基于微纳结构精度调控的微流控芯片构建及其肺癌外泌体检测研究

Exosomes are of great value in the diagnosis and treatment of tumors. However, there is a lack of efficient separation and quantitative detection techniques in clinical practice. Microfluidic chip is a promising detection technology. But the existing manufacturing technology can not achieve precise control of micro-nano structure. As a result, the microstructure of the chip is single, the specific surface area of the chip is low, and the optical signal can not be mediated or sensitized by the chip. Thus, microfluidic chip have not achieved efficient separation and detection for exosomes. In view of the above technical problems, this project focuses on the research of high-precision regulation methods for micro-nano structure. Based on the applicant's previous work, this study mainly explores the rule and mechanism of the combined regulation on micro-nano structure of precious metals using electrohydrodynamic jet printing and nano-synthesis technology. And then the enhancement mechanism of high-precision micro-nano structure on surface-enhanced Raman spectroscopy (SERS) is studied. By regulating the micro-nano structure of the substrate, the types of micro-nano structures for the efficient detection of exosomes are explored. The exosome size sorting chip based on deterministic lateral displacement and quantitative detection chip based on SERS signals were constructed to detect EGFR mutant exosomes in lung cancer. This subject is expected to develop novel micro-nano manufacturing technology. The efficiency of microfluidic microarray in separation and quantitative detection of exosomes was improved. It is of great significance to promote the clinical diagnosis and treatment of lung cancer.

外泌体在肿瘤诊疗中具有极高的应用价值，但临床中缺乏高效的分选和检测技术。微流控芯片是极具前景的检测方法，但现有的制造技术难以实现高精度微纳结构的有序调控，导致芯片存在微观结构单一、比表面积低、无法介导或增敏光学信号等缺陷，因而未能实现对外泌体的高效分选和检测。针对上述技术难题，本项目重点研究高精度微纳结构的调控方法。在前期工作基础上，具体探索微纳电喷印技术联合纳米合成技术调控贵金属三维基底微纳结构的规律和机制，并研究高精度微纳结构对表面增强拉曼光谱（SERS）的增强机制。进而通过调控芯片的加工精度，探索外泌体高效分选和鉴定所需的微纳结构类型及影响因素，构建出基于确定性侧向位移原理的尺寸分选芯片和基于SERS信号的定量检测芯片，将其用于肺癌EGFR突变型外泌体的检测。本课题有望研发出新的微纳制造工艺，并据此提升微流控芯片分选富集和定量检测外泌体的效率，对推动肺癌的临床诊疗具有重要意义。

肿瘤的微量检测一直是临床中亟需解决的关键难题，针对血液ctDNA、外泌体等标志物的探针敏感度和特异度难以满足实际的临床需求。外泌体等标志物在肿瘤诊疗中具有极高的应用价值，但临床中缺乏高效的检测技术。微流控芯片是极具前景的检测方法，但现有的制造技术难以实现高精度微纳结构的有序调控，导致芯片存在微观结构单一、比表面积低、无法介导或增敏光学信号等缺陷，因而未能实现对外泌体的高效分选和检测。针对上述技术难题，本项目重点研究高精度微纳结构的调控方法。在前期工作基础上，具体探索微纳电喷印技术联合纳米合成技术调控贵金属三维基底微纳结构的规律和机制，并研究高精度微纳结构对表面增强拉曼光谱（SERS）的增强机制。进一步，在金纳米材料生物探针构建方面，本项目利用多种茎环状核酸适配体先后构建了金纳米核酸SERS探针、比例化金纳米核酸SERS探针、多组分球形核酸探针，三类探针分别在临床肺癌、膀胱癌血液ctDNA和尿液mRNA中获得较高的检出效率，检测敏感度70-80%、特异度达100%，同时能够准确显示肿瘤组织的细胞学边界，为肿瘤超敏检测和临床手术提供了有效的工具，具有重要的科学意义和潜在的临床价值。上述研究成果及相关工作在Nano Research、Nano Research、Journal of Pharmaceutical and Biomedical Analysis、Advanced Materials Interfaces等发表项目资助标注的SCI学术论文4篇，其中高水平论文2篇（IF>10）。申请发明专利1项，培养博士生2名，硕士生2名。项目申请人获得陕西青年科技奖1项和陕西省科技进步奖一等奖1项。