Biosensors and Bioelectronics

Continuous monitoring of proteins in complex biological fluids is essential for advancing personalized medicine, yet existing biosensors are often limited by instability, single-use designs, and insufficient sensitivity. Here, we describe a detailed protocol for the fabrication and operation of molecular pendulum (MP) electrochemical sensors integrated with an active-reset mechanism to enable real-time, reversible, and ultrasensitive protein detection. The protocol is described in two parts. First, we describe the microfabrication of gold microelectrodes and their nanostructured modification, followed by assembly of DNA-based pendulum probes with redox reporters and affinity receptors that translate binding into kinetic electron-transfer shifts. Second, we detail the sensing and active-reset approach, which detects the target analyte and applies tunable oscillatory potentials to accelerate its dissociation, regenerate sensor surfaces, and extend operational lifetime. The protocol includes detailed guidance on device fabrication, surface functionalization, sensing and reset cycles, and data analysis. When implemented, MP sensors with active-reset achieve pg/ml sensitivity, rapid equilibration, and robust performance across biofluids and in situ models, enabling continuous protein monitoring over extended periods. This combined technology represents a biosensing platform with significant potential, opening new avenues for wearable and implantable molecular monitoring, early disease detection, and personalized therapeutic guidance.

Non-small-cell lung cancer (NSCLC) diagnosis is challenged owing to the need for reliable, non-invasive biomarkers. Circulating cell-free microRNAs (ccf-miRNAs) are promising candidates, but their low levels and short sequences in blood complicate detection. In this study, a novel nano-hybrid fluorescent array was developed to enable rapid and sensitive identification of NSCLC-related ccf-miRNAs. The array consists of poly(T)-modified fluorescent nano-polystyrene beads for capturing target miRNAs, and fluorophore-labeled, sequence-specific locked nucleic acid (LNA) probes for their detection. This design forms a sandwich structure with the target miRNA. Nano cytometry and fluorescence microscopy assessed both the capture and specificity of detection. The assay was tested on plasma samples for miR-16-5p and U6, evaluating selectivity, sensitivity, and reproducibility. The results showed that the poly(T)-modified beads efficiently captured the target miRNAs, and the LNA probes accurately distinguished the sequences. The assay enabled direct detection of miR-16-5p and U6 from plasma without amplification, demonstrating high selectivity, sensitivity, and reproducibility. Combining enrichment with nano-polystyrene beads and sequence-specific LNA probes addressed the main challenges of ccf-miRNA detection, namely their low abundance and short sequence length. As ccf-miRNAs are linked to NSCLC progression, this method could assist in early diagnosis, disease monitoring, and assessment of treatment response. The target-independent nature of the poly(T) capture layer allows for easy calibration to new miRNAs by changing the LNA probes, rendering it suitable for multiplex detection in clinical settings. However, further preclinical and clinical studies are needed before adoption in routine practice. This nano-hybrid fluorescent array presents a rapid and reliable approach for detecting low-abundance ccf-miRNAs in plasma. Its encouraging performance and flexible design show potential for future application in NSCLC diagnosis and monitoring, subject to additional confirmation.

Surface plasmon resonance (SPR) and biolayer interferometry (BLI) are well-established label-free analytical technologies used to determine biomolecular interaction rates (ka, kd) and equilibrium constants (KD). While assays historically rely on multi-cycle kinetics (MCK), this approach requires intermediate surface regeneration, risking sensor degradation and increasing reagent consumption. Single-cycle kinetics (SCK) avoids regeneration by sequentially injecting increasing analyte concentrations. However, implementing SCK workflows on biosensor platforms like the OctetRR8 BLI instrument (Sartorius) remains a challenge due to the lack of built-in or online data analysis tools. To address this, we introduce SCKAnalyzer, a free, user-friendly web application designed for processing and fitting SCK datasets. SCKAnalyzer accepts column-based text files and native raw BLI data (.frd format) from Sartorius instruments, performing data pre-processing and non-linear fitting to a 1:1 interaction model. Additionally, it offers advanced simulation capabilities that allow variable association and dissociation durations across sequential injection steps, a feature absent in existing platforms such as BiacoreTMSimul8. The performance of SCKAnalyzer was experimentally validated using a 3-step SCK method (SCK3) for DNA/RNA duplex formation on a Biacore T200, as well as 4-step (SCK4) and 5-step (SCK5) methods for DNA/DNA duplex formation on an OctetRR8 BLI instrument. Binding parameters (ka, kd, KD) derived from SCKAnalyzer showed excellent agreement with values from established proprietary softwares such as Biacore T200 Evaluation Software v3.2.1 and BIAevaluation v4.1.1. SCKAnalyzer provides a practical alternative to MCK, reducing probe consumption and extending SCK utility across biosensor platforms. SCKAnalyzer is available at sck.iecb.u-bordeaux.fr (source code: github.com/synthaze/sckanalyzer).

Antimicrobial resistance (AMR) represents a major global health threat, with plasmid-borne mcr genes driving colistin resistance and exposing critical gaps in One-Health surveillance across human, animal, and environmental reservoirs. The most prevalent variant, mcr-1, remains difficult to monitor in resource-limited settings due to the lack of rapid, affordable, and field-deployable molecular tools. Here, we developed C12amcr, an integrated molecular toolbox that combines pre-amplification PCR with a fluorescent CRISPR-Cas12a assay targeting a conserved region of mcr-1 and a custom low-cost, hand-held 3D-printed portable fluorometer. Under optimized conditions, the assay achieved a limit of detection of 630 cells/mL. In poultry feces spiked with mcr-1-positive E. coli, C12amcr detected as few as 1,800 cells/mL. When tested on 22 community-derived E. coli isolates, the assay showed 100% concordance with both next-generation sequencing for mcr-1 detection and phenotypic colistin susceptibility testing by broth microdilution. The accompanying portable fluorometer performed equivalently to a laboratory microplate reader while enabling fully decentralized workflows compatible with portable PCR platforms. By integrating locally produced molecular reagents, straightforward protocols, and an accessible field-ready fluorescence reader, C12amcr overcomes key barriers to decentralized AMR surveillance and provides a practical, scalable solution for One-Health monitoring in resource-limited settings.

Rapid, selective detection of bacterial pathogens remains a central challenge. Here we report a label-free electrochemical biosensing approach that leverages protein-protein interaction (PPI)-derived peptides as recognition elements for rapid detection of Listeria monocytogenes (LM). The sensor design is inspired by the interaction between the LM virulence factor Internalin A (InlA) and the human host receptor E-cadherin (E-Cad1). Peptides derived from the InlA-binding domain of E-Cad1 were engineered as molecular recognition elements, with the E-Cad1(15-24) peptide displaying micromolar affinity and selective binding towards LM. Immobilization of these peptides on gold electrodes enabled bacterial detection by electrochemical impedance spectroscopy within 10 minutes, without labels or external signal amplification. A low peptide surface density was associated with enhanced binding-site accessibility and may facilitate multivalent interactions between the bacterial surface and the immobilized peptides. The platform produced a detectable response at experimentally tested concentrations as low as 1 CFU mL {superscript 1} and exhibited excellent selectivity under the conditions examined. This work introduces a chemically programmable, PPI-inspired biosensing paradigm that uses a reductionist approach and could potentially be extended to other pathogen targets.

Fermentation is a process through which microorganisms are used to transform basic molecules into high value substances in various applications such as biopharma, food production, and biofuel generation. In such processes, monitoring the health of microbial cells is essential, as it is a key factor in the final product yield. Traditional methods of assessing the health of fermenter populations have involved manually extracting samples from the fermenter for benchtop testing, which lacked the accuracy and efficacy of real-time continuous measurements. This has opened the door for inline sensors to be integrated into fermenters for continuous measurement; including microfluidic based Electrochemical Impedance Spectroscopy (EIS). EIS utilizes a range of frequencies to gain insight into the characteristics of microbial populations, such as cell size and membrane capacitance. EIS cytometry, in particular, has emerged as a relevant tool, as it enables single-cell level detection in real-time. However, current EIS cytometry devices cannot be deployed directly within fermenters due to the high cell density conditions. This has limited EIS analysis in fermenters to bulk measurements averaged across a population. Here, we present a microfluidic chip that uses passive cell-flux-partitioning geometry to direct cells from high-density culture through a single-cell-constrained flow path containing integrated impedance-sensing electrodes. The proposed chip combines geometric channel design and embedded electrodes to enable continuous impedance measurements from cell-containing flow while operating at input cell densities on the scale of 10^9 CFU/mL. This work demonstrates a first step toward integrating single-cell-constrained microfluidic handling with impedance sensing for inline monitoring in industrial biomanufacturing.

Square-wave voltammetry (SWV) is widely used for electrochemical biosensing because it enables sensitive, temporally resolved measurement of redox reporter signals. However, automated quantification of SWV signal remains challenging for long duration and in vivo measurements, where voltammograms can exhibit changing baselines, heterogeneous noise, peak drift, outliers, and interfering faradaic processes. Here, we introduce the Adaptive Square-Wave Voltammetry Iterative Fitting Toolkit (ASWIFT), an automated method for robust SWV signal extraction based on iteratively reweighted regularized smoothing. ASWIFT is available as both an open-source Python package and a downloadable desktop application. The method adaptively estimates the baseline, selects regularization strengths, fits the redox peak, and reports peak height without trace-specific parameter tuning. Across simulated datasets spanning diverse baseline, peak, noise, and concentration-response conditions, ASWIFT produced less systematic bias and more consistent signal estimates than existing methods. We further evaluated ASWIFT using in vitro doxorubicin measurements and in vivo DNA-based kanamycin sensor measurements collected in rat blood and interstitial fluid, demonstrating agreement with established methods. These results support ASWIFT as a robust framework for automated SWV analysis in real-time electrochemical biosensing.

Background: Rapid and accurate measurement of procalcitonin (PCT) is useful for diagnosing bacterial infections and guiding antibiotic therapy, yet current laboratory-based immunoassays require centralised infrastructure, delaying clinical decision-making and limiting access in low-resource settings. We developed a battery-free, smartphone-connected electrochemical lateral flow assay with linked analytics for the detection of PCT (ELLA-PCT) enabling quantitative testing at the point of care without conventional laboratory instrumentation. Methods: We designed a competitive electrochemical lateral flow assay using gold nanoparticles co-functionalised with a PCT-specific DNA aptamer and ferrocene hexanethiol as a redox reporter. A miniaturised near-field communication (NFC) potentiostat embedded within a disposable cassette enabled wireless electroanalytical measurements using a smartphone. Analytical performance was assessed in buffer and serum, including limit of detection (LOD), linearity, specificity, and stability. Clinical evaluation was performed on 27 serum samples from nine adults undergoing antibiotic treatment for suspected bacterial infection, with results compared against the reference Time-Resolved Amplified Cryptate Emission (TRACE) assay. Findings: ELLA-PCT achieved an LOD of 46 pg/mL and a linear detection range of 0.5-100 ng/mL. The ELLA-PCT assays remained stable for three months under ambient storage conditions, and cross-reactivity with calcitonin, C-reactive protein, and interleukin-6 remained below clinically relevant thresholds. Clinical results showed strong correlation with TRACE (R2 = 0.979, p < 0.0001), with a mean bias of 0.05 ng/mL and narrow 95% limits of agreement (-0.69 to 0.89 ng/mL). Importantly, ELLA-PCT delivered results in only 30 minutes without requiring external power or clinical laboratory instrumentation. In comparison to TRACE, the total turnaround time was reduced by at least 50%, which typically requires at least one hour from sample collection to results at a hospital setting. Interpretation: ELLA-PCT is an antibody-free, smartphone-connected test that provides laboratory-grade quantitative PCT measurement using only an NFC-enabled electroanalytical sensor and a mobile phone. The platform has a strong potential to decentralise diagnosis of infectious diseases, support antibiotic stewardship, and enable remote, real-time monitoring in outpatient and resource-limited settings. Larger studies are needed to evaluate the use of whole-blood samples and the integration of the platform into digital clinical workflows.

Monitoring pH and extracellular acidification rate (ECA) in biological samples containing live mammalian cells can provide valuable information on the glycolytic activity and bioenergetic status of cells. Compared to pH electrodes, optochemical pH sensors look more advantageous, since they allow rapid, non-invasive parallel analysis of multiple samples with stable readout of pH. We have developed new fluorescent pH sensors based on hydrophobic protonable metal-free porphyrins,OEP and OEPK, embedded in a plasticized PVC matrix containing a proton transfer agent. These pH sensors provide internally-referenced calibration-free operation, both in ratiometric intensity and lifetime-based detection modes. Sensor development included optimization of the indicator dye and its photophysical characteristics, screening of different proton transfer agents to minimize sensor toxicity, tuning of the protonation range and pKa, long-term storage stability and response time studies. Optimised pH sensor coatings were then deposited on plastic substrates (96-well microplates) and used for real-time monitoring of Extracellular Acidification Rate (ECAR) for cultured cancer cells and 3D spheroid structures on standard laboratory equipment (multi-label plate reader and confocal FLIM microscope). The advanced pH sensors tailored for use with biological samples have high potential for cell analysis and related applications.

Organic electrochemical transistors (OECTs) enable high transconductance at low operating voltages through the volumetric charging of organic mixed ionic/electronic conductors (OMIECs), making them attractive transducers for biosensing. However, the same ionic -electronic coupling makes OMIECs, such as poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS), sensitive to different ions and various environmental fluctuations, so drift and memory effects may compromise the quantitative readout in complex biofluids. In this work, we introduce a novel OECT-based sensor configuration that minimises drift by addressing a single channel with two gates: a functionalised sensing gate and a reference gate that allows real-time decoupling of environmental and channel-state fluctuations. A pulsed gate-biasing protocol is introduced to promote channel conductivity recovery and mitigate state accumulation. Using enzymatic glucose sensing as a proof of concept, the platform suppresses time-dependent drift by ~96%, eliminates temperature-induced variations up to 99% across the 20-70 {degrees}C range, improves calibrated sensitivity by 55% and reduces interferent-induced deviations under physiologically relevant conditions. By improving both signal stability and analytical reliability, this strategy addresses a central limitation of OECT biosensors and advances their use in high-sensitivity point-of-care biosensing in complex biological environments.

Continuous tracking of cortisol is central to understanding human stress physiology, yet direct electrochemical detection without biological receptors remains challenging despite its promise for stable and dynamic sensing. Electrochemical reduction of cortisol typically occurs at highly negative potentials, where parasitic interfacial currents, hydrogen evolution, and substantial capacitive background overlap with the cortisol reduction signal, preventing accurate quantification. We overcome this barrier through an integrated material-measurement strategy combining a defect-engineered graphene interface with a transition-resolved interrogation (TRI) measurement strategy. First, we engineered polybenzimidazole-derived laser-induced graphene containing nitrogen-rich defects while suppressing oxygen-derived functionalities which reduced parasitic background currents within the same cathodic potential regime. Next, we designed TRI to leverage differences in the time-dependent evolution of overlapping cathodic processes to isolate a localized cortisol-associated electrochemical reduction transition. Derivative-domain projection coupled with background estimation enables its reliable quantification. This integrated sensing architecture enables sensitive, selective, and dynamic cortisol detection in both artificial and biological interstitial fluids at low nanomolar concentrations. The response remains reproducible across physiologically relevant variations in pH, ionic strength, temperature, and repeated cycling. Together, these capabilities provide a basis for continuous electrochemical cortisol monitoring for future study of stress physiology.

Paper-based colorimetric sensors offer a low-cost and accessible platform for point-of-care (POC) analysis, but enzyme activity loss during coating and drying can weaken analytical signals and require high enzyme loadings or complex immobilization procedures. Although surfactants are widely used to improve wettability in paper-based assays, their potential contribution to colorimetric performance beyond these effects remains unclear. Here, we investigated surfactant-assisted colorimetric signal enhancement in a glucose assay implemented on a 96-puddle paper plate (96-PPP) and identified Tween 20 as the most effective surfactant. Its effect on detection performance became more pronounced as glucose oxidase (GOx) loading decreased; at 0.1 mg/mL GOx, Tween 20 lowered the limit of detection (LoD) from 0.113 to 0.034 mg/mL (approximately 3.3-fold) over a working range of 0-5 mg/mL, despite no statistically significant change in the measured contact angle at this loading. Tween 20 had no appreciable effect on the reaction in solution but preserved 95% of the apparent reaction rate constant after drying, compared with 11% without it, and atomic force microscopy (AFM) revealed a more dispersed dried enzyme morphology on mica. Tween 20-containing sensors also showed slower signal decay during repeated wetting-drying cycles and thermal stress, retained 77% (vs 26%) of the response at 400 mM NaCl, and exhibited within-PPP and between-batch coefficients of variation (CVs) below 10% (vs 12.3-19.5%), while maintaining glucose selectivity over potentially interfering molecules. These results indicate that Tween 20 enhances paper-based glucose sensing beyond wettability, in part by retaining enzyme cascade activity during drying, although the contributions of the individual enzymes and the underlying mechanism remain to be established.

Accurate, noninvasive biomarkers for diagnosis and longitudinal monitoring of metabolic dysfunction-associated steatohepatitis (MASH) remain an urgent unmet clinical need. Here, we describe an electrochemical immunoassay that involves capturing hepatic extracellular vesicles (EVs) on antibody-modified electrodes followed by detecting EV-borne biomarkers using redox active gold nanoparticles (AuNPs). The intensity of electrochemical redox signal represents a quantitative measure of biomarker abundance. Three MASH-associated biomarkers-S100A11, TNF-, and TGF-{beta}1 were analyzed on hepatic EVs to assess steatotic, inflammatory, and fibrotic states of the diseased liver. The electrochemical signals for individual biomarkers were added into a composite EVMASH score. Plasma samples from 28 patients with MASH, 22 healthy controls, and 27 patients with non-MASH liver disease were scored in this manner, revealing 100% accuracy in the training cohort and >90% accuracy in an independent validation cohort. Our electrochemical immunosensor represents a minimally invasive liquid liver biopsy, offering a promising alternative to solid liver biopsies for screening and monitoring of MASH.

Continuous biomarker monitoring with affinity biosensors remains limited by probe saturation, slow dissociation, signal carryover, and regeneration methods that require chemical washing, harsh buffers, or external reset modules. Here, we report a flow-programmed, self-regenerative electrochemical aptamer-based biosensor for continuous 17{beta}-estradiol monitoring. The platform restores the sensing interface by tuning buffer flow through the device, enabling repeated sensing-regeneration cycles without chemical stripping or active-reset hardware. The biosensor achieves a 1.46 pM limit of detection, and a log-linear dynamic range from 100 pM to 1 M in real human sweat, covering physiologically relevant estradiol concentrations. It exhibits high selectivity, with responses to potential molecularly similar interferents remaining below 10% of the 17{beta}-estradiol signal. Under dynamic flow operation, the device shows reversible signal recovery and stable electrochemical performance, with higher buffer-wash flow reducing baseline carryover between measurements. A coupled binding-transport simulation captures the flow-dependent regeneration trends and maps how wash-flow rate, wash duration, and apparent aptamer affinity affect regeneration efficiency. Ex vivo testing in real human sweat, including samples from pregnant women, supports translational relevance. This work establishes buffer-flow programming as a reagent-free route to regenerable electrochemical aptamer sensing for automated, wearable-compatible estradiol monitoring.

Studying algae requires specialized equipment to characterize culture growth and physiological performance and non-invasive monitoring of these cultures typically relies on optical density (OD) measurements. Currently, OD is often measured using standard spectrometers that suffer from detector saturation at high cell densities, which requires time consuming sample dilutions and lacks compatibility with flat-walled cell culture flasks like Roux bottles. Here we describe a 3D-printable device to measure optically dense algal cultures directly within these cultivation vessels. This instrument combines light attenuation and scattering, resulting in a dual-sensor system that utilizes near-infrared light (950 nm) to precisely quantify biomass without interference from changing photosynthetic pigments. Cultures that reach high cell densities can be assumed to be accurately quantified in situ using the scattered signal, preserving the integrity of the culture and eliminating the need for subsampling. This method is adaptable to the most common geometries of Roux bottles (T-25, T-75, and T-175) and could be used to monitor small-scale batch cultures without contamination risks. A cyanobacteria algal species (Limnospira platensis) and three flask sizes were analysed on light attenuation linearity and scattering detection limits at different cell concentration, demonstrating that the light attenuation module can reliably measure biomass up to an OD of 3.1 (equivalent to >1.5 g/L) without requiring dilution.

Perovskite quantum dots (PQDs) are exceptionally promising next-generation optical materials for bioimaging applications. This paper presents an optical PQDs nanocomposite method to identify bacterial species associated with breast cancer, employing a LSTM deep learning model. The results indicate that the developed PQD-based nano-sensor offers impressive sensitivity, selectivity, and practical application. The four bacterial species identified in the study, Pseudomonas aeruginosa, Vibrio parvula, Acinetobacter baumannii, and Streptococcus vestibularis, were considered the most relevant indicators of breast cancer. With rapid detection, an intuitive design, and improved reliability, the PQD sensor could be an excellent breast cancer diagnostic tool for a point-of-care application. Furthermore, it has the potential to be integrated into healthcare systems, specifically in resource-limited settings, thereby improving the accessibility and efficiency of early breast cancer detection.

Rapid tests with visual readout can quickly help identify and triage at-risk patients. However, multiplexed visual tests (MVTs) for targets with clinical thresholds above the limit of detection -- e.g., rogue autoantibodies (raAbs) -- are lacking because test interdependency and cross-reactivity make optimization intractable. Here, we introduce a conceptual and experimental framework to synchronize visual readout and clinical thresholds for multiple, cross-reacting targets simultaneously, and illustrate it with a 3D-printed, structurally-preprogrammed capillaric MVT for anti-interferon (IFN)- and -{omega} raAbs and anti-SARS-CoV-2 spike protein (anti-SCoV2) antibodies. Using design of experiments, we sought and identified parameters that collectively govern the background of all tests (buffer composition, ionic strength), and ones that individually govern assay signal and sensitivity (capture probe density, sample volume), thus enabling both collective background reduction and independent tuning of test line visual threshold. The instrument-free MVT is highly sensitive (pg-ng mL-1), reproducible (CV<10% in plasma), and completed in <1 h. We benchmarked the threshold-calibrated MVTs to microplate ELISA with 41 COVID-19 patient plasma samples yielding ROC-AUCs of 0.97, 1.00, and 0.98 for anti-IFN-, -IFN-{omega} and -SCoV2 tests, respectively. The proposed framework for synchronizing multiple visual readouts with respective clinical thresholds, combined with capillarics, opens the door to instrumentation-free MVTs for point-of-care use.

Electrical stimulation (ES) and transepithelial/transendothelial electrical resistance (TEER) measurements are essential techniques in cell biology and tissue engineering, yet commercial devices for these applications cost between USD 2,500-9,000 and typically offer only one functionality. We present LATEER (Low-cost Arduino-based TEER and Electrical stimulation device), an open-source hardware platform that combines both ES and TEER measurement capabilities at a total cost below USD 100. The device features four independent channels, configurable pulsatile signals (amplitude up to 8.2 V, frequency 0.1-500 Hz, pulse width [&ge;]0.1 ms), and a resistance measurement range of 300 {Omega} to 1 M{Omega}, with <5% error for R {gtrsim} 4.7 k{Omega}. LATEER uses commercially available graphite pencil leads as electrodes ([~]USD 2 vs. USD 350 for commercial Ag/AgCl electrodes), which demonstrated excellent biocompatibility in cell culture. The system includes 3D-printed electrode holders compatible with standard 12-well and 24-well plates, allowing microscope visualization without electrode removal, and a Python-based graphical user interface for parameter configuration and real-time data acquisition. Because the electrodes remain fixed in the plate lid and only a single cable enters the incubator, both stimulation and resistance measurement can run continuously under standard culture conditions (37 {degrees}C, 5% CO2) without removing the plate or repositioning the electrodes, avoiding the temperature excursions and placement variability inherent to manual chopstick measurements. Validation with human pluripotent stem cell-derived cardiomyocytes demonstrated reliable frequency capture (electrical pacing) of the contracting monolayer, with a capture threshold between 250 and 400 mV/mm and controlled pacing across the 0.5-5 Hz range. TEER functionality was verified with mesenchymal stem cells, where the device resolved cell-density-dependent differences in electrical resistance in real time. All design files, firmware, and software are freely available under the CERN-OHL-S v2 license, enabling replication and customization by research laboratories worldwide. HighlightsO_LIAn open-source device combines electrical stimulation and TEER measurement under $100 C_LIO_LIGraphite electrodes offer biocompatibility at 0.6% cost of commercial alternatives C_LIO_LIFour independent channels with configurable parameters and real-time data logging. C_LIO_LIContinuous run setup in-incubator; no electrode repositioning needed C_LIO_LIValidated with stem cell-derived cardiomyocytes, achieving frequency capture (threshold 250-400 mV/mm) C_LIO_LI3D-printed holders enable microscope visualization without electrode removal C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/743263v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@98f9deorg.highwire.dtl.DTLVardef@13c73aborg.highwire.dtl.DTLVardef@1cdf099org.highwire.dtl.DTLVardef@16ed0cf_HPS_FORMAT_FIGEXP M_FIG C_FIG Specifications Table O_TBL View this table: org.highwire.dtl.DTLVardef@4ef802org.highwire.dtl.DTLVardef@7c651borg.highwire.dtl.DTLVardef@d20013org.highwire.dtl.DTLVardef@102fc89org.highwire.dtl.DTLVardef@111b927_HPS_FORMAT_FIGEXP M_TBL C_TBL

Gold electrodes are attractive substrates for bioelectronic and cell-free synthetic biology platforms because they are conductive, chemically stable, biocompatible, and readily functionalized through thiol-gold chemistry. Here, gene-length DNA monolayers assembled on planar gold electrodes as reusable templates for cell-free protein expression are investigated. Using thiol-modified sfGFP genes, DNA surface density is shown to be tunable by changing the DNA concentration during incubation, with the immobilized genes able to support cell-free sfGFP expression directly from the electrode surface. Further, the effects of applied voltage, storage, repeated reactions, reducing agents, and protein fouling on monolayer stability and expression output are examined. While some conditions lead to loss of reusable expression activity, dense chemisorbed monolayers can retain partial function under neutral, non-reducing conditions and are relatively robust to protein exposure. In contrast, low-density physisorbed monolayers show a stronger relationship between DNA loss and expression output. Finally, when using gold-mediated fluorescence quenching to monitor changes in DNA conformation, surface-bound DNA demonstrates electrophoretic addressability. Together, these results establish DNA- functionalized planar electrodes as a promising foundation for modular, addressable cell-free expression platforms.

Hormone dynamics are central to a multitude of conditions in women's health; however, limitations in the cost, convenience, and multiplexing capacity of current hormone assays preclude routine longitudinal hormone testing. This limitation severely restricts our understanding in this historically neglected field, presenting a need for improved bioanalytical tools to support hormone-informed care. Silicon photonic resonator sensors have excellent multiplexing capacity and scalable fabrication, and offer strong potential to meet this important need. Here we demonstrate, for the first time, multiplexed silicon photonic detection of two urinary hormone biomarkers, follicle-stimulating hormone (FSH) and pregnanediol-3-glucuronide (PdG), using sub-wavelength grating ring resonators and a polydopamine-based, regenerable surface chemistry. We combine a sandwich immunoassay for FSH and an antigen-down competitive immunoassay for PdG on the same chip, both amplified using a commercially available quantum-dot-streptavidin conjugate and with on-chip regeneration over multiple measurement cycles. In artificial urine, this proof-of-principle multiplexed assay achieves limits of detection of 0.594 ng/mL for FSH and 19.1 ng/mL for PdG. This architecture is inherently scalable to tens of hormone targets on a millimetre-scale chip (a >10x multiplexing improvement over typical lateral-flow immunoassays) enabling future low-cost, quantitative at-home hormone panels that could transform research, diagnostics, and treatment in women's health.