【摘要】：納米材料獨特的結(jié)構(gòu)賦予其量子尺寸、小尺寸和宏觀量子隧道等特殊效應(yīng)。發(fā)光金屬納米材料作為納米材料的一個分支,由于具有合成簡單、量子產(chǎn)率高、生物相容性好、不易光漂白等優(yōu)點而在生物傳感領(lǐng)域備受關(guān)注。細(xì)胞色素C(Cyt c)是一種含血紅素的金屬蛋白,能夠接受電子,其等電點約為10.0,在緩沖介質(zhì)p H低于它的等電點時帶正電,這兩大特點讓Cyt c在生物傳感領(lǐng)域得到廣泛應(yīng)用。本論文基于Cyt c和納米材料中的銀簇、銅納米粒子,建立了三種新型的熒光生物傳感平臺。具體內(nèi)容如下:(1)第2章,我們利用銀簇與Cyt c間的電子轉(zhuǎn)移作用構(gòu)建了一種新的方法用于胰蛋白酶檢測。銀簇是以寡核苷酸為模板一步合成得到的,由于磷酸基團(tuán)的存在,銀簇帶負(fù)電。完整的Cyt c等電點約為10.0,在本實驗條件下帶正電。當(dāng)銀簇與Cyt c在溶液中同時存在時,兩者由靜電作用形成復(fù)合物,接著通過電子轉(zhuǎn)移作用,銀簇的熒光被Cyt c中的血紅素淬滅。胰蛋白酶可水解Cyt c,水解產(chǎn)物中連著血紅素的短肽片段等電點在7.0左右,帶少量負(fù)電荷,對銀簇的作用很弱,于是銀簇的熒光不會被淬滅。通過測熒光強(qiáng)度,實現(xiàn)對胰蛋白酶的檢測。另外,胰蛋白酶抑制劑可抑制胰蛋白酶活性,因此這種方法可進(jìn)一步實現(xiàn)對胰蛋白酶抑制劑的篩選。該方法無需標(biāo)記、探針合成簡單、成本低、選擇性好。(2)第3章,我們基于Cyt c以及核酸外切酶Ⅲ(Exo Ⅲ)輔助的信號放大策略構(gòu)建了一種新型的DNA傳感器。這種傳感器主要依賴于Cyt c對標(biāo)記在DNA鏈上的熒光染料與單個熒光染料的親和力不同從而引起淬滅程度不同而建立的。我們選擇5′端修飾了熒光基團(tuán)的單鏈DNA為熒光探針,在沒有目標(biāo)鏈時,探針不會被Exo Ⅲ水解,由于靜電作用而與Cyt c結(jié)合,接著染料的熒光被Cyt c中的血紅素淬滅。有目標(biāo)鏈存在時,目標(biāo)鏈與探針雜交形成雙鏈,這種情況下,探針可被Exo Ⅲ水解成單個核苷酸,并釋放出游離的染料。而目標(biāo)鏈則進(jìn)入下一個循環(huán)。游離染料與Cyt c間的作用較弱,熒光不會被淬滅。實驗結(jié)果顯示,這種方法可區(qū)分單堿基錯配,而且能夠?qū)崿F(xiàn)雙組份DNA的同時檢測。(3)第4章,我們利用三聚氰胺與胸腺嘧啶能形成穩(wěn)定的氫鍵從而抑制熒光銅納米粒子形成構(gòu)建了一個三聚氰胺傳感平臺。實驗中選擇的核酸序列T30由30個胸腺嘧啶組成,T30保護(hù)的銅納米粒子能夠形成的前提是Cu~(2+)可在DNA上吸附,接著在抗壞血酸鈉還原下沿著DNA骨架成簇。三聚氰胺與胸腺嘧啶形成氫鍵,一方面阻礙了Cu~(2+)與T30作用,另一方面阻礙了Cu0在T30骨架上成簇,而且在一定范圍內(nèi)這種阻礙作用是隨著三聚氰胺濃度的增大而增大的,于是合成的銅納米粒子的熒光強(qiáng)度也隨之減低,根據(jù)熒光強(qiáng)度的變化,實現(xiàn)三聚氰胺的定量分析。該方法快速、可靠、簡單。
[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，

本文編號：2404202