基于自阵列化表界面的复杂样品中细胞因子检测及疾病诊断新方法研究

The generation and secretion of cytokines in human body are closely related with disease development and progression, therefore the direct detection of cytokine concentration from blood, serum and body fluid samples has significant importance for disease diagnosis and evaluation of treatment efficiency. However, current detection strategies suffer from several limitations, such as the lack of sensitivity, the requirement of pretreatment, and the deficiency for real-time measurement. To solve these problems, here we propose to design a self-patterning microarray via click chemistry and photopolymerization, apply on-site polymer growth and nanomaterial synthesis for signal amplification, develop novel aptamer probes and perform real-time measurement based on their configuration changes, improve surface anti-fouling property by various hydrophilic polymer coatings, therefore achieve direct cytokine detection in complex samples without pretreatment process. In the meanwhile, we propose to study the mechanisms of immune reaction and signal transduction in micro size reaction areas, establish new principles and strategies for disease diagnosis based on cytokine secretion. This project will increase detection sensitivity and simplify operation process for cytokine detection and disease diagnosis, as well as provide new strategies for the fabrication of microarrays and functional interfaces, therefore would have broad application in bioanalysis and disease diagnosis.

细胞因子在体内的合成与分泌同疾病的发生发展进程密切相关，检测细胞因子的含量对于疾病的临床诊断与疗效评价意义重大。针对目前细胞因子检测法中灵敏度不足、前处理过程复杂、实时性欠缺的问题，本项目拟通过点击化学、光聚合与表面微纳加工技术构建自阵列化亲水微区域表界面，采用原位高分子生长与纳米材料合成技术发展信号放大新方法，利用核酸适配体探针的杂交与变形发展即时检测新手段，并通过亲水高分子修饰提高表面抗非特异性吸附特性，建立复杂样品中免前处理过程的细胞因子检测新策略。同时探讨生物探针在微区域传感界面上的免疫识别与信号传导机制，建立基于细胞因子分泌进行疾病诊断的新原理与新平台。本项目工作不仅可以为细胞因子检测及临床疾病诊断提供高效灵敏且操作方便的新方法，而且可以为生物分析中功能表界面的制备及检测策略的开发提供新思路，在生物分析与临床诊断领域具有广阔的应用前景。

细胞因子在体内的合成与分泌同疾病的发生发展密切相关，检测细胞因子的含量对于疾病的临床诊断与疗效评价意义重大。本项目针对当前细胞因子分泌检测及与之相关的疾病诊断灵敏度欠缺、检测步骤繁琐、在复杂生物样品中应用受限的缺点，开展了以下4个方面的研究工作：1) 利用光影印、光聚合技术开发憎水基底上的亲水液滴自阵列化表界面，制备阵列化传感平台；2) 利用局域空间增大反应物浓度的特性提出高效信号放大新方法，实现了活细胞中mRNA的高效灵敏成像与小数量细胞裂解液中miRNA的高灵敏检测，检测限低至fM数量级，为实现微区域内细胞因子的高效灵敏检测奠定了基础；3) 同时结合基底印刷三电极体系，在亲水液滴中实现了细胞因子的定量分析，并开发纳米线生长基底电极表面以提高细胞捕获效率与检测灵敏度，通过阵列化器件开发，实现了IFN-γ与TNF-α两种细胞因子分泌的同时检测，为临床疾病诊断提供普适高效的检测平台。除此之外，4) 在目标待测物高效灵敏检测新策略开发的基础上，本项目进一步将靶标mRNA响应信号放大方法的应用由检测拓展至治疗，利用DNA自组装结构与上转换纳米材料开发了细胞内siRNA原位合成的新方法及药物高效递运与释放的新途径，在肿瘤高特异性、高可控性诊断与治疗研究中具有应用前景。