生物分子修饰的荧光纳米簇材料的制备及在生化分析中的应用

发布时间：2018-06-15 07:49

本文选题：荧光 + 金纳米簇　；参考：《青岛科技大学》2017年硕士论文

【摘要】：近年来荧光纳米材料特别是荧光金纳米簇已成为国内外科研工作者研究的热点。荧光金纳米簇作为一种简单易制备、水分散性好、荧光信号强、斯托克位移大、生物相容性良好的纳米材料已被广泛应用于生物传感及生物医学等领域。本论文采用较温和的方法合成荧光金纳米簇,以此构建新型生物传感器,并将其应用于疾病小分子、肿瘤标志物的检测,成为临床诊断及治疗的一种辅助手段。主要工作概括如下:(1)使用球蛋白包覆合成了一种新型的、稳定的荧光金纳米簇,并利用它实现了对无机焦磷酸盐以及无机焦磷酸酶的快速灵敏检测。其检测原理是利用Cu2+可以猝灭金纳米簇荧光的特性,设计构筑多重荧光检测生物分子的体系。首先向Cu2+/Au NCs荧光猝灭体系中加入无机焦磷酸盐(PPi),由于PPi可以与Cu2+发生强络合作用,可使猝灭的金纳米簇荧光恢复,实现了对生物分子PPi的定量检测,线性检测范围是0.05-218.125μM,检测限为0.02μM。向该混合体系中加入无机焦磷酸酶(PPase),可将无机焦磷酸盐PPi水解为磷酸分子,Cu2+重被释放,纳米簇的荧光又被猝灭,从而实现对PPase的定量检测。实验结果表明,该检测方法的线性范围为0.1-8 mU,检测限为0.04 mU,并且选择性较好,有望应用于实际检测。(2)利用单一激发双发射金纳米簇设计了一种新型“关开”式FRET传感器用于同时检测多种肿瘤标志物—甲胎蛋白(AFP)、癌胚抗原(CEA)。AFP适配体修饰的绿色荧光金纳米簇(510 nm)以及CEA适配体修饰的红色荧光金纳米簇(650 nm)作为能量供体,二硫化钼(MoS2)作为能量受体,构建新型FRET传感器,加入目标CEA和AFP后记录510 nm和650 nm处的荧光强度变化同时定量检测这两种肿瘤标志物。AFP线性检测范围为0.5到60 ng/m L,CEA的线性检测范围0.5到120 ng/mL检测限(3σ)分别为0.16 ng/m L、0.21 ng/m L。此外该生物传感器不仅可以实现准确定量测定多种肿瘤标志物也可用于可视化的半定量检测。更重要的是激光共聚焦荧光显微镜实验证明该生物传感器可以定性的区分健康与肝癌病人的血清,此实验方法具有临床诊断试验的潜力。
[Abstract]:In recent years, fluorescent nanomaterials, especially fluorescent gold nanoclusters, have become the focus of research at home and abroad. As a kind of simple and easy preparation, gold nanoclusters with good water dispersion, strong fluorescence signal, large Stoke shift and good biocompatibility have been widely used in biosensor and biomedical fields. In this paper, fluorescent gold nanoclusters were synthesized by a mild method, and a new biosensor was constructed. The biosensor was applied to the detection of disease small molecules and tumor markers, and became an auxiliary method for clinical diagnosis and treatment. The main work is summarized as follows: (1) A novel stable fluorescent gold nanocluster was synthesized by globulin coating, and the rapid sensitive detection of inorganic pyrophosphate and inorganic pyrophosphatase was achieved by using it. The detection principle is to design and construct a multiplex fluorescence detection system for biomolecules by using Cu _ 2 to quench the fluorescence characteristics of gold nanoclusters. Firstly, the inorganic pyrophosphate PPian was added to the fluorescence quenching system of Cu2 / au NCs. The fluorescence of the quenched gold nanoclusters could be restored due to the strong complexation of PPi with Cu2, and the quantitative detection of PPi was realized. The linear detection range was 0.05-218.125 渭 m, and the detection limit was 0.02 渭 M. When the inorganic pyrophosphatase PPaseau was added to the mixed system, the inorganic pyrophosphate PPi could be hydrolyzed into phosphoric acid molecule, Cu2, which was rereleased, and the fluorescence of the nanoclusters was quenched, thus the quantitative detection of PPase could be realized. The experimental results show that the linear range of the method is 0.1-8 mU, the detection limit is 0.04 mU, and the selectivity is good. A novel "closed" fret sensor has been designed for simultaneous detection of a variety of tumor markers, such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA) CEA, AFP aptamer modified green. Color fluorescent gold nanoclusters (510 nm) and CEA aptamer modified red fluorescent gold nanoclusters (650 nm) were used as energy donors. Molybdenum disulfide (MoS _ 2) as an energy receptor, a novel fret sensor was constructed. The fluorescence intensity changes at 510nm and 650nm were recorded after the addition of target CEA and AFP. The linear detection range was 0.5-60 ng/m / mL. The linear detection limit of CEA was 0.16 ng/m / L 0.21 ng/m / mL, respectively. In addition, the biosensor can not only accurately and quantitatively determine many tumor markers, but also can be used for visualized semi-quantitative detection. More importantly, laser confocal fluorescence microscopy has proved that the biosensor can qualitatively distinguish the serum of healthy and liver cancer patients. This experimental method has the potential of clinical diagnosis.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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