Massad-Ivanir Naama, Shtenberg Giorgi, Segal Ester
Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology.
J Vis Exp. 2013 Nov 20(81):e50805. doi: 10.3791/50805.
A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g. by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-Pérot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a 'real time' observation of bacteria attachment. The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 10(4) cells/ml) in less than an hour.
一种基于纳米结构多孔硅的无标记光学生物传感器被设计用于快速捕获和检测作为模型微生物的大肠杆菌K12。该生物传感器依赖于目标细菌细胞直接结合到其表面，而无需对研究样本进行预处理（例如通过细胞裂解）。介孔硅薄膜用作生物传感器的光学传感元件。在白光照射下，多孔层在其反射光谱中显示出清晰分辨的法布里-珀罗条纹图案。对反射率数据应用快速傅里叶变换（FFT）会产生一个单峰。监测FFT峰强度的变化。因此，通过抗体-抗原相互作用，目标细菌捕获到生物传感器表面会导致FFT峰强度发生可测量的变化，从而可以“实时”观察细菌附着情况。通过电化学阳极氧化工艺制备的介孔硅薄膜与针对目标细菌的单克隆抗体结合。通过荧光标记实验确认了抗体的固定化、免疫活性和特异性。一旦生物传感器暴露于目标细菌，细胞就会直接捕获到抗体修饰的多孔硅表面。这些特异性捕获事件会导致生物传感器薄膜光学干涉光谱的强度变化。我们证明这些生物传感器能够在不到一小时的时间内检测到相对较低的细菌浓度（检测限为10(4) 个细胞/毫升）。
