High-throughput microscopy system for ultrasensitive and multiplexed nanoarray analysis

决定许多疾病结果的最重要的一个因素是疾病的进展和诊断。不幸的是，疾病通常是在疾病已经处于晚期时出现症状时被检测到的。疾病的一个标志是细胞中蛋白质的失调，这些蛋白质被释放到血液中，因此可以作为疾病诊断的生物标志物。因此，如果能够检测到疾病的蛋白质指示物，疾病的早期诊断可能是可能的。尽管目前的血液检测只能检测到只占血液总蛋白100亿份中的一种蛋白质，但为了早期疾病诊断，需要能够同时检测多种蛋白质的技术，而且灵敏度要高出一千到一百万倍。近年来出现了超灵敏的蛋白质分析技术，但它们都很复杂，而且多重蛋白质测量要么是不可行的，要么是以牺牲灵敏度和可靠性为代价的。 我们建议开发新的技术，对血液中循环的蛋白质生物标志物以及癌细胞分泌的单个纳米尺寸囊泡(称为外体)的蛋白质含量进行超灵敏、多路检测，这些囊泡可用于识别不同类型的癌症。我们将开发由数字纳米点阵列制成的新型平台，这些纳米点阵列由数百万个纳米点组成，每个点都可以使用我们开发的低成本图案化方法捕获蛋白质/单个外切体。捕获的蛋白质/单个外显体将以单分子灵敏度进行检测，并对信号进行数字计数，用于蛋白质测量和单个外显体上的蛋白质含量分析。为了对疾病生物学进行更基础的研究，我们将利用我们在构建纳米点阵列方面的专业知识，利用定义明确但复杂的参数来创建细胞外基质蛋白的人造景观，以跟踪细胞迁移和细胞内信号。 我们的研究涉及与健康、物理和工程研究人员的合作，属于纳米阵列制造、表征和超灵敏信号检测的整体主题。NSERC-RTI的这项提案要求一种自动化和高灵敏度的显微系统，这对纳米阵列的高通量成像至关重要。拟议的显微镜系统将是自动化的，高度灵敏，能够快速和高通量成像，将涉及费力的手动调整的一整天缩短为一个小时的自动图像采集。该仪器加速了将超敏感蛋白质分析专家、细胞和癌症生物学家以及生物医学工程师聚集在一起的研究，创建了一种独特的、最先进的显微镜设备，可供许多跨学科的学员使用。

The single most significant factor that determines the outcome of many diseases is disease progression and diagnosis. Unfortunately, diseases are typically detected when symptoms appear when the diseases are already at an advanced stage. A hallmark of diseases is the dysregulation of proteins in cells, which are released into the blood and thus can be used as biomarkers for disease diagnosis. Early diagnosis of disease might therefore be possible if protein indicators of disease can be detected. Whereas current blood tests can detect a protein present as little as one part in 10 billion of total blood proteins, for early disease diagnosis, technologies that can measure multiple proteins simultaneously and are a thousand to a million times more sensitive are needed. Ultrasensitive protein analysis technologies have emerged recently, but they are complex, and multiplex protein measurement is either not feasible, or only possible at the expense of sensitivity and reliability. We propose to develop new technologies for ultrasensitive, multiplex detection of protein biomarkers that circulate in blood, as well as the protein content of individual nanometer-sized vesicles secreted by cancer cells, known as exosomes, that could be used to identify different types of cancers. We will develop novel platforms made of digital nanodot arrays that comprise millions of nanoscale-dots, each can capture proteins/single exosome using a low-cost patterning method we developed. Captured proteins/single exosome will be detected at single-molecule sensitivity, and the signal counted digitally for protein measurement and profiling of protein content on individual exosomes. For more fundamental study of the biology of disease, we will leverage our expertise in building nanodot arrays to create an artificial landscape of extracellular matrix proteins with well-defined yet complex parameters, to track both cell igration and intracellular signaling. Our research involves collaboration with heath, physics and engineering researchers, and fall under the integral theme of nanoarray fabrication, characterization and ultrasensitive signal signal detection. This NSERC-RTI proposal requests an automated and high sensitivity microscopy system that is critical for high-throughput imaging of nanoarrays. The proposed microscopy system will be automated, highly sensitive, and capable of rapid and high-throughput imaging, shortening a whole-day session that involves laborious manual adjustments into an hour of automatic image acquisitions. This instrument accelerate research that brings together experts in ultra-sensitive protein analysis, cell and caner biologists, and biomedical engineers, creating a unique, state-of-the-art microscopy facility to be used by many interdisciplinary trainees.