基于金复合微纳米材料和靶向噬菌体构筑的电化学细胞传感器检测结肠癌细胞研究

The early and accurate diagnosis of cancer is very important to its prompt treatment. However, great issues existed for conventional assays, such as low sensitivity, false positive results, the difficult preparation of target cells, high-cost and long-term process. To date, antibody-based electrochemical cytosensors also experienced insensitivity and instability. In this project, the colon cancer cells will be chosen as the target to biopan with f8/8 landscape phage library. Through several rounds of biopanning, the colon cancer-specific phages (proteins) will be obtained in a high-throughput screening fashion. On the other way, the Au@BSA composites micro/nano spheres with the uniform sizes will be synthesized by using Bovine Serium Albumin (BSA) as template. Subsequently, the phages displaying cancer-targeting peptide will be conjugated onto Au@BSA composites via the covalent bond interaction. In this way, a functional nano/bio-sensing interface will be successfully fabricated on the Au electrode. By using the [Fe(CN)6]3−/4− as a redox probe, through the electrochemical measurements such as cyclic voltammometry, electrochemical impedance spectroscopy and differential pulse voltammetry, the elecrochemical cytosensor will be developed. The strategy described in this study can be applicable to designing elecrochemical cytosensors for other cancers. It is expected that the novel biosensor will play a key role in the field of biomedicine such as the early diagnose and monitoring of cancers, anticancer drug screening and cell adhesion, etc.

癌症的准确诊断对其治疗十分重要，但是，常规的检测方法存在灵敏度低、易出现假阳性、靶细胞制备困难、成本高和周期长等缺点。迄今，基于抗体的电化学细胞传感器，其检测灵敏度和稳定性都不理想。本项目拟以结肠癌细胞为研究对象，与f8/8风景噬菌体展示文库相互作用，经多轮生物淘选，高通量筛选出与结肠癌细胞靶向特异性结合的噬菌体单克隆; 以牛血清白蛋白（BSA)为模板合成粒径均一的金复合微纳米材料Au@BSA。将具有靶向结合特性的噬菌体单克隆与电极表面固定的Au@BSA纳米复合物通过共价偶联，构筑新颖的功能化纳米生物传感界面。然后以铁氰酸钾为氧化还原探针，利用循环伏安、差分脉冲伏安和交流阻抗等电化学技术，构建电化学癌细胞传感器，旨在实现对结肠癌细胞的灵敏、特异检测。该电化学细胞传感器构筑策略可以推广到开发其它癌细胞传感器，并可望在癌症的早期诊断和监测、抗癌药物筛选和细胞吸附等生物医学研究领域发挥重要作用。

癌症的早期确诊在临床上具有重要的地位。常规的检测方法存在灵敏度低、易出现假阳性、抗体制备困难、成本高和周期长等缺点。迄今，基于抗体的电化学细胞传感器，其检测灵敏度和稳定性都不理想。现有大量的数据表明，唾液酸（一种含有9个碳原子骨架的糖蛋白）的含量与许多癌症（直结肠癌、乳腺癌、肝癌和卵巢癌等）具有密切的关系。因此，设计简单快速、准确、低成本和高灵敏的技术来研究细胞表面的唾液酸对了解和认识疾病的发展具有重要的意义。本项目合成了生物相容性好，比表面积大，三维多孔结构的Ag@BSA“纳米花”，并将其作为电化学生物传感界面，进而通过化学键偶联接骨木凝集素（对唾液酸具有高亲和力，易合成和标记，稳定性好，耐储存等优点），构筑了电化学细胞传感器，来检测结肠癌细胞并评估其上的唾液酸含量。.结果表明，电子阻抗值与被捕获到电极上的结肠癌细胞浓度（范围从1.35×102-1.35×107 cells/mL）的对数呈良好的线性关系，检测限为 40 cells/mL。此外，该传感器具有很好的特异性（可明显区分出肿瘤细胞和非肿瘤细胞）、稳定性（放置20天峰电流仅降低8.7%）和重现性（5次测定，相对标准偏差是4.3%）。进一步，利用紫外-可见光谱技术，我们构建了崭新的定量和灵敏检测结肠癌细胞表面唾液酸的方法，可以检测到单个结肠癌细胞表面的唾液酸数量大约为2.16×1012个分子。基于Ag@BSA仿生界面和接骨木凝集素-唾液酸构筑的电化学结肠癌细胞传感器策略具有线性范围宽，检测限低，重现性和准确性好等优点，对唾液酸高度表达的癌细胞具有高特异性，可以推广到开发其它癌细胞传感器，并有望在癌症的早期诊断和监测、抗癌药物筛选和细胞吸附等生物医学研究领域发挥重要作用。