Nanoplasmonic microarray–based solid-phase amplification for highly sensitive and multiplexed molecular diagnostics: application for detecting SARS-CoV-2

IF 5.3 2区化学 Q1 CHEMISTRY, ANALYTICAL

Microchimica Acta Pub Date : 2024-10-29 DOI:10.1007/s00604-024-06723-4

Ji Young Lee, Hyowon Jang, Sunjoo Kim, Taejoon Kang, Sung-Gyu Park, Min-Young Lee

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Abstract

A novel approach is introduced using nanoplasmonic microarray–based solid-phase recombinase polymerase amplification (RPA) that offers high sensitivity and multiplexing capabilities for gene detection. Nanoplasmonic microarrays were developed through one-step immobilization of streptavidin/biotin primers and fine-tuning the amplicon size to achieve high plasmon-enhanced fluorescence (PEF) on the nanoplasmonic substrate, thereby improving sensitivity. The specificity and sensitivity of solid-phase RPA on nanoplasmonic microarrays was evaluated in detecting E, N, and RdRP genes of SARS-CoV-2. High specificity was achieved by minimizing primer-dimer formation and employing a stringent washing process and high sensitivity obtained with a limit of detection of four copies per reaction within 30 min. In clinical testing with nasopharyngeal swab samples (n = 30), the nanoplasmonic microarrays demonstrated a 100% consistency with the PCR results for detecting SARS-CoV-2, including differentiation of Omicron mutations BA.1 and BA.2. This approach overcomes the sensitivity issue of solid-phase amplification, as well as offers rapidity, high multiplexing capabilities, and simplified equipment by using isothermal reaction, making it a valuable tool for on-site molecular diagnostics.

Graphical abstract

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基于纳米质子微阵列的固相扩增技术用于高灵敏度和多重分子诊断：在检测 SARS-CoV-2 中的应用

本文介绍了一种基于纳米质谱微阵列的固相重组酶聚合酶扩增（RPA）新方法，该方法具有高灵敏度和多重基因检测能力。纳米质子微阵列是通过一步固定链霉亲和素/生物素引物和微调扩增子大小来开发的，以在纳米质子基底上实现高质子增强荧光（PEF），从而提高灵敏度。在检测 SARS-CoV-2 的 E、N 和 RdRP 基因时，对纳米质子微阵列上固相 RPA 的特异性和灵敏度进行了评估。通过最大限度地减少引物二聚体的形成和采用严格的洗涤过程，实现了高特异性；在 30 分钟内，每个反应的检测限为 4 个拷贝，实现了高灵敏度。在鼻咽拭子样本（n = 30）的临床测试中，纳米质谱微阵列检测 SARS-CoV-2 的结果与 PCR 结果的一致性达到 100%，包括区分 Omicron 突变 BA.1 和 BA.2。这种方法克服了固相扩增法的灵敏度问题，而且速度快、复用能力强、使用等温反应简化了设备，是现场分子诊断的重要工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Microchimica Acta 化学-分析化学

CiteScore

9.80

自引率

5.30%

发文量

410

审稿时长

2.7 months

期刊介绍： As a peer-reviewed journal for analytical sciences and technologies on the micro- and nanoscale, Microchimica Acta has established itself as a premier forum for truly novel approaches in chemical and biochemical analysis. Coverage includes methods and devices that provide expedient solutions to the most contemporary demands in this area. Examples are point-of-care technologies, wearable (bio)sensors, in-vivo-monitoring, micro/nanomotors and materials based on synthetic biology as well as biomedical imaging and targeting.