Abstract

The growing interest in microfluidic biosensors has led to improvements in the analytical performance of various sensing mechanisms. Although various sensors can be integrated with microfluidics, electrochemical ones have been most commonly employed due to their ease of miniaturization, integration ability, and low cost, making them an established point-of-care diagnostic method. This concept can be easily adapted to the detection of biomarkers specific to certain cancer types. Pathological profiling of hepatocellular carcinoma (HCC) is heterogeneous and rather complex, and biopsy samples contain limited information regarding the tumor and do not reflect its heterogeneity. Circulating tumor DNAs (ctDNAs), which can contain information regarding cancer characteristics, have been studied tremendously since liquid biopsy emerged as a new diagnostic method. Recent improvements in the accuracy and sensitivity of ctDNA determination also paved the way for genotyping of somatic genomic alterations. In this study, three-electrode (Au-Pt–Ag) glass chips were fabricated and combined with polydimethylsiloxane (PDMS) microchannels to establish an electrochemical microfluidic sensor for detecting c.747G > T hotspot mutations in the TP53 gene of ctDNAs from HCC. The preparation and analysis times of the constructed sensor were as short as 2 h in total, and a relatively high flow rate of 30 µl/min was used during immobilization and hybridization steps. To the best of our knowledge, this is the first time a PDMS-based microfluidic electrochemical sensor has been developed to target HCC ctDNAs. The system exhibited a limit of detection (LOD) of 24.1 fM within the tested range of 2–200 fM. The sensor demonstrated high specificity in tests conducted with fully noncomplementary and one-base mismatched target sequences. The developed platform is promising for detecting HCC-specific ctDNA at very low concentrations without requiring pre-enrichment steps.

Graphical Abstract

中文翻译：

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通过 PDMS 和基于 MEMS 的微流体传感器对肝细胞癌特异性 ctDNA 进行预富集检测

摘要

人们对微流控生物传感器的兴趣日益浓厚，导致各种传感机制的分析性能得到改善。尽管各种传感器都可以与微流体集成，但电化学传感器因其易于小型化、集成能力强和低成本而被最常用，使其成为一种成熟的护理点诊断方法。这个概念可以很容易地适用于某些癌症类型特有的生物标志物的检测。肝细胞癌 (HCC) 的病理学特征具有异质性且相当复杂，活检样本包含的有关肿瘤的信息有限，不能反映其异质性。自从液体活检作为一种新的诊断方法出现以来，循环肿瘤 DNA (ctDNA) 包含有关癌症特征的信息，已经得到了大量研究。最近 ctDNA 测定准确性和灵敏度的提高也为体细胞基因组改变的基因分型铺平了道路。在本研究中，制作了三电极（Au-Pt-Ag）玻璃芯片，并将其与聚二甲基硅氧烷（PDMS）微通道结合，建立了电化学微流体传感器，用于检测 HCC ctDNA TP5​​3 基因中的 c.747G > T 热点突变。构建的传感器的制备和分析时间总共短至2小时，并且在固定和杂交步骤中使用了30μl/min的相对较高的流速。据我们所知，这是首次开发出基于 PDMS 的微流体电化学传感器来靶向 HCC ctDNA。该系统在 2-200 fM 的测试范围内表现出 24.1 fM 的检测限 (LOD)。该传感器在使用完全非互补和一碱基错配的目标序列进行的测试中表现出高度特异性。开发的平台有望在极低浓度下检测 HCC 特异性 ctDNA，而无需预富集步骤。

图形概要