Integrated Acoustofluidic Plasmonic Molecular Diagnostic System for Detecting MicroRNA Biomarkers

摘要 本项目的目标是开发一个集成的声流控等离子体分子诊断系统 用于循环胞外体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记物。经常观察到miRNAs的调节失调 治疗各种疾病，包括癌症、心血管疾病和传染病。例如，最近 研究还表明，miRNAs可以通过外体从肿瘤细胞分泌到血液中， 它们的表达谱可以用来对癌症类型进行分类。因此，循环的外体miRNAs 被认为是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 由于分离涉及的技术困难，尚未应用于临床实践 外体和外体miRNAs分析。因此，有必要开发替代分析 可以提供比传统方法更多优势的策略。在这方面，我们提出了一个综合的 用于快速分离和多重检测的声流控等离子体分子诊断系统 外体miRNA生物标记物。跨学科的方法将提供新的能力来推动 癌症的精确临床诊断。该系统集成了两种独特但经过验证的技术：(1) 声流控技术可以高产率和高纯度地快速分离外切体，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。 虽然与结直肠癌相关的miRNAs将被用作模型系统，但拟议的项目将 导致开发一种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)开发和集成用于外体miRNA分离的声流控系统； (2)开发用于多重检测外体miRNA生物标志物的IMS纳米探针；及(3)技术 SERS-声流控系统的评价。我们将以我们的联合知识为基础 跨学科团队，并建立必要的技术验证，使这项集成技术做好投入使用的准备 在临床环境中。在整个项目中，将开发一个集成的声流等离子体系统，并 可用于临床样本中多种外体miRNA的原位分析，而不需要miRNA 提取和扩增。在技术验证阶段，将结果与使用 常规检测方法(如qRT-PCR)。拟议的系统将导致无样品制备和“样品-- 基于对现有子组件的集成，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 能够在各种癌症的早期发现和筛查领域加强研究和翻译 超越了CRC，并最终非常适合于护理点。建议的分子分析系统代表了 在生物医学研究、诊断和筛查方面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.