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

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.

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。

表面等离子体共振成像(SPRI)是理想的高通量免疫分析技术之一，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。本项目针对这一研究现状，提出了发展特异性高、定量关系好的串联信号放大方法，提高免疫检测的灵敏度，并用于检测不同肿瘤标志物。项目实施3年来，进行了如下研究工作：.1）开发了兼具高抗体固定量和抗非特异性蛋白吸附的PEGMA-co-GMA聚合物刷，并发展了基于该聚合物刷的表面修饰方法和微纳复合材料，在此基础上构筑了几类高效SPRI和荧光蛋白质芯片。.2）制备了石墨烯－抗体复合物，利用石墨烯对银离子独特的催化沉积特性，对SPRI免疫检测信号进行串联放大，实现了对肿瘤标志物甲胎蛋白的灵敏检测，在10%血清中的检测限达到了100 pg mL-1；且特异性良好、定量关系明确。.3）采用聚多巴胺修饰石墨烯，并固定抗体蛋白，用于一次SPRI信号放大；在此基础上使用氯金酸流过芯片表面，让金纳米粒子在聚多巴胺修饰石墨烯的表面生长，从而实现第二次信号放大。相比2），该方法更方便快捷，因为聚多巴胺即充当了还原剂的角色，无须额外加入还原剂；并且它对金属粒子（离子）具有高螯合能力，使得生长出的金属粒子都在其表面定域生长，保证了信号放大的定量关系。在10%血清中对癌胚抗原的检测限可低至500 pg mL-1。.4）利用表面引发原子转移自由基聚合（SI-ATRP）稳定可控的优点，发展了一种新型的SPRI串联信号放大方法。制备抗体－引发剂修饰的纳米金粒子，并用于第一次SPRI信号放大，而在芯片表面流过含有聚合单体和催化剂的水溶液即可使聚合物链在纳米金表面生长，实现第二次信号放大，在10%血清中对甲胎蛋白的检测限可低至1.0 ng mL-1；.5）在SPRI检测小分子毒素、荧光免疫微阵列芯片、比色免疫分析和生物分析复合材料等方面开展了一些有益探索，并取得了较好成绩。.综上，本项目发展了一系列基于串联放大思路的适用于SPRI检测平台的信号放大方法，实现了SPRI芯片在大浓度范围内灵敏检测不同肿瘤标志物。这些工作为解决SPRI在免疫检测中灵敏度较低这一难点提供了新思路和新方法，同时，本项目所发展的免疫界面构造方法和串联信号放大方法也能为其它免疫检测和生物分子间相互作用研究提供一定的参考意义。