基于生物放大技术与新型结构纳米贵金属表面增强拉曼免疫分析新方法研究

Highly sensitive determination of cancer biomarkers is critical to many areas, such as disease diagnosis and biomedical research. Surface-enhanced Raman scattering (SERS) immunoassay has the highly sensitive and multiplexing detection capability due to its narrow-band Raman spectral signature and its wide excitation wavelength. In general, the sensitivity of SERS immunoassay is attributed to the SERS effect of the molecules absorbed on the metal nanoparticle. It is well known that the response ability of the SERS is critically related to the size of the gold nanoparticles.Bigger gold nanoparticle s will result in greater SERS signals. But their stability may decrease significantly with the increasing sizes when they are functinalaized with Raman reporters.Fortunately,the surface enhancement effect can be remarkable generated by the assemlby of metal nanoparticle.DNA biological amplification is great suit to assembly for metal nanoparticle though biotin-streptavidin.In view of the characteristic of highly sensitive SERS immunoassay, this project proposes some novel strategy for protein by some novel SERS highly enhanced nanoprobes, the surface of the immunosensor and DNA biological amplification. Firstly, we intend to controlled synthesis some novel nanomaterials , investigate the relationship between SERS enhancement with morphology, size and surface roughness, clarify SERS enhancement mechanism, and prepare high SERS probes. Secondly, we intend to construction the surface of SERS immunosensor using LB technologies. The low background noise of SERS immunosensor'surface will be obtain though adjusting the physical and chemical properties and antibody immobilization methods. Finally, combining with DNA biological amplification technologies, such as rolling circle amplification, Surface-initiated enzymatic polymerization, and exonuclease III-aided DNA recycling amplification, some ultrasensitive SERS immunoassay methods for ultra-low abundance cancer biomarkers will be developed. The proposed system will be shown great promise for clinical application,and open a new avenue for sensitive immunoassay and other biochemical anlaysis based on SERS.

高灵敏检测癌症蛋白的免疫分析在疾病诊治、生物医药研究等领域至关重要。表面增强拉曼散射(SERS)免疫分析法具有灵敏度高、抗干扰能力强等优点，为蛋白的多组分超灵敏检测提供可能。通常，SERS免疫分析的灵敏度主要来自于纳米金对拉曼活性分子的SERS增强。存在纳米金粒径小，SERS增强弱，粒径大，探针不稳定的矛盾。本项目从构建高灵敏SERS免疫分析方法的特点出发，从SERS纳米探针、免疫传感界面及DNA生物放大三方面出发，拟可控合成结构新型的纳米材料，考察其形貌、尺寸、表面粗糙度对SERS信号的影响，制备出SERS高响应的纳米探针；利用LB组装技术，通过调节界面理化性质和改变抗体固定方法，构建超低背景噪音的SERS界面，结合DNA的生物放大技术，如滚环扩增、表面酶促聚合、核酸剪切酶放大等，对SERS探针进行有序组装，实现信号的多重放大，建立对肿瘤蛋白低丰度的检测方法，为临床诊断提供潜在应用。

表面增强拉曼散射(SERS)免疫分析法具有灵敏度高、抗干扰能力强等优点，为蛋白的多组分超灵敏检测提供可能。通常，SERS免疫分析的灵敏度主要来自于纳米金对拉曼活性分子的SERS增强。存在纳米金粒径小，SERS增强弱，粒径大，探针不稳定的矛盾。本项目从构建高灵敏SERS分析方法的特点出发，从SERS纳米探针、传感器界面及DNA生物放大三方面出发，引入SERS增强的多种机制，发展了多种SERS增强的分析方法。提高单个纳米粒子的SERS增强性能，通过调节氯金酸的量，可控合成比单纯纳米金SERS信号增强10倍多的银核介孔金纳米粒子，同时，通过电沉积的方法，研究了界面的润湿性以及拉曼信号的关系，在此基础上构建了纳米粒子拉曼增强免疫分析；基于纳米粒子SERS的远程效应，通过SERS增强粒子与拉曼信标分子之间的距离调控，构建了对葡萄糖的分析方法；基于银核介孔金具有人工模拟HRP酶的特性，研究了其催化性能，结合其拉曼增强性能，发展了一种针对过氧化氢的SERS检测方法，同时，利用银核介孔金的催化和拉曼增强的双功能热性，构建了高灵敏的SERS免疫分析；基于拉曼热点增强效应，通过调节pH值，使巯基DNA修饰在CTAB裸露的-金纳米棒两端，有汞离子作用下，发生DNA杂交，实现金纳米棒的有序组装，从而实现对痕量汞离子SERS检测分析；利用DNA的放大技术，通过DNA扩增技术，实现了金银纳米粒子的聚集形成了SERS热点面增强，构建了高灵敏的SERS免疫分析；同时，利用Te材料的可控制备和原位转换贵金属，发展出了一种简易可控的制备拥有热点面SERS增强材料。