hanyiwen-20140523修改_new详解.doc

摘 要 细胞表面的多聚糖在许多至关重要的生理和生物过程中起着重要的作用，其中唾液酸（SA）在细胞表面的过表达已被公认为是恶性肿瘤的一个典型特征。凝集素是一种可以和糖类特异性结合的天然生物提取蛋白。具有特定亲和性凝集素的使用为靶细胞不同成长阶段多聚糖表达水平的检测提供了一种温和、非破坏性的定量分析方法。各种基于凝集素的检测方法中，电化学方法和生物传感器由于它们操作简便灵活、灵敏度高等特点，能够有效克服传统方法的局限性，因此得以被广泛研究应用。实验中我们设计以接骨木凝集素（SNA）、3-氨基苯硼酸（APBA）和辣根过氧化物酶（HRP）作为糖类识别和信号放大源来实现对细胞表面糖的定量分析。我们用化学还原的氧化石墨烯和纳米金（rGO/PDDA/Au纳米复合物）作为凝集素固定的基底平台，以此构建生物传感器。然后，制备APBA修饰的碳球，在弱碱性的条件下，APBA基团能够与细胞表面的唾液酸作用而固定在细胞表面，同时APBA又可以与HRP上的甘露糖进行结合，从而将酶固定到碳球上。通过固定酶产生的电化学信号，希望能用于MCF-7（人乳腺癌细胞）的分析检测。 关键词：凝集素；生物传感器；电化学方法；唾液酸 Abstract The sophisticated conformations of glycoproteins on cell surface play essential roles in a broad range of crucial physiological and biological processes, including growth, differentiation, cell-cell communication, cellular adhesion, immune response, disease and progression of cancer. The overproduction of sialic acid on glycan chains has been recognized as a characteristic feature of malignant properties. Glycan assays based on lectin-glycan interactions have attracted much attention recently due to the relatively high efficiency of lectin arrays and the availability of diverse engineered natural lectins. The use of lectins with defined specificities offers alternative nondestructive ways to profile glycan expression on cell surface at different developmental stages of diseases. Among the various lectin-based tools, lectin-based electrochemical strategies and carbohydrate biosensors have been studied extensively in complementing the limitation of conventional methods for cell surface glycan analysis owing to their operational simplicity, flexibility, and acceptable sensitivity. In our work, a biosensor for glycan assay on cell surface was constructed. Chemically reduced graphene oxide and gold nanoparticles (rGO/PDDA/Au nanocomposite) were used as the sensor platform for lectin loading. Sambucus nigra agglutinin (SNA), a sialic acid binding protein was selected as a model. Then, a highly selective strategy integrating specific carbohydrates identification an