DNA损伤传感器制备及其用于饲料中黄曲霉毒素B1的快速检测

Preparation of a Biosensor for DNA Damage Detection and its Application in Rapid Detection of Aflatoxin B1 in Feed

摘要:

本文利用巯基化单链DNA（ssDNA）自组装修饰在金电极表面，制备了ssDNA修饰金电极（ssDNA/Au）；在最优化条件下，与互补片段杂交制备了相应的双链DNA修饰金电极（dsDNA/Au），构建了一种快速、简便、价廉的检测黄曲霉毒素B1（AFB1）的新型电化学DNA生物传感器。采用循环伏安法（CV）和示差脉冲伏安法（DPV）等电化学方法表征了电子媒介体铁氰化钾和亚甲基蓝（MB）在ssDNA/Au和dsDNA/Au界面上的电化学行为。一定浓度的黄曲霉毒素B1（AFB1）诱导dsDNA/Au造成DNA损伤，使得MB电化学信号降低，实现了快速检测AFB1。在最优化条件（在4 ℃下，ssDNA在金电极上自组装14 h，与cDNA在37 ℃下杂交2 h，制备了dsDNA/Au电极；37 ℃下，AFB1溶液诱导损伤DNA 22 min后），该方法对AFB1的线性检测范围为10~500 ng/mL，加标回收率在95.99 %~104.57 %。建立的AFB1诱导DNA损伤方法可以实现对AFB1快速、简便、准确的定量检测。

Abstract:

In this study, a single-stranded DNA/Au (ssDNA/Au) electrode was prepared by aligning a self-assembled monolayer of thiolated ssDNA onto an Au electrode surface. Under optimal conditions, ssDNA/Au hybridized with the complementary DNA (cDNA) fragment to produce a double-stranded DNA/Au (dsDNA/Au) electrode, which was a useful and cost-effective electrochemical DNA biosensor for the rapid detection of aflatoxin B1 (AFB1). Electrochemical methods such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were applied to characterize the electrochemical behavior of ferricyanide and methylene blue (MB) (as electron mediators) on the surface of ssDNA/Au and dsDNA/Au electrodes. A certain concentration of AFB1 reacted with dsDNA/Au to induce DNA damage, thereby reducing the electrochemical signals from MB and allowing for rapid detection of AFB1. Under optimized conditions (ssDNA was self-assembled on gold electrodes for 14 h at 4 ℃ and hybridized with cDNA at 37 ℃ for 2 h to produce the dsDNA/Au electrode; AFB1 solution induced DNA damage for 22 min at 37 ℃), the linear detection range for AFB1 was 10~500 ng/mL, with a satisfactory recovery ranging from 95.99% to 104.57%. This method of AFB1-induced DNA damage detection could be explored as a useful, quantitative method for accurate and rapid detection of AFB1.