基于核酸扩增技术-3D石墨烯高灵敏、免标记和普适性电化学检测海水与海产品中食源性弧菌

In this project, novel 3D graphene will be integrated with nucleic acid amplification technique and electrochemical biosensor for highly sensitive, label-free and universal detection of vibrios originated from seawater and seafoods. Physical and chemical routes will be adopted to prepare a series of 3D graphene sensing platforms. We will study the effect of different preparation schemes and experimental parameters on the morphology, size, and electrochemical signals of the sensing platforms. The sensing platforms possess plenty of electrochemical sites, high specific surface area, powerful mechanical properties, etc, which endow sensing platforms with super conductivity, highly electrochemical activities and stability in different interfaces and phases. The immobilization of the probe will be mainly conducted via covalent or noncovalent methods for universal detection. The noncovalent method depends on the π-π interaction between rich-conjugated structures on 3D graphene sensing platform and DNA bases. The reversible specific covalent link between phenylboronic acid and 1,2-hydroxy functinal groups also insures the freely switchable tests. In the presence of target, the binding hybrids will disturb the interaction between probes and 3D graphene, accompanied with the electrochemical signal change of the nanointerfaces (signal-on and signal-off). The signal change will be served as a powerful tool to investigate the effect of different morphologies and sizes of sensing platforms on the loading of the probe, sensitivity, recovery of sensing platform. The synergistic effect for improving the sensitivity of the target detection will be pursued. Above 3D graphene sensing platforms will be integrated with nucleic acid amplification technique to highly sensitively and universally detect the gene sequences originated from real sample of the vibrios originated from seawater and seafoods.

本项目拟将3D石墨烯、核酸扩增技术和电化学DNA传感器技术相结合，开展海水及海产品中食源性病原弧菌高灵敏、免标记和普适性电化学检测研究。设计一系列3D石墨烯及其复合材料传感界面，对比各种制备方案对界面形貌、电化学信号等的影响。借助传感界面受DNA固定与杂交的影响而产生界面电化学信号的变化（"信号关"或"信号开"），成功实现对DNA探针的固定及目标扩增产物的杂交识别。传感界面优异的导电性、丰富的边缘缺陷，高的比表面积与强的机械性能等优点增强纳米界面的电化学响应与稳定性，结合核酸扩增技术，提高检测灵敏度。各种电化学活性功能团，丰富的共轭结构和DNA碱基之间的非共价π-π堆积作用，确保无损、免标记与普适性检测。比较不同传感界面的组成、尺度等因素对电极稳定性，探针固定效率、灵敏度以及再生性能的影响。有效整合核酸扩增模块与电化学检测模块，最终实现高灵敏、免标记、普适性检测多种食源性弧菌。

本课题组围绕开发高灵敏、免标记和普适性弧菌基因检测的新材料、新技术、新方法和新系统开展研究。采用小分子插层-超声剥离、化学聚合、电沉积与电聚合方法等技术，分步或者同步构建与主要食源性病原弧菌基因片段检测相匹配的3D（类）石墨烯及其复合纳米材料的电化学传感平台；探讨3D（类）石墨烯基界面的组成、空间取向、有序度、间隙度等因素对DNA检测的影响；优化扩增参数与条件。以电化学活性分子信号的变化，特别是整合界面自身信号的变化作为比率型信号，构建了新型“信号开”-“信号关”的双信号比率型电化学DNA传感平台，有力地提高检测灵敏度与可信度，实现了对食源性弧菌特定基因片段的高精度、稳定、普适性的检测。在国内外高水平学术期刊如ACS Appl Mater Inter, Biosens Bioelectron, ACS Sustain Chem Eng, J Mater Chem B, J Phys Chem C, Electrochim Acta, Sensor Actuat B Chem等上面发表和接受发表了28篇SCI收录论文（均标明基金资助）。其中影响因子大于5的15篇，中科院大类一区14篇，这些文章目前为止被Chem Soc Rev, Mater Today, Biosens Bioelectron, Anal Chem等国际权威、重要刊物他引300余次。获第十届青岛市青年科技奖，山东省科学技术奖(2015年，自然科学类)二等奖。作为指导老师获山东省2015年度研究生优秀科技创新成果一等奖。培养硕士研究生13人，其中8人已获得硕士学位。