微液滴电化学阻抗传感芯片用于核酸扩增检测的研究

As the development of microfluidic and Micro-Electro-Mechanical System, nucleic acid analysis based on droplets is focusing on chip and high-throughput, and becoming an effective method in research and application of quantitative determination of rare DNA. However, detection of nucleic acid amplification is dominated by the use of fluorescence, which suffers from the cost and time of preparing probes, the errors brought by fluorescence saturation and photo bleaching, and the huge and expensive optical equipment. These shortcomings hamper the on chip integration and application of nucleic acid amplification in droplets. On the other hand, DNA analysis, especially the DNA sequencing, get a great leap by making use the low cost and high sensitivity of electrochemical biosensor and large scale integration in semiconductor field. In this project, we design and fabricate chips and electrodes with high sensitivity using semiconductor manufacturing to achieve the impedance-based detection of nucleic acid amplification in droplets. The chip for DNA amplification integrates with temperature control, thus all the DNA analyses will be operated on chip. We will study the impedance signal of PCR droplets and carry out the research on the optimization of PCR reaction system to achieve the maximum sensitivity. We will also study the sensitization mechanism of buffer and nanoparticles in PCR reagent to achieve the high sensitive detection of nucleic acid amplification in droplets using impedance integrated biosensor. Eventually, both the chip and sensitization mechanism could be achieved with the accomplishment of this project, which would be a new ideas and technology platform for droplets detection.

随着微流控和微机电技术的发展，基于微液滴的核酸分析技术及设备逐渐芯片化和高通量，成为疾病预防和早诊、检验检疫、痕量核酸定量等研究和应用领域的重要手段。然而，微液滴内核酸扩增产物的检测多依赖荧光标记，不仅需要对样品进行荧光衍生前处理，受荧光过饱和及光漂白影响；还需搭载精密和昂贵的检测光路，成为微液滴核酸分析芯片化和高通量检测以及推广应用的最大障碍之一。电化学传感器和大规模集成电路的快速、灵敏、低能耗和低成本已为核酸分析带来重大飞跃。本项目将发挥电化学传感优势，利用半导体微纳加工技术，设计并研制集成温控的微液滴核酸扩增芯片和高灵敏度的微液滴阻抗检测电极；研究适用于阻抗检测的核酸扩增体系，以及低缓冲条件和金纳米材料对扩增产物阻抗检测的增敏机理和特异性应用；最终实现微液滴核酸扩增的高灵敏度、集成化电阻抗检测。本项目的实施不仅提供具有重要应用价值的研究技术和芯片，还将促进阻抗检测增敏机理研究。

单细胞核酸分析可突破常规群体细胞分析的平均结果掩盖个别重要信息的局限性，掌握更准确、更全面的细胞信息。本项目研制了硅基微孔、微液滴生成、核酸扩增及温度控制等多种芯片，对芯片和片上PCR反应体系进行了优化，实现微孔阵列式和微液滴式单细胞的高通量核酸检测分析，研制出便携式核酸现场快速荧光检测装置。对疾病的早期预防和诊断、细胞异质性基础研究具有重要意义。