免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿

引用本文: 吴文伟, 王翌, 刘可鑫, 李天松, 杨咏洁. 免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿[J]. 色谱, 2020, 38(11): 1332-1339. doi: 10.3724/SP.J.1123.2020.04010 shu

Citation: WU Wenwei, WANG Yi, LIU Kexin, LI Tiansong, YANG Yongjie. Simultaneous and rapid determination of malachite green and leucomalachite green by a label-free colorimetric aptasensor[J]. Chinese Journal of Chromatography, 2020, 38(11): 1332-1339. doi: 10.3724/SP.J.1123.2020.04010 shu

免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿

吴文伟 ^1, ,
王翌 ^2, ,
刘可鑫 ^1, ,
李天松 ^{2,
,} ,
杨咏洁 ^{1,
,}

1.
延边大学农学院, 吉林延吉 133002;
2.
北华大学理学院, 吉林吉林 132001

通讯作者: 李天松, E-mail:lts0309@163.com; 杨咏洁, E-mail:yjyang@ybu.edu.cn

基金项目:
国家自然科学基金项目（31602126）；吉林省教育厅基金项目（JJKH20180901KJ）；延边大学青-基金项目（602017020）.

摘要: 研究以双特异性核酸适配体A3作为传感探针、纳米金（AuNPs）为指示剂、NaCl溶液为聚集诱导剂，构建了一种新型的免标记AuNPs比色生物传感器，可实现水产品中孔雀石绿（MG）和无色孔雀石绿（LMG）的同步、快速、可视化检测。该方法的检测原理是核酸适配体A3对MG和LMG有双特异性识别能力，可作为MG和LMG理想的识别受体。它可通过静电作用吸附到AuNPs表面，保护AuNPs并抑制高盐溶液诱导的聚集，AuNPs溶液颜色不变，即为红色；当加入靶标MG或LMG后，该核酸适配体能够与靶标特异性结合，并从AuNPs表面上解离，AuNPs失去保护作用而在高盐溶液诱导下发生聚集，溶液颜色由红变蓝。根据颜色变化，可通过肉眼定性或通过光谱仪定量分析MG和LMG的残留量。该方法首先将50 μL的核酸适配体A3（终浓度150 nmol/L）与150 μL的AuNPs（终浓度1.25 nmol/L）混合，室温孵育6 min。随后加入50 μL待测液，室温孵育30 min。最后加入50 μL NaCl（终浓度150 mmol/L），4 min后观察溶液颜色变化，并分别测定MG和LMG在520 nm和650 nm下的吸光度值。结果表明，在最佳反应条件下，该方法能够特异性检测MG和LMG，而对磺胺嘧啶（SDZ）和硝基呋喃妥因（NFT）无交叉反应；当MG、LMG的浓度为0~17.5 μmol/L时，吸光度比值与靶标浓度呈现良好的线性关系，相关系数（R²）分别为0.9938和0.9715。MG和LMG的检出限分别为6.93 nmol/L和6.38 nmol/L，加标回收率分别为88.60%~93.30%和101.80%~107.00%，相对标准偏差（RSD）分别为2.27%~3.55%和2.62%~3.75%。该方法操作简单，快速和灵敏，可为水产品中MG和LMG的同步快速检测提供一种新方法。

关键词:

English

Simultaneous and rapid determination of malachite green and leucomalachite green by a label-free colorimetric aptasensor

WU Wenwei ^1, ,
WANG Yi ^2, ,
LIU Kexin ^1, ,
LI Tiansong ^{2,
,} ,
YANG Yongjie ^{1,
,}

1.
Agricultural College of Yanbian University, Yanji 133002, China;
2.
College of Science of Beihua University, Jilin 132001, China

Corresponding author: LI Tiansong, ; YANG Yongjie,

Received Date: 17 April 2020
Fund Project:
National Natural Science Foundation of China (No. 31602126)

Abstract: A label-free colorimetric aptasensor, using a bispecific aptamer (A3) as a sensing probe, gold nanoparticles (AuNPs) as an indicator, and NaCl solution as an aggregation inducer, was successfully developed for the simultaneous, rapid and visual detection of malachite green (MG) and leucomalachite green (LMG) in aquatic products. This method is based on the aptamer A3 having bispecific binding ability with MG and LMG, making it an ideal recognition receptor for MG and LMG. It can adsorb on the AuNPs and protect AuNPs against salt-induced aggregation, maintaining the red color of the solution. When MG or LMG was added to a solution, aggregation of AuNPs was specifically induced by desorption of aptamer from the AuNPs surface upon formation of the aptamer-target complex. Therefore, the salt could trigger aggregation of AuNPs and the solution color was changed from red to blue. This color change allowed the qualitative determination of MG and LMG visually, and quantitative determination by measuring the ratio of the absorbances at 520 nm and 650 nm. In this study, 50 μL of the nucleic acid aptamer A3 (final concentrations 150 nmol/L) and 150 μL of AuNPs (final concentrations 1.25 nmol/L) were incubated at room temperature (RT) for 6 min, then 50 μL of the sample was added and incubated at RT for 30 min, and finally 50 μL NaCl solution (final concentrations 150 mmol/L) was added. After 4 min, the solution color change was observed, and the absorbances at 520 nm and 650 nm were measured. Under the optimal conditions, MG and LMG could be detected specifically without any cross-reactivity with sulfadiazine (SDZ) and nitrofurantoin (NFT). The absorbance were related to the concentrations of MG and LMG, and a good linear relationship was obtained in the range of 0-17.5 μmol/L. The correlation coefficients (R²) were 0.9938 and 0.9715, respectively. The limits of detection of MG and LMG were 6.93 nmol/L and 6.38 nmol/L, respectively. The spiked recoveries of MG and LMG ranged from 88.60% to 93.30% and 101.80% to 107.00%, respectively. The relative standard deviations (RSDs) of MG and LMG ranged from 2.27% to 3.55% and 2.62% to 3.75%, respectively. This colorimetric method is simple, rapid, sensitive, and allows visual, and it can provide a new method for the simultaneous and rapid determination of the MG and LMG in aquatic products.

Key words:

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沈阳化工大学材料科学与工程学院沈阳 110142

免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿

通讯作者: 李天松, E-mail:lts0309@163.com; 杨咏洁, E-mail:yjyang@ybu.edu.cn

English

Simultaneous and rapid determination of malachite green and leucomalachite green by a label-free colorimetric aptasensor

Corresponding author: LI Tiansong, ; YANG Yongjie,

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿

通讯作者: 李天松, E-mail:lts0309@163.com; 杨咏洁, E-mail:yjyang@ybu.edu.cn

English

Simultaneous and rapid determination of malachite green and leucomalachite green by a label-free colorimetric aptasensor

Corresponding author: LI Tiansong, ; YANG Yongjie,

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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