一种新型异质结卫星结构超荧光增强生物免疫探针及检测薄膜

Finite difference time domain numerical calculation demonstrates that the enhancement factor of local electric field around the gold satellite nanostructure could realize thousands of times, two orders of magnitude higher than that of single nanoparticle, and heterogenous satellite nanostructure presents much higher enhancement factor. In allusion to the fast and synchronous clinical detection of liver cancer markers such as AFP-L3 and AFU with high specificity, we put forward a novel fluorescence enhancement probe based on core-satellite nanostructure and microarray film. We mainly focus on the microscopic mechanism and preparation method of high specific immune fluorescent probe with antibody covalent coupling by using the long chain mercaptoundecanoic acid and biological modified (Au-Ag core-shell nanorod)-(Au nanosphere) core-satellite nanostructure. This kind of heterogeneous core-satellite nanostructure could achieve the fluorescence enhancement with more than one hundred thousand times theoretically. We firstly investigate the preparation method of heterogeneous core-satellite nanostructure with ultra-great enhancement by using the method of self-assembly at liquid phase under the ultrasonic condition. Then, we study the fluorescence spectrum of two kinds of molecular markers with different heterogeneous core-satellite.nanostructure by coupling interaction, and investigate the probes with high specificity and ultra-sensitive detection both theoretically and experimentally. Furthermore, the micro jet printing technology and molecular blotting method are used to study the preparation technology of microarray film by integrating two tumor markers fluorescent probes in the same chip.

时域有限差分数值计算表明，金纳米卫星结构周围的电场可以实现上千倍增强，比单个纳米颗粒的增强因子提高了两个数量级，而异质结纳米卫星结构的增强因子更高。本项目针对肝癌标志物甲胎蛋白异质体、α-L-岩藻糖苷酶的高特异性超高灵敏同步快速检测，首次采用金银核壳纳米棒金纳米球的异质结核-纳米卫星结构，研究其经巯基烷酸生物修饰形成长链抗体共价偶联高特异性超高灵敏免疫荧光探针的机理及制备方法。这种异质结核-纳米卫星结构理论上可实现十万倍以上的荧光增强。课题首先从理论和实验上研究采用超声波条件下液相自组装的方法制备具有超高荧光增强特性的不同异质结核-纳米卫星结构；其次从微观耦合关系的角度研究两种标志物分子与不同异质结核-纳米卫星结构相互作用的荧光光谱特性，探索制备高特异性超高灵敏检测探针的理论基础和技术方法；最后采用微喷印技术和分子印章法研究针对两种标志物检测的荧光探针集成于同一芯片的微阵列薄膜制备技术。

随着社会、经济的发展进步，公众对以肿瘤为代表的重大疾病精准早期诊断的需求在不断增强，而血清标志物检测在肿瘤早期精准诊断方面显示出巨大的潜力。基于贵金属金、银纳米结构局域表面离体子共振及光学局域场增强效应的生物光谱传感技术在血清标志物检测方面具有独特优势。因此，设计制备具有超高局域场增强效应的纳米光学结构具有重要意义。本项目围绕构建新型异质结超荧光增强卫星纳米结构及其检测应用，开展并完成了以下研究内容：1.成功制备出了中空多孔金银核壳-金纳环、金银核壳-金纳球以及金纳米棒-金纳米球三种异质结纳米卫星结构，并对制备过程进行了优化，掌握了制备异质结纳米卫星结构的关键工艺参数；2.利用FDTD和DDA计算方法，从理论上优化了异质结纳米卫星结构的几何参数，发现中空金纳米环作为周围偶联体的纳米卫星结构具有优良的局域场增强效果，实现了具有超强局域场增强的纳米卫星结构的设计和制备；3.基于制备和优化的异质结纳米卫星结构，制备完成了针对肝癌血清标志物检测的生物免疫光学探针，并构建具有荧光/拉曼增强的检测薄膜，实现了以AFP及CEA为代表的一系列生化目标分子的高灵敏光谱检测，检测限最低可达fg/mL级。本项目的研究成果为研发肿瘤标志物光学检测仪器提供了坚实的基础。