Biology subjects

Soh, H. T.

Publications and source records attributed to Soh, H. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Charge-screening dominates signal transduction in electrochemical nucleic acid sensors for aminoglycosides

Electrochemical aptamer-based (EAB) sensors have emerged as a promising framework for reagentless, real-time molecular monitoring in complex biological matrices. Signal transduction in a prominent benchmark system--targeting aminoglycoside antibiotics--has been nearly universally attributed to target-specific, binding-induced conformational changes that modulate the distance between a redox reporter and the electrode surface. Here, using a suite of interfacial electrochemical and optical characterization methods, we systematically show that charge-screening, not structure switching, dominates the signaling mechanism in the case of the aminoglycoside EAB sensor. First, we demonstrate that the legacy aminoglycoside sensor sequence exhibits apparent affinities and relative electron kinetic modulations similar to unrelated control sequences, including generic stem-loop hairpins, unstructured poly-T sequences, and short duplexes. Multi-modal validation via bio-layer interferometry (BLI), circular dichroism (CD), and fluorescence spectroscopy confirms that this promiscuous target association occurs across disparate DNA sequences and without detectable conformational reconfiguration. Further investigation via charge-scaling polyamine assays and on-electrode fluorescence imaging demonstrate that the dominant signal transduction mechanism in these sensors is instead driven by macro-scale electrostatic backbone charge-screening.

bioengineering↗

bioRxiv2026

Robust Regularization Enables Automated, Real-Time Square-Wave Voltammetry Signal Quantification

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.

bioengineering↗

bioRxiv2026

Rationally designed split Lettuce aptamer based on large scale mutational analysis

Split aptamer biosensors offer exceptionally low background by assembling only in the presence of a target analyte; however, their performance is frequently limited by the lack of robust design rules for selecting effective split sites. Existing approaches largely rely on heuristic, structure-based assumptions that are poorly validated and often yield suboptimal signal. Herein, we introduce a systematic, data-driven strategy for identifying high-performance split sites within fluorogenic DNA aptamers. Using our massively-parallel aptamer performance analyzer (MAPA) platform, we performed comprehensive single- and double-mutant analysis of the DFAME-binding region of the fluorogenic DNA aptamer Lettuce, informed by its three-dimensional structure. Dimensionality reduction and clustering of the resulting sequence-function landscape revealed mutation-tolerant elements within the binding domain that are suitable for splitting while preserving fluorophore activation. Sensors constructed using these non-intuitive split sites, which are unconventional by standard design principles, exhibited a nearly four-fold improvement in fluorescence signal-to-background ratio for SARS-CoV-2 RNA detection compared to a prior split-Lettuce design. The same split architecture also enabled robust detection of high-pathogenicity H5Nx avian influenza RNA. These results demonstrate that large-scale, data-driven interrogation of aptamer sequence-function relationships can identify non-intuitive split sites and provide a proof-of-concept framework for developing measurement-based design principles for split-aptamer biosensors.

bioengineering↗

bioRxiv2026

Aptamer charge-amplified field-effect transistor biosensors achieve picomolar detection limits for small-molecule biomarkers in complex biological matrices

Aptamers are attractive receptors for small-molecule biomarker detection in complex samples because of their high stability, affinity, and specificity, but aptamer-based sensors generally lack the sensitivity to detect low-abundance analytes. As a solution, we developed the charge-amplified FET (CAFET) aptamer biosensor, which is designed to amplify the net charge variation within the Debye length that occurs as a consequence of aptamer-target binding. Our sensor utilizes a strand-displacement aptamer switch, which releases an initially-hybridized displacement strand (DS) upon target binding and thus induces a measurable net charge variation within the Debye length that is amplified to a large FET current response as signal readout. This signal can be further enhanced by adding a charge label to the DS. As a consequence, our sensor can achieve far greater sensitivity than previously described aptamer-FET sensors, where the binding-induced local charge variation is modest. We demonstrate 3-hydroxykynurenine and progesterone detection with a picomolar limit of detection in undiluted human plasma--four orders of magnitude lower than the dissociation constant (KD) of the aptamer component. The CAFET sensor design is modular and should be adaptable for the detection of a wide range of clinically-informative low-abundance analytes in complex samples.

bioengineering↗

bioRxiv2025

Algorithm-Powered Analyzer for Continuous Electrochemistry: A Toolkit for Real-Time Electrochemical Data Analysis

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.

bioengineering↗

bioRxiv2025

Discovery of high-specificity DNA aptamers for progesterone using a high-throughput array platform

Aptamer-based biosensors offer several advantages for detecting small molecules, including chemical stability and compatibility with diverse sensing formats. However, developing highly specific DNA aptamers that can distinguish between structurally similar small-molecule analytes remains a major challenge. Steroid hormones share a common four-ring scaffold, and even small modifications in functional groups can lead to distinct biological activities. Although DNA aptamers targeting the female reproductive hormone progesterone have been previously reported, many exhibit substantial cross-reactivity with other steroid hormones. In this work, we report the discovery of DNA aptamers with high affinity and specificity for progesterone using our aptamer array platform, which can characterize millions of aptamer candidates in a single, automated experiment. This platform allows us to profile aptamer libraries from earlier rounds of systematic evolution of ligands by exponential enrichment (SELEX), while preserving sequence diversity. Using this strategy, we identified multiple aptamers with nanomolar affinity for progesterone and minimal cross-reactivity to structurally related steroids. These high-specificity aptamers provide a strong foundation for the development of biosensors applicable to both clinical diagnostics and biological research.

bioengineering↗

bioRxiv2025

Accelerated Discovery of Aptamer Beacons via Massively Parallel Screening

Aptamer beacons are unimolecular probes that undergo a reversible conformational change upon target binding, making them a promising tool for the real-time detection and monitoring of molecular analytes. However, the development of such sensors has been impeded by the lack of generalizable tools for the efficient discovery and optimization of aptamer beacons for diverse molhhtmlecular targets. Here, we present a scalable approach for converting existing strand-displacement aptamer switches into aptamer beacons by introducing targeted mismatches within their non-target-binding stem domain, destabilizing the aptamer structure to an extent that it can only refold upon binding its target. In order to perform this screening in a high-throughput fashion, we have developed the Massively-parallel Aptamer Performance Analyzer (MAPA), an automated, fluorescence-based screening platform based on a reconfigured sequencing instrument that can functionally evaluate millions of aptamer variants in parallel. Using MAPA, we identified multiple aptamer beacons for glucose, serotonin, and dopamine, and demonstrated that these beacons retain their sensing performance when translated from the on-chip surface-based MAPA format to a solution-based assay. We performed all three aptamer beacon experiments on a single MAPA chip, and even greater multiplexing should be possible, greatly accelerating the discovery of aptamer-based sensors for real-time molecular detection.

bioengineering↗

bioRxiv2025

Continuous FACS sorting of double emulsion picoreactors with a 3D printed vertical mixer.

High-throughput screening and directed evolution using microfluidic picoreactors have produced high-activity enzymes. In this approach, a substrate is co-encapsulated with a candidate enzyme and individual picoreactors are sorted based on an activity reporter. While many approaches use water-in-oil droplets (single emulsions) for fluorescence-activated droplet sorting (FADS) on custom-fabricated microfluidic devices that require integrated optics and electronics, recent approaches have lowered the engineering barriers to adoption by using simple microfluidic droplet generators to produce water-in-oil-in-water droplets (double emulsion picoreactors, DEs) that can be sorted with commercial FACS (fluorescence-activated cell sorting). Despite the simplified engineering requirements, high variability in loading rates and low yield during loading are barriers to efficient DE FACS sorting. Here, we optimized surfactants to enhance DE stability and demonstrated that a 3D-printed corkscrew on the sample line acts as a vertical mixer to enable more continuous loading. With these optimized loading conditions, we analyzed 1.17 million DEs in four 10-minute sorting rounds with a mean frequency of 480 Hz (390 Hz including sample exchanges); in a mock sort of 10% fluorescent DEs, we achieved 89.2{+/-}1.1% accuracy and 78{+/-}0.9% recovery with our optimized loading protocol. Overall, improved ease-of-use and throughput for FACS sortable DEs should expand the accessibility of directed evolution in controlled in vitro environments.

bioengineering↗

bioRxiv2025

Direct Optical Detection of Factor Xa Activity in Minimally Processed Whole Blood

The ability to measure factor Xa activity directly in whole blood samples offers a path toward point-of-care monitoring and personalized anticoagulant dosage, potentially reducing bleeding risk and other anticoagulant-associated complications. We present a strategy to enable direct optical detection of factor Xa in minimally processed whole blood samples. Our strategy relies on a custom FRET-pair labeled DNA-peptide substrate, allowing FRET ratio to be monitored as an indicator of factor Xa activity. Substrates are tethered to a tapered-fiber sensor to allow evanescent detection of fluorescence directly at the sensor surface, minimizing background media interference and enabling detection directly in blood samples. After characterizing the custom substrate and demonstrating the correlation of fiber-based measurements to an existing chromogenic assay, we demonstrate the detection of endogenous factor Xa activity in >85% whole blood. Finally, we demonstrate the detection of therapeutic concentrations of enoxaparin, a widely used anticoagulant, directly in 90% whole blood in less than an hour and correlate these measurements to activated partial thromboplastin time (aPTT) testing. Together, these results indicate a promising strategy to achieve point-of-care factor Xa detection, enabling personalized anticoagulant treatment and reducing adverse outcomes.

bioengineering↗

bioRxiv2024

Peptide sequencing via reverse translation of peptides into DNA

Scalable methods that can accurately sequence peptides at single-amino acid resolution could significantly advance proteomic studies. We present a protein sequencing method based on the "reverse translation" of peptide sequence information into DNA barcodes that document the identity, position, and the originating peptide of each amino acid. We employ a modified Edman degradation process that converts peptides into DNA-barcoded amino acids, which are subsequently detected by proximity extension assay, yielding multi-barcoded DNA outputs that can be PCR amplified and sequenced. Using our method, we sequenced multiple consecutive amino acids within a model peptide. This method also enables the differentiation of single amino acid substitutions, and the identification of post-translational modifications and their positions within multiple peptides simultaneously. With further development, we anticipate that this method will enable highly parallel de novo protein sequencing with single-molecule sensitivity.

bioengineering↗

bioRxiv2024

A multiplexed and quantitative assay for small-molecule detection via photo-crosslinking of structure switching aptamers

There is an unmet need for molecular detection assays that enable the multiplexed, quantification of smallmolecule analytes. We present xPlex, an assay that combines aptamer switches with ultraviolet-crosslinkable complementary strands to record target-binding events. When the aptamers small-molecule target is present, the crosslinkable strand is displaced, enabling PCR amplification and detection of the relevant aptamer. In the absence of that target, the aptamer is readily cross-linked to the strand, preventing amplification from happening. The resulting aptamer-specific amplicons can be detected and quantified in a multiplexed fashion using high-throughput sequencing. We demonstrate quantitative performance for a pair of small-molecule analytes, dopamine and glucose, and show that this assay retains good specificity with mixtures of the two molecules at various concentrations. We further show that xPlex can effectively evaluate the specificity of cross-reactive aptamers to a range of different small-molecule analytes. We believe the xPlex assay format could offer a useful strategy for achieving multiplexed analysis of small-molecule targets in a variety of scenarios.

bioengineering↗

bioRxiv2023

Theoretical framework and experimental validation of multiplexed analyte quantification using cross-reactive affinity reagents

Gold standard immunoassays depend on specific affinity reagents for accurate molecular quantification. Any cross-reactivity of affinity reagents, wherein the reagent non-specifically binds to unintended molecules, can create false positive binding signals and result in inaccurate quantification of analytes. Mitigating cross-reactivity represents one of the greatest challenges in molecular diagnostics, and remains an unsolved problem. To instead overcome the effects of cross-reactivity, we present a mathematical framework that uses generalized binding equations and noise estimation to enable the use of multiple cross-reactive reagents for multiplexed molecular quantification. As a proof-of-concept, we experimentally demonstrate accurate quantification of a small molecule for which no specific affinity reagents are available, even at high concentrations of a cross-reactive molecule. Furthermore, this robust schema yields well-defined bounds of quantification that make it easier to assess the quality of assay results and predicts under which conditions assay performance is likely to break down. This work turns cross-reactive affinity reagents, which were previously a liability, into an asset for achieving accurate quantification of analytes.

bioengineering↗

bioRxiv2023

Aptamer-antibody chimera sensors for sensitive, rapid and reversible molecular detection in complex samples

The development of receptors suitable for the continuous detection of analytes in complex, interferent-rich samples remains challenging. Antibodies are highly sensitive but difficult to engineer in order to introduce signaling functionality, while aptamer switches are easy to construct but often yield only modest target sensitivity. We present here the programmable antibody and DNA aptamer switch (PANDAS), which combines the best features of both systems by using a nucleic acid tether to link an analyte-specific antibody to an internal strand-displacement (ISD)-based aptamer switch that recognizes the same target. The monoclonal antibody mediates initial analyte binding due to its higher affinity; the resulting increase in local analyte concentration then leads to cooperative binding and signaling by the ISD switch. We developed a PANDAS sensor for the clotting protein thrombin and show that this design achieves 100-fold enhanced sensitivity compared to using an aptamer alone. This design also exhibits reversible binding, enabling repeated measurements with temporal resolution of [~]10 minutes, and retains excellent sensitivity even in interferent-rich samples. With future development, this PANDAS approach could enable the adaptation of existing protein-binding aptamers with modest affinity into sensors that deliver excellent sensitivity and minute-scale resolution in minimally prepared biological specimens.

bioengineering↗

bioRxiv2023

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, we have developed an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time. As a proof of concept, we performed real-time imaging of Dictyostelium discoideum cells, a well-studied amoeba model wherein inter-cellular communication is performed though cAMP signaling. We engineered a set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals. By combining multiple switches with different dynamic ranges, we can measure cAMP concentrations spanning three orders of magnitude in a single experiment. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, we achieved the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements. Using different aptamer switches, this approach could be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

bioengineering↗

bioRxiv2023

A rapid ELISA platform with no sample preparation requirement

Since its invention in the 1970s, the enzyme-linked immunosorbent assay (ELISA) has served as the "gold-standard" for blood and plasma protein biomarker quantification. However, ELISAs require significant amounts of sample preparation entailing multiple reagent additions, incubations, and washing steps, limiting their clinical usefulness in the context of diagnosis and prognosis of rapidly evolving medical conditions. In this work, we describe the instant ELISA biosensor platform, a probe that can be exposed directly to blood or other biological samples and quantifies protein biomarkers within 15 minutes. The sensor leverages a novel affinity reagent termed monolithic dual-antibody clamp (MDAC) which preserves the specificity, sensitivity, and generalizability of ELISA while also enabling rapid analysis of unprocessed blood and other complex matrices. Using MDAC in chicken media, we demonstrate picomolar quantification of the inflammatory marker tumor necrosis factor alpha (TNF), as well as monocyte chemotactic protein (MCP)-1, a useful prognostic indicator of cytokine release syndrome (CRS) during chimeric antigen receptor (CAR) T-cell immunotherapy. Finally, we demonstrate MCP-1 quantification in plasma samples from patients who had undergone CAR T-cell treatment.

bioengineering↗

bioRxiv2023

An Antibody-Based Molecular Switch for Continuous Biosensing

We present a generalizable approach for designing biosensors that can continuously detect specific biomarkers in real time and without sample preparation. This is achieved by converting existing antibodies into target-responsive "antibody-switches" that enable continuous optical biosensing. To engineer these switches, antibodies are linked to a molecular competitor through a DNA scaffold, such that competitive target binding induces scaffold switching and fluorescent signaling of changing target concentrations. As a demonstration, we designed antibody-switches that achieve rapid, sample-preparation-free sensing of digoxigenin and cortisol in undiluted plasma. We showed that, by substituting the molecular competitor, we can further modulate the sensitivity of our cortisol switch to achieve detection at concentrations spanning 3.3 nM to 3.3 mM. Finally, we integrated this switch with a fiber-optic sensor to achieve hours-long continuous sensing of cortisol in buffer with <5-minute time resolution. We believe this modular sensor design can enable continuous biosensor development for many biomarkers.

bioengineering↗

bioRxiv2023

Continuous optical detection of small-molecule analytes in complex biomatrices

Current technology for measuring specific biomarkers - continuously in complex samples, without sample preparation - is limited to just handful of molecules such as glucose and blood oxygen. In this work, we present the first optical biosensor system that enables continuous detection of a wide range of biomarkers in complex samples, such as human plasma. Our system employs a modular duplex-bubble switch (DBS) architecture that converts aptamers into structure-switching fluorescence probes whose affinity and kinetics can be readily tuned. These DBS constructs are coupled to a fiber-optic detector that measures the fluorescence change only within an evanescent field, thereby minimizing the impact of background autofluorescence and enabling direct detection of analytes at physiologically relevant concentrations even in interferent-rich sample matrices. Using our system, we achieved continuous detection of dopamine in artificial cerebrospinal fluid for >24 hours with sub-second resolution and a limit of detection (LOD) of 1 {micro}M. We subsequently demonstrated the systems generalizability by configuring it to detect cortisol with nanomolar sensitivity in undiluted human plasma. Both sensors achieved LODs orders of magnitude lower than the KD of the DBS element, highlighting the potential to achieve sensitive detection even when using aptamers with modest affinity.

biophysics↗

bioRxiv2023

Extending the dynamic range of biomarker quantification through molecular equalization

Precision medicine requires highly scalable methods of multiplexed biomarker quantification that can accurately describe patient physiology. Unfortunately, contemporary molecular detection methods are generally limited to a dynamic range of sensitivity spanning just 3-4 orders of magnitude, whereas the actual physiological dynamic range of the human plasma proteome spans more than 10 orders of magnitude. Current methods rely on sample splitting and differential dilution to compensate for this mismatch, but such measures greatly limit the reproducibility and scalability that can be achieved--in particular, the effects of non-linear dilution can greatly confound the analysis of multiplexed assays. We describe here a two- pronged strategy for equalizing the signal generated by each analyte in a multiplexed panel, thereby enabling simultaneous quantification of targets spanning a wide range of concentrations. We apply our EVROS strategy to a proximity ligation assay and demonstrate simultaneous quantification of four analytes present at concentrations spanning from low femtomolar to mid-nanomolar levels. In this initial demonstration, we achieve a dynamic range spanning seven orders of magnitude in a single 5 {micro}l sample of undiluted human serum, highlighting the opportunity to achieve sensitive, accurate detection of diverse analytes in a highly multiplexed fashion.

bioengineering↗