用于三核苷酸重复疾病早期诊断的高灵敏电化学生物传感体系的构建和应用研究

Trinucleotide repeats are disease gene of neurodegenerative diseases, known as Trinucleotide Tepeat Disease (TRD). The detection of trinucleotide repeats have been a very important significance for early diagnosis and warning of the genetic diseases. Currently, there are few effective methods to detect with rapid, high sensitivity, simplification and low-cost. The project would apply low-cost and convenient electrochemical method to detect trinucleotide repeats. Here especial for trinucleotide repeats such as CGG)n, (CAG)n, (CTG)n and so on, the novel electrochemical function-nano-probes would be designed, prepared and optimized through integration of three functions: molecule recognition, signal amplification and reuse, by combining gold-magnetism nanomaterials with DNA recognition molecules (naphthyridine derivatives) and the horseradish peroxidase enzyme reaction or electrochemical active molecule (ferrocene et al.). Based on the novel electrochemical function-nano-probes, we would construct the electrochemical biosensor system for the detection of trinucleotide repeat DNA, and coordinate all segments such as modification of electrodes, capture of targets, and role of function-nano-probes et al. These studies would provide new methods and devices for the detection of trinucleotide repeat DNAs with rapid, high sensitivity and high selectivity, and provide a feasible clinical diagnostic method for early diagnosis and warning of neurodegenerative diseases—Trinucleotide Repeat Disease (TRD).

三核苷酸重复序列的异常增多是导致一系列神经性遗传病的根本原因，这类疾病也叫三核苷酸重复性疾病，因此三核苷酸重复序列的检测分析对于疾病早期诊断和预警有着非常重大的意义。而目前对其快速、高灵敏、低成本检测缺乏有效的手段。本项目率先针对三核苷酸重复序列，采用低成本、便捷的电化学方法；结合核酸识别分子萘啶衍生物、金磁纳米材料、二茂铁电活性探针修饰的纳米颗粒或辣根过氧化酶等酶促反应扩大电信号；融合分子识别、信号扩大和重复利用等功能域，发挥最优功能和作用，制备新型电化学功能纳米探针。基于这些新型电化学功能纳米探针，构建三核苷酸重复序列纳米电化学生物传感体系；研究电极的修饰、目标物的捕获、电化学功能纳米探针的捕获等环节的协调优化；为三核苷酸重复序列的快速、高选择性、高灵敏的分析检测提供新方法和新器件，为实现三核苷酸重复疾病的早期诊断和预警提供一种可行的临床诊断方法。

电化学生物传感器是目前人们寄予最大期望的一类疾病标志物方法之一，是一种非常理想的POC（point of care）临床诊断方法。本项目主要针对神经遗传病标志物三核苷酸重复序列，开展了一系列的电生物传感研究。通过修饰核酸识别分子萘啶衍生物到电极表面，构建了阻抗型和电流型电化学传感。结合核酸识别分子萘啶衍生物、磁纳米材料、二茂铁电活性探针等制备了功能磁纳米探针，并成功的应用于电化学传感和荧光传感分析检测核酸，该方法操作简便、快速、灵敏。结合双信号技术、Exo III辅助循环扩增、电化学分子信标、原位杂交链式反应以及石墨烯对单链DNA的选择性等方法和技术，构建了双信号的比率型电化学生物传感探究核酸标志物，该传感不仅检测了CAG三核苷酸重复序列，并能够检测出扩增长度。同时，基于石墨烯的优异电性能和晶体管自身的信号放大功能，构建了新型溶液门控石墨烯晶体管生物传感研究核酸葡糖糖等标志物，选择性高，灵敏度高。本项目能够较好地协调融合分子识别和信号放大技术等功能，使他们各自发挥作用，降低干扰、同时也提高灵敏度；构建的这些电化学生物传感和晶体管生物传感选择性高、灵敏度高，并具有较好地稳定性和一定的实用性。这些研究为疾病标志物三核苷酸重复序列的快速灵敏分析检测提供了电化学新方法和新器件，为实现神经遗传病三核苷酸重复疾病和癌症的早期诊断和预警提供了一种可行性的技术和方法。