Biology subjects

Raut, B.

Publications and source records attributed to Raut, B..

8 recordsLinked to original sources

bioRxiv2026

A rapid, field-deployable paper-based biosensor for the detection of African swine fever virus in whole blood

African swine fever virus (ASFV) poses a major transboundary threat to global swine production, underscoring the need for rapid and field-deployable diagnostic tools. Although quantitative polymerase chain reaction (qPCR)-based assays are the standard molecular assay for ASFV detection, their reliance on centralized laboratory infrastructure, multi-step sample preparation, and trained personnel limit their utility for timely decision-making at the point of need (PON). Here, we report a portable molecular diagnostic platform that enables colorimetric quantitative loop-mediated isothermal amplification (qLAMP) directly from diluted whole blood on microfluidic paper-based analytical devices ({micro}PADs). The assay targets the conserved ASFV viral protein 72 (VP72) and topoisomerase II (TOPII) genes and incorporates objective image-based colorimetric signal analysis to reduce user-dependent interpretation. Using plasmid DNA spiked into whole blood diluted to 5% (v/v) in 5% D-mannitol, the {micro}PAD-LAMP assay achieved a limit of detection (LOD) of 25 copies per reaction (67 copies/{micro}L of whole blood sample) for VP72 targets with no observed cross-reactivity against nine common swine pathogens, demonstrating 100% analytical sensitivity and specificity during in-house testing and 90% and 92% analytical sensitivity and specificity respectively in an external laboratory evaluation. The complete assay was performed within 60 minutes using a portable heating and imaging platform. Together, these results demonstrate a simple, DNA extraction-free molecular diagnostic approach that enables rapid and reliable ASFV detection from whole blood applicable to field-relevant conditions.

bioengineering↗

bioRxiv2026

Bacteroidales on Harvesters: Baseline Prevalence and Abundance

Fresh produce encounters pathogens at various stages of production and supply, with the harvesting process serving as one of these stages. To evaluate contamination associated with harvesting, we systematically swabbed zone 1 harvester surfaces and quantified Bacteroidales as a fecal biomarker using quantitative polymerase chain reaction (qPCR). Baseline contamination was dominated by non-detects, with occasional low-level detections (<25 copies/cm2) near the assay limit of detection (LoD). Detection occurred more frequently post-harvest (overall [~]4% pre-harvest and 10% post-harvest), while microbial loads remained low, indicating that harvesting primarily affected the likelihood of low-level contamination rather than increasing contamination abundance. Additionally, we developed and field-deployed a portable loop- mediated isothermal amplification (LAMP) assay for rapid harvester hygiene assessment and benchmarked its field performance against qPCR. Together, these results support a practical molecular tool for monitoring fecal contamination and informing cleaning and sanitization decisions.

bioengineering↗

bioRxiv2026

A solid-state heater-imager for quantitative evaluation of colorimetric isothermal nucleic acid amplification on paper

Maintaining precise isothermal conditions in portable nucleic acid amplification tests (NAATs) is critical for reproducible results but remains challenging with conventional single-sided thin-film heaters, which exhibit temperature gradients and strong dependence on ambient conditions. To close this gap, we engineered ThermiQuant VitroMini, a dual-sided heater design that achieves volumetric-level temperature uniformity using thin-film heaters while preserving optical transparency for real-time colorimetric loop-mediated isothermal amplification (LAMP) analysis on microfluidic paper-based analytical devices ({micro}PADs). The device integrates two independently regulated indium tin oxide (ITO) heaters (8 {Omega} each) controlled by independent proportional-integral-derivative (PID) algorithms. Heaters were evaluated under controlled ambient environments of 4 {degrees}C (refrigerated), 23 {degrees}C (room temperature), and 50 {degrees}C (oven). Analytical tests were performed using a colorimetric LAMP assay targeting the SARS-CoV-2 orf7ab gene on {micro}PADs preloaded with dried LAMP reagents, with time-lapse images (30 seconds interval) analyzed via Amplimetrics software. VitroMini maintained 65 {+/-} 0.5 {degrees}C across 4 to 50 {degrees}C ambient conditions and achieved a limit of detection of 34 copies/reaction (4.5 copies/{micro}L) and limit of quantification of 1000 copies/reaction (133 copies/{micro}L), with quantification time (Tq) linearly correlated with log10 DNA concentration. Dual-sided heating eliminated temperature bias, condensation artifacts, and ambient-dependent variability while preserving optical transparency for real-time quantitative LAMP reaction. ThermiQuant VitroMini bridges the gap between benchtop volumetric heaters and portable diagnostic devices, offering a compact, low-power platform for quantitative colorimetric molecular analysis on paper with potential for decentralized and field-deployable applications.

bioengineering↗

bioRxiv2026

ThermiQuantTM AquaStream: A Portable Instrument for Quantitative Colorimetric Isothermal Nucleic Acid Amplification Reactions in Paper and Tube Formats

Microfluidic paper-based analytical devices ({micro}PADs) are an attractive format for colorimetric nucleic acid amplification tests (NAATs) because they enable low-cost, portable diagnostics in resource-limited settings. However, researchers often optimize colorimetric assays in liquid reactions in tubes before translating them to {micro}PADs. Since both formats require separate instruments for incubation and real-time sensing, direct comparison of reactions between the two formats is difficult. To address these cross-platform limitations, we developed ThermiQuant AquaStream, a portable benchtop device (15 x 20 x 16 cm, [~]5 kg; cost: USD 327) that supports seamless colorimetric loop-mediated isothermal amplification (LAMP) reactions in both {micro}PADs and tubes under a common workflow. The system enables real-time reaction tracking (every 30 seconds) through onboard image processing, precise isothermal control ({+/-}0.5 {degrees}C) using a repurposed consumer-grade sous-vide heater, and medium-throughput (24 tubes or 42 {micro}PADs). Testing with synthetic SARS-CoV-2 orf7ab DNA fragments demonstrated a limit of detection corresponding to a 95% probability of detection (LOD95) of 110 copies per reaction in tube (22 copies/{micro}L) and 39 copies per reaction in {micro}PADs (5 copies/{micro}L), estimated using probit regression. In both formats the limit of quantification (LOQ), defined as the lowest concentration yielding a coefficient of variation (CV) of Tq [≤] 10%, was 250 copies/reaction resulting in a strong linear (R2 = 0.98 & R2 = 0.96 respectively for tube and {micro}PADs) standard calibration curves. ThermiQuant AquaStream provides an affordable and versatile benchtop platform capable of supporting both tube- and {micro}PAD-based colorimetric LAMP assays, serving as a proof-of-concept research tool for assay development and molecular diagnostics in One Health settings such as clinics, farms, and field environments.

bioengineering↗

bioRxiv2026

ThermiQuant(TM) MegaScan: High-throughput isothermal reactor with quantitative colorimetric readout for paper-based nucleic acid amplification tests

Isothermal nucleic acid amplification tests (NAATs), such as loop-mediated isothermal amplification (LAMP) implemented on microfluidic paper-based analytical devices ({micro}PADs), enable inexpensive and rapid ([≤]60 min) colorimetric molecular diagnostics; however, no existing instrument supports high-throughput (>100 reactions) quantitative analysis of colorimetric isothermal assays on paper substrates under controlled laboratory conditions. To address this gap, we developed ThermiQuant MegaScan, a scanner- and water-bath-based platform that accommodates a 160-reaction {micro}PAD cartridge, maintains uniform incubation at 65 {+/-} 0.5 {degrees}C, and enables real-time imaging every 30 s. We also developed accompanying software, Amplimetrics, for automated {micro}PAD detection and kinetic colorimetric analysis. Using paper-based colorimetric LAMP targeting the SARS-CoV-2 orf7ab region, the assay achieved a limit of detection at 95% probability (LOD95) of 34 copies per reaction (5 copies/{micro}L) and a limit of quantification (LOQ) of 250 copies per reaction (33 copies/{micro}L) using purified synthetic DNA targets, and achieved 72% sensitivity and 100% specificity relative to digital PCR (dPCR) for diluted human nasopharyngeal (NP) swab virus samples. We further evaluated the effects of viral and universal transport media (VTM/UTM) on assay performance and found that linear calibration derived from synthetic targets do not reliably translate to clinical samples in these media. Together, these results establish ThermiQuant MegaScan as a high-throughput laboratory research platform for standardized evaluation, optimization, and benchmarking of paper-based colorimetric nucleic acid amplification assays.

bioengineering↗

bioRxiv2025

Evaluation of Paper-based Loop-Mediated Isothermal Amplification

Colorimetric loop-mediated isothermal amplification (LAMP) on microfluidic paper-based analytical devices (PADs) offers a low-cost, disposable, and equipment-free alternative to liquid LAMP assays. However, amplification on PADs is consistently slower, by 5-46%, than reactions in tubes. To identify the origin of this delay, we evaluated heat transfer, diffusion in porous cellulose, and nonspecific adsorption of LAMP components across both high- and low-copy input regimes. Our results show that once thermal equilibrium is reached, reduced effective diffusion is the dominant contributor to the kinetic lag at low copy numbers, whereas nonspecific adsorption becomes the primary barrier at higher template concentrations. Pre-coating the paper with bovine serum albumin (BSA) mitigates adsorption. It narrows the tube-to-paper gap, thereby accelerating amplification of the SARS-CoV-2 ORF7ab synthetic gene by an average of 6 minutes, from 1E3 to 1E5 copies per reaction. These findings provide a mechanistic basis for the copy-number-dependent behavior of PAD LAMP and offer simple, low-cost strategies to improve the speed and reliability of PAD nucleic acid assays.

bioengineering↗

bioRxiv2023

A portable, easy-to-use paper-based biosensor for rapid in-field detection of fecal contamination on fresh produce farms

Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations target clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detecting Bacteroidales as a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies of Bacteroidales per cm2) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation.

bioengineering↗

bioRxiv2023

A drop dispenser for simplifying on-farm detection of foodborne pathogens

Rapid nucleic-acid biosensors are useful for on-farm detection of foodborne pathogens on fresh produce during pre-season and pre-harvest stages. Such tools aim to be user-friendly so that a producer could operate them in a few simple steps and detect multiple targets. Currently, an easy-to-use device for on-farm applications does not exist commercially. One of the bottlenecks is the delivery of a prescribed amount of sample to the reaction sites of the biosensor using a simple and precise approach. Here, we developed drop dispensers using 3D printing and a hydrophilic surface chemistry treatment to generate precise drops (DNA/bacterial samples) of a few micro-liters ([~]20 to [~]33 {micro}L). We tested multiple copies of these devices over time of repeated application to estimate their shelf-lives which is about one month. In addition to drop generation tests, we employed these devices in nucleic-acid testing. The tests used loop-mediated isothermal amplification (LAMP) to detect DNA or whole cells of Shiga-toxin-producing Escherichia coli O157:H7. These tests were performed to simulate the on-farm sample collection (using collection flags that we previously designed) and delivery using the drop dispensers. Our results showed that these devices performed similarly to standard commercial pipettors in LAMP assays, providing a limit of detection of 7.8x106 cell/mL for whole-cell detection. This drop dispenser will eventually be part of a user-friendly consumable kit that will enable performing LAMP assays by non-specialist users for a cost of USD 4 per test.

bioengineering↗