Table of contents

My brilliant former student NJ Tao, to whose memory this symposium is dedicated, showed in a remarkable single-author paper¹ that the conductance of a molecule containing a redox center was strongly dependent on surface potential, being a maximum at the redox potential. Some years later, we showed² that defined contacts and statistical analysis were the keys to measuring molecular conductance quantitatively. NJ Tao came up with a simpler way of implementing our method, now the gold standard in single molecule conductance measurements.³ Gating of single-molecule conductance has now been reliably demonstrated.⁴ Despite many advances, there have been few real applications for single-molecule electronic devices. Proteins make excellent electronic conductors, as demonstrated by measurements under potential control that eliminate Faradaic contributions to the current.⁵ A remarkably long electron mean-free path and means for self-assembly give proteins advantages over conventional molecular wires.⁶ Furthermore, enzyme activity may be followed directly via conductance fluctuations, a possible basis for a new DNA sequencing technology.⁷ This talk will look at the physics behind these phenomena.

1. Tao, N. J., Probing Potential-Tuned Resonant Tunneling through Redox Molecules with Scanning Tunneling Microscopy. Physical Review Letters 1996,76 (21), 4066-4069.

2. Cui, X. D.; Primak, A.; Zarate, X.; Tomfohr, J.; Sankey, O. F.; Moore, A. L.; Moore, T. A.; Gust, D.; Harris, G.; Lindsay, S. M., Reproducible Measurement of Single-Molecule Conductivity. Science (New York, N.Y.) 2001,294 (5542), 571.

3. Xu, B.; Tao, N. J., Measurement of Single-Molecule Resistance by Repeated Formation of Molecular Junctions. Science (New York, N.Y.) 2003,301 (5637), 1221.

4. Song, H.; Kim, Y.; Jang, Y. H.; Jeong, H.; Reed, M. A.; Lee, T., Observation of molecular orbital gating. Nature 2009,462 (7276), 1039-1043.

5. Zhang, B.; Song, W.; Pang, P.; Lai, H.; Chen, Q.; Zhang, P.; Lindsay, S., The Role of Contacts in Long-Range Protein Conductance. Proc Natl Acad Sci U S A 2019,116, 5886-5891.

6. Lindsay, S., Ubiquitous Electron Transport in non-Electron Transfer Proteins. Life 2020,10, 72

7. Zhang, B.; Deng, H.; Mukherjee, S.; Song, W.; Wang, X.; Lindsay, S., Engineering an Enzyme for Direct Electrical Monitoring of Activity. ACS Nano 2020,14, 1630-1638.

While cancer is mostly viewed as a genetic disease and characterized by genetic markers and expression of mutant proteins, there is considerable evidence that there is more to cancer than somatic mutations. For example, the first signature looked for by a pathologist is a grossly aberrant cell nucleus. Chromatin compaction and structure play a major role in the overall nuclear structure. We compared chromatin compaction, structure and gene expression for two esophageal cell lines, EPC2 (non-cancerous) and CP-D (cancerous) by using a combination of salt fractionation, DNA quantification by spectroscopy, atomic force microscopy, and sequencing.

Salt fractionation is believed to be an efficient method for quantitative extraction of intact chromatin fragments from cell nuclei. We found that this method is not quantitative unless the supernatant fraction is included. For EPC2 and CP-D cells, about half of the genomic content is solved in the supernatant fraction. Further, we found significant differences for DNA amounts, and chromatin morphology for the cancerous and non-cancerous cell lines, as well as variations in the nucleosome partitioning. We anticipate that our results will help to get insights into the mechanisms of cell phenotype changes from normal to cancerous.

Rapid tests of COVID-19 have played a critical role in controlling the virus spread during the pandemic. Lateral flow assays (LFA), a paper-based technique, are widely adopted as a platform for detecting SARS-CoV-2 antigens, anti-SARS-CoV-2 antibodies, and viral RNA in combination with other technologies such as CRISPR. Due to its rapidness, cheapness, and visual readout, LFA is especially suitable for the at-home test. However, LFA has manifested a low sensitivity for antigen-based COVID-19 test. It has been reported that the sensitivity was as low as 12–18% when Ct values of RT-PCR were between 30 and 37, which limits LFA as a diagnostic tool.

We intend to develop a sensitive LFA for at-home use, like a pregnancy test strip, with a configuration shown in Figure 1a. Our first efforts are to lower its limit of detection (LoD) by enhancing the efficiency of molecular capture on the nitrocellulose membrane. As shown in Figure 1b, a test line on the strip comprises a hydrogel polymer, which would provide a biologically friendly environment for molecular interactions. We have fabricated a polyacrylamide test line containing streptavidin on a nitrocellulose strip using photolithography. Thus, a test strip can be produced by printing biotinylated affinity molecules on the test line. We have used gold nanoparticles as a colorimetric readout of LFA. Our results show that the immobilization of an antibody in the hydrogel polymer would enhance the sensitivity of LFA's detection. Compared to those by physical adsorption of antibodies directly to the nitrocellulose membrane, our LFA reduced the LoD by ~ 2 orders of magnitude. We are currently applying our LFA to detect neutralizing antibodies and variants of concern (VOC) of SARS-CoV-2. In this presentation, we will report the current progress of our project.

Figure 1. Illustration of (a) general configuration of lateral flow strips (b) a lateral flow strip with hydrogel polymer embedded in the test line for antibody testing

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Recently, Lindsay et al. showed in a series of papers (summarized in Lindsay, S. Life2020, 10, 72.) that single proteins coupled to metal electrodes have unexpectedly good conductance properties. It became clear that much of the resistance drop happens at the metallic contacts, and the internal conductivity of the protein itself can be very close to the quantum limit 2e^2/h for short proteins. The localization length can be as large as ten nanometers for longer ones. Cahen et al. showed that the conductance is temperature independent in the range of 30-300 Kelvins in a series of measurements. All this indicates that proteins support fast and at least partially coherent electron transport at room temperature. According to electronic structure calculations, proteins have significant HOMO-LUMO gaps surrounded by localized orbitals, a typical arrangement for insulators ruling out efficient single-electron quantum transport even in the presence of photon mediation. However, due to the repetitive nature of the basic peptide backbone of the proteins, there are states deeper below the HOMO energy, which are intermediary between extended states typical in metallic conductance bands and the localized ones. These so-called critical states (Vattay G. et al. 2015 J. Phys.: Conf. Ser.626 012023) have fractal density distributions and show large electron density correlations between other orbitals in the same energy region. It has been shown that in the presence of electron-electron attraction, these states are especially suited for pair formation of electrons, and an insulator-superconductor transition can occur even at high temperatures. Here we argue that polarizable sidechains of amino acids can create an attractive force between electrons propagating along the backbone. We show that the typical size and polarization of amino acids make the proper magnitude of pairing energy to maintain superconducting patches even at room temperature. We calculate and display the geometry of the superconducting gap over the protein structure and propose this new mechanism as a possible explanation for the observed coherent transport.

A specially engineered biological nanopore directly resolves a large variety of analytes including inorganic ions, small molecules, nucleic acids or even proteins. This high resolution enables the biological nanopore as an ideal sensor to directly monitor single molecule analytes, which include but not limited to the probing of carcinogenic DNA damages, cisplatin modifications to DNA and small molecule interactions with ion channels. O6-carboxymethyl guanine, which is a highly carcinogenic base damage related to the development of rectum cancer, were identified by a nanopore sequencing assay with no ambiguity. By taking a tetrachloroaurate(III) as an atomic adaptor inside a biological nanopore, we report a novel concept of nanopore fabrication with a bio-inorganic interface, which immediately discriminate between cysteine, homocysteine and glutathione via direct nanopore readouts. Emerging optical single channel recording (oSCR) techniquesand its simplified form DiffusiOptoPhysiology (DOP) has also significantly improved the measurement throughput of nanopore via fluorescence microscopy instead of a patch clamp which lacks a desired throughput.

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Electronically detecting and identifying four DNA nucleosides offers great opportunity in DNA sequencing. By placing DNA nucleosides in tunneling gap, the conductance of four DNA nucleosides can be obtained through analyzing the switching tunneling current signals and might be used to distinguish four different bases. However, the conductance of A, T, C, G measured in a bare electrode gap is heavily overlapped, posing great challenge in separating them. By introducing a cage-shaped molecule-cucurbituril (CB) into the tunneling junction, we show that all four DNA nucleoside can be readily detected and separated, with a conductance sequence of T<A~C<G. The confinement effect of the CB cavity might restrain the conformation variation of the DNA bases in the tunneling gap, giving rise to the better distinction of four bases. This works suggests a new approach in detecting DNA molecules with tunneling signals.

This is a continuing tribute from various collaborators and former postdocs/students to Prof. Stuart Lindsay

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The rapid progress of precision/personalized medicine requires new sensing, analysis, and imaging tools, especially at the nanoscale. In this talk, I will briefly describe our recent progress in this direction. I will first show the nanopore-nanoelectrode nanopipette based electrochemical single-entity technique for single nanoparticle and single molecule detections. I will then introduce our works on single cell analysis and imaging. Finally, I will discuss our efforts to develop and apply nanoscale tools for engineered cardiac tissues.

The aggregation of amyloid beta (Aβ) is a self-assembly process that results in the production of fibrillar structures along with neurotoxic aggregates. However, in the vast majority studies in vitro the required Aβ concentrations is several orders higher of the physiological relevant concentrations of Aβ; no aggregation is observed at physiological low nanomolar range of Aβ. This suggests that the assembly of Aβ in aggregates in vivo utilizes pathways different from those used in experiments in vitro. We have discovered recently that surface plays a role of catalyst allowing the self-assembly of amyloid aggregates to occur at physiologically relevant concentrations. We proposed a model in which the monomers transiently immobilized on the surfaces work as nuclei for the next aggregation step. The model was verified by experimental time-dependent AFM measurements. AFM studies of aggregation of Aβ on supported phospholipid bilayer revealed a strong effect of the membrane composition on the surface aggregation catalysis. We combined AFM experimental studies with all-atom molecular dynamic (MD) simulations to characterize the on-surface self-assembly process of amyloid proteins. MD simulations show that the surface-protein interactions induce a conformational transition of the monomer facilitating binding of another molecule. Importantly, even transient interactions of amyloid beta protein with the membrane bilayer facilitate sampling of the energy profile allowing for the monomer to adopt the β rich conformations responsible for the assembly of conformationally stable oligomers. A membrane-mediated aggregation catalysis explains a number of observations associated with the development of Alzheimer's disease. The affinity of Aβmonomers to the membrane surface is the major factor defining the aggregation process rather than Aβ concentration. Therefore, the development of potential preventions for the interaction of monomeric amyloids with membrane can help control the aggregation process.

Non-equilibrium (NE) energy routes underpin fundamental bio-physical mechanisms like cell migration, proliferation and signaling. Yet, our predictability of such NE pathways in biology are still at its infancy. This is because of our general lack of understanding of the role of heterogeneities in NE kinetics. Here we study differentiated-Cath-a cancer cells on engineered polydopamine (PDA) surfaces employing transitional-tapping atomic force microscopy (AFM) to map the surface energy heterogeneities of the cells relative to the engineered soft PDA surface on which they are allowed to proliferate for different lengths of time. Transitional tapping AFM enables us to determine deformation dependent heterogeneity maps by decoupling the heterogeneous and homogeneous loss mechanisms at the tip-surface interface without necessarily indenting the surface at near contact conditions. A unique Lévy-like distribution in surface energy and cell-height is observed deviating from standard Boltzmann distribution as typically observed by optical methods. The technique and results of the Lévy-like distributions revealed in this work have broad implications in terms of understanding bio-physical origins of heterogeneities in soft-bio-matter and can inspire further work in determining role of heterogeneity and loss pathways in protein-folding, tumor progression, and soft-tissue regeneration.

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The emergence of tools at the forefront of advanced atomic force microscopy (AFM) holds great potential to bolster our fundamental understanding of heterogeneous and dynamic systems at the nanoscale, of prime interest for material science, chemistry and life sciences. Developments using multi-frequency AFM provide more sensitive detection and a wider frequency span to explore the morphology, mechanical and chemical properties of the sample volume. However how these performances compare to more conventional modes is not well established.

Here, we will present the implementation of multifrequency AFM nanoscale infrared imaging and spectroscopy. We will first discuss how the nonlinear nature of the tip-sample interaction in AFM can be used as a sensitive channel to access infrared (IR) fingerprints of materials at the nanoscale. We will compare the IR signatures obtained with conventional nanoscale IR spectroscopy, both on homogeneous and heterogeneous materials. In doing so, we will estimate the volume probed as a function of the data acquisition parameters and materials properties. Finally, we will illustrate how the high sensitivity and spatial resolution of nanoscale IR spectroscopy provide new insights in the molecular arrangements of complex systems, especially when guided by macro- and micro-scale studies.

The strength of the humerus bone is evaluated for 17 species of extant birds with varying mass by measuring its section modulus. The least massive bird is Regulus calendula (0.0058 kg) while the most massive is Cygnus olor (8.959 kg), a range in mass spanning more than three orders of magnitude. The humeral section modulus is found to be proportional to the mass of the bird. This relationship is used to determine the flight abilities of 19 extinct dinosaurs from Dromaeosauridae, Troodontidae and Avialae. Though all the avialans could fly, only four dromaeosaurids (Microraptor gui, Graciliraptor lujiahunensis, Buitreraptor gonzalezorum, and Changyuraptor yangi) and one troodontid (Jianianhualong tengi) are found to have had humeri that were consistent with extant volant birds. The humeri of the remaining dromaeosaurids and troodontids were too weak for flight.

Through the (rapidly) approaching 30 years since departing graduate school at Arizona State University – I have been on an intellectually gratifying journey that can trace its origins to influencers in my life not only limited to my Family, but, also to professional mentors/colleagues of mine throughout. These include a subset of those organizing this event as well as a those we are honoring today – 'N.J.' and 'Big S'. It is my intent through the time that I have in this forum to share some experiences and give a trace to them and their connectedness to the success I have been privileged to have in my career. Along the way – I will interject some jocular tidbits as well as some real-world technical problems that I have been able to attack and, in some cases, contribute to in my journey – of Life.

Over the last few years, the U.S. Army has embarked on a revolutionary journey to reinvent its ability to conduct, support and fund basic and applied Science and Technology. The U.S. Army has established the Army Futures Command (its first 4 star command since 1973) to accelerate and improve its agility and robustness of the U.S. Army's Science and Technology internal and external investments to insure "overmatch" against near peers in the near, mid, and far term. Discussed will be the Army internal and external investment and research strategies to support near, mid, and far term National priorities.

The applications of lithium-ion batteries (LIBs) have rapidly embedded into almost every aspect of our life and society. In the LIB industry, a well-controlled and lengthy process is proceeded before the LIBs are brought into markets. The aim of this process is to form a "good" solid electrolyte interphase (SEI) on the graphite anode in a LIB to prevent continuous electrolyte decomposition and graphite exfoliation, which greatly influence the performance and cycle life of a LIB. The SEI formation process usually takes one or two weeks, is the major bottleneck and the most expensive component for mass production of LIBs. Efforts have been devoted to accelerating the SEI formation process by increasing charging rates or skipping the high state-of-charge region. However, the shortened SEI formation time usually comes with a large decrease in capacity retention of a LIB.[1-3] Improving our understanding on the structure and property evolution of SEI during the SEI formation process is urgently required for the development of faster formation protocols without sacrificing the high quality SEIs.[4-7]

Aiming at better understanding of the initial stage of SEI formation process in LIB production, in-situ electrochemical atomic force microscopy (EC-AFM) has been employed to monitor the morphological evolution of the electrode materials in nano-scale levels. While different from most of the previous works, this work applied Galvanic constant current (CC) control to closely mimic the industrial SEI formation processes of different formation rates. For comparison, linear potential sweep (LS) controlled SEI formation of various potential sweeping rates, which is commonly applied in fundamental studies of SEI formation, was also performed in parallel. For the first time, it is revealed that the onset potentials for co-intercalation and electrochemical reduction of solvated lithium-ion complexes, the two competitive and/or sequential processes during SEI formation, vary as a function of potential sweeping rates. The onset potential for co-intercalation shifts positively as the potential changing rate increases, which is in high contrast to the onset potential of their electrochemical reduction. It has been well established that the onset potential of their electrochemical reduction is largely determined by the lowest unoccupied molecular orbital (LUMO) level of the solvent molecules in the solvated lithium ion complexes,[6] so that it may remain largely constant or shift negatively, due to electrode polarization.[2] As a result, the onset potential for co-intercalation becomes more positive than that of the electrochemical reduction as the potential sweeping rate increases. Our experimental results demonstrate that this relationship applies to both CC and LS-controlled SEI formation processes. However, the difference between these two onset potentials is much greater in CC than that of LS controlled processes, due to the associated faster potential changing rates at the beginning of CC controlled lithiation. The difference becomes even more prominent when high charging current is applied to shorten the SEI formation time and lower LIB production cost. This understanding combined with the clear visible evidence of different structural evolution under CC and LS controls should provide more practical guidance to develop protocols for faster formation of SEI with higher quality, which is intensively pursued in industry.

[1] Lee, H. H.; Wang, Y. Y.; Wan, C. C.; Yang, M. H.; Wu, H. C.; Shieh, D. T., A fast formation process for lithium batteries. Journal of Power Sources 2004,134 (1), 118-123.

[2] Bhattacharya, S.; Alpas, A. T., Micromechanisms of solid electrolyte interphase formation on electrochemically cycled graphite electrodes in lithium-ion cells. Carbon 2012,50 (15), 5359-5371.

[3] An, S. J.; Li, J. L.; Du, Z. J.; Daniel, C.; Wood, D. L., Fast formation cycling for lithium ion batteries. Journal of Power Sources 2017,342, 846-852.

[4] Wang, L. N.; Menakath, A.; Han, F. D.; Wang, Y.; Zavalij, P. Y.; Gaskell, K. J.; Borodino, O.; Iuga, D.; Brown, S. P.; Wang, C. S.; Xu, K.; Eichhorn, B. W., Identifying the components of the solid-electrolyte interphase in Li-ion batteries. Nature Chemistry 2019,11 (9), 789-796.

[5] Cai, W. L.; Yao, Y. X.; Zhu, G. L.; Yan, C.; Jiang, L. L.; He, C. X.; Huang, J. Q.; Zhang, Q., A review on energy chemistry of fast-charging anodes. Chemical Society Reviews 2020,49 (12), 3806-3833.

[6] Xu, K., Nonaqueous liquid electrolytes for lithium-based rechargeable batteries. Chemical Reviews 2004,104 (10), 4303-4417.

[7] Xu, K., Electrolytes and Interphases in Li-Ion Batteries and Beyond. Chemical Reviews 2014,114 (23), 11503-11618.

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Developing in situ Optical Imaging Techniques to Understand Battery Solid Electrolyte Interface Formation

Xiaonan Shan. Xu Yang, Guangxia Feng, Yaping Shi, ECE, University of Houston

Increasingly complex and heterogeneous chemical reactions on the battery and catalytic electrode surfaces require the characterization methods to provide a complete picture of molecular interactions across the interface in situ. For example, the biggest challenge in accelerating the Li metal batteries towards higher energy density is the lack of fundamental understanding of interfacial chemical reaction on the electrode surface. During the initial lithiation cycles, a solid electrolyte interphase (SEI) forms on the electrode surface due to the electrochemical instability of the electrolyte. Inhomogeneity in reaction activity/deposition rate, chemical compositions, and ionic and electrical conductivity on the battery electrode will cause the non-uniform Li-ion diffusion, and lead to inhomogeneous nucleation and dendrite formation. The traditional imaging and measurement techniques have experienced challenges to characterize these complicated interfacial chemical reactions. For example, most of the methods only provide rich information at a certain time point in the dynamic process, or measure an average result over a large area during the reaction. On the other hand, these interfacial reactions are highly dynamic and spatially varied, and the signals at different time points or different locations could be totally different.

We have developed a multimodal optical imaging platforms to image the battery electrode reaction dynamics across the interfaces throughout the entire reaction process. The platform includes surface plasmon microscope, optical reflection microscope, and Raman spectrometry. The platform provides us important interfacial chemical information including SEI formation and chemical compositions: 1) The plasmonic imaging technique provide us information of localized SEI thickness and localized charging status during the reactions; 2) optical reflection microscope allow us to study the SEI and Li nucleation dynamics; and 3) Raman spectrometry provide us chemical composition information. The integration of all the information obtained from this multimodal imaging platform will allow us fundamentally understand SEI formation and electrochemical reaction on the anode electrode.

The nanoscale manipulation and characterization of individual molecules is necessary for understanding the intricacies of molecular structure which ultimately governs phenomena such as reaction mechanisms, catalysis, local effective temperatures, surface interactions, and charge transport phenomenon. Along these lines, the field of molecular-scale electronics has evolved tremendously in recent years: from the initial experiments claiming single-molecule conductance measurements to the development of robust and reproducible platforms that have begun providing a detailed understanding of the charge transport properties of these systems. It has now become possible to probe the chemical, structural, mechanical, and electrical properties of single-molecule devices to explore unique functional paradigms for applications. However, continued advances in in situ characterization of a molecular junction are needed to provide detailed information about the molecular configuration and its impact on the charge transport, reactions, and device behavior. Single-molecule conductance and Raman spectroscopies each provide unique perspectives into the behavior of molecular systems and reactions at the single-molecule level. In this talk we will discuss the development and implementation of system designed to simultaneously obtain conductance information and Raman spectra from a molecular junction to provide this information. This will include the development of a MEMS-based break junction system that allows facile integration with a Raman microscope to maximize the solid-angle and photon collection.

This multi-dimensional information yields repeatable, self-consistent, verification of single-molecule resolution, and allows for detailed analysis of structural and configurational changes of the molecule in situ. We will discuss the correlation between single-molecule binding events and changes in Raman spectra (intensity, modes, etc.) and conductance to explore the possibility of obtaining single-molecule spectra from the molecule bound between two electrodes. We will further explore the utility of this system for in situ characterization of active single-molecule devices including electrically-active single-molecule switches and memory devices.

Single molecule study, where science and engineering met, applies the tools and measurement techniques of nanoscale physics and chemistry to generate remarkable new insights into how physical, chemical, and biological systems function. It permits direct observation of molecular behavior that can be obscured by ensemble averaging and enables the study of important problems ranging from the fundamental physics of electronic transport in single molecule junctions and biophysics of single molecule interactions, such as the energetics and nonequilibrium transport mechanisms in single molecule junctions and the energy landscape of biomolecular reactions, associated lifetimes, and free energy, to the study and design of single molecules as devices-molecular wires, rectifiers and transistors and high‐affinity, anti‐cancer drugs.

In this talk, we present two of our recent researches using the legendary scanning probe microscope Dr. Stuart Lindsay and the Late Dr. Nongjian Tao pioneered. One studies the small molecule-DNA interactions electronically by STM-breakjunction measurements of site-specific intercalation of small molecules (coralyne) into a custom-designed 11-base-pair DNA duplex. The results show that a single molecule DNA electrical rectifier is realized and the coralyne-induced spatial asymmetry in the electron state distribution caused the observed rectification. The other study elucidated the in situ single-molecule interaction of heparan sulfate (HS) with antithrombin (AT) on the endothelial cell membrane surface under near-physiological conditions, especially the role of sulfate groups in specific binding sites of HS for AT, using AFM recognition imaging and dynamic force spectroscopy measurements.

In the realm of the Single-Molecule Electronics, a suite of advanced electrical characterization approaches have emerged allowing measuring charge transport in an electric contact made out of an individual molecule[1]. The field has (and is still) drawn(ing) an scenario where individual molecules can be chemically modified to deliver a particular electrical function in a nanoscale circuit, e.g. variable resistors[2], diodes[3], switches[4], etc. Along this excursion, we have observed that single molecules trapped in a nanoscale tunneling junction experience conformational structural changes and changes in molecule/electrode contact geometries, which are usually accompanied by large conductance variations and can be easily detected electrically[1]. Such changes are induced by the imposed forces fields experienced by the molecules within the nanoscale gap, namely, a mechanical force and/or an electric field. In the past decade, we have learnt that under certain force field conditions, the individual molecules wired in a nanoscale junction undergo chemical transformations. Here, we will present a couple of illustrative examples of the use of single-molecule junction to study and control reactivity at the nanoscale using electric fields, a concept that is inherent in the natural enzymatic molecular machinery[5]. Tunable, well-oriented electric fields can be easily delivered along the main junction axis of a nanoscale electrical device[6]. We exploited the latter to study; (1) a simple monomolecular cis-trans isomerization reaction[7], and (2) a bi-molecular Diels-Alder reaction[8]. Along with the experimental design and fundamental chemistry aspects, we will discuss the advantages these examples can bring to possible technological applications.

[1]. L. Sun et al. Chem. Soc. Rev., 2014,43, 7378-7411.

[2]. A. C. Aragones et al. Chem. Eur.J.2015, 21,7716 –7720.

[3]. I. Diez-Perez et al. Nature Chemistry 1, 635–641 (2009).

[4]. N. Darwish et al. Nano Lett. 2014, 14, 12, 7064–7070.

[5]. Stephen D. Fried et al. Annu Rev Biochem. 2017; 86: 387–415.

[6]. N. Darwish et al. "Principles of Molecular Devices Operated by Electric Fields" in Effects of Electric Fields on Structure and Reactivity: New Horizons in Chemistry, Royal Society of Chemistry, Ed. Sason Shaik.

[7]. C. S. Quintans et al. Appl. Sci. 2021, 11(8), 3317.

[8]. A. C. Aragones et al. Nature 531, 88–91 (2016).

The sensitivity enhancement of biosensors can be achieved by increasing the active surface area using nanoparticle-functionalized electrodes. However, these enhancements have certain limitations due to a lack of information about the properties of nanoparticles and their behavior under certain conditions arising due to polydispersity, orientation, and surface roughness.

Nanoparticles (NPs) have broad spectra of their applications in electronics. In most sensing methods, the deposition of high-density NPs causes polydispersity. Hence, the distribution achieved on the electrode surface conceals the effects of their size-dependent properties. Moreover, the tethered patterning of recognition molecules on the electrode surface is always represented using simple sketches, which fail to consider the non-idealities in the target-surface arrangement. The data obtained in these cases are averaged over these phenomena is challenging to interpret.

The interparticle interactions play an active role in enhancing the sensitivity of the biosensors; these interactions are controlled by the Van der Waals force of attraction and the Electrostatic double layer (EDL) on the surface. According to DLVO mode, the Van der Waals force and EDL on the surface of nanoparticles are dependent on average nanoparticle size, the distance between interacting surfaces, absolute temperature, stern layer thickness, and zeta potential. In this work, we present an experimental setup to control the nanoparticle distribution and the surface charges. The experimental methods consist of depositing nanoparticles by drop-casting, aerosol spraying, and atmospheric plasma-assisted aerosolized deposition (Figure 1).

The studies are carried out to investigate the impact of atmospheric plasma treatment on the morphology and optical properties of gold nanoparticles (Au NPs). The morphological changes such as size reduction observed due to the surface plasma treatment of AuNPs are attributed to the symmetric partial oxidation. The plasma deposited films were used as SERS substrate to detect R6G peaks. The intensity of the SERS signal of R6G from plasma-treated AuNP substrate was ~95 times as high as that of the untreated substrate. This enhancement is attributed to the surface roughness, presence of nanogaps, and size reduction after the treatment of NPs on the substrates. The morphological changes are explained by a redox reaction induced by the energy and concentration of reactive species in the plasma environment.

Surface architectures of electrochemical biosensors have complex structures which facilitate the effective capture of probes (analytes). A minor defect in the architecture can cause an error in probe-target interaction. In such electrochemical sensors, the primary assumption made is that the sensor has an ideal architecture. Nevertheless, these sensors drive the detection of analytes as the focus is on the probe-target interaction. The underlying surface properties due to the resultant architecture are overlooked while analyzing the detection phenomenon. Hence, assessing the dependence of the sensing process on the non-idealities present is required to achieve reliable measurements.

This study also presents the plasma-induced enhancement of electrical properties of Au NPs at room temperature. The distribution of plasma-assisted aerosolized deposited NPs showed the promising architecture for surface modification of the electrochemical sensor that has improved the electrochemical sensing, which is validated by detecting the cortisol molecules (cortisol is a stress biomarker found in interstitial fluids, saliva, and sweat). This improved electrochemical response is due to plasma-assisted surface activation of NPs. Another reason for the enhancement is the increase in the density of NPs on the substrate leading to a larger electroactive surface area (Figure 2A, 2B).

These NP functionalized electrodes were then modified using DTSP SAM (dithiobis(succinimidyl propionate) self-assembled monolayer) and cortisol antibodies to detect cortisol molecules. The enhancement in the sensitivity was observed for plasma-assisted AuNPs functionalized electrodes as against drop-casted and aerosol sprayed electrodes (Figure 3A, 3B, 3C, 3D).

In summary, a novel technique of plasma-assisted NPs deposition, which could maintain the inter-particle repulsive barrier to obtain the uniform monolayer of NPs, is reported. This barrier potential was achieved due to the de-agglomeration of NPs during deposition using nebulizer in experimental assembly and providing the surface excitation and surface charge tuning of NPs during plasma-assisted deposition. The properties of plasma-treated NP films at room temperature are comparable to the thermally-treated films at higher temperatures. This unique feature can be used for processing metal nanoparticles at room temperatures for designing sensitive SERS substrates on flexible materials such as polymers, papers, and textiles.

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The detection of nucleic acid molecules plays important role in clinical diagnosis of diseases and fundamental biomedical research. The widely used polymerized chain reaction technique requires hours of amplification process, and is prone to aerosol pollution. Here, we presented the amplification-free digital sensing of nucleic acid molecules with the interferometric plasmonic microscopy (iPM). A sandwich assay was performed with the primary DNA probes coated on the plasmonic sensing surface and the secondary DNA probes tethered Au nanoparticles. Hybridization between DNA probes and single analyte molecules was monitored by the imaging of single Au nanoparticle with iPM system. By digitally counting the events of hybridization, the detection limit at the fM level could be achieved. Without amplification, this assay could be done within one hour. Furthermore, by analyzing the real-time binding behaviro during single hybridization event, non-specific bindings could be excluded from the specific ones, resulting in the improved specificity. This technique could be further improving by a careful design of the kinetic binding properties between DNA probes and analyte molecules.

Measuring individual protein properties is the most critical and challenging tasks for protein analysis. We show that it is possible to quantify single protein size and binding kinetics by plasmonic scattering imaging (PSM). The interference between the protein scattered light and the background light scattered by the rough substrate significantly enhances the signal intensity and contrast, which allows PSM to image single proteins, measure their sizes, and identify them based on their specific binding to antibodies. In addition, PSM can quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. Furthermore, PSM can distinct specific and nonspecific binding processes by quantifying the mass and binding dynamics of individual bound analyte molecules, thus allowing the binding kinetic analysis in complex media such as serum. PSM can be implemented on top of an objective or a prism coupled surface plasmon resonance system, providing a convenient solution to realize high sensitivity single molecule imaging. We anticipate that PSM will become an important tool for single protein analysis, especially for low volume samples, such as single cells.

This is a continuing tribute from various collaborators and former postdocs/students to Prof. Nongjian Tao

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To produce next-generation, shelf-stable biosensors for point-of-care diagnostics, it is imperative to achieve rugged biomolecular recognition elements as well as innocuous deposition approaches for efficient encapsulants. Moreover, to maintain the sensitivity and specificity that is inherent to biological recognition elements in solid-state biosensing systems, site-specific immobilization chemistries must be invoked such that the function of the biomolecule remains unperturbed. In this work, we present a universally applicable strategy to establish robust solid-state biosensors using emergent nanobody (Nb) recognition elements with a vapor-deposited polymer encapsulation layer. As compared to conventional immunoglobulin G (IgG) antibodies, Nbs have multiple advantages, including higher thermal stability and pH tolerance, greater ease of recombinant production, and capable of binding antigens with high affinity and specificity. Photoinitiated chemical vapor deposition (piCVD) provides thin, protective polymer barrier layers over immobilized Nb arrays that allow for retention of biosensing activity and specificity after both storage under ambient conditions and complete desiccation. Most importantly, we also demonstrate that vapor-deposited polymer encapsulation of Nb arrays enables specific detection of target proteins, while suppressing nonspecific binding interactions in complex heterogeneous samples, such as unpurified cell lysate, which is otherwise challenging to achieve with bare Nb arrays.

Figure 1

The occasional use of chemical warfare agents (CWAs) by rogue states in current conflicts provides a reminder that these hazards are a real threat. Although hazmat suits made of fully impermeable barrier materials provide an effective means of protecting against CWAs, they also inhibit evaporative cooling which can cause rapid hyperthermia. This conundrum has motivated a search for novel materials that allow water vapor but not CWA permeation. Here we show that, at least for aerosolized CWA, this can also be achieved using a highly breathable composite fabric that self-seals only when exposed to target chemicals. Our approach is based on the use of selectively superabsorbing polymer (SAP) microbeads that are dispersed on a highly breathable fabric. Many CWAs, especially nerve and blistering agents, have low vapor pressure and can only be dispersed as a "fog" from aerosolization. We show that upon contact with an example organic aerosol (o-xylene) the proposed SAP microbeads dispersed on a nylon mesh swell highly, seal pores, and inhibit passage of the microdroplets. In contrast, in normal conditions the SAP microbeads do not absorb or swell upon contact

with water and provide over 10 kg m⁻² day⁻¹ water permeation rate that is comparable to a cotton shirt.

Figure 1

Recently, non-invasive diagnosis of human diseases using gas samples from human has become increasingly important due to its simple way to make a person less reluctant. The exhaled breath or urine vapors from patients can contain VOCs, which have been reported to be clinically meaningful biomarkers. However, it is difficult to make a diagnosis accurately due to very low gas concentration level of disease related VOCs, which is 1~100 ppb level. To overcome the detection limit of various commercialized gas sensors or detectors, the preconcentration of gas samples from human is required. On the other hand, gas chromatography (GC), which is extensively applied for gas mixture analysis, is necessary to separate and identify the gas components.

A miniaturized gas analysis platform for the analysis of the low concentration and multiple disease related VOCs has been developed using a micro gas preconcentrator chip, a micro gas chromatography chip, a thermal conductivity detector chip, etc. Its design and fabrication process will be introduced and its basic performance will be presented for various clinically important VOCs.

Precision medicine is moving cancer treatment from etiological and histological parameter-based treatments to those that target specific key molecular drivers of disease in a time-resolved fashion. To arrive at actionable end points, representative subsamples of the disease tissue and multiparameter measurements are now being optimized to accurately profile human specimens at the single cell level to describe the relevant biological unit of disease. As these new biosensing and nanoscale measurement technologies mature and transition from basic research use to clinical research, it is important to determine pre-analytical variables that may significantly contribute to changes in readouts that can be optimized with appropriate quality control verification criteria versus those that cannot be further optimized in a clinical setting. The term pre-analytical variables refer to all factors that may affect a specimen or sample before it enters the analytical process. No matter how complete analytical and clinical validation for an assay is, if there is not confidence that the sample being analyzed actually reflects what is happening in the patient, then the results will be meaningless. Often times, especially in oncology and related fields, specimen collection, handling, and processing (CHP) variables are the most important of these pre-analytical variables to consider. This presentation will provide perspectives and lessons learned from national initiatives and public-private partnership efforts.

Disorders in iron metabolism are endemic globally, affecting more than several hundred million individuals and often resulting in increased mortality rates or general deterioration of quality of life. To prevent and monitor iron-related disorders, we present a point of care multiplex system to measure four clinically relevant iron biomarkers: blood iron levels (iron bound to transferrin), total iron-binding capacity (TIBC), percent transferrin saturation, and blood ferritin. This system leverages three distinct channels: two colorimetric and one electrochemical emerging from the same sample injection port designed to accommodate 50 ul of whole blood, filter out cellular components, and transport the filtered sample to the three specified channels' capillary action. The first channel measures iron levels. It uses a membrane impregnated with the working reagents that reduce iron (III) to iron (II) and chelate the reduced iron with ferene forming a blue- complex. A custom smartphone app quantifies the Fe-ferene complex and provides outputs iron levels in whole blood. The second channel measures TIBC. This channel uses the same membrane and detection method that the first channel but requires an extra preconditioning step of saturating the blood sample with iron standard and precipitating excess unbound iron with a specific binding agent (magnesium carbonate). Using the ratio of total iron (output of channel 1) and TIBC (output of channel 2) enables calculating the percentage of transferrin saturation. The two colorimetric channels were created at Forzani's Team at Arizona State University, while the electrochemical channel is created by Diez-Perez's Team at King's College London. The detection of Ferritin consists of a novel method that combines the selectivity of antibodies with the electrochemical properties of Ferritin for high sensitivity detection. The sensor components are all 3D-printed and require a finger-prick sample for complete measurement of these clinically relevant iron biomarkers. Iron biomarkers. Comparative studies of results obtained by the new sensing device and the reference method in actual samples were performed to determine the device's capacity to detect iron parameters' concentrations. Correlation plots with a slope of ~ 1 and regression coefficient of higher than 0.82 were obtained for detection of blood iron levels, total iron-binding capacity, and percentage of transferrin saturation. This indicated that the new device is substantially equivalent to the reference method. With detection times of five minutes, fingerpick sample, and sensor cost less than 10 cents; the device shows excellent promise for point care testing of iron disorders.

Since the first use of the surface plasmon resonance (SPR) technology for biosensing more than two decades ago, SPR has become a powerful tool for characterizing and quantifying biomolecular interactions, especially in pharmaceutical industry for drug discovery. It has made great strides both in instrumentation developments and applications. One of the most significant developments is the SPR microscopy, which integrates the optical microscopy with SPR technology to enable many new studies such as direct measurements of small drug molecules interacting with GPCR proteins on cell membrane in their native state, and direct measurements of drug response over hundreds of cells with different phenotypes and growth stages to reveal their heterogeneity and other important functionalities. Here I will present some of the latest developments in SPR technology along with its applications in diverse fields.

The intrinsic electronic properties of biomolecules with their self-assembly capability has sparked a revolution in developing biomedical devices for both diagnosis and therapy. Living cells are associated with electrical characteristics due to the cell membrane transport processes and are thus responsive to and even generate electric fields and currents. Electrical properties of cells determine most of the cellular functions it has been shown that the electrical properties of cancer cells differ from normal proliferating cells. Electric fields may induce differential effects in normal and cancer cells thus providing a powerful electrotherapy option for the treatment of cancer with limited toxic chemicals and possible immunogenic responses in the host tissue.

Cell on a chip-based biosensor is valuable tool for monitoring cell behavior because they can provide information about the total physiological responses of cells to external stimuli. We successfully used cell impedance sensing system to monitor the real-time consequences on cellular viability under the electric field stimuli. We demonstrated that cancer cell proliferation can be modulated by externally applied alternating electric fields in the intermediate frequency range of 100 kHz - 200 kHz. Interestingly, we demonstrated that different types of cancer cells are affected by different optimal frequencies of these electric fields. We also observed a decrease in proliferation with the addition of HER2-gold nanoparticles (AuNps) to target the cancer cells and enhance the effects of the electric field towards the cells without affecting the non-cancerous cells. With the attached nanoparticles, the zeta potential of the SKOV3 and the MCF7 before and after incorporation of the HER2-AuNPs decreased compared to their non-cancerous counterparts. The decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells and define optimal parameters of electrotherapy for clinical and drug delivery applications.

Accumulation of excessive bilirubin induces hyperbilirubinemia and neonatal jaundice, and is a signal of adult liver diseases. Currently, the mainstream bilirubin detection is conducted in hospital pathological laboratories, which is expensive and time-consuming. To release the heavy burden of laboratory-based tests and to provide point-of-care testing and feedback to patients, recently some handheld optical devices have been developed for blood bilirubin measurement; however, they still require professional operation and uneconomical for home use. In this regard, a lower-cost and non-powered paper-based analytical device is developed for fast and point-of-care quantitative measurement of blood bilirubin levels in jaundiced neonates and hepatopathy adults. The design achieves plasma separation from whole blood and optical quantification of the chromogenic results by a specially designed smartphone APP. Especially, for jaundiced neonates, the APP not only calculates the bilirubin levels but also suggests the essential therapy if the detected bilirubin level versus the age of the baby is at risk. It is believed that this low-cost design provides a home-use solution for parents/patients to monitor the health conditions of newborn babies/themselves.

Abstract:

Bacterial cells are found everywhere and can be both beneficial or harmful to our diverse ecosystem, from plants to animals and humans. As such, studying them to better understand their interactions with the surrounding environment is important from both fundamental and applied perspectives. The existing analytical methods for studying bacterial cells require bulky readout equipment and complex sample preparation, are cell-destructive, or involve excessive sample preparation (1–3) which make real-time and in situ analysis hardly possible. In addition, probing bacterial growth has been traditionally done using colony counting, which is an end-point method and takes at least 24 hours for results.

In situ monitoring of bacterial phenotypes (e.g. motion, respiration, adhesion, virulence factors) and how they change in response to environmental perturbation will have a broad impact in food safety, microbial fuels, studying gut-brain interaction, and fighting antibiotic resistance. This talk presents our recent works on developing novel biosensing tools for in situ and non-destructive probing and monitoring of bacterial phenotypes – from electrochemical sensors for reagent-free monitoring of metabolic activity or measuring biofilm virulence factors, to impedimetric sensors to decipher the mechanism of action of new antibiotics or activation of osmoregulatory transporters (4–7), to machine learning-enabled dynamic laser speckle imaging for monitoring bacterial motion (8), and others.

References:

1. M. Cambronel, et al., Influence of Catecholamines (Epinephrine/Norepinephrine) on Biofilm Formation and Adhesion in Pathogenic and Probiotic Strains of Enterococcus faecalis. Front. Microbiol.11, 1–13 (2020).

2. V. M. Rekdal, E. N. Bess, J. E. Bisanz, P. J. Turnbaugh, E. P. Balskus, Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism. Science (80-. ).364 (2019).

3. D. Villageliú, M. Lyte, Dopamine production in Enterococcus faecium: A microbial endocrinology-based mechanism for the selection of probiotics based on neurochemical-producing potential. PLoS One13, 1–10 (2018).

4. D. Butler, N. Goel, L. Goodnight, S. Tadigadapa, A. Ebrahimi, Detection of bacterial metabolism in lag-phase using impedance spectroscopy of agar-integrated 3D microelectrodes. Biosens. Bioelectron.129, 269–276 (2019).

5. A. Bolotsky, et al., Organic redox-active crystalline layers for reagent-free electrochemical antibiotic susceptibility testing (ORACLE-AST). Biosens. Bioelectron.172, 112615 (2021).

6. A. W. Simonson, et al., Pathogen-specific antimicrobials engineered de novo through membrane-protein biomimicry. Nat. Biomed. Eng., https://doi.org/10.1038/s41551-020-00665-x (2021).

7. A. Ebrahimi, M. A. Alam, Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability. Proc. Natl. Acad. Sci. U. S. A.113 (2016).

8. K. Zhou, et al., Dynamic Laser Speckle Imaging Meets Machine Learning to Enable Rapid Antibacterial Susceptibility Testing (DyRAST). ACS Sensors (2020) https:/doi.org/10.1021/acssensors.0c01238.

We report the development of an electrochemical biosensor platform based on multiplex micro-/nano- electrode arrays toward cancer diagnosis based on rapid profiling of protease activities. Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and treatment monitoring of diseases such as cancers. This study demonstrates the fabrication and characterization of a nanoelectrode array platform based on embedded vertically aligned carbon nanofibers for electrochemical detection of protease activities and further development into an individually addressed 3x3 gold thin-film microelectrode array for rapid profiling of multiple protease activities.

Peptides with specific sequences of 4 to 8 amino acids are designed and synthesized as the substrates for selective proteolysis by the cognate proteases, which are attached to the electrode surface as the specific probes. The far end is covalently attached with a ferrocene (Fc) group as the redox moiety for electrochemical measurements. The quantity of the intact peptides on the electrode surface can be sensitively detected with an AC voltammetry method and presents as the peak current at the specific potential for Fc oxidation into ferrocenium (Fc⁺). As the peptide is cleaved by the cognate protease, the peak current decreases exponentially in the continuously repeated AC voltammetry measurements. The recorded kinetic proteolytic curve can be quantitatively described by a surface-based heterogeneous Michaelis-Menten model. The inverse of exponential decay time constant, 1/t, is found to represent the protease activity which equals to [E](k_cat/K_M), where [E] is the protease concentration and k_cat/K_M is the specificity constant defined by the kinetic proteolytic reaction constant k_cat and the Michaelis equilibrium constant K_M. We have systematically investigated the factors that affect the value of k_cat/K_M, including the peptide sequence, peptide length, temperature and buffer composition, and optimized the conditions for highly sensitive detection of protease activity of cathepsin B, a potential cancer biomarker.

Toward rapid profiling of multiple proteases, we have developed a multiplex electrochemical sensor chip based on a 3x3 gold microelectrode array. The nine individual gold microelectrodes are partially buried underneath the surrounding SiO₂ thin film, which show highly consistent cyclic voltammetric signals in gold surface cleaning experiments and detecting benchmark redox species in solution. Upon selectively functionalizing the individual gold microelectrodes with the specific ferrocene-labeled peptide probes, simultaneous detection of the proteolytic curves of the target proteases can be obtained over 9 channels by monitoring the decay of the AC voltammetry signal of the ferrocene-labeled peptide molecules. So far, the algorithm for fitting the kinetic proteolytic curves to accurately derive the activity of cathepsin B has been established. Simultaneous detection of the proteolysis of cathepsin B on the microelectrode array functionalized with three different hexapeptides has been demonstrated, showing the potential of this sensor platform for rapid detection of the activity profiles of multiple proteases.

Interestingly, the above-discussed electrochemical method detects the activity of the proteases, which reflects the true biological function and is fundamentally different from the concentration derived from commonly used affinity biosensors or assays such as enzyme-linked immunosorbent assay (ELISA). The direct comparison has shown that ELISA measurements will give the same results no matter the original proenzyme form (zymogen) in the sample is activated or not, while the electrochemically measured activity shows dramatic increase after being chemically activated. In addition, in the current practice, each protease is measured separately in its optimal buffer with the pH value varying from 5 to 9. It is not possible to measure them in a common buffer simultaneously. We have demonstrated that the electrochemical method can be applied for protease activity profiling in a buffer at pH = 7.4 that is compatible to the physiology conditions. This enables measuring the activity of multiple proteases in human serum, which is a critical step toward protease activity for cancer diagnosis.

The figure shows the optical images of (a) the whole 3x3 Au microelectrode array chip and (b) the active electrode area, and the schematic illustration of (c) the mechanism of proteolysis of the peptide probes, (d) the change of the AC voltammetry signal before and after proteolysis and (e) the kinetic proteolytic curves, i.e. the exponential peak current decay over time, at different protease concentration [E].

Figure 1

New biomarkers are always needed for the fast and early diagnosis of gastric cancer. Lately, maspin was used for diagnosis of gastric cancer. Most of the standard methods are only used for qualitative assay of maspin. Its quantification is very important for the correct diagnosis of gastric cancer. 2D disposable sensors as well as 3D microsensors based on graphene modified with nanoparticles were proposed for the fast screening tests – based on molecular recognition and quantification of maspin in biological samples such as whole blood, tumor tissue, saliva, and urine. High sensitivity and low limits of determinations were recorded for the assay of maspin. High reliability was obtained for the assay of maspin in biological samples.

The mechanism of fluorescence resonance energy transfer (FRET) was studied after inorganic nanomaterials have replaced organic dyes as energy donors and acceptors. Quantum dots and graphene oxide have been used for construction of FRET sensors. Next the fluorophores are incorporated to paper-based lateral flow assays toward point-of-care testing. The fluorescence sensors have sued for detection of heavy metal ions and protein biomarkers.

The development of highly sensitive biosensors has received increasing attention recently. Due to the unique physicochemical characteristics, using functional nanomaterials to enhance biosensor performance has made some major breakthroughs. However, sensitivity and selectivity of biosensors still need to be improved. Single atom catalysts (SACs) show unique advantages in terms of catalytic activity and selectivity to various catalytic reactions. Therefore, SAC-enabled signal amplification strategies have broad prospects in biosensors. By adjusting the metal-carrier interaction, the coordination environment and the geometric/electronic structure of the active sites, satisfactory sensitivity and selectivity have been demonstrated for SAC-based biosensors. In this presentation, first, I will discuss the structural advantages of SAC. Then, I will discuss the various design and synthesis strategies of SACs. Finally, I will discuss the catalytic mechanisms at the atomic scale, the enzyme-like properties of SACs and their applications in improving the sensitivity and selectivity of the biosensors.

References:

1. L. Jiao, H. Yan, Y. Wu, W. Gu, C. Zhu, D. Du, Y. Lin. When Nanozymes Meet Single-Atom Catalysis. Angew. Chem. Int. Ed. 2020,132, 2585-2596

2. C. Zhu, S. Fu, Q. Shi, D. Du, Y. Lin. Single-Atom Electrocatalysts. Angew. Chem. Int. Ed. 2017, 56, 13944-13960.

3. N. Cheng, J. Li, D. Liu, Y. Lin, D. Du. Single-Atom Nanozyme Based on Nanoengineered Fe-N-C Catalyst with Superior Peroxidase-Like Activity for Ultrasensitive Bioassays. Small, 2019, 1901485.

4. X. Niu, Q. Shi, W. Zhu, D. Liu, H. Tian, S. Fu, N. Cheng, S. Li, J. N Smith, D. Du, Y. Lin. Unprecedented Peroxidase-mimicking Activity of Single-atom Nanozyme with Atomically Dispersed Fe-Nx Moieties Hosted by MOF Derived Porous Carbon. Biosensors & Bioelectronics, 2019, 142, 111495.

5. L Jiao, W Xu, H Yan, Y Wu, C Liu, D Du, Y Lin, C Zhu. Fe-NC Single-Atom Nanozyme for the Intracellular Hydrogen Peroxide Detection. Anal. Chem., 2019, 91, 11994-11999

6. L. Jiao, W. Xu, Y. Wu, H. Yan, W. Gu, D. Du, Y Lin, C. Zhu, Single-atom Catalysts Boost Signal Amplification for Biosensing. Chemical Society Reviews 2021, 50, 750-765

7. Z Lyu, S Ding, N Zhang, Y Zhou, N Cheng, M Wang, M Xu, Z Feng, X Niu,D. Du, Y. Lin, Single-Atom Nanozyme Linked Immunosorbent Assay for Sensitive Detection of Aβ 1-40: A Biomarker of Alzheimer's Disease. Research, 4724505, 2020

Electrochemical, aptamer-based sensors (E-ABs) achieve selective, continuous, and real-time molecular measurements in vivo. Their selectivity and modularity originate from the use of nucleic acid aptamers – DNA oligos selected in vitro for their ability to bind specific molecular targets. To create E-ABs, redox-reporter-modified aptamers are attached to gold electrodes via self-assembly. These modified aptamers are designed to have two thermodynamic states: one unbound, slow electron transferring state in the absence of target, and one bound, fast electron transferring state in the presence of target. The two states exist in dynamic equilibrium (the aptamers reversibly bind to and dissociate from their targets at rates of milliseconds); thus, their fractional populations depend on target concentration. This sensor architecture readily allows continuous sensing in vivo via serial electrochemical interrogation. Unfortunately, E-AB signals degrade within hours in the complex environments found in the body. This is in part due to biofouling but also to progressive changes in the chemical interface of these sensors driven by kinetic and biological processes. In response, the Arroyo lab is investigating the different mechanisms driving E-AB signal decay in vivo. This presentation will discuss strategies we have explored to reengineer the E-AB interface to enable continuous, multiday E-AB sensing.

This oral presentation will describe of a new type of immunoassays (i.e., sink/float magnetic immunoassays) that could be performed in the field with a simple analytical protocol that does not include washing steps and does not need any analytical instrument (just a small permanent magnet). Sink/float magnetic immunoassays combine elements of traditional immunoassays, immunomagnetic separations, and density-based sink/float tests in a single analytical platform that can provide sensitive results with a mix and observe protocol. Sink/float magnetic immunoassays use a polymeric sphere (that is visible to bare eye) as the solid substrate of the immunoassay, detection antibodies labeled with magnetic nanoparticles, and a simple sink/float test to detect the analyte-mediated binding of magnetic nanoparticles on the polymeric sphere; if the sphere sinks, then the target analyte is present in the tested solution. In this talk the detection mechanism of sink/float magnetic immunoassays will be explained in detail. Examples of the use of sink/float magnetic immunoassays for the detection of proteins, disease-specific antibodies and bacteria will be also demonstrated.

Electrochemical, DNA-based sensors represent a powerful tool to selectively detect and quantify molecular targets in highly complex media such as whole blood in living animals. These sensors consist of a biointerface containing redox reporter-modified DNA molecules attached to the surface of (typically) gold electrodes via sulfur-gold bonds. They also employ short chain alkanethiols for the purpose of passivating the electrode surface, thus preventing undesired electrochemical reactions and achieving antifouling characteristics to, for example, prevent non-specific protein binding. Despite the huge success and promising applications of electrochemical, DNA-based sensors and the development of this platform for over 15 years, the DNA sensing architecture has been almost exclusively limited to the use of thiol-based monolayers on gold electrodes. This limits the performance of such sensors by the surface stability of alkanethiols, which are labile, and excludes the use of electrode materials other than gold, considerably reducing potential fields of application for DNA-based sensors. Motivated to expand the diversity of monolayer chemistries and electrode materials that can be used to fabricate DNA-based sensors, our group is currently exploring different approaches to chemically functionalize the surface of carbon-based electrodes. In this work, I will discuss the different chemical approaches we are pursuing in this regard and their performance in terms of monolayer packing, extent of surface passivation, and long-term stability in physiological buffers and biological fluids. I will also discuss how these chemistries compare with benchmark thiol-on-gold monolayers.

It is imperative to prevent from the spread of the SARS-CoV-2 virus to end the global pandemic. Rapid and field-deployable testing of acute infection is one of the effective ways to control the spread of the virus. The gold standard is polymerase chain reaction (PCR)-based nucleic acid testing, which takes time and requires lab personnel and equipment. Testing of viral antigens is simpler and faster, which can be used for rapid initial screening. This talk will show a fluorescent paper-based test strip for measuring COVID-19 viral antigens in saliva.

Atomic force microscopy (AFM) recognition imaging is a label-free technique which can identify specific molecules from compositionally complex samples while mapping the topography of the samples simultaneously in a liquid environment. It required a sensitive imaging mode. Typically, the so-called MAC mode is used, where the AFM tip is driven directly by a magnetic field which will reduce the movement of liquid surrounding the tip and increase signal to noise ratio. The AFM tips for MAC mode need to have a ferromagnetic coating like nickel in order to be driven by a magnetic field. Commercial MAC mode probes are typically silicon nitride or quartz. We developed a protocol to coat sharp commercial silicon probes with nickel, making them reliable MAC mode tips. We applied our new probes to samples prepared from the supernatant fraction of a nuclear extract of EPC2 (non-cancerous) and CPD (cancerous) esophageal cell lines. Salt fractionation is an efficient method for the extraction of intact chromatin fragments from cell nuclei, but the supernatant fraction was not studied in previous work. With anti-H3 antibody modified MAC mode tips, we identified histones H3 in samples of both cell lines, indicating that this fraction contains chromatin. We proved the specificity by blocking with a peptide mimicking the H3 binding site for our antibody. Further, we demonstrated the dependence of the recognition signals on the oscillation amplitude of the probe, indicating the robustness of our recognition signal. We also analyzed the nuclear extracts for the presence of SMC2, a structural maintenance protein for chromosomes. We applied AFM recognition imaging with anti-SMC2 antibody modified probes to the prepared chromatin samples and identified SMC2 proteins in samples of both cell lines. Western blots confirmed the presence of SMC2 proteins in the samples. Overall, AFM recognition imaging provides a robust way to analyze the structure and molecular components of nuclear extracts from human cells, providing insights into the link between chromatin structure and cell phenotypes.

Rapid identification of different plastics for recycling is very important in overcoming the plastic waste challenge our society is facing today. An ideal plastic detector should be able to discern different plastics at the molecular level in a standoff fashion where the detector does not come in contact with the target plastic. We have demonstrated a standoff technique for the rapid identification of plastics based on their mid-IR absorption spectra. Since mid-IR region is free of overtones, this region is known as molecular finger print regime This technique is based on a variation of photoacoustic spectroscopy (PAS), where the light from a tunable infrared source, a quantum cascade laser (QCL), is scattered/reflected off the target plastic object. The returning scattered light is collected and made to create acoustic waves on a microfabricated cantilever sensor. Our experimental results show that the cantilever response, resonance frequency, amplitude, and bending, changes with infrared absorption characteristics of the target. A plot of cantilever response as a function of illuminating wavelength mimic the infrared absorption peaks of the plastics material producing a mechanical spectrum. The generated spectrum obtained for different plastics is then compared with that conventional spectrum for polymer identification.

Electrochemistry single-entity methods are of fundamental importance and show promise for ultrasensitive chemical and biological sensing applications. Recently, we have demonstrated that various nanoparticles can be detected individually based on the open circuit potential (OCP) changes induced by their collision events on a floating carbon nanoelectrode. Unlike the widely used amperometry approach, the potentiometric method provides the label-free detection of individual nanoscale entities without redox mediators in solution. However, the carbon nanoelectrode is chemically inert and limited in surface functionalization. Here, I will present our recent progress of using surface functionalized gold nanoelectrode (GNE) to replace the carbon nanoelectrode for potentiometric single-entity studies. By utilizing the advantage of mixed surface functionalization of GNE with the Raman reporter molecule and polyethylene glycol or zwitterionic molecules, we formed near 'stealth' GNE surface and demonstrated that the non-specific adsorptions of nanoparticles to the GNE surface can be minimized, allowing continuous hit-n-run events for over 30 minutes. The surface functionalized GNE also enabled surface enhanced Raman spectroscopy (SERS) measurements. By using simultaneous time-resolved OCP and SERS measurements, both the OCP and SERS signals induced by the hit-n-run type of collision events can be better understood.

Microchannel cantilevers, one of promising physical microelectromechanical systems (MEMS) devices that have embedded microchannels, have been widely used in gravimetric sensing applications of liquids and particles introduced into the channel or dispensing/patterning applications with liquid phase materials when a dispensing nozzle is additionally configured near their free ends. Although there are numerous potential applications at elevated temperatures such as material synthesis, calorimetric measurements, and phase change mediated manipulation or control, to name a few, microchannel cantilevers are mostly used at or near room temperature mainly due to the absence of integrated active heating elements. Only for a few studies, an off-chip heater has been employed to slightly increase temperature of amicrochannel cantilever as well as its packaging/mounting structures thus both speed and range of temperature modulation were significantly limited. As an alternative, photothermal heating relying on a focused laser was recently employed but laser alignment was not user-friendly and precise quantitative heating was not straightforward due to the uncertain absorption of the incident laser. Towards various applications under fast and quantitative temperature modulation, we have developed fluidic resonators with integrated heating capability.

First, we have used stainless steel tubes for proof-of-concept. By simply clamping a straight stainless steel tube on CNC-machined jigs along with piezoelectric chips, a doubly clamped tube resonator with piezoelectric actuation and detection was completed. After fabrication, basic resonant characteristics were investigated with various liquid samples at fundamental and higher flexural bending modes at room temperature. Then, the stainless-steel tube resonator under Joule heating was employed for boiling point measurements of water. The onset of the boiling inducing liquid-to-vapor transition could be detected by monitoring the resonance frequency with the aid of the phase-locked loop. After the feasibility study with the simple stainless-steel tube resonator, we have switched to microfabricated channel resonators with an integrated heater for exquisite sensitivity. Heater-integrated microchannel cantilevers with or without a dispensing nozzle were batch-fabricated via sacrificial process, ion implantation, and other typical microfabrication processes. Then, fabricated heater-integrated microchannel cantilevers were thoroughly calibrated and characterized in a variety of coupled physical domains. Upon pulsed operations, electrothermomechanical time constants extracted from the transient resonance frequency provided a new measurement modality for thermophysical properties of the fluid contained in the microchannel. When the reference solution, glycerol-water binary mixture, was pulsed heated above its boiling point, atomized droplets could be spray-ejected out of the integrated nozzle.

The unravelling of a double stranded DNA into separate strands is crucial to DNA transcription, the integral basis of life, and thus remains a well explored bio-physics problem. In a double stranded DNA, the nucleotides are stacked and held together by hydrogen bonds in paired A-T and G-C sequence. With increasing temperature, the hydrogen bonds break and the strands separate base by base over a narrow temperature range. Melting of DNA samples causes measurable changes in their physical properties such as viscosity, buoyant density, UV260 nm absorption, optical rotation, etc. At molecular level unravelling of a dsDNA into individual strands when heated, occurs through an entropy-driven conformational transition. The unravelling typically proceeds through conformations that have locally denatured single stranded bubbles bound in-between double stranded helical segments. Investigations carried out using pico-liter solutions of micro molar concentrations of DNA molecules confined in a resonating microfluidic cantilever reveal interesting mechanical characteristics of denaturation process. As the strands unravel, the nanomechanical dissipation response sensitively follows the base-pair binding-unbinding energy landscape, revealing the characteristics of a two-stage transition curve. The fluctuation mediated transition pathway suggests an intermediate nucleation stage to the two-principal DNA denaturation pathways: the molecular zipper transition and the bubble state cooperativity. In fact, for both melting and pre-melting states, the cantilever response provides a framework to calculate the specific heat capacity and the storage and loss modulus the cantilever-DNA solution system, thereby establishing a platform for quantifying thermomechanical behavior of confined DNA molecules. Extremely small sample volume a hollow channel cantilever together with its high thermal sensitivity due to bi-material effect may open further avenues for studying fluctuation kinetics in DNA transcription and other complex bio-molecular phenomena.

Temperature, being one of the fundamental parameters in the physical characterization of a material, temperature sensors with high spatial, temporal, and thermal resolution are on great demand in the field of fundamental research, protein blotting, DNA melting, drug development, biochemistry, biomedical devices, and analytical systems. Sensitive real time measurements unveil the dynamics of the analytes in a lab-on-a-chip platform where miniaturized volumes of samples are characterized when they are either rare or volume-limited. Mechanical resonators with microscale fluidic channel integrated are widely adopted as micro/nanoscale sensing platform where the resonance frequency shift of the resonators can detect minute fluctuation of physical, chemical, and biological stimulus within medium of fluidic channel at the picogram level. Even though microfluidic channels are successfully implemented for measuring thermal properties, integration with complicated QCL and static response of resonators based on static deflection delivered the results that are prone to be affected by mechanical drift and higher noise level of the photodetector. Hence the need of a simpler experimental model and dynamic response is urged. This paper reports analysis of thermal characteristics of liquid analytes using photothermal heating effect of high-speed microfluidic cantilever. While a channel-integrated resonator is modulated by a diode laser, real-time tracking of resonance frequency shift of the microfluidic resonator allows estimation of thermo-physical properties of liquid samples where the local laser irradiation results in photothermal heating of the resonator. The heating results expansion of the liquid inside the channel induces thermal stress on the walls of the channel. This thermal stress contributes to rise of the resonance frequency of the microfluidic resonator and, therefore, the frequency shift is linearly dependent of the volumetric coefficient expansion of the liquid. Also, the heating time constant is inversely proportional to the thermal diffusivity of the liquid. In addition, a higher flexural mode displays more than 3 times improved sensitivity for thermal characterization of liquids via photothermal heating.

Continuous monitoring of soil health is important in precision agriculture. Microbial activities can be an indicator for soil health, where the metabolism of the soil microbiota associated with plants plays a crucial role in plant development. These plant-associated microbiomes can also influence other traits such as disease resistance, growth, flowering, and abiotic stress tolerance. Activities of these microbial colonies affect all aspects of plant life because of their symbiotic relationship. Therefore, a thriving microbiota is directly related to soil health. By detecting and analyzing the variations of emitted volatile metabolites, it is possible to monitor the activities of the microbiota. These data can provide a deeper understanding of the relationship between activities of microbial communities and plant health. However, presently available low-cost, in situ sensors used in agriculture only detect a limited number of physical parameters such as moisture, pH, electrical conductivity, temperature, etc. Here we demonstrate microcantilever based photothermal spectroscopic sensors for detecting vapor phase analytes related to microbial activities such as CO₂, methane, etc. Photothermal spectroscopy combines the temperature sensitivity of a bi-material cantilever with the selectivity of mid-infrared spectroscopy. Nanomechanical photothermal spectroscopy has sensitivity in the ppb range, fast response time, and requires no chemical coating for selectivity. Since the mid-infrared spectroscopy is free from overtones, it is extremely selective even in the presence of interfering compounds. Multiple IR peaks are monitored and analyzed using pattern recognition techniques for uniquely identifying the analyte molecules in vapor phase in the presence of interfering chemical compounds.

In this talk, I will present our ongoing efforts on developing sensitive gamma rays dosimeter using two sensing techniques namely, metal oxide-coated microcantilever and Quartz tuning forks. The primary objective of these studies is to exploit the appealing characteristics (i.e. low in cost, sensitive and rapid) of the abovementioned techniques in developing gamma radiation dosimeters. In this first study, we have investigated the potential of exploiting TiO₂ thin films as a sensing layer on Silicon microcantilevers for detection of gamma-ray radiations. Silicon microcantilever and wafers are both used for this purpose. All samples were exposed to gamma rays produced by ⁶⁰Co with different doses ranging between 0 kGy and 40 kGy. The optical properties of silicon-coated by TiO₂ at 200°C ALD grown and 100 nm thickness were studied before and after irradiation using X-ray diffraction (XRD) spectrum, scanning electron microscopy (SEM) and spectroscopic ellipsometry (SE) and Atomic force microscopy (AFM). The optical studies by X-ray diffraction showed that change of our film from the nanocrystalline mixture of rutile and anatase phases to anatase by irradiation, this change appeared as (Δf) in AFM whereas the response of sensors to the doses between 0KGy and 20KGy was very sensitive and linear. The results of the spectroscopic ellipsometry measurements showed the same linearity change in the thickness, roughness and optical constants with irradiation doses were fitted using the Cauchy model. In the second study, gold coated quartz tuning forks were exposed to gamma radiations and we found that QTFs underwent a clear change in response to increasing gamma dosage. Our results show that both techniques are excellent candidates for dosimetry applications.

Ultra-thin MEMS Thermoelectric Chip and Its Applications

Zhiyu Hu

Institute NanoMicroEnergy, Department of Micro/Nano-Electronics

Shanghai Jiao Tong University, Shanghai, China, 200240

Email: zhiyuhu@sjtu.edu.cn

Abstract:

For a long time, mankind has been seeking inexhaustible energy in nature. There is an urgent need to obtain new types of sustainable, environmentally friendly and green energy to reduce our dependence on petrochemical energy. The thermoelectric generator (TEG) can directly convert heat into electrical energy through the Seebeck effect, thereby obtaining electrical energy, which makes it a promising environmentally friendly energy conversion method. TEG has many special advantages, such as no moving parts, no pollution, no noise, no mechanical vibration, etc. At present, all TEGs on the market is made with traditional method which yields high cost, low efficiency, not suitable for massive and economic production.

Thermoelectric generator (TEG) can directly convert heat into electrical energy. Due to its special advantages, such as no pollution, no noise, no mechanical vibration, etc., TEG has been widely used in vehicles, wearable devices, solar energy systems, and industrial waste heat recovery systems. In order to promote the application of thermoelectric devices, it is necessary to improve the performance of thermoelectric materials through nanostructures. Generally, thermal resources are concentrated on increasing the temperature at the hot end of the TEG. In this article, the heat in the environment is pumped into the external space through radiant cooling (RC), thereby generating the cold end of the TEG and a continuous self-powered system was obtained.

Taking advantages of micro/nano-fabrication and the scale effects of heat theory, we are able to design and build a thermoelectric chip in a large array and maintain a stable temperature difference between sub-micrometer-thick of a thermoelectric unit on a silicon wafer. The submicron-thick TEG containing more than 46,000 thermoelectric modules in series was fabricated on SiO₂/Si. The combined Seebeck coefficients of Sb₂Te₃ and Bi₂Te₃ were adopted the average value which were evaluated to be 250 μV/K. The details of the fabrication method were proposed first and demonstrated in our previous work.

Nanostructured multilayer thermoelectric films have been developed to improve thermoelectric efficiency. The effects of interface microstructure on the cross-plane thermal conductivities of the multilayer thin films have been extensively examined and the thermal transfer mechanism has been explored. Experimental results indicate that ultrathin thermoelectric device works stable and reliable, and is suitable for fabrication of thinner and higher integrality devices in mass production.

We have demonstrated a novel energy utilization that MEMS-based thermoelectric chip can produce electricity by radiative cooling (RC) in day and night. Thermal emitter composed evaporated Ag layer and 12 alternating SiO₂ and Si₃N₄ layer periods deposited on a silicon by plasma enhanced chemical vapor deposition (PECVD). The absorptivity of the thermal emitter is 80.8%. Here, the emission is caused by phonon-polaron excitation in the Si₃N₄ layer. SiO₂ was chosen to fully reduce the adverse radiation loss because it has an absorption peak close to 9 μm in the atmospheric window due to its phonon-polaron resonance. The silver-plated layer can enhance the electromagnetic wave reflection without resonance.

In addition, the MOST material that collected solar energy in Gothenburg，Sweden has been delivered to Shanghai, China by express mail, and successfully generated electricity on the MEMS TE chip a few months later.

MEMS-based thermoelectric chips can use ultra-low thermal energy to directly generate electricity with a very small temperature difference. In future, when such advanced energy technology is wildly available and at very low costs, we then will obtain truly sustainable, completely environmentally friendly clean energy, and greatly reducing our dependence on petrochemical energy. MEMS-based thermoelectric chip can also be used as an ultra-sensitive temperature sensor, several testing examples will be introduced and with further discussions.

There is an ever increasing demand for nanosensors with enhanced characteristics such as miniature size, low power consumption, higher sensitivity and selectivity, ease of manufacturing, decreased cost, simultaneous detection of multi-analytes, etc. As a result, the area of nanoscale sensing remains an area of rich opportunity to develop new ideas for immediate applications ranging from health and environmental monitoring to military and homeland defense. Therefore, these sensor technologies have great potential to revolutionize science and to influence major economic, agricultural, environmental, social, and health issues. Recent advances in our understanding of 1-D nanomaterials are paving the way for developing novel platforms for sensors and devices based on multi-physics, multi-modal approaches. Optically induced surface state (or defect states) population-depopulation in nanomaterials with extremely small thermal mass changes its electrical resonance and serves as a bridge to nano-world for developing next-generation, miniature spectrometers and molecular sensors with unprecedented selectivity and sensitivity. Modulating the defect state population in wide bandgap materials using optically-induced thermal pathways offers a new approach for designing advanced sensors and devices based on nanowire resonators with extremely low thermal mass. Resonant excitation of molecules adsorbed on these nanowires using tunable mid-infrared radiation heats the nanowires during the de-excitation process affecting the surface state population which can be sensitively monitored as changes in the dissipation at its electrical resonance frequency as a function of illumination wavelength and mimics the infrared absorption spectra of the physisorbed molecules offering excellent molecular selectivity. Since the mid-IR spectra are free of overtones (molecular fingerprint regime) and the spectrum of individual molecular species are linearly independent, this approach offers a new paradigm for chemical vapor sensing with high sensitivity and selectivity. Further, monitoring the dissipation variations at resonance as a function of temperature can provide information on thermally induced desorption and polarization or depolarization of adsorbed chemical species. The temperature response of the nanowire at resonance can be used to discriminate different vapors based on differential calorimetry due to a difference in the dipole moments.

In surgical procedures, it is vital to keep the anesthetic agent concentration appropriate as a slight variation can cause either an overdose or awareness in the surgery. Most of the anesthesia apparatuses in current practice do not have a system for monitoring the anesthetic agent's concentration; therefore, a convenient anesthetic monitor to continuously measure the concentration is highly expected. The widely used inhalational anesthesia, isoflurane (1-chloro-2,2,2-trifluoromethyl difluoromethyl ether) is colorless, volatile, and nonflammable. Therefore to maintain the anasthesia, complex machines equipped with vaporizers and infrared (IR) sensors are used to deliver the required dose of isoflurane. However, existing IR analyzers for the isoflurane have to be operated with highly precise optical alignments, which find limited deployment for critical care in low resource environments due to size, cost, and complexity. Results from controlled or uncontrolled administration of the anesthetic bring serious negative health outcomes. There is an urgent unmet clinical need to cost-effectively ensure the patient's safety and the personnel exposed in the operating room. We are developing a transcutaneous isoflurane biosensor device that can accurately measure volatile anesthetic gas concentration in blood at a much lower cost/unit to address this unmet need. Our approach consists of using micro-fuel cell Transcutaneous Anesthesia Monitoring Systems (TAMS). Micro-fuel cell TAMS is the simplest form of an electrochemical device composed of a Proton Exchange Membrane (PEM) sandwiched between two metal electrodes. In micro-fuel cell TAMS sensing application, an electrical current is directly generated to the isoflurane concentration. Comparing to the existing IR analyzers, micro-fuel cell TIMS is much smaller but more robust. It can be easily deployed for applications under various environmental applications. This easy-to-use, low-cost anesthetic sensing system will provide the patients' safety during surgical procedures, even in low-resource settings in the developing world. We propose the micro-fuel cell TAMS for cost-effectively and continuously accurate anesthesia detection.