Integrated Acoustofluidic Plasmonic Molecular Diagnostic System for Detecting MicroRNA Biomarkers

摘要 该项目的目标是开发集成声流控等离子体分子诊断系统 设计用于循环外泌体 microRNA (miRNA) 的快速分离、检测和多重鉴定 人类临床样本中用于疾病早期检测的生物标志物。经常观察到 miRNA 的失调 多种疾病，包括癌症、心血管疾病和传染病。例如，最近 研究还表明，miRNA 可以通过外泌体从肿瘤细胞分泌到血液中， 它们的表达谱可用于对癌症类型进行分类。因此，循环外泌体 miRNA 被认为是用于癌症早期检测和诊断的有前景的生物标志物。然而，这些小分子 由于分离技术困难，尚未应用于临床实践 外泌体和外泌体 miRNA 的分析。因此，需要开发替代分析 与传统方法相比，这些策略可以提供更多优势。对此，我们提出综合 声流控等离子体分子诊断系统，设计用于快速分离和多重检测 外泌体 miRNA 生物标志物。跨学科方法将提供新的能力来推进 癌症的精确临床诊断。该系统集成了两项独特但经过验证的技术：(1) 声流控技术可以快速分离高产量和高纯度的外泌体，以及（2）表面增强 基于拉曼散射 (SERS) 的“反向分子哨兵”(iMS) 纳米探针，用于直接检测 miRNA。 虽然与结直肠癌（CRC）相关的 miRNA 将被用作模型系统，但拟议的项目将 导致开发出适用于其他类型疾病的普遍适用的护理点诊断技术。 具体目标是：（1）开发并集成用于外泌体miRNA分离的声流系统； (2) 开发iMS纳米探针用于外泌体miRNA生物标志物的多重检测； (3) 技术 SERS 声流系统的评估。我们将建立在我们的综合知识之上 跨学科团队并建立准备使用该集成技术所需的技术验证 在临床环境中。在整个项目中，将开发一个集成的声流体等离子体系统 经验证可对临床样本中的多种外泌体 miRNA 进行原位分析，无需 miRNA 提取和扩增。在技术验证阶段，结果将与使用获得的结果进行比较 常规检测（例如 qRT-PCR）。所提议的系统将导致无样品制备和“样品- to-answer”分析方法基于现有子组件的集成，其中许多子组件具有 已经在类似的应用中单独进行了测试。这种新的集成分子诊断系统是 能够加强各种癌症早期检测和筛查领域的研究和转化 超越 CRC，并且最终非常适合即时护理。所提出的分子分析系统代表 这是一项在生物医学研究、诊断和筛查领域具有变革潜力的重大创新。

ABSTRACT The goal of this project is to develop an Integrated acoustofluidic plasmonic molecular diagnostic system designed for rapid isolation, detection and multiplexed identification of circulating exosomal microRNA (miRNA) biomarkers in human clinical samples for early detection of diseases. Dysregulation of miRNAs is often observed in various diseases including cancer, cardiovascular illnesses, and infectious diseases. For instance, recent studies have also demonstrated that miRNAs can be secreted from tumor cells into bloodstream via exosomes, and their expression profiles can be used to classify cancer types. Therefore, circulating exosomal miRNAs are considered promising biomarkers for early detection and diagnosis of cancer. However, these small molecules have not been adopted into clinical practice because of the technical difficulties involved in the isolation of exosomes and the analysis of exosomal miRNAs. Thus, there is a need to develop alternative analyzing strategies that could offer more advantages over conventional methods. In this regard, we propose an integrated acoustofluidic plasmonic molecular diagnostic system designed for rapid isolation and multiplexed detection of exosomal miRNA biomarkers. The cross-disciplinary approach will provide new capabilities to advance the precise clinical diagnosis of cancer. This system integrates two unique but proven technologies: (1) the acoustofluidic technology that can rapidly isolate exosomes with high yield and purity, and (2) surface-enhanced Raman scattering (SERS)-based “Inverse Molecular Sentinel” (iMS) nanoprobes for direct miRNA detection. Although miRNAs related to colorectal cancer (CRC) will be used as the model system, the proposed project will lead to the development of a generally applicable point-of-care diagnostic technology for other types of diseases. The specific aims are: (1) Develop and integrate an acoustofluidic system for exosomal miRNA isolation; (2) Develop iMS nanoprobes for multiplexed detection of exosomal miRNA biomarkers; and (3) Technical Evaluation of the SERS-acoustofluidic system. We will build upon the combined knowledge of our interdisciplinary team and establish the technical validation required to ready this integrated technology for use in clinical settings. Throughout this project, an integrated acoustofluidic plasmonic system will be developed and validated for in-situ analysis of multiple exosomal miRNAs from clinical samples without the need for miRNA extraction and amplification. In the technical validation phase, the result will be compared to that obtained using conventional assays (e.g. qRT-PCR). The proposed system will lead to a no-sample preparation and “sample- to-answer” analysis approach that is based on the integration of existing sub-components, many of which have already been separately tested in similar applications. This new integrated molecular diagnostic system is capable of enhancing research and translation in the areas of early detection and screening of various cancers beyond CRC, and is ultimately well suited for point-of-care. The proposed molecular analysis system represents a major innovation that has a transformative potential in biomedical research, diagnostics and screening.