CAREER: Integrated Optofluidic Chips towards Label-Free Detection of Exosomal MicroRNA Biomarkers

癌症是全球发病率和死亡率的主要原因。随着癌症中观察到的异质性和复杂性的增加，对疾病进展的准确诊断和分子监测的需求变得比以往任何时候都更加重要。从人体体液中循环的细胞中提取微小囊泡进行液体活检是一种有前途的、廉价的、微创的癌症诊断和个性化医疗方法。特别是，检测这些囊泡携带的生物分子，包括编码遗传信息的核酸，已经成为早期诊断的一种有希望的策略。然而，这些技术的现有诊断工具缺乏临床可行所需的灵敏度、特异性、速度和成本效益。本CAREER方案充分利用纳米光子学、纳米流体学和生物传感等小规模技术的前沿发展，为临床样品诊断性核酸检测提供新的解决方案，提高灵敏度，减少样本量，缩短分析时间。该技术的成功将对疾病的早期诊断、预后和管理产生重大影响，包括心血管疾病、自身免疫性综合征、神经退行性疾病和传染病。通过整合研究和教育，该项目将促进公众对纳米生物技术在卫生保健中的重要性的认识，并培养纳米技术和生物传感领域的下一代科学家和工程师，以应对负担得起的便携疾病诊断方面的重大挑战。此外，该项目旨在吸引K-12学生和代表性不足的个人（例如女性和美洲原住民学生）参与STEM职业。本CAREER提案的研究目的是验证集成和多路光流平台可以准确检测外泌体mirna的假设。为了实现这一目标，一种纳米流控芯片（ExoMiRChip）将被设计成功能集成无标记外泌体纯化、片上外泌体miRNA提取和等离子体miRNA传感。将理论与实验相结合，解决实现高分辨率和高通量外泌体纳米颗粒分选，高灵敏度和高特异性miRNA检测以及纳米流体系统多功能集成的基本挑战。本项目将在纳米尺度上探索外泌体纳米颗粒流体动力学的科学未知数，旨在全面阐明片上外泌体纯化的限制因素。该项目将创新用序列特异性锁定核酸（LNA）探针功能化的光耦合超灵敏等离子体纳米传感器，并利用它们确定影响外泌体miRNA准确检测的关键因素，包括等离子体传感器设计、纳米结构制造、miRNA分子浓度和miRNA选择性。ExoMiRChip上成功的纳米流体集成将显著减少诊断中的样本量（从毫升到微升），最大限度地减少偏差和污染，提高诊断速度（估计从几天/几周到几小时），并有可能实现多路生物标志物检测。我们期望该项目在未来的生物传感领域具有变革性，并适用于各种生物分子。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Cancer is a major global cause of morbidity and mortality. With the increasing heterogeneity and complexity observed in cancers, the need for accurate diagnosis and molecular monitoring of disease progression has become more important than ever. Liquid biopsy of microscopic vesicles from our cells circulating in human bodily fluids is a promising, inexpensive, and minimally invasive approach for cancer diagnosis and personalized medical treatment. Particularly, detecting the biomolecules carried by these vesicles, including nucleic acids encoding genetic information, has emerged as a promising strategy for early diagnosis. However, the existing diagnostic tools for such technologies lack the needed sensitivity, specificity, speed, and cost-effectiveness necessary to become clinically viable. This CAREER proposal fully exploits the cutting-edge development in small-scale technologies such as nanophotonics, nanofluidics, and biosensing to provide novel solutions for the detection of diagnostic nucleic acids from clinical samples with an improved sensitivity, reduced sample volume, and decreased analysis time. The success of the proposed technology will have significant impact on early-stage diagnosis as well as prognosis and management of diseases, including cardiovascular diseases, autoimmune syndromes, neurodegenerative disorders, and infectious diseases. By integrating research and education, the project will promote public awareness of the importance of nanobiotechnology in health care, and to cultivate the next-generation of scientists and engineers in nanotechnology and biosensing to address grand challenges in affordable and portable disease diagnosis. Further, this project aims to attract the participation of K-12 students and underrepresented individuals (e.g., female and Native American students) in STEM careers. The research objective of this CAREER proposal is to validate the hypothesis that an integrated and multiplexed optofluidic platform can accurately detect exosomal miRNAs. In pursuit of this goal, a nanofluidic chip (ExoMiRChip) will be designed to functionally integrate label-free exosome purification, on-chip exosomal miRNA extraction, and plasmonic miRNA sensing. Theories and experiments will be combined to address fundamental challenges in achieving high-resolution and high-throughput exosome nanoparticle sorting, high-sensitivity and high-specificity miRNA detection, and multi-functional integration of nanofluidic systems. This project will explore scientific unknowns in exosome nanoparticle fluidic dynamics at the nanometer scale, and aim to comprehensively elucidate the limiting factors in on-chip exosome purification. The project will innovate optically coupled ultrasensitive plasmonic nanosensors functionalized with sequence-specific locked nucleic acid (LNA) probes, and use them to identify the critical factors affecting accurate detection of exosomal miRNA, including the plasmonic sensor design, nanostructure fabrication, miRNA molecular concentration, and the miRNA selectivity. Successful nanofluidic integration on the ExoMiRChip will significantly reduce sample volume in diagnosis (from milliliters to microliters), minimize bias and contamination, improve diagnosis speed (estimated from days/weeks to hours), and potentially enable multiplexed biomarker detection. We expect the project to be transformative in future biosensing and applicable to a wide variety of biomolecules.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.