芯片式微管电极3-D微纳协同传感界面构建及用于肿瘤外泌体高效捕获与原位分析

Exosomes are emerging as new-generation tumor biomarkers. Nevertheless, the high-efficiency separation and sensitive detection of exosomes are challenging. Herein, we engineer a monolithic micro-nanostructured biosensing interface to enable highly efficient capturing of exosomes and in-situ detection of exosomal molecules on the basis of our previous advancement in microfluidics and electrochemical biosensors. This synergic micro-nanoscale functional interface was constructed by combined strategies, including polymer-assisted metal deposition, electrochemical deposition and self-assembling of DNA nanostructures, based on the templates of the inherent three-dimensional (3D) structure of microfluidic channels. Importantly, the surface-attached biomolecular probe-target interaction confined in the 3D microchannel is significantly enhanced as compared to that on 2D planar electrode surface, due principally to the increased collision frequency and enlarged accessibility of probes. The biomolecular assembling, folding and binding dynamics on the sensor surface will be investigated along with the controlled fabrication and functionalization of surface-confined gold nanostructures and DNA nanostructured probes to modulate the effective biorecognition between tumor exosomes and the capturing probes. In light of the deep understanding of surface-confined molecular recognition on micro-nanostructured surface, we aim to developing a high-performance, low-cost microtubular electrochemical biosensor for in-situ detection of exosomes and exosomal nucleic acids by using an integrative mode of capturing-lysing-quantifying with improved efficiency and accuracy. This project is expected to construct micro-nanostructured biosensing with high capturing efficiency and high sensitivity towards precise capturing and in-situ detection of exosome at different levels, which is significant to early diagnosis and treatment in cancers.

外泌体逐渐成为新一代肿瘤标志物，但其分离检测面临挑战。基于前期在微流控及电极传感界面设计的工作基础,本项目提出采用溶液相无电沉积技术、DNA纳米结构自组装和电化学调控策略，以微通道为模板，构建微流控整体柱式3D微纳协同传感界面,其特点是限域微腔纳米曲面的探针排布可显著增强界面分子相互作用，解决了传统2D平面电极表面分子碰撞效率低，空间位阻大等局限,从而实现外泌体的高效捕获与原位分析。深入研究微纳界面上的生物分子组装、折叠和结合动力学等过程，设计并构筑形貌可控、表面功能化的纳米结构，调控传感界面高效识别肿瘤外泌体。研制高性能、廉价的微管电化学生物传感器，建立针对被捕获外泌体内含分子的原位检测策略和一体化捕获-裂解-原位定量的分析模型以提高外泌体检测的准确性和效率。预期结果将构筑高捕获效率、高灵敏度的微纳协同传感界面，实现外泌体的精准捕获和原位多水平检测,对癌症的早期诊断和治疗具有重要意义。

循环外泌体因携带大量其来源细胞表面及胞内分子信息，逐渐发展为新的疾病标志物，但如何实现人体复杂样本中外泌体及内容物的高灵敏、高特异性定量分析依然面临挑战。本项目按计划：1）发展了基于2D纳米材料界面的核酸适体ExoAPP技术，精准靶向纳米囊泡表面蛋白组，监测药物诱导的肿瘤外泌体EMT表型变化；开辟了病毒启发的3D多价分子识别和自驱动多色ExoADM技术，用于外泌体PD-L1指导的肿瘤免疫治疗疗效监测；2）设计协同电中性肽核酸和高电荷球形核酸的SEEmiR传感器，用于肿瘤外泌体microRNA的高灵敏、高特异性定量检测。在此基础上，发展了框架核酸超电荷纳米标记增敏的电化学生物传感器用于外泌体microRNA的超灵敏检测（检测限：49 aM）；3）利用跨尺度界面工程原理，构建金属纳米结构调控的芯片式微纳协同分子界面，实现阿摩尔水平microRNA的高灵敏、高特异性检测。进一步构建基于同轴碱基堆积效应的Bsee-miR传感器，通过程序化调控多级纳米结构，增强界面核酸碱基堆积效应，建立通用型的循环microRNA检测新方法。在此基础上，发展了3D纳米阵列界面，研制了电化学草本传感器（nanoE-herb sensor），可克服长链DNA扩散速率低、目标序列隐蔽的不足，增强界面核酸杂交，实现了草本药用植物及伪品的快速鉴定。本项目发展了多种面向外泌体的靶向检测技术（ExoAPP、ExoADM技术）和分析方法（SEEmiR、Bsee-miR、eTDN、nanoE-herb、电子参杂放大器）。本项目在肿瘤外泌体分析领域获得一系列重要进展和研究发现，提出了仿生跨尺度生物传感界面，程序化调控分子界面性能，突破了纳米传感界面的尺寸障碍，揭示了跨尺度界面生物分子作用机制；发现了多级纳米结构增强界面核酸碱基堆积效应。