用于肝癌诊断的多种标志物同时识别检测新方法研究

In the development of bio-detection assays that could be useful in hepatocellular carcinoma (HCC) clinical diagnosis, improving assay sensitivity might be the foremost important task, but doing so without deteriorating other factors such as assay cost, time, complexity, dynamic range and reproducibility is as important as improving sensitivity for practical applications of developed bioassays. To address this assay sensitivity issue, it is necessary to synthesize highly functional nanomaterials and used their structures and functions to amplify detection signals. On the other hand, alphafetoprotein (AFP) has served as a diagnostic test for HCC since the 1970s, when most patients with HCC were diagnosed at an advanced stage with clinical symptoms. However, AFP levels are elevated both in patients with HCC and in those with chronic liver diseases, and there is a wide overlap between the two groups. Thus, the role of AFP as a diagnostic test is getting smaller. To date, many tumor markers have been proposed as a complement or substitute for AFP in the diagnosis of HCC, such as des-gamma-carboxyprothrombin, lens culinaris agglutinin-reactive fraction of AFP, α-L-fucosidase, golgi protein 73, and various cytokines. The absence of correlation between these markers suggests that each marker is related to a different aspect of HCC, therefore the use of these markers in combination however may significantly improve the diagnostic accuracy of HCC diagnosis. In this project, we propose various signal amplification and bio-detection strategies based on electrochemical, surface plasmon resonance, fluorescence resonance energy transfer and magnetic properties of functional nanomaterials for the ultrasensitive and simultaneous detection of multi-targets in samples. The combination and simultaneous determination of the combined tumor markers will aid in the assessment of their diagnostic utility, and provide an objective basis for the assessment of their clinical utility.

本项目拟发展生物识别分子在纳米材料表面的定向偶联和有序组装方法，构筑对多种特定靶标分子具有高效识别性能的纳米生物探针和生物传感界面。利用纳米生物探针和纳米传感界面上生物分子与样品中肿瘤标志物靶标分子特异性识别过程引起的界面电化学、荧光、表面等离子共振特征变化，结合电化学、表面等离子体共振和多种谱学手段，实时跟踪监测传感界面上生物识别分子/靶标分子之间相互作用的动力学过程，揭示界面识别过程中生物量和化学量通过界面效应转换成物理量的机制，获取传感体系的多靶标特异性同时识别传感规律，建立基于电化学、表面等离子共振和荧光共振能量转移原理的多种标志物同时识别检测新技术和新方法。筛选肝癌"最佳肿瘤标志物组合"，开发用于肝癌早期诊断的多种标志物同时识别检测传感器件，为提高肝癌诊断的特异性、灵敏性和准确性提供可靠途径，对肝癌的早期筛查、早期诊断、早期治疗以及提高患者存活率具有重要意义。

基于生物识别分子与功能纳米材料之间的特殊相互作用构筑了多种纳米生物探针和生物传感界面，利用生物识别过程中纳米材料的光电效应，构建了几类基于电化学、表面等离子体共振以及荧光技术的多种标志物同时识别传感方法，用于对甲胎蛋白、癌胚抗原以及致病基因等标志物的分析检测。围绕以上内容，本项目主要开展了以下方面的研究工作：（1）以氧化还原电位存在明显差异的电子介体为信号探针，通过共价偶联技术将生物识别分子与对应的电子介体修饰于纳米材料表面制备纳米生物信号探针，通过DNA杂交、生物素反应、免疫识别等方式将纳米生物信号探针连接到传感界面上，采用电化学方法记录并区分传感界面上电子介体的氧化峰/还原峰的差异，建立基于纳米生物信号放大和电子介体响应的多种标志物同时识别检测方法。（2）将对目标分子有特异性识别作用的DNA、生物素、适配体、抗体等生物分子组装到SPR金片表面，利用SPR技术实时监测传感界面上生物分子的相互作用，建立基于免疫识别、DNA链置换反应、杂交链扩增放大以及纳米粒子耦合放大效应的SPR识别传感新方法，实现了甲胎蛋白以及生物小分子的高灵敏识别检测。（3）选择不同颜色的半导体量子点以及金属纳米簇为信号探针，目标物与识别分子之间的作用使得量子点聚集以及金属纳米簇解体而荧光改变，在单一激发光源下得到可区分的多色量子点以及金属纳米簇的发射光谱，建立了基于量子点聚集以及金属纳米簇解体而荧光猝灭原理的多种目标物同时识别传感平台。（4）制备了集样品反应、富集和分离等功能于一体的微芯片分析系统，结合磁性表面分子印迹技术以及自聚合反应，对芯片通道进行修饰，实现了多种类物质的同时分离分析。项目已按计划完成，在本项目的资助下，在Anal. Chem., Chem. Commun., Biosens. Bioelectron., Anal. Chim. Acta，J. Chromatogra. A等刊物上发表SCI收录论文20余篇，授权发明专利5项，申请发明专利6项。