CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics
CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics
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DOI：
10.1016/j.bios.2020.112887
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发表时间：
2021-01-22
影响因子：
12.6
通讯作者：
Dincer, Can
中科院分区：
文献类型：
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作者：
Bruch, Richard;Johnston, Midori;Dincer, Can
最近，使用 microRNA (miRNA) 作为多种疾病的生物标志物对于临床和护理点诊断具有重大意义。然而，在其他挑战中，它的核心要求是必须在其他因素的背景下评估给定 miRNA 的浓度，以便明确诊断一种特定疾病。就诊断方法和设备的开发而言，这意味着不可避免地需要多重分析，以便能够并行测量患者样本中多种感兴趣成分的丰度。在这项研究中，我们设计并实现了不同的多重版本的电化学微流体生物传感器，将其通道分为几个部分，创建了四种新颖的芯片设计，用于在 CRISPR-Biosensor X 上对多达 8 个 miRNA 进行无扩增和同时定量（“X”突出了该设备的多重方面）。然后，我们使用一步模型测定，然后结合电流读数和 2 分钟停流方案来探索不同版本设备的流体和机械特性以及局限性。该传感器显示出最佳性能，随后用于对 miRNA-17-92 簇中的两个 miRNA（miRNA-19b 和 miRNA-20a）进行 Cas13a 驱动的概念验证测量，这在儿科髓母细胞瘤患者的血液中失调。后者的量化以及同时的阴性对照测量是在同一设备上完成的。因此，我们确认了我们的平台对于无扩增、平行检测多种核酸的挑战的适用性。
Recently the use of microRNAs (miRNAs) as biomarkers for a multitude of diseases has gained substantial significance for clinical as well as point-of-care diagnostics. Amongst other challenges, however, it holds the central requirement that the concentration of a given miRNA must be evaluated within the context of other factors in order to unambiguously diagnose one specific disease. In terms of the development of diagnostic methods and devices, this implies an inevitable demand for multiplexing in order to be able to gauge the abundance of several components of interest in a patient's sample in parallel. In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X ('X' highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-min-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17-92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.