182, 733-740.

7. Lach, P.; Cieplak, M.; Majewska, M.; Noworyta, K. R.; Sharma, P. S.; Kutner, W., "Gate Effect" in p-Synephrine Electrochemical Sensing with a Molecularly Imprinted Polymer and Redox Probes. Anal. Chem. 2019,91 (12), 7546-7553.

Figure 1

Urinary tract infections (UTIs) are one of the most ubiquitous bacterial infection diseases affecting half of the global population at least once in their lifetime. Given the occurrence of the false positive result in dipstics and the time-consuming identification of bacteria using culturing method to detect UroPathogenic Escherichia coli (UPEC), there is the need for a point-of-care diagnostic platform that is reliable, sensitive, specific, cost-effective, fast and easy to use. With the advent electrochemical methods coupled with the specific biorecognition elements, the detection of any biomarker has become possible with one touch. Among many biorecognition molecules, DNAzymes are gaining attention due to their robustness, stability, specificity, and the ease of selection without prior knowledge of the biomarker. Their structure switching and functionality (RNA cleaving) can be utilized with redox active species (like methylene blue) to design highly sensitive and selective electrochemical assays for pathogen detection. In this work, we have developed a dual signaling electrochemical DNAzyme based platform for specific and sensitive detection of Escherichia coli (E.coli) in UTI patient urine samples. The two on-chip integrated working electrodes named as release channel and capture channel are integrated with DNAzyme-redox barcode and DNA probe respectively. In the presence of the bacterial target the DNAzyme catalyzes the cleavage of the redox DNA barcode in the release channel, which is subsequently translated into a redox signal upon hybridization with the probe on the capture channel. The differential signal generated by the two channels in the sensor demonstrated the sensitive detection of 10 CFU for E.coli in buffer and urine. Additionally, the sensor specifically detected E. coli in the presence of a panel of other gram positive and negative bacteria. Clinical validation of the assay was performed using 45 E.coli+ and E.coli- patient urines delineating a clinical sensitivity of 100% and specificity of 78% in a 30 minute testing time. This cost effective, reagent-free, time-efficient, and amplification-free electrochemical assay is suitable for detecting a wide range of pathogens, also alleviates the additional knowledge requirement of the biomarker as compared to other protein and aptamer-based assay. With the advancement in the chip fabrication and microfluidics, this assay can also be multiplexed with other pathogen for simultaneous detection of multiple pathogens in a single urine sample.

Tau is a neuronal protein responsible for microtubule formation [1]. It is also the pathological protein active in the Alzheimer's Disease (AD) pathway. When Tau begins to aggregate as a result of misfolding, the neuronal cells die due to the lack of microtubule formation and can take on prion-like properties [2]. Therefore, detection of aggregated Tau can serve as a powerful tool in the early diagnosis of AD and dementia related diseases. As has been previously demonstrated, Tau 441 can be detected with an electrochemical immunoassay based on the electrochemical impedance spectroscopy (EIS) by using gold electrode [3]. The EIS was also used to monitor interactions between Tau protein and Heparin [4]. Herein, we describe use of EIS to monitor and detect Tau and its aggregates by using gold electrode modified to include a biotinylated aptamer to tau and streptavidin-Au sensor through a series of surface modifications. Surface modifications were characterized using EIS and CV. After modifying the surface of the electrode, the detection of Tau was achieved using EIS. Aggregation of tau was also monitored as a function of Tau or Heparin concentrations, and incubation time and temperature. The control experiments included, Tau-free surface, Heparin-free solution, buffer solution in order to minimize aggregation. The charge transfer resistance, R_ct, was determined by fitting the experimental data to the equivalent circuit. The R_ct values for Tau-films on gold surface were compared prior and post aggregation. The R_ct values were highly dependent on the experimental conditions. Data indicate that this electrochemical sensor holds great promise for detection of neurodegenerative biomarkers which undergo aggregation.

References

[1] M.D. Weingarten, A.H. Lockwood, S. Hwo, M.W. Kirschner, A Protein Factor Essential for Microtubule Assembly, Natl. Acad Sci. 72 (1975) 1858–1862.

[2] F. Clavaguera, J. Hench, M. Goedert, M. Tolnay, Invited review: Prion-like transmission and spreading of tau pathology, Neuropathol. Appl. Neurobiol. 41 (2015) 47–58.

[3] N. Carlin, S. Martic-Milne, Anti-tau antibodies based electrochemical sensor for detection of tau protein biomarkers, J. Electrochem. Soc. 165 (2018) G3018–G3025.

[4] H. Trzeciakiewicz, J. Esteves-Villanueva, R. Soudy, K. Kaur, S. Martic-Milne, Electrochemical characterization of protein adsorption onto YNGRT-Au and VLGXE-Au surfaces, Sensors (Switzerland). 15 (2015) 19429–19442.

Proteases are a class of enzymes responsible for selectively breaking down proteins and thus they are involved in many biological processes ranging from apoptosis to hormone regulation. Overexpression of proteases is often indicative of several health conditions, such as muscle atrophy, bone loss and cancer progression. Consequently, the measurement of protease activity can serve as a biomarker for diagnosis as well as the efficacy of treatment regimens. Developing rapid low-cost techniques that can quantitatively detect multiple proteases is essential for these applications.

Here we show the electrochemical characterization of a microfabricated sensor consisting of nine individually addressable microelectrodes. Subsequently, we demonstrate the application of this microelectrode array for the simultaneous, multiplex detection and quantification of protease activity. To accomplish this, each electrode is functionalized with a specific ferrocene labelled hexapeptide substrate and the electrochemical signal is monitored by AC voltammetry. In the presence of the target protease, the AC voltammetry signal decays as the target protease cleaves the peptide substrate, causing the ferrocene tags to leave the electrode surface. The proteolytic activity is determined by fitting the decay in signal to an exponential curve in accordance with the Michaelis-Menten model of heterogeneous kinetics. Our results show that each electrode of the MEA can be selectively modified with little or no contamination from adjacent electrodes. Additionally, we demonstrate that three different peptide substrates can be simultaneously used to quantify the protease activity of a sample. This microelectrode array sensor will be used to develop a portable instrument to track muscular atrophy progression during long duration space habitation in microgravity and to evaluate potential physiological and pharmaceutical countermeasures.

Development of rapid tests for quantitative detection of analytes in complex matrices, plays an important role in medical diagnostics, prognostics and therapeutics. Current methods for molecular diagnostics are time-consuming and multiple-step reliant on well-trained technicians and fully-equipped laboratories to provide quantitative detection of biomarkers. In this case, the challenges to overcome the sensitivity and selectivity of working directly in biological fluids in a timely manner have always been the main step for the development of such rapid tests.

Electrochemical sensors, analogous to glucometer, are known to be rapid with the ability to provide direct electronic signal without any interference from the biological phenomena. When functionalized with specific biological substances, they intend to provide the required specificity in addition to signal selectivity in biological fluids. Inspired by nature, we design biorecognition probes that are combined with newest advancement in the engineering for the development of electrochemical immune-biosensors. These molecular recognition probes are designed using the ability of antibodies to bind antigens, and DNA construct to hybridize to the complementary construct, to create specificity for direct detection in complex media. On the other hand, we rely on the innovative approaches in nanomaterials, surface sciences, and self-assembly techniques to improve the conductivity and sensitivity of electrode surface. This includes the incorporation of high-curvature nanostructured microelectrodes that helps with the efficiency of molecular interactions at the surface and the transfer of electrons for the ease of signal transduction.

In this regard, we fabricate small-scale nanostructured electrodes and engineer the surface for the immobilization of the biomolecular monolayer at the interface to provide the desired specificity for biomarkers in real biological samples, e.g. whole blood. We adapted our nano-bio-sensors for translational applications such as (1) at-line monitoring of signaling proteins in hematopoietic stem cell expansion, (2) rapid diagnosis of pathogenic infections through quantitative detection of antibodies directly in patient samples, and (3) rapid diagnosis of inner ear disorders based on the detection of blood biomarkers. We showed that our electrochemical immune-biosensors are capable of detecting at therapeutic concentrations with tunable ranges while performing in a 10 µL sample within less than 30 minutes.

Our goal is to make electrochemical sensors for measuring oral fluids and tissues to aid in more accurate diagnosis and monitoring of oral diseases. In this work, we developed a sensor platform capable of measuring simultaneously pH and temperature for detecting inflammation at specific sites in the mouth. Miniaturized, planar pH sensing and reference electrodes were combined on a flexible polymer platform along with a platinum resistance temperature detector (RTD) using a combination of microfabrication and electrodeposition techniques. The resistance of the platinum RTD was linear between 20 and 60 °C, with a precision of ±0.1 °C. The pH response and reproducibility of sputtered iridium oxide (IrOx) was compared with those of electrochemically deposited IrOx. The voltage of the pH sensor was measured in solutions of varying pH, chloride concentration, and temperature mimicking the oral environment. At room temperature, sputtered IrOx sensors exhibited a linear response (-47 to -55 mV/pH) for pH 2-10 whereas electrodeposited IrOx was linear (-72 to -82 mV/pH) over pH 4-8, which is within the range expected in the oral cavity. The sensitivity of the sputtered IrOx was more reproducible between individual sensors than electrochemically deposited IrOx; however, sputtered IrOx electrodes were more difficult to fabricate and had a lower sensitivity to pH. Voltage stabilized (within 1-2 mV) in 10-120 s and reached 90 % of this stable value within 5 s at each pH value. The electrodeposited Ag/AgCl reference responded linearly to [Cl^-] as well asvaried by <2 mV over pH 4-8. Repetitions of reproducible sensors at one pH were ≈0.1 pH unit. Preliminary measurements indicated that pH sensitivity of the IrOx sensors was ≈0.1 mV/pH/°C, producing an apparent change of ≈0.2 pH between room temperature (21 °C) and the mouth (35 °C); therefore, the voltage will need to be calibrated based on temperature.

Recent studies shows laser-induced graphene (LIG) based electrodes can be very effective for electrochemical sensing applications. In this work, we aim to develop a stable ion selective membrane (ISM) based sweat sensor using laser-induced graphene (LIG) as the electrode material. However, the surface of pristine LIG is hydrophobic. Hence, coating these hydrophobic LIG films with ISM produces low sensitivity in sensing applications. In this study, UV-Ozone irradiated laser-induced graphene (LIG) based electrodes were explored for sensing biomarkers like ion in sweat. Solid-state ion-selective electrodes (ISEs) sensitive to ions were fabricated by a two-step drop-coating process of polyvinyl chloride solution containing plasticizer, ionophore, and ion-exchanger on the LIG electrode. We found the ozone treatment process significantly increased the electrochemical active surface area (EASA) and porosity of the pristine LIG. Hydrophobic to hydrophilic transition induced by UV-Ozone treatment reduced the contact angle (CA), resulting in better permeation of the ion-selective membrane (ISM) into the ozonized LIG film. Raman spectroscopy and IR absorption studies indicate physisorption of ozone on LIG. The concentration of the ISM solution has a great influence on attachment to the O-LIG film. Here, we report the applicability of O-LIG as an electrochemical sensor towards the detection of sodium ion () using the open circuit potential (OCP) method. The performance of ozonized LIG (O-LIG) electrodes was much better than pristine LIG and screen printed carbon electrodes. The sensitivity of 60.2 ± 0.9 mV/ decade to Na⁺ ions and a lower limit of detection of 1 × 10^-6 M was achieved using O-LIG based ISEs. Response time of the O-LIG based electrodes were around 1 min. The current study demonstrates that the LIG-based electrodes can be used as a flexible electrochemical sensing platform and is suitable for future wearable sensing devices.

Keywords

Laser-induced graphene; ion-selective electrode; wearable sensors; electrochemical sensor; sweat sensor.

The development of additive manufacturing techniques to manufacture sensors rapidly and cost-effectively would benefit healthcare, environmental monitoring, food, and cosmetic industries. Because of the inherent complexity of sensing devices, new fabrication methods that utilizes recent advances in additive manufacturing to create sensors on a large scale efficiently are necessary. Techniques like 3D printing and 3D bioprinting enable printing different biomaterials into intricate 3D architectures that could serve as sensing platforms. This presentation will describe 3D printed hydrogel-based sensors' development and optimization with incorporated receptor molecules and transduction interfaces for UV sensing¹. To 3D print these sensors, an extrusion-based 3D bioprinter and a computer-aided designing software were used along with a novel bio-ink formulation. The bio-ink formulation contains nanoparticles with photocatalytic properties and degradable dyes dispersed homogeneously within a mechanically stable hydrogel network. The optimum hydrogel composite provides excellent mechanical properties to the printed sensors, enabling them for many applications. The proposed sensors are reagent-less and highly portable, making them ideal for wearable devices and printed platforms for portable bioelectronics. Apart from their ability to be used as wearable sensors, these sensors also have the potential to be used simultaneously with UV-based workspace sterilizing devices to guarantee that surfaces are adequately exposed to UV. The sensors are cheap, durable, robust, biodegradable, and simple to use. The tunable, biocompatible, and printable properties of the ink offer great potential for developing advanced 3D printing methods that, in addition to UV sensors, can be applied more broadly to fabricate other sensing technologies for various other applications.

1. A. S. Finny, C. Jiang, and S. Andreescu, ACS Applied Materials & Interfaces, 12, 43911–43920 (2020).

Figure 1

Alzheimer disease (AD) is a chronic neurodegenerative disease threating the health of human being. The clinical symptoms mainly consist of severe cognitive impairment, comprehensive dementia, memory loss and other mental behavioral problems. According to data that collected in 2018, AD has become the sixth fatal disease in the worldwide. Nevertheless, the pathogenesis of AD has not been clarified, except for two main pathological features: from extracellular, the amyloid plagues appear in the dendrites which is mainly caused by the formation of the toxic amyloid-β oligomers (AβOs); from intracellular, it appears the accumulation of neurofibrillary tangles (NFTs) which is formed by hyperphosphorylated tau protein. Recent years, olfactory dysfunction has been reported to be an early symptom of AD. From the research of clinical experiment, olfactory dysfunction happens much earlier than other clinical symptoms. Besides, there have been plentiful methods for olfactory dysfunction diagnosis, which indicates olfactory test for disease diagnosis has been paid more and more attention. Many researches have indicated olfactory bulb is the first region of the olfactory system to be damaged in AD. AβOs has been confirmed in early olfactory dysfunction in AD, but how the AβOs lead to the olfactory dysfunction is still not exactly realized. Consequently, we chose the OB region as the research object and proposed to establish an olfactory dysfunction model of AD, by the means of a directly electrophysiology method.

AβOs-induced olfactory bulb neural network cultured on MEA as dysosmia model of AD at early-stage in vitro is established to investigate that AβOs is detrimental to the processing of olfactory information over time. Firstly, olfactory bulb tissues were extracted and then were digested so that we got the single cell suspension. On the one hand, OB neurons were plated on the surface of MEA chip so that we can detect the extracellular signal in time after AβOs induction. On the other hand, they were cultured on 24-well plate for immunofluorescence to observe the changes of the neuron morphology and cellular proteins. When the OB neural network grew to 13 days, the typical extracellular signals of OB neurons in spontaneous firing state can be recorded. After induced by AβOs, the firing rate of OB neuron disappeared temporarily. After 1 h, the firing rate increased gradually, but after 2 h, the firing rates neurons decreased significantly. Moreover, the firing rate of OB neuron disappeared completely after 3 h.

To observe the changes of the cell morphology and cellular proteins after treated with AβOs, we used immunofluorescence method to verify the validity of the dysosmia model in AD. After being induced by AβOs for 3 hours, the neuron plaques increase significantly. This indicates the fact that neurites degeneration and dendrites impairment. As previously described in the mechanics section, phosphorylated tau protein(p-tau) is hyperphosphorylated and abnormally accumulates in Alzheimer's disease. We performed the staining of p-tau antibody, and analyzed the mean fluorescence intensity to quantify p-tau protein levels. The statistical graph shows the great significant difference between the control and AβOs-treated group. Our results validated that the AβOs can make tau protein phosphorylate, which is similarly to those observed in the degenerating neurons of AD. This also verifies the effectiveness of the model established in this study.

Then we try to explain why the spike firing changes after the AβOs induction. When the AβOs was added, it caused N-methyl-D-aspartate (NMDA) receptor and L-type calcium channels opening so that the calcium flew into the cell largely. Eventually, the intracellular calcium overloaded, which then resulted in abnormally improving spontaneous activity. However, the large amount of calcium caused potassium channels opening, and impaired mitochondrial and endoplasmic reticulum (ER) metabolism. Eventually, the whole cellular metabolism disordered, and the neurons became inactive till death.

The study will contribute to understanding the cellular and synaptic mechanisms of the early-stage olfactory dysfunction in AD and providing a potential strategy for diagnosis and treatment. And then discuss what it is for and what will we do next by the means of dysosmia model of AD established in the research. Recent study shows that intranasal drug administration is expected to be a targeted therapy method for treating the cerebral diseases. Because it is close to the central nervous system so that the drugs can be transmitted to the cerebral area and be absorbed quickly. Therefore, applying the early-stage dysosmia model of AD to screening the intranasal drug of treating AD is of great significance.

Early-stage tumor detection is one of the most coveted goals for the world research community. An effective prevention, associated to reliable screening protocols, is crucial to favor a early diagnosis, allowing doctors and surgeons to intervene with high probabilities of success on tumor affected patients. The main purpose of this study is cancer detection, exploiting the volatile organic compounds (VOCs) produced by cancer cells as tumor biomarkers [1][2], by investigating tumor tissue and blood samples. Tumor biomarkers exhalation is attributed to two main cell involving biological mechanisms: altered metabolism and cellular membrane peroxidation [3]. This experimental work was carried on using an innovative, fast-responding and reliable patented device, named SCENT B1 [4], entirely designed and assembled in the Sensor Laboratory of the University of Ferrara. It hosts an array of four specific metal-oxide (MOX) sensors able to detect gases in low concentration (up to 10 ppm) with high stability and repeatability, chosen after many tests on different types of biological samples (feces, blood, cell cultures, etc.) [5,6]; the chosen sensors are based on different mixtures of tin, titanium, tungsten, niobium, tantalum and vanadium oxides (ST25Au, W11, STN, TiTaV). Moreover, SCENT B1 is gifted of a power supply and sensor signal transduction electronic boards, a pneumatic air system (necessary to direct the VOCs contaminated air from the sample chamber to the sensors) and an ad hoc management and data acquisition software (LSS4) [6]. The sensor output signal is a voltage directly proportional to the sensor conductance, depending on the chemical redox reactions taking place on the surface of the sensor sensing material. The sensor response is: R=V_gas/V_air , where V_gas and V_air are the sensor voltage in gas presence and in dry air after the steady state achievement respectively [6].Sensor responses were further elaborated, using different statistical approaches as principal component analysis and receiver operating characteristics methods.

Measurements have been performed on colorectal cancer and healthy tissues surgically removed from the same operatory piece (directly and far enough from tumor mass respectively) (Figure 1a), and on blood samples collected from tumor affected subjects and healthy ones as controls (Figure 1b). with the future aim of extending the study to other tumor types. All four sensors gave larger responses (although with different amplitudes) to the tumor tissue with respect to the healthy one. Smaller and reproducible responses were given by the breeding ground (DMEM) only, confirming that it does not alter the measurements. The results are consistent with the stronger and altered metabolism of tumor cells, leading them to emit a larger amount of volatile biomarkers with respect to the healthy ones;.

All the four sensors hosted by SCENT B1 proved to be capable of distinguishing between healthy and tumor tissue and blood samples, although with different discrimination power. The encouraging results of this feasibility study are the basis of a new in-depth study, which includes also a follow-up protocol based on post surgery blood monitoring of patients. Our future aims foresee the extension of this study to other tumor types and the obtainment of the SCENT B1 clinical validation as oncologic screening device.

Figure 1

Introduction

Extensive research over the past half a century indicates that reactive oxygen species (ROS) play an important role in cancer. ROS are small molecules from the partial reduction of molecular oxygen during oxygen consumption and cellular metabolism. Increased ROS levels have been observed in various cancers. The abnormal concentration of ROS can activate signaling pathways about cancer cell survival, tumor progression, drug resistance, and driven DNA damage and genetic instability [1]. Precisely regulated antioxidant defense system in cancer cells can detoxify elevated ROS levels while maintaining pro-tumorigenic signaling and resistance to apoptosis. However, if the balance of redox status was disturbed, ROS may exert a cytotoxic effect which induces malignant cell death. It has been explored to selectively kill cancer cells without damage normal cells by manipulating ROS levels and antioxidant systems [2]. ROS-generating and elimination agents have been applied and found effective in many cases. Arsenic trioxide can impair the respiratory chain and increase superoxide production while doxorubicin can induce ROS production by intracellular chelation of iron and trigger a Fenton-type reaction leading to the generation of the highly reactive hydroxyl radical. Depletion of the GSH pool, which is the major ROS-scavenging system in cells, can increase oxidative stress and cell death. To maximize the therapeutic effect, a combination of arsenic trioxide and ascorbic acid-mediated GSH depletion has been exploited and showed clinically effective in the treatment of myeloma. Thus the quantitative determination of ROS in cells is in great demand in clinical cancer treatment and basic research. The non-enzymatic electrochemical sensor is excellent stable, reproducible, and cost-effective with no need to immobilize enzymes on the electrode surface [4]. To increase the activity and selectivity, Au-based nanoparticles are of particular interest for their great conductivity and superior electrochemical properties. By introducing the MWCNTs as supporting material due to their excellent electrical conductivity and large surface area, the electrocatalytic activity can be further improved.

Method

An electrochemical biosensor consists of gold nanoparticles/multi-walled carbon nanotubes (AuNP/MWCNTs) decorated screen printing electrodes and lung cancer cells were developed to determine the change of ROS levels induced by chemical drugs. The nanocomposite of AuNPs/MWCNTs was prepared following the previous method with a slight modification [5]. 20 μL of the well-dispersed suspension was dropped onto the working electrode of the screen printing electrode. The AuNPs or MWCNTs modified electrodes were prepared under the same conditions as a control. A CHI660E electrochemical workstation with a three-electrode system was purchased from Chenhua Technology Co. Ltd. A 3D cell culture of GelMA/graphene oxide (GelMA/GO) was used to encapsulate A549 lung cancer cells [6]. The H₂O₂ levels of cancer cells under different drug treatments were detected and calculated with peak current in DPV analysis as a representative for oxidant stress. Conventional biological assays, including fluorescence detection, and cell apoptosis assay were applied to detect the ROS levels as control.

Results and Conclusions

The biosensor demonstrates good sensitivity towards hydrogen peroxide at PH 7 solution at a working potential of -0.50 V with a linear response range from 2.0 μM to 53.8 μM. We found that combining applying arsenic trioxide and ascorbic acid can effectively elevate oxidative stress level in A549 cells and induce cell apoptosis. This system is sensitive and simple to operate with the advantage to evaluate the oxidative stress level of different drugs by the detection of H₂O₂ in the cell membrane. In summary, this work describes a novel method for assessing the effect of ROS-target drugs using cell-based electrochemical signaling with a rapid screening pattern.

References

[1] Prasad S, Gupta S C, Tyagi A K. Reactive oxygen species (ROS) and cancer: Role of antioxidative nutraceuticals[J]. Cancer letters, 2017, 387: 95-105.

[2] Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?[J]. Nature reviews Drug discovery, 2009, 8(7): 579-591.

[3] Grad J M, Bahlis N J, Reis I, et al. Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells[J]. Blood, The Journal of the American Society of Hematology, 2001, 98(3): 805-813.

[4] Dhara K, Mahapatra D R. Electrochemical nonenzymatic sensing of glucose using advanced nanomaterials[J]. Microchimica Acta, 2018, 185(1): 49.

[5] Maluta J R, Canevari T C, Machado S A S. Sensitive determination of nitric oxide using an electrochemical sensor based on MWCNTs decorated with spherical Au nanoparticles[J]. Journal of Solid State Electrochemistry, 2014, 18(9): 2497-2504.

[6] Xing L, Ge Q, Jiang D, et al. Caco-2 cell-based electrochemical biosensor for evaluating the antioxidant capacity of Asp-Leu-Glu-Glu isolated from dry-cured Xuanwei ham[J]. Biosensors and Bioelectronics, 2018, 105: 81-89.

Figure 1

The blood glucose concentration is the basis of diabetes mellitus "chronic and metabolic disease" diagnosis. The elevated levels of blood sugar "glucose" lead to many health problems, such as chronic kidney failure, stroke, cardiovascular disease, eyes retina damage, and foot ulcers. Hence, early diagnosis is crucial to prevent and avoid life-threatening complications caused at abnormal glucose levels. In recent, different methods (such as electrochemical, colorimetric, piezoelectric, and thermoelectric based biosensors) have been utilized for glucose concentration detection. Among these methods, the electrochemical based biosensors were extensively employed; however, most of the biosensors were enzyme-based. The enzyme-modified electrodes suffered from some drawbacks, for instance, complicated immobilization procedures, high cost, instability, and low sensitivity. Therefore, it becomes crucial to develop novel electrode nanomaterials that work as electrocatalyst for glucose oxidation and also result in high sensitivity and stability. The performance of nonenzymatic glucose biosensors depends on the morphology of the electrode material. Hence, a variety of nanostructured nanomaterials are utilized to fabricate nonenzymatic biosensors with a high working electrode surface area.

To fabricate nonenzymatic biosensors for glucose detection, significant efforts have been made to synthesize nanomaterials and use them as an electrocatalyst, for example, metals, metal oxides, and their hybrid nanostructures^16-18. Among different catalysts, nanostructures of copper oxide (CuO) have received considerable interest^{19, 20}. CuO is the best candidate to fabricate electrochemical based nonenzymatic glucose biosensors, as CuO based biosensors directly oxidizes glucose on the working electrode surface. Moreover, CuO nanostructures possess advanced properties, which are beneficial for designing biosensors.

Research has been concentrated on the shape/dimensions controlling during synthesis of (nano)materials, which have better structural properties that results in enhanced electrochemical performance due to high specific surface area. Previously, a variety of morphologies of CuO nanomaterials have been produced (i.e. nanoparticles, nanowires, nanowhiskers, nanoneedles, nanorods, nanoshuttles, nanoribbons, and nanotubes) using solution-based approach, sonochemical deposition, vapor phase growth, high temperature synthesis, double-jet precipitation, micro-emulsion synthesis, etc. Among these synthesis methods, hydrothermal method presents an environmentally friendly, simple, cost-effective, high-yield, and low-temperature method to synthesize various CuO nanostructures.

Herein, we report hierarchical CuO nanoleaves synthesis in large-quantity by a low-temperature hydrothermal method. Hierarchical CuO nanoleaves were synthesized using a low-temperature (95 °C) hydrothermal method (Fig. 1a). For synthesis, 0.25g Cu(CO₂CH₃)₂·H₂O and 0.77g CTAB were added in 40 mL deionized (DI) water. Next, 4 mL of 50 mM NaOH was added in the above solution while stirring. Then, the above solution was poured into a refluxing pot on a heating mantle and refluxed at 95 °C for 5h. Finally, black colored precipitates were washed using methanol and DI water to remove impurities and dried at room temperature. FESEM images and EDS analysis of CuO nanoleaves are shown in Fig. 1b-d. The low- and high-magnification images confirm that the CuO nanostructures prepared bear nanoleaves like morphology, and the nanoleaves are uniformly grown in large quantity. EDS analysis of CuO nanoleaves shows that nanoleaves are made of copper (Cu) and oxide (O) elements only (Fig. 1d, inset). EDS spectra shows an additional peak of Si, which is originating from Si substrate used to spread CuO nanoleaves sample for FESEM and EDS analysis. To fabricate hierarchical CuO nanoleaves based nonenzymatic glucose biosensor; first, the slurry of engineered CuO nanoleaves was prepared after mixing with conducting butyl carbitol acetate binder (8:2 v/v ratio) (Fig. 1a). The prepared slurry (2-6 μL) was cast on cleaned glassy carbon electrode (GCE, 0.071 cm²) and kept for drying. Finally, Nafion (5 μL) was coated on the electrode (CuO nanoleaves/GCE) surface and kept for overnight at 4 °C.

The electrochemical behavior of fabricated biosensor towards glucose was analzed with cyclic voltammetry (CV) and amperometry (i-t) techniques. Owing to the high electroactive surface area, hierarchical CuO nanoleaves based nonenzymatic biosensor electrode shows enhanced electrochemical catalytic behavior for glucose electro-oxidation in 100 mM sodium hydroxide (NaOH) electrolyte. The nonenzymatic biosensor displays a high sensitivity (1467.32 mA/(mM cm²)), linear range (0.005-5.89 mM), and detection limit of 12 nM (S/N = 3). Moreover, biosensor displayed good selectivity, reproducibility, repeatability, and stability at room temperature over three-week storage period. Further, as-fabricated nonenzymatic glucose biosensors were employed for practical applications in human serum sample measurements. The obtained data were compared to the commercial biosensor, which demonstrates the practical usability of nonenzymatic glucose biosensors in real sample analysis. Therefore, we believe our engineered CuO nanoleaves based nonenzymatic biosensor can pave the way to detect glucose in low glucose level samples (i.e., saliva, tear, sweat).

References:

1. Marunaka, World Journal of diabetes, 6, 125-135 (2015).

2. Ahmad, M. Vaseem, N. Tripathy, Y.-B. Hahn, Anal. Chem,. 85, 10448-10454 (2013).

3. Xiao, Y. Liu, L. Su, D. Zhao, L. Zhao, X. Zhang, Anal. Chem., 91, 14803-14807 (2019).

4. Ahmad, M. Khan, M. R. Khan, N. Tripathy, M. I. R. Khan, P. Mishra, M. A. Syed, Ajit Khosla, Microsyst. Technol., 1-6, (2020).

5. Ahmad, M. Khan, N. Tripathy, M. I. R. Khan, A. Khosla, J. Electrochem. Soci., 167, 107504 (2020).

6. Ahmad, T. Mahmoudi, M.-S. Ahn, Y.-B. Hahn, Biosens. Bioelectron., 100, 312-325, (2018).

7. Ahmad, N. Tripathy, J.-H. Park, Y.-B. Hahn, Chem. Commun., 51, 11968-11971 (2015).

8. Yoon, S. N. Lee, M. K. Shin, H.-W. Kim, H. K. Choi, T. Lee, J.-W. Choi, Biosens. Bioelectron., 140, 111343 (2019).

9. Puttananjegowda, A. Taksi, S. Thomas, J. Electrochem. Soci., 167, 037553 (2020).

Figure 1. (a) Schematic representation of CuO nanoleaves synthesis, (b-d) FESEM images and EDS spectra (inset d) of engineered hierarchical CuO nanoleaves.

Figure 1

Electrochemical immunosensors combine the advantages of electrochemical transducers such as low cost, potential for miniaturization, and quantified readout with the selective properties of immunochemical recognition elements. Here we describe a simple, label-free electrochemical immunosensor for the rapid detection of traumatic brain injury (TBI) by quanitifcation of a cytokine interleukin-6 (IL-6) in cerebrospinal fluid (CSF). The sensor system consists of gold interdigitated electrode arrays (IDEAs) that are modified with self-assembled monolayers (SAMs) and IL-6 antibodies (IL-6 mAb) for antigen recognition and capture. Electrode modification was confirmed using surface characterization (FT-IR, fluorescence) and electrochemically via EIS. The sensor performance was evaluated in IL-6 spiked PBS solution: the LOD was determined to be 1.63 pg/mL, with a linear range across 4 orders of magnitude. The sensor exhibits selectivity against non-target cytokines TNF-α and IL-10. The detection of CSF and serum IL-6 using the outlined system will be discussed for use as a point-of-care (POC) diagnostic and prognostic tool for TBI.

Introduction

Recently, fast response humidity sensors are an active area of research for applications such as industrial, agricultural and point of care devices. Among various types of humidity sensor materials, thin film based resistive humidity sensors have been actively studied because those exhibit high surface area and the sensor configuration is simple. However, most of these sensors are relatively slow with long response time (over 60 seconds) and narrow detection limits (30-50 RH%), making them difficult to use in many applications [1-2]. Besides, the need of an external heater for fast response increases power consumption and complicates the manufacturing process of the sensor. Functionalized graphene, the most popular humidity sensor material, offers significant advantages due to its unique 2D structure and oxygen-containing functional groups, providing a large surface reaction site. However, it also has limitations such as poor stability in a moist environment due to its hydrophilic nature and weak adhesion with the substrate, complex synthesis procedure, makes it difficult to develop practical sensing devices.

Here, we developed the low-cost and fast-response humidity sensors by integration of thermally decomposed carbon (TDC) thin film and carbon nano-sized interdigitated electrodes (IDEs). Carbon IDEs were used as a sensor platform due to its excellent thermal compatibility with TDC films and it provides low-cost wafer-level fabrication of three-dimensional nanoelectrodes [3], resulting in the miniaturized sensor configuration at a wafer level. Shellac, a low-cost biopolymer, was used as a precursor for synthesizing TDC films via single-step pyrolysis. The obtained film showed conformal coating with no grain boundaries or defects and formed high sp² hybridized carbon network on the carbon nano-sized IDEs, thus providing excellent stability, rapid response/recovery and low power consumption as a room temperature humidity sensor. Our results demonstrate the potential application of TDC film as an alternative sensing material for environmental humidity, instant calibration for gas sensing measurements and human respiration in real time.

Methods

We integrated a nanometer-thick TDC film onto the carbon nano-IDEs, using two-step process, to facilitate a simple and cost-effective humidity sensor. Firstly, microscale interdigitated polymer patterns were fabricated using photolithography and then pyrolyzed into nanoscale carbon IDEs due to volume reduction in pyrolysis. The interdigitated carbon nanoelectrodes were annealed at 1000 ^°C using a rapid thermal annealing process to enhance the electrical conductivity. The next step is the integration of a TDC film on the carbon IDEs. For that, shellac solution (4 wt.%) was uniformly spray-coated on the carbon IDEs and air-dried (1 hour). This shellac film was pyrolyzed at 600 ^°C to obtain a uniform TDC thin film.

Results and Conclusions

Figure a shows a scanning electron microscopy (SEM) image of the TDC coated nano-sized carbon IDEs (width = 800 nm, height = 300 nm, gap = 2.5 μm). The film displays a conformal coating with no signature of defects or grain boundaries. The electrical properties of TDC film were evaluated using a two-probe I-V technique (Figure b), which indicated the excellent ohmic contact with carbon IDEs. Raman spectra of the TDC film (Figure c) indicated the presence of local intrinsic defects and disorder (intense D band) due to the presence of oxygen functional groups, especially at the edges and basal plane of the TDC film. X-ray photoelectron spectroscopy (XPS) for carbon (Figure d) also confirmed the presence of oxygen-containing functional groups (C-OH and C=O) and a high sp² hybridized carbon network. Higher dynamic response (40 %) is attributed to the presence of oxygen functional groups (Figure e), whereas sp² carbon network induce hydrophobicity, which lowers the condensation of water molecules without using any external heating source, resulted in fast response (37s) and recovery time (8s) (Figure f), compared to previously reported rGO-based sensors [4]. The obtained thin film humidity sensor also displayed the linear response over the wide humidity range (10-90 RH%), with high sensitivity > 0.7/RH%. The dynamic response of the sensor revealed that the protonic conduction mechanism is dominant in TDC film.

References

[1] M. Packirisamy, I. Stiharu, X. Li and G. Rinaldi, A polyimide-based resistive humidity sensor, Sensors Review. 25 (2005) 271-276.

[2] M. Ueda, K. Nakamura, K. Tanaka, H. Kita, and K. Okamoto, Water-resistant humidity sensors based on sulfonated polyimides, Sensors and Actuators B. 127 (2007) 463-470.

[3] C. Wang, G. Jia, L. H. Taherabadi and M. J. Madou, A novel method for the fabrication of high-aspect-ratio C-MEMS structures, J. Micro electro mech. Syst. 14 (2005) 348–358.

[4] D. T. Phan and G. S. Chung, Effect of rapid thermal annealing on humidity sensor based on graphene oxide thin films, Sensors and Actuators B. 220 (2015) 1050-1055.

Figure 1

A biosensor is a device that can determine the concentration or presence of chemicals, such as neurotransmitters and neuropeptides. Certain excitatory neurotransmitters and neuropeptides can be measured by Methylene blue (MB), a synthetic dye that stains to negatively charged cell components. For the modification process in this work, aptamers are used because they possess the advantage of binding to any given target molecule and, when they bind, they undergo conformational changes, which is where MB comes into play. This research focuses on the modification of the surface of Gold (Au) and Highly Oriented Pyrolytic Graphite (HOPG) electrodes using MB as a redox label probe for the detection of neurotransmitters and neuropeptides in aptamer-modified electrodes. Electrochemical Impedance Spectroscopy (EIS) was used to understand the adsorption properties of molecules that stick to the surface of the electrode and Atomic Force Microscopy (AFM) was used to measure the topography of the electrodes before and after the aptamer modification. As results, we were able to prove that AFM cannot monitor the changes on the surface of the Au electrodes with the different modifications due to the roughness of the surface, and that, electrochemically, the capacitance of the gold electrode decreases at higher concentrations of NPY. Different NPY measurements were made, ranging from femtograms to picograms, and different molecules such as dopamine, epinephrine, and PYY were also tested to determine if the sensor system has the same affinity with different types of molecules. These results give us a better understanding of the use of these techniques for the monitoring of biomolecules.

Introduction

In addition to electrophysiological signals [1-2], mechanical properties such as contraction and relaxation of cardiomyocytes are also important properties. They are essential for the evaluation of drug efficacy and toxicity, and they can change the viscoelasticity of cells, thus providing the possibility for quantitative detection. The acoustic wave sensors are able to detect this slight change in viscoelasticity, which may not be easily noticed under the microscope in the early stages. At present, it has been reported that QCM-D can be used to detect viscoelastic properties of pre-osteoblast cells [3]. Besides, shear-horizontal surface acoustic wave sensor has been applied for the monitoring of cell spreading and attachment [4]. However, no study has realized the label free, real-time and quantitative detection of cardiac viscoelasticity for drug evaluation based on acoustic wave sensors.

Recently, the surface acoustic wave (SAW) sensor has been greatly developed as a well-performed mass and viscoelasticity sensing device for label-free immunoassay. In particular, the Love Wave sensor has shown its advantages in biosensor applications due to its high sensitivity and stability in the liquid phase [5]. Hence, it is a promising device capable of monitoring viscoelastic properties of a population of cells in a label free and real-time way.

As shown in Fig.1a, a self-designed Love Wave biosensor was used to monitor the viscoelastic properties of HL-1 cardiomyocytes in this study. Our previous studies have shown that insertion loss (IL) of Love Wave biosensor is a more suitable parameter for the quantitative detection of viscoelasticity than phase. Then, two different drugs were added to observe the effects on the viscoelastic properties of HL-1 cells.

Method

In this study, the Love Wave biosensor is constructed based on a piezoelectric quartz substrate. Interdigitated transducers (IDTs) with Ti/Au (20/200 nm) are deposited to generate acoustic waves. The input and output IDTs electrodes consist of 50 split-finger pairs with a preset wavelength λ=28 μm, which determines the center frequency of the sensor to be about 152 MHz. The distance between IDTs centers is 200λ and the acoustic aperture is 75λ. Afterwards, the IDTs patterned substrate is guided with a 3 μm SiO₂ film deposited by plasma enhanced chemical vapor deposition (PECVD). Au layer (200 nm) is deposited on top of guiding layer to improve the cell attachment on the sensor surface.

A polydimethylsiloxane (PDMS) chip (Fig.1b) was designed and used as the cell culturing chamber. The height and volume of this cavity are about 10 mm and 110 μL, respectively. A portable multi-channel detection system was developed to collect both the IL and phase signals of Love Wave biosensors. Then the HL-1 cardiac cells with the density of 50,000 cells/well were seeded on the surface of Love Wave biosensor (Fig.1c). To test the function of the detection system, isoprenaline (ISO) and verapamil (VRP) with different concentrations were used to induce changes in the viscoelastic properties of HL-1 cells.

Results and Conclusions

In Fig.1c, HL-1 cells attach and grow well on the pre-coated Love Wave biosensor, which demonstrates the biocompatibility of the sensor. The insertion loss signals of sensor after treated by ISO and VRP are shown in Fig.1d. It can be found that the insertion loss of the control group decreases slightly, which may be due to cell proliferation. VRP is a calcium channel antagonist that blocks the L-type calcium channel and prevents calcium influx into the cell, thereby decreasing the cardiomyocyte contraction force. With the addition of VRP solutions, the insertion loss of Love Wave biosensors increases with the increase of VRP concentration within the dosage range tested. In contrast to VRP, ISO is positive inotropic drug. After ISO treatment, HL-1 cells tend to be contractive due to the traction forces. Therefore, the higher the ISO concentration, the tighter the contact of the cells with the sensor substrate. Thus, insertion loss decreases after ISO treatment which changed positively correlated with concentration. Moreover, the optical microscope photos also proved that the cells after ISO treatment seems to be smaller than control group, while the cells after VRP treatment seemed to be larger.

According to our preliminary experimental results, the affection on cell viscoelasticity of different drugs can be monitored by recording the insertion loss value of the SAW biosensor. Our results demonstrated that the proposed cell-based SAW biosensor is a potential, convenient and quantitative platform for in vitro cardiac viscoelasticity evaluation, especially in the early stages. It has promising applications in cell monitoring, drug evaluation and many other fields.

References

[1] Wei X, Qin C, Gu C, et al. A novel bionic in vitro bioelectronic tongue based on cardiomyocytes and microelectrode array for bitter and umami detection[J]. Biosensors and Bioelectronics, 2019, 145: 111673.

[2] Wei X, Gu C, Li H, et al. Efficacy and cardiotoxicity integrated assessment of anticancer drugs by a dual functional cell-based biosensor[J]. Sensors and Actuators B: Chemical, 2019, 283: 881-889.

[3] Shoaib S, Tabrizian M. A QCM-D sensing strategy for investigating the real-time effects of oxidative stress on the viscoelastic properties of pre-osteoblast cells[J]. Sensors and Actuators B: Chemical, 2019, 293: 235-246.

[4] Brugger M, Schnitzler L, Nieberle T, et al. Shear-horizontal surface acoustic wave sensor for non-invasive monitoring of dynamic cell spreading and attachment in wound healing assays[J]. Biosensors and Bioelectronics, 2021, 173: 112807.

[5] Matatagui D, Moynet D, Fernández M J, et al. Detection of bacteriophages in dynamic mode using a Love-wave immunosensor with microfluidics technology[J]. Sensors and Actuators B: Chemical, 2013, 185: 218-224.

Figure 1

Wearable sensors have received a major recent attention owing to their considerable promise for monitoring the wearer's health and wellness. The medical interest for wearable systems arises from the need for monitoring patients over long periods of time. These devices have the potential to continuously collect vital health information from a person's body and provide this information to them or their healthcare provider in a timely fashion. Unlike early efforts aimed at monitoring mobility and vital signs, our recent efforts aim at filling the gaps by providing continuous biochemical information. The new chemical sensing wearable platforms provide new avenues to continuously and non-invasively monitor individuals and can thus tender crucial real-time information regarding a wearer's health. This presentation will discuss recent developments in the field of wearable electrochemical sensors integrated directly on the epidermis or within the mouth for various non-invasive biomedical monitoring applications [1-3]. Particular attention will be given to non-invasive monitoring of metabolites and electrolytes using flexible amperometric and potentiometric sensors, respectively, along with related materials, energy and integration considerations. Microneedle sensor arrays for multiplexed ISF monitoring will also be discussed also with wearable energy harvesters aimed to power these devices. The preparation and characterization of such wearable electrochemical sensors will be described, along with their current status and future prospects and challenges.

REFERENCES

[1] "Wearable Chemical Sensors: Present Challenges and Future Prospects" A. J. Bandodkar, I. Jeerapan, J. Wang, ACS Sensors, 2016, 1, 464.

[2] A. J. Bandodkar and J. Wang, "Non-invasive wearable electrochemical sensors: a review", Trends Biotechnol., 2014, 32, 363.

[3] "Wearable biosensors for healthcare monitoring", J. Kim, A. S. Campbell, B. Esteban-Fernández de Ávila, and J. Wang, Nature Biotechnology, 2019, 37, 389.

Artificial intelligence (AI) and wearable sensors are two fields that are instrumental in realizing the goal of precision medicine—tailoring the best treatment for individual patients. Recent development between these two fields is enabling better patient data acquisition and improved design of wearable sensors for monitoring the wearers' health, fitness, and their surroundings. The growing field of wearable sensors aims to tackle the limitations of centralized, reactive healthcare by giving individuals insight into the metrics of their own physiology. However, assessing the effectiveness of a therapeutic platforms on disease is extremely complex, due to the massive quantities of data generated by biomedical devices. Integration of AI approaches can bridge this gap, using pattern analysis and classification algorithms for improvement of diagnostic and therapeutic accuracy. The future AI-biosensors (AI wearable sweat biosensor, AI- eatable biosensor, AI-glass biosensor, AI-implantable biosensor et al.) mainly have the function of AI-diagnosis (Diagnostic algorithms in the microprocessor can verify the output of the sensor and present the diagnostic information), Big data processing (Use of self-contained space for historical data and various necessary parameters of data storage to greatly improve the performance of the controller.) and Self-learning/adaptive (Embedded microprocessor with advanced programming function. In the working process, the AI-biosensor can reconstruct the structure and parameters according to certain behavioral criteria, and has adaptive functions)

First, surface-enhanced Raman scattering (SERS) probes have been developed. Subsequently, SERS probes have been coupled to a nano-array to form chip-based enzyme-linked immunosorbent assays (ELISA). Next, the SERS probes and ELISA have been integrated into paper-based microfluidic strips to form an automated and miniaturized optofluidic device for point-of-care testing.

Apparel with embedded self-powered sensors can revolutionize human behavior monitoring by leveraging everyday clothing as the sensing substrate. The key is to inconspicuously integrate sensing elements and portable power sources into garments while maintaining the weight, feel, comfort, function and ruggedness of familiar clothes and fabrics. We use reactive vapor coating to transform commonly-available, mass-produced fabrics, threads or premade garments into comfortably-wearable electronic devices by directly coating them with uniform and conformal films of electronically-active conjugated polymers. By carefully choosing the repeat unit structure of the polymer coating, we access a number of fiber- or fabric-based circuit components, including resistors, depletion-mode transistors, diodes, thermistors, and pseudocapacitors. Further, vapor-deposited electronic polymer films are notably wash- and wear-stable and withstand mechanically-demanding textile manufacturing routines, enabling us to use sewing, weaving, knitting or embroidery procedures to create self-powered garment sensors. We will describe our efforts in monitoring heartrate, breathing, joint motion/flexibility, gait and sleep posture using loose-fitting garments.

We have built a multianalyte microphysiometer that detects multiple analytes involved in the cellular bioenergetics simultaneously. Monitoring the uptake of glucose and oxygen, and the production of lactate and acid gives a more complete picture of the metabolic processes within the cell than just acidification microphysiometry alone. Metabolic processes such as glycolysis, mitochondrial ATP generation, and glycogenesis are all directly related to the flux of these analytes. Temporal resolution of metabolic responses is much faster than conventional well-plate studies, leading to dynamic metabolic data. Electrochemical sensors are easily adapted into microfluidic formats for microphysiology readouts.

Our newest instrumental designs include a complete overhaul of the microfluidic chambers, screen printed electrodes, and microfluidic pumps, valves, and drivers that will enable easy adoption of this methodology in the development of 3D cell and tissue studies using 8 separate electrochemical measurements at a time. In the first design, 8 separate amperometric sensors are developed using a screen printed electrode with isolated microfluidic channels. In the second design, 4 amperometric sensors are combined with 4 potentiometric sensors using the newly created CH1440 created by CH Instruments. The adaption of this technology to instrumenting organs-on-a-chip is currently underway.

The rising research interest in personalized medicine promises to revolutionize traditional medical practices. This presents a tremendous opportunity for developing wearable devices toward predictive analytics and treatment. In this talk, I will introduce our recent advances in developing fully-integrated skin-interfaced flexible biosensors for non-invasive molecular analysis. Such wearable biosensors can continuously, selectively, and accurately measure a wide spectrum of sweat analytes including metabolites, electrolytes, hormones, drugs, and other small molecules. These devices also allow us to gain real-time insight into the sweat secretion and gland physiology. The clinical value of our wearable sensing platforms is evaluated through multiple human studies involving both healthy and patient populations toward physiological monitoring, disease diagnosis, and drug monitoring. This talk will also feature our very recent works on laser-engraved lab on the skin and biofuel powered battery-free electronic skin toward metabolic/nutritional management as well as dynamic stress monitoring. These wearable and flexible devices could open the door to a wide range of personalized monitoring, diagnostic, and therapeutic applications.

Introduction

Biochemical sensors are one of the essential tools for cell-based assays used in major research fields including drug discovery, cancer research, and immunology. Although significant efforts have been made to develop new technologies and tools, most existing systems in this field are still not suitable for "ready‐to‐use in-field" applications, as the cells inside the assay need vital parameters, such as oxygen, nutrients, and temperature and therefore will not survive during a transportation to an end user. Nevertheless, providing these parameters is not practicable during conventional transportation either. Those systems need to be assembled and prepared on-site, which often requires a cell culture laboratory setting and trained staff. Thus, there is an urgent need for preserving the whole optimized assay system to utilize them for concepts like "ready-to-use" or "on-demand-use".

Cryopreservation, the process of freezing and preserving the cells at sub-zero temperatures is a well-known technique for cell culturing. It could be one possible solution to solve the transportation and storage challenges in the fields mentioned above. However, freezing of adherent cells is more prone to the cell membrane damage and cell detachment, due to ice crystal formation and a mismatch in the linear thermal expansion between the frozen cell membrane and the rigid substrate [1]. Therefore, innovative methods and tools must be implemented to overcome these challenges.

In this work, for the first time, the surface of a field-effect-based sensor was modified with flexible electrospun fibers to allow on-sensor cryopreservation. Furthermore, a protocol was developed to freeze and to thaw adherent cells in a microfluidic channel.

Method

The light-addressable potentiometric sensor (LAPS), a field-effect-based biochemical sensor, can detect the extracellular acidification of the cells due to its pH sensitive transducer layer (Ta₂O₅). The LAPS was fabricated as a structure of Al/p-Si/SiO₂/Ta₂O₅ as described in ref. [2]. The sensor surface was modified with flexible polyethylene vinyl acetate (PEVA) fibers using an electrospinning method. The modified LAPS chip was then fixed to the underside of a bottomless microfluidic slide to form a miniaturized device (cryo-chip) (Figure 1(a) and (b)). A flexible polymer coverslip and a conventional LAPS were used as control samples. All samples were sterilized by 70% ethanol for one hour under aseptic conditions in a laminar flow cabinet to maintain the sterility. Chinese hamster ovary (CHO-K1) cells were adherently cultured inside the channels of the device with the mixture of Ham's Nutrient Mixture F-12/Dulbecco's Modified Eagle Medium (Ham's F-12/DMEM, 1:2 mixture, pH 7) supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin. After the cell culture inside the channels were obtained for 48h under cell culture conditions in an incubator, the entire device was placed in a 3D-printed freezing container and frozen at -80^oC using a cryoprotectant solution (90% fetal calf serum supplemented with 10% dimethyl sulfoxide). The samples were kept in the freezer overnight. The frozen device was then thawed rapidly by transferring them directly to the incubator (37^oC). The cryoprotectant solution inside the channels was removed by washing with the fresh medium (37^oC) four times, as soon as the ice crystals disappeared. Cell recovery and cell viability were analyzed before and after cryopreservation using a dye exclusion assay and a cell counting kit (CCK)-8 assay. In addition, as proof-of-concept measurements, the extracellular acidification of the cells was monitored by the LAPS chip, after the cryopreservation.

Results and Conclusions

The results showed that the sensor surface modified with the electrospun PEVA fibers exhibits enhanced biocompatibility, which promotes cell proliferation and spreading. In addition, the novel cryo-chip system using this hybrid structure (rigid/flexible) is effective compared to an un-modified LAPS surface for keeping cells viable during on-sensor cryopreservation. These findings showed, for the first time, that a cryopreservation on-chip is possible and that microfluidic systems including semiconductor based sensors can survive the cryopreservation treatment. Thus, this kind of cryo-chip systems enable on-sensor cryopreservation and measurement which can open up a new possibility for "ready-to-use, in field" applications. Being able to prepare the entire system at the manufacturing stage and to send them via a cold-chain transport further eliminate potential run-to-run and operator-to-operator variability, resulting in more reproducible results.

Acknowledgements

Dua Özsoylu (DÖ) would like to acknowledge the PhD research scholarship grant from Scientific and Technological Research Council of Turkey (TÜBITAK) under BIDEB 2214-A. The authors also gratefully thank the Federal Ministry of Education and Research of Germany (Opto-Switch FKZ: 13N12585). This study is a part of PhD thesis study of DÖ (thesis number DEU. HSI. PhD-2013970198) in Dokuz Eylül University, Institute of Health Sciences, Turkey.

References

[1] T. Rutt, N. Eskandari, M. Zhurova, J.A.W. Elliott, L.E. McGann, J.P. Acker, J.A. Nychka, Thermal expansion of substrate may affect adhesion of Chinese hamster fibroblasts to surfaces during freezing, Cryobiology. 86 (2019), 134–139. doi.org/10.1016/j.cryobiol.2018.10.006.

[2] D. Özsoylu, S. Kizildag, M.J. Schöning, T. Wagner, Effect of plasma treatment on the sensor properties of a light-addressable potentiometric sensor (LAPS), physica status solidi (a). 216 (2019), 1900259. doi.org/10.1002/pssa.201900259.

Figure 1: Photos of the cryo-chip system for on-sensor cryopreservation. a) The modified LAPS chip integrated in a microfluidic chip system and its complementary connections; b) culturing of the CHO-K1 cells inside the channels on the LAPS surface, which are modified by electrospun PEVA fibers.

Figure 1

Endotoxins, also known as lipopolysaccharides (LPS), are important macromolecules in outer membrane of Gram-negative bacteria, which are generated when the bacteria are in growing and stationary phases, and disintegrate after death. While endotoxins induce a strong immune response in hosts, it also can serve as an important biomarker to diagnose various bacterial diseases. Among various pathogenic bacteria, Porphyromonas gingivalis is attracting much attention in recent years. P. gingivalis is a major Gram-negative bacterium that is responsible for chronic periodontitis, among > 500 bacteria species found in subgingival plaque. Periodontal disease presents a chronic inflammation caused by bacterial infection, leading to gum disease and even loss of teeth. LPS from P. gingivalis (PG LPS) induces significant host responses in gingival tissue by increasing the production of inflammatory biomarkers, such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor alpha (TNF- a) in gingival fibroblasts.[1] Furthermore, because of the close relationship between P. gingivalis and other important systemic diseases such as cardiovascular diseases (e.g. atherosclerosis[2]) and neurodegenerative Alzheimer's disease[3] that has emerged in recent years, quantitative evaluation of PG LPS has become a key measure in oral health as well as in the whole body health system.

Considering the increasing significance of P. gingivalis LPS, high demand for a novel point-of-care device has emerged for both early diagnosis and frequent monitoring PG LPS levels. Traditional qualitative detection of bacterial biomarkers is carried out mostly utilizing the well-known PCR method, which requires trained personnel and relatively longer time span in a laboratory setting. Lateral flow assay is one of the most promising POC platforms with many benefits, such as low cost, simple fabrication process, user-friendly colorimetric interface, rapid turnaround time, and no need for external instruments.[4] We have developed two different approaches for LFA-based POC devices to detect and quantify PG LPS concentrations. First, traditional immunoassay based LFA was developed to detect the PG LPS. Because PG LPS is a large macromolecule, multiple binding sites can be present on PG LPS. The sandwich assay mechanism was adopted using two different antibodies against PG LPS (Fig. 1a). Monoclonal antibodies are conjugated on the surface of gold nanoparticles (AuNP) to capture PG LPS molecules in the sample solution, and polyclonal antibodies are printed on the nitrocellulose membrane to form a test line by capturing PG LPS bound AuNP conjugate from the sample solution. After dispensing the sample solution on LFA, the test line is formed based on the PG LPS presence in the sample solution (Fig. 1b). Higher LPS concentration leads to a stronger test line. The second approach is to replace antibodies with aptamers as a bio-recognition molecule for target analytes. Aptamer-based assay uses a short synthetic DNA or RNA molecules that replace the traditional antibody/antigen immunoassay. Aptamer can be designed and synthesized in vitro with excellent sensitivity and selectivity to various target molecules. In addition, excellent long-term and thermal stability can also be provided because aptamers are chemically synthetized molecules. As shown in Fig. 2a, duplex aptamer was covalently conjugated to AuNPs. When LPS is introduced, the aptamer binds to the target LPS and is released from the duplex aptamer. In the competitive binding approach (Fig. 2b), a stronger test line is formed when less LPS is present. As shown in Fig. 2c, an increasing difference in test line intensity is observed as the LPS concentration is varied between 0 and 1 ng/mL. Interestingly, although usually the competitive assay is less sensitive than sandwich assay, in this case so far the competitive aptamer-based LFA presented higher sensitivity than that of sandwich immunoassay-based LFA test results. To further increase the assay sensitivity, future work will focus on enhancing the sandwich aptamer based LFA device using two different aptamers binding to the different sites on PG LPS.

1. Wang, P.-L. and K. Ohura, Porphyromonas gingivalis Lipopolysaccharide Signaling in Gingival Fibroblasts–CD14 and Toll-like Receptors. Critical Reviews Oral Bio & Med, 2002. 13(2): 132-142.

2. Mougeot, J.L.C., et al., Porphyromonas gingivalis is the most abundant species detected in coronary and femoral arteries. J. Oral Microbiology, 2017. 9(1): p. 1281562.

3. Dominy, S.S., et al., Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Science Advances, 2019. 5(1): p. eaau3333.

4. Dalirirad, S., D. Han, and A.J. Steckl, Aptamer-Based Lateral Flow Biosensor for Rapid Detection of Salivary Cortisol. ACS Omega, 2020.doi: 10.1021/acsomega.0c03223

Figure 1

Introduction

Lab-on-a-Chip systems are innovative tools which can be used in the field of life sciences. They find applications e.g. in single cell analysis or cytotoxicity tests [1]. Despite the growing popularity of the use of microsystems in biological research, there is a lack of microsystems that may be used in the electrochemotherapy (ECT) studies. ECT is a antitumor therapy, based on an application of electroporation (EP) during standard chemotherapy (CT). EP uses external electric field to form hydrophilic pores in the cells membrane. Electropores are additional migration pathway for molecules which enhanced their delivery into cells [2]. ECT allows to use lower concentration of drug and reduces side effects in comparison to standard chemotherapy [3]. We develop a Lab-on-a-Chip microsystem for cell electroporation that could be used to examine the effectiveness of chemotherapy as well as for evaluation the effectiveness of electrochemotherapy.

Microsystem fabrication

The microsystem is made of polydimethylsiloxane (PDMS) and glass. Casting method was used to obtained the microchannels and microchambers in PDMS layer. Pairs of gold electrodes were arranged parallelly along the microchannel with microchambers at the distance of 2 mm. The microsystem allows simultaneous culturing of normal and tumor cells. There are four rows of microchambers for each cell line: I - cells not exposed to compound or electric field (control), II - electroporated cells not exposed to compound (control for EP), III – cells electroporated with compound (simulating condition of ECT), IV - cells incubated with compound (simulating condition of CT). In this way, it is possible to evaluate and compare the effectiveness of two types of therapeutic procedures.

Method

The microchip was sterilized using 70% ethanol and UV radiation. After that, the cell suspension was introduced using a peristaltic pump at a speed of 3.5 µl/min. After 24 h of incubation, the cells medium (control) and the solution of the test compound were respectively introduced into the microsystem, nextly the cells were electroporated. Cell observations were performed using an inverted fluorescence microscope. In addition, fluorescence intensity measurements of the introduced molecules were carried out using a multi-well plate reader. The AlamarBlue test was performed to determine cell viability. For this purpose, a 10% AlamarBlue solution was introduced into the microsystem, than fluorescence intensity was measured at λex=558 nm and λem= 585 nm.

Results and Conclusions

To determine the optimal electroporation parameters (pulse length, their number, voltage) preliminary experiments were led using propidium iodide (PI). Tests were carried out for two skin cell lines: normal HaCaT and tumor A375. Two sets of parameters were examined: 1 pulse 10 ms and 8 pulses 0.1 ms, each in three voltage variants: 150, 180 and 200V. Cell viability after electroporation was determined. It was found that there were no significant changes in the cell viability after electroporation with the voltage lower than 200V. The efficiency of PI delivery into cells was confirmed by microscopic observation as well as determined by fluorescence intensity measurements. Significantly lower PI level inside cells (at the level of 30%) using 8 pulses 0.1 ms for both cell lines was observed. The best efficiency of PI delivery (about 90%) was observed when 1 pulse of 180V lasting 10 ms was applied. In addition, cell morphology was observed and cells parameters such as: shape factor, sphericity, convexity and elongation were determined. It was confirmed that the electroporation of cells does not change their morphology. Based on the obtained results it was concluded that the optimal electroporation conditions for HaCaT and A375 cell lines are: 1 pulse 10 ms 180V.

References

[1] Grabowska-Jadach I., Haczyk M., Drozd M., Fischer A., Pietrzak M., Malinowska E., Brzózka Z., "Evaluation of biological activity of quantum dots in a microsystem", Electrophoresis, 2016, 35, 165-177.

[2] Yarmush M. L., Golberg A., Sersa G., Kotnik T., Miklavcic D., "Electroporation-based Technologies for Medicine:Principles, Applications and Challenges", Annual Review of Biomedical Engineering, 2014, 16, 295-320.

[3] Calvet Ch. Y., L. Mir M., "The promising allianceof anti-cancerelectrochemotherapywithimmunotherapy", Cancer Metastasis Review, 2016, 35, 165-177.

This work was realized with the frame of project Preludium no 2018/31/N/ST4/02922.

Introduction

Molecularly imprinted polymers (MIPs) are recognition elements with specific cavities designed for a particular target molecule. Recently, MIPs have been used in various applications explicitly requiring molecular recognition. In MIPs, the crosslinking monomers and functional monomers, having an affinity with a target molecule, are copolymerized in the presence of the target or the template molecule. Upon selective removal of the template molecule, imprinted cavities are formed. MIPs provide a wide range of benefits, including mechanical reliability, cost-effectiveness, and rapid mass production, and have recently been used for various applications, especially in biosensing areas [1]–[3].

Theophylline (THO) is a drug commonly used for the therapy of respiratory diseases. However, owing to its highly toxic nature, an overdose can induce paralysis, seizures, and even death.[4] Additionally, the therapeutic window of THO is relatively small (5-15 mgmL^-1) [4],[5], and therefore, the therapeutic drug monitoring (TDM) of theophylline is highly significant [5]. In this study, molecularly imprinted polymers grafted carbon pastes were prepared for THO sensing. The commonly used functional monomer, methacrylic acid, crosslinking monomers, N, N'-methylenebisacrylamide (MBAA), and ethylene glycol dimethacrylate (EDMA) were used, and the MIPs thus formed were evaluated using differential pulse voltammetry on a paper chip.

Methods

Fig. 1 shows the scheme of the paper chip along with the sensing response of theophylline MIP. The paper chip composed of a photo-paper base with electrodes printed using conductive ink through an inkjet printer. The holes for electrodes (working electrode, a reference electrode, and counter electrode) and reservoir are cut on laminating sheets using a laser cutter. The three parts are then attached using the laminator. The reference electrode is packed with Ag/AgCl ink and dried overnight at 60 ℃. The counter electrode with the printed conductive ink is used as it is. The MIP-grafted carbon paste is packed in the working electrode with the help of a glass tube. The theophylline imprinted poly(methylene bisacrylamide-co-ethylene dimethacrylate-co-methacrylic acid) was grafted on the graphite particle surface by a procedure similar to our previous work [3], [6]. This grafted graphite was mixed with ferrocene containing silicone oil to make a paste used as the sensing material. We performed the differential pulse voltammetry with theophylline sample solution (in saline buffer with pH 7.4, 0-40 mgmL^-1 and whole bovine blood) filled in the sample-reservoir.

Results and Conclusions

The execution time of each voltammetry was 2 min only. The plot in Fig. 1 shows the influence of the theophylline concentration on the redox current at 0.8 V at the MIP-carbon paste electrode in both buffer saline and whole bovine blood. It is quite evident from the figure that MIP is sensitive towards theophylline in both buffer and whole blood. It indicates that MIP activates the electrocatalytic behavior of the carbon-paste electrode. The dynamic range at the MIP electrode versus the concentration of theophylline is seen in the range of 0-40 mgmL^-1, which covers the therapeutically effective theophylline concentration level in plasma varying from 5 to 15 mgmL^-1. The DPV measurements at each concentration has been done using a new chip every time, i.e. 'single-use' of the chip. Additionally, no reagents have been added during the analysis, thus making the procedure of drug level measurement simple. From the results of this study, it can be concluded that the proposed method is useful for quick and straightforward real-time TDM to prevent the toxic side effects of an overdose.

References

[1] T. Sakata, et al.RSC Adv., 10(29),16999–17013, 2020; doi:10.1039/d0ra02793f.

[2] Y. Yoshimi, et al.Sensors, 19(10), 2415, 2019; doi:10.3390/s19102415.

[3] Y. Yoshimi, et al.Sensors Actuators, B Chem., 259, 455–462, 2018; doi:10.1016/j.snb.2017.12.084.

[4] R. Vassallo, et al.Mayo Clin. Proc., 73(4), 346–354, 1998; doi:10.1016/s0025-6196(11)63701-4.

[5] A. Peng, Int. J. Electrochem. Sci., 12(1), 330–346, 2017; doi:10.20964/2017.01.03.

[6] Aaryashree et al., Sensors, 20(20), 5847, 2020; doi:10.3390/s20205847

Figure 1

Introduction

At present precise tracking of pesticide has become very important for safeguarding the environment and food resources owing to their very high toxicity [1]. The development of sensitive and convenient sensors for the sensitive detection of pesticides is imperative to overcome practical limitations encountered in conventional methodologies, which require skilled manpower at the expense of high cost and low portability [2–3]. However, there are increasing bottlenecks in terms of poor stability caused by recognition unit and false positive result induced by single-modal readout, especially the bad performance in on spot monitoring. Herein, taking advantage of all-in-one enzyme-inorganic hybrid nanoflowers (ACC-HNFs) to fabricate high-performance artificial enzyme cascade system, we newly designed a ultrasensitive and affordable lab-on-paper device that incorporated disposable screen-printed carbon electrode (SPCE) and colorimetric test strips, which brought about the dual-modal readout (electrochemical and colorimetric signal) for on-site monitoring of pesticide, achieving an "on-demand" tuning of the detection performance.

Method

Typically, 200 mmol L^–1 PBS (pH 8.0) contained AChE (1.0 U mL⁻¹) and ChO (2.0 U mL⁻¹) was added to deionized water, then 200 mmol L^–1 CuSO₄ was added to the mixture, followed by incubation at 4.0 °C for 18 h. Finally, ACC-HNFs were obtained. ACC-HNFs modified paper was directly added into the detection zone. Then, ACh, TMB, and stop solution was added into different regions in sequence. After paraoxon was introduced into detection zone, region Ⅰ, Ⅱ, and Ⅲ of the detection zone was folded and the fold of each region was maintained for 30 min, then a discernible colorimetric signal was observed with naked eyes in the hollow region. Meanwhile, the entire paper was folded above the electrodes, and the generated electrons can be better connected to the working electrode through the hollow region. All electrochemical measurement processes were performed at room temperature and each measurement was performed in a new disposable SPCE. Before the analysis of the paraoxon, parameters, such as ACh concentration, ACC-HNFs concentration, and incubation time, were further optimized. Amperometric i-t curves were recorded at +100 mV during 30 s. The detection mechanism of this dual-modal biosensor is based on inhibiting the activity of AChE, thus the inhibition rate (I%) can be expressed as a linear relationship with the concentration of paraoxon. I% was analyzed by the following relation:

I% = (I no inhibitor – I inhibitor) / (Ino inhibitor ) ×100%

Where I no inhibitor and I inhibitor represented the response of ACC-HNFs-TMB and ACC-HNFs-TMB-OPs system, respectively.

Results and Conclusions

Using paraoxon as a model analyte, the ACC-HNFs-based lab-on-paper platform could reach a limit of detection down to the picogram/mL level (0.06 pg mL⁻¹), which is over 10-fold lower than that of conventional electrochemical assay. This proposed platform possesses the following advantages: (1) In the artificial enzyme cascade system, ACC-HNFs exhibited high catalytic activity by integrating nanozyme and natural enzyme, which remarkably amplified detection signal. (2) This approach integrates electrochemistry and colorimetric patterns into one system for "on-demand" detecting pesticide. The two groups of results can mutually authenticate, which can effectively avoid false positive and negative detection. (3) This portable lab-on-paper device dispenses with complex sample pretreatment or sophisticated instruments, which makes it suitable for on-site monitoring. We anticipated that the meticulous design of ACC-HNFs provided a versatile approach for constructing artificial enzyme as a recognizer and amplifier to fill the gap in constructing robust artificial enzyme systems, making it practically functional for on-site applications in the contamination monitoring and biological diagnosis, just through changing the building blocks.

References

[1] M. J. Palmer, C. Moffat, N. Saranzewa, J. Harvey, G. A. Wright, C. N. Connolly, Cholinergic pesticides cause mushroom body neuronal inactivation in honeybees. Nat. Commun. 4 (2013) 1634. doi: 10.1038/ncomms2648.

[2] E. Cequier, A. K. Sakhi, L. S. Haug, C. Thomsen, Development of an ion-pair liquid chromatography–high resolution mass spectrometry method for determination of organophosphate pesticide metabolites in large-scale biomonitoring studies. J. Chromatogr. A. 1454 (2016) 32–41, doi: https://doi.org/10.1016/j.chroma.2016.05.067.

[3] F. Zhao, J. Wu, Y. Ying, Y. She, J. Wang, J. Ping, Carbon nanomaterial–enabled pesticide biosensors: Design strategy,biosensing mechanism, and practical application. TrAC-Trend. Anal. Chem. 106 (2018) 62–83, doi: https://doi.org/10.1016/j.trac.2018.06.017.

Introduction

Diabetes mellitus is a group of metabolic diseases characterized by elevated blood sugar resulting from a defect in the production of insulin by pancreatic β-cells or tissue insensitivity for this peptide hormone [1]. In 1980s, there were approximately over 100 million diabetic adults in the world. Nowadays this number is 4 times bigger and by 2030 it will probably reach 1 billion [2]. The most common form of the disease is diabetes mellitus type 2 (insulin-dependent), which is diagnosed in about 80% of all patients. In patients with this type of diabetes, both the efficiency and the secretion of insulin are disturbed [3]. The most important cells for this disease are insulin secreting β-cells and their antagonist - glucagon-secreting α-cells. The pancreatic islet consists of 5 cell types, the largest percentage is occupied by β-cells, constitute about 60% (in the core) and α-cells constitute about 25% (located on the periphery) of all cells in the islet [4]. In our research, we focused on imitate the pancreatic islet structure, which can be a universal model for testing the impact of various environmental factors on disease development. The idea of using a Lab-on-a-chip system for three-dimensional islet cells culture came from the possibility of mimicking in vivo conditions. This can give a wider and more accurate view of the pancreatic islets function than the cell culture in static conditions.

Lab-on-a-chip system

In our research we present a Lab-on-a-chip system in which a pancreatic "pseudoislet" model will be developed. This system is composed of biocompatible, non-toxic and transparent materials, such as: poly(dimethylsiloxane) (PDMS) and thin glass. Thanks to this, it is possible to conduct cell culture and observe the results in various types of microscopes. The geometry of Lab-on-a-chip system consist of two elliptical cell culture chambers, one for the test sample and second for the reference sample. In in each of the chambers there are 15 round microtraps. Each of the microtraps is made of 7 micropillars. This solution forces the dense packing of cells and their aggregations. The geometry of the developed system is consistent with the culture wells on a standard multi-well plate, which allows measurements in a multi-well plate reader.

Methods

All experiments were performed using two commercially purchased pancreatic islets cell lines: β-cells (INS-1E) and α-cells (α-TC1-6), which are a model cell lines for the study of diabetes mellitus type 2. Appropriate α-cells and β-cells ratios were selected to reflect the morphology and composition of the pancreatic islet found in the body. The cells were introduced through the inlet to the Lab-on-a-chip system and placed for 24h in an incubator to form spherical aggregates. In the next stage immunostaining protocol using primary and secondary antibody solutions was developed. Due to the use this method and confocal microscope it was possible to confirm the correct distribution of cells in the obtained aggregate. To confirm cells viability previously prepared solution of Calcein AM and Propidium Iodide were introduced through the microsystem inlet with a flow rate 5µL/min for 10 min. After 10 min incubation (37^oC, 5% CO₂) the viability of the cells was determined using fluorescence microscope. Cells proliferation was measured using spectrofluorimetric multi-well plate reader after 1 hour incubation with AlamarBlue solution. Moreover, to confirm functionality of the α- and β-cells aggregates, insulin secretion tests were performed 24 h after introducing the cells into the microfluidic system. The ELISA test was performed after stimulating the aggregates with low (2.75 mM) and high (16.5 mM) glucose concentrations.

Results and Conclusions

It was confirmed that 15 spherical α and β cell aggregates with diameters of 160-180 µm were obtained using the method described earlier. As was expected the participation of β-cells in the core and α-cells on the periphery of the aggregate was also confirmed by the use of immunostaining method. Thanks to this, it was confirmed that the obtained three-dimensional models correspond to the distribution/arrangement and cellular composition of the pancreatic islet located in the body. Moreover, the compatibility of the designed microsystem with the multi-well plate reader allows to monitor cell proliferation and viability in situ during the cultures. Due to using Calcein AM and Propidium Iodide dye a high viability after 24 and 48 hours of culture in the microchip was confirmed and was 97% and 95% respectively. The cells maintained their morphology, function and high level of proliferation for up to 48 hours of culture. Moreover, functionality of the α- and β-cells aggregates was confirmed. The level of insulin secretion in the developed microfluidic system was 1.38 ng per pseudoislet after low glucose stimulation. For high glucose, we detected 2.75 ng insulin per aggregate. At this stage, a research model corresponding to the model in vivo conditions was obtained. This study presents basic research and, in the future, this model could be utilized to simulate diabetes, testing new drugs and therapy in diabetes mellitus treatment.

References

[1] Selby J.V., Ray G.T., Zhang D., Colby C.J., Excess costs of medical care for patients with diabetes in a managed care population. Diabetes Care 1997; 20: 1396–1402.

[1] World Health Organization. Global report on diabetes. World Health Organization, 2016.

[2] American diabetes association Diagnosis and Classification of Diabetes Mellitus. Diabetes care 2008, 31:1

[3] Brissova, M., Fowler, M. J., Nicholson, W. E., Chu, A., Hirshberg, B., Harlan, D. M. & Powers, A. C.Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J.Histochem. Cytochem. 2005; 53:1087–1097.

Introduction

Pesticide contamination has severely attacked the ecosystem and food safety, gradually becoming an escalating threat to public health [1]. Current laboratory-based pesticides-analyzing conventional apparatus comprising of chromatography [2] and mass spectrometry [3] with satisfactory precision and resolution were limited by on-site monitoring. Hence, the development of convenient sensors for pesticide is of great significance. Herein, we constructed a handheld smartphone-assisted fluorometric platform for on-site quantification of pesticide with excellent selectivity and high selectivity based on the optical features of red-emission carbon dots (CDs) and signal amplification of alkaline phosphatase (ALP). The red emissive CDs whose maximum emission center was located at 605 nm with high stability could shield photodamage and autofluorescence interference and enhance anti-interference capability toward matrix. For accurate quantification, the corresponding images were captured by smartphone, together with 3D-printed auxiliary equipment. Using a self-designed application, color intensity was directly relevant to the concentration of analyte pesticide. It is believed that the proposed smartphone-based handheld platform could afford experience for designing neotype sensing strategy in pesticides monitoring.

Method

For smartphone-based quantitative analysis 2, 4-D, 50 μL various concentrations of 2, 4-D and 50 μL of ALP (7.0 U L^-1) were equilibrated at 37 ºC for 10 min. Following, 120 μL of ascorbic acid-2-phosphate trisodium salt (AAP) (100 μmol L^-1) containing Tris-HCl buffer were added for incubating another 10 min at 37 ºC and 160 μL CDs/CoOOH composite solution was added to the mixture. After equilibrated for 10 min, the cuvette was inserted into the 3D-printed smartphone reader to obtain the FL photo under the excitation of 532 nm laser. Then, the displayed photo image was directly analyzed by App installed on smartphone.

Results and Conclusions

Given the design and exploration above, we can directly detect 2, 4-D concentrations in real-time by collecting the optical image of sample. The collected images brightness of the probe solution was in negative connection with 2, 4-D concentrations. Coupling with the FCSMP software, the smartphone readout gray values of various analyte concentrations were acquired in Figure 1. The linear relationship (R² = 0.990) between smartphone readout gray value and the concentration of 2, 4-D was I = 46.14825 – 2.26776 [2, 4-D], with a LOD of 100 μg L^-1 (3σ). Although weaker than the FL spectrometry method on the basis of identical system, that the integrated smartphone-connected sensor displayed comparable liner range and LOD compared with other reported methods was worth noting. The results of 2, 4-D detection demonstrated that the smartphone-assisted fluorometric sensing platform possessed a low requirement for sample volume (50 μL), rapid (30 min) and easy readouts for POC testing.

In summary, we have constructed an innovative and portable smartphone-based POC platform for precise quantification of 2, 4-D in a good sensitivity and excellent selectivity manner based on CDs/CoOOH composite. Employing CDs/CoOOH composite as signal indicator, a simple but potentially promising biosensor with outstanding anti-interference capability and good sensitivity have been established. The anti-interference ability was ascribed to the red emissive CDs that could shield background interference. In virtue of introducing ALP, the biosensor performed specific recognition capacity toward 2, 4-D, endowing it high-performance of selectivity. The proposed approach which integrated commercial smartphone, 3D model smartphone accessory and self-designed App into one platform simplified image processing and minimized detection device, effectively circumventing the drawback of bulk instrument with inaccessibility in carrying and complex computer-assistant data analysis. More significantly, the sample-to-answer analysis time of smartphone-based platform is 30 minutes, which was shorter or comparable than that of the immunoassay, chromatograph and enzyme-based strategies. What's more, such a smartphone optical-sensing system has been triumphantly applied to biological and environmental samples, indicating its robust performance in biological/chemical analysis. Thus, the proposed smartphone-based handheld device provided a promising platform for on-site monitoring pesticide, possessing potential applications in environmental screening, health monitoring, and disease prevention.

References

[1] G. Aragay, F. Pino, A. Merkoci, Nanomaterials for sensing and destroying pesticides, Chem. Rev. 112 (2012) 5317-5338. doi: 10.1021/cr300020c.

[2] Y. Wu, L. Jiao, W. Xu, W. Gu, C. Zhu, D. Du, Y. Lin, Polydopamine-capped bimetallic AuPt hydrogels enable robust biosensor for organophosphorus pesticide detection, Small 15 (2019) e1900632. doi: 10.1002/smll.201900632.

[3] Ł. Rajski, M.J. Martínez-Bueno, C. Ferrer, A.R. Fernández-Alba, LC-ESI-QOrbitrap™ MS/MS within pesticide residue analysis in fruits and vegetables, TrAC, Trends Anal. Chem. 118 (2019) 587-596. doi: 10.1016/j.trac.2019.06.032.

Figure 1

Introduction

Novel and robust point-of-care (POC) biosensors were designed and developed for the detection of two inherited blood disorders; glucose-6-phosphate dehydrogenase (G6PD) and phenylketonuria (PKU), with PreQuine Systems, Fig. 1a. The G6PD enzyme performs a critical function in human biochemistry. G6PD deficiency is among the most common enzyme pathology that affects 400 million people worldwide and is mainly found in malaria-endemic regions. Malaria affects over 200 million people yearly with 440,000 deaths. Malaria caused by Plasmodium vivax and by Plasmodium oval threatens over 2 billion people globally and sickens tens of millions annually. While treatments such as primaquine and tafenoquine provide a cure, over 8% of the global population are contraindicated due to inherited G6PD deficiency, as these treatments can cause serious, and often life-threatening, anemia. Therefore, a robust, user-friendly, and reliable test is necessary for diagnosing the G6PD deficiency to prevent hemolytic disorders while treating malaria. PreQuine is a novel platform for simultaneous quantification of G6PD and hemoglobin (Hgb) concentrations.

PKU is a genetic disorder that causes a build-up of amino acid, specifically L-phenylalanine (Phe), in the blood. PKU is the most common amino acid metabolic disorder and occurs in 1 out of every 8,000 newborns globally. Currently, whole blood is collected in EDTA tubes or spotted onto Dried Blood Spot (DBS) Cards by parents, patients, or caregivers. These samples are sent to laboratories for measurement by tandem mass spectrometry, and results can take days to weeks. This complicated process for monitoring and controlling Phe levels results in non-compliance, a decrease in quality of life, as well as increased healthcare costs for treating complications. Once diagnosed with PKU, Phe concentration levels must be monitored and maintained within acceptable limits between 2 – 6 mg/dL. If left untreated, infants can develop intellectual disabilities or suffer from other common side effects such as seizures, delayed development, behavioral problems, and psychiatric disorders. We have developed a vertical flow colorimetric assay for the quantitative determination of Phe in biological specimens. The assay requires only 15 µL of blood, the Phe within the specimen reacts with the reagent layer to produce an end-point color. This POC platform can be used at home, in the hospital, or at a clinician's office to measure Phe concentrations and diagnose a patient with PKU.

Methods

The PreQuine Platform is a novel POC diagnostic test for simultaneous quantification of G6PD and Hgb in malaria patients, Fig.1b. A 10μL finger-stick blood sample is collected via a small capillary tube. The capillary tube is placed directly into a micro-centrifuge tube containing a fixed volume of lysing solution to lyse red blood cells, and a test strip is directly placed into a hand-held meter. The G6PD enzyme and Hgb are liberated into the buffer. Sodium nitrite is immobilized on the transport membrane, which is used to oxidize the hemoglobin. The transport membrane removes cellular debris, and the oxidation of the Hgb is required to eliminate it as a G6PD assay interference. The hemolysate solution migrates laterally over the two detection zones, vertically into the Hgb detection pad and vertically through the optical membrane and into the G6PD detection pad. The optical membrane, above the G6PD detection pad, inhibits the transmittance of light from the LED. The G6PD test strip contains an immobilized substrate, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP⁺), diaphorase (electron mediator), and a tetrazolium salt indicator.

A novel vertical flow assay was also developed for the detection of Phe in blood, Fig. 1c. A 15μL finger-stick blood sample is collected via a small capillary tube and dispensed on the test strip. The test strip is composed of a series of membranes that separate plasma from whole blood. The primary blood separation membrane is composed of glass microfiber with a pore size of 3 – 4 µm, which size excludes 85 to 90 % of cells through immobilized hemagglutinating agents (lectin). The secondary blood separation membrane with a pore size of 2 µm is necessary to remove 100 % cell separation. This membrane also preconditions the plasma to a pH of 7.0 to eliminate interference from tyrosine. The third and final reagent membrane is composed of an anisotropic polysulfone membrane coated with an immobilized tetrazolium salt (WST-5), enzyme (phenylalanine dehydrogenase), electron mediator (diaphorase), and NAD⁺.

Results & Discussion

The end-color of the G6PD and Hgb detection pads are measured at 670 nm and 570 nm, with read times of 6 and 1 minute(s), respectively. The PreQuine Platform versus spectrophotometric values revealed an excellent agreement with an R² = 0.98. Over the analytical range, the average bias was less than 0.75 Unit G6PD per gram of Hgb. The %R was then respectively interpolated to G6PD U/g Hgb to obtain calculated UG6PD/g Hgb.

The PKU test results demonstrated exceptional performance over the analytical range of 0-25 mg/dL of Phe (0-6 mg/dL, normal range of Phe in the blood) and showed an excellent correlation with the mass specs values in the range of (0-25 mg/dL) with an R² = 0.99. The high sensitivity and specificity achieved by the coupling of a diaphorase performs extremely well at low pH needed for the suppression of L-tyrosine interference, along with a highly sensitive tetrazolium salt indicator that acts as a good substrate for the preferred diaphorase. The harmonization of the diaphorase, at a given pH with a specific tetrazolium salt, provided the necessary sensitivity in the analytical range of Phe in blood.

Figure 1

Manganese (Mn) is one of the most common elements in the earth's crust, and its compounds are widely used in consumer products including gasoline, cell phone batteries, fertilizer, pigments, fireworks, etc. Although a trace metal in the human body and under tight homeostatic control, elevated levels of Mn due to occupational or environmental exposure can cause serious adverse health effects, such as manganism (a Parkinsonian-like syndrome), liver cirrhosis, and Behcet disease. The normal range of Mn in blood is 4.7-18.3 µg/L (5-20 ppb), and values greater than 36 µg/L correlate with disease. The current method for analyzing Mn in blood is via inductively coupled plasma mass spectrometry (ICP-MS) in centralized labs, which requires a venipuncture sample, can take 3-6 months for analyses, and can be costly. Electrochemical analysis can offer an attractive alternative, leading to developing a point-of-care system for Mn monitoring in blood, with the advantages of being inexpensive and easily miniaturized. Herein, we report the first Indium Tin Oxide (ITO) electrochemical microsensor capable of accurately measuring Mn levels in human blood with cathodic stripping voltammetry (CSV). As shown in Fig. 1 (a), the ITO microsensor was developed by integrating a screen-printed carbon auxiliary electrode and an Ag/AgCl reference electrode on an ITO coated glass slide (1 cm × 4 cm). A circular ITO film with 3 mm diameter was exposed and used as the working electrode. To start electrochemical analysis, blood sample was first microwave digested with nitric acid and hydrogen peroxide to release Mn²⁺ bound to the proteins. The acid-digested blood sample was then titrated with sodium hydroxide to pH 5. Square wave cathodic stripping voltammetry (SWCSV) with optimized parameters was carried out to construct the calibration curve in the digested & pH adjusted blood sample with the Mn concentration ranging from 1.2 ppb to 25 ppb (confirmed by ICP-MS). The voltammogram in Fig. 1(b) shows an increasing trend of peak height and area with Mn concentration; and the calibration curve in Fig. 1(c) shows a sensitivity of 38.6 nC/ppb and linearity of 0.997 from the sensor's measurements. We used the same stripping parameters, while applying a 3-point standard addition method, to determine the Mn concentration in two blood samples. The concentrations of 10.5 ppb and 26.3 ppb from our measurements lead to an average accuracy of 90% in comparison to the ICP-MS results of 8.9 ppb and 27.6 ppb. Four 150 µl sample droplets with a total volume of 600 µl were used in the measurement. Considering the 10X dilution from the blood digestion and pH adjustment process, only 60 µl of whole blood is needed to determine the Mn level with our approach. The favorable results and the small blood volume required in this work suggests the feasibility of point-of-care monitoring Mn in blood and is expected to be very useful for monitoring Mn exposure in vulnerable populations like children.

Figure 1

Colorectal cancer (CRC) is a worldwide diffused pathology for both men and women and it is the third most common cancer for both genders, in the United States[1] However, if promptly diagnosed, CRC is also one of the most curable tumor types (90% curability rate at stage I), therefore prevention is fundamental to avoid degeneration. Here the clinical validation outcomes of a preventive screening method based on a patented device, SCENT A1, composed by a core of nanostructured MOX sensors [2]. SCENT A1 is capable of detecting the presence of CRC from fecal odor with an in-vitro, inexpensive and non-invasive methodology. Fecal odor can be affected by the presence of CRC-biomarkers produced by membrane peroxidation and metabolic alterations [3,4]. The screening analysis adopted by NHS of Italy and of other countries is fecal occult blood test (FOBT). In Italy, FOBT is performed every two years on subjects from 50 to 69 years old and shows a huge percentage of false positives (about 65% according to data). FOBT-positives are then invited to undergo colonoscopy. SCENT A1 is capable of reducing FOBT false positives and so the total number of non-operative colonoscopies with related risks (e.g. bowel perforation). The device, presently undergoing instrumental certification, is composed by a core of two chemoresistive MOX sensors, chosen and calibrated for this specific aim [5-11]. Sensors (Figure 1) have been synthesized at Sensor Laboratory of the University of Ferrara and are composed by a nanostructured semiconductor film screen-printed onto an alumina substrate and a platinum heater to modulate operating temperature. The first sensor is composed by Iron and Samarium oxides while the second one by Tin and Titanium oxides. The data collection and analysis software employs support vector machine (SVM) [9]. The clinical validation protocol of this device started in May 2016 and ended in July 2019, in collaboration with Hospital St. Anna of Ferrara and Department of Public Health of Ferrara. A total of 398 fecal samples of FOBT-positive subjects have been analyzed by SCENT A1 system with the k-fold cross validation method. Samples have been grouped into two macro categories, depending on the gold-standard (colonoscopy) outcomes: 260 healthy subjects (negative to colonoscopy); 138 colorectal adenomas and carcinomas (positive to colonoscopy), 54 low-risk adenomas and 84 high-risk adenomas and carcinomas. The system has been capable of distinguishing among these two categories with a sensitivity (TPR: true positive rate) and specificity (TNR: true negative rate) respectively of 84,1% and 82,4%, defined as follows:

• TPR=TP/P=TP/(FN+TP);

• TN/N=TN/(FP+TN);

where TP, TN, FP and FN are the abbreviations that indicate respectively true positives, true negatives, false positives and false negatives. The choice to group carcinomas, high- and low-risk adenomas into a single class and not into two diverse classes as before [10], has simplified the test and ensured greater doctor protection, despite a small loss in specificity. Low-risk adenomas are, in fact, often similar to healthy tissues and have a risk of evolving into cancer comparable to the risk of colonoscopy complications. As already emerging from a previous study [10-11], in fact, the major error of classification (only 57%) concerned just low-risk tumors that emit gases halfway between healthy and tumoral samples. What emerges is that, if SCENT A1 test is carried out on FOBT-positives subjects as a second check, it would guarantee a reduction of about two thirds of the colonoscopies performed on healthy patients. A new collaboration, involving private clinic Quisisana, started in 2020 with some laboratory tests on sensors reproducibility [12] will continue in January 2021 with tests on patients directly inside the clinic, in order to test the new shape of the device under certification. Moreover, if employed by the NHS of countries without screening system, this method will significantly reduce CRC-mortality rate.

[1] [https://www.cancer.org/cancer/colon-rectal-cancer/about/key-statistics.html

[2] Italian #: RM2014A000595, European #: 3210013 (Germany, UK);

[3] B. Szachowicz-Petelska et al., NMR in Biomedicine V5,226–233, 1992;

[4] T. G. de Meij et al., International Journal of Cancer (2014), 134, 1132–1138;

[5] C. Malagù et al., Sensors, 14, 18982-18992, 2014;

[6] G. Zonta et al., Sensors and Actuators B, 218, 289-295, 2015;

[7] N. Landini et al., Scholars' Press, ISBN-13: 978-3-639-76538-0;

[8] G. Zonta et al., Sensors and Actuators B, 238, 1098–110, 2016;

[9] G. Zonta et al., Sensors and Actuators B 262 (2018) 884–891;

[10] G. Zonta et al., Sensors and Actuators B: Chemical, Volume 301, 12 December 2019, 127062;

[11] G. Zonta et al., Cancers 2020, 12(6), 1471;

[12] G. Zonta et al., Ceramics International Volume 46, Issue 5, 1 April 2020, Pages 6847-6855.

Figure 1: a) external view of the patented prototype SCENT A1, composed by flowmeters, a flow diverter and a sample box; b) one of the chemoresistive sensors composing the core of the instrument; c) magnificated scheme of the sensor.

Figure 1

Infectious diseases are worldwide a major cause of morbidity and mortality. Fast and specific detection of pathogens such as bacteria is needed to combat these diseases. Optimal methods would be non-invasive and without extensive sample-taking/processing. Here, we developed a set of near infrared (NIR) fluorescent nanosensors and used them for remote fingerprinting of clinically important bacteria. The nanosensors are based on single-walled carbon nanotubes (SWCNTs) that fluoresce in the NIR optical tissue transparency window, which offers ultra-low background and high tissue penetration. They are chemically tailored to detect released metabolites as well as specific virulence factors (lipopolysaccharides, siderophores, DNases, proteases) and integrated into functional hydrogel arrays with 9 different sensors. These hydrogels are exposed to clinical isolates of 6 important bacteria (Staphylococcus aureus, Escherichia coli, ...) and remote (≥25 cm) NIR imaging allows to identify and distinguish bacteria. Sensors are also spectrally encoded (900 nm, 1000 nm, 1250 nm) to differentiate the two major pathogens P. aeruginosa as well as S. aureus and penetrate tissue (>5 mm). This type of multiplexing with NIR fluorescent nanosensors enables remote detection and differentiation of important pathogens and the potential for smart surfaces.

Introduction

Precise and sensitive detection of protein biomarkers is of vital importance in clinical diagnosis and biomedical research. To date, enzyme-linked immunosorbent assay (ELISA) remains the gold standard in detecting protein biomarkers due to the robust properties of simple operation, acceptable sensitivity, and automated high throughput [1, 2]. Despite desirable results from ELSA, pursuing high sensitivity and accuracy to meet diagnosis demands is still an ongoing endeavor. For that, nanomaterials behaving with favorable physical properties and biocompatibility may serve as a solution, especially the carbon nanosphere (CN) or gold nanoparticles (AuNPs) that feature strong stability, easy manufacture, low cost, and environment-friendly, and abundant active sites [3, 4].

Heart failure (HF) is one of the most threatening cardiovascular diseases. B-type natriuretic peptide (BNP), a vital cardiac biomarker, has been acknowledged as one of the principal biomarkers for HF [5]. Individuals at risk of HF demonstrate raised BNP in serum and real-time quantification of HF biomarkers, identifying those at highest risk of HF early, and ensuring they receive appropriate treatment can effectively prevent premature death [6]. Although current ELISA-methods for detecting BNP have been developed, there are urgent needs for providing more efficiently and reliably in vitro diagnostic (IVD) methods or point-of-care (POC) devices in BNP clinical measurement.

Most of the previous studies for ELISA-based BNP detection required large instruments with high costs such as microplate readers, spectrophotometers. In this work, we adopted a self-developed portable bionic electronic eye (Bionic E-eye) system using a smartphone (iPhone 4S and iPad 3) integrated with simple accessories including a piece of electroluminescent, a wide-angle lens, and a dark hood instead of the commercial microplate reader to make the entire analysis process more intelligent and convenient. In addition, the CN-AuNPs nanocomposites (CGN) we prepared was used as a signal amplifier to develop a high-throughput immunocolorimetric method that sensitively identified BNP through the color change of antibody-antigen reactions using enzyme-substrate and horseradish peroxidase-antibody (pAb-HRP) linked CGN immunoprobe (CGNs@ pAb-HRP). The combination of Bionic E-eye and high-throughput immunocolorimetric system aimed to establish a general and reliable strategy for POC testing (Figure. 1A).

Method

Monoclonal antibody against BNP were immobilized on the surface of the 96-well plate as capture antibody. Then the different concentrations of BNP were added to the wells. Finally, the HRP-labeled polyclonal BNP (pAb-HRP) antibody or the pAb-HRP modified CGNs (pAb-HRP-CGNs) were added to bind with BNP. After 3,3',5,5'-Tetramethylbenzidine (TMB) and HCl were added, different shades of yellow color related to BNP concentration appeared. Combining with the Bionic E-eye, BNP concentration could be analyzed by image processing.

Results and Conclusions

To achieve optimally sensitive signal output and fabricate a well-performance immunoprobe, the synthesized conditions were optimized. Under optimal conditions, we have successfully synthesized stable and homogeneous CGNs validated by TEM (Figure. 1B) and XPS. The performance for BNP detection by a commercial microplate reader (Figure. 1C) and Bionic E-eye were evaluated and compared. The detection range of both was comparable (7.8 ppb~125 ppb) while the Bionic E-eye system had a lower limit of detection (LOD) than a commercial microplate reader (4.3 ppb < 7.2 ppb) (Figure. 1D), indicating that compared with a commercial colorimetric reader the self-developed system achieved higher sensitivity which could be attributed to the superiority of saturation channel in HSV or RGB color model the Bionic e-Eye used. In addition, the signal amplifying effect of the pAb-HRP-CGNs probe was also confirmed in the comparison of LOD and detection range with pAb-HRP. Figure. 1E showed that the LOD in the method using pAb-HRP-CGNs reached 0.92 ppb and the wider detection range of 3.9 ppb~250 ppb was achieved compared with that of 7.8 ppb~125 ppb in the non-CGNs method. Based on the above results, this proposed CGNs based detection of BNP with Bionic e-Eye shows promising application prospect but more experimental optimizations should be performed to further improve its performance.

References

[1] Shi, C., et al., Nanoscale technologies in highly sensitive diagnosis of cardiovascular diseases. Frontiers in Bioengineering and Biotechnology, 2020. 8.

[2] Jiao, L., et al., Au@Pt nanodendrites enhanced multimodal enzyme-linked immunosorbent assay. Nanoscale, 2019. 11(18): p. 8798-8802.

[3] Xu, T., et al., Triple tumor markers assay based on carbon–gold nanocomposite. Biosensors and Bioelectronics, 2015. 70: p. 161-166.

[4] Zeng, S., et al., A review on functionalized gold nanoparticles for biosensing applications. Plasmonics, 2011. 6(3): p. 491-506.

[5] Szunerits, S., et al., Electrochemical cardiovascular platforms: current state of the art and beyond. Biosensors and Bioelectronics, 2019. 131: p. 287-298.

[6] Savonnet, M., et al., Recent advances in cardiac biomarkers detection: From commercial devices to emerging technologies. Journal of Pharmaceutical and Biomedical Analysis, 2020: p. 113777.

Figure 1

The ability to fabricate incredibly sophisticated integrated circuits at low cost rendered modern electronics more affordable, efficient, and functional. This monumental advance in technology would usher in the age of the Internet-of-things and transform wearable electronics from a largely academic pursuit into an everyday reality around which a robust and rapidly growing industry has formed. However, even the highly connected wearable electronics of today leave much to be desired with respect to their compatibility with soft tissues such as skin. More specifically, the poor compliance of the rigid, silicon-based integrated circuits conventionally used in the construction of these devices with the soft, curvilinear surfaces of the human body can result in high impedances at the skin-sensor interface which in turn contribute to low signal-to-noise ratios as well as poor sensitivity and accuracy. Motion artifacts originating from the poor conformability of such devices only exacerbate these problems. This mechanical mismatch represents a significant hurdle in the effort to move away from the conventional reactive approach to healthcare towards a more proactive approach that leverages 'truly' wearable sensors to improve health outcomes.

Transistors are the building blocks upon which the vast majority of modern electronics are built. Although single transistors can be used to amplify or otherwise control electronic signals using only a small input signal, most of their functionality comes in the form of integrated circuits (ICs) consisting of many connected transistors. Conventional techniques for fabricating ICs exploit the rigidity of the semiconducting silicon substrate to ensure the device performance is consistent throughout the circuit. In contrast, integrated circuits for highly compliant wearable electronics that can conform even to microscopic features of the skin[1,2] face the inherent challenge of maintaining their electrical performance under mechanical deformations. As is the case with most sensors, the front-end circuitry for these devices (responsible for conveying the information contained in the signal – either wirelessly or without wires – to the end user) requires reliable and performant integrated circuits. Of course, integrating soft, ultra-thin sensors with the rigid silicon-based integrated circuits results in a mechanical mismatch with mechanical failure at the interface where the two components meet being the most likely scenario.

Instead, here we present an approach which uses two-dimensional (2D) transistors in a specially designed strain-neutralizing configuration which circumvents much of the difficulties associated with the aforementioned mismatch. The extraordinary properties of 2D materials—particularly their excellent mechanical flexibility, ultimate thinness, optical transparency, and favorable transport properties for realizing electronics—make them prime candidates for replacing conventional silicon-based transistor circuits in next-generation wearable applications. Using finite-element analysis we have designed and simulated a compliant, strain-insensitive substrate which can resist uniaxial applied strains upwards of 35%. Moreover, simulations of the electronic band structure of the transistors under applied strain is used to optimize the circuit design and further minimize the effect of strain in the transistors. These electronic and mechanical simulations are used together in the configuration and subsequent fabrication of fully integrated strain-neutralized 2D transistors compatible with state-of-the-art soft stretchable wearable sensors.

[1]S. Kabiri Ameri et al., "Graphene electronic tattoo sensors," ACS Nano, 11, 7634–7641, 2017.

[2] S. Kabiri Ameri et al., "Imperceptible electrooculography graphene sensor system for human–robot interface", npj 2D Materials and Applications, 2, 1-7, 2018.

Introduction

For the last years, low-cost microfluidics and microarrays on polyester substrates have gained applications in a variety of fields, including analytical Point-of-Care systems. The use of a 2D platforms brings a significant reduction in assay costs, both due to a lower consumption of materials and reagents, as well as a simplicity of manufacturing process in comparison to current approaches [1]. Commercial applicability of immunoassays requires the development of efficient strategies for antibody immobilization in bulk quantities on a wide variety of readily available substrates. Other factors, which influence the usefulness of antibody immobilization approach are scalability, reproducibility and cost-efficiency. Simple, one-step methods such as passive adsorption on hydrophobic surfaces or direct protein immobilization with the use of functionalized silanes are particularly appreciated in fabrication of immunosensing platforms [2]. The use of masking properties of cured toner as an intermediate for passive and covalent immobilization of antibodies on poly(ethylene terephtalate) (PET) seems to be an attractive and still undiscovered path for development of immunoassays on versatile, printable substrates [3].

Fabrication of toner-basedplatforms for Ab-capturing and immunosensing

Throughout presented research two general approaches for immobilization of antibody and antibody-binding protein (protein A/G) were examined: 1) adsorption on pristine PET@toner foil via hydrophobic interactions, 2) amine-dependent, covalent binding on glycidyloxypropyl tromethoxysilane (GTPMS)- coated surface. Print pattern (at various grayscale levels) was designed in ChemSketch 2.0 and printed on PET surface using HP 100 LaserJet P1006 printer at resolution of 1200 dpi. Pristine PET@toner foils were used for passive adsorption. For GTPMS coating, hydrophilization of toner was performed by a 10-minute treatment with the use of UV/ozone cleaner. Then, 3% GTPMS solution in 50% ethanol was dispensed on freshly-oxidized foil for 30 min. Both multi-purpose substrates characterized by different surface properties were then incubated with rabbit anti-CRP antibody or protein A/G followed by surface blocking.

Method

To fully characterize the analytical performance in a role of immunosensing platforms, two types of immunoreactions were carried out on prepared 2D Ab/protein arrays. Direct immuno-labelling of rabbit anti-CRP antibody with anti-rabbit IgG-alkaline phosphatase (ALP) conjugate aimed at comparative evaluation of specificity and efficiency of antibody coating. Indirect sandwich immunoassay for C-reactive protein was selected as a model to determine basic working parameters of toner-based immunoassay. Antibody-capturing properties of protein A/G surface were evaluated by determination of monoclonal antibody-ALP conjugate binding capacity. For quantitative analysis, enzymatic reaction was carried out by dispensing p-nitrophenyl phosphate as chromogenic ALP substrate.

Results and Conclusions

Results of immunolabeling confirmed the usefulness of cured toner as an intermediate for efficient antibody and protein A/G coating via hydrophobicity-driven adsorption. Due to an increase of surface area and a hydrophobicity higher than for pristine PET, the densest layers were observed for fully printed surfaces (100% black level). The differences in the surface properties were also reflected in kinetics of antibody binding. UV/ozone treatment resulted in permanent oxidation and thus hydrophilization of toner surface. The introduction of hydroxyl groups favored reaction with GTPMS. Epoxy-silane coating of oxidized toner enabled a rapid fabrication of protein-reactive surface characterized by a high binding capacity in a single step immobilization. Thanks to good adhesion and thus tight bonding of laser-cut adhesive tape to PET@toner, the microchannel architecture was obtained. It opened up the possibility to carry out immunoassays in microfluidic systems. It was proven that presented methodologies, due to the design and fabrication simplicity, use of commonly available materials and up-scalability, could be successfully applied in immunodiagnostics or flexible microfluidics for Lab-on-a-Foil technology.

References

[1] E.F.M.Gabriel, B.G.Lucca, G.R.M.Duarte, W.K.T.Coltro, Recent advances in toner-based microfluidic

devices for bioanalytical applications, Anal. Methods. 10 (2018) 2952-2962. doi:10.1039/c8ay01095a

[2] A.I.Barbosa, A.s.Barreto, N.M.Reis, Transparent, Hydrophobic Fluorinated Ethylene Propylene Offers Rapid, Robust, and Irreversible Passive Adsorption of Diagnostic Antibodies for Sensitive Optical Biosensing, ACS Appl. Bio. Mater. 2 (2019) 2780–2790. 10.1021/acsabm.9b00214

[3] B.L.Thompson, C.Birch, J.Li, J.A.DuVall, D.LeRoux, D.A. Nelson, A-C.Tsuei, D.L.Mills, S.T. Krauss, B.E.Root. J.P.Landers, Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device, Analyst. 141 (2016) 4667-4675 doi:10.1039/c6an00209a

Acknowledgement

This work has been financially supported by the National Centre for Research and Development in Poland (grant no. POIR.04.01.04-00-0027/17).

Over the past 40 years, miniaturized potentiometric and amperometric sensors for ions (K⁺, Ca⁺⁺, Na⁺, Mg⁺⁺, Cl^-, H⁺), gases (O₂ and CO₂), and nutrients/metabolites (glucose, lactate, creatinine, urea) have revolutionized the practice of critical care medicine by providing tools to measure an array of these physiologically important species, simultaneously, in small volumes of undiluted whole blood. Indeed, all modern point-of-care whole blood analyzers used in hospitals worldwide now employ electrochemical sensor arrays as either single-use or multi-use devices for near-patient testing, especially in operating rooms, emergency rooms, intensive care units, etc. Some of the same or similar chemistries have also been adapted to create either single-use or continuous monitoring optical sensors (fluorescence, etc.). Further, nearly all blood glucometers as well as newer implantable subcutaneous glucose sensors now use electrochemical measurement principles to provide accurate glucose concentrations for millions of diabetic patients each and every day. A brief overview of these existing electrochemical/optical sensor technologies that have already had such a great impact in medicine will be provided during the introductory portion of this lecture.

At the same time, there remain a number of unmet needs in medicine where electrochemical/optical chemical sensing devices could still play important analytical roles. Therefore, in the major portion of this presentation, the following research projects ongoing in our laboratories at the University of Michigan will be highlighted: 1) recent efforts to utilize electrochemical sensors for measurement of polyionic drugs and associated contaminants (including the anticoagulant heparin, low-molecular weight heparin, and inflammatory over-sulfated chondroitin sulfate (OSCS) contaminants in biomedical heparin preparations);^1-2 2) research aimed at utilizing the same chemistry employed to make ion and polyion selective electrochemical sensors to create optical sensing films as well as optical sensing microfluidic devices that are able to quantitate species in volumes of << 1 µL;³ 3) research related to the development of implantable electrochemical sensors for ions, gases, glucose, lactate, etc. that emit low levels of nitric oxide (NO) (a potent anti-platelet and antimicrobial agent) and that can potentially be used to continuously monitor critical care species intravenously in ICU patients with improved accuracy (see Fig. 1);⁴ and 4) efforts to use improved electrochemical/amperometric gas phase sensors for detecting nitric oxide (NO) in exhaled nasal/oral breath and also the use of NO and nitrogen dioxide (NO₂) sensors to monitor and feedback control the levels of therapeutic gas phase NO being generated by novel photochemical and electrochemical gas phase generators.^5-6 These gas phase NO generators can potentially replace use of very high cost compressed gas cylinders containing NO to provide controlled levels of NO gas on-demand for inhalation therapy of patients with pulmonary hypertension (including infants) and for use in the sweep gas of oxygenators to prevent clotting and Systemic Inflammatory Response Syndrome (SIRS) in patients undergoing open heart surgery.

Literature Cited:

1. L. Wang, S. Buchanan and M. E. Meyerhoff, "Rapid Detection of High Charge Density Polyanion Contaminants in Biomedical Heparin Preparations Using Potentiometric Polyanion Sensors," Anal. Chem., 80, 9845-9847 (2008).

2. K. Gemene and M. E. Meyerhoff, "Reversible Detection of Heparin and Other Polyanions by Pulsed Chronopotentiometric Polymer Membrane Electrode," Anal. Chem., 82, 1612-1615 (2010).

3. X. Wang, M. Sun, S. A. Ferguson, J. D. Hoff, Y. Qin, R. C. Bailey, and M. E. Meyerhoff, "Ionophore-Based Biphasic Chemical Sensing in Droplet Microfluidics," Angew. Chem. Int. Ed., 58, 1-6 (2019).

4. M. Frost and M. E. Meyerhoff, "Real-Time Monitoring of Critical Care Analytes in the Bloodstream with Chemical Sensors: Progress and Challenges," Ann. Rev. Anal. Chem., 8, 171-192 (2015).

5. J. Zajda, N. J. Schmidt, Z. Zheng, X. Wang and M. E. Meyerhoff, "Evaluation of Amperometric Platinized-Nafion-Based Gas Phase Sensor for Determining Nitric Oxide (NO) Levels in Exhaled Human Nasal Breath," Electroanalysis 30, 1602-1607 (2018).

6. Y. Qin, J. Zajda, H. Ren, J. Toomasian, T. Major, A. Rojas-Pena, B. Carr, T. Johnson , J. Haft, R. H. Bartlett, A. Hunt, N. Lehnert, and M. E. Meyerhoff, "Low Cost Portable Gas Phase Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications in Inhaled NO Therapy and Within the Sweep Gas/Cardiotomy Suction Air During Cardiopulmonary Bypass Surgery," Mol. Pharmaceutics, 14, 3762-3771 (2017).

Figure 1. Comparison of average error of PO₂ levels using amperometric IV-oxygen sensing catheters with (n=4) and without (n=4) NO release chemistry placed into the arteries of pigs for 20 h. % deviation calculated vs. measurements made on discrete whole blood samples (heparinized syringe) on Radiometer blood-gas/electrolyte analyzer.

Figure 1

Diamond is grown in some laboratories by either HPHT process or Plasma-Enhanced Chemical Vapor Deposition (MP-CVD) since a few decades. Single crystal diamond exhibits outstanding properties including a high optical transparency over a broad electromagnetic spectrum, high thermal conductivity approx. five times higher than copper, and acoustic wave velocity close to 19 000 m.s^-1. It displays also remarkable mechanical properties with e.g. a Young's modulus exceeding 1000 GPa along with high resistance to fracture, to name a few. Some of these properties remain also remarkable in its polycrystalline form when compare to most other materials. Furthermore, diamond can be doped with nitrogen or boron during growth, offering electrical properties from semiconducting to quasi-metallic regimes. When heavily doped with boron (~2.10²¹ cm^-3), the so-called Boron Doped Diamond (BDD) electrodes become attractive electrodes featuring a high potential window > 3V in water and low double-layer capacitance. Moreover, diamond is extremely resilient to corrosion and more generally to chemical attacks. It is also biocompatible, which makes it very attractive for in-vivo sensing applications. Finally, the carbon nature of the diamond offers wide opportunities for surface grafting of chemical or biochemical functional groups through highly stable covalent carbon-carbon bonding. These properties can be exploited advantageously to enhance the analytical performances and stability of chemical/biochemical sensors and have motivated our research over the last 15 years. Our work focuses mainly on polycrystalline diamond thin films that can be grown typically on inches silicon substrates, thus offering access to some clean-room processes and potentially large-scale production.

Several processes were elaborated to micro-pattern diamond layers in order to design chemical transducers such as gravimetric MEMS devices, electrodes, field effect transistors, etc. Diamond microstructures may also be transferred to flexible parylene or polyimide substrates, thus making them attractive e.g. for wearable sensors and implantable medical electrodes. Further techniques have also been developed to enhance the active surface area of diamond transducer surfaces at the nanoscale and thus increase drastically the sensitivity of the resulting sensors by multiplying the number of active sites. Here diamond is typically grown onto well-chosen high aspect ratio templates that can withstand the growth conditions of diamond in high-density hydrogen plasma. Most of these methods have been "standardized". They involve clean-room processing including dry etching, photolithography, and so on and forth.

As examples, heavily doped diamond electrodes were developed successfully both as macro- and micro-electrodes for biomedical, pharmaceutical or foodstuff analysis applications. These applications benefit both from the high analytical performances of diamond electrodes in particular due to their low background signals and high reactivity, and high stability and reliability. BDD electrodes may also be modified with transition metal nanoparticles to enhance their catalytic behavior. From this concept, diamond multi-electrode arrays were designed for chemical patterns identification, for instance for sensory analysis of coffee, or for the monitoring of environmental pollutants. BDD electrodes offer also significant advantages in electrochemiluminescence (ECL) techniques, which are being investigated for various applications ranging from foodstuff analysis to narcotics detection. A key benefit of BDD electrodes for all of the above applications is certainly that they can be electrochemically reactivated following fouling, sometimes directly in the analytical medium, to maintain high reactivity thus opening the way to reusable sensors and online monitoring. Besides, BDD microelectrode arrays have also been transferred to flexible substrates for neural stimulation and recording, along with in-vivo neuromodulators measurements. Finally, diamond based MEMS devices (microcantilevers, SAW sensors) take advantage both of the mechanical properties of diamond, along with steady carbon interface for convenient bio-functionalization. Our work here focused mainly on the detection of odorant molecules, using biomolecular receptors involved in olfaction in Nature as sensitive layers, including Odorant Binding Proteins (OBPs), Major Urinary Proteins (MUPs) and Olfactory Receptors (OR). Multisensor array instrumentations were developed around this concept, for applications ranging from breathe analysis to security applications.

Biosensors are devices that combine biorecognition with signal transduction to analyze biologically-relevant targets. In order for these devices to be used in disease management, clinical decision making, and health monitoring, they must deliver sufficient sensitivity, specificity, and speed at the point-of-care and rely on minimal sample processing. This work is focused on developing integrated biorecognition and signal transduction strategies for enhancing the performance metrics of biosensors.

Barcode-based biorecognition systems translate analyte capture to the release of specific signal transducing barcodes. We have focused on developing barcode-based systems that are compatible with photoelectrochemical and electrochemical readout for targeting proteins and nucleic acids. These systems translate molecular recognition elements using functional nucleic acids and antibody-DNA complexes for releasing an electroactive or a photoelectrochemical DNA barcode. We have combined these barcode-based systems with nanostructured electrochemical transducers for increased signal-to-noise-ratio and sensitivity.

By combining barcode-based biorecognition with electrochemical readout, we have developed handheld biosensors for clinical analysis of urinary tract infections. We have also used these strategies for detecting prostate cancer biomarkers. Integration of barcode-based biorecognition with photoelectrochemical readout presents a new modality for biosensing and opens the route for ultrasensitive and programmable biosensing.

There is a significant need for development of low-cost and portable diagnostics for non-invasive monitoring of biochemical markers. Among various biosensors, electrochemical sensors are at the forefront of point of care diagnostics, due to simple and real-time readout, low cost, high sensitivity, and portability, as evident from the dominance of enzymatic glucose sensors in the healthcare sector.(1) Despite their significant success, one of the main challenges with enzyme-based sensors is the limited (or none) choice of enzymes for analytes beyond glucose and their incompatibility with device engineering processes.(2)

Over the past few years, there have been significant advances in flexible sensors, printed electronics, and wireless devices based on graphene and other 2D materials.(2–5) Being atomically thin, with high surface-to-volume ratio, 2D materials are extremely sensitive to surface perturbation, making them suitable for highly sensitive and selective electrochemical sensors. Moreover, their physical, electronic, and electrochemical properties can be modified via doping, defects, and heterostructures to enable new functionalities for biochemical sensing. We have recently made significant progress in developing non-enzymatic electrochemical sensors based on 2D materials and hybrids as 'artificial enzymes', with special focus on device scalability and ease of fabrication. In this regard, we have studied the role of substrate in material-analyte interface characteristic,(6) the effect of post-deposition low-temperature annealing of 2D inks on material-analyte interaction, and the importance of defect configuration in tuning the material-analyte band alignment.(7) In this talk, I will present our recent progress and undergoing research in this area.

References:

1. C. Chen, et al., Current and emerging technology for continuous glucose monitoring. Sensors (Switzerland)17, 1–19 (2017).

2. A. Bolotsky, et al., Two-Dimensional Materials in Biosensing and Healthcare: From In Vitro Diagnostics to Optogenetics and Beyond. ACS Nano, acsnano.9b03632 (2019).

3. S. Khan, S. Ali, A. Bermak, Recent developments in printing flexible and wearable sensing electronics for healthcare applications. Sensors (Switzerland)19 (2019).

4. X. Zhang, et al., Two-dimensional MoS2-enabled flexible rectenna for Wi-Fi-band wireless energy harvesting. Nature566, 368–372 (2019).

5. D. W. Park, et al., Graphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications. Nat. Commun.5, 1–11 (2014).

6. A. Ebrahimi, et al., FeSx-graphene heterostructures: Nanofabrication-compatible catalysts for ultra-sensitive electrochemical detection of hydrogen peroxide. Sensors Actuators, B Chem. (2019) https:/doi.org/10.1016/j.snb.2018.12.033.

7. Y. Lei, et al., Single-atom doping of MoS2 with manganese enables ultrasensitive detection of dopamine: Experimental and computational approach. Sci. Adv.6, eabc4250 (2020).

Diagnostics of pathogenic and genetic disease (such as cancer) at the point of need, in particular at early-stage, requires dynamic manipulation and concentration of a small number of target molecules at individual single-molecule level, which currently limit microfluidic technologies. In my lab, we focus on engineering of new approaches in lab-on-chip technology via synergistically combining nanostructured materials with fluidic sample delivery systems to enhance the sensitivity and selectivity of the detection. Nanostructured materials boost the sensor resolution and show higher biochemical sensitivity and selectivity by significant amplification of the detection sites. We investigate 1) fabrication of novel nanostructured platforms based on 3D materials such as gold and 2D materials such as graphene and molybdenum disulfide, 2) integration of nanostructures with fluid sample delivery and biological assays (based on DNA/antibody) and 3) implementation of the device for detection of small molecules, pathogenic disease, and cancer genomics. In this regard, we address fundamental questions including: optimal interface of nanostructures with fluidic devices; target isolation, preparation and concentration in fluidic devices. We have successfully implemented the nanosurface fluidic devices for rapid and quantitative detection of bacteria such as Escherichia coli (E.coli) and Methicillin-resistant Staphylococcus aureus (MRSA), electrochemical detection of small molecules such as Dopamine and optical detection of extracellular vesicles (EVs).

While developing low-cost and multiplexing electrochemical (EC) devices for bioassay is very attractive, the miniaturization of reference electrodes (RE) is a critical issue for fabricating EC devices. Herein, a polymer-based EC device, named EC 6-well plate, is proposed and fabricated using a non-photolithography method. Polyethylene terephthalate glycol (PETG) is used as a substrate and laser-cut polyester (PET) film is used as a mask for patterning the electrodes. Acrylic mold with wells (60 μL) was bonded to the PETG substrate. Schematic diagrams (Figure 1) are used to illustrate the preparation of electrochemical 6-well plates. The diameter of the working electrode (WE) is 900 μm, and each WE-modifying step only requires 1 μL of reagent.

Both electrochemical (HCl method) and chemical methods (FeCl₃ method) were proposed to prepare the solid-state Ag/AgCl RE. Precise time control is critical for the preparation procedure. It is hard to control the time in 1 s or under 1 s using the chemical method. Hence, the electrochemical method was selected for continued work since the reaction time can be precisely controlled. The stability of solid-state Ag/AgCl and drift of open circuit potential (OCP) can be affected by anions present in the background electrolyte. Herein, 0.1 M KCl, 1 M KCl, and 0.1 M KNO₃ containing 5 mM K₃[Fe(CN)₆]/K₄ [Fe(CN)₆] were compared as the electrolytes. Considering that 0.01 M PBS is the most commonly used buffer solution for bioassay, 0.01 M PBS containing 5 mM K₃[Fe(CN)₆]/K₄ [Fe(CN)₆] was also included. OCP was recorded in these electrolytes for 1 hour. These results revealed that 0.1 M KCl is the best choice for the background electrolyte. The solid-state Ag/AgCl RE-based three-electrode system, the all Au three-electrode system (3E), and all Au two-electrode system (2E) were used to develop the immunosensor forClostridium difficile toxin B detection. Differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were applied to test the stability of the EC immunosensor. The three Au electrodes system showed just a minor DPV peak position shift and the standard deviation (SD) of OCP is about 0.0026 mV. The two Au electrodes system showed no DPV peak position shift and the SD of OCP is 0.32 mV. The solid-state Ag/AgCl RE-based three-electrode system showed a relatively large DPV peak position shift and the SD of OCP is about 4.6 mV. It is demonstrated that the all Au three-electrode system is superior to the solid-state Ag/AgCl RE-based three-electrode system for developing an immunosensor.

Figure 1

Nowadays, stress is one of the major causes of pathologies in the human body. In India, most of the time around 87% of women feels stressed. World Health Organization reported that globally 450 million people suffer from mental health related problems while in India the morbidities due to mental health showed an increasing trend from 9.5 to 102.8 per 1000 persons. Current techniques used to measure stress levels consist of self-reporting method and multimodal physiological analysis. Self-reporting consists of physical interviews and self-response questionnaire. Stress-response questionnaire lacks in direct link to stress response while physical interviews are time intensive process and also require trained interviewees. Furthermore, these cannot be used for the continuous monitoring. Multimodal physiological analysis involves the monitoring of the heart rate variability, blood pressure; brain activity and skin conductance. Major advantage in using multimodal physiological analysis is the non-invasive measurements. Still, continuous monitoring is challenging and lacking direct link with the stress. Since stress varies with time, it is also difficult to quantify it with these techniques as there is no standard available. For the monitoring of stress, the monitoring of change in concentration in biochemical marker in biofluids is the need of an hour. Cortisol, a steroid hormone and major glucocorticoid in human body is the key biomarker for stress and hence also known as stress hormone. There are many techniques to detect cortisol such as Enzyme Linked Immuno Assay (ELISA), Electro Chemi Luminescence Immuno Assay (ECLIA), chromatography and other immunoassays but these techniques are limited only up to the laboratory levels, required large volume of samples and skilled personnels. Hence to overcome these limitations, Biosensor came into picture. In biosensor, various types of bio-receptors have been used for the sensing like antibody, aptamer and molecularly imprinted polymers (MIP) etc. Due to more stability at room temperature and more specificity, aptamers are preferred as bio-receptor over antibody and MIP. Aptamers are RNA or ssDNA molecules generated by an in vitro evolution process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). For cortisol, a 61-mer long sequence of aptamer has been generated and used for sensing but in principle only a portion of aptamer participates in its interaction with the analyte. Hence in the present work, we have rationally truncated long sequence of aptamer into a smaller variant of 14-mer by considering the secondary structure of the aptamer. Since nanomaterials are the suitable candidates for the immobilization of the bioreceptor. Hence, we have synthesized and characterized graphene quantum dots(GQDs) using various characterization such as UV, PL, FTIR and RAMAN. For the electrochemical sensing of cortisol, Aptamers were immobilized on the GQDs modified electrodes. Performance of both the aptamers in terms of binding affinity, limit of detection and specificity were compared. Both the aptamers exhibited same limit of detections of 0.1 pg/ml but in terms of specificity 14-mer aptamer showed better results than the parent 61-mer aptamer. Parent aptamer showed interferences with the structural analogues of the cortisol i.e. cortisone, corticosterone and triamcinolone while truncated aptamer showed almost negligible interferences with cortisone and triamcinolone. Also, on truncation of the aptamer, binding affinity was improved by ~1900 fold which indicate the superiority of truncated aptamer (14-mer) over the parent aptamer (61-mer).

Introduction

Insufficient or elevated hormone cortisol levels triggered by physical or psychological stress can cause several health problems, such as Addison's disease, Cushing's syndrome, and chronic fatigue syndrome. High serum cortisol levels are also related to increased mortality in COVID-19 patients. Thus, sensitive cortisol detection can provide an early indication for critical patients and monitor treatment efficacy. The physiological range of salivary cortisol fluctuates in a circadian rhythm between sub-nanomolar to nanomolar range. Late-night salivary cortisol concentration in the range of 2.76-166 nM was determined for clinically confirmed Cushing's patients. A proper cortisol test requires mapping a patient's cortisol levels over cycle of 24 hours. Most of the reported cortisol immunosensors involve biological receptors, such as antibodies, which are expensive, require refrigerated transportation and storage, and suffer from steric hindrance arising from antibody-small molecule interactions [1]. Cortisol immunoassays also suffer from a slow response (30 min to 5 hr) and cross-reactivity with structurally similar hormones, such as prednisolone and progesterone.

Alternatively, the biorecognition can be realized using molecularly imprinted polymer (MIP), which provides artificial binding sites and cavities in polymer for target-specific binding. MIP holds numerous advantages over antibodies, including longer lifetimes, higher stability, and cost-effectiveness [2]. Yet, there remain several issues in the MIP-based biosensors. For instance, conventional particle-based MIP requires a time-consuming and tedious polymerization process. The coating of such MIP tends to result in an uncontrolled thickness, slow diffusion process for targets, and inefficient signal transduction and limited sensitivity. We overcome these shortcomings by creating controllable MIP thin films with target selective binding sites through electropolymerization. We also dope the MIP with nano gold to enhance the sensitivity of cortisol detection.

Method

The MIP was formed directly onto a gold nanoparticle-deposited glassy carbon electrode (GCE) through one-step voltammetric electropolymerization (inset to Figure 1a) with a mixture of o-phenylenediamine (o-PD) and HAuCl₄ in the presence of cortisol template. Imprinted cortisol molecules were eluted by washing in an ethanol solution to create cortisol specific imprinting sites into the Au-poly-o-PD composite film. For the control experiment, a non-imprinted polymer (NIP) film was also prepared under the same process conditions in the absence of templates. We controlled the process conditions (e.g., the ratio of monomer/ HAuCl₄/ template, number of polymerization cycles, pH, elution buffer, elution, and reaction time) to optimize the performance of the MIP sensor. The sensor's electrochemical responses were performed with cyclic voltammetry (CV) and square wave voltammetry (SWV).

Results and Discussion

The MIP film can be produced by electropolymerization within 20 min to achieve reproducible and controllable characteristics. During electropolymerization, AuNPs grow and anchor to the polymeric framework, possibly through the abundant amine groups on o-PD. This hybrid metal-polymer composite film could increase the electron transfer rate for the reaction with the redox reagents and thus enhance the sensitivity of cortisol detection. During electrodeposition, cortisol molecules were imprinted into composite film, which can be attributed to the H-bond between the hydroxyl groups on cortisol and amine groups on o-PD. The sensing mechanism relies on the change in redox current peak measured in the presence of the redox reagents, K₃Fe(CN)₆ and K₄Fe(CN)₆. The current decreases with the increase of the target cortisol binding on the MIP that hinders the redox reagents' access to the electrode. The CV curves in Figure 1a characterize the sensor electrode during MIP fabrication and cortisol sensing. The proposed sensor responds to cortisol detection within 8 min. The peak currents decrease with the increase in cortisol concentration (Figure 1b) due to the reduced number of imprinted cavities. The sensor exhibits a dynamic range of detection covering the physiological cortisol level from 10 pM to 100 nM with a detection limit of 1.5 pM, while the NIP shows negligible change under various cortisol concentrations (Figure 1c). Figure 1d shows that the cortisol MIP sensor also exhibits a highly selective detection against several structurally similar hormones, including estriol, estrone, progesterone, and β-estradiol.

Conclusions

We demonstrate nano gold-doped MIP electrode for sensitive, rapid, and selective detection of the hormone cortisol. The cost-effective, robust sensor provides an alternative to the conventional immunoassay and holds great potential for point-of-care cortisol detection.

References

[1] Zea M, Bellagambi FG, Halima HB, Zine N, Jaffrezic-Renault N, et al. TrAC Trends in Analytical Chemistry, 132 (2020) 116058.

[2] Beluomini MA, da Silva JL, de Sá AC, Buffon E, Pereira TC, Stradiotto NR. Journal of Electroanalytical Chemistry, 840 (2019) 343-66.

Figure 1

Monitoring the endocrine disrupting lipid-compounds such as progesterone (a gonadal steroid hormone) is becoming vital in environmental as well as clinical samples because of the deleterious effects of its excessive consumption. Herein, our present work illustrates a label free electrochemical detecting approach employing Magnetic Graphene Oxide as electrode casting nanomaterials which offered the characteristics of enlarged surface area and plenty of sites for bio receptor immobilization. Further we also characterized the synthesized nanomaterials via FT-IR, HR-TEM, XRD and RAMAN. Implementing the electrochemical techniques such as Cyclic Voltammetry and electrochemical impedance spectroscopy along with the nanomaterials generated a simplified, fast and sensitive determination of the target. With optimized parameters, this developed bio sensing platform demonstrates a wide linearity ranging from 10^-6 M to 10^-14 M with 0.173 pM as detection limit, excellent selectivity, sensitivity and a high recovery in real water samples. The stated work successfully signified the potential of developed immunosensor in real applications by giving good recovery for detecting progesterone in tap water samples.

Figure 1

Surface imprinted polymers (SIPs) are materials able to act as biomimetic receptors for different biological targets.[1] Their recognition ability is attributed to the presence of three-dimensional cavities on their surfaces that complement the target in shape.[2] Furthermore, the polymer binds to the analyte throughout interactions of covalent or non-covalent nature.[3], [4] Such interactions can be converted into measurable analytical signals by a transducer.[5]–[7] The combination of SIPs as recognition elements with different transducing elements make these sensing platforms a versatile alternative detecting tool in comparison to conventional diagnostic techniques.

In this work, a novel polyurethane-urea SIP for the real time detection of pathogen Escherichia coli is presented. The ability of the synthetic receptor to bind the analyte was assessed optically as well as quantitatively. The integration of the SIP into a flow cell allowed the detection of the analyte in one simultaneous read-out platform that combines electrochemical impedance and thermal measurements derived from the interactions at the solid-to-liquid interface. The results show that upon the exposure of the target in buffer within the flow cell, it re-binds to the polymer, resulting in an increase of the thermal resistance and a decrease of impedance, allowing the generation of a dose response curve for each transducer.

This study shows that the prepared polyurethane-urea SIPs are suitable to be implemented into a combined thermal and impedometric platform. Moreover, the results highlight the possibility of detecting quantitatively in real time the pathogenic analyte with the proposed sensor. This device could possess relevance in fields in which bacterial testing is required, such as food safety and medical diagnosis.

Bibliography

[1] K. Eersels et al., "Selective Identification of Macrophages and Cancer Cells Based on Thermal Transport through Surface-Imprinted Polymer Layers," ACS Appl. Mater. Interfaces, vol. 5, no. 15, pp. 7258–7267, Aug. 2013.

[2] D. Yongabi et al., "Cell detection by surface imprinted polymers SIPs: A study to unravel the recognition mechanisms," Sensors Actuators B Chem., vol. 255, pp. 907–917, Feb. 2018.

[3] P. S. Sharma, M. Dabrowski, F. D'Souza, and W. Kutner, "Surface development of molecularly imprinted polymer films to enhance sensing signals," TrAC - Trends Anal. Chem., vol. 51, pp. 146–157, 2013.

[4] B. Sellergren and C. J. Allender, "Molecularly imprinted polymers: A bridge to advanced drug delivery," Advanced Drug Delivery Reviews. 2005.

[5] M. M. Peeters, B. Van Grinsven, C. W. Foster, T. J. Cleij, and C. E. Banks, "Introducing thermal wave transport analysis (TWTA): A thermal technique for dopamine detection by screen-printed electrodes functionalized with Molecularly Imprinted Polymer (MIP) particles," Molecules, vol. 21, no. 5, 2016.

[6] F. Cui, Z. Zhou, and H. S. Zhou, "Molecularly imprinted polymers and surface imprinted polymers based electrochemical biosensor for infectious diseases," Sensors (Switzerland), vol. 20, no. 4, 2020.

[7] K. Eersels, P. Lieberzeit, and P. Wagner, "A Review on Synthetic Receptors for Bioparticle Detection Created by Surface-Imprinting Techniques - From Principles to Applications," ACS Sensors, vol. 1, pp. 1171–1187, 2016.

Figure 1

Bacteria are one of the most common sources of illness, responsible for a wide range of infections. Bacterial infections can be caused from a variety of sources, with thousands of deaths attributed to waterborne outbreaks alone [1]. Detection of both infectious and non-infectious bacteria is important to ensure commercial goods and water are safe for consumers. Current detection of bacterial contamination typically uses cell culture plates to measure the number of colony forming bacteria (CFU/ml) in a liquid media. This technique is widely used due to its high sensitivity and easy visual readout. Unfortunately, culture counting technique is time consuming (1-3 days), requires skilled lab technicians, can be contaminated during preparation, and cannot be integrated into the desired testing media directly.

To overcome these limitations, many bacterial sensors and detection techniques have been developed over the last few decades [2]. A recent sensor for bacteria detection is utilizing the organic electrochemical transistor (OECT). OECTs are polymer-based transistors that utilize ionic solutions to de-dope and re-dope a conducting channel. These devices are known for the high transconductance, bio-compatibility, low operating voltage, low cost, small size, ease of integration into measurement systems, and ability to function in aqueous environments [3]. Detection of a single bacteria is typically desired and achieved through the capture of bacteria cells onto the OECT channel area [4]. This approach allows for rapid detection of a specific pathogen with high sensitivity. However, for detecting contamination from a variety of bacteria a different approach must be used.

We have investigated the OECT response to the presence of non-captured bacteria present within the OECT operating media. In this approach cells are detected both in solution and on the surface of the source-drain channel and on the gate electrode. Presence of bacteria between the gate and channel region will cause a shift in effective gate voltage, leading to a change in source-drain current. Detection and characterization of (non-bacteria) whole cells has previously been reported utilizing this approach [5, 6]. In our work we have focused on the bacteria pseudomonas fluorescens (p. fluorescens) due to its size (~1µm) and potential as a water borne pathogen. P. fluorescens was cultured in Luria-Bertani (LB) and Dey-Engley (D/E) media, diluted to different concentrations in broth, and used as the operating media for OECT. Concentrations of 1.9e5 to 6.8e8 CFU/ml were found to cause a shift in effective gate voltage between 36.8 to 97.5mV.

1. Craun, G.F., Statistics of waterborne outbreaks in the US (1920–1980). Waterborne diseases in the United States, 2018: p. 73-159.

2. Ahmed, A., et al., Biosensors for whole-cell bacterial detection. Clinical microbiology reviews, 2014. 27(3): p. 631-646.

3. Strakosas, X., M. Bongo, and R.M. Owens, The organic electrochemical transistor for biological applications. Journal of Applied Polymer Science, 2015. 132(15).

4. Demuru, S., et al. Flexible Organic Electrochemical Transistor with Functionalized Inkjet-Printed Gold Gate for Bacteria Sensing. 20th International Conference on Solid-State Sensors, Actuators and Microsystems & Eurosensors, 2019.

5. Liao, J., et al., Organic electrochemical transistor based biosensor for detecting marine diatoms in seawater medium. Sensors and Actuators B: Chemical, 2014. 203: p. 677-682.

6. Ramuz, M., et al., Monitoring of cell layer coverage and differentiation with the organic electrochemical transistor. Journal of Materials Chemistry B, 2015. 3(29): p. 5971-5977.

Figure 1

Introduction

Light-addressable potentiometric sensor (LAPS) is a field-effect chemical sensor with the ability of spatial resolution proposed by Hafeman [1] and has an electrolyte-insulator-semiconductor (EIS) structure. With the illumination of focused light, photocurrent can be generated locally, providing the information of surface potential. As a member of potentiometric sensors, technologies for ion-selective electrodes (ISEs) and ion-sensitive field-effect transistor (ISFET) are also applicable to LAPS. For example, by depositing the ion-sensitive membrane (ISM) on the surface of the LAPS, an ion-sensitive LAPS (ISLAPS) can be obtained. Conventional ISEs which contains liquid contacts have some limitations such as requiring maintenance, proper handling, leakage of inner filling solution and primary ion diffusion, which will deteriorate limit of detection. In this work, a multiplex ISLAPS detection system has been proposed combined with Na⁺, K⁺, and Ca²⁺ all-solid-state ISM and a conventional pH-LAPS. The matrix of the plasticizer free ISM is the silicone-rubber, which has not only the same sensitivity of PVC-based ISM, but also better adhesion and longer lifetime. With the help of a program-controlled two-axis translation stage, the detection sites of the sensor were sequentially illuminated by modulated light from the backside. Different from the conventional multi-channel potential sensors, LAPS system essentially uses a single-channel instrument to realize multi-parameter detection, and can be expanded easily by adding other ISMs only. The multiplex ISLAPS can meet the ion detection requirements, and it is a promising physiology detection platform.

Method

Figure 1a shows the structure of LAPS chip, the fabrication is similar to the previous report [2], except the thinned illumination area in the center of backside. The sensing material on the oxide layer is 30 nm Al₂O₃ layer deposited by atomic layer deposition (ALD) [3] for pH detection, and silicone-rubber ISMs for Na⁺, K⁺, and Ca²⁺ detection. The chip was cut into 1 cm×1 cm with a 4 mm×4 mm thinned area, in turn washed by acetone, ethanol and deionized water to store and use.

Preparation of the ISM is as follows: Firstly, about 300mg silicone-rubber (RTV 730) was evenly dissolved in 1.5mL THF and centrifuged. Then the supernatant was mixed evenly with the ionophores and ion additives in 30min ultrasonic bath. Before deposition of ISM, a P3OT layer was applied on the surface of SiO₂-LAPS chip. After that the ISM mixture was spin-coated on the P3OT layer and dried overnight at room temperature. The three ISM-coated LAPS chips and a Al₂O₃-LAPS chip were placed in a PMMA chamber, pasted with conductive silver glue on one bonding pad of PCB, with four holes to expose the thinned area of each site. The edges of chips were encapsulated by epoxy adhesive.

The schematic of the multiplex ISLAPS system is shown in Figure 1b. The modulated light with collimator was fixed on the translation stage, illuminating the thinned area of sensor. The data acquisition (DAQ) device performed bias voltage output, signal acquisition, and was controlled by LabVIEW software together with the translation stage. The ISMs are sequentially illuminated and the response of four ions can be obtained in one measurement.

Results and Conclusions

Sensitivities of the multiplex ISLAPS were calibrated with series of concentration gradient solutions. The background electrolyte for Na⁺, K⁺ and Ca²⁺ is 0.1M CH₃COOLi, and PBS solutions adjusted with HCl/NaCl were used in pH sensitivity determination. The ISMs were conditioned in the solutions of 10^-2 M corresponding ions for 1h. The I-V curves and the bias-concentration fit lines are shown in Figure 1c. The sensitivity was obtained from the maximum slope points of I-V curves. The limits of detection (LODs) of the Na⁺, K⁺ and Ca²⁺ were about 10^-6M, and the sensitivities were 57.37mV/pH, 56.9mV/pNa, 58.4mV/pK, and 25.3mV/pCa respectively, close to the Nernst theoretical value. The LOD and linear ranges can meet the requirements of physiological ions detection.

The real DMEM samples were tested with standard addition method. The light spot moved with the stage and stayed at the center of each site for 10s. The responses of standard and spiked samples are shown in Figure 1d and results are listed in Table 1, indicating that the multiplex ISLAPS can be applied in physiological ions detection.

Table 1 The results of DMEM samples detection

In summary, we have firstly combined silicone-rubber ISM with LAPS and proposed a multiplex ISLAPS system for Na⁺, K⁺, Ca²⁺ and H⁺ with good performance. The ISM-coated chips are packaged in one detection channel, and the response of multiple ions can be recorded sequentially. It seems to be a promising physiology detection platform.

References

[1] Hafeman D G, Parce J W, McConnell H M. Light-addressable potentiometric sensor for biochemical systems[J]. Science, 1988, 240(4856): 1182-1185.

[2] Liang T, Gu C, Gan Y, et al. Microfluidic chip system integrated with light addressable potentiometric sensor (LAPS) for real-time extracellular acidification detection[J]. Sensors and Actuators B: Chemical, 2019, 301: 127004.

[3] Ismail A B M, Harada T, Yoshinobu T, et al. Investigation of pulsed laser-deposited Al2O3 as a high pH-sensitive layer for LAPS-based biosensing applications[J]. Sensors and Actuators B: Chemical, 2000, 71(3): 169-172.

Figure 1

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) is the main cause of various acute infections, especially in patients with severely burned and chronic infections. Moreover, P. aeruginosa can be found in lungs of patients with cystic fibrosis and in implanted devices.¹ Biofilm of P. aeruginosa usually shows increased tolerance to antimicrobial agents.^2,3 Among main virulence factors secreted by P. aeruginosa are pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA) which belong to a group of quorum sensing molecules called phenazines. These phenazines also play an important role in the formation and growth of P. aeruginosa biofilms. As specific biomarkers of P. aeruginosa, detection of PYO and PCA can enable diagnosis of P. aeruginosa infection. Existing methods for detection of phenazines include high performance liquid chromatography-mass spectrometry (HPLC-MS),⁴ fluorescence measurements⁵, and electrochemical methods.^6–8 But bulky and expensive instruments and the complex sample preparation process limit the broad application of the MS method. In addition, neither MS and fluorescence detection method can achieve in-situ monitoring of biofilm formation and growth. Compared to these methods, electrochemical sensors are low-cost, user-friendly, and compact. They also show the potential for in vivo monitoring.⁹

In this work, we have developed an all-in-one electrochemical sensor for selective identification of PYO and PCA down to 1 µM and 10 µM respectively in bacterial culture medium (brain heart infusion, BHI) using square wave voltammetry (SWV). The sensor working electrode (WE) is comprised of platinum (Pt) nanocrystals electrodeposited on laser-induced graphene (LIG). The counter (CE) and pseudo-reference electrode (RE) are based on LIG and electrodeposited silver (Ag) on LIG, respectively. We studied the effect of various electrodeposition conditions to optimize the material stability and sensor response. The fabrication process is facile, scalable, and user-friendly. The schematic of the fabrication process is shown in Figure 1a. The inset shows a picture of a fabricated sensor on polyimide. Figure 1b and Figure 1c show the SWV results for different concentrations of PYO and PCA in BHI. The oxidation peak for PYO and PCA are located at -0.15 V and -0.25 V, respectively, which enable selective identification of these phenazines. We studied the effect of SWV frequency on the sensor signal and found that 15 Hz and 10 Hz are the optimum frequency with the highest ratio of peak current to the baseline current for PYO and PCA, respectively. Compared to other electrochemical sensors based on gold^7,10 or carbon,¹¹ our sensor is flexible and integrates all three electrodes in a compact and scalable design.

In conclusion, we successfully developed a printable flexible sensor based on LIG, modified using electrodeposition of platinum and silver to create a portable and compact three-electrode electrochemical device for selective detection of two phenazines (PYO and PCA) directly in bacterial culture broth. A limit of detection of 1 µM was achieved for PYO and 10 µM for PCA. To achieve a high sensitivity, the SWV frequency was optimized. The ability to identify both phenazines can advance our knowledge on how the interplay between these molecules affect biofilm phenotypes. Future works include improving the sensitivity through electrode engineering, growing P. aeruginosa biofilms on the sensors, and in situ monitoring of the pyocyanin level during biofilm growth. The flexibility of this sensor also promises its application in wearable technology or medical devices (e.g. for smart catheters, smart wound dressings, etc.).

REFERENCES

1. S. L. Percival et al., Wound Repair Regen., 20, 647–657 (2012) http://doi.wiley.com/10.1111/j.1524-475X.2012.00836.x.

2. S. Subramanian, R. C. Huiszoon, S. Chu, W. E. Bentley, and R. Ghodssi, Biofilm, 2, 100015 (2020) https://www.sciencedirect.com/science/article/pii/S2590207519300152.

3. S. E. Darch and D. Koley, Proc. R. Soc. A Math. Phys. Eng. Sci., 474, 20180405 (2018) https://royalsocietypublishing.org/doi/10.1098/rspa.2018.0405.

4. M. Kushwaha et al., ACS Chem. Biol., 13, 657–665 (2018) https://pubs.acs.org/sharingguidelines.

5. S. Fulaz et al., ACS Appl. Mater. Interfaces, 11, 32679–32688 (2019) https://pubs.acs.org/doi/abs/10.1021/acsami.9b09978.

6. F. A. Alatraktchi et al., Y. K. Mishra, Editor. PLoS One, 13, e0194157 (2018) https://dx.plos.org/10.1371/journal.pone.0194157.

7. L. Liu et al., Sensors Actuators B Chem., 327, 128945 (2021) https://linkinghub.elsevier.com/retrieve/pii/S0925400520312922.

8. J. Elliott, O. Simoska, S. Karasik, J. B. Shear, and K. J. Stevenson, Anal. Chem., 89, 6285–6289 (2017) https://pubs.acs.org/sharingguidelines.

9. F. A. Alatraktchi, W. E. Svendsen, and S. Molin, Sensors (Switzerland), 20, 1–15 (2020) https://www.mdpi.com/1424-8220/20/18/5218.

10. F. A. Alatraktchi, H. K. Johansen, S. Molin, and W. E. Svendsen, Nanomedicine, 11, 2185–2195 (2016) https://www.futuremedicine.com/doi/10.2217/nnm-2016-0155.

11. R. Burkitt and D. Sharp, Electrochem. commun., 78, 43–46 (2017).

Figure 1

Introduction

SO₂ concentration detection in harsh environment is still a challenge because the present technology is based on large-scale instrument, which is expensive and can't monitor in real time. Recently, gas sensor with low cost and the ability of in-situ measurement has been used to SO₂ detection. Due to high sensing performance, reliable stability and acceptable humidity resistance, the mixed potential sensor based on YSZ was expected to achieve the real-time detection in harsh environment [1].

Method

Fabrication and measurement of the gas sensor

The sensor was fabricated utilizing 8mol% Y₂O₃-doped YSZ plate (2 mm×2 mm square, 0.3 mm thick, provided by Anpeisheng Corp. China). A point-shaped and a narrow stripe-shaped Pt electrode (PE) were formed on two ends of the YSZ plate using a commercial Pt paste (Sino-platinum Metals Co. Ltd.), and sintered at 950°C. The paste which was mixed by a minimum quantity of deionized water and the sensing materials ZnGa₂O₄. Next, the resultant paste was applied on the point-shade Pt to form stripe-shaped ZnGa₂O₄ electrode (ZE). Afterward, the device was annealed at 800°C to make a good contact between the sensing electrode and electrolyte. A Pt heater and a linear DC Power Supply were used to provide required heat to regulate the operating temperature of the sensor.

Sulfuration process

In order to further investigate the sensing mechanism of the sensor, we added a sulfuration experiment. The sulfuation process is as following: The sensor was put into a 1L chamber with a mixture of air and 100 ppm SO₂ for 24 hrs, the temperature of the sensor maintain at 650°C during this process. When time is up, put the sensor into a 1L chamber with fresh air and begin to measure the sensing performance of the sensor after sulfuration.

Results and Conclusions

YSZ-based mixed potential sensor with ZnGa₂O₄ and Pt electrodes (ZE and PE) was developed for the SO₂ detection. The effects of long-term aging and continuous test on various properties of the electrode materials had been investigated and characterized. Some interesting phenomenon were found that when the sensor exposed to the relative high concentration SO₂ with air, the response direction was opposite to that exposing to low concentration SO₂ as shown in Fig. 1. Moreover, with the last of the testing time, the responses of the sensor to all concentration SO₂ were opposite to that in the original state. Hence, giving a reasonable explanation is one of the most important purposes in this paper. We have proved that the PtS was produced during the testing and aging process. According to the Y. shimizu's work [2], most of metal mono-sulfides performed a high electrochemical catalytic activity to SO₂. Hence, we speculated that the mono-sulfides PtS also has a high electrochemical catalytic activity to SO₂. In this case, when measuring lower concentration SO₂, the diffusion rate is a slow value. Due to the Pt's high catalytic activity to SO₂, SO₂ react with Pt electrode and changed to PtS, which adhere to the Pt electrode surface. The process was clearly exhibited in the bottom half of Fig. 1. This process sharply decreased the SO₂ concentration reaching TPB of PE, and then caused the ΔV_PE approaching 0. On the other hand, ZnGa₂O₄ electrode had a response to SO₂ and according to the mixed potential mechanism, the ΔV_ZE should be a negative value. However when measuring higher SO₂ concentration, Pt has changed to PtS, which decreased the contact area, and then decreased the catalytic activity to SO₂. Because of the much higher electrochemical catalytic activity of PtS at PE to SO₂ than that of ZnGa₂O₄, the response at PE was much higher than that at ZE. Hence, the reversed response characteristics occurred as shown in the second response and recovery transient of Fig. 1. Furthermore, with continuous testing and aging, more and more sulfur element was deposited to the Pt electrode and the critical concentration deceased, finally, all of the ΔV were changed to the positive values as shown in the third of Fig. 1. Moreover, the sensor after sulfuration process (the detail of the sulfuration process was given in the last part) also performed similar sensing properties to the sensor with continuous testing and aging process, which indicated that the produced PtS should be the reason for that the sensor performed reversed sensing performances. In addition, the sensor after sulfuration can detect 0.05-500 ppm SO₂ with the sensitivity being 5 mV/decade to 0.05-1 ppm and 41 mV/decade to 1-500 ppm. The sensor also had a reliable stability during the continuous measurement.

References

[1] X. Liang, T. Zhong, B. Quan, B. Wang, H. Guan, Solid-state potentiometric SO₂ sensor combining NASICON with V₂O₅-doped TiO₂ electrode, Sens. Actuators B: Chem. 134 (2008) 25-30. doi:10.1016/j.snb.2008.04.003.

[2] N. Souda, Y. Shimizu, Sensing properties of solid electrolyte SO₂ sensor using metal-sulfide electrode, Journal of Materials sciences 38 (2003) 4301-4305. doi: 10.1023/A:1026378931023.

Ackownledgments

This work was supported by the National Nature Science Foundation of China (Nos. 61973134)

Figure 1

Early detection of cancer can noticeably increase the survival chance of many cancer patients. Quantifying cancer biomarkers detectable from blood is an efficient way for early detection of cancer diseases. Among various discovered cancer biomarkers, platelet-derived growth factor-BB (PDGF-BB) is an essential biomarker for early detection of cancer and monitoring cancer patients. This biomarker plays a vital role in developing and lymphatic metastasis of solid malignant tumors such as brain, lung, breast, and liver; which enlightens the importance of developing point-of-care (POC) biosensors for the detection of PDGF-BB. In recent years, the application of synthetic DNA or RNA-based bio-recognizers (i.e., aptamers) in cancer biomarker sensor development has been vastly investigated. Electrochemical label-free aptamer-based biosensors (also known as aptasensors) are highly suitable for POC. In this study, the application of bipolar exfoliated (BPE) graphene for developing PDGF-BB label-free aptasensors is investigated. Graphene as a single, two-dimensional layer of carbon atoms has very interesting features suitable for biosensor applications. The common graphene synthesis methods require complicated and costly processes and excessive use of harsh chemicals, as well as complex subsequent deposition procedures. In this study, a single setup has been used for exfoliation, reduction, and deposition of graphene nanosheets on a conductive electrode based on the principle of bipolar electrochemistry of graphite in deionized water. We investigated the properties of aptasensors based on graphene oxide (GO) deposited on a positive electrode feeding electrode and reduced-GO (rGO) deposited on a negative electrode feeding electrode. The PDGF-BB affinity aptamers were covalently immobilized by binding amino-tag terminated aptamers and carboxyl groups of GO and rGO surfaces. In this study, Fourier-transform infrared spectroscopy (FTIR) was used to study the surface characteristics of synthesized graphene and developed aptasensors. The scanning electron microscopy (SEM) was used to study the morphology of the bipolar exfoliated GO and rGO. The SEM study demonstrated that the rGO has a porous vertically aligned structure with pore sizes of around 100 nm, while GO has bulky flattened plates with random cracks. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used for characterizing the BPE graphene-based aptasensors in different stages of development and their sensing performances. The turn-off sensing strategy was implemented by measuring the peak-currents from DPV plots. The optimized aptasensor based on rGO showed a wide linear range of 0.75 pM-10 nM, high sensitivity of 7.83 A Logc^-1 (unit of c, pM), and a low detection limit of 0.53 pM. However, the optimized aptasensor based on GO reached to its saturation point around 150 pM. This study demonstrated that bipolar electrochemistry is a simple yet efficient technique that could provide high-quality graphene for biosensing applications. Considering the BPE technique's simplicity and efficiency, this technique is highly promising for developing feasible and affordable lab-on-chip and point-of-care cancer diagnosis technologies.

Melatonin(N-acetyl-5-methoxy tryptamine) is a paracrine hormone secreted by pineal gland in the brain. This endogenous hormone has the ability to maintain the body circadian rhythm.¹ 3,4-dihydroxy-L-phenylalanine (L-Dopa) is the intermediate precursor of the Neurotransmitter dopamine. L-dopa, as one of catecholamines, is widely used as a source of dopamine in the treatment of Parkinson's disease and epilepsy.²

Here, we report a sensor based on Platinum electrode modified with hydrothermally synthesized RGO and spinel nanoferrate for the simultaneous determination of L-Dopa and Melatonin.

Graphene oxide (GO)is a graphene derived material with oxygen containing functional groups. Graphene is a material with honey comb like structure of carbon atoms having sp² bonding character. It exhibits high electrical conductivity, thermal conductivity and mechanical strength. GO containing functional groups like –OH, -COOH etc., acts as the site for the nucleation of metal oxide nanoparticles. It improves direct electron transfer between the electrode and the redox species by accelerating the electron transfer and enlarging the effective surface area.³ GO was synthesized by modified Hummers method. Chemically converted graphene RGO has been prepared by various reduction methods such as chemical, microwave, electrochemical, thermal, solvothermal/hydrothermal, by reducing its oxygen content.⁴ Green synthetic pathway of hydrothermal method was adopted for RGO synthesis to avoid hazardous chemical species and vigorous reaction conditions. Spinel ferrate like Copper Cobalt ferrate (CuCoFe₂O₄) was synthesized by sol-gel method and RGO/Spinel nanocomposite was prepared by physical mixing.⁵ The synthesized materials were characterized using various techniques like FTIR, XRD, FESEM etc. The electro catalytic activity of RGO/Spinel nanocomposite anchored platinum electrode for the simultaneous determination of melatonin and L-Dopa was studied using several voltammetric techniques. Bare platinum electrode was able to sense melatonin and L-Dopa both individually, but not simultaneously. Compared to RGO modified Platinum electrode, RGO-Spinel ferrate/Pt electrode exhibited good response in terms of lower potential of electrooxidation of L-Dopa and Melatonin with enhanced catalytic current with a peak separation of 0.48V. This enhancement in electrochemical performance is due to the increase in surface area as well as conductivity of the electrode upon modification with RGO-spinel nanocomposite which act as an excellent electrochemical oxidant for L-Dopa and melatonin.

References:

1. Levent, A.J. Diam. Relat. Mater.2012, 21, 114-119

2. Yan, X.; Pan, D.; Wang, H.; Bo, X.; Guo, L.J. Electroanal. Chem.2011, 663, 36-42

3. Pei, S.; Zhao, J.; Du, J.; Ren, W.; Cheng, H.J. Carbon.2010, 48, 4466-4474

4. Luo, D.; Zhang, G.; Liu, J.; Sun, X.J. Phys. Chem. C.2011, 115, 11327-11335

5. Elkholy, A. E.; Heakal, F. E.; Allam, K. A.J. RSC Adv.2017, 7, 51888

Due to a relation between glucose in sweat and blood, there is an opportunity to monitor patients' glucose levels non-invasively through sweat. There is a high demand for developing highly selective and sensitive biosensors in order to sense biomarkers like glucose and, therefore, the diseases based on those biomarkers. However, enzymatic sensors are selective to specific biomarkers; they are suffering from high sensitivity to the fluctuation of temperature, oxygen, pH, humidity, detergents, organic reagents, and toxic chemicals, affecting their stability and sensitivity, and reproducibility. Therefore, developing non-enzymatic glucose (or other biomarkers) sensors is getting significant attention to fulfill higher sensitivity and selectivity as well as minimized susceptibility to fouling by enzyme-ageing and adsorbed intermediates. Here we have proposed a combination of two sensors that can help us improve these non-enzymatic sensors' selectivity. Two electrochemical arrayed sensors have been developed. The first electrochemical sensor has been achieved by controlled growth of cobalt nanowire and copper nanoparticles on carbon substrate in order to measure the glucose level at low concentrations, and the second electrochemical sensor has been modified by MWCNT-CO-NH-cyanuric-NH₂ and Fe₃O₄ in order to measure the uric acid and eliminate the interference of it in glucose measurement results. In order to show the morphology of the glucose sensor, the SEM and EDX have been conducted. Also, the FT-IR test has been shown to confirm the functionalization of MWCNT. The electrocatalytic and electrochemical performance of each sensor have been evaluated in the presence of the various contaminants of sweat. The glucose sensor showed less than 5% interference toward ascorbic acid, sodium bicarbonate, and the lactic acid at their max range of presence in the sweat. For eliminating the interference of uric acid, the second sensor has developed, which has no response to the glucose and high sensitivity to the uric acid. The calibration curve of each sensor has been provided in the 3D form, and a simple method of arraying has been applied to improve the sensors' selectivity. The arrayed sensors showed a highly glucose-selective sweat-based sensor with minimized error imposed by the uric acid interference. The glucose sensor's reproducibility and durability have also been tested, which showed less than 5% and 10% variations, respectively. In the end, the arrayed sensor's performance has been evaluated by real sweat samples of a male and a female, analyzed by Clarke's error grid analysis showing less than 20% deviation from the glucose levels measured by the commercial glucometers.

Parkinson's disease is one of the most frequent diseases of the central nervous system with rather severe consequences for patients. Since the disorder can not be cured, the dopamine level in the brain needs to adjusted. Here several key enzymes of the dopamine metabolism [e. g. catechol-O-methyl transferase (COMT)] are targets for administered drugs. However, the efficiency of the treatment is decreasing with time. Consequently a monitoring of the actual activity of these enezmyes and an adjustment of the pharmaceutical treatment would be beneficial to prolong the treatment effects.

In this study, an electrochemical approach is introduced for activity determination of the COMT enzyme. As detection method differential pulse voltammetry (DPV) has been used exploiting an improved signal to noise ratio. Activity determination is based on the selective detection of one substance in the reaction scheme of the enzyme – here dopamine. As electrode material fluorine doped tin oxide (FTO) has been elucidated since it is characterized by a clear discrimination between the substrate and the product of the COMT [1]. This can be achieved without additional layers on top of the electrode. The response is characterized by a rather high stability of the dopamine signal during consecutive measurements and a linear dependency of the peak currents on the dopamine concentration in the range of the maximum reaction rate of COMT [2]. Furthermore it has been detected that redox active interferences such as ascorbic acid do not disturb the analysis.

Despite these advantageous results, it has been found that the electrochemical dopamine oxidation in the complete activity assay is influenced by each of the added essential assay components, even though none of the added components reveal a current signal at the FTO electrode itself. After adjusting the potential range of the electrochemical analysis and the solution composition, these effects can be circumvented.

Consequently, by following the dopamine concentrations during COMT action, it can be shown that the

activity of the enzyme can be detected by using DPV at a FTO electrode. Experiments with different amounts of the enzyme further demonstrate that activity quantification is feasible.

References

[1] G. Göbel, A. Talke, F. Lisdat, Electroanalysis, 2018, Vol. 30 (2), p. 225-229

[2] G. Göbel, A. Talke, U. Ahnert, F. Lisdat, ChemElectroChem, 2019, Vol. 6 (17)

Biosensors based on functionalized metal oxide nanostructures, such as zinc oxide (ZnO) and Nickel Oxide (NiO), and have been demonstrated using electrochemical methods. Those nanostructures can be deposited on both rigid and flexible substrates at low temperature using rather simple and low-cost processes. Recently, it was discovered that decorating the metal oxide nanostructures with noble metal nanoparticles opens the doors to fabricating biosensors to detect volatile organic compounds.

The aim of this work is to improve the knowledge and understanding of a novel composite material structure where wide bandgap semiconductor metal oxide nanostructures are decorated with gold nanoparticles that their surface can be either decorated by functional biological material or converted to catalytic Pt (or Pd) shell. The metal nanoparticle surface functionalization poses some material-related questions regarding the behavior of that nano-structured material, especially when exposed to biological electrolytes or volatile organic compounds (e.g. Ethylene). This will allow us to develop a novel family of biosensors that enable sensitivity, rapidity, and selectivity for the detection of a wide range of target analytes, from pathogens to volatile organic gases, compared to existing bio-electrochemical sensors.

The final goal is a flexible all-in-one , highly sensitive and selective biosensors using relatively simple and low cost materials and processing. The sensors will use recent advances in functionalized Gold NP decorated ZnO and NiO electrodes based sensors.

ZnO nanowalls was deposited both on rigid (Silicon/ SiO2) substrate and on flexible (Polyimide) substrates. A Scanning Electron Microscopy study of the nucleation and growth of the ZnO nanowalls, and of the NP decoration was conducted. Different conditions such as growth temperature, concentration and time were used. RBS (Rutherford backscattering spectrometry) was performed on the ZnO nanowalls on Polyimide substrate.

After mastering the best methods for ZnO nanowalls growth and decoration with Au NP, decoration with different NP such as Platinum or Palladium will be compared. We believe that decoration of metal oxide nanostructures with noble metal nanoparticles will enhance the electrochemical performance of the sensors, for example - highly sensitive detection of VOCs such as ethylene.

This new structure should be further explored to reveal its full potential for flexible bio-sensor applications.

Figure 1

Infectious diseases remain a major cause of morbidity and mortality in the low income countries. Patients present with fever and non-specific symptoms, which are difficult to diagnose without specialist laboratory tests.This results in presumptive diagnosis and treatment, which may be incorrect. Without tools to identify infection, antibiotic administration is an attractive generic treatment. Chronic non-communicable disease is also under monitored in low income countries (LICs). For example, in a study in Mozambique only 6% of facilities could carry out a blood glucose analysis and personal monitoring is not generally available.

Thus, instead of having accurate diagnosis, patients are more often managed based on probability and clinical judgment. The healthcare economics can make antibiotic administration an attractive generic treatment, but now antibiotic resistance is so serious that in some regions half the patients with pneumonia do not respond to the first-line antibiotics. There have been numerous attempts to propose low cost diagnostics for low and middle income countries (LMICs), but a barrier to low-cost diagnostics, especially in LICs, arises through purchases from the west, without Purchasing Power Parity. When diagnostics are produced in high income countries they remain at high cost when taken in the local affordability context. The required biological reagents for a diagnostic are often the largest proportion of its total cost (eg >80% in the Philippines for a polymerase in a nucleic acid test, and a similar price to US/ Europe, despite the average household income being 80-90% lower).

We have revisited the 'unaffordable' diagnostics and created a design with reagents that can be manufactured locally, with only basic infrastructure. For example, we have used synthetic biology to design a polymerase fusion for nucleic acid amplification, that incorporates locally resourced materials and is targeted to easy local production in resource poor areas.

We will report on a 'gene to diagnostic' approach with a multifunctional fusion enzyme as the central reagent in point-of-care diagnostics. The components of the fusion are a functional assay protein reagent, a visualising unit and an in-built immobilisation peptide. This reduces downstream isolation steps and eliminates expensive coupling chemicals for integration in a diagnostic. In-built production monitoring and QA for the analytical reagent product is shown with the visualising protein.

The platform is demonstrated in nucleic acid tests for leptospirosis and malaria as well as a device for sarcosine determination in urine (as a marker of early-stage prostate cancer). We have the first step towards providing low cost diagnostics in resource poor areas, which could deliver a sustained improvement in healthcare, while also developing the local economy. Data from a clinical trial of a nucleic acid amplification test, designed for malaria screening in Africa will be presented and the first reports of the latter nucleic acid test which is beginning trials in Ghana for Covid-19.

Toxic pollutants such as polychlorinated biphenyls, organic pesticide, food additives, plasticizer and dyes have been producing increasing harm to the environment and human health. Surface-enhanced Raman scattering (SERS) spectroscopy is a powerful method for sensing these small molecule pollutants for the advantages of fast response, high sensitivity and fingerprint effect.1 The crucial technique is to design and fabricate uniform and highly sensitive SERS substrates with high affinity to the analytes. Patterned templates, such as anodic aluminum oxide (AAO), polystyrene nanosphere array and lithographic templates were used to fabricate uniform metal nanostructure array.2-5 Meanwhile, the combination of electromagnetic enhancement from metal nanostructures and chemical enhancement from semiconductors or graphene was used to improve the detection limit.6-8 On the other hand, strategies such as surface functionalization of metal nanostructures by decanethiol or cyclodextrin based on the "like dissolves like" principle,9,10 physical trapping mechanism based on the electrostatic attraction, and mechanical limitation based on temperature or humidity-sensitive materials were used to capture more analytes to the surface of the SERS substrate.11-14 For practical application, simple sensors and devices were designed to detect the harmful food additives in the milk or pesticide residue on the surface of orange or in juice.15-17

Figure 1. Highly sensitive SERS substrates and prototype devices. (a) Ag nanorods array with tunable gaps (hot spots) fabricated by AAO template [Ref 9]. (b) Decoration of cyclodextrin to improve the affinity to polychlorinated biphenyls [Ref 9]. (c) Ordered Ag nanorod bundles with highly efficient hot spots [Ref 5]. (d) A pipet-like SERS sensor for detection of melamine in milk [Ref 17]. (e) A cut-and-paste SERS substrate (flexible transparent Ag-nanocube@ polyethylene film) for rapid in situ detection of pesticide residue on the surface of orange [Ref 15].

References

(1) Tang, H. B.; Zhu, C. H.; Meng, G. W.; Wu, N. Q. J Electrochem. Soc. 2018, 165, B3098.

(2) Chen, B.; Meng, G.; Huang, Q.; Huang, Z.; Xu, Q.; Zhu, C.; Qian, Y.; Ding, Y. ACS Appl. Mater. Interfaces 2014, 6, 15667.

(3) Huang, Z.; Meng, G.; Huang, Q.; Yang, Y.; Zhu, C.; Tang, C. Adv. Mater. 2010, 22, 4136.

(4) Li, Z.; Meng, G.; Huang, Q.; Hu, X.; He, X.; Tang, H.; Wang, Z.; Li, F. Small 2015, 11, 5452.

(5) Zhu, C.; Meng, G.; Zheng, P.; Huang, Q.; Li, Z.; Hu, X.; Wang, X.; Huang, Z.; Li, F.; Wu, N. Adv. Mater. 2016, 28, 4871.

(6) Tang, H.; Meng, G.; Huang, Q.; Zhang, Z.; Huang, Z.; Zhu, C. Adv. Funct. Mater. 2012, 22, 218.

(7) Wang, X. J.; Meng, G. W.; Zhu, C. H.; Huang, Z. L.; Qian, Y. W.; Sun, K. X.; Zhu, X. G. Adv. Funct. Mater. 2013, 23, 5771.

(8) Liu, J.; Zhu, C. H.; Pan, Q. J.; Meng, G. W.; Lei, Y. Chemistryselect 2020, 5, 8338.

(9) Huang, Z.; Meng, G.; Huang, Q.; Chen, B.; Zhu, C.; Zhang, Z. J Raman Spectrosc. 2013, 44, 240.

(10) Zhu, C. H.; Wang, X. J.; Sho, X. F.; Yang, F.; Meng, G. W.; Xiong, Q. H.; Ke, Y.; Wang, H.; Lu, Y. L.; Wu, N. Q. ACS Appl. Mater. Interfaces 2017, 9, 39618.

(11) Zhou, Q.; Meng, G.; Liu, J.; Huang, Z.; Han, F.; Zhu, C.; Kim, D.-J.; Kim, T.; Wu, N. Adv. Mater. Technol. 2017, 2, 1700028.

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Figure 1

Introduction

A non-invasive monitoring is important not only for medical inspection in hospitals but also healthcare management in daily life. For 30 years, we have developed several types of detachable sensors into human cavitas sites (conjunctiva, pharyngeal, oral cavity, etc.) with biocompatible materials and harmless device-techniques (Fig. 1). "Cavitas sensors" attached to body cavities such as a contact lens type and a mouthguard ("no implantable", "no wearable") are attracted attention for preventive medicine [1]. In this contribution, the cavitas sensors (soft contact-lens [SCL] and mouthguard [MG] biosensors) in human body cavities will be introduced as novel non-invasive biosensing devices.

Soft contact lens type glucose sensor

The soft contact lens type biosensor was designed for continuous glucose monitoring in tear fluids (Fig. 2) [2]. Biocom-patible poly(MPC-co-PMEH) (PMEH) (Fig. 3) [3,4] and polydimethyl siloxane (PDMS) were employed as the biosensor material. The biosensor consists of a flexible Pt working electrode and an Ag/AgCl reference/counter electrode, which were formed by micro-electro-mechanical systems (MEMS) technique. Glucose oxidase (GOD) was immobilized on the sensitive area by entrapping with PMEH polymer. The contact lens biosensor showed a good relationship between the output current and glucose concentration in a range of 0.03–5.0 mM, covering the reported tear glucose concentrations in normal and diabetic patients. Also, the biosensor was applied to a rabbit for the purpose of tear glucose monitoring. The basal tear glucose was estimated to 0.14 mM. Also, the change of tear glucose induced by the change of blood sugar level was assessed by the oral glucose tolerance test. As a result, tear glucose level increased with a delay of few min from blood sugar level (Fig. 4). The result showed that the contact lens biosensor is expected to provide further detailed information about the relationship between dynamics of blood glucose and tear glucose.

Invisible Mouth guard type sensors with BLE transmitter

The telemetric mouthguard-biosensor was developed with dental materials integrated with Bluetooth low energy (BLE) wireless module for measurement of saliva glucose [5]. The electrodes were formed on the thinner mouthguard surface which made of a polyethylene terephthalate glycol (PETG) using the same method as the contact lens one (Fig. 5). The BLE module, small potentiostat and battery were mounted and heat-sealed between upper and lower mouth guards. In the investigation of in-vitro characterization, the transparent biosensor showed excellent relationship between the output current and the glucose concentration (Fig. 6). In artificial saliva consisting of salts and proteins, the glucose sensor exhibits high-sensitive detection in a range of 5-1000 µmol/L including glucose concentration of human saliva. We demonstrated the capability of the sensor and wireless communication module to characterize an inclusion in oral phantom that imitative structure of human oral cavity. Stable and long-term monitoring (more than 1 day) using telemetry system was established. Monitoring of real-time changes of glucose concentration in artificial saliva with evaluation system of phantom jaw was demonstrated. The mouthguard device would be useful not only for monitoring of the oral chemical and physical information but also for unconstrained controlling of external device and system [6].

Future potential of cavitas sensors

The self-detachable cavitas sensors are expected to allow the non-invasive bio/chemical monitoring, thus improving quality of life in view of the aging of society in near future.

References

[1] K. Mitsubayashi, T. Arakawa, Cavitas Sensors:Contact Lens Type Sensors & Mouthguard Sensors, Electroanalysis, 28(6), 1170-1187, 2016. (10.1002/elan.201600083)

[2] K. Mitsubayashi, Cavitas Biosensors: Noninvasive Approaches to Blood Glucose Monitoring for Diabetes Mellitus, Sensors and Materials, 30(10), 2313–2320, 2018. (10.18494/SAM.2018.2011)

[3] S. Iguchi, H. Kudo, T. Saito, M. Ogawa, H. Saito, K. Otsuka, A. Funakubo, K. Mitsubayashi, A flexible and wearable biosensor for tear glucose measurement, Biomedical Microdevices, 9, 4, pp.603-609, 2007

[4] M.X. Chu, K. Miyajima, D. Takahashi, T. Arakawa, K. Sano, S. Sawada, H. Kudo, Y. Iwasaki, K. Akiyoshi, M. Mochizuki, K. Mitsubayashi, "Soft contact lens biosensor for in situ monitoring of tear glucose as non-invasive blood sugar assessment", Talanta 83, 960–965, 2011. (10.1016/j.talanta.2010.10.055)

[5] Arakawa T, Kuroki Y, Nitta H, Chouhan P, Toma K, Sawada S, Takeuchi S, Sekita T, Akiyoshi K, Minakuchi S, Mitsubayashi K, Mouthguard biosensor with telemetry system for monitoring of saliva glucose: A novel cavitas sensor, Biosens Bioelectron, 84, 106-111, 2016. doi:10.1016/j.bios.2015.12.014.

[6] Toma K, Tomoto K, Yokota K, Yasuda N, Ishikawa T, Arakawa T, Mitsubayashi K, Mouthguard controller for unconstrained controlling of external devices. Sensors and Materials, 30, 3053-3060, 2018. (10.18494/SAM.2018.2014)

Figure 1

On the surface of graphene, energy transfer occurs when molecules are located close to the surface. The energy transfer yield depends on the degree of molecular interaction between the adsorbed molecules and the graphene surface. For example, when a fluorescent molecule such as a dye is located very close to the graphene surface, the dye does not exhibit fluorescence because of the efficient fluorescence resonance energy transfer (FRET). The degree of energy transfer varies when the molecules on the surface changes their molecular states and structures. This allows us to visualize biological/chemical reactions by converting those invisible molecular interactions into the measurable physical quantities such as light and electricity. This makes graphene a promising material for a novel biosensor.

We have created a unique type of biosensor, which works on a graphene surface by modifying it with a specific DNA called an aptamer, for the detection of biologically important proteins such as cancer markers. One end of the aptamer is labeled with a fluorescent dye and the other end is connected to a pyrene linker molecule, which shows a strong affinity to the graphene. Thus, the aptamer is firmly fixed to the graphene surface. The graphene aptasensor detection mechanism is as follows. In the initial stage, the dye-conjugated aptamer is adsorbed on the graphene surface via physical adsorption (π-π interactions), and thus the dye is located close to the graphene surface. Here, the fluorescence of the dye is well quenched by graphene via FRET and is barely observable (Fig. 1(a)). If the target of the aptamer is present in the system, the aptamer forms a complex with the target and leaves the graphene surface. At the same time, the dye molecule also leaves the graphene surface and the dye recovers its fluorescence (Fig. 1(b)). We can detect the target molecule by observing the fluorescence [1]. The system allows us to perform molecular detection on a solid surface, which is a powerful tool to realize a two-dimensional (2D) on-chip sensor. By using the on-chip sensor, detection of the target protein is possible simply by adding a sample smaller than 1 μL to a sensor chip and is completed in about a minute. The system also allows us to perform real-time molecular detection, the simultaneous detection of multiple target molecules on a single chip [2], and the molecular design of a probe for enhancing the sensitivity by using single-stranded DNA spacer (Fig. 2)[3].

We then extend the biosensing platform from a 2D plane to a hollow three-dimensional (3D) space by building the protein detection system on the inner surfaces of flexible layered polymer films. The different strain gradients of the polymeric bilayer are used as the driving force behind the 3D transformation [4]. Detection of human serum albumin (HAS) in a 3D aptasensor has been successfully demonstrated (Fig. 1(e-f)). Since the formation of the micro-roll avoids any cytotoxic processes, they can be used for encapsulating cells in vitro [4, 5]. Our sensor is expected to be applied to spatiotemporal detection of several secreted proteins such as growth factors in near future.

References

[1] K. Furukawa, Y. Ueno, M. Takamura, H. Hibino, Graphene FRET Aptasensor, ACS Sensors, 1 (2016) 710-716. doi:10.1021/acssensors.6b00191.

[2] Y. Ueno, K. Furukawa, K. Matsuo, S. Inoue, K. hayashi, H. Hibino, On-chip graphene oxide aptasensor for multiple protein detection, Anal Chim Acta, 866 (2015) 1-9. doi: 10.1016/j.aca.2014.10.047.

[3] Y. Ueno, K. Furukawa, K. Matsuo, S. Inoue, K. Hayashi, H. Hibino, Molecular design for enhanced sensitivity of a FRET aptasensor built on the graphene oxide surface, Chem. Commun. 49 (2013) 10346-10348. doi:10.1039/C3CC45615C.

[4] T. F. Teshima, H. Nakashima, Y. Ueno, S. Sasaki, C. S. Henderson, S. Tsukada, Cell Assembly in Self-foldable Multilayered Soft Micro-rolls, Sci. Rep., 7 (2017) 17376. doi:10.1038/s41598-017-17403-0.

[5] K. Sakai, T. F. Teshima, H. Nakashima, Y. Ueno, Graphene-Based Neuron Encapsulation with Controlled Axonal Outgrowth, Nanoscale, 11 (2019) 13249-13259. doi:10.1039/C9NR04165F.

Ackowledgements

This work was supported by JSPS KAKENHI Grant Number JP17H02759.

Figure 1

When the body is under stress, a lot of physiological processes work towards bringing the body back to its normal state. Cortisol, a stress hormone and TNF-alpha, a protein related to inflammation, are direct products of these physiological processes. The goal of this work is the demonstration of a biosensor that is capable of tracking these molecules in human sweat. The significance lies in simplifying chronic disease diagnostics, i.e. technique used to detect and monitor diseases in a non-invasive manner. This system is designed to be a wearable device that will track those physiologica process and use cortisol and TNF-alpha levels as indicators of user's health status.

Background: The HPA (Hypothalamic-Pituitary-Adrenal) axis is involved in maintaining homeostasis by engaging with the parasympathetic nervous system. During the process of chronic disease affliction to the body, this relationship is disturbed and there is an imbalance driven response observed with creation of disease related symptoms. This is of prime clinical importance when the body is chronically exposed to high levels of stress. By monitoring the two key components, cortisol, a direct product of the HPA axis and TNF-alpha, a pro-inflammatory cytokine, the manifestations of chronic stress on the homeostasis and diseased state of the body can be evaluated in a holistic/comprehensive manner. Materials and Methods: This work describes a novel sweat based sensing system which uses electrochemical modality to perform detection of cortisol and TNF-alpha. Results and Conclusion: This work highlights the development of an electrochemical detection system for the two biomarkers through human sweat. Limit of detection of the system is 1 ng/ml for cortisol and 1 pg/ml for TNF-alpha and dynamic range is 1-200ng/ml and 1-1000 pg/ml for cortisol and TNF-alpha respectively. This wearable system is designed to be a point of use, patient centric, chronic disease self-monitoring and management platform.

Introduction

We report development of a prototype transdermal ethanol (EtOH) monitor, in wristwatch format, to provide a modern device for consumer, clinical and law enforcement applications. Our vision is an attractive, comfortable smart watch, which will measure transdermal alcohol concentration (TAC) and predict blood alcohol concentration (BAC) during drinking. Current commercially available alcohol monitors are bulky, ankle-fixed devices, predominantly used in court-ordered monitoring and some research studies [1]. There has been little advancement in wearability, comfort and analytical performance of these devices. Our long-term goal is to realize a new generation of wearable monitor that combines a sensitive ethanol measurement and smartwatch capabilities in a comfortable and attractive package. This device could greatly expand the possible applications employing this technology.

Experimental

Watch Fabrication. The sensor platform was fabricated based on a scaled down version of our dual gas sensor platform and printed amperometric gas sensor [2, 3]. The simple prototype device contains a single EtOH sensor, analog front end, 24 bit ADC, Bluetooth LE chip and T/P/RH sensor. The board is contained in a commercially available wristwatch enclosure (BodyCase B1606117) as in Fig. 1. Initial tests of linearity and S/N showed that the circuit was linear over at least 0-300 ppm EtOH at 25 C (R² = 0.9996 – 0.9998 for 4 boards) with sensitivity 23 ± 2 nA/ppm (for 4 boards) and limit of detection (LOD) 0.26 ± 0.06 ppm.

Laboratory Testing. A flow cell was designed to deliver 0 – 0.4 w/o EtOH (simulated BAC) through a Strat-M membrane, which served as a skin surrogate [4]. EtOH solutions in phosphate buffered saline pH 7.1 were used. The cell was configured such that EtOH permeated the Strat-M, with vapor entering the headspace above to which the watch was interfaced.

Results and Conclusions

Flow Cell Tests. EtOH steps between 0 and 0.4 %EtOH (simulated BAC) were applied with 30 min exposure at each step. A 6 hr stability test was included. High stability and S/N were demonstrated. The EtOH concentrations in simulated BAC solutions were standardized and calibrated to headspace vapor phase concentrations, giving a calibration curve for %EtOH in the PBS buffer (simulated %BAC) as a function of measured EtOH (ppm EtOH) in headspace above the membrane. For two devices, we obtained highly linear calibrations (R² = 0.9983, 0.9992, respectively) with slope of 0.0013% and 0.0011% BAC/ppm EtOH, with near zero intercepts (-0.009 and -0.006 %BAC, respectively). From these two tests and the baseline noise we determined a limit of detection (LOD) of ±0.005% BAC. In later tests (e.g., Fig. 2) the LOD was considerably improved. The 6 hr stability test at 0.12% EtOH showed very stable output of 0.116 ±0.003 % EtOH.

Human Tests. An example of real transdermal alcohol measurement using the watch prototype on a human subject is shown in Fig. 2. A male volunteer ingested six 6 oz. aliquots of 5 v/o alcoholic beverage over ca. 1 hr. Two different watches were used, one fixed to each wrist. The measured temperature at the devices was typically in the range 28 – 34 C during tests. The data in Fig. 2 are not temperature compensated. A commercial BACtrack Trace breathalyzer was used to collect contemporaneous breath alcohol data (BrAC).

The TAC measurements taken on two wrists correlated quite well with each other. The TAC values tracked BrAC trend qualitatively, but with a shift to longer times, with the peak BrAC occurring ca. 1.5 hours before the peak TAC value. In other tests we have also observed lags on the order 30 min to 2 hr for TAC vs. BrAC measures. This lag is well-known for transdermal ethanol measurements [5] and presents a challenge for reliable instantaneous BAC prediction from TAC data. We note that the lag can depend on many factors including person-to-person variability of skin permeability, presence of sweat (humidity), food consumption and variable rates of alcohol consumption and metabolism, to name a few examples. Mechanical effects such as sudden movements and impacts can also affect the measurement.

We demonstrated use of a printed amperometric sensor for TAC measurement. Controlled clinical trials are currently being performed in collaboration with Boston Medical Center. The ultimate goal is to reliably predict BAC from TAC data across a broad range of individuals and use cases.

References

1. R. Marques, A. S. McKnight, Field and Laboratory Alcohol Detection with 2 Types of Transdermal Devices, Alcoholism: Clinical and Experimental Research, 33, 703-711 (2009). doi: 10.1111/j.1530-0277.2008.00887.x

2. R. Stetter, A. G. Shirke, B. J. Meulendyk, V. Patel, G. O'Toole , M. T. Carter, Health and Environmental Applications of Integrating Low Power Sensors with Wireless Technology, ECS Transactions, 53, 7-12 (2013). doi: 10.1149/05318.0007ecst

3. T. Carter, J. R. Stetter, M. W. Findlay, B. J. Meulendyk, V. Patel, D. Peaslee, Amperometric Gas Sensors: From Classical Industrial Health and Safety to Environmental Awareness and Public Health, ECS Trans., 75, 91-98 (2016). doi: 10.1149/07516.0091ecst

4. A. Imran, U. Anand, R. Agu, "Human Skin Substitute (Strat-M) as an alternative for Testing Transdermal Delivery of Levothyroxine (T4), Paper MON-0474, The 16^th International Congress of Endocrinology and the Endocrine Society, ICE/ENDO 2014.

5. M. Dougherty, N. E. Charles, A. Acheson, S. John, R M. Furr, Comparing the detection of Transdermal and Breath Alcohol Concentrations During Periods of Alcohol Consumption Ranging from Moderate Drinking to Binge Drinking, Exp. Clin. Psychopharmacology, 20, 373-381 (2012). doi: 10.1037/a0029021

Acknowledgements

The program is supported by NIH NIAAA grant no. 5R44AA024651-03.

Figure 1