基于新一代量子点探针的心肌多标志物POCT联合诊断研究

Developing high-efficient point-of-care testing (POCT）technique for myocardial biomarkers, is of great importance for on-site and rapid diagnosis of cardiovascular disease such as acute myocardial infarction. This project may develop highly luminescent and narrow-emissive CsPbX3 perovskite quantum dots based immuno-labeling probes, for the great demands of high sensitive detection and multi-analysis using immunochromatography technique. To overcome the drawback of polar-environment-sensitivity from colloidal sized CsPbX3 quantum dots, this project would develop dendritic mesoporous carriers derivated by ammonium halide type affinity ligands, for templated and affinity assembly of CsPbX3 quantum dots in hydrophobic environment driven by ligand-exchange, aiming to address the nanoscale controlled assembly issue of CsPbX3 quantum dots; Realizing gradually modification of CsPbX3 quantum dots from hydrophobic to hydrophilic by means of deep-embedding into radial pore channels, coating by hydrophobic polymer layer, silanization and organosilica shell encapsulation, aiming to address the photo stability issue of CsPbX3 quantum dots. Constructing immunochromatography platform based on highly luminescent and spectra-resolved multi-color CsPbX3 quantum dots probes. Realizing high sensitive and combined POCT diagnosis of multiple myocardial biomarkers in blood, by means of efficient signal amplification of microsphere probe in single immuno-reaction, multiple immuno-recognition and controlling in chromatography strip, and multi-spectra intensity collection from fluorescence emissions.

发展高效的心肌标志物即时检验(POCT)技术，对于急性心肌梗死等心血管疾病的现场、快速诊断具有重要意义。本项目拟发展高发光及窄发射CsPbX3钙钛矿量子点免疫标记探针，应对当前免疫层析技术的高灵敏检测及多元分析重大需求。为解决胶体尺寸CsPbX3量子点对极性环境的敏感性问题，拟发展卤化铵类亲和配体改性树状介孔载体，以配体交换驱动疏水环境中CsPbX3量子点的模板化亲和组装，解决CsPbX3量子点的纳米尺度可控组装问题；以放射状孔道深层包埋、聚合物疏水层包覆、硅烷化及有机硅壳层包封等策略，实现CsPbX3量子点由疏水至亲水的逐步改性，以期解决CsPbX3量子点的光学稳定性问题。构建基于高发光及光谱分辨型多色CsPbX3量子点探针的免疫层析平台。借助微球探针对单次免疫信号的有效放大、层析试纸多重免疫识别及质控、荧光发射多波段强度采集等方式，实现以POCT对血液中心肌多标志物的高灵敏联合诊断。

CsPbX3作为新一代量子点材料，具备优异的发光性能及其可调谐性。在液相胶体分散状态下对CsPbX3量子点进行组装及修饰，并应用于生物医学领域，是目前国际上尚未解决的难题。本项目发展了中心-径向孔道结构树状介孔二氧化硅模板，并设计合成了两性离子配体硅烷化试剂用于孔道表面的亲和配体改性；通过有机相直接组装将CsPbX3量子点高效填入亲和模板内部获得高亮度及荧光稳定的组装体；以亲和配体ZW对量子点进行表面修饰剂替换进行两性离子硅烷分子层包覆，并经过在水解缩合形成一层有机硅包覆层。所获得的SiO2/CsPbX3-ZW复合材料在醇等极性有机溶剂中可胶体分散并且荧光稳定。对组装结构高温热处理进行二氧化硅封口操作，获得了SiO2完全包封且高密度负载CsPbX3量子点的水分散性荧光微球。本项目所提出的以深孔道模板有机相亲和组装、有机硅-致密二氧化硅复合表面改性理念，有效地解决了稳定性难题，在确保组装体尺寸处于纳米级以及良好胶体分散的前提下，其CsPbX3量子点负载量高、荧光亮度高、发光窄且在水相体系中长期稳定发光，并基本实现了颜色在可见光区内可调，为CsPbX3钙钛矿量子点的水溶性修饰提供了重要的方法参考。为将其拓展至生物传感领域，发展了CsPbX3组装结构的表面化学改性及其生物功能化，获得了性能理想的免疫标记探针。CsPbX3组装结构以其亮度与色纯度优势，可获得更多可辨识色度区间以及更高检测灵敏度。发展了基于量子点组装结构的荧光标记探针、荧光免疫层析检测试纸条以及便携式免疫层析定量检测装置（检测卡读条机以及3D打印荧光成像装置）用于定量分析。开展了以荧光免疫层析对心型脂肪酸结合蛋白（h-FABP）、肌钙蛋白I（cTnI）、N端前脑钠肽（NT-proBNP）的高灵敏即时诊断。通过裸眼辨识、便携式仪器定量分析，实现了对h-FABP、cTnI及NT-proBNP的有效检出，其裸眼与定量检测限分别为1 ng mL-1/0.21 ng mL-1、0.2 ng mL-1/0.062 ng mL-1以及0.025 ng mL-1/0.013 ng ml-1，均达到了对以上三种心肌标志物的临床诊断浓度阈值，可为进一步判断有无发生心肌梗死提供必要的科学依据。