基于纳米材料和细胞色素C的生物传感新方法研究
发布时间：2019-01-07 21:42
【摘要】：纳米材料独特的结构赋予其量子尺寸、小尺寸和宏观量子隧道等特殊效应。发光金属纳米材料作为纳米材料的一个分支,由于具有合成简单、量子产率高、生物相容性好、不易光漂白等优点而在生物传感领域备受关注。细胞色素C(Cyt c)是一种含血红素的金属蛋白,能够接受电子,其等电点约为10.0,在缓冲介质p H低于它的等电点时带正电,这两大特点让Cyt c在生物传感领域得到广泛应用。本论文基于Cyt c和纳米材料中的银簇、铜纳米粒子,建立了三种新型的荧光生物传感平台。具体内容如下:(1)第2章,我们利用银簇与Cyt c间的电子转移作用构建了一种新的方法用于胰蛋白酶检测。银簇是以寡核苷酸为模板一步合成得到的,由于磷酸基团的存在,银簇带负电。完整的Cyt c等电点约为10.0,在本实验条件下带正电。当银簇与Cyt c在溶液中同时存在时,两者由静电作用形成复合物,接着通过电子转移作用,银簇的荧光被Cyt c中的血红素淬灭。胰蛋白酶可水解Cyt c,水解产物中连着血红素的短肽片段等电点在7.0左右,带少量负电荷,对银簇的作用很弱,于是银簇的荧光不会被淬灭。通过测荧光强度,实现对胰蛋白酶的检测。另外,胰蛋白酶抑制剂可抑制胰蛋白酶活性,因此这种方法可进一步实现对胰蛋白酶抑制剂的筛选。该方法无需标记、探针合成简单、成本低、选择性好。(2)第3章,我们基于Cyt c以及核酸外切酶Ⅲ(Exo Ⅲ)辅助的信号放大策略构建了一种新型的DNA传感器。这种传感器主要依赖于Cyt c对标记在DNA链上的荧光染料与单个荧光染料的亲和力不同从而引起淬灭程度不同而建立的。我们选择5′端修饰了荧光基团的单链DNA为荧光探针,在没有目标链时,探针不会被Exo Ⅲ水解,由于静电作用而与Cyt c结合,接着染料的荧光被Cyt c中的血红素淬灭。有目标链存在时,目标链与探针杂交形成双链,这种情况下,探针可被Exo Ⅲ水解成单个核苷酸,并释放出游离的染料。而目标链则进入下一个循环。游离染料与Cyt c间的作用较弱,荧光不会被淬灭。实验结果显示,这种方法可区分单碱基错配,而且能够实现双组份DNA的同时检测。(3)第4章,我们利用三聚氰胺与胸腺嘧啶能形成稳定的氢键从而抑制荧光铜纳米粒子形成构建了一个三聚氰胺传感平台。实验中选择的核酸序列T30由30个胸腺嘧啶组成,T30保护的铜纳米粒子能够形成的前提是Cu~(2+)可在DNA上吸附,接着在抗坏血酸钠还原下沿着DNA骨架成簇。三聚氰胺与胸腺嘧啶形成氢键,一方面阻碍了Cu~(2+)与T30作用,另一方面阻碍了Cu0在T30骨架上成簇,而且在一定范围内这种阻碍作用是随着三聚氰胺浓度的增大而增大的,于是合成的铜纳米粒子的荧光强度也随之减低,根据荧光强度的变化,实现三聚氰胺的定量分析。该方法快速、可靠、简单。
[Abstract]:The unique structure of nanomaterials gives them special effects such as quantum size, small size and macroscopic quantum tunneling. As a branch of nanomaterials, luminescent metal nanomaterials have attracted much attention in the field of biosensor due to their advantages of simple synthesis, high quantum yield, good biocompatibility and poor photobleaching. Cytochrome C (Cyt c) is a heme-containing metalloprotein capable of receiving electrons. Its isoelectric point is about 10.0, with a positive charge when the buffer medium pH is lower than its isoelectric point. These two characteristics make Cyt c widely used in the field of biosensor. In this paper, three novel fluorescent biosensors are established based on Cyt c and silver clusters and copper nanoparticles in nanomaterials. The main contents are as follows: (1) in Chapter 2, we construct a new method for the detection of trypsin by using the electron transfer between silver clusters and Cyt c. Silver clusters were synthesized by using oligonucleotides as template. Due to the presence of phosphoric acid groups, silver clusters were negatively charged. The complete Cyt c isoelectric point is about 10.0, with positive charge under this experimental condition. When the silver cluster and Cyt c exist at the same time in the solution, they form a complex by electrostatic interaction, and the fluorescence of the silver cluster is quenched by heme in Cyt c by electron transfer. Trypsin can hydrolyze Cyt c, and the isoelectric point of the short peptide fragment with heme in the hydrolyzed product is about 7.0, with a little negative charge, so the fluorescence of the silver cluster will not be quenched. The detection of trypsin was realized by measuring the fluorescence intensity. In addition, trypsin inhibitors can inhibit trypsin activity, so this method can further screen trypsin inhibitors. The method is simple, low-cost and selective. (2) in Chapter 3, we construct a novel DNA sensor based on the signal amplification strategy of Cyt c and nucleic acid exonuclease 鈪，
本文编号：2404202
[Abstract]:The unique structure of nanomaterials gives them special effects such as quantum size, small size and macroscopic quantum tunneling. As a branch of nanomaterials, luminescent metal nanomaterials have attracted much attention in the field of biosensor due to their advantages of simple synthesis, high quantum yield, good biocompatibility and poor photobleaching. Cytochrome C (Cyt c) is a heme-containing metalloprotein capable of receiving electrons. Its isoelectric point is about 10.0, with a positive charge when the buffer medium pH is lower than its isoelectric point. These two characteristics make Cyt c widely used in the field of biosensor. In this paper, three novel fluorescent biosensors are established based on Cyt c and silver clusters and copper nanoparticles in nanomaterials. The main contents are as follows: (1) in Chapter 2, we construct a new method for the detection of trypsin by using the electron transfer between silver clusters and Cyt c. Silver clusters were synthesized by using oligonucleotides as template. Due to the presence of phosphoric acid groups, silver clusters were negatively charged. The complete Cyt c isoelectric point is about 10.0, with positive charge under this experimental condition. When the silver cluster and Cyt c exist at the same time in the solution, they form a complex by electrostatic interaction, and the fluorescence of the silver cluster is quenched by heme in Cyt c by electron transfer. Trypsin can hydrolyze Cyt c, and the isoelectric point of the short peptide fragment with heme in the hydrolyzed product is about 7.0, with a little negative charge, so the fluorescence of the silver cluster will not be quenched. The detection of trypsin was realized by measuring the fluorescence intensity. In addition, trypsin inhibitors can inhibit trypsin activity, so this method can further screen trypsin inhibitors. The method is simple, low-cost and selective. (2) in Chapter 3, we construct a novel DNA sensor based on the signal amplification strategy of Cyt c and nucleic acid exonuclease 鈪，
本文编号：2404202
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