Biosensors and Bioelectronics

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.

Conventional molecular recognition elements, such as antibodies, present issues for the development of biomolecular assays for use in point-of-care devices, implantable/wearables, and under-resourced settings. Additionally, antibody development and use, especially for highly multiplexed applications, can be slow and costly. We developed a perception-based platform based on an optical nanosensor array that leverages machine learning algorithms to detect multiple protein biomarkers in biofluids. We demonstrated this platform in gynecologic cancers, which are often diagnosed at advanced stages, leading to low survival rates. We investigated the platform for detection in uterine lavage samples, which are enriched with cancer biomarkers compared to blood. We found that the method enables the simultaneous detection of multiple biomarkers in patient samples, with F1-scores of ~0.95 in uterine lavage samples from cancer patients. This work demonstrates the potential of perception-based systems for the development of multiplexed sensors of disease biomarkers without the need for specific molecular recognition elements.

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.

Microorganisms account for most of the biodiversity on earth. Yet while there are increasingly powerful tools for studying microbial genetic diversity, there are fewer tools for studying microorganisms in their natural environments. In this paper, we present recent advances in CMOS electrochemical imaging arrays for detecting and classifying microorganisms. These microscale sensing platforms can provide non-optical measurements of cell geometries, behaviors, and metabolic markers. We review integrated electronic sensors appropriate for monitoring microbial growth, and present measurements of single-celled algae using a CMOS sensor array with thousands of active pixels. Integrated electrochemical imaging can contribute to improved medical diagnostics and environmental monitoring, as well as discoveries of new microbial populations.

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.

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).

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers due in part to the cancer being diagnosed is at a late stage when effective treatment options are limited. Early detection of PDAC via liquid biopsy would revolutionize survival from the disease. To address the lack of effective non-invasive detection assays for PDAC, we developed a protease activity-based assay using a magnetic nanosensor (PAC*MANN). The PAC*MANN assay leverages protease activity in blood to amplify the signal of the target-probe based sensor. An initial screening revealed that the PAC*MANN assay could reliably differentiate patients with PDAC from healthy subjects and patients at high risk of PDAC. Finally, in two cohorts: training (n=145) and blinded validation (n=72), we demonstrated that the PAC*MANN assay had high specificity (86%) and sensitivity (78%) for detection of PDAC compared to healthy subjects. This performance was enhanced when combined with the current standard of care assay, CA19-9 (100% specificity, 84% sensitivity). Our results demonstrate a novel assay that is rapid, high-throughput, and requires low specimen volume, which may not only improve cancer detection but could be useful for monitoring of at-risk patients and could be deployed in low resource settings. One sentence summaryA high-throughput, non-invasive, rapid protease-activated nanosensor identifies pancreatic cancer from a small volume of blood

Real-time biosensors offer significant potential for continuous monitoring of biomolecules. However, their practical application and further development face challenges on data analysis, including poor signal-to-noise ratio when effective sensing area decreases due to signal degradation by biofouling, time-consuming and subjective process of manual or semi-automated peak identification, and inconsistencies in data interpretation, thereby complicating reproducibility and cross-comparison of biosensing results. In this study, we introduce the Algorithm-Powered Analyzer for Continuous Electrochemistry (A-PACE), an open-source toolkit providing streamlined and automated data analysis protocol optimized for real-time electrochemical data analysis. A-PACE comprises three modules: (1) Change point detection for automated identification of peak regions; (2) Baseline fitting with multiple algorithms to handle diverse electrochemical signals and baseline screening to eliminate unreasonable fits; (3) Large dataset input with user-friendly interface for data processing, exporting and visualization. To ensure optimal performance, we curated a training set of 2000 electrochemical curves from an extensive electrochemical dataset (>100,000 curves) collected over the past five years under varied electrochemical measurement conditions. These curves were analyzed using 2046 algorithm sets to identify a default algorithm set, compared to existing tools, demonstrating its capability in peak detection and baseline fitting across a broad spectrum of electrochemical data. Case studies reveal the A-PACE can analyze month-long in vitro serum data and week-long in vivo intravenous blood data, extending the operational lifespan of real-time sensors. This cross-platform compatible toolkit supports both real-time and post-processing analysis, reducing the processing time from minute level per signal by human labeling to second level by A-PACE and subjectivity associated with electrochemical signal processing. By providing this solution for continuous electrochemical data analysis, A-PACE enhances biosensors applications in medical diagnostics and continuous monitoring with high throughput analysis.

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

The COVID-19 pandemic has underscored the critical need for rapid and accurate screening and diagnostic methods for potential respiratory viruses. Existing COVID-19 diagnostic approaches face limitations either in terms of turnaround time or accuracy. In this study, we present an electrochemical biosensor that offers nearly instantaneous and precise SARS-CoV-2 detection, suitable for point-of-care and environmental monitoring applications. The biosensor employs a stapled hACE-2 N-terminal alpha helix peptide to functionalize an in-situ grown polypyrrole conductive polymer on a nitrocellulose membrane backbone through a chemical process. We assessed the biosensors analytical performance using heat-inactivated omicron and delta variants of the SARS-CoV-2 virus in artificial saliva (AS) and nasal swabs (NS) samples diluted in a strong ionic solution. Virus identification was achieved through electrochemical impedance spectroscopy (EIS) and frequency analyses. The assay demonstrated a limit of detection of 40 TCID50/mL, with 95% sensitivity and 100% specificity. Notably, the biosensor exhibited no cross-reactivity when tested against the influenza virus. The entire testing process using the biosensor takes less than a minute. In summary, our biosensor exhibits promising potential in the battle against pandemic respiratory viruses, offering a platform for the creation of rapid, compact, portable, and point-of-care devices capable of multiplexing various viruses. This groundbreaking development has the capacity to significantly bolster our readiness and response to future viral outbreaks.

The integration of transepithelial/transendothelial electrical resistance (TEER) measurement into organ-on-chip (OoC) platforms provides a unique opportunity to monitor the integrity of biological barriers in real-time. This is particularly important for detecting rapid changes in the temporal dynamics of intercellular junctional complexes in response to drug compounds, changes in host-microbiota interactions, and pathological disease states. Conventional TEER systems usually require special cell culture components or complex measurement technology that must be operated by experienced users. In this work, we present an innovative approach that represents an extension of the existing and well-established Dynamic42 chip platform by integrating semi-transparent TEER electrodes with fixed positions in combination with a measurement device. Remarkably, this system works in a plug-and-play manner and can continuously measure TEER inside the incubator without user intervention or invasive manipulation. Other important features of OoC, such as the microfluidic perfusion, multicellular cell colonization, and the possibility of microscopic examination, are not compromised by the integrated TEER electrodes. To demonstrate the performance of this new TEER system, we leveraged a 3D intestine-on-chip (IoC) model and investigated TEER during model assembly, barrier disruption, and recovery as a proof-of-concept. Moreover, we compared and discussed this with data from a conventional end-point fluorescence permeability assay to demonstrate the benefits of real-time measurements with higher sensitivity.

Highly sensitive protein detection is critical in research and healthcare diagnostics but remains limited to resource intensive environments. In contrast, lateral flow immunoassay (LFIA)-based diagnostics are popular point-of-care tests due to their simplicity and user-friendly format but lack sensitivity for detecting the low concentrations of proteins that are present early in infections and among asymptomatic individuals. To overcome these limitations, we developed a novel biosensor platform utilizing antibody-initiated loop-mediated isothermal amplification (ai-LAMP) with an LFIA readout for protein detection. This platform integrates protein-specific antibody-antigen binding with the robust signal amplification of LAMP. By conjugating pairs of antibodies to overlapping DNA strands, the presence of a target protein brings the DNA strands into proximity, completing a DNA target to directly initiate the LAMP reaction without any additional ligation step. This approach facilitates the rapid detection of low concentration proteins with a clear visual readout and can be performed in 3 steps from sample to answer. Using ai-LAMP we detected HIV-1 p24 on commercially available LFIAs at a limit of detection (LOD) of 20 fM (0.53 pg/mL), demonstrating 46x improvement over existing HIV p24 LFIAs. The use of ai-LAMP eliminates the need for sophisticated laboratory equipment to detect protein targets in low concentrations, paving a new way for rapid and accessible biomarker detection in clinical and research settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/678293v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5b587forg.highwire.dtl.DTLVardef@10f3587org.highwire.dtl.DTLVardef@1833eeborg.highwire.dtl.DTLVardef@197d1bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionHead and neck squamous cell carcinoma (HNSCC) is associated with high morbidity and mortality due to late detection. Tapered optical fiber sensors (TOFS) are biosensors with the potential application as a point-of-care device for detection of HNSCC biomarkers. TOFS uses optical fibers as transduction elements and antigen-antibody binding for the detection of target biomolecules. The present TOFS system was designed to achieve high specificity, sensitivity, and repeatability. To explore its application in HNSCC, we targeted the proinflammatory cytokine IL-8, known for its role in promoting tumorigenicity, metastasis, and angiogenesis in HNSCC. To validate our proof-of-concept experiment, a viral surrogate of SARS-CoV-2, Human Coronavirus OC43 (HCoV-OC43), was also tested. MethodsOur TOFS system contains four main parts: the tapered optical fiber, reservoir (cell), laser light source, and photodetector. The fiber is bitapered with a narrow waist region that anchors antibodies to detect target biomolecules. Light is transduced by the laser to the fiber where it travels through the down-taper region to the tapered waist followed by the up-tapered region and ultimately transmitted to the photodetector. The tapered waist facilitates the transformation of a single mode light to multiple modes, which reverts to a single mode of light as it continues through the up-taper region. Biomolecules introduced to the waist region alter the effective refractive index, which is reflected in the phase change of the wavelength. This is detected by the photodetector and quantified using Fouier analysis. Our tapered fibers were designed with antigen-antibody complexes targeting IL-8 and HCoV-OC43 to validate our TOFS design across two different antigens. ResultsTOFS with tethered mouse anti-human IgG (28nm) bound to dissolved IL-8 (14 nm) produces a measurable phase shift. As little as 10pg/ml or 7.1X105 IL-8 molecules/l was detected by the prototype device and the phase change represented in real-time the binding dynamic of IL-8 to the tethered IgG. To validate our proof-of-concept experiment, HCoV-OC43 dissolved in saliva was detected with a sensitivity of 50 viruses/mL. ConclusionsTOFS device is a highly sensitive system capable of detecting proteins, viruses, and other biomolecules. Selectivity of the system is guaranteed by specific antigen-antibody binding, supported by the detection of IL-8 and HCoV-OC43 at the femtomolar level. The long-term goal for TOFS is its application as a point-of-care device used for detection, monitoring, and surveillance of HNSCC as well as detection of other pathogens in the clinical setting.

Plants are non-equilibrium systems consisting of time-dependent biological processes. Phenotyping of chemical responses, however, is typically performed using plant tissues, which behave differently to whole plants, in one-off measurements. Single point measurements cannot capture the information rich time-resolved changes in chemical signals in plants associated with nutrient uptake, immunity or growth. In this work, we report a high-throughput, modular, real-time chemical phenotyping platform for continuous monitoring of chemical signals in the often-neglected root environment of whole plants: TETRIS (Time-resolved Electrochemical Technology for plant Root In-situ chemical Sensing). TETRIS consists of screen-printed electrochemical sensors for monitoring concentrations of salt, pH and H2O2 in the root environment of whole plants. TETRIS can detect time-sensitive chemical signals and be operated in parallel through multiplexing to elucidate the overall chemical behavior of living plants. Using TETRIS, we determined the rates of uptake of a range of ions (including nutrients and heavy metals) in Brassica oleracea acephala. We also modulated ion uptake using the ion channel blocker LaCl3, which we could monitor using TETRIS. We developed a machine learning model to predict the rates of uptake of salts, both harmful and beneficial, demonstrating that TETRIS can be used for rapid mapping of ion uptake for new plant varieties. TETRIS has the potential to overcome the urgent "bottleneck" in high-throughput screening in producing high yielding plant varieties with improved resistance against stress.

Current diagnostic approaches for myocardial infarction (MI) rely on blood-based cardiac biomarker analysis by centralized instruments, often delaying timely clinical decisions. We present a microneedle-based capacitive biosensor (MiCaP) for in situ, minimally invasive monitoring of cardiac troponin I (cTnI) in interstitial fluid (ISF) for point-of-care (POC) applications. MiCaP is a label-free biosensor operating based on non-faradaic sensing by monitoring electric double layer capacitance at the microneedle-ISF interface. We extracted a simplified equivalent circuit model for MiCaP inserted into skin, confirming that the measured capacitance variations originate from cTnI binding to surface-immobilized antibodies. MiCaP was fabricated using a scalable process and functionalized with anti-cTnI antibodies. In vitro measurements showed a dynamic detection range of 10 pg/mL to 10 ng/mL, a limit of detection (LOD) of 3.27 pg/mL, and a total assay turnaround time of less than 15 minutes. A spike-and-recovery test using cTnI-spiked human serum yielded a recovery accuracy exceeding 93%. In vivo studies in rats demonstrated ISF cTnI levels of 3 {+/-} 0.4 pg/mL in controls and 912 {+/-} 683 pg/mL in experimental animals, indicating an increasing trend consistent with serum concentrations measured using a clinical immunoassay. These results support the potential of MiCaP as a minimally invasive biosensing platform for cardiac biomarker monitoring, with possible extension to multiplexed ISF-based diagnostics in POC.

Breast cancer is a substantial source of morbidity and mortality worldwide. It is particularly more difficult to treat at later stages, and treatment regimens depend heavily on both staging and the molecular subtype of the tumor. However, both detection and molecular analyses rely on standard imaging and histological method, which are costly, time-consuming, and lack necessary sensitivity/specificity. The estrogen receptor (ER) is, along with the progesterone receptor (PR) and human epidermal growth factor (HER-2), among the primary molecular markers which inform treatment. Patients who are negative for all three markers (triple negative breast cancer, TNBC), have fewer treatment options and a poorer prognosis. Therapeutics for ER+ patients are effective at preventing disease progression, though it is necessary to improve the speed of subtyping and distribution of rapid detection methods. In this work, we designed a near-infrared optical nanosensor using single-walled carbon nanotubes (SWCNT) as the transducer and an anti-ER antibody as the recognition element. The nanosensor was evaluated for its response to recombinant ER in buffer and serum prior to evaluation with ER- and ER+ immortal cell lines. We then used a minimal volume of just 10 {micro}L from 26 breast cancer biopsy samples which were aspirated to mimic fine needle aspirates. 20 samples were ER+, while 6 were ER-, representing 13 unique patients. We evaluated the potential of the nanosensor by investigating several SWCNT chiralities through direct incubation or fractionation deployment methods. We found that the nanosensor can differentiate ER-from ER+ patient biopsies through a shift in its center wavelength upon sample addition. This was true regardless of which of the three SWCNT chiralities we observed. Receiver operating characteristic area under the curve analyses determined that the strongest classifier with an AUC of 0.94 was the (7,5) chirality after direct incubation and measurement, and without further processing. We anticipate that further testing and development of this nanosensor may push its utility toward field-deployable, rapid ER subtyping with potential for additional molecular marker profiling.

Tumor Mutational Burden (TMB) has emerged as a crucial biomarker to guide patient eligibility for immunotherapy. However, whole exome sequencing, the gold-standard method for TMB measurement, remains limited in accessibility due to its high costs, operational complexity, and lengthy processing times. To address these limitations, we investigated whether Ultra-High-Frequency (UHF) technology could serve as a novel approach to assess TMB by analyzing the crossover frequencies or electromagnetic signature (EMS) of cancer cells on a lab-on-a-chip biosensor, integrating microfluidics and dielectrophoresis. In a panel of 12 cancer cell lines with varying TMB levels, we observed that EMS showed an upward shift correlating with higher TMB, particularly in solid tumor cell lines. This finding suggests a potential relationship between TMB and EMS. To further explore this hypothesis, we artificially increased mutation levels by treating cells with the highly mutagenic compound N-ethyl-N-nitrosourea (ENU). Results showed that EMS captured significant TMB variations in ENU-treated cells with enhanced proliferative capacity compared to their parental counterparts. These results underscore the importance of matched control samples for reliable EMS measurements. Altogether, our findings highlight the potential of EMS to detect TMB variations associated with proliferative activity, a key hallmark of cancer cells, thereby enabling a more precise stratification of cancer cells. HighlightsO_LIWe propose a new biosensor to improve patient stratification for ICI eligibility C_LIO_LIHigh frequency fields and dielectric spectroscopy can estimate TMB in cancer cells C_LIO_LISignificant changes in UHF-DEP signatures correlate with varying TMB levels C_LIO_LIUHF-DEP provides a cheap, rapid and label-free method to predict ICI response C_LIO_LIOffers an innovative and complementary marker to conventional diagnostic approaches C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/622085v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@703983org.highwire.dtl.DTLVardef@1cfdf46org.highwire.dtl.DTLVardef@4b9285org.highwire.dtl.DTLVardef@1809255_HPS_FORMAT_FIGEXP M_FIG C_FIG

There are various protein assays for specific and quantitative detection and widely used for laboratory and clinic purposes, but current methods still have limitations. Immunoassays based on antibodies, like ELISA, suffer from slow response and a long antibody-screening period, while physical or electrochemical methods are generally restricted by high cost or the stringent requirement of equipment or operating skills. In this study, we developed an in vitro sensitive protein quantification method: Captamer. The Captamer system comprises a molecular switch derived from aptamer sequence and an exponential fluorescence signal amplification pathway based on recombinase polymerase amplification (RPA). We demonstrated the Captamer for SARS-CoV-2 nucleocapsid protein detection and obtained results from samples within 30 min, displaying a wide detection window from 0.2 pg/mL to 200 pg/mL with high specificity. Furthermore, we tested the Captamer for Tau441 protein (a potential Alzheimers disease biomarker) and thrombin (a classic aptamer-protein interaction model), showing the limit of detection as low as 1 ng/mL and 0.02pg/mL respectively, which suggested the capacity of Captamer to be applied to various aptamer-protein pairs. Compared with the most commonly used and recently reported protein quantification methods, Captamer stands out for its high sensitivity, short response time, low cost, and simplicity, indicating its great potential to be widely used in protein quantification. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/686180v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e38ffaorg.highwire.dtl.DTLVardef@1019e72org.highwire.dtl.DTLVardef@1498b68org.highwire.dtl.DTLVardef@15f5ea2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extracellular vesicles (EVs) play important roles in cell-cell communication but they are highly heterogeneous, and each vesicle has dimensions smaller than 200 nm thus encapsulates very limited amounts of cargos. We report the technique of NanOstirBar (NOB)-EnabLed Single Particle Analysis (NOBEL-SPA) that utilizes NOBs, which are superparamagnetic nanorods easily handled by a magnet or a rotating magnetic field, to act as isolated "islands" for EV immobilization and cargo confinement. NOBEL-SPA permits rapid inspection of single EV with high confidence by confocal fluorescence microscopy, and can assess the colocalization of selected protein/microRNA (miRNA) pairs in the EVs produced by various cell lines or present in clinical sera samples. Specific EV sub-populations marked by the colocalization of unique protein and miRNA combinations have been revealed by the present work, which can differentiate the EVs by their cells or origin, as well as to detect early-stage breast cancer (BC). We believe NOBEL-SPA can be expanded to analyze the co-localization of other types of cargo molecules, and will be a powerful tool to study EV cargo loading and functions under different physiological conditions, and help discover distinct EV subgroups valuable in clinical examination and therapeutics development.

Rapid detection of pathogens at the point-of-need is crucial for preventing the spread of human, animal and plant diseases which can have devastating consequences both on the lives and livelihood of billions of people. Colorimetric, lateral flow assays consisting of a nitrocellulose membrane, are the preferred format today for low-cost on-site detection of pathogens. This assay format has, however, historically suffered from poor analytical performance and is not compatible with digital technologies. In this work, we report the development of a new class of digital diagnostics platform for precision point-of-need testing. This new versatile platform consists of two important innovations: i) A wireless and batteryless, microcontroller-based, low-cost Near Field Communication (NFC)-enabled potentiostat that brings high performance electroanalytical techniques (cyclic voltammetry, chronoamperometry, square wave voltammetry) to the field. The NFC-potentiostat can be operated with a mobile app by minimally trained users; ii) A new approach for producing nitrocellulose membranes with integrated electrodes that facilitate high performance electrochemical detection at the point-of-need. We produced an integrated system housed in a 3D-printed phone case and demonstrated its used for the detection of Maize Mosaic Virus (MMV), a plant pathogen, as a proof-of-concept application.