Department of Pathology, Brigham and Women's Hospital, Harvard Medical School and Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts 02115, United States.
ACS Nano. 2023 Mar 28;17(6):5412-5420. doi: 10.1021/acsnano.2c09889. Epub 2023 Mar 6.
In recent years, nanopore-based sequencers have become robust tools with unique advantages for genomics applications. However, progress toward applying nanopores as highly sensitive, quantitative diagnostic tools has been impeded by several challenges. One major limitation is the insufficient sensitivity of nanopores in detecting disease biomarkers, which are typically present at pM or lower concentrations in biological fluids, while a second limitation is the general absence of unique nanopore signals for different analytes. To bridge this gap, we have developed a strategy for nanopore-based biomarker detection that utilizes immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. These DNA fragment reporters produce sets of nanopore signals that form distinctive fingerprints, or clusters. This fingerprint signature therefore allows the identification and quantification of biomarker analytes. As a proof of concept, we quantify human epididymis protein 4 (HE4) at low pM levels in a few hours. Future improvement of this method by integration with a nanopore array and microfluidics-based chemistry can further reduce the limit of detection, allow multiplexed biomarker detection, and further reduce the footprint and cost of existing laboratory and point-of-care devices.
近年来，基于纳米孔的测序仪已成为具有独特优势的强大工具，可应用于基因组学领域。然而，将纳米孔作为高度敏感、定量诊断工具的应用进展受到了几个挑战的阻碍。一个主要的限制是纳米孔在检测疾病生物标志物方面的灵敏度不足，这些生物标志物通常在生物体液中的浓度为 pM 或更低，而另一个限制是通常不存在针对不同分析物的独特纳米孔信号。为了弥补这一差距，我们开发了一种基于纳米孔的生物标志物检测策略，该策略利用免疫捕获、等温滚环扩增和产物的序列特异性片段化，释放多个用于纳米孔检测的 DNA 报告分子。这些 DNA 片段报告分子产生一组纳米孔信号，形成独特的指纹或簇。因此，这种指纹特征允许鉴定和定量生物标志物分析物。作为概念验证，我们在几个小时内以低 pM 水平定量检测人附睾蛋白 4 (HE4)。通过与纳米孔阵列和基于微流控的化学集成来改进这种方法，可以进一步降低检测限，允许多重生物标志物检测，并进一步减小现有实验室和即时检测设备的占地面积和成本。
