基于核酸适体检测 ATP 的酶联分析新方法

摘要

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 concentra-tion ranged from 50 to 400 nmol/ L, and the detection limit is 26 nmol/ L.%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.