基于核酸适体检测ATP的酶联分析新方法

doi:10.7503/cjcu20140129

摘要/Abstract

摘要：

基于核酸适体对靶标的特异性识别和辣根过氧化物酶(HRP)的高效催化反应, 发展了一种用于检测三磷酸腺苷(ATP)的酶联核酸适体分析新方法. 核酸适体和靶标的特异性结合导致与核酸适体杂交的短链DNA解链, 解离的DNA通过杂交被固定在另一酶标板的DNA捕获. 解离的DNA预先标记了异硫氰酸荧光素(FITC)基团, FITC特异性结合HRP标记的FITC抗体, HRP作为信号传导元素催化四甲基二苯胺(TMB)底物显色, 通过颜色变化及450 nm波长处吸光度的变化检测ATP. 该方法对ATP具有良好的选择性, 检测不受其它物质如GTP, UTP和CTP的干扰, 且检测能在较复杂的试样(体积分数10%和50%的血清)中进行. 实验结果表明, 在ATP浓度为50~400 nmol/L范围内, 具有良好的线性关系, 检出限为26 nmol/L.

关键词: 核酸适体, 酶联吸附分析, 三磷酸腺苷(ATP), 选择性, 血清

Abstract:

A novel aptamer-based enzyme-linked assay for the detection of adenosine triphosphate(ATP) was established based on the specific recognition of aptamer towards target and the efficient catalytic ability of horseradish peroxidase(HRP). Aptamer binding with targets caused that duplex DNA containing short chain DNA and aptamer dissociated and dissociated DNA was captured by another DNA immobilized on other plate through hybridization. A fluorescein isothiocyanate(FITC) was tagged on dissociated DNA, a HRP as signal transduction element was linked on microplate through specific interactions with anti-FITC-HRP and catalyzed methyenedianiline(TMB) substrate to produce color change. The detection of ATP was achieved by color change and absorbance change at 450 nm. This method has good selectivity for ATP recognition interfering from other proteins such as GTP, UTP and CTP and the detection can be carried in complex sample such as 10% and 50% serum. Experimental results show that there is a good linear relationship with ATP concentration ranged from 50 to 400 nmol/L, and the detection limit is 26 nmol/L.

Key words: Aptamer, Enzyme-linked assay, Adenosine triphosphate(ATP), Selectivity, Serum

中图分类号:

O657.1

TrendMD:

赵秋伶, 刘玲玲, 杨丽娜. 基于核酸适体检测ATP的酶联分析新方法. 高等学校化学学报, 2014, 35(6): 1161.

ZHAO Qiuling, LIU Lingling, YANG Lina. Novel Aptamer-based Enzyme-linked Assay for the Detection of ATP. Chem. J. Chinese Universities, 2014, 35(6): 1161.

图/表 7

Table 1 DNA sequences used in this experiment

Name	DNA sequences(5'-3')
A1	Biotin-AGCT ACC TGG GGG AGT ATT GCG GAG GAA GGT
B	GTC ACC TTC CTC CGC AAT
B1	FITC-GTC ACC TTC CTC CGC AAT
C1	Biotin-ATT GCG GAG GAA GGT GAC

Fig.1 Sensing principle of aptamer based enzyme-linked assay

Fig.2 Fluorescence spectra of A1+B/SYBR Green I system upon analyzing different concentrations of ATPATP concentration/(nmol·L-1), a—g: 0, 50, 100, 150, 200, 250, 300. λex=497 nm.

Fig.3 Photograph of ATP concentration detection in Tris-HCl buffer

Fig.4 Corresponding calibration plot of A450 vs. ATP concentration(A) and linear part of the plot in a range of 0—400 nmol/L(B) The experiment was performed under optimal conditions. Error bars: SD, n=3.

Fig.5 Selectivity of the ELAA assay for ATP over CTP, GTP and UTP

Fig.6 Detection results of ATP in the complex sample systemsError bars: SD, n=3.

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