基于双纳米金探针增强免疫传感器对牙周炎症因子水平与冠心病生物学相关性的研究

Periodontal disease as inflammation of periodontal issues caused by dental plaque is believed to be potential factor leading to coronary heart disease and inflammatory factors are very likely to be the bond of the relevance. Therefore, it is of great necessity to examine the level of inflammation factors in gingival crevicular fluid and serum, clinically explore its biological relevance to periodontal disease and coronary heart disease in the control of infection, health care of periodontal issues, and prevention of coronary heart disease as well as periodontal disease. By a joint application of nano-materials, protein microarray and biosensor technique, this project aims to accomplish the design and making of a nanogold-supported probe signal detecting system, the building of protein microarray immunosensor based on the multi-nanogold-supported probe, so as to realize a multi-sampling and multi-target synchronous detection with high sensitivity and specificity, and thus find a new clinical method with high efficiency and rapidity in the detection of the inflammatory markers in gingival crevicular fluid. Meanwhile, the project aims to fulfill the variance analysis of periodontal disease indexes, levels of blood lipid metabolism, levels of coronary heart disease biomakers, and levels of inflammatory biomarkers in gingival crevicular fluid, and further explore the linear correlation between the level of periodontal inflammation and that of the systemic inflammation. The above work is of great scientific significance and high clinical value in that it is to provide empirical data for the establishment of a non-invasion, non-trauma diagnosis method against periodontal disease and related coronary heart disease.

牙周炎是由菌斑微生物引起的牙周组织的炎症性疾病，也是冠心病的潜在危险因素，炎症因子可能是二者相互关联的纽带，因此，检测牙周龈沟液及外周血清中炎症因子的水平，探索其与牙周炎和冠心病的临床和生物学相关性对于有效控制感染、保存牙周组织健康、预防牙周炎相关冠心病的发生具有重要意义。本项目拟将纳米材料，蛋白芯片和生物传感技术等联合应用，设计并制备双纳米金探针标记信号检测系统，结合蛋白微阵列芯片免疫传感技术，构建双纳米金复合探针增强蛋白芯片免疫传感器，实现多样本、多靶标的高灵敏度和特异性的同步检测，建立牙周龈沟液炎症标志物的临床椅旁高效、快速定量检测新方法。完成牙周炎症指数、血脂代谢水平、冠心病标志物水平及龈沟液炎症标志物水平的差异分析，进而探索牙周局部炎症水平和系统性炎症水平的线性关系，为建立非侵入式、无创伤的牙周炎及相关冠心病诊断方法提供实验依据，具有重要的科学意义和临床实用价值。

本研究分别采用纳米金复合探针结合蛋白芯片技术、纳米金和免疫磁珠作为纳米颗粒的免疫层析技术、及基于叉指电极的电容芯片以及PDMS微流控通道技术构建了针对冠心病标志物及相关牙周炎性因子的多种生物检测芯片平台。以冠心病患者外周血为样本，通过DNA杂交的方法，形成双金探针链接放大信号，完成C反应蛋白（CRP）等炎性标志物的检测，检测限CRP为326pg/ml，IL-6为8pg/ml，显示出纳米金复合探针结合蛋白芯良好的特异性、选择性和低信噪比，具有稳定的可重复性和良好的准确度。以肌红蛋白、癌胚抗原和角蛋白作为目标抗原，在检测线上形成有三明治结构的“纳米金/免疫磁珠-检测抗体-检测抗原-捕获抗体”复合物，完成定量检测。构建用于冠心病标志物及牙周炎症因子CRP检测的电容芯片生物传感器，微流控通道中完成羧基化、活化等工艺，实现电极表面CRP抗体的修饰和验证，通过检测样品-电极-目标蛋白-电容值的系列性反应和电容值变化量监测目标蛋白CRP的浓度，检测限可达到1 pg/mL。.同时，本研究完成了冠心病患者临床数据信息库和生物样本库的建立，针对高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)、总胆固醇(TC)、甘油三酯(TG)、空腹血糖(FPG)等冠心病血清学指标及牙周病相关的CPR、TNF-α、IL等冠心病的炎症标志物的统计学分析，证实，TG、HDL和LDL对C反应蛋白，白介素-6和肿瘤坏死因子的影响都是负向的，总胆固醇对三个解释变量的影响都是正向的，而且影响程度很大，三个解释变量与其各个影响因素之间是密切相关的。由此建立的冠心病血脂水平与血清炎性因子之间的线性关系，完成了牙周炎症指数、血脂代谢水平、冠心病标志物水平及龈沟液炎症因子水平的差异分析，为用于牙龈沟液检测的生物检测芯片平台的设计和指标提供技术要求和数据基础，力求实现一种非侵入式、无创伤的牙周炎及相关冠心病诊断平台的构建，争取“早发现、早诊断、早治疗”，对有效保存牙周组织健康，预防牙周炎相关冠心病的发生具有重要的科学意义和临床实用价值。