Next Generation Disease Screening using Nanosensors'; Highly multiplexed label-free detection of soulble proteins and miRNA in biofluids

对于能够同时检测复杂生物体液中的多种可溶性蛋白质和miRNA的分析方法有着巨大的需求。一项能够实现这一目标的技术有望在从神经退行性疾病到几种主要癌症等多种医疗保健重大挑战中产生深远影响。该项目旨在开发一种基于牛津纳米孔小工具的生物体液中可溶蛋白质和miRNA的多重免标记检测方法。Minion是一个成熟的平台，能够同时探测512个频道的高通量测序。通过定制和使用分子载体，我们相信这样的平台可以用于以无标记的方式同时检测多达44(256)个蛋白质和miRNA生物标志物。分子载体不仅能够有效地将生物标记物运输和检测到纳米孔，而且还将结合由4个碱基组成的独特的DNA条形码识别符，该识别码可用于确认特定生物标记物的浓度和存在。每个载体将包含用于检测miRNA的互补序列或用于检测蛋白质的适体序列。作为概念的证明，我们选择了与一种主要的神经退行性疾病(帕金森氏症、阿尔茨海默氏症)有关的2种关键蛋白质和10个在患者中上调或下调的miRNA序列。开发的方法是通用的，如果成功，这项试点工作将为检测复杂的未经修饰的样品中的蛋白质和小分子(如miRNA和神经递质)的一般方法奠定基础。

There is an enormous need for analytical methods that can achieve simultaneous detection of multiple soluble proteins and miRNA in complex biological fluids. A technology that can achieve this holds the promise of far-reaching impact in multiple healthcare grand challenges ranging from neurodegenerative disease to several major cancers. This project aims to develop a multiplexed label-free detection of soluble proteins and miRNA in biofluids based on the Oxford Nanopore MinION. The MinION is an established platform capable of high-throughput sequencing capable probing 512 channels simultaneously. Via customisation and use of molecular carriers, we believe that such a platform can be used for the detection of up to 44 (256) protein and miRNA biomarkers simultaneously in a label-free manner. The molecular carriers will not only enable efficient transport and detection of the biomarker to the nanopore but will also incorporate a unique DNA barcode identifier consisting of 4 bases which can be used to confirm concentration and presence of a particular biomarker. Each carrier will contain a complementary sequence for the detection of miRNA or an aptamer sequence for detection of protein. As a proof of concept, we have selected 2 key proteins linked to a major neurodegenerative disease (Parkinson's, Alzheimer's) and 10 miRNA sequences which are either up or down regulated in patients. The developed method is universal and if successful this pilot work will build the basis for a general approach for the detection of proteins and small molecules such as miRNA and neurotransmitters in complex unmodified samples.