一种生物合成碳点荧光微球的构建及其在高灵敏度病原体检测和疾病诊断中的应用研究

The rapid development of fluorescent nanomaterials has made it possible to realize high-sensitivity bio-detections and early disease diagnosis. Carbon dots (CDs) are one of the nanomaterials with superior optical properties and low cytotoxicity. However, there are few applications of CDs in rapid bioassays. Based on the widely reported ability of CDs to enter bacteria cell, this study will develop a biosynthesized CDs-based fluorescent microsphere, through using CDs as the reporter and the bacteria (Staphylococcus aureus) as the carrier. This fluorescent bacterial microsphere lit up by CDs is also called “CDs-Firefly”. In the study, S. aureus cell is chosen as the carrier of CDs-Firefly, because it contains a large number of SPA proteins that can easily and efficiently combine the IgG antibodies. In this way, the CDs-Firefly could be used as detecting probes with high biological affinity in the immunoassays. Furthermore, the study will explore the mechanism of CDs entering into bacterial cells, which could provide theoretical basis and instructions for the preparation of CDs-Firefly. By combining the CDs-Firefly and immunomagnetic beads, the study will develop a novel high-sensitivity assay for the rapid detection of pathogens (Escherichia coli O157: H7) and clinical diagnostic biomarkers (N-terminal pro-B-type natriuretic peptide, NT-proBNP), providing a new method for early diagnosis of the infectious diseases and other major diseases.

荧光纳米材料技术的快速发展为超敏生物检测与早期疾病诊断的实现提供了可能。碳点是其中一种兼具优越光学性质和低细胞毒性的纳米材料，但在生物检测领域内的应用目前仍较少报道。基于碳点能够进入细菌体内实现荧光染色的材料性能，本研究拟采用一种生物合成方法，即以碳点为荧光分子、以金黄色葡萄球菌菌体为载体，通过碳点染色金黄色葡萄球菌来制备一种细菌性荧光微球（称为“碳点囊萤”）；并利用金黄色葡萄球菌表面富含SPA蛋白的特性，实现碳点囊萤与检测抗体的亲和偶联，进而构建一种可直接用于生物检测的免疫荧光探针。通过探究碳点的细菌染色机制和关键影响因素，本项目将发展一种高荧光强度和高生物亲和性碳点囊萤的制备方法；并结合免疫磁珠富集技术建立一种可对病原体大肠杆菌O157：H7和心衰诊断标志物NT-proBNP进行高灵敏度分析的快速检测方法，从而为传染病和其他重大疾病的早期诊断提供一种新方法。

纳米材料的快速发展为超敏生物检测与早期疾病诊断的实现提供了多种可能。碳点是其中一种兼具优越光学性质和低细胞毒性的荧光纳米材料，但在生物检测领域内的应用目前仍较少报道。本研究通过微波辅助合成法制备得到一种能够实现细菌荧光染色成像的固态发光碳点材料，并基于该特性构建了一种以细菌（金黄色葡萄球菌）为载体、碳点为荧光报告分子的荧光微球（“碳点囊萤”）制备方法。在此基础上，进一步研究了碳点染液浓度、染色时间和染色温度等多种因素对微球荧光强度的影响，并推测碳点可能主要通过扩散途径进入细菌体内。通过多种表征测试发现，碳点荧光微球的荧光强度约为单个碳点粒子的1000倍，而且具有较好的荧光均一性和储存稳定性。利用微球载体表面天然存在的金黄色葡萄球菌A蛋白（SPA），可不借助其他化学修饰手段而实现碳点荧光微球与抗体Fc端的定向偶联，从而可将所得微球作为一种特异性荧光探针应用于生物检测。本研究将碳点荧光微球和免疫磁珠相结合，基于双抗体夹心免疫检测原理，建立了一种可在20min内检测牛奶样品中大肠杆菌O157:H7的定量检测方法，最低检出限为24CFU/反应，其检测灵敏性与荧光PCR方法相当。本研究将碳点材料特性和细菌生理特性相融合，建立了一种高荧光强度和高生物活性碳点荧光微球的制备方法，不仅进一步拓展了碳点材料在生物检测领域的应用，而且为实现超敏生物检测和早期疾病诊断提供了一种新的检测示踪材料及检测方法。