Biosensors and Bioelectronics

Since the early 2000, regulation agencies have encouraged viral vaccine manufacturers to implement in-process and real-time monitoring tools in production processes. Even if more assays have been recently developed, none of the novel viral particle quantification technologies can monitor virus levels and their secretion kinetics within production vessels. Vaccine manufacturers still rely on offline cell-based infectivity assays and antigen amount quantification to monitor their processes. The present study describes the development of the first automated biosensor for at-line monitoring of influenza virus production. It involves coupling a fetuin-based SPRi quantitative biosensor with an automated sampler of culture broth and a consecutive clarification setup via an acoustic filter. The SPRi response of different viral strains produced in two distinct cell production platforms was qualified. We demonstrated that fetuin-based quantitative SPRi is a robust, potency-indicating, and universal analytical technology for quantifying bioactive influenza virus particles. It was validated with both purified and complex matrices. Finally, an influenza viral production kinetic was monitored online for three days. This novel online tool enabled the access in real-time to total bioactive viral particles from early production phases (8hpi).

Graphene Field-Effect Transistors (GFETs) are increasingly employed as biochemical sensors due to their exceptional electronic properties, surface sensitivity, and potential for miniaturization. A critical challenge in deploying GFETs is determining the optimal electrical readout strategy. GFETs are typically operated with either of two modalities: one measuring current in real time (amperometric) and the other monitoring the change in voltage for charge neutrality (potential potentiometric). Here, we undertake a systematic study of the two modalities to determine their relative advantages/disadvantages towards guiding the future use of GFETs in sensing. We focus on viral proteins in wastewater, given the matrixs complexity and the growing interest in the field of wastewater surveillance. Our results show that transconductance offers far superior limits of detection (LOD) but suffers from limited reproducibility, a narrower dynamic range, and is ineffective for some viral proteins. In comparison, we find that Dirac point tracking offers higher reproducibility and superior robustness, but at a higher LOD. Interestingly, both techniques exhibit similar sensitivity, highlighting the importance of the aptamers employed. Systematic experiments also help explain differences in dynamic range and limited functionality in detecting some proteins, resulting from hidden electrophoresis, and shifting the high transconductance point away from the active region. Thus, our findings provide crucial considerations for designing and operating resilient graphene biosensors suitable for real-time pathogen monitoring in environmental scenarios.

Real-time multi-channel measurements of tonic serotonin (5-hydroxytryptamine, 5-HT) concentrations across different brain regions are of utmost importance to the understanding of 5-HTs role in anxiety, depression, and impulse control disorders, which will improve the diagnosis and treatment of these neuropsychiatric illnesses. Chronic sampling of 5-HT is critical in tracking disease development as well as the time course of pharmacological treatments. Despite their value, in vivo chronic multi-site measurements of 5-HT have not been reported. To fill this technological gap, we batch fabricated implantable glassy carbon (GC) microelectrode arrays (MEAs) on a flexible SU-8 substrate to provide an electrochemically stable and biocompatible device/tissue interface. Then, to achieve multi-site detection of tonic 5-HT concentrations, we incorporated the poly(3,4-ethylenedioxythiophene)/functionalized carbon nanotube (PEDOT/CNT) coating on the GC microelectrodes in combination with a new square wave voltammetry (SWV) approach, optimized for selective 5-HT measurement. In vitro, the PEDOT/CNT coated GC microelectrodes achieved high sensitivity towards 5-HT, good fouling resistance in the presence of 5-HT, and excellent selectivity towards the most common neurochemical interferents. In vivo, our PEDOT/CNT-coated GC MEAs were able to successfully detect basal 5-HT concentrations at different locations of the CA2 hippocampal region of mice in both anesthetized and awake head-fixed conditions. Furthermore, the implanted PEDOT/CNT-coated MEA achieved stable detection of tonic 5-HT concentrations for one week. Finally, histology data in the hippocampus shows reduced tissue damage and inflammatory responses compared to stiff silicon probes. To the best of our knowledge, this PEDOT/CNT-coated GC MEA is the first implantable flexible multisite sensor capable of chronic in vivo multi-site sensing of tonic 5-HT. This implantable MEA can be custom-designed according to specific brain region of interests and research questions, with the potential to combine electrophysiology recording and multiple analyte sensing to maximize our understanding of neurochemistry. HighlightsO_LIPEDOT/CNT-coated GC microelectrodes enabled sensitive and selective tonic detection of serotonin (5-HT) using a new square wave voltammetry (SWV) approach C_LIO_LIPEDOT/CNT-coated GC MEAs achieved multi-site in vivo 5-HT tonic detection for one week. C_LIO_LIFlexible MEAs lead to reduced tissue damage and inflammation compared to stiff silicon probes. C_LI

We present a rapid, label-free, and highly sensitive platform for characterising recombinant protein functionality using Ion Channel Switch (ICS) technology. This method enables precise evaluation of binding specificity, oligomeric state discrimination, and real-time analyte detection, addressing key challenges in protein engineering and bioprocess quality control. Using engineered single-chain variable fragments (scFv), ICS reliably distinguishes monomeric from multimeric forms, facilitates wash-free detection of analytes ranging from small molecules to larger biomolecules, and enables quantitative biosensing within seconds in real-time and continuous format. These capabilities establish ICS as a powerful tool for streamlining recombinant protein screening, with broad applications in diagnostics, therapeutic quality control, and automated bioprocess workflows.

KRAS mutations are among the most prevalent oncogenic alterations in colorectal, lung, and pancreatic cancer, yet their detection remains analytically challenging in the presence of an overwhelming wild-type (WT) background. Here, we report a photoelectrochemical (PEC) genotyping platform that integrates clamp-inhibited loop-mediated isothermal amplification (C-LAMP) with enzyme-free singlet oxygen (1O2)-driven PEC transduction for mutation-selective KRAS detection. Locked nucleic acid (LNA) clamp probes selectively suppress WT amplification during isothermal amplification, enriching mutant alleles and enabling single-nucleotide variant (SNV) discrimination with high selectivity. Amplified products are magnetically captured and transduced into photocurrent via visible-light-induced 1O2 redox cycling, eliminating enzymatic reporters and reducing background interference. The C-LAMP/PEC platform achieves a limit of detection of 35 copies {micro}L-1 (58 aM) and a minimum detectable variant allele frequency (VAF) of 4.8% in heterogeneous mutant/WT genomic DNA mixtures. Analytical performance was validated in cancer cell lines and in patient-derived fresh frozen tissues, showing complete concordance with Nanopore sequencing and droplet digital PCR (ddPCR) within the evaluated cohort (n = 16). This work introduces a robust and modular PEC biosensing strategy that combines molecular WT suppession with enzyme-free photoelectrochemistry, offering an economically competitive and instrumentation-simplified approach for clinically relevant KRAS mutation analysis toward decentralized testing.

1The growing threat of antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), underscores the urgent need for rapid, sensitive, and field-deployable diagnostic technologies. Here, we present a silicon micropillar-enhanced CRISPR biosensor that integrates high-aspect-ratio microstructures with a one-pot RPA/CRISPR-Cas12a assay for ultrasensitive and specific detection of MRSA. Micropillar arrays with fixed diameters and varying heights (100 {micro}m, 300 {micro}m, and 500 {micro}m) were fabricated via deep reactive ion etching and functionalized for surface probe immobilization. Suboptimal crRNA design was employed to modify Cas12a activation kinetics, enabling declined trans-cleavage and enhanced end-point signal accumulation. The 500 {micro}m micropillar configuration demonstrated a tenfold improvement in sensitivity compared to the 100 {micro}m array, with a limit of detection reaching 103 CFU mL-1. The platform also showed high specificity against non-target bacterial strains. These findings highlight the potential of combining microstructured chips with one-pot CRISPR diagnostics to advance next-generation point-of-care tools for infectious disease monitoring. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/694528v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@5f5f7corg.highwire.dtl.DTLVardef@175cb35org.highwire.dtl.DTLVardef@7043e5org.highwire.dtl.DTLVardef@79aa15_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rapid identification of bacteria based on nucleic acid amplification allows dealing with the detection of pathogens in clinical, food, and environmental samples. Amplification product must be detected and analyzed by external devices or integrated complicated optical systems. Here, we developed a solid-state pH electrode based on iridium oxide (IrO2) films to measure released hydrogen ions (H+) from isothermal nucleic acid (NA) amplification of bacterial samples. By recombinase polymerase amplification (RPA), we achieved rapid (< 15 min) and sensitive (<30 copies) detection with an accuracy of about 0.03 pH. The RPA-based hydrogen ion sensing assay shows higher specificity, sensitivity, and efficiency as the same polymerase chain reaction (PCR) methods. We initially used the RPA-based sensor to detect E. coli species in laboratory samples. Among, 27 random laboratory samples of E. coli samples, 6 were found to be DH5alpha, 9 BL21, 3 HB101, 6 TOP10, and 3 JM109. The electrical detection of amplification provides generally applicable techniques for the detection of nucleic acid amplification, enabling molecular diagnostic tests in the field and integrating data transmission to the mobile device. These results can be future developed into an efficient tool for rapid on-site detection of bacterial pathogens in clinical samples.

Detecting small amounts of analytes, especially antibodies, presents a significant challenge in high-throughput methods. Fortunately, nucleic acid amplification techniques provide a promising solution. We have successfully developed and tested an innovative technology for detecting anti-glycan antibodies, utilizing a printed glycan array combined with a rolling circle amplification reaction based on the ABO blood group antibody model. This breakthrough has dramatically enhanced the sensitivity of our immunoassay, improving it by over an order of magnitude and allowing us to detect concentrations as low as 1 ng/ml. This advancement opens new approaches for research and clinical applications, making previously undetectable analytes accessible for study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or nCovid-19) outbreak has become a huge public health issue due to its rapid transmission and global pandemic. Currently, there are no vaccines or drugs available for nCovid-19, hence early detection is crucial to help and manage the outbreak. Here, we report an in-house built biosensor device (eCovSens) and compare it with a commercial potentiostat for the detection of nCovid-19 spike antigen (nCovid-19Ag) in spiked saliva samples. A potentiostat based sensor was fabricated using fluorine doped tin oxide electrode (FTO) with gold nanoparticle (AuNPs) and immobilized with nCovid-19 monoclonal antibody (nCovid-19Ab) to measure change in the electrical conductivity. Similarly, eCovSens was used to measure change in electrical conductivity by immobilizing nCovid-19 Ab on screen printed carbon electrode (SPCE). The performances of both sensors were recorded upon interaction of nCovid-19Ab with its specific nCovid-19Ag. Under optimum conditions, the FTO based immunosensor and eCovSens displayed high sensitivity for detection of nCovid-19Ag, ranging from 1 fM to 1 M. Our in-house developed device can successfully detect nCovid-19Ag at 10 fM concentration in standard buffer that is in close agreement with FTO/AuNPs sensor. The limit of detection (LOD) was found to be 90 fM with eCovSens and 120 fM with potentiostst in case of spiked saliva samples. The proposed portable eCovSens device can be used as a diagnostic tool for the rapid (within 10-30 s) detection of nCovid-19Ag traces directly in patient saliva in a non-invasive manner.

In vitro models have now become a realistic alternative to animal models for cardiotoxicity assessment. However, the cost and expertise required to implement in vitro electrophysiology systems to study cardiac cells poses a strong obstacle to their widespread use. This study presents a novel, cost-effective approach for in vitro cardiac electrophysiology using fully-printed graphene-based microelectrode arrays (pGMEAs) coupled with an open-source signal acquisition system. We characterized the pGMEAs electrical properties and biocompatibility, observing low impedance values and cell viability. We demonstrated the platforms capability to record spontaneous electrophysiological activity from HL-1 cell cultures, and we monitored and quantified their responses to chemical stimulation with noradrenaline. This study demonstrates the feasibility of producing fully-printed, graphene-based devices for in vitro electrophysiology. The accessible and versatile platform we present here represents a step further in the development of alternative methods for cardiac safety screening.

Acute myocardial infarction (AMI) remains one of the most prevalent and fatal cardiovascular disease. Given the critical diagnostic significance of cardiac troponin I (cTnI) and myoglobin (Myo) in AMI, there is an urgent clinical demand for rapid and accurate detection methods to improve patient outcomes and reduce mortality. To meet this need, we developed a sensing platform that integrates functionalized gold nanoparticle-based lateral flow immunoassay (AuNPs-LFIA) with a machine vision model, enabling rapid quantitative detection of cTnI and Myo. By covalently conjugating antibodies to gold nanoparticles (AuNPs@Ab), we achieved greater probe specificity and stability. The integration of machine vision algorithms allowed quantitative readouts within 8 minutes, a 46.7% improvement of the detection time compared to conventional methods (15 minutes). The platform achieved limits of detection of 0.224 ng/mL for Myo and 0.071 ng/mL for cTnI, with excellent correlation to commercial kits (R2 > 0.99). Overall, these results demonstrate that machine vision-enhanced AuNPs-LFIA offers an efficient, sensitive and reliable strategy for point-of- care testing (POCT) in cardiovascular diagnostics, particularly in resource-limited settings.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a highly contagious disease with several variants, continues to spread as part of the global pandemic. With the roll-out of vaccines and development of new therapeutics that may be targeted to distinct viral molecules, there is a need to screen populations for viral antigen-specific SARS-CoV-2 antibodies. Here, we describe a rapid, multiplexed, electrochemical (EC) platform with on-chip control that enables detection of SARS-CoV-2 antibodies in less than 10 min using 1.5 {micro}L of a patient sample. The EC biosensor demonstrated 100% sensitivity and specificity, and an area under the receiver operating characteristic curve of 1, when evaluated using 93 clinical samples, including plasma and dried blood spot samples from 54 SARS-CoV-2 positive and 39 negative patients. This EC biosensor platform enables simple, cost-effective, sensitive, and rapid detection of anti-SARS-CoV-2 antibodies in complex clinical samples, which is convenient for monitoring host humoral responses to vaccination or viral infection in broad population testing, including applications in low-resource settings. We also demonstrate the feasibility of using dried blood spot samples that can be collected locally and transported to distant clinical laboratories at ambient temperature for detection of anti-SARS-CoV-2 antibodies which can be used for serological surveillance and demonstrate the utility of remote sampling.

1In many biological systems, pH can be used as a parameter to understand and study cell dynamics. However, measuring pH in live cell culture is limited by the sensor ion specificity, proximity to the cell surface, and scalability. Commercially available pH sensors are difficult to integrate into a small-scale cell culture system due to their size and are not cost-effective for disposable use. We made PHAIR - a new pH sensor that uses a micro-wire format to measure pH in vitro human airway cell culture. Tungsten micro-wires were used as the working electrodes, and silver micro-wires with a silver/silver chloride coating were used as a pseudo reference electrode. pH sensitivity, in a wide and narrow range, and stability of these sensors were tested in common standard buffer solutions as well as in culture media of human airway epithelial cells grown at the air-liquid interface in a 24 well cell culture plate. When measuring the pH of cells grown under basal and challenging conditions using PHAIR, cell viability and cytokine responses were not affected. Our results confirm that micro-wires-based sensors have the capacity for miniaturization, and detection of diverse ions while maintaining sensitivity. This suggests the broad application of PHAIR in various biological experimental settings.

Cassava brown streak disease (CBSD) severely threatens food security in East Africa and the livelihoods of hundreds of millions globally. Effective control requires large-scale surveillance in resource-limited settings, which is currently lacking. We present ELLA (Electrochemical Lateral Flow Assay with Linked Analytics), a portable, battery-free, low-cost digital diagnostic platform integrating lateral flow assays with near-field communication and electrochemical readouts for cloud-based data storage and analytics. Validated through extensive field trials in East Africa and smaller studies in Brazil, ELLA achieved 89% agreement with RT-qPCR and 95% with ELISA, often surpassing ELISA sensitivity at a material cost below US$1 per test. Leveraging ELLAs molecular results, we trained a deep-learning model (DeepELLA) for rapid, image-based diagnosis of CBSD, enabling scalable surveillance of this and potentially emerging plant pathogens. By combining electrochemical sensing, digital connectivity, and AI-driven analytics, ELLA offers a powerful tool to strengthen plant disease monitoring and food security. Its modular design also allows adaptation to other chemical and biological targets, creating opportunities for novel datasets and new insights into plant and environmental health.

By engineering extracellular electron transfer (EET) to be dependent on an analyte, researchers have developed whole cell bioelectronic sensors that sense hazards to human and environmental health1. However, these sensors regulate a single electron transfer pathway as an electrochemical channel, limiting the sensing information to a single analyte. To increase information content, we developed a multichannel bioelectronic sensor through which different chemicals regulate distinct extracellular electron transfer pathways within a single Escherichia coli cell. One channel utilizes the flavin synthesis pathway from Bacillus subtilis2 and the other a set of cytochromes constructing the Mtr pathway from Shewanella oneidensis3. We demonstrate an arsenite responsive promoter can control the Mtr pathway through activation of cytochrome CymA expression and a cadmium responsive promoter can control the flavin synthesis pathway4,5. The redox potential of flavin-mediated EET is different from that of CymA-mediated one6. This allowed for development of a redox-potential-dependent algorithm that distinguishes variable input signals of each analyte mediated by two EET pathways in vivo. This approach enables a 2-bit binary signal readout for real-time tracking throughout the entire sensing duration. Our multichannel bioelectronic sensor was able to accurately sense and distinguish different heavy metals in Brays Bayou water samples with a response time comparable to that in clean water. This multichannel bioelectronic sensors allow for simultaneous detection of different chemicals, significantly expanding information transmission and helping to safeguard human and environmental health.

Rapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention e.g., drug therapy, quarantine, no action etc. when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2 (the pathogen causing COVID-19), and shows no or similar symptoms to other common infections. We report a silicon-based integrated Point-of-Need (PoN) transducer (TriSilix) that can chemically-amplify and detect pathogen-specific sequences of nucleic acids (NA) quantitatively in real-time. Unlike other silicon-based technologies, TriSilix can be produced at wafer-scale in a standard laboratory; we have developed a series of methodologies based on metal-assisted chemical (wet) etching, electroplating, thermal bonding and laser-cutting to enable a cleanroom-free low-cost fabrication that does not require processing in an advanced semiconductor foundry. TriSilix is, therefore, resilient to disruptions in the global supply chain as the devices can be produced anywhere in the world. To create an ultra-low-cost device, the architecture proposed exploits the intrinsic properties of silicon and integrates three modes of operation in a single chip: i) electrical (Joule) heater, ii) temperature sensor (i.e. thermistor) with a negative temperature coefficient that can provide the precise temperature of the sample solution during reaction and iii) electrochemical sensor for detecting target NA. Using TriSilix, the sample solution can be maintained at a single, specific temperature (needed for isothermal amplification of NA such as Recombinase Polymerase Amplification (RPA) or cycled between different temperatures (with a precision of {+/-}1.3{degrees}C) for Polymerase Chain Reaction (PCR) while the exact concentration of amplicons is measured quantitatively and in real-time electrochemically. A single 4-inch Si wafer yields 37 TriSilix chips of 10x10x0.65 mm in size and can be produced in 7 hours, costing ~US $0.35 per device. The system is operated digitally, portable and low power - capable of running up to 35 tests with a 4000 mAh battery (a typical battery capacity of a modern smartphone). We were able to quantitatively detect a 563-bp fragment (Insertion Sequence IS900) of the genomic DNA of M. avium subsp. paratuberculosis (extracted from cultured field samples) through PCR in real-time with a Limit-of-Detection of 20 fg, equivalent to a single bacterium, at the 30th cycle. Using TriSilix, we also detected the cDNA from SARS-CoV-2 (1 pg), through PCR, with high specificity against SARS-CoV (2003).

COVID-19 pandemic has caused tens of thousands of deaths and is now a severe threat to global health. Clinical practice has demonstrated that the SARS-CoV-2 S1 specific antibodies and viral antigens can be used as diagnostic and prognostic markers of COVID-19. However, the popular point-of-care biomarker detection technologies, such as the lateral-flow test strips, provide only yes/no information and have very limited sensitivities. Thus, it has a high false negative rate and cannot be used for the quantitative evaluation of patients immune response. Conventional ELISA (enzyme-linked immunosorbent assay), on the other hand, can provide quantitative, accurate, and sensitive results, but it involves complicated and expensive instruments and long assay time. In addition, samples need to be sent to centralized labs, which significantly increases the turn-around time. Here, we present a microfluidic ELISA technology for rapid (15-20 minutes), quantitative, sensitive detection of SARS-CoV-2 biomarkers using SARS-CoV-2 specific IgG and viral antigen - S protein in serum. We also characterized various humanized monoclonal IgG, and identified a candidate with a high binding affinity towards SARS-CoV-2 S1 protein that can serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. Furthermore, we demonstrated that our microfluidic ELISA platform can be used for rapid affinity evaluation of monoclonal anti-S1 antibodies. The microfluidic ELISA device is highly portable and requires less than 10 L of samples for each channel. Therefore, our technology will greatly facilitate rapid and quantitative analysis of COVID-19 patients and vaccine recipients at point-of-care.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. However, current methods are limited by the low sensitivity, long turn-around time or high cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 VOCs by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 204 clinical samples, showing 99% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and {Delta}H69-V70 were discriminated by RRCd, demonstrating 100% accuracy in clinical samples with Ct < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which may facilitate point-of-care testing of SARS-CoV-2 and its variants.

The lateral flow assay is one of the oldest and most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the tedious and inefficient hybridization steps. Here, we have introduced a new version of the lateral flow assay, termed Cas9-mediated lateral flow nucleic acids assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify Listeria monocytogenes, genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a sensitivity of hundreds of copies of genome samples with high specificity within 1 h. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for analyzing genes in resource-poor or nonlaboratory environments.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has emphasized the urgent need for accurate and readily available diagnostic tools. Conventional diagnostic methods, such as reverse transcription real-time polymerase chain reaction (RT-qPCR), are often labor-intensive and time-consuming, which highlights the necessity for rapid point-of-care diagnostic solutions. This study introduces an innovative, low-cost, and highly sensitive diagnostic platform for swift COVID-19 detection. Our platform utilizes the mass sensing properties of thickness shear mode (TSM) transducers to detect and quantify the SARS-CoV-2 nucleocapsid protein through polyethylene glycol (PEG)-based chemistry (1). To confirm surface functionalization and evaluate the effects of the virus lysis buffer, we employed surface characterization techniques including Digital Holographic Microscopy (DHM), Scanning Electron Microscopy (SEM) with Energy-Dispersive X-ray spectroscopy (EDX), and Raman spectroscopy. Sensitivity tests with heat-inactivated SARS-CoV-2 samples demonstrated a sensitivity of about 0.256 Hz/TCID50/mL and a limit of detection (LOD) of roughly 150 TCID50/mL. Specificity was verified through cross-reactivity testing. Our detailed characterization and sensitivity analysis underscore the platforms reliability, making it a promising candidate for efficient and accessible COVID-19 diagnosis at the point of care.