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Bioengineering

光学检测<em> E。大肠杆菌</em>由细菌介孔硅生物传感器

Published: November 20, 2013
doi:
10.3791/50805
, ,
1Department of Biotechnology and Food Engineering,Technion – Israel Institute of Technology, 2The Inter-Departmental Program of Biotechnology,Technion – Israel Institute of Technology, 3The Russell Berrie Nanotechnology Institute,Technion – Israel Institute of Technology

Summary

一个无标记光学生物传感器快速检测细菌的介绍。该生物传感器是基于纳米结构的多孔硅，被设计成直接捕获目标细菌的细胞在其表面上。我们使用的单克隆抗体，固定在所述多孔性换能器，作为捕获探针。我们的研究表明这些生物传感器用于与事先没有样品处理（如细胞裂​​解）分钟内低细菌浓度的检测的适用性。

Abstract

基于纳米结构的多孔Si一种不用标记的光学生物传感器被设计为大肠杆菌 K12细菌的快速捕获和检测，作为一个模型的微生物。在生物传感器依赖于直接结合的靶细菌细胞在其表面上，同时研究了样品的无预处理（ 例如，通过细胞裂解）是必需的。的中孔硅薄膜被用作生物传感器的光学换能器元件。在白光照明下，多孔层显示在它的反射率光谱很好分辨的法布里 – 珀罗干涉条纹图样。应用快速傅立叶变换（FFT），以在一个单一的峰值反射率数据的结果。变化的FFT峰的强度进行监测。因此，目标捕捉细菌到生物传感器表面，通过抗体 – 抗原相互作用，诱导的FFT峰的强度可测量的变化，允许一个“实时”观察细菌附着。

NT“>介孔硅薄膜，通过电化学阳极氧化工艺制造，缀合的单克隆抗体，特异于靶细菌的固定化，免疫活性和抗体的特异性是由荧光标记的实验证实，一旦生物传感器暴露于靶细菌，细胞被直接捕获到的抗体修饰的多孔硅表面。这些特定的捕获事件导致在生物传感器的薄膜光学干涉光谱强度的变化。我们表明，这些生物传感器可以检测到相对低的细菌浓度（检测限制在不到一个小时10 4个细胞/ ml）。

Introduction

早期准确鉴定致病菌的是食物和水安全，环境监测，并点护理诊断1极为重要。由于传统的微生物学技术是费时，费力，缺乏检测微生物的“实时”或在实验室环境之外的能力，生物传感器不断发展，以应对这些挑战2-5。

近年来，在多孔硅（PSI）已经成为一种很有前途的平台，用于传感器和生物传感器6-20的设计。在过去的十年里许多关于PSI为基础的光学传感器和生物传感器的研究发表21,22。纳米结构PSI层典型地制作由单晶Si晶片电化学阳极蚀刻。所得到的PSI纳米材料表现出许多有利的特性，例如大的表面和自由体积，孔径大小，可控制和可调谐OPTI校准性能10,16。 PSI的层，例如光致发光8,11和白光反射型干涉7,19的光学特性，强烈受到环境条件的影响。捕获在多孔层产生的薄膜，观察到的光致发光光谱的调制或者作为反射率光谱10的波长偏移的平均折射率的变化范围内的客体分子/靶分析物。

尽管在PSI光学生物传感器技术的巨大创新，也只有在PSI的平台为细菌检测6,8,20,23-29报道很少。此外，大多数证明概念的这些研究已证实“间接”细菌检测。因此，该细胞的一般现有的裂解是必需的，提取该目标蛋白质/ DNA片段，特征为研究细菌29。我们的做法是直接捕捉目标细菌细胞到PSI生物传感器。因此，单克隆抗体，其特定于针对细菌，被固定在所述多孔表面上。通过抗体-抗原相互作用的结合细菌细胞的，固定在生物传感器的表面上引起的变化的反射光谱24-26的振幅（强度）。

在这项工作中，我们报告的光学PSI为基础的生物传感器的构造，并证明了其作为无标记生物传感平台， 大肠埃希氏菌的检测（ 大肠杆菌 ）K12菌（作为模型微生物），监控的应用光信号是从PSI纳米结构反映由于法布里-珀罗薄膜干涉（ 图1A）的光。改变光的振幅/强度是相关的，以在目标细菌细胞的特异性固定到生物传感器表面上，从而允许快速检测和细菌的定量。

Protocol

1。氧化多孔SiO 2的制备蚀刻Si晶片（单面抛光的<100>面与重掺杂的p型，0.0008Ω·cm的）在HF水溶液和无水乙醇中进行30秒的3:1（V / V）溶液在恒定电流密度为385毫安/厘米2。请注意，HF是高腐蚀性的液体，它应该格外小心。 冲洗所得的多孔Si（PSI）膜与无水乙醇的表面数次;干燥在干燥氮气气体的薄膜。 氧化在管式炉的新鲜蚀刻PSI的样品在800℃下1小时，在环境?…

Representative Results

氧化PSI（PSIO 2）膜作为在协议文本部分中所述制备。 图1B示出的热氧化后得到的PSI膜的高分辨率扫描电子显微照片。该PSIO 2层的特征在于良好定义的圆柱形孔，其直径在30-80纳米的范围内。 的单克隆抗体（IgG）分子是通过使用一个完善的硅烷化技术，加上生物素-SA系统接枝到PSIO 2的表面。详细的合成方案在图2中所概述。首先?…

Discussion

一种不用标记的光学免疫传感器的基础上，PSIO 2纳米结构体（法布里-珀罗薄膜）的制造，并且其潜在适用性作为生物传感器用于细菌检测被确认。

修改和故障排除

一个设计免疫传感器时的主要关注是抗体的敏感性沉积和图案化期间经受不期望的构象变化到固体基板上，这可能导致在生物传感器灵敏度31,32下降。为了尽量减少这种问题，抗体…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由以色列科学基金会（批准号：1118年至1108年，并批准号1146至1112年）和明娜克罗尔纪念研究基金的支持。 ES衷心感谢罗素贝里纳米技术研究所的资金支持。

Materials

Name of Reagent/Material Company Catalog Number Comments
Si wafer Siltronix Corp. Highly-B-doped, p-type, 0.0008 Ω-cm resistivity, <100> oriented
Aqueous HF (48%) Merck 101513
Ethanol absolute Merck 818760
PBS buffer solution (pH 7.4) prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in Milli-Q water (18.2 MΩ)
Saline 0.85% w/v prepared by dissolving 0.85 g NaCl in 100 ml Milli-Q water (18.2 MΩ)
95% (3-Mercaptopropyl)trimethoxysilane (MPTS) Sigma Aldrich Chemicals 175617
PEO-iodoacetyl biotin Sigma Aldrich Chemicals B2059
Streptavidin (SA) Jackson ImmunoResearch Labs Inc. 016-000-114
Fluorescein (DTAF)-streptavidin Jackson ImmunoResearch Labs Inc. 016-010-084
Biotinylated-rabbit IgG Jackson ImmunoResearch Labs Inc. 011-060-003
Fluorescently tagged anti-rabbit IgG Jackson ImmunoResearch Labs Inc. 111-095-003
Fluorescently tagged anti-mouse IgG Jackson ImmunoResearch Labs Inc. 115-095-003
Biotinylated E. coli antibody Jackson ImmunoResearch Labs Inc. 1007
E. coli (K-12) was generously supplied by Prof. Sima Yaron, Technion
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Tags

Optical BiosensorMesoporous SiliconEscherichia ColiLabel-free DetectionAntibody-antigen InteractionFabry-Pérot InterferenceFast Fourier TransformReal-time MonitoringElectrochemical AnodizationFluorescent Labeling

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Massad-Ivanir, N., Shtenberg, G., Segal, E. Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors. J. Vis. Exp. (81), e50805, doi:10.3791/50805 (2013).
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