色谱

色谱 ›› 2020, Vol. 38 ›› Issue (11): 1332-1339.DOI: 10.3724/SP.J.1123.2020.04010

• 研究论文 • 上一篇    下一篇

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. 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
  • 收稿日期:2020-04-17 出版日期:2020-11-08 发布日期:2020-12-11
  • 通讯作者: 李天松,杨咏洁
  • 作者简介:李天松.E-mail:lts0309@163.com (李天松)
    杨咏洁.E-mail:yjyang@ybu.edu.cn (杨咏洁);
  • 基金资助:
    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)

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

Wenwei WU1, Yi WANG2, Kexin LIU1, Tiansong LI2,*(), Yongjie YANG1,*()   

  1. 1 Agricultural College of Yanbian University, Yanji 133002, China
    2 College of Science of Beihua University, Jilin 132001, China
  • Received:2020-04-17 Online:2020-11-08 Published:2020-12-11
  • Contact: Tiansong LI,Yongjie YANG
  • Supported by:
    National Natural Science Foundation of China(31602126);Foundation of Educational Commission of Jilin Province(JJKH20180901KJ);Youth Foundation of Yanbian University(602017020)

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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（R 2 ）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: 纳米金比色法, 同步快速检测, 双特异性核酸适配体, 孔雀石绿, 无色孔雀石绿

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 (R 2 ) 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: gold nanoparticles (AuNPs) colorimetry, simultaneous and rapid detection, bispecific aptamer, malachite Green (MG), leucomalachite Green (LMG)