构建快速检测降钙素原的高灵敏量子点免疫层析技术的研究

A highly sensitive immunoassay is required for detection of procalcitonin (PCT) in human serum, using as an important infection disease biomarker. However, low sensitivity and non-quantitative test results of traditional colloidal gold immunochromatography assay restricted its application for detection of low-abundance biomarkers in human serum. This proposal aims to establish a highly sensitive point-of-care test based on quantum dots labeling, for detection of low-abundance biomarker PCT in human serum. Our previous studies indicated that the background fluorescence of nitrocellulose membrane, the ratio of antibody to labels and conjugation method of antibody and labels all affected the sensitivity of immunochromatography assay. In order to improve the sensitivity of quantum dots immunochromatography assay, we’d like to construct a high sensitivity quantum dots immunofluorescent probe, via using red-near infrared fluorescent quantum dots to reduce the background fluorescence, micro/nano sphere signal amplification strategy to improve the signal/noise ratio, and oriented antibody conjugation method to keep the activity of antibody. Further, the highly sensitive quantum dot probe was used to establish a point-of-care test for detection of PCT in human serum by combination with immunochromatography technique. The sensitivity, specify, precision, dynamic range and stability of the immunoassay were respectively evaluated. At last, the feasibility of this technique was tested using the clinical serum samples. This proposal will provide a new method for rapid detection of low-abundance biomarker in human serum, and promote the development of point-of-care test industry.

降钙素原（PCT）是一种重要的感染性疾病血清标志物对免疫检测灵敏度要求较高。而传统胶体金免疫层析技术检测灵敏度低、定量效果差等缺点限制了其用于低丰度血清标志的检测。本项目基于量子点免疫层析技术，以PCT为检测目标，构建高灵敏的快速免疫诊断方法。前期研究发现，免疫层析技术中的硝酸纤维素膜背景荧光、抗体-标记物比例和抗体-标记物偶联方式都影响免疫层析技术的检测灵敏度。为提高量子点免疫层析技术检测灵敏度，本项目拟综合采用红光-近红外荧光量子点降低背景荧光、微球信号放大提高信噪比以及抗体定向偶联技术保持抗体免疫活性，构建得到高性能量子点免疫荧光探针。进一步与免疫层析技术结合，建立血清中PCT的快速免疫诊断方法，评估其灵敏度、特异性、精密度、线性范围和稳定性等关键参数，并在临床样本中验证该技术的可行性。本项目的开展，为快速检测低丰度血清标志物提供一种新途径，促进快速免疫诊断技术的发展。

本项目主要开展了高灵敏度快速检测降钙素原的量子点免疫层析技术的研究，项目基本按照任务书中规定的考察指标完成，取得的主要研究成果包括：（1）基于量子点纳米球的信号放大技术，通过乳液溶剂挥发法制备得到量子点纳米球，然后抗体与纳米球表面的羧基共价偶联得到信号放大型量子点免疫探针，斑点免疫检测乙肝表面抗原，与传统量子点免疫探针比较，信号放大型量子点免疫荧光检测的灵敏度提高约10倍。（2）建立一种量子点与抗体的定向柔性偶联技术，选用含有PEG柔性链段的定向偶联剂，通过抗体铰链区的巯基与表面带氨基的量子点定向共价偶联，与非定向偶联方法制备的量子点免疫探针相比较，可以显著提高量子点免疫探针的检测灵敏度，拓宽检测动态范围。（3）荧光量子点免疫探针与免疫层析技术结合，比较了不同表面性质量子点免疫探针的免疫活性，进一步与免疫层析技术结合，建立快速检测C反应蛋白和降钙素原的快速免疫诊断方法。（4）采用两种颜色的量子点分别标记，C反应蛋白（CRP）和降钙素原（PCT）抗体，通过量子点-胶体金间形成的荧光共振能量转移（FRET）体系，实现对两种感染性疾病血清标志物CRP和PCT的同时检测。（5）基于项目的相关研究成果，发表SCI论文3篇，获得发明专利1项。本项目是具有明显应用前景的基础研究课题，项目的开展建立一种新型的快速免疫诊断技术平台，项目成果可促进体外诊断相关企业的发展，提升体外诊断行业的产业水平。