基于二维有序球腔阵列微电极的蛋白质电化学传感器研究

Cancer is a serious disease that jeopardizes human life and health, however, a fundamental breakthrough in human cancer treatment is still missing. Early detection and early treatment are still the basic countermeasures to treat cancer patients. Therefore, to achieve the ultra-sensitive detection of cancer biomarkers, in this project, an electrochemical-based protein sensor with a novel “super-sandwich” structure, which consists of two-dimensional highly-ordered nanocavity array and signal amplified immunocomplex on gold nanoparticles, will be designed and synthesized for early diagnosis of cancer biomarkers. Firstly, a two-dimensional ordered polystyrene template will be prepared on the surface of ITO electrodes through self-assembly technique, combined with electrochemical deposition and a sacrificial-template method, series of two-dimensional ordered insulators (such as SiO2) and semiconductors (such as WO3) microcavity array will be constructured, serving as the electrochemical microreactor on the the electrode surface. Secondly, a long DNA concatemer with redox-active probe molecules will be desiged using hybridization chain reaction (HCR) technique, which accordingly achieves significant in situ signal amplification to enhance the detection sensitivity. Using several typical cancer protein markers as research models, through control group tests，the diagnosis value of the proposed protein chip will be evaluated. Besides the controllable preparation of the novel electrochemical sensor for cancer biomarker detection, the microstructure-control mechanism and the electrochemical properties of such microcavity array electrodes will be clarified; the model for electrochemical detection on such electrodes will also be established. Moreover, the suitable micro-environment for keeping protein activity and for electrons transfer will be investigated through the modification of the inner surface of the as-prepared microcavity array. Additionally, the internal connection between signal sensitivity and the competitive adsorption behavior (occurred between the antibody and the labeled–molecule-amplification system) will be revealed.

为实现对癌症标志蛋白的超高灵敏检测，本项目拟通过构筑一种具有二维有序球腔结构的微电极阵列，结合杂交链式反应技术及免疫识别形成“超级三明治”式的免疫复合物，以电化学手段为信号读出方法来加以解决。首先，利用自组装在电极表面制备二维有序的聚苯乙烯模板，进而通过电化学沉积结合牺牲模板法构筑系列底部开放的二维有序绝缘体（如SiO2）及半导体（如WO3）微腔阵列，以形成电极表面电化学微反应区。其次，通过杂交链式反应在引入带有标记分子的DNA链节连环，构建实现标记分子电化学信号的原位扩增，提高检测灵敏度。本课题拟在解决新型蛋白质电化学传感器可控制备的基础上，阐明此类球腔微电极阵列的的微结构控制机制及电化学特性，建立电化学检测模型；通过微球腔内表面的修饰，探寻有利于保持蛋白分子活性及电子传递的微环境体系；揭示标记分子扩增体系及抗体在金纳米颗粒表面的竞争吸附行为与信号灵敏度及重现性之间的内在联系等基础问题。

癌症是严重危害人类健康的三大疾病之一，已成为我国城市居民第一位死亡原因。早期癌症患者一般无明显症状，传统的癌症诊断方法，如病理、影像学检查往往无法给出准确的早期诊断结果。为了早期发现、诊断和治疗癌症，临床已广泛开展肿瘤标志物的检测。酶联免疫测定(ELISA)是检测蛋白分子的“金标准”，此类方法利用抗体的多样性和特异性识别对应的抗原，然而灵敏度与分辨率一直是该方法亟待突破的瓶颈。血液样品中与早期癌症相关的蛋白标志物浓度仅为10-16~10-12 mol/L，而目前临床使用的免疫分析方法的检测限却只能达到皮摩尔水平，无法满足癌症早期诊断的迫切需要。因此，构建灵敏可靠的高通量新型蛋白质芯片对生物医学基础研究，以及癌症的早期/预后诊断都具有重大意义。在本项目的研究过程中，我们采用二维有序的球腔微电极阵列为固相基底，结合免疫分析方法、HCR扩增技术，构建了高灵敏的蛋白质电化学传感器用于癌症标志蛋白的检测。为此，我们首先构建了具有限域效应的微电极阵列。以密堆积的聚苯乙烯微球为模板，利用电化学方法于模板间隙内沉积二氧化硅，从而在导电玻璃表面构成底部开放的微电极阵列，通过改变聚苯乙烯微球粒径实现对电极间距的调控以优化其微电极特性，进而对该微电极的电化学特征和电荷选择性进行了理论研究。通过精心设计的DNA之间的链式反应（HCR）形成DNA链节连环，极大地增加了标记分子的标记量，从而提高检测灵敏度，最后结合免疫识别反应成功构建了癌症标志蛋白电化学传感器，并完成对癌症标志蛋白的检测。研究结果表明，模板法无需使用昂贵的仪器，极大地简化了微电极的制备过程；所得二维有序的球腔电极阵列具有明显的微电极特征，能明显降低电化学过程中的充电电流，其电流密度相对于习用裸金电极提升了至少两个数量级，且由于球腔表面的负电荷特性，使得其在免疫复合物构建过程中起到明显的富集作用。HCR反应能明显提高免疫复合体系中信标分子的标记量；基于微电极阵列和HCR反应的免疫电化学传感器用于癌症标志蛋白的检测，其最低检测限可达5×10-15 mol/L，用于实际血液样品的检测，具有令人满意的灵敏度和选择性。. 本课题的实施为生物医学基础研究及癌症早期诊断提供新的技术手段，不仅可为电化学领域的理论及应用研究提供实践基础，而且对开发稳定性好、灵敏度高的新一代生物芯片也有重要的参考价值。