SPRI微阵列芯片串联信号放大新策略及其肝癌早期诊断应用

表面等离子体共振成像(SPRI)是理想的高通量分析技术之一，但其灵敏度较差。现有信号放大方法普遍存在特异性差和定量关系差两个弊端，难以在大浓度范围准确定量目标物，因此亟待发展可靠的信号放大方法。本项目在前期开发的抗污SPRI微阵列芯片（胡卫华等，Adv.Funct.Mater.2010.20,3497）的基础上，拟发展基于氧化石墨烯催化银沉积的特异性高、定量关系好的串联信号放大方法，提高芯片灵敏度，并用于多组分检测肝癌标志物，实现早期诊断。具体来说：先采用抗体偶联氧化石墨烯与探针抗体/目标物组成免疫复合物，形成第一次信号放大；再通过银离子在氧化石墨烯表面的催化沉积实现第二次信号增敏，使SPRI芯片能在大浓度范围内灵敏检测多种肝癌标志物。本项目定位于建立一种可靠的SPRI芯片信号放大方法，亦将有助于科学认识"金属/聚合物刷/水溶液"这一三相体系及生物大分子、金属离子在其中的行为规律。

Surface plasmon resonance imaging (SPRI) is a powerful tool for high-throughput analysis of biomolecular interactions and multiplexed immunoassay of targets with the advantages of label-free and real time. Sensitivity, however, is a major disadvantage of SPRI, restricting its applications in a wide variety of practical fields. To enhance the sensitivity, some signal amplification strategies such as surface-initiated polymerization, enzyme-catalyzed deposition and bioconjugated nanomaterials have been developed, but their specificities are not satisfying due to the nonspecific protein adsorption on sensing surface and/or poor specificity of the bio/chemical reactions used. Moreover, in some cases the amplification is not quantitative, making it impossible to accurately quantify the target over a wide concentration range, which is very important for some applications, e.g., the biomarker detection for early diagnosis, where the concentration of a biomarker may elevates for 3-4 orders of magnitude upon the tumor outbreak. In this project a tandem signal amplification strategy based on bioconjugated graphene oxide (GO) is proposed to specifically and quantitatively enhance the SPRI signal and extend SPRI's application to early diagnosis of liver cancer. In details, water-dispersible GO sheets are first conjugated with antibodies and used to form immunocomplexes on the sensing surface to generate the first signal amplification; subsequently, the silver enhance solution (silver nitrate and hydroquinone) is flowed through the sensing surface, where the captured GO catalyzes the silver reductive deposition to further amplify the SPRI signal. Most importantly, both amplification steps are conducted on an anti-biofouling SPRI sensing surface based on poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-co-GMA) polymer brush previously reported by us (Hu W.H. et al., Adv. Funct. Mater.,2010,20,3497), which demonstrates excellent resistance to nonspecific protein adsorption; also, the dense polymer brush is able to efficiently suppress the silver deposition on the underlying gold surface according to our preliminary results, guaranteeing exclusive silver deposition on the GO, and in turn high specificity of immunoassay and signal amplification. Based on this tandem signal amplification strategy, multiple biomarkers related with liver cancer including alpha fetoprotein (AFP), Carcinoembryonic antigen (CEA), CA125, CA199, Hepatitis B surface antigen (HBsAg) and Hepatitis C virus antibody (HCV antibody）in human serum will be quantitatively analyzed on the SPRI microarray chip. This project aims to develop a novel tandem signal amplification strategy for SPRi microarray chip, and get more scientific insights into the interfacial behaviors of biomolecules and metal ions in the gold/polymer brush/aqueous solution three-phase system.