Name: optical detection of e. coli bacteria by mesoporous silicon biosensors
Uploaded: 2013-11-20T17:00:00
Description: Watch this Scientific Journal Video about Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors at JoVE.com

This work was supported by the Israel Science Foundation (grant No. 1118/08 and grant No. 1146/12) and the Minna Kroll Memorial Research Fund. E.S gratefully acknowledges the financial support of the Russell Berrie Nanotechnology Institute.

1. Preparation of Oxidized Porous SiO2
<ol>
	<li>Etch Si wafers (single side polished on the &#60;100&#62; face and heavily doped, p-type, 0.0008 &#937;&#183;cm) in a 3:1 (v/v) solution of aqueous HF and absolute ethanol for 30 sEC at a constant current density of 385 mA/cm2. Please note that HF is a highly corrosive liquid, and it should be handled with extreme care.</li>
	<li>Rinse the surface of the resulting porous Si (PSi) film with absolute ethanol several times; dry the films under a dry ni...

<ol>
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A label-free optical immunosensor, based on a PSiO2 nanostructure (a Fabry-P&#233;rot thin film) is fabricated, and its potential applicability as a biosensor for bacteria detection is confirmed.
Modifications and troubleshooting
One of the major concerns when designing an immunosensor is the susceptibility of antibodies to undergo undesired conformation changes during deposition and patterning onto the solid substrate, which may lead to a decrease in the bi...

Early and accurate identification of pathogenic bacteria is extremely important for food and water safety, environmental monitoring, and point-of-care diagnostics1. As traditional microbiology techniques are time consuming, laborious, and lack the ability to detect microorganisms in &#34;real-time&#34; or outside the laboratory environment, biosensors are evolving to meet these challenges2-5.
In recent years, porous Si (PSi) has emerged as a promising platform for the design of sensors and biosensors6-20. Over the past decade numerous studies regarding PSi-based optical sensors and biosensors were published21,22. The nanostructured PSi layer is typically fabricated by electrochemical anodic etching from a single-crystal Si wafer. The resulting PSi nanomaterials exhibit many advantageous characteristics, such as large surface and free volume, pore sizes that can be controlled and tunable optical properties10,16. The optical properties of the PSi layer, such as photoluminescence8,11 and white light reflectance-based interferometry7,19, are strongly influenced by environmental conditions. Capture of guest molecules/target analytes within the porous layer results in a change in the average refractive index of the film, observed as a modulation in the photoluminescence spectrum or as a wavelength shift in the reflectivity spectrum10.
Although the vast innovation in PSi optical biosensor technology, there are only few reports on PSi-based platforms for bacteria detection6,8,20,23-29. In addition, most of these proof-of-concept studies have demonstrated &#34;indirect&#34; bacteria detection. Thus, generally prior lysis of the cells is required to extract the targeted protein/DNA fragments, characteristic to the studied bacteria29. Our approach is to directly capture the target bacteria cells onto the PSi biosensor. Therefore, monoclonal antibodies, which are specific to target bacteria, are immobilized onto the porous surface. Binding of bacteria cells, via antibody-antigen interactions, onto the surface of the biosensor induce changes in the amplitude (intensity) of the reflectivity spectrum24-26.
In this work, we report on the construction of an optical PSi-based biosensor and demonstrate its application as a label-free biosensing platform for the detection of Escherichia coli (E. coli) K12 bacteria (used as a model microorganism).The monitored optical signal is the light reflected from the PSi nanostructure due to Fabry-P&#233;rot thin film interference (Figure 1A). Changes in the light amplitude/intensity are correlated to specific immobilization of the target bacteria cells onto the biosensor surface, allowing for rapid detection and quantification of the bacteria.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Siltronix Corp.</td>
			<td></td>
			<td>Highly-B-doped, p-type, 0.0008 &#937;-cm resistivity, &#60;100&#62; oriented</td>
		</tr>
		<tr>
			<td>Aqueous HF (48%)</td>
			<td>Merck</td>
			<td>101513</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck</td>
			<td>818760</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>Saline 0.85% w/v</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 0.85 g NaCl in 100 ml Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>95% (3-Mercaptopropyl)trimethoxysilane (MPTS)</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-iodoacetyl biotin</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>B2059</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (SA)</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-000-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein (DTAF)-streptavidin</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-010-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>011-060-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>111-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>115-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated E. coli antibody</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>1007</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli (K-12)</td>
			<td></td>
			<td></td>
			<td>was generously supplied by Prof. Sima Yaron, Technion</td>
		</tr>
	</tbody>
</table>

optical detection of e. coli bacteria by mesoporous silicon biosensors

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.&#160;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-P&#233;rot 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 &#39;real time&#39; 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 104 cells/ml) in less than an hour.

Oxidized PSi (PSiO2) films are prepared as described in the Protocol Text section. Figure 1B shows a high-resolution scanning electron micrograph of the resulting PSi film after thermal oxidation. The PSiO2 layer is characterized by well-defined cylindrical pores with a diameter in the range of 30-80 nm.
The monoclonal antibody (IgG) molecules are grafted onto the PSiO2 surfaces by using a well-established silanization technology coupled with a...

Watch this Scientific Journal Video about Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors at JoVE.com

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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 ...

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A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...