微流控芯片和比色法双模同时检测副溶血性弧菌和鼠伤寒沙门氏菌

引用本文: 何立勇, 俞佳乐, 李天华, 陈荣恒, 林建原, 干宁. 微流控芯片和比色法双模同时检测副溶血性弧菌和鼠伤寒沙门氏菌[J]. 分析化学, 2021, 49(1): 50-59. doi: 10.19756/j.issn.0253-3820.201354 shu

Citation: HE Li-Yong, YU Jia-Le, LI Tian-Hua, CHEN Rong-Heng, LIN Jian-Yuan, GAN Ning. Simultaneous Detection of Vibrio Parahaemolyticus and Salmonella Typhimurium Based on Dual Mode Analysis Using Microfluidic Chip and Colorimetric Method[J]. Chinese Journal of Analytical Chemistry, 2021, 49(1): 50-59. doi: 10.19756/j.issn.0253-3820.201354 shu

微流控芯片和比色法双模同时检测副溶血性弧菌和鼠伤寒沙门氏菌

何立勇 ^1,2, ,
俞佳乐 ^2, ,
李天华 ^2, ,
陈荣恒 ^1, ,
林建原 ^{1,
,} ,
干宁 ^{2,
,}

1.
浙江万里学院生物与环境学院, 宁波 315100;
2.
宁波大学材料科学与化学工程学院, 宁波 315211

通讯作者: 林建原,E-mail:linjianyuan33@163.com; 干宁,E-mail:ganning@nbu.edu.cn

基金项目:
国家自然科学基金项目（No.21974074）、浙江省"生物工程"一流学科开放基金项目（A类）（No.KF2019001）、宁波市高校"食品科学与工程"重点学科自设课题（No.2019FSEKDNB007）、浙江省基础公益研究计划项目（No.LGN18H300001）和浙江省大学生创新创业训练计划项目（No.S201910876019）资助。

摘要: 由病原微生物引起的食源性疾病日益引起全球关注，迫切需要开发同时快速检测多种食源性致病菌的方法。本研究建立了一种基于磁性适配体捕获探针的微流控芯片-比色双模式传感器，实现了副溶血性弧菌（V.P）和鼠伤寒沙门氏菌（S.T）的快速定性和同时定量分析。此探针包括磁珠及其表面修饰的含ATP、待测物适配体及重复G碱基核苷酸片段的DNA长链。当探针和目标物结合后，会释放出上述DNA长链，形成与氯高铁血红素（Hemin）结合的G四链体，具有类过氧化物酶活性，可催化四甲基联苯胺（TMB）-H₂O₂体系的反应，产生肉眼可见的蓝色，实现两种致病菌的现场分析。释放的DNA长链经过EcoRV核酸内切酶剪切后，得到不同长度DNA片段，分别对应V.P和S.T的数量。在微流控芯片中分析和测定各片段，可实现两种细菌的定量分析。在最佳实验条件下，传感器对两种细菌检测的线性范围为10²~10⁷ CFU/mL，检出限可达30 CFU/mL。此传感器为现场定性筛查致病菌污染水平和实验室准确检测其含量提供了可靠的方法。

关键词:

English

Simultaneous Detection of Vibrio Parahaemolyticus and Salmonella Typhimurium Based on Dual Mode Analysis Using Microfluidic Chip and Colorimetric Method

1.
College of Biology and Environment, Zhejiang Wanli University, Ningbo 315100, China;
2.
Faculty of Material Science and Chemical Engineering, Ningbo Univeristy, Ningbo 315211, China

Corresponding author: LIN Jian-Yuan, ; GAN Ning,

Received Date: 18 June 2020
Revised Date: 22 November 2020
Fund Project:
Supported by the National Natural Science Foundation of China (No.21974074).

Abstract: Foodborne diseases caused by pathogenic microorganisms have attracted increasing attention in the world. It is urgent to develop efficient analytical methods for simultaneous and rapid detection of foodborne pathogens. In this regard, a dual-mode sensor based on the colorimetric principle coupled with microfluidic chip was developed using magnetic aptamer capture probes. It could realize rapid qualitative and simultaneous quantitative determination of Vibrio parahaemolyticus (V.P) and Salmonella typhimurium (S.T). The probe consisted several long DNA strands containing adenosine triphosphate (ATP), analyte aptamer and repeated G-base nucleotide fragments which were labeled on the surface of magnetic beads. When the probe reacted with the target analytes, the above long DNA strands would be released. The G quadruplet formed by hemin exhibited catalytic effect on tetramethylbenzidine (TMB)-H₂O₂ system, which could produce visible blue color and be used for on-site analysis of two pathogenic bacteria. After the long strand of released DNA was cut by EcoRV endonuclease, different lengths of DNA were obtained corresponding to V.P and S.T respectively. By analyzing each DNA in microchips, quantitative analysis of two kinds of bacteria V.P and S.T could be realized. Under the best experimental conditions, the detection linear range for the two bacteria was 10²-10⁷ CFU/mL, and the detection limit was 30 CFU/mL. The sensor provided a reliable method for screening contamination of pathogenic bacteria and quantification of each target accurately in the laboratory.

Key words:

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1.
沈阳化工大学材料科学与工程学院沈阳 110142

微流控芯片和比色法双模同时检测副溶血性弧菌和鼠伤寒沙门氏菌

通讯作者: 林建原,E-mail:linjianyuan33@163.com; 干宁,E-mail:ganning@nbu.edu.cn

English

Simultaneous Detection of Vibrio Parahaemolyticus and Salmonella Typhimurium Based on Dual Mode Analysis Using Microfluidic Chip and Colorimetric Method

Corresponding author: LIN Jian-Yuan, ; GAN Ning,

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

微流控芯片和比色法双模同时检测副溶血性弧菌和鼠伤寒沙门氏菌

通讯作者: 林建原,E-mail:linjianyuan33@163.com; 干宁,E-mail:ganning@nbu.edu.cn

English

Simultaneous Detection of Vibrio Parahaemolyticus and Salmonella Typhimurium Based on Dual Mode Analysis Using Microfluidic Chip and Colorimetric Method

Corresponding author: LIN Jian-Yuan, ; GAN Ning,

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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