基于2D层状金属(或共价)有机骨架纳米片-等温扩增技术探索转基因生物比率型电化学快速检测

In this project, rising 2D layered metal-organic framework (MOF) or covalent organic framework (COF) nanosheets and their interfaces will be prepared, which will be further integrated with simple isothermal nucleic acid amplification techniques and novel ratiometric electrochemical DNA sensing strategy for exploring the highly sensitive and rapid detection of genetically modified organisms. 2D MOF or COF nanosheets possess ordered porous structure, rich functional groups, artificially controllable sites for DNA immobilization, which can realize the ordering and maximization of the DNA immobilization. Their excellent conductivity, large specific surface area, as well as strong mechanical properties and stability will help to improve the electron conduction performance and electrochemical responses of obtained sensing interfaces, which result in the improvement of DNA detection sensitivity and stability. A double-signal ratiometric electrochemical sensing platform will be constructed based on the inherent redox activity or electrocatalysis of 2D MOF or COF, which fundamentally overcomes the intrinsic systematic errors of electrochemical sensors that come from environmental and personal factors and thus leads to remarkably enhanced reproducibility and reliability. We will study the effect of different preparation schemes and experimental parameters on the morphology, size, and ratiometric electrochemical signals of the sensing platforms. In addition, simple and novel isothermal nucleic acid amplification techniques will be integrated for exploring high-sensitive, high-precision and rapid detection of various genetically modified organisms. We will comparatively study the difference between the electrochemical sensors based on various 2D MOF or COF nanosheets and different isothermal nucleic acid amplification techniques on the sensitivity, speed, reproducibility etc. investigation factors considering practical application.

本项目拟制备新兴的2D层状金属有机骨架或共价有机骨架纳米片，结合等温核酸扩增技术和蓬勃兴起的比率型电化学DNA传感策略，探索转基因生物高灵敏、快速检测。借助2D有机骨架丰富的功能团、规则有序的孔洞结构、人为可控的固定位点，实现DNA固定的有序化与最大化，有力提高杂交效率；利用其高的导电性、高的比表面积以及强大的机械性能和稳定性提高传感界面的电子传导性能与电化学响应，进而提高DNA检测灵敏度与稳定性；依靠2D有机骨架优异的自身电化学活性或电催化性能，构建双信号比率型电化学传感平台，从基本上克服来自环境和个人因素固有的系统误差，增强检测重现性和可信度；有效整合简单、新颖的等温核酸扩增技术，探索对多种转基因生物高灵敏、高精度和快速检测。对比各种制备方案对界面形貌、比率型信号等的影响，比较基于各种2D有机骨架以及各种等温扩增技术的电化学传感器在检测灵敏度、速度和重现性等实际应用方面考察因素的差异。

随着二维尺度的石墨烯蓬勃发展，二维类石墨烯层状纳米材料，如层状金属(或共价)有机骨架，MoS2纳米片，也日益受到人们的广泛关注。另一方面，由于采用探针信号和参比信号的比值作为测量信号，避免采用传统方法仅利用目标带来的单一的“信号开”或“信号关”模式，比率型电化学传感平台从基本上降低了来自环境和个人因素固有的系统误差，也被人们广泛采用。本课题组基于开发以上新型类石墨烯的二维纳米材料基传感界面，进行比率型生物电化学快速检测研究。根据“自下而上”的组装策略，合成了多种层状MOFs纳米片，并参考其他（类）石墨烯复合传感界面的制备方法，采用小分子插层-超声剥离、电沉积与电聚合方法等技术，设计一系列具有与比率型生物电化学快速检测相匹配的（类）石墨烯及其复合纳米材料的电化学传感平台；利用复合传感界面丰富的功能团、高导电性、大的比表面积以及强大的机械性能和稳定性，提高传感界面的电子传导性能与电化学响应。进行了形貌、有序程度等物理要素与电化学功能团电化学活性之间关系研究。基于新筛选的内参分子的电化学信号变化，特别是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”的比率型电化学传感平台，有力地提高检测灵敏度与可信度，实现了对特定靶标的高精度、稳定检测。并借助监测扩增前后的dGTP浓度变化，开发快速判断序列特异性DNA是否存在的定性检测方法。在国内外高水平学术期刊如Anal Chem, ACS Appl Mater Inter, ACS Sustain Chem Eng, J Hazard Mater, Sensor Actuat B Chem, Compos Part A-Appl S,化学学报等上面发表了15篇论文（均标明基金资助），其中14篇SCIE收录论文，中科院大类一区9篇。授权发明专利1项。培养博士研究生2人，硕士研究生8人，其中4人已获得硕士学位。所开发的比率型电化学传感平台能够克服电化学传感器的重现性或者稳定性较差的这一重要缺陷，有望推动电化学检测实际应用。