功能化纳米复合材料hCEs SERS探针的构建及在血清与细胞中检测羧酸酯酶活性的应用研究

Human carboxylesterases (hCEs) have many important physiological functions. Meanwhile, hCEs is one of the tumor markers of liver cancer, and can also be used as a drug target for the screening of hCEs inhibitors. The development of rapid and sensitive hCEs detection method is of great significance for the study of physiological processes, hCEs inhibitors screening and even early diagnosis of clinical liver cancer. In this research, we intend to develop sensitive, stable and reproducible, renewable functionalized nanocomposites by integrating non-metallic elements doped semiconductor nanomaterials with Au/Ag nanoparticles, to overcome the inherent shortcomings of instability, poor biocompatibility and nonrepeatability in SERS substrate which is currently composed by single component of Au/Ag nanoparticles. On this basis, the SERS tag substrate was preparated, to construct the highly integrated hCEs SERS tag. And a method for the detection and activity analysis of hCEs in serum and cells was established. Additionally, the charge transfer enhancement and electromagnetic interaction mechanisms of the prepared nanocomposites will be extensively investigated. Furthermore, we explored the inherent principles of the carboxylesterase SERS detection and cell imaging in the cell, tissue or in vivo. And subcellular localization of hCEs was studied. Moreover, the SERS tag was used for the early screening of liver cancer and the screening of hCEs inhibitors. This work can expand the application potential of SERS tags in field of biomedicine, pharmacy and clinical applications.

人体内羧酸酯酶(hCEs)具备多种重要生理作用。同时，hCEs是肝癌血清标志物之一，也可作为药物靶点进行hCEs抑制剂筛选。发展其快速、灵敏的检测方法对于生理过程研究、hCEs抑制剂筛选、肝癌早期诊断具有重要意义。本项目拟采用非金属元素掺杂半导体纳米材料与纳米金（银）复合制备成灵敏、稳定、重现性好的功能化纳米复合材料，以改善单一成分Au/Ag纳米颗粒构成的SERS基底性质不稳定、结果难以重复的缺点。在此基础上构建高度集成的hCEs SERS探针，建立血清中、细胞中hCEs检测和活性分析方法。探讨非金属元素掺杂半导体材料时电荷转移增强、纳米复合材料中相互影响的电磁增强效应机理；研究该探针在细胞、组织或活体中羧酸酯酶SERS检测、细胞成像的一般规律及hCEs的亚细胞定位情况。并应用该探针进行肝癌早期筛查和hCEs抑制剂筛选，拓展SERS探针在生物医学基础研究、药学领域及临床上的应用潜力。

人类羧酸酯酶（hCEs）通过水解各种内源性和外源性酯类，参与了若干生理过程以及多种药物和环境毒素的解毒和代谢。快速、灵敏的hCEs检测方法对于研究肝损伤和肝细胞癌的发展过程、hCEs抑制剂筛选及药效评价具有重要意义。当前，传统方法样品预处理复杂、操作繁琐，而hCEs荧光探针也存在对pH敏感、激发波长短，组织穿透能力弱等缺点。项目采用磁性材料、金属-有机框架材料（MOF）与纳米金（银）组装成功能化纳米复合材料作为SERS探针的基底或标签。通过hCEs抗体特异性识别hCEs，构建高度集成的功能化纳米复合材料hCEs SERS探针。研究了功能化纳米复合材料SERS增强机制以及靶标刺激信号响应机制，提高了SERS探针的灵敏度、选择性及稳定性。建立了人血清、HepG-2细胞及其培养基上清液中hCEs的三明治夹心检测和SERS-ELISA分析方法，并应用该hCEs SERS探针进行了临床肝癌病人的早期筛查和hCEs抑制剂筛选的初步研究。同时，采用活细胞成像与激光共聚焦显微分析，明确了hCE1的亚细胞定位于HepG-2细胞的高尔基体与内质网中，为深入理解hCE1的作用机制及生理功能提供了新的研究方向。