A High-Sensitivity Colorimetric Dot-Blot Aptamer Assay Platform for SARS-CoV-2 Nucleocapsid Protein Detection in Resource-Limited Settings

IF 4.5 2区医学 Q2 CHEMISTRY, MEDICINAL

ACS Infectious Diseases Pub Date : 2026-07-10 Epub Date: 2026-06-05 DOI:10.1021/acsinfecdis.5c01125

Kauser Banu*, Bhairab Mondal*, S. Madhan Kumar, C. Sai Sudha and Raju H,

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Abstract

We developed a colorimetric dot-blot aptamer assay to address the urgent need for accessible, high-performance SARS-CoV-2 diagnostics in resource-limited settings. Our in-house 96-well dot-blot platform featuring optimized PVDF membrane fluidics and standardized clamping combines nucleocapsid protein (N)-specific monoclonal antibody capture with biotinylated DNA aptamer detection, achieving high sensitivity (0.5 ng/mL limit of detection) and exceptional specificity (0/36 false positives) for the conserved nucleocapsid protein. The assay demonstrates 2-fold greater sensitivity than conventional ELISA (p < 0.001). Clinical validation with 230 patient specimens showed 100% sensitivity (95% CI: 96.4–100%) and 100% specificity (95% CI: 96.4–100%), with results in 100 ± 5 min without specialized instrumentation. The assay enables semiquantitative classification of viral load (low, high) based on signal intensity without providing absolute quantitative measurements. The platform merges laboratory-grade performance with point-of-care practicality, supporting high-throughput processing, minimal infrastructure, low cost (<$1 per test), and heat-inactivation compatibility.

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资源有限条件下检测SARS-CoV-2核衣壳蛋白的高灵敏度比色点印迹适体检测平台

我们开发了一种比色点印迹适体测定法，以满足资源有限环境下可获得的高性能SARS-CoV-2诊断的迫切需求。我们的内部96孔点印迹平台具有优化的PVDF膜流体和标准化夹夹，结合了核衣壳蛋白(N)特异性单克隆抗体捕获和生物素化DNA适体检测，对保守的核衣壳蛋白实现了高灵敏度（0.5 ng/mL检测限）和卓越的特异性（0/36假阳性）。该方法的灵敏度是常规ELISA的2倍（p < 0.001）。230例患者标本的临床验证显示100%的敏感性（95% CI: 96.4-100%）和100%的特异性（95% CI: 96.4-100%），无需专门仪器，结果在100±5分钟内得到。该分析能够基于信号强度对病毒载量（低、高）进行半定量分类，而不提供绝对定量测量。该平台将实验室级性能与护理点实用性相结合，支持高通量处理、最小基础设施、低成本(

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来源期刊

ACS Infectious Diseases CHEMISTRY, MEDICINALINFECTIOUS DISEASES&nb-INFECTIOUS DISEASES

CiteScore

9.70

自引率

3.80%

发文量

213

期刊介绍： ACS Infectious Diseases will be the first journal to highlight chemistry and its role in this multidisciplinary and collaborative research area. The journal will cover a diverse array of topics including, but not limited to: * Discovery and development of new antimicrobial agents — identified through target- or phenotypic-based approaches as well as compounds that induce synergy with antimicrobials. * Characterization and validation of drug target or pathways — use of single target and genome-wide knockdown and knockouts, biochemical studies, structural biology, new technologies to facilitate characterization and prioritization of potential drug targets. * Mechanism of drug resistance — fundamental research that advances our understanding of resistance; strategies to prevent resistance. * Mechanisms of action — use of genetic, metabolomic, and activity- and affinity-based protein profiling to elucidate the mechanism of action of clinical and experimental antimicrobial agents. * Host-pathogen interactions — tools for studying host-pathogen interactions, cellular biochemistry of hosts and pathogens, and molecular interactions of pathogens with host microbiota. * Small molecule vaccine adjuvants for infectious disease. * Viral and bacterial biochemistry and molecular biology.