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-minute-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.

最近，将microRNA（miRNA）用作多种疾病的生物标志物已在临床以及即时诊断中获得了重要意义。然而，在其他挑战中，它具有中心要求，即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发展而言，这意味着对复用的必然需求，以便能够并行地测量患者样品中感兴趣的几种组分的丰度。在这项研究中，我们通过将其通道划分为多个小部分，设计并实现了电化学微流体生物传感器的不同多路复用版本，创建了四个新颖的​​芯片设计，用于在CRISPR-Biosensor X上实现多达8个miRNA的无扩增和同时定量（'X'突出了设备的多重性）。然后，我们使用一步模型分析，然后使用安培计读数结合2分钟停止流量协议来探索流体和机械特性以及该设备不同版本的局限性。表现出最佳性能的传感器随后用于由Cas13a技术提供的概念验证，用于测量小儿血液中失调的miRNA-17〜92簇中的两个miRNA（miRNA-19b和miRNA-20a）。髓母细胞瘤患者。后者的量化以及同时的阴性对照测量均在同一设备上完成。