Editors’ Choice—Challenges and Opportunities for Developing Electrochemical Biosensors with Commercialization Potential in the Point-of-Care Diagnostics Market

There is a plethora of electrochemical biosensors developed for ultrasensitive detection of clinically relevant biomarkers. However, many of these systems lose their performance in heterogeneous clinical samples and are too complex to be operated by end users at the point-of-care (POC), prohibiting their commercial success. Integration of biosensors with sample processing technology addresses both of these challenges; however, it adds to the manufacturing complexity and the overall cost of these systems. Herein, we review the different components of a biosensor and avenues for creating fully integrated systems. In the context of integration, we focus on discussing the trade-offs between sensing performance, cost, and scalable manufacturing to guide the readers toward designing new electrochemical biosensors with commercialization potential.

2][3] Biosensors have been designed for detecting a wide range of disease biomarkers; [4][5][6][7][8] however, the clinical translation and commercialization of many researched technologies are still pending due to the challenges related to scalable manufacturing, patient-to-patient sample variability, and the large amount of capital needed for pushing these technologies through the manufacturing, quality control, and regulatory approval pipelines.
0][11][12] The amperometric glucose monitor developed by Leland Clark Jr in 1962 13 evolved into three landmark technologies-enzyme electrode glucose test, glucose test strip, and the continuous glucose monitor-commercialized in 1970, 14 1987, 15 and 2017, 16 which have inspired the electrochemical biosensors research for over seventy years (Fig. 1).Other commercial success stories in electrochemical biosensing include the portable i-STAT analyzers (currently manufactured by Abbott POC), first commercialized for detecting electrolytes in 1992 17 and advanced into a digitalized user-friendly format in 2016. 18The i-STAT has been customized to detect a wide range of analytes such as blood gases, ions, electrolytes, as well as biomarkers for cardiac, blood coagulation, and endocrinology management. 18Even though the i-STAT uses a portable analyzer, its cost and complexity limit its use to medical facilities and prohibit its extension to home healthcare. 19he conventional chemistry of glucose sensing is dependent on the use of specific enzymes for selectively generating redox products in response to the target.As such, this approach is not directly translatable to other non-enzymatic biomarkers such as nucleic acids, 20 proteins, 21 extracellular vehicles, 22 and cells, 23 which are all important for disease prognosis and diagnosis.Over the past thirty years, biorecognition elements beyond enzymes such as nucleic acids, 2,24 antibodies, 25 antigens, 26,27 and peptides [28][29][30] have been successfully integrated into electrochemical platforms for sensing the aforementioned biomarkers.However, the translation of such assays from the research laboratory to the market relies on the scalable manufacturing of stable and reproducible biorecognition elements, ensuring extended shelf life and sustained performance in real patient samples.Many of such technologies require rigorous pre-processing to allow them to tackle the variability of clinical samples, which further adds to the cost and complexity of the biosensing technology and presents new commercialization challenges.In this review, we will shed light on the technical challenges for the commercial translation of electrochemical biosensors, intended for in vitro diagnostics, and present suggested approaches that can be used to overcome them.2][33][34] Additionally, challenges related to nonspecific binding in biosensors are of critical importance; however, these have been reviewed thoroughly elsewhere. 35,36ectrochemical Chips for Biosensing An electrochemical biosensor is composed of three major components: a biorecognition element for selective identification of the target, a conductive transducer on which biorecognition interactions are measured, and an electrical reader that converts biorecognition events into an electrical signal. 37The analyte interaction with the biorecognition element causes a measurable change in electrical properties of the electrochemical transducer (Fig. 2).
Electrochemical transducers are commonly a three-electrode system, which includes working, counter, and reference electrodes.Scalable manufacturing is essential for translating technologies from the laboratory to the market and is often associated with reduced costs and improved reproducibility. 37,38Electrodes fabricated using screen printing, printed circuit board, laser-ablation, and microfabrication are commercially scalable and will be discussed in this section.These methods offer trade-offs in terms of material purity and pattern resolution versus cost, 39 and should be selected based on specific assay requirements (Table I). 40,41reen-printed electrochemical chips.-Screen-printedelectrodes (SPEs) were first manufactured in the 1990's and have been heavily employed in the fields of electrochemistry, 42 microelectronics, 43 and energy storage, 44 with recent efforts focused on their application for POC diagnostics.45 The market for the production of SPEs and printed electronics is competitive and is set to expand rapidly, from an estimated market value of USD 41.2 billion to USD 74 billion by 2030.41 The blood glucose test strip, ExacTech by Genetics International (MediSense), was the first commercial technology that used screen printing technology.46 These SPEs are fabricated by pushing conductive inks through a formulated screen mesh that is placed on a substrate, 47 followed by thermal curing for up to an hour (Fig. 3a).42,45 The type and viscosity of the conductive material impact the cost and analytical performance of SPEs.45 The use of conductive inks in SPEs has mostly relied on carbon-based materials, including carbon ink, 48 graphene quantum dots, 49 graphene, 50 and carbon nanotubes, 51 given their wide voltage stability and minimal background current compared to other materials.41 Nevertheless, the ease of onestep thiol assembly on gold and platinum materials offers facile immobilization for thiolated receptors.55 However, its scalable application in SPEs is limited because of its laborious and expensive synthesis. 50This can be overcome by using varying ratios of carbon ink to graphene for mass production of SPEs and sensitivity enhancement, and has already shown promising results for the electroanalytical detection of small molecules like glucose, 56 dopamine, 50 uric acid, 50 and serotonin.57 For instance, at a 1:9 graphene to carbon ink ratio, the limit-of-detection (LOD) for dopamine and uric acid detection was significantly improved by 9.6 and 3.8 folds respectively, compared to the case when only carbon ink was used. 50Another crucial factor impacting the quality and scalability of SPEs is the type of substrate housing the conductive ink patterns.Ceramic substrates are commonly used in non-flexible SPEs; 58 whereas, polyimide, 59 polyethylene terephthalate, 60 and paper 61 are used as flexible substrates. Poyimide has a high thermal resistance of up to 400°C and does not require pre-treatment procedures (e.g., oxygen plasma treatment).45 Ceramics also offer high thermal and mechanical stability; however, they require intermediate barrier layers to help with deposition of conductive films.62,63 Paper offers a cost-effective and eco-friendly choice for the fabrication of SPEs; however it suffers from sample leakage, affecting the reproducibility of sensing platforms.64 Common filter and office papers have been used to develop biosensing platforms, a recent example being of an enzymatic-inhibition biosensor for the detection of paraoxon nerve agent stimulant.61 The leakage of the target analyte (i.e., paraoxon) and reagents can be addressed by encasing the test area with melted wax.The authors reported a low LOD of 2 parts per billion of paraoxon.In addition to ink material and substrate, factors relating to ink rheology and viscosity (determined by solvent) 41 and their resultant impact on printing resolution need to be considered for the scalable production of SPEs for biosensing.41 Printed circuit board electrochemical chips.-Printed Cicuit Board (PCB) technology, integrating various electronic components into a single board, 65 was introduced in early to mid-20th century.66 Major improvements were made with the development of throughhole technology in the 1950's and surface-mount technology in the 1980's, leading to miniaturization and cost reduction of Printed Circuit Boards (PCBs).67 Conventionally, PCB design starts by transferring a patterned mask corresponding to the electrode design onto a copper clad board containing a photoresist film followed by ultraviolet (UV) light treatment in a cleanroom.Several etching processes are then repeated to obtain the designed copper traces.68 An epoxy solder mask is then used to passivate the circuit lines to protect the copper layer from oxidation, which is finally followed by the baking of the PCBs to cure the solder mask (Fig. 3b).65,69 Conventionally, hot air solder level (HASL) was used to submerge the boards in a tin/lead mix, followed by the removal of excess soldering material with hot air prior to baking. 70However, the toxicity of lead and uneven coatings of HASL has raised concerns with this method.
Surface finishes relying on electroless nickel immersion gold (ENIG), immersion silver, electroless nickel electroless palladium  immersion gold (ENEPIG), and hard gold electroplating methods have been used for creating electrodes suitable for electrochemical measurements. 65,71ENIG uses a nickel layer on copper prior to gold deposition, while ENEPIG uses an intermediate palladium layer between nickel and gold to prevent long-term corrosion. 65,72Hard gold plating offers a thicker layer of gold compared to ENIG to protect electrochemical chips from sliding wear, but ENIG plating offers more resistance to corrosion. 72,73The thick layer of hard gold in high-wear areas of PCBs makes hard gold plating expensive. 67herefore, the most commonly used method for fabricating electrochemical chips is ENIG. 65,72,73Immersion silver is directly electroplated from the electrolyte solution onto the copper layer, and lacks a nickel layer, making it weaker than immersion gold and requiring more care for long-term storage. 68ndustrial plating processes that use ENIG, ENEPIG, and hard gold introduce organic and inorganic impurities such as watersoluble and rosin-based flux residues, 74 copper traces, 75 and in rare cases, nickel 76 on electrodes, which could interfere with functionalization and consequently, the electroanalytical performance of biosensors manufactured using PCB technology. 40It is crucial to reduce impurities introduced during plating by employing wet pretreatment or plasma cleaning techniques. 77Several strategies for PCB pre-treatment have been explored to achieve the desirable sensitivity.A pre-treatment protocol involving sonication in acetone, ethanol, and water, followed by a thirty-minute sonication in a NH 4 OH/H 2 O 2 solution demonstrates improved glucose sensitivity compared to an untreated PCB. 78An increase of current density from 4.6 μA mm −2 to 18.4 μA mm −2 , 4.6 μA mm −2 to 16.6 μA mm −2 , and 4.6 μA mm −2 to 19.3 μA mm −2 was achieved for plasma treatment, KOH/H 2 O 2 , and NH 4 OH/H 2 O 2 , respectively while detecting procalcitonin and E. coli DNA. 77The observed decrease in copper reduction peaks, as highlighted by the authors, indicates that wet processing is notably more effective than just plasma treatment.
Because of its scalable and inexpensive production cost, PCB electrodes are becoming key alternatives to SPEs and have been employed for POC detection of various target analytes including infectious agents, 72,79 disease biomarkers, 65 and small molecules. 80,81Nevertheless, the oxidation of copper in the tracks of PCB electrodes introduces a challenge for electrochemical biosensors as it interferes with their analytical performance, hindering their wider acceptance. 68,80,81ser-ablated electrochemical chips.-Laser-ablationuses highly focused laser systems (such as CO 2 ) to engrave the exposed part of a substrate coated with or containing conductive carbon-rich precursors to obtain the desired electrode patterns (Fig. 3c).Laserablation is commonly employed on synthetic polymers such as polyimide, 82 polysulfone, 83 and polyetherimide, 84 with recent studies aiming to make hybrid nanoparticle and polymers with laserinduced graphene (LIG).85,86 Commercially available polyimide tapes have been used to develop inexpensive and flexible hierarchical and hydrophilic graphitic laser-ablated carbon electrodes for urea detection with a LOD of 10 −4 M, which is a hundred times lower than the average concentration in blood serum of a healthy adult.87 LIG is commonly produced by laser-ablation on Kapton tape, which allows the photothermal reduction of polyimide into a conductive graphene structure.However, the inert nature of polyimide foil substrates makes it difficult to utilize them for microfluidic applications, creating adhesion issues.This challenge can be overcome by transferring the LIG structure to a different substrate using a hot-pressing method.82 The analytical performance of electrochemical biosensors created through laser-ablation depends on the power and fluence used during electrode fabrication.88 The high throughput 89 and single-step nature of laser-ablation make it suitable for large scale manufacturing.Microfabricated electrochemical chips.-Microfabricatedchips contain thin layers of conductive films that are commonly deposited using physical vapour deposition and patterned using photolithography and etching (Fig. 3d).90 Microfabricated chips are used in the commercial Abbot Freestyle Libre system, and consist of gold working electrodes with a 40-120 nm thickness.91 Miniaturization of the electrode geometry, enabled by microfabrication, 92 can be used to enhance mass transport, decrease ohmic drop, and increase the signal-to-noise ratio of electrochemical systems.93 The small size of microfabricated electrodes also makes them suitable for integration with microfluidic devices.
The combination of microfabrication with bottom-up fabrication methods such as electroless 94 or electrodeposition 95 is commonly used to increase the surface area and tune the micro/nanostructure of microfabricated electrodes.Previous studies investigated the impact of supporting electrolyte concentration, applied voltage, and deposition time on the micro/nanostructure of electrodeposited electrodes created on microfabricated seed layers.It was found that the degree of nanostructuring directly affects the time it takes to reach the maximum signal in a DNA biosensor. 96Similarly, we have used electrochemical deposition to fabricate nano-sized gold patterns on gold electrodes for E. coli detection in unprocessed urine or prostate specific antigen in unprocessed plasma. 95,97The nanostructured gold electrodes increased the surface area of the microfabricated chips, enabling a hundred-fold enhancement in LOD compared to planar electrodes for E. coli detection. 95,98ven though microfabrication is adapted by the semiconductor industry for high throughput manufacturing of electronic chips at a low cost; the high cost of vacuum-based equipment remains a barrier to prototype development and low-volume fabrication that is needed at early stages of biosensing device commercialization.Modern maskless photolithography techniques using electron beam 99 and scanning probe 100 are capable of producing high-resolution patterning well below 100 nm, but are low-throughput and are expensive for commercial production.

Biorecognition Elements and Sensor Functionalization
Given the vital role of biorecognition elements in identifying target analytes and translating their presence or concentration into detectable signals, 37 their scalable manufacturing for electrode functionalization is crucial for biosensor commercialization.Three classes of biorecognition elements are extensively used in electrochemical biosensors, including proteins, nucleic acids, and molecularly imprinted polymers (MIPs).Other biorecognition elements such as ion-selective membranes are also used in biosensors; however, considering our focus on affinity-based or biocatalytic biosensors, we will not discuss these herein and refer the readers to other reviews on this topic. 108,109oteins.-Assaysusing antibodies and receptors such as lectins rely on bio-affinity, while assays using enzymes rely on bio-catalysis to catalyze redox chemical reactions. 2Antibodies are the most common bio-affinity recognition probes, 2 and have been shown as powerful tools for the detection of various targets including small molecules, 110 proteins, 111 and infectious agents. 112,113Antibody production begins with injecting antigens into laboratory or farm animals (e.g., mice, rats, or rabbits) and recovering antigen-specific antibodies in serum in ten to fourteen days. 1146][117] The production of mAbs using the hybridoma method is more time-consuming and costly compared to pAbs; 118 however, they have the advantage of being highly specific towards a single epitope on the target antigen. 114Commercially available mAbs cost $100-2000 mg −1 , whereas, pAbs cost $100-400 mg −1 . 119Researchers aim to reduce antibody cost by enhancing the yield of antibody production in bioreactors.This is achieved by optimizing mammalian cell culture conditions, focusing on waste removal, pH balance, and nutrient supply. 120nzymes are biocatalytic biorecognition elements, which detect specific targets by converting bioanalytes into measurable products. 121Enzymes are commercially produced from a wide range of biological sources including microorganisms (e.g., bacteria, yeast, fungi), plants, and animals; 121,122 however, more than 80% of enzymes are manufactured from microbial sources due to their lower cost of production. 123Enzymes cost $200-2,000 kg −1 for bulk (>1000 tonnes per year) to $10,000-100,000 kg −1 for smaller market segments (<100 tonnes per year). 1246][127][128][129] They are selected for binding specific targets by screening large random peptide libraries generated using in vitro display techniques, including phage display, mRNA display, ribosome display, bacteria display, and yeast display.This step is followed by characterization, where the kinetics and thermodynamics of the peptide-target are assessed and the peptide is synthesized using solution-based or solidphase synthesis. 130There is a bottleneck for selecting peptides for small molecule targets due to the need for modification of the peptide structure for improved immobilization. 131Reducing the amino acid sequence length of natural receptors such as enzymes, transmembrane proteins, and antigens for peptides may help to overcome this bottleneck in selecting peptides for small molecules. 132,133Detailed use of peptides as biorecognition elements for biosensing is reviewed elsewhere. 131,132,134here are several strategies employed to immobilize antibodies and enzymes on the surface of electrodes including crosslinker-mediated, site-directed capture, and physical adsorption/passivation.Crosslinkermediated immobilization involves using crosslinkers, a class of chemicals with a reactive centre at their termini, which can bind two functionalities.Commonly used crosslinkers for antibody immobilization include glutaraldehyde, dimethyl pimelimidate (DMP), dithiobis (succinimidyl propionate), and bis(sulfosuccinimidyl) suberate (BS3). 135ite-directed capturing for functionalization involves using an intermediate layer with an affinity towards a region of the antibody or enzyme.Commonly reported strategies include protein A/G-mediated immobilization, 136,137 biotin-streptavidin, 138,139 poly-histidine-tags, 140 and oligonucleotide-mediated immobilization. 141,142Physical adsorption or passivation does not require any modification of the antibody, enzyme, or the surface of the electrode, relying on hydrophobic, van ECS Sensors Plus, 2024 3 011601 der Waals, and pi-pi stacking interactions. 143Site-directed immobilization enables oriented immobilization, minimizing random orientation reported in physically-adsorbed antibodies (Fig. 4a: (i)). 144Enzymes are typically physically entrapped in polymeric membranes, gels, or nanolattice materials.Commercial glucose sensors are created by entrapping glucose oxidase (GOx) in a hydrophilic transducing gel, which consists of a dense redox polymer mediator with a modified poly(vinylpyridine) backbone linked together with a bifunctional epoxide crosslinker. 145cleic acids.-Nucleicacids used in electrochemical biosensors as biorecognition probes can be categorized into capture probes and functional nucleic acid probes.[146][147][148][149] Capture probes are short (<30 nucleotides) oligonucleotides that are designed for either specific detection of the target, 96,150 or reporter barcodes released in response to the target.95,97 Functional nucleic acids are longer nucleic acid sequences with specific secondary structures that can be used for specific binding (aptamers) or catalysis of chemical reactions (DNAzymes).149,151,152 Nucleic acids are manufactured using solid-phase synthesis, employing phosphoramidite chemistry.Prior to use in electrochemical assays, the synthesized products are purified either by high-performance liquid chromatography (HPLC) or gel purification. 153 Nuceic acids are less expensive to produce ($0.3/mg for nucleic acid 154 versus $100-2000 mg −1 for mAbs 119 ) and are more resistant to changes in environmental pH or temperature compared to proteins.155 DNA aptamers, the nucleic acid analogue of antibodies, are produced using in vitro selection or systemic evolution of ligands by exponential enrichment (SELEX), where a large DNA pool (up to 10 15 sequences) 156 is used to identify aptamer sequences that bind specifically to the target of interest through multiple selection cycles.147 The time required for aptamer selection is highly dependent on the number of SELEX cycles used.Commercial selection involves the use of capillary electrophoresis-based SELEX, an automated and high-throughput method that requires eight to ten weeks.This method shortens the selection time by using three to five selection rounds compared to the ten to fifteen rounds used in conventional methods. 157 A umber of signal transduction strategies have been developed to convert target-aptamer binding into measurable signals.These include conformational changes, change in impedance on the transducer, strand displacement, or forming sandwich-based structures (Fig. 4b).One of the early examples of aptasensors was developed for detecting thrombin, where a conformational change in the redox-labelled thrombinbound aptamer enabled detection. 158 Oer the past twenty years, numerous aptamers have been selected and incorporated into electrochemical assays for detecting different classes of targets, including small molecules (e.g., glucose, adenosine), [159][160][161][162] nucleic acids, 146,163 proteins (e.g., infectious agents, hormones, receptors), 147,[164][165][166][167][168][169] extracellular vesicles, 170,171 and cancer cells.172,173 Even though such advancements are promising, the degradation of aptamers by nucleases in biological samples is an ongoing concern.174,175 Performing SELEX in biological samples, as well as chemical modifications using Click-SELEX can minimize such challenges associated with aptamers.176 Additionally, aptamer discovery using automation and microfluidic technology 177 shows great promise for increasing the throughput of aptamer discovery.
DNAzymes are also selected in vitro using methods similar to SELEX, which screen for DNA sequences that function as bond-cleaving or bond-formation catalysts. 149,151,178DNAzymes have been selected for detecting metal ions, 179 infectious agents, 95,98,149,180 disease biomarkers 181,182 or used for transducing an electrochemical signal in response to target binding, which is mostly demonstrated by peroxidase-mimicking enzymes. 183The number of DNAzyme sequences selected for different targets is lower than aptamers, limiting their potential commercial success. 184,185he performance of nucleic acid-based sensors depends on the density, quantity, and orientation of probe molecules immobilized at the electrode surface. 1869][190] In addition, linkers such as trithiols can promote the stability of the self-assembled monolayers on the electrode (Fig. 4a: (ii)). 188[193][194][195][196][197][198] Molecularly imprinted polymers.-MIPsare synthetic polymer analogues of antibodies that are designed to bind specific antigens or a group of related targets.MIPs are manufactured by polymerizing monomers in the presence of a target or a template, followed by template removal, leaving cavities for target binding. 199MIPs are produced using free-radical polymerization, controlled-radical polymerization, electropolymerization, emulsion polymerization, precipitation polymerization, core-shell grafting and polymerization, solid-phase synthesis, and high-dilution polymerization. 200These polymerization strategies can be combined with patterning strategies such as soft lithography, 201 microcontact printing, 202 and surface imprinting. 203MIPs are currently commercially produced using target proteins as templates. 204Commercial MIPs are less expensive ($0.1-0.5 mg −1 ) 205 compared to mAbs ($100-2000 mg −1 ); 119 however, the recognition heterogeneity and non-specific adsorption of bulk MIPs remain challenging for their use in biosensing platforms. 205unctionalization of electrodes with MIPs can be accomplished either in situ or ex situ; the former involves direct polymerization on the electrode surface; whereas the latter decouples the synthesis from immobilization.In situ functionalization reduces the required processing steps; 206 however, ex situ functionalization allows for the incorporation of metallic (e.g., gold nanoparticles) and carbonbased nanomaterials (e.g., carbon nanotubes) during deposition (Fig. 4a: (iii)) for improving the conductivity of the sensor and adsorption of the MIP to the electrode. 207

Device Integration
Sample collection and pre-processing.-There is an ongoing need for clinical assays that can be operated using simple methods performed by individuals with minimal training.Conventionally, clinical sampling for biomarkers is done from bodily fluids such as tears, nasal discharge, saliva, sweat, blood, urine, and stool, which need to be collected from the patient and processed to either preserve or extract the respective biomarkers before analysis.Sample collection typically involves the use of a capillary tube or paper (Schirmer strips) for tears; 208 cotton or nylon swabs for nasal, spit for saliva; 162 gauze/filter paper or the Macroduct system for sweat collection, 209 arterial/venipuncture and lancet pin-pricking for blood; 210 and rectal swabs for voiding and indwelling catheter collection for stool/urine (Fig. 5a). 211These samples need to be transported within a given timeframe in proper transport media to preserve the analytes.Sample processing and preparation with dilution in transport or storage buffers serves to reduce the matrix effect and preserve the sample. 212ample preparation methods include blood fractionation to separate blood components, 191 heating/chemical treatment to inactive nuclease and proteolytic enzymes that may interfere with detection methods; 213,214 freezing to reduce analyte degradation; sub-micron filtration or high-speed centrifugation to extract the target analyte; and/or, homogenization to disrupt cellular structures and release their components (Fig. 5b). 215The successful translation of biosensing devices into commercial POC diagnostics, relies on the successful integration of sample collection and processing with bioanalytical sensing, signal acquisition, and processing to improve the simplicity of assay operation by the end users.
Successful integration of sample processing and preparation with microfluidic platforms allows for low sample volumes to minimize wastage, automation of manual clinical steps with concurrent biosensing for fast turnaround times, and realizing portability. 233,234otably, an integrated biosensing platform must be carefully designed to minimize cross-contamination in POC settings to avoid false positives and/or false negatives and eliminate the probability of spread for infectious diseases.6][237] Additionally, fully-integrated systems need to incorporate methods by which errors in sample processing or the presence of contaminations are detected, indicating the need for re-testing or alternative testing. 238,239Importantly, individual components of the integrated platform need to be cost-effective for their successful commercialization.
Recent advances in 3D printing as an additive manufacturing method serve to minimize the cost of prototype development needed for the integration and validation of biosensing platforms.4][245][246] 3D printed valves can replace commercial four-port injection valves, offering advantages such as low dead volumes as well as adjustable volumes for biosensing assays. 247By incorporating conductive carbon nanomaterials into commercially available polylactic acid and polyamide filaments, electrochemical chips can achieve comparable electron transfer kinetics with only 30% infill. 248Interestingly, functional components and miniaturized lab equipment can be fully-integrated into a 3D printed platform or holder, housing miniaturized peristaltic pumps, heat blocks, tubes, and devices for sample handling and detection. 247,2494][245][246] Other methods such as selective laser sintering, two-photon polymerization, and laminated object manufacturing are also applicable to microfluidic systems as reported elsewhere. 250,251lly-integrated non-electrochemical biosensing platforms.-Aseries of technologies, based on manual valves, microfluidics, paperbased microfluidics, immiscible-filtration, manual centrifugation, and bio-inspired liquid repellency have been developed to simplify and streamline the operation of biosensors (Figs.6a-6e).An example of such a device is a valve-enabled and paper-based device integrated with viral lysis, RNA enrichment and purification, and reverse transcription loop-mediated isothermal amplification (RT-LAMP), which eliminates the need of pipetting for the sequential delivery of reagents (Fig. 6a: (i)). 252In this device, the addition of nasal and saliva samples triggers the discharge of lysis buffer by sliding the mixing unit, which allows pins to align with ball-based valves at the bottom, pushing them up for controlled release.As the solution goes through the detection unit, binding buffer is discharged to facilitate RNA absorption onto paper pads.After full absorption of the sample solution onto paper, the mixing unit is slid again, allowing for pre-loaded washing buffers to mix in for RNA purification.Lastly, the detection units are removed and transferred to a commercial battery-powered coffee mug with water pre-set to 62.5 °C for RT-LAMP.The signal readout is performed by adding SYBR green I for colorimetric detection with the naked eye or a smartphone camera (Fig. 6a: (ii)).The LOD of inactivated SARS-CoV-2 and influenza A (H1N1) viral samples are 2 and 6 genomic equivalents with a sample-to-result time of fifty minutes, respectively.This device does not provide quantitative viral analysis; however, it can be integrated with other nucleic acid amplification platforms and readout strategies such as electrochemical signal transduction, for integrating sample processing with analysis. 252nother system integrates viral RNA extraction and purification into a lab-on-chip (LOC) microfluidic system. 253This integrated system consists of oligonucleotide-functionalized magnetic beads for capturing viral RNA, a series of oil/aqueous barriers for washing and separating viral RNA, and a final reservoir for RT-LAMP and colorimetric detection (Fig. 6b: (i)).Within this system, the sample is moved between different zones using a magnet, followed by the addition of RT-LAMP reaction mixture in the last reservoir.The LOC platform is placed on a heat block for thirty to forty minutes at 65 °C for nucleic acid amplification and colorimetric detection based on pH-dependent colour change (Fig. 6b: (ii)). 253This system offers a sample-to-result time of one hour with LOD of 470 copies/mL for detecting SARS-CoV-2.The cost of the LOC system is estimated to be 10 USD, where 18% is attributed to device fabrication via CNC milling; however, this does not include the cost of the magnetic and heating units.
Microfluidic paper-based analytical devices (μPADs) are commonly used for integrating sample preparation and analysis in biosensors because they are inexpensive, enable capillary and pump-free transport, and are easily stackable. 257,258Most μPADs employ qualitative optical methods for detection; however, recent attempts combine these with electrochemical readout platforms for quantitative analysis. 259,260The biggest challenge in integrated μPADs lies in the controlled flow of the sample and bioreagents through the paper channels and timed incubations at the various steps of the bioassay.Recently, a novel integrated free energy paperpowered microfluidic system aimed to resolve this issue. 254In this system, algorithm-driven liquid handling sequentially releases 300 sample aliquots across chained interconnected monolithic chips (Fig. 6c: (i)). 254The heart of this microfluidic chain reaction (MCR) technology lies in its 3D printed capillary circuit, which consists of an intricate set of capillary valves. 254The capillary pump is made from commercial filter and absorbent papers to create a negative pressure for drawing aqueous solutions from the microfluidic circuit.The MCR technology is applied for the detection of SARS-CoV-2 antibodies in saliva, eliminating the eight-step sequence needed in conventional enzyme-linked immunosorbent assay (ELISA). 254The sample is first loaded, followed by the MCR's sequential loading of four reagents (loaded sample, detection antibody, horse-radish peroxidase (HRP) enzyme, and 3,3ʹ-diaminobenzidine substrate) and four washing buffers in between, allowing for naked-eye visualization and quantification of the substrate precipitate (Fig. 6c: (ii)).Furthermore, MCR was applied to a thrombin generation assay, referred to as the "Thrombochip."The "Thrombochip" has defibrinated coagulation-activated plasma samples and reagents loaded in reservoirs, with controlled one-minute release time intervals, enabling fluorescence measurement of thrombin concentration for reliable assessment of coagulation cascades. 254This intriguing design can be applied for controlled reagent handling inside LOC and biosensing systems, regardless of the readout method used.
Given the vital role of centrifugal g-force as a conventional method of sample fractionation in laboratories, a fidget-spinner inspired device, referred to as the diagnostic fidget spinner (Dx-ECS Sensors Plus, 2024 3 011601 FS), was fabricated for the colorimetric detection of E. coli.Dx-FS includes an inlet for sample loading, a sample holder, and a nitrocellulose filter membrane (Fig. 6d). 255Upon loading the unprocessed urine and spinning the device, the sample passes through the nitrocellulose filter due to the centrifugal force, leading to bacterial cell enrichment.Interestingly, the bottom of the chamber is pre-loaded with a fluid-assisted separation technology to enable uniform spreading of the bacterial samples onto the porous filter.The detection solution, loaded with water-soluble tetrazolium 8 dye, displays a color code that is correlated to the bacterial concentration.The system has a sample-to-result time of forty five minutes after spinning and can detect bacterial cells in the range of 10 2 -10 6 CFU ml −1 . 255The hand-powered nature of this device makes it suitable for facile sample processing in POC and resource-limited settings.
Bio-inspired technologies can be integrated into LOC devices to leverage their repellency and anti-biofouling properties.A recent droplet-based system, slippery liquid-infused porous surface laboratory (SLIPS-LAB), was developed for reporting on the level of calcium, citrate, uric acid, oxalate, and pH within thirty minutes by running parallelized colorimetric assays (Fig. 6e). 256Solutions are flown into the device by dipping from the bottom inlet or via capillary force from the top.The device is then laid on a flat surface, a tape is removed from an air hole on the top of the device, causing the sample droplets to move along the SLIPS by the Laplace pressure gradient, inspired from shorebirds, spider silk, and cactus. 256It is possible to conduct autonomous liquid handling using the SLIPS-LAB, without the need for any complex microfluidic circuitry or external pumps for integrating sample processing, transport, and sensing.Even though most of the aforementioned studies integrate creative sample processing methods with optical detection systems, we believe they offer viable, cost-effective, and facile approaches for automating the operational workflow of electrochemical biosensing systems, facilitating their use for POC diagnostics and health monitoring.
Fully-integrated electrochemical biosensing platforms.-Electrochemicalbiosensors that are compatible with integrated circuits and microfluidic technologies are well-positioned for creating fullyintegrated systems (Figs.7a-7i).A battery-operated electrochemical system with built-in microfluidics uses a fingertip-sized peristaltic pump for transporting saliva samples between different on-chip reservoirs (Fig. 7a: (i)-(ii)). 261This system is used for the concurrent detection of SARS-CoV-2 viral RNA and antibodies.The device consists of four reservoirs for sample preparation, LAMP, clustered regularly interspaced short palindromic repeats (CRISPR) reaction, and antibody detection.The last component is a four-electrode microfabricated chip, functionalized with antigens for antibody detection and peptide nucleic acid probes for RNA detection.The device works by manually adding saliva into the sample preparation reservoir for RNA detection, and the antibody-detection reservoir for antibody detection.For detecting viral RNA, saliva is combined with proteinase K and heated in the sample preparation reservoir, followed by pumping into the reaction chamber and heating to denature the potential reactioninhibitors.The LAMP solution is then pumped into the chamber and incubated for amplification, followed by pumping of the CRISPR solution.Lastly, the amplicon mixture and the saliva in the antibodydetection reservoir are directly pumped into the readout chip in a single-step, where polystreptavidin-HRP and tetramethylbenzidine (TMB) are added to generate the electrochemical signal.The sensing mechanism is signal-on for antibody detection, but signal-off for the viral RNA.Specifically, the presence of viral RNA leads to the cleavage of a nearby single-stranded DNA (ssDNA) reporting probe, reducing the binding of polystreptavidin-HRP, which consequently reduces TMB precipitation deposited on the electrode surface. 261This integrated system has a LOD of 0.8 copies μl −1 with 100% specificity and sensitivity.However, the use of an external pump is a limiting factor of this technology, which can potentially be addressed using finger-actuated microfluidics. 262revious work by our group uses anti-biofouling microgel magnetic beads (MMBs) modified with DNAzymes, together with chips containing hierarchical electrodes for ultrasensitive detection of E. coli in unprocessed urine.This system uses a 3D printed kit that houses a built-in magnet for MMB separation and a slot for inserting the electrochemical chip into a potentiostat that is connected to a smart phone for signal readout (Fig. 7b). 263The assay works by adding samples to a tube containing MMBs, which leads to the release of methylene blue-tagged barcodes from DNAzymes.The tube is incubated in the kit for 30 minutes and moved to the magnetic separation slot to separate the beads from the released barcodes.The barcode solution is then inputted onto the electrochemical chip that is connected to a smart phone.The chip contains electrodes functionalized with DNA sequences that are designed to capture the released barcodes.In less than one hour, this assay detects E. coli with a LOD of 138 CFU/mL in unprocessed urine of patients suspected of having urinary tract infection. 263The anti-biofouling properties in this work can be extended to other microfluidic platforms that use magnetic beads for sample separation in clinical samples.
A fully integrated electrochemical assay was developed for detecting vascular endothelial growth factor 165 (VEGF 165 ) in whole blood, utilizing a centrifugal microfluidic system with builtin detection electrodes.The system can analyze four blood samples in parallel and features blood/plasma separation, reagent addition, and detection zones, all integrated on a single centrifugal chip created on a glass substrate.(Fig. 7c). 264The assay uses a sandwich strategy using dual aptamers, where a nanoprobe made of polythionine (PThi), gold nanoparticles (AuNPs), capture aptamer, and bovine serum albumin (BSA) captures VEGF 165 in plasma, which is then detected by another aptamer functionalized on the electrode for VEGF capture forming a sandwich sensing film.The process begins by adding blood and nanoprobes into the blood cell chamber and using centrifugal force to separate plasma from blood cells, extracting VEGF 165 into the plasma chamber.The device is spun again to generate a siphon flow from the plasma chamber at a programmed speed, driving the nanoprobe-captured VEGF 165 into the detection chamber.An electrochemical signal is generated in the presence of VEGF 165 by differential pulse voltammetry upon redox reaction of PThi, and a LOD of 0.67 pg ml −1 is achieved. 264Despite the novel design for on-chip blood fractionation, the use of glass adds to the set-up costs and requires additional cleaning steps for reuse with blood and other clinical samples.Paper provides a more cost-effective alternative for single-use disposable integrated platforms.
A paper origami biosensor has been developed to integrate sample processing on a screen-printed electrode for the analysis of DNA base-pair mismatches, delivering a sample-in-result-out system.The device consists of three layers of folded paper including hydrophilic areas for sample loading or housing pre-loaded reagents and hydrophobic regions for holding the liquid in pre-defined areas (Fig. 7d). 265The sample is first added to the top layer of the paper, where it is mixed with a pre-loaded biotin-DNA reporter forming a target/reporter complex; the complex is then moved to the next paper layer by folding, where it is hybridized with the immobilized capturing probes, which are either ssDNA or tetrahedral DNA nanostructures, forming a sandwich-like structure.Lastly, avidin-HRP is transported to the reaction solution.The readout solution containing TMB and H 2 O 2 is added to the reaction chip for electrochemical signal readout using cyclic voltammetry.The system displays results within one hour of incubation with a LOD of 1 pM for ssDNA.This paper origami-based concept introduces a viable method for eliminating the manual steps associated with reagent addition, step-wise incubation, and washing.The test is low cost (<$1/test) 265 and provides a suitable avenue for creating commercialization-ready electrochemical tests; however, the relatively long test time (one hour) and the need to manually add the readout solution should be integrated to allow the test to compete with commercial lateral flow assays.
An all-in-one microfluidic system combines sample extraction from a cotton swab, sample delivery to an electrode, and electrochemical readout for detecting cotinine.The device consists of a 3D printed cover that allows a cotton swab to be inserted into the device, allowing the saliva to be delivered to the electrode surface using a peristaltic pump. 266The cover sits on a 3D printed holder which houses a screen-printed electrode at the bottom and microfluidic channels for sample transport (Fig. 7e). 266Prior to the extraction of the saliva from the swab by squeezing into the collector, thionine and H 2 O 2 are drop-casted on the electrode.The working electrode of the device is modified by cotinine-HRP (c-HRP), BSA, anti-cotinine antibody, and 3-mercaptopropionic acid (MPA), which upon binding with cotinine and forming a cotinine/BSA/anti-cotinine/MPA immunocomplex, reduces the binding of c-HRP, which in turn decreases the electrochemical current.The LOD of this assay is 6 × 10 −2 pg ml −1 and is in good agreement with cotinine measured in saliva using liquid chromatography-tandem mass spectrometry. 266he use of a peristaltic pump in this work adds to the device's complexity for POC applications, which can be miniaturized or replaced with a controlled suction-based mechanism for reagent delivery.
A pipette-based integrated electrochemical system has been developed in which sample collection, detection, washing, and regeneration are all performed using a single hand. 267Three prototypes of this tubular tip-like sensor (TTLS) technology are developed that contain one (TTLS1), three (TTLS2), or eight working electrodes (TTLS3).TTLS has three components-(1) cylindrical electrode(s) positioned in the middle of the pipette tip, (2) a cone-shaped bottom piece for suction, and (3) a connector for attaching the upper pipette with the electrode tube and the potentiostat cable to the inner electrode (Fig. 7f). 267TTLS1-3 is used for detecting nitrite, uric acid, and three liver biomarkers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin (BIL)).For nitrite, varying sampling volumes are suctioned into the TTLS1 for characterizing the impact of volume on the sensitivity and performance of the TTLS platform as a whole, followed by signal readout using cyclic voltammetry, enabling a LOD of 0.03 μM/U.Detection of liver biomarkers relies on the suction of substrate solutions (i.e., NADH/Prussian blue for ALT and AST detection, and methylene blue for BIL), which serve as a reactive medium for enzymatic reaction and detection.This step is followed by an incubation for two minutes for ALT/AST samples and five minutes for the BIL sample, and a final signal readout using square wave voltammetry (SWV).A LOD of 0.1, 0.2, and 0.05 μM/ U was achieved for ALT, AST, and BIL, respectively.Lastly, direct analysis of uric acid is used in a multichannel pipette, resulting in a 3.5% relative standard deviation value among the eight sensors, providing promising reproducibility of this integrated technology.Interestingly, the system requires only a small sample volume (5 μl), which together with its compact design and portability make it ideally suited for on-site and POC diagnostics.
A magnetic-bead based system was developed for high-throughput detection of extracellular vesicles (EVs) used as biomarkers for detecting colorectal cancer, termed high-throughput integrated magneto-electrochemical extracellular vesicle (HiMEX). 268HiMEX is composed of 96 electrode sets inside a 96-well plate with a built-in potentiostat and push-pin connectors below the electrodes for electrical contacts, as well as magnets for capturing bead-EV complexes (Fig. 7g). 268The overall assay involves two major steps, which begins by enrichment of EVs in whole blood with immunomagnetic beads for fifteen minutes, followed by labelling of bead-EV immunocomplex with biotin-modified antibodies.After subsequent washing of the beads, streptavidin-HRP is added, which binds to the biotin-labeled antibody.This process is followed by a final wash before adding TMB for detection using chronoamperometry.The device can deliver results within an hour with a LOD of 10 4 EVs ml −1 , which is more than a thousand-fold lower compared to ECS Sensors Plus, 2024 3 011601 ELISA. 268The ability of HiMEX to integrate electrochemical readout circuitry in a 96-well format, a widely used platform for performing biological assays, opens the route of delivering commercial laboratory analyzers based on electrochemical readout.
The Integrated Biosensor for Sepsis (IBS) is an example of a fully-integrated biosensor combining magnetic separation technology, smart pipette design, and electrochemical readout for the diagnosis of sepsis by analyzing the interleukin 3 (IL-3) biomarker.This device uses a magnetic pipette and a series of linear wells to facilitate and streamline the device operation (Fig. 7h). 269The magnetic pipette, consisting of a sheathed magnet attached to a mechanical plunger, is used to extract the target from blood using antibody-modified magnetic beads and releasing them into the next wells for labeling with detection antibodies and oxidizing enzymes (HRP, and introducing electron mediators for signal generation (TMB and H 2 O 2 ).The solution from the final well is then inputted on the electrode surface for signal readout.Electrochemical currents are measured on the electrode via TMB redox reaction catalyzed by HRP-coated magnetic beads that carry the target analyte.The platform is reported to be ten times more sensitive and five times faster than a conventional ELISA assay, with an accuracy of 86.0%, sensitivity of 91.3%, and specificity of 82.4%. 269This is a versatile integrated platform which can be further applied to other biomarkers, for which commercial antibodies are available.In recent years, the market for wearable sensors has been revolutionized by progress in sweat-based sensors, providing end-users with convenient monitoring solutions.
InflaStat is a wearable immunosensor developed for the real-time detection of C-reactive protein (CRP) in sweat, allowing for automated sweat sampling and reagent handling on a flexible patch.This platform consists of an iontophoresis module for sweat induction, LIG electrodes decorated with AuNPs-capturing antibody conjugates, AuNPs-thionine-detector antibody conjugates, a skininterfaced microfluidic component that aids with washing, and a flexible PCB (FPCB) for iontophoretic sweat induction, data acquisition, and wireless data transfer (Fig. 7i). 270Specifically, FPCB is housed on top of the microfluidic module and the sensor patch, and is powered by a small onboard lithium battery for communication with a user interface via Bluetooth.This platform consists of a reagent reservoir, a mixing channel, and a detection reservoir, and uses five steps of reconstitution, mixing, incubation, refreshment, and detection.Reconstitution begins with concurrent passage of the induced sweat sample and the AuNPs-thioninedetector antibody conjugates through a serpentine mixer (mixing channel) to facilitate the capture of CRP.This step is followed by incubation, which allows for the inputting of mixed components into the detection reservoir, filling the chamber, and exiting through an outlet.The refreshment process involves passive label removal using fresh sweat streaming through the microfluidics.Lastly, SWV is used to quantify the amount of bound thionine as a direct correlation to the amount of CRP target.This assay achieves a LOD of 8 pM in CRP-spiked buffer with high selectivity towards CRP in the presence of interferents such as cortisol, glucose, lactate, and urea. 270InflaStat is a novel platform that incorporates automation and sample processing for providing personalized inflammatory information to the end-user, paving the way toward health monitoring using fully-integrated wearable sensing. 271,272

Future Outlook and Conclusions
Half a decade has passed since the commercialization of the first glucose enzymatic electrode.Yet, 85% of the biosensor market is still dominated by glucose sensors.The widespread commercial translation of biosensors for other biomarkers is still lagging. 6Part of this problem is related to identifying technologies that address large enough commercial markets, like the glucose monitoring market, that justify the high development and regulatory costs of creating a new POC diagnostic device.The other part has to do with technical challenges in translating biosensing devices from the laboratory to the market, which add to the development timeline and cost of technology translation, further increasing the risk of investing in diagnostic technologies even if a large market potential exists.
The major technical challenge identified by many academic laboratories and companies focused on commercializing POC diagnostics is related to technology integration.Much of the analytical chemistry community is focused on developing new ultrasensitive signal transduction schemes for improving the LOD of biosensors; however, many of these methods are developed in clean buffers and evaluated with purified targets.These assays are often creative and elegant; however, they are often not adaptable to clinical samples and/or clinical workflows.Assay performance deteriorates significantly when many of these assays are challenged with heterogeneous clinical samples, which causes variability in both biorecognition and signal readout, resulting in large error bars and indistinguishable response between positive and negative samples or positive samples of varying concentrations.The proposed solution is often the use of sample processing methods that reduce interference of the sample matrix on the sensing mechanism; however, it is often not realized that the integration of sample processing technologies, such as those highlighted in this paper, is not straightforward and can add several months and even years of optimization and iteration to the development cycles of a new POC diagnostic device.Additionally, it is sometimes forgotten that these systems need to be manufactured using scalable methods to create a marketable product.
The key to overcoming the translation and integration challenges would be to focus on developing fully-integrated systems from an early stage in the development cycle; rather than dealing with integration in the aftermath.Another promising avenue is to use the built-in capabilities of molecules such as nucleic acids-cleavage, recognition, base pairing, in situ synthesis, and enzyme-enabled degradation-to develop one-pot assays 95,97,98,273 to limit (but most likely not eliminate) the need for sample processing steps such as washing and reagent addition; even though, steps such as dilution or target extraction might be inevitable.Finally, increasing the number of data points acquired from a biosensor would enable more rigorous calibration using artificial intelligence and machine learning techniques, 274 which could in turn help the biosensor look past the heterogeneity presented by clinical samples to visualize identifying features in measured signals.Overall, the COVID-19 pandemic has shed light on the urgency of the need for creating commercializationready platforms and has raised the bar on the requirements of bioanalytical assays used for POC diagnostics within the analytical chemistry community, which we believe will expedite the development cycle of commercial biosensors in the future.

Figure 1 .
Figure 1.Historical timeline: Timeline of advancements in commercial electrochemical biosensors.

Figure 2 .
Figure 2. The major components of an electrochemical biosensor: (a).Biorecognition elements interact with specific target analytes for translating their concentration into a measurable signal.(b).Charge transfer at the transducer surface is monitored by measuring an electrochemical signal.(c).Amperometric, voltametric, and impedance-based electrochemical techniques are applied using a potentiostat for signal readout.

Figure 3 .
Figure 3. Fabrication of scalable electrochemical chips: (a).Screen-printed electrode fabrication uses a soft squeegee to push ink through patterned masks.(b).PCB chips undergo several patterning processes prior to electrode material deposition and final solder mask coverage.(c).Laser-ablation uses a focused laser beam to ablate the carbon-rich substrate to form the desired pattern.(d).Microfabricated chips rely on photolithography and lift-off for electrode patterning.

Figure 5 .
Figure 5. Overview of samples used for diagnostics and health monitoring and the required laboratory processing methods: (a).Sample collection methods for various bodily fluids for disease diagnostics.(b).Sample processing and preparation can rely on dilution, separation, filtration, heating, homogenization, or additional steps of storage prior to conducting diagnostic assays.

Figure 6 .
Figure 6.Integrated devices with non-electrochemical output: (a).A multilayered valve-based sample preparation system 2-plex VLEAD for SARS-CoV-2 detection.(i) The device contains separate units for mixing and detection.(ii) Images of the paper-based detection units under room light (top panel) and blue LED (bottom panel) with samples containing SARS-CoV-2 (S), influenza A (F), CoV-OC43 (O), and no RNA (N).Adapted and reprinted with permission from Ref. 252.Copyright 2021 American Chemical Society.(b).A LOC device for SARS-CoV-2 RNA colorimetric detection under 40 mins.(i) The detection comprises of three consecutive steps of bead mediated RNA extraction, separation, and purification of the captured RNA and lastly RT-LAMP based detection.(ii) Negative sample (spiked with HCoV-OC43 and H1N1) turns red and positive sample (spiked with SARS-CoV-2, HCoV-OC43, and H1N1) turns yellow.Adapted and reprinted from Ref. 253.Copyright (2021), with permission from Elsevier.(c).A capillary chain reaction device with (i) assembled chip showing different regions for the bio-reagents.(ii) The four bio-reagents and buffers for the SARS-CoV-2 assay are supplied sequentially.Adapted from Ref. 254.Springer Nature Limited.(d).A fidget-spinner device integrating filtration and sample preparation for colorimetric detection of E. coli in urine.Adapted from Ref. 255.Springer Nature Limited.(e).SLIPS-LAB integrates volume metering, liquid handling, and reaction time control for colorimetric detection of urinary stone-associated analytes.Adapted from Ref. 256 ©.The Authors, some rights reserved, exclusive licensee AAAS.Distributed under a CC BY-NC 4.0 license http://creativecofmmons.org/licenses/by-nc/4.0/.Reprinted with permission from AAAS.

Figure 7 .
Figure 7. Integrated devices with electrochemical output: (a).A LOC device for detecting SARS-CoV-2 RNA and antibodies (i) Device workflow and photograph.(ii) The multiplexed system includes a heater, a sealed microfluidic chip, and a multiplexed electrochemical sensor chip for signal readout.Adapted from Ref. 261.Springer Nature Limited.(b).Microgel magnetic bead assay using RNA-cleaving DNAzymes and a smartphone with a portable potentiostat for detecting E. coli in patients suspected of having urinary tract infection.Adapted with permission from Ref. 263.Copyright 2022 American Chemical Society (c).Centrifugal disk chip used for blood plasma separation and on-chip detection of VEGF 165 using microfabricated electrodes.Adapted with permission from Ref. 264.Copyright 2022 American Chemical Society.(d).3D paper origami device using molecular threading for sample flow for detecting DNA via a series of folding, sequential sample addition, and readout.Adapted with permission from Ref. 265.Copyright 2019 American Chemical Society.(e).An integrated platform which consists of a commercial cotton-swab-type collector, 3D printed housing, and microfluidic channels integrated with an electrochemical competitive immunosensor to determine the level of salivary cotinine.Reproduced and adapted from Ref. 266 with permission from the Royal Society of Chemistry.(f).Handheld commercially available pipette with a tubular tip enclosed with electrodes for detection of nitrite, uric acid, and liver biomarkers in as low as 5 μl volume with full integration with a potentiostat.Reprinted and adapted with permission from Ref. 267.Copyright 2021 American Chemical Society.(g).High-throughput integrated magneto-electrochemical extracellular vesicle (HiMEX) platform using electrochemical cells integrated into a 96-well plate for parallel readout of magnetic bead/extracellular vesicle immunocomplexes.Adapted from Ref. 268.Springer Nature Limited.(h).An integrated biosensor for sepsis combines magnetic separation, smart pipette design, and electrochemical readout for analyzing the sepsis biomarker, IL-3.Reprinted and adapted with permission from Ref. 269.Copyright 2018 American Chemical Society.(i).A flexible patch integrated with graphene sensor array electrodes, a microfluidic module, and a flexible PCB (FPCB) for non-invasive and real-time monitoring of the inflammatory C-reactive protein in (CRP) biomarker in sweat.Adapted from Ref. 270.Springer Nature Limited.

Table I .
Strengths and limitations of electrode fabrication methods.