Pathogen, prevalent in both natural and human environments, cause approximately 4.95 million deaths annually, ranking them among the top contributors to global mortality. Traditional pathogen detection methods, reliant on microscopy and cultivation, are slow and labor-intensive and often produce subjective results. While nucleic acid amplification techniques such as polymerase chain reaction offer genetic accuracy, they necessitate costly laboratory equipment and skilled personnel. Consequently, isothermal amplification methods like recombinase polymerase amplification (RPA) have attracted interest for their rapid and straightforward operations. However, these methods face challenges in specificity and automated sample processing. In this study, we introduce a self-interferometric digital optofluidic platform incorporating asymmetric direct solid-phase RPA for real-time, label-free, and automated pathogen genotyping. By integration of digital microfluidics with a DNA monolayer detection method using hyperspectral interferometry, this platform enables rapid, specific, and sensitive pathogen detection without the need for exogenous labeling or complex procedures. The system demonstrated high sensitivity (10 CFU·mL^–1), specificity (differentiating four Candida species), detection efficiency (fully automated within 50 min for Gram-negative bacteria), and throughput (simultaneous detection of four indices). This integrated approach to pathogen quantitation on a single microfluidic chip represents a significant advancement in rapid pathogen diagnostics, providing a practical solution for timely pathogen detection and analysis.

中文翻译：

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用于集成和自动化无标记病原体检测的自干扰数字光流体基因分型


病原体普遍存在于自然和人类环境中，每年导致约 495 万人死亡，是导致全球死亡的主要因素之一。传统的病原体检测方法依赖于显微镜和培养，速度慢且劳动强度大，并且经常产生主观结果。虽然核酸扩增技术（如聚合酶链反应）提供了遗传准确性，但它们需要昂贵的实验室设备和熟练的人员。因此，重组酶聚合酶扩增 （RPA） 等温扩增方法因其快速直接的操作而引起了人们的兴趣。然而，这些方法在特异性和自动化样品处理方面面临挑战。在这项研究中，我们介绍了一个自干涉数字光流体平台，该平台结合了不对称直接固相 RPA，用于实时、无标记和自动化的病原体基因分型。通过将数字微流体技术与使用高光谱干涉测量法的 DNA 单层检测方法相结合，该平台可实现快速、特异性和灵敏的病原体检测，而无需外源性标记或复杂的程序。该系统表现出高灵敏度 （10 CFU·mL^–1）、特异性 （区分 4 种念珠菌）、检测效率 （革兰氏阴性菌在 50 分钟内完全自动化）和通量 （同时检测 4 个指标）。这种在单个微流体芯片上进行病原体定量的集成方法代表了快速病原体诊断的重大进步，为及时的病原体检测和分析提供了实用的解决方案。