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Bioengenharia

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Published: June 03, 2018
doi:
10.3791/56585
, , ,
1Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security,Universiti Putra Malaysia, 2Laboratory of Functional Device, Institute of Advanced Technology,Universiti Putra Malaysia, 3Department of Chemistry, Faculty of Science,Universiti Putra Malaysia, 4La Trobe Institute for Molecular Science,La Trobe University

Summary

本文提出了一种电化学 DNA 生物传感器的研制方案, 包括聚乳酸稳定、金纳米粒子修饰、丝网印刷碳电极检测副溶血性弧菌。

Abstract

副溶血性弧菌 (副溶血性)是一种常见的食源性致病菌, 在全球范围内造成很大比例的公共卫生问题, 严重影响人类死亡率和发病率。常规的检测v. 副溶血性的方法, 如基于文化的方法、免疫学化验和基于分子的方法, 需要复杂的样品处理, 耗时、繁琐、费用高昂。近年来, 生物传感器已被证明是一种有前景的综合检测方法, 具有快速检测、性价比高、实用性强等优点。本研究的重点是利用 DNA 杂交原理开发一种快速检测具有高选择性和灵敏度的v. 副溶血性方法。在工作中, 采用 x 射线衍射 (XRD)、紫外可见光谱 (紫外-可见光)、透射电镜 (TEM)、场发射等方法, 对合成聚乳酸稳定金纳米粒子 (PLA-AuNPs) 进行了表征。扫描电镜 (FESEM) 和循环伏安法 (CV)。并对 PLA-AuNPs 的稳定性、灵敏度和重现性进行了进一步的测试。研究发现, PLA-AuNPs 在水溶液中形成了稳定纳米粒子的良好结构。我们还观察到, 由于较小的电荷转移电阻 (Rct) 值和活动表面积 (0.41 厘米2) 的增加, 灵敏度得到了改善。我们的 DNA 生物传感器的发展是基于对 AuNPs 和以亚甲基蓝 (MB) 为氧化还原指标的丝网印刷碳电极 (SPCE) 的改性。用差分脉冲伏安法 (DPV) 对固定化和杂交事件进行了评估。我们发现, 互补, 非互补, 和不匹配的寡核苷酸是专门区分的捏造的生物传感器。它还显示了对各种食物传播病原体的交叉反应性研究和在新鲜的蛤中鉴定v. 副溶血的可靠敏感检测。

Introduction

近年来公众和科学争论的一个主要话题, 食物中毒主要与3剂有关: 微生物1、化学试剂2和寄生虫3。受污染的食物会对人类造成严重的健康后果, 特别是在免疫系统薄弱、老年人、孕妇、婴儿和年幼儿童的高危人群中,4。由于非洲、亚洲和拉丁美洲5岁以下儿童每年发生100万多例急性腹泻, 食物中毒是全球主要疾病5,6 , 世界卫生组织已建立微生物作为最重要的贡献者7。副溶血弧菌在最广泛公认的毒株中脱颖而出。通常在沿海、河口和海洋环境中发现8, 它是一种革兰阴性菌, 在高盐环境中活跃, 在未充分烹调、处理不当或未加工的海洋中进食时引起严重的人胃肠炎。产品9。此外, 一些人现有的医疗条件使他们容易受到伤口感染、败血症或耳部感染, 这是由v 副溶血症10引起的。溶血素的毒力因子可分为两种类型, 分别为致病致病机制: TDH 基因编码的耐热直接溶血素 (TDH) 和 trh 基因11编码的 TDH 相关溶血素. 在临床标本中, 而不是在环境标本中, tdh 的毒力标志物 (trh 基因) 主要表现为副溶血性。

v. 副溶血性具有在广泛的条件下生存的能力, 迅速响应环境变化12。它的增殖机制随着细胞质量的增加, 其毒性也随之增大13, 从而提升其危险电位。更糟糕的是, 气候变化给这些细菌提供了充足的条件来加速他们的细胞数量增长14。由于其频率高, 需要在食品供应链上对v. 副溶血性进行监测, 特别是在海鲜的贸易和生产方面, 因为这些产品在很大数量上被发现,15,16在世界各地。目前, 细菌的识别和隔离使用了一系列的方法, 包括生物化学试验, 浓缩和选择性培养基17, 酶联磁吸附剂检测 (ELISA)18, 脉冲场凝胶电泳 (PFGE)19、乳胶凝集试验和聚合酶链反应 (PCR) 测试20。这些方法通常需要合格的人员, 先进的仪器, 和费力的技术, 没有立即提供污染的信息。这严重限制了及时检测有害污染和现场应用的可能性。快速检测工具仍然是一个突出的挑战。

生物传感正在成为检测食源性病原体的一个有希望的选择, 因为它提供了节省时间、经济高效、实用和实时分析方法 21, 22, 23, 24.然而, 尽管在尖刺样品和使用生物传感器的标准溶液中分析物检测有许多积极的结果, 但在水混合物或有机萃取物中, 仍缺乏对实际样品的研究25。最近, 利用直接和/或间接脱氧核糖核酸 (DNA) 检测的电化学生物传感器在科学家中得到了越来越多的关注, 因为它们通过杂交事件对互补靶的具体检测26,27,28,29. 与基于酶的生物传感器相比, 这些独特的方法更加稳定, 从而为小型化和商业化提供了一个有前景的技术。这项研究的目标是构建一个快速的工具, 能够检测出具有高选择性、灵敏性和实用性的、基于 DNA 序列特异性的杂交法. 识别策略包括聚乳酸稳定的金纳米粒子 (PLA-AuNPs)30和丝网印刷碳电极 (SPCEs) 在存在杂交指示剂, 亚甲基蓝 (MB)。利用细菌 DNA 裂解液和新鲜的蛤样, 进一步探讨了所研制的检测结构的潜力。

Protocol

注: 所有使用的化学药剂和生化试剂均应采用分析级, 无需进一步纯化。使用无菌去离子水制备所有溶液。灭菌前的所有玻璃器皿。 警告: 在进行实验室活动时, 请使用所有适当的安全做法, 包括使用工程控制 (油烟机、glovebox) 和个人防护设备 (安全眼镜、手套、实验室大衣、全长长裤、闭合脚趾鞋)。 1. 用 PLA-AuNPs 修饰电极的制备与表征 PLA-AuN…

Representative Results

通过柠檬酸钠的水溶液的颜色变化, 揭示了 AuNPs 的形成。这使颜色从淡黄色变为深宝石红色。从紫外可见光谱 (图 1) 中证实了 PLA-AuNPs 的产生, 其中表面等离子体共振 (SPR) 峰值的增长约为 540 nm。AuNPs 的形成和存在是在 500-600 nm 波长范围内, 这取决于粒子大小37。AuNPs 和 PLA-AuNPs 的 XRD 模式显示在图 2中。在31.7、38…

Discussion

这类电化学生物传感器成功发展的框架中的关键步骤是为传感器 (核酸或 DNA) 选择合适的生物识别元件;构造传感器传感层的化学方法转导材料;DNA 固定化与杂交的优化研究并通过实际样品对所研制的生物传感器进行验证。

核心到成功开发的敏感和选择性电化学 DNA 生物传感器, 是优化的固定化和杂交条件。在这项工作中, 我们用 MB 作为氧化还原络合物。有多种原则的 mb DNA 相互…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者希望感谢马来西亚马来西亚的支持。

Materials

Acetic acid Merck 100056
Chloroform Merck 102445
Diaminoethane tetraacetic acid Promega E5134
Dibasic sodium phosphate  Sigma-Aldrich S9763
Disodium hydrogen phosphate Sigma-Aldrich 255793
Ethanol  Sigma-Aldrich 16368
Gold (III) chloride trihydrate Sigma-Aldrich 520918
Hydrochloric acid Merck 100317
Methylene blue Sigma-Aldrich M44907
Monobasic sodium phosphate, monohydrate Sigma-Aldrich S3522
Phosphate-buffered saline Sigma-Aldrich P5119
Poly(lactic acid) resin, commercial grade 4042D NatureWorks 4042D
Potassium chloride R&M Chemicals 59435
Potassium dihydrogen phosphate Sigma-Aldrich P9791
Potassium hexacyanoferrate III R&M Chemicals 208019
Sodium acetate anhydrous salt Sigma-Aldrich S2889
Sodium chloride Sigma-Aldrich S9888
Trisodium citrate Sigma-Aldrich S1804
Tris(hydroxymethyl) aminomethane Fisher Scientific T395-100
Tris-Base Fisher Scientific BP152-500
2X PCR Master Mix with Dual-Dye Norgen Biotek 28240
Agarose gel Merck 101236
Bolton Agar Merck 100079
Bolton Broth Merck 100079
CHROMagar Vibrio CHROMagar VB910
dNTPs Promega U1511
Nuclease-free water Thermo Scientific R0581
Eosin methylene blue agar  Merck 101347
GelRed Biotium 41001
Glycerol Merck 104092
Go Taq Buffer Promega M7911
Loading dye 100 bp DNA ladder Promega G2101
Loading dye 1kb DNA ladder Promega G5711
Magnesium chloride Promega 91176
Mannitol egg yolk polymyxin agar Merck 105267
McConkey Agar Merck 105465
Nutrient Broth Merck 105443
Taq polymerase Merck 71003
Trypticase Soy Broth Merck 105459
Trypticase Soy Agar Merck 105458
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Keywords: Electrochemical DNA BiosensorFoodborne Pathogen DetectionGold NanoparticlesPolylactic AcidScreen Printed Carbon ElectrodeMethylene BlueDifferential Pulse VoltammetryHybridizationUV-vis SpectroscopyXRDTEMFESEM-EDXElectrochemical Characterization
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Nordin, N., Yusof, N. A., Radu, S., Hushiarian, R. Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen. J. Vis. Exp. (136), e56585, doi:10.3791/56585 (2018).
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