三维纸芯片细胞微环境下的弗林蛋白酶活性原位分析

In vitro studies of cells play a significant role in revealing life phenomena, disease prevention, drug screening, etc. In this project, by simulating three-dimensional culture of tumor cells under closing to real in vivo microenvironment on paper chip, in situ accurate analysis of high expression furin activity during tumor progression will be achieved. The simple superposition of cellulose paper in the polydimethylsiloxane culture tank completes the conversion of the two-dimensional and three-dimensional configuration of the chip, simulating the in vitro generation and transformation of the stereoscopic tumor tissue. Then the different issue between the conventional cell culture mode and the real environment of the human body could be solved. Adhesive factors are coated on paper materials to construct a hydrogel network scaffold that could fix, protect and nourishe the implanted cells, and weaken the damage of the external environment, realizing the three-dimensional culture of cells under the condition of reproducible in the microenvironment. By covalently immobilizing the amino acid recognition sequence labeled with electroluminescent reagent and the resonance energy transfer group, an electroluminescence detection platform based on a three-dimensional paper-based flexible sensing interface is fabricated. Through the transformation of paper chip configuration from three-dimensional to two-dimensional, in situ sensitive analysis of furin activity from breast cancer cell in simulated environment was realized by employing furin to identify and cut specific amino acid sequences. With the controllable splitting of superimposed paper, the “slice” and “biopsy” of tumor tissue could be completed, which lays a certain theoretical and technical basis for the diagnosis, treatment and prognosis of tumor diseases.

细胞的体外研究在揭示生命现象、疾病防治、药物筛选等研究中起着重要作用，本项目在纸芯片上模拟接近真实在体微环境条件的肿瘤细胞三维培养，实现对肿瘤进展过程高表达弗林蛋白酶活性原位精准分析。利用纤维纸在聚二甲基硅氧烷培养池中的叠加完成细胞培养模型二维与三维构型的互换，模拟立体肿瘤组织的体外生成转化，解决常规细胞培养模式与人体真实环境的差异问题；引入黏附因子包被纸材料，构筑对种植细胞起固定、保护和营养作用的水凝胶网络支架，实现在体微环境重现细胞的三维生长；共价固载标记电致发光试剂和共振能量转移基团的氨基酸识别序列，构建基于三维纸基柔性传感界面的电致发光检测平台；通过纸芯片三维构型到二维的转换，利用弗林蛋白酶对特定氨基酸序列的识别剪切，实现仿真环境下乳腺癌细胞弗林蛋白酶活性的原位灵敏分析；借助叠加纸的可控拆分完成肿瘤组织“切片”与“活检”，为肿瘤疾病诊断、治疗和预后奠定一定的理论与技术基础。

针对重大疾病对无创伤、低成本、易推广筛查手段的需求，本项目将电化学发光、光致电化学技术引入纸基微流控芯片建立了低成本、多功能纸基传感平台。采用种子原位生长、无电沉积等方法制备了高性能纸基铜电极，降低制备成本的同时提高了电极的导电性；制备纸基功能纳米材料，构筑高效识别且局域空间内可发挥多功能的协同纳米界面；利用纳米材料类酶特性、光电转换特性实现传感信号方法，结合DNA步行马达等分子生物学技术提高传感平台的灵敏度；开展卟啉/苝酰亚胺类有机大环半导体材料的可控性制备方法学研究及其在气体传感器件方面的应用，实现了对气体分子的灵敏响应以及电信号的高效传输；将塑料封装技术与纸基微流控器件相结合，开发了全密封的纸基电化学发光传感器，保持传感器件微型化的同时有效避免了环境中氧气的干扰，成功实现了对肿瘤标志蛋白、核酸的灵敏检测，为重大疾病的早期筛查与预后评价提供了新策略，为无创和可穿戴医疗诊断设备的开发提供了新方法。