基于SERS免疫编码探针对多组分肝癌标志物同时定量检测及其早期临床应用研究

Because a single liver cancer marker is not sufficient to accurate diagnosis the occurrence of liver cancer, it is very important to develop a highly sensitive simultaneous quantitative detection technology of multi-component markers for the early accurate diagnosis of liver cancer. Surface-enhanced Raman spectroscopy (SERS) has become an ideal method for the detection of tumor markers due to its advantages of ultrasensitivity and fingerprint recognition. This project aims at the problem that it is difficult to realize simultaneous and accurate quantitative detection of multi-component liver cancer markers by SERS technology, and proposes to combine SERS coding probe, internal standard (IS) quantitative analysis and magnetic bead (MB) separation technology. Based on the basic principles of SERS coding probe “multi-component detection”, IS “quantitative analysis”, MB “high efficiency separation and enrichment” and specific immune reaction, we adopt a new method of magnetic capture substrate and SERS coding probe modified with specific antibody to capture liver cancer markers, and develop a new sandwich structure marker sensor. To construct a SERS detection mode with high separation enrichment, high enhancement and high stability, and study the relationship between the Raman signal ratio of SERS coding probe and IS molecule with the concentration of multi-component liver cancer markers. To reveal the law of simultaneous and accurate quantitative detection of multi-component markers in complex clinical samples, so as to provide new technologies and methods for early clinical diagnosis of liver cancer.

由于单一肝癌标志物不足以精确诊断肝癌的发生，开发多组分标志物的高灵敏同时定量检测技术，对肝癌的早期精准诊断至关重要。表面增强拉曼光谱（SERS）技术兼具超灵敏和指纹识别的优势已成为肿瘤标志物检测较为理想的手段。本项目针对目前SERS技术难以实现多组分肝癌标志物同时精准定量检测的问题，提出将SERS编码探针、内标（IS）定量分析与磁珠（MB）分离技术相结合。基于SERS编码探针“多组分检测”、IS“定量分析”、MB“高效分离富集”和特异性免疫反应的基本原理，采用对新型磁性捕获基底和SERS编码探针修饰特异性抗体来捕获肝癌标志物的方法，开发一种夹心结构的新型标志物传感器。构建高分离富集、高增强和高稳定的SERS检测模式，研究SERS编码探针和IS分子的拉曼信号比值与多组分肝癌标志物浓度关系的问题。揭示对复杂临床样品中多组分标志物同时精准定量检测的规律，为肝癌的早期临床诊断提供新技术和方法。

本项目聚焦癌症的早期临床精准诊断新技术和方法，从多组分肝癌标志物的同时定量检测出发，基于三明治夹心策略构建表面增强拉曼光谱（SERS）生物传感器，以满足临床血液样本中低浓度的多组分标志物高灵敏检测的需求。通过构建高富集、高增强和高稳定的磁辅助SERS编码探针获得了高SERS增强的最佳复合结构，阐明了磁珠技术“高效分离富集”、拉曼光谱“指纹识别”、SERS编码探针“多组分检测”和内标“定量分析”的基本原理。建立了多组分肝癌标志物编码探针、内标分子信号比值与标志物浓度的定性定量关系，筛选和优化了血液中多组分肝癌标志物同时检测与定量分析的条件。构建的生物传感器用于肝癌标志物检测时，实验结果表明，SERS信号分子、内标分子强度比与标志物浓度的对数值呈现良好的线性关系，实现了临床血液样本中多组分肝癌标志物的同时、准确、定量和超灵敏检测，检测限低至fg/mL（低于其他检测技术）。此外，14例AFP阴性患者、52例巴塞罗那不同临床肝癌分期（BCLC）患者的检测结果显示，与医院检测方法（化学发光）相比，该多组分标志物SERS检测方法的准确率更高(>85%)，尤其是对AFP阴性被漏诊的肝癌患者，提供了一种更精准的筛查方案。总之，该方法为肝癌的快速筛查、准确评估、预警和精准诊断提供了一个高灵敏的传感平台。相关研究在Biosens.Bioelectron.,Sensor.Actuat.B-Chem.,Anal.Chem.等国际权威期刊发表SCI论文7篇，其中影响因子大于10的2篇。