生物条形码模式电化学发光免疫分析及其超高灵敏检测肿瘤标志物的应用研究

Bio-barcode immunoassay has an ultrahigh sensitivity with a few shortcomings such as cumbersome process, long detection time and poor stability. On the other hand, electrochemiluminescence immunoassay has the advantages of simple process, short detection time and high stability, but its sensitivity is limited. Therefore, neither method could meet the requirement of detecting very low concentrations of tumor markers (TM) in pre-cancer or early-cancer patients. Based on our previous research on biosensors for the determination of TM, we propose to develop a novel method and system of electrochemiluminescence immunoassay using bio-barcode mode. The innovation of this method lies in the use of functional biological nanomaterials in separation device and probe-labeling means. In other words, TM antibody surface-coated on the nanomagnetic bead acts as the first antibody. While, TM antibody and electrochemiluminescence probe, which are both surface-coated on the porous hollow nanosphere, act as the second antibody and signal probe, respectively. In addition, an integrated instrumental system is to be preliminarily constructed by integrating the three key steps, i.e., incubating, cleaning and testing. The method to be developed is expected to be ultrahighly sensitive, label-free, simple, rapid and stable; thus facilitating the ultrasensitive and rapid determination of TM at very low concentrations. Besides, the lung cancer early-warning mechanism is to be explored preliminarily through studying the concentration variation of TM in different stage of lung cancer. Electrochemiluminescence immunoassay using bio-barcode mode to be developed in this project would provide a technological platform for early diagnosis and early warning of pre-cancer or early-cancer patients. Thus, this proposal has high scientific significance and application prospect.

生物条形码免疫分析灵敏度超高，但检测步骤繁琐、耗时长、稳定性差；电化学发光免疫分析步骤简单、耗时短、稳定性高，但灵敏度有限；故二者均无法满足癌前病变、肿瘤早期患者体内极低浓度肿瘤标志物(TM)的检测要求。在本研究组开发TM检测生物传感器的前期研究基础上，本项目拟基于功能化生物纳米材料，创新分离器件和探针标记手段，以纳米磁珠表面包被的TM抗体为第一抗体，纳米空心多孔球表面同时包被的电化学发光探针、TM抗体分别为信号检测探针、第二抗体，创立生物条形码模式电化学发光免疫分析，并初步构建集成孵育、清洗、检测三个步骤的一体化仪器系统，具有灵敏度超高、免标记、步骤简单、耗时短、稳定性高的特点，可实现极低浓度TM的超高灵敏快速检测，并通过不同癌变时期的TM浓度变化初步探索肺癌预警机制。本项目创立的生物条形码模式电化学发光免疫分析，可为癌前病变、肿瘤早期诊断与预警提供技术平台，兼具重要科学意义和应用前景。

生物条形码免疫分析灵敏度超高，但检测步骤繁琐、耗时长、稳定性差；电化学发光免疫分析步骤简单、耗时短、稳定性高，但灵敏度有限；故二者均无法满足癌前病变、肿瘤早期患者体内极低浓度肿瘤标志物的检测要求。因此，我们构建了兼具生物条形码免疫分析和电化学发光免疫分析优点的生物条形码模式电化学发光免疫分析新方法和新体系。基于功能化生物纳米材料、创新分离器件和探针标记手段，以功能纳米材料表面包被的肿瘤标志物抗体为第一抗体，功能化纳米材料同时包被的电化学发光探针、肿瘤标志物抗体分别为信号检测探针、第二抗体，创立了生物条形码模式电化学发光免疫分析。通过分析生物条形码模式电化学发光免疫分析的局限性，如标记手段不合适、制备方法不合适、激发方法不合适等，采用比表面积大、导电性好的功能化纳米材料构建捕获探针、信号探针，通过形成“第一抗体—抗原—第二抗体”免疫复合物二者包合在一起，创立了基于生物条形码模式的全新概念的“电极内”“法拉第笼式”电化学发光免疫分析模式和升级版金标银染免疫分析，实现了高灵敏、准确、简单、快速、廉价的肿瘤标志物检测与监测，检测限达到ag/mL水平，克服了现有分析方法的复杂、低灵敏度等问题，为进一步构建新型肿瘤标志物传感平台建立了坚实的基础，为癌前病变、肿瘤早期的诊断与预警提供了技术平台。

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