Biology subjects

Gidi, Y.

Publications and source records attributed to Gidi, Y..

7 recordsLinked to original sources

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

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↗

bioRxiv2024

Automated Synthesis of Wireframe DNA Nanotubes

DNA nanotechnology has revolutionized our ability to position matter at the nanoscale, but the preparation of DNA-based architectures remains laborious. To facilitate the formation of custom structures, we report a fully automated method to produce sequence- and size-defined DNA nanotubes. By programming the sequential addition of desired building blocks, rigid DX-tile-based DNA nanotubes (and flexible wireframe DNA structures) are attained, where the total number of possible constructs increases as a power function of the number of different units available. Using single-molecule fluorescence imaging, the kinetics and yield of each synthetic step can be quantitatively determined, revealing differences in self-assembly dynamics as the nanotube is built up from the solid support and providing new insights into DNA self-assembly. The exploitation of automation for both assembly and analysis (tthrough an ad-hoc developed K-means clustering algorithm) facilitates a workflow wherein the synthesis parameters may be iteratively improved upon, demonstrating how a single-molecule assembly-analysis-optimization sequence can be used to generate complex, non-covalent materials in good yield. The presented synthetic strategy is generalizable, making use of equipment already available in most standard laboratories and represents the first fully automated supramolecular assembly on a solid support.

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

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↗