微芯片双电化学整体分析系统：设计、性能及应用研究

Detector is crucial to microfluidic chemical analysis systems. In general, a single detector is only suitable for specific analytes; whereas, dual detection techniques can be complementary, thus broadening the analytical capacity and improving the qualitative identification for the targets. Up to now, dual electrochemical modes consisted of contactless conductivity detection (CCD) and amperometric detection (AMD) on microchip have largely been achieved by using different electrode materials and manufacturing process, wherein the sensing electrodes are fabricated separately, thereby making the total system fairly complicated yet impairing the integrity. More importantly, these researches fail to meet the inherent demands of microfluidic, consequently impose a huge restriction for applications in the analytical community. Herein, we make efforts to develop monolithic microfluidic analysis systems that incorporate CCD and AMD detectors to electrophoresis microchips based on indium tin oxide (ITO) glass by means of standard photolithography, screen printing and chemical etching. This leads to the miniaturization and integration of separation function and detection unit as a result of the identical procedures involved in the preparation of sensing electrodes and electrophoresis microchips. Specifically, the developed systems would reap the analytical universality and detection sensitivity, due principally to the perfect alliance of two kinds of electrochemistry protocols in a dexterous manner. The present work mainly focuses on: 1) designing multiple CCD/AMD configurations, such as sequential detection in a common separation channel, simultaneous detection at one point, and synchronous detection in parallel channels, with the aim to ameliorating the spatiotemporal resolution of CCD and AMD schemes; 2) preparing chemically modified ITO electrodes by in-situ methods to enhance the sensitivity and stability of AMD, and improving the analytical performance of CCD through rational design; 3) investigating the potential “cross-talk” effect and competitive phenomenon, to reveal the essential principles of dual electrochemical response; 4) exploring the methodology toward clinical biochemical analysis. This project is expected to offer facile and integrative microchip-based dual electrochemistry systems, and to enrich the analytical strategy and application realm of multiplex detection on microfluidic platform.

检测器是微芯片化学分析系统的关键；单一检测器的分析对象较为有限，双检测可以相互补充，拓宽分析范围和容量，并能增强组分的定性识别。迄今，电化学双检测如非接触电导法（CCD）和安培法（AMD）均采用不同的材质和工艺，分别制作检测电极，工艺复杂、集成度差，不符合微芯片的内在要求，且实际应用价值不足。本项目拟采用氧化铟锡（ITO）玻璃，通过光刻、丝网印刷和化学刻蚀等手段，同步制作电导电极和安培电极，兼容CCD的通用性和AMD的灵敏性，并实现分离和检测单元的微型化和集成化。研究内容包括：1）设计单通道序列检测、同点同时双检测和平行双通道检测，增强双电化学信号的时空分辨；2）原位制备ITO修饰电极，提高安培检测的灵敏度和稳定性，并优化CCD的设计和性能；3）探讨双响应的交互效应和竞争现象，揭示其内在规律；4）临床生化检验。本项目有望实现芯片双电化学系统的一体化，从而丰富芯片多检测的分析策略和应用价值。

本项目针对纳米孔-双电化学分析系统，以及纳米电化学展开。主要内容包括：（1）纳米孔-双电化学整体分析系统的设计及用于区分不同区域功能探针对离子门控的影响。（2）集成式纳米孔道电化学阵列系统的构建及生物检测应用。（3）基于功能纳米材料和界面的电化学传感（葡萄糖、肼、尿酸）、荧光分析（6-巯基嘌呤）、比色法检测（卡那霉素）等。重要结果和科学意义：（1）以阳极氧化铝纳米孔为基材，通过可控的金属喷镀，形成不同的金属修饰界面，结合DNA自组装技术，构建了对ATP分子具有特异性响应的仿生离子门控通道。该研究首次实现了两种电化学信号即离子电流和电解电流在纳米孔上的集成，阐明孔道不同区域功能分子对离子门控的贡献，即单独的孔道外表面探针分子不具备离子门控作用，但能够与孔道内探针分子产生协同增强的门控效应。（2）制备了集成有工作电极和辅助电极的纳米孔电化学阵列，将流体输运、分子识别和信号激发等功能集于纳米孔道上，实现了非标记生物大分子的超灵敏检测。该纳米孔电化学平台显著提升了电化学检测的集成度和灵巧性。（3）基于功能纳米材料和界面的电化学传感和荧光分析。具体包括：（i）制备了一种可弃式非酶型葡萄糖电化学传感器铜镍纳米粒/化学接枝碳纳米管/ITO电极。揭示了在碱性条件下，Cu2+可直接参与葡萄糖电化学氧化，并用于尿液中葡萄糖的定量分析。（ii）报道了一种层层自组装氮掺杂石墨烯/金纳米粒复合物制备方法，用于尿液中尿酸的检测。（iii）报道了一种绿色环保、一步电化学法制备多层还原氧化石墨烯-金/钯纳米粒复合物，用于水样中肼的灵敏检测。该制备方法无需任何还原剂、稳定剂、表面活性剂等，为制备其他石墨烯/双金属纳米材料提供了一种技术参照。（iv）报道了一种灵敏度高、选择性好的CdTe/SiO2/叶酸/银纳米粒比率型荧光探针，用于抗癌药6-巯基嘌呤尿液浓度的检测。核-壳结构消除了介质环境对荧光发射的影响，提高了分析的稳定性和准确性。