用于肝癌早期诊断的多通道纸基微流控生物传感器的研究

Enzyme linked immunosorbent assay and electrochemiluminescence were used to determine the level of serum alpha-fetoprotein (AFP) in early diagnosis of liver cancer, but there were still some limitations in the analysis of time, sample volume and cost etc. The nonspecific of AFP in clinical diagnosis of liver cancer can lead to false-positive and false-negative, which not only increased the probability of misdiagnosis and missed diagnosis, but also increased the burden of patients. Therefore, the project intends to design an instant, portable and accurate electrochemical immunofiltration biosensor based on the combined detection of AFP, alpha-L-fucosidase (AFU) and gamma glutamyl transpeptidase (GGT), which could reduce the probability of false-positive and false-negative in the early diagnosis of hepatocellular carcinoma. Firstly, a three-dimensional paper-based microfluidic electrode with three separated channels is designed. Secondly, some electrochemical markers such as ferrocene derivatives or biological enzymes are used to label the anti-AFP (AFU or GGT) monoclonal antibody, so as to prepare high-sensitive electrochemical probes. Thirdly, a paper-based microfluidic electrochemical immunosensor with three channels was constructed based on the combined detection of AFP, AFU and GGT. Finally, the new electrochemical immunoassay can be applicated in clinical diagnosis to establish a miniature, portable, low cost and highly sensitive technology platform for early diagnosis of liver cancer.

目前，临床常用酶联免疫吸附法或电化学发光法测定血清甲胎蛋白(AFP)水平来进行肝癌早期诊断，但是这些方法在分析时间、样品量及检测成本等方面仍存在一定局限性，并且由于AFP诊断肝癌的非特异性，临床还经常出现假阳性和假阴性，不仅会增加误诊、漏诊的概率，还会增加病人负担。鉴于此，本项目拟设计一种基于AFP、α-L-岩藻糖苷酶(AFU)和γ-谷氨酰转肽酶(GGT)联合即时、便携、准确检测的电化学免疫渗滤生物传感器，以降低肝癌早期诊断假阳性和假阴性的发生概率。研究内容包括：设计一种具有三通道的三维(3-dimensional，3D)纸基微流控免疫检测基底电极；制备以二茂铁衍生物或生物酶等为电活性物质的高灵敏度电化学探针；构建基于AFP、AFU和GGT联合检测的三通道纸基微流控电化学免疫传感器；开展本项目所建立的检测方法在临床诊断中的应用研究，建立微型、便携、低成本、高灵敏的肝癌早期检测通用技术平台。

肝癌是影响人类生命健康的重大疾病之一，中国是肝癌高发区。它属于中国的重大公共卫生问题，间接影响了中国未来几十年发展。在早期阶段肝癌不易发现，往往到了中晚期才确诊，导致患者的存活率大大降低。因此，对于肝癌早期的发现和治疗是很有必要的。目前临床上用于肝癌早期诊断方法，如ELISA和ECL等仍然存在众多问题。例如，检测耗费时间较长、检测中花费的成本高昂以及需要专业人员和场地等。因此，基于生物传感器分析速度快、准确度高、操作简单和成本低等独特的优势，开发了用于检测肿瘤标志物的生物传感器，从而实现高灵敏、高特异性的肝癌早期诊断的意义和目的。.本项目构建了基于肝癌标志物微量检测的生物传感技术。结果表明，基于无标记抗体的电化学传感器对CEA检测极限是0.06 pg/mL, 通过对抗体结合方式及纳米材料的的优化，CEA传感器的检测极限可以达到1.42 pg/mL。同时，基于此免疫结合方式，开发了一种双重信号放大的夹心型电化学AFP传感器，其检测极限是2.2 pg/mL.另外，随着光刻技术，纳米材料信号放大技术的引入，开发了一种基于内滤效应的纸基荧光GP73传感器检测极限达到了6 pg/mL。通过对同一样本的检测，能够达到高灵敏度、高特异性的肝癌早期诊断目的。开发出的生物传感技术为肝癌早期临床诊断提供了检测方法，具有重要的研究价值。