基于金磁微粒的即时检测用免疫探针的构建及微结构调控

The major target of point-of-care testing (POCT) is to get higher sensitivity, specifity and reach quantitative level. Composite Fe3O4/Au nanoparticle have arracted considerable interest in medicine due to their unique physical and chemical properties, including optical properties, superparamagnetic properties and flexibility to biofunctionalization for downstream application. In this study, we explore Fe3O4/Au nanoparticle as carriers, and simultaneously cardiac marker N-terminal pro-B-type natriuretic peptide (NT-proBNP) as model to realize simple, inexpensive, sensitive, quantitative lateral flow immunoassay. Firstly, Fe3O4/Au nanoparticles with different surface properties and mono-dispersion are fabricated by harnessing the surfactant, ligand and polymer synergistic cooperation. Secondly, the immunoassay probes with regulated micro-structure can be synthesized by conjugating monoclonal antibodies (NT-proBNP) to the surface of gold magnetic nanoparticle. At last, the probes are used in lateral flow immunoassay for NT-proBNP quantitative detection. In the study, we investigate the stability mechanism of composite Fe3O4/Au nanoparticles before and after surface modification, reveal how surface characteristics and composition effect on the regulation of micro-structure of the immunoassay probes in detail, establish thermodynamic and kinetic equations for the formation of probes’ micro-structure, investigate the relationship between micro-structure and detection results, including sensitivity, specificity and magnetic signal intensity. The study will not be of benefit to POCT detection based on gold magnetic nanoparticles, but also the design and development of native vitro diagnostic reagents.

针对即时检测的高灵敏度、高特异性及定量的需求，本研究以兼具光学、磁学双重特性的核壳Fe304/Au纳米粒为载体，以氨基末端脑尿肽前体（NT-proBNP）为检测目标，制备微结构可控的金磁免疫探针，建立简单、经济、快速的免疫层析定量检测系统。首先，通过多种表面改性方法，获得具备不同表面特性的单分散性的Fe304/Au纳米粒；再通过“纳米-生物界面控制”偶联单抗NT-proBNP，制备微结构可控的金磁免疫探针；最后在免疫层析系统实现待检物的磁定量检测。本课题在研究Fe304/Au纳米粒的稳定性及机理的基础上，揭示Fe304/Au纳米粒的表面特性对金磁免疫探针微结构调控的影响规律，并建立探针微结构形成的热力学及动力学方程；揭示“结构-检测性能”关系规律，量化“结构-磁信号强度”的内在联系，建立POCT免疫定量检测模型，为基于金磁纳米微粒的即时检测及体外诊断试剂的设计与开发提供新方法及理论支持。

为了获得具有高灵敏度、高特异性的及能实现定量检测的即时检测方法。首先，本研究基于多种表面特性及形貌的超顺磁性的Fe3O4磁性纳米粒子的制备，实现了氨基末端脑尿肽（NT-proBNP）抗体的定性及定量检测，定性检测限按临床检测需求检测限调制，磁性检测信号值与待检测特浓度在三个数量级内呈线性关系。其次，在前期研究基础上，还采用高压静电纺法调节纺丝纤维直径、形貌和功能基团，得到膜孔径、厚度、不同蛋白承载力及层析速度的NC膜，更好的实现“结构-检测性能”的相关关系，实现免疫定量检测系统的灵敏度可控调节。再次，基于抗体和抗原之间的特异性结合形成大体积复合物，继而会干扰取向排列的液晶分子的取向，提出了一种简单的基于液晶（LC）构建生物传感的方法，基于直接免疫法及竞争免疫法，实现了天蚕素B与人β防御素的定性检测。通过液晶池光学图像平均灰度强度与天蚕素B浓度之间的相关性，可实现两种靶标物质的半定量检测。与传统分析方法相比，液晶生物传感器实施的目视化检测具有无需标记、高灵敏度、特异性的特点。最后，本研究尝试了制备稳定性良好的水性聚苯胺（PANI），制备了具有良好电导率、拉伸强度的三维均匀的PANI导电膜，为后续纸基检测装置结合电学性能的研究提供了研究基础。. 综上所述，本研究完成了多种纳米粒子的制备及表面改性工作、免疫探针的构建及结构调控、实现了磁定量免疫层析系统的构建。同时，研究扩展至NC膜结构调控、液晶生物传感及纸基与电学性能结合的领域。获批1项中国博士后科学基金第61批面上资助项目，获2017年度陕西省优秀博士论文奖。已培养4名研究生，7名本科学生。已发表学术论文5篇，还有多篇论文在投稿及整理过程中。申请国家发明专利2项，获授权国家发明专利1项；指导学生参加“大学生创新创业计划”、“互联网+”等科技竞赛项目2项；参加国内的学术会议9人次，作口头报告1次。