基于核酸适配体及胶体金可视化检测水中孔雀石绿

doi:10.3964/j.issn.1000-0593(2020)03-0831-06

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摘要：孔雀石绿（MG）是一种有毒的三苯甲烷类物质，由于其价格低廉，抑菌效果好，曾在水产养殖中被作为抑菌剂广泛使用。但是长期大量的使用孔雀石绿将会对人体产生致癌、致畸、致突变的危害。传统检测水中孔雀石绿的方法需要复杂的前处理，花费大量时间，且需要昂贵的仪器设备，技术难度高，因此发展一种快速简便的MG检测方法十分必要。核酸适配体是一种能与靶标分子特异性结合的DNA或RNA片段，它具有高特异性、高亲和力、易于化学合成和修饰、稳定性高等特点，是比抗体更为有潜力的靶标识别元素，目前被广泛应用于传感检测中。胶体金（AuNPs）具有高消光系数和表面等离子体共振现象，可用于可视化检测体系中。研究了一种基于胶体金和RNA适配体的可视化快速检测孔雀石绿的方法。当有盐（NaCl）存在时，AuNPs会受到盐的作用而发生团聚，其吸收光谱峰由520 nm处移到690 nm处，溶液颜色由红色变成蓝色。由于RNA适配体可以通过静电作用吸附在AuNPs表面，对AuNPs起到保护作用，可使AuNPs在盐溶液中不发生聚集而呈红色；而当体系中有MG存在时，由于MG与RNA适配体的特异性结合，使得RNA适配体从AuNPs表面脱离，游离的AuNPs遇盐发生聚集呈蓝色。随着MG浓度的升高，520 nm处吸光度值逐渐降低，690 nm处吸光度值逐渐升高，且溶液颜色逐渐由红色变为蓝色。因此，目标物MG的含量可通过肉眼观察溶液颜色或通过可见吸收光谱来确定，整个检测过程不超过1小时。以有或无MG时AuNPs于520及690 nm处吸光度比值的差值Δ（A₆₉₀/A₅₂₀）作为检测信号，发现在NaCl浓度为0.2 mol·L^-1、RNA浓度为10 μmol·L^-1及AuNPs的浓度为7 nmol·L^-1时，MG浓度的线性范围为0.6~12.5 μmol·L^-1[线性方程为Δ(A₆₉₀/A₅₂₀)=0.06c-0.01，R²为0.993]，检出限为0.04 μmol·L^-1(3α/κ，n=9)。该方法对MG检测具有良好的选择性，将此方法应用于养殖水样中孔雀石绿的检测，加标回收率为92%~108%，说明该方法能够准确、灵敏、快速检测水产养殖中的孔雀石绿。

关键词：可视化检测；孔雀石绿；胶体金；RNA核酸适配体

Abstract：Malachite green (MG), one of the toxic triphenylmethane chemicals, has been used worldwide in aquaculture because of its low cost and high efficacy in bacteriostasis. However, the residue of MG may cause carcinogenic, teratogenic and mutagenic effects. Traditional methods for MG detection are relatively complex, which require complicated operations of pretreatment, take a lot of time -, and need expensive instruments. Thus a rapid and simple detection method needs to be developed. Aptamers are the functional single stranded DNA or RNA molecules which have high-affinity and specificity in binding capabilities towards a vast range of targets. Therefore, aptamers have been widely applied as recognition elements for biosensor in recent years. Gold nanoparticles (AuNPs) with high extinction coefficient and surface plasmon resonance have been used in colorimetric detection systems. In this work, a facile colorimetric aptasensor for highly sensitive detection of MG based on aptamer and AuNPs was developed. In the absence of salt, AuNPs agglomerated in salt with the absorption peak of UV-vis spectrum shifted from 520 nm to 690 nm, and the solution color changed from red to blue. However, the complexation of RNA aptamer with AuNPs prevented the nanoparticles from aggregation in a high-salt solution because of the electrostatic interaction between RNA aptamer and AuNPs. In the presence of MG, RNA aptamer could specifically bind with MG, causing AuNPs aggregation with blue in color in salt. With the increases of MG concentrations, the values of A₅₂₀ decreased and the values of A₆₉₀ increased, and the color of the solution gradually changed from red to blue. Taking advantage of this sensing technique, MG could be detected by naked eyes or UV-Vis spectroscopy within one hour. The differences of A₆₉₀/A₅₂₀ with and without MG in the detection systems were used as the detection signals. The results showed that the linear range of MG was 0.6 to 12.5 μmol·L^-1 at the optimum conditions of 0.2 mol·L^-1 NaCl, 10 μmol·L^-1 RNA and 7 nmol·L^-1 AuNPs. A linear equation of Δ(A₆₉₀/A₅₂₀)=0.06C-0.01 was obtained with a correlation coefficient (R²) of 0.993. The detection limit was 0.04 μmol·L^-1 (3α/κ, n=9). The method had good selectivity for MG detection. Furthermore, the developed method was successfully applied to the detection of MG in aquaculture water with excellent accuracies. The results indicated that the developed method has significant potentials for trace MG detection in real samples.

Key words：Colorimetric detection; Malachite green; AuNPs; RNA aptamer

收稿日期: 2019-01-09 修订日期: 2019-04-25

中图分类号:

O657.32

基金资助: 国家自然科学基金项目（21804050），厦门市科技计划项目（3502Z20183031），福建省基金项目（2018J01432）资助

通讯作者: 黄志勇 E-mail: zhyhuang@jmu.edu.cn

作者简介: 赵晨，女，1994年生，集美大学食品与生物工程学院硕士研究生 e-mail: 690197388@qq.com

引用本文:

赵晨，洪诚毅，林郑忠，黄志勇. 基于核酸适配体及胶体金可视化检测水中孔雀石绿[J]. 光谱学与光谱分析, 2020, 40(03): 831-836.
ZHAO Chen, HONG Cheng-yi, LIN Zheng-zhong, HUANG Zhi-yong. Colorimetric Detection of Trace Malachite Green by Label-Free RNA-Aptamer and AuNPs. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2020, 40(03): 831-836.

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https://www.gpxygpfx.com/CN/10.3964/j.issn.1000-0593(2020)03-0831-06 或 https://www.gpxygpfx.com/CN/Y2020/V40/I03/831