基于高通量单颗粒LSPR成像的胰腺癌早期标志物检测研究

To validate protein biomarker candidates for pancreatic cancer is a prerequisite for developing early cancer screening methods and a key of increasing early detection rate of pancreatic cancer. Current approaches used for quantifying low abundant proteins in blood more or less suffer from the detection limitation, the cost, and the time. Here, we are going to develop a novel method to determine low abundant proteins in blood based on single particle surface localized plasmon resonance spectral imaging for validating biomarker candidates for ealry pancreatic cancer. Using the array single Au nanoparticles modified with partial antibody as sensors, we build a platform to detect 3 model candidates by LSPR peak shift with spectral imaging at single particle level. The platform is integrated with microfluidics in order to accelerate mass transportation of antigen and parallelly measure multi proteins. We have great chance to develop an approach for quantifying low abundant proteins in blood with the advantages including label-free, low cost, easy operation, low detection limitation, high speed and throughput, and low consumption of samples and reagents.

从大量候选蛋白标志物中验证出临床上可用的胰腺癌肿瘤标志物，是建立早期肿瘤筛查方法的前提，也是提高胰腺癌早期诊断率的关键。现有低丰度蛋白质直接定量技术在检测限、检测成本、检测时间等方面存在或多或少的问题，难以满足临床需求。本项目拟发展一种基于单颗粒局域表面等离子体共振光谱成像的蛋白质定量方法，并用于胰腺癌早期肿瘤标志物的临床验证研究。该方法以在表面修饰了不完整抗体的单金纳米粒子为传感器，以单颗粒光谱成像技术为手段，以LSPR光谱变化为抗原分子检测参数，针对三种胰腺癌早期候选蛋白标志物，搭建检测平台，建立检测方法，优化平台性能。该方法同时尝试借助微流体控制技术，一方面加速抗原分子的质量传递过程，缩短分析时间；另一方面实现多种蛋白质的同时检测，提高分析通量。本项目有望开发出一种高灵敏蛋白质测试平台，具有免标记、成本低、操作简便、检测限低、分析速度快、分析通量高、样品及试剂消耗量少的优点。

在单颗粒水平上，建立了两种蛋白质的超灵敏检测平台。其中一种以检测抗体和捕获抗体分别修饰量子点，对应抗原存在时，生成以抗原为中心的量子点二聚体。连续激发下，团聚的量子点发生不同步的光谱蓝移，表现为团聚体的一级条纹发生分裂。通过一级条纹分裂与否来判断抗原是否存在，实现了蛋白质的超灵敏检测。以癌胚抗原为例，其线性范围为13.6 fM-1399.3 pM, 检测限为6.7 fM。我们的方法突破了以往均相免疫分析方法灵敏度差的局限性，为疾病的早期诊断打下了坚实的基础。