基于高亮度、高稳定的CuInS2核壳量子点的心肌标志物H-FABP和cTnI的联合即时检测方法研究

The mortality rate of cardiovascular disease is the highest in China and even in the world. Among the cardiovascular disease, acute myocardial infarction (AMI) is very dangerous and the most immediately life threatening. So it is of great clinical significance to realize the rapid and accurate diagnosis of AMI. The problems of low sensitivity and imprecision results for point-of-care testing (POCT) of heart-type fatty acid binding protein (H-FABP) and cardiac troponin I (cTnI) are the key bottlenecks that restrict the early detection of AMI. The lateral flow immunoassay based on CuInS2 quantum dots (QDs) can greatly improve the sensitivity and stability of detection while ensuring rapid detection. In this project, we intend to develop a sensitive, stable and simultaneous POCT method of the two biomarkers based low-toxic CuInS2 QDs as a label material. The highly stable, high quantum yield CuInS2 core/shell QDs is synthesized by precisely controlling the shell coating with the method of “interval drop addition, in situ growth", and microsphere encapsulating multiple CuInS2 QDs is obtained by multi-step silica coating, which will greatly improve the sensitivity and stability of POCT for H-FABP and cTnI. A novel simultaneous POCT method based cadmium-free QDs for detection of H-FABP and cTnI will be constructed, which not only can help for diagnosis and treatment of AMI and then reducing the mortality rate, but also can lay an important foundation for the cadmium-free QDs to become the promising candidates for cadmium-based QDs to construct a novel in vitro diagnostic technique.

心血管疾病已成为我国乃至全球致死率第一的疾病，其中急性心肌梗死（AMI）发病凶险，实现其“快而准”的早期发现和诊断具有重要的意义。目前，心肌标志物心脏脂肪酸结合蛋白（H-FABP）和心肌肌钙蛋白I（cTnI）的即时检测灵敏度不高、准确度不够是AMI诊断亟待解决的关键问题。以低毒性的CuInS2荧光量子作为标记材料构建免疫层析即时检测体系在实现快速检测的同时，可有效提高检测的灵敏度和稳定性。本项目拟采用“间隔滴加，原位生长”方式精确调控壳层包覆以合成高亮度、高稳定的CuInS2核壳量子点，并结合多步二氧化硅包覆制备量子点微球，以提高基于非镉量子点的免疫层析即时检测体系的稳定性和灵敏度，构建H-FABP和cTnI的灵敏、稳定的联合即时检测新方法。这不仅有助于实现AMI的早期发现并降低死亡率，同时也将为非镉量子点成为含镉量子点的替代标记材料构建新型的体外诊断技术奠定重要的基础。

心肌标志物-心脏脂肪酸结合蛋白（H-FABP）和心肌肌钙蛋白I（cTnI）的即时检测灵敏度不高、准确度不够是急性梗死（AMI）诊断亟待解决的关键问题。本项目以低毒的CuInS2核壳量子点作为标记材料构建H-FABP和cTnI的灵敏、稳定的即时联合检测方法为目标。首先通过精确调控CuInS2量子点的壳层生长过程，制备出了厚壳层、大尺寸、量子产率约80%的高稳定的绿色CuInZnS/ZnS核壳量子点；同时基于性能优异的CdSe/ZnS量子点分别通过微乳液法、组装法和生物分子修饰法制备出了各种高亮度、稳定性好的镉基量子点荧光微球，并以它们为标记材料构建免疫层析传感器和三维阵列传感器分别实现了三种心肌标志物cTnI、肌酸激酶同工酶（CK-MB）和肌红蛋白（Myo）的联合即时检测，以及炎症标志物C反应蛋白（CRP）和血清淀粉样蛋白A（SAA）的联合检测，灵敏度和稳定性都得到了很大的提高。针对表面具有巯基配体的高质量CuInZnS/ZnS核壳量子点难以进行水相转移的问题，我们通过采取预包硅再通过反相微乳液分步包硅的方法得到了稳定性较好的二氧化硅包覆单个量子点的水相CuInZnS/ZnS@SiO2量子点，以它为标记材料对H-FABP进行定量检测。为了进一步提高检测的灵敏度，我们采用微乳液和表面活性剂取代的两步法得到了硅球中包覆多个量子点的具有更高亮度的荧光微球，以荧光微球为标记材料制备的荧光探针构建荧光免疫吸附法实现了对H-FABP的灵敏检测，最低检出限为0.48 ng/mL。该方法大大提高了基于非镉量子点的生物检测的灵敏度，且检测方法具有良好的准确度和稳定性。不仅为AMI的早期诊断开辟了新的机遇，同时为非镉量子点成为含镉量子点的替代标记材料构建新型的体外诊断技术提供了重要的研究基础。