Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Summary

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).

Abstract

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 10⁴ cells/ml) in less than an hour.

Introduction

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 "real-time" or outside the laboratory environment, biosensors are evolving to meet these challenges^2-5.

In recent years, porous Si (PSi) has emerged as a promising platform for the design of sensors and biosensors^6-20. Over the past decade numerous studies regarding PSi-based optical sensors and biosensors were published^21,22. 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 properties^10,16. The optical properties of the PSi layer, such as photoluminescence^8,11 and white light reflectance-based interferometry^7,19, 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 detection^6,8,20,23-29. In addition, most of these proof-of-concept studies have demonstrated "indirect" 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 spectrum^24-26.

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-Pérot 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.

Protocol

1. Preparation of Oxidized Porous SiO₂

1. Etch Si wafers (single side polished on the <100> face and heavily doped, p-type, 0.0008 Ω·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.
2. Rinse the surface of the resulting porous Si (PSi) film with absolute ethanol several times; dry the films under a dry nitrogen gas.
3. Oxidize the freshly-etched PSi samples in a tube furnace at 800 °C for 1 hr in ambient air (place the sample in the furnace at room temperature, heat the furnace to 800 °C, leave in furnace for 1 hr, turn off the furnace and remove the samples from the furnace only at room temperature).

2. Biofunctionalization of PSiO₂ Scaffolds

1. Incubate an oxidized PSi (PSiO₂) sample for 1 hr in mercaptopropyl(trimethoxysilane-3) 95% (MPTS) solution (108 mM in toluene).
2. Rinse the silane-treated PSiO₂ sample with toluene, methanol, and acetone; dry under a dry nitrogen gas.
3. Incubate the silane-modified PSiO₂ sample for 30 min in 1 ml of 100 mM PEO-iodoacetyl biotin solution.
4. Rinse the biotin-treated PSiO₂ sample with 0.1 M phosphate buffered saline (PBS) solution several times.
5. Incubate the biotin-modified PSiO₂ sample for 30 min in 1 ml of 100 μg/ml streptavidin (SA) solution.
6. Rinse the SA-treated PSiO₂ sample with 0.1 M PBS solution several times.
7. Incubate the resulting SA-modified PSiO₂ sample for 30 min in 1 ml of 100 μg/ml biotinylated E. coli monoclonal antibody (Immunoglobulin G, IgG) solution or with 100 μg/ml biotinylated-rabbit IgG (as a model for a monoclonal antibody).

3. Fluorescent Labeling and Fluorescence Microscopy

1. Incubate the IgG-modified surfaces with 15 μg/ml fluorescein (FITC) tagged anti rabbit IgG for 40 min and with 15 μg/ml fluorescein (FITC) tagged anti mouse IgG as a control.
2. Rinse the modified samples with 0.1 M PBS solution several times.
3. Examine the samples under a fluorescence microscope.

4. Bacteria Culture

1. Cultivate E. coli K12 bacteria in a 10 ml tube with 5 ml of Luria Bertani (LB) medium (medium composition in 1 L of deionized water: 5 g of NaCl, 5 g of yeast extract, and 10 g of tryptone). Incubate the bacteria overnight shaking at 37 °C.
2. Monitor the bacteria concentration by reading the optical density (OD) at a wavelength of 600 nm. After overnight growth in LB medium, read the OD₆₀₀ using a spectrophotometer to determine the bacterial concentration. The number of cells is directly proportional to the OD₆₀₀ measurements (1 OD₆₀₀ = 10⁸ cells/ml).

5. Bacteria Sensing

1. Place the IgG-modified PSiO₂ and neat PSiO₂ (as the control) samples in a custom-made Plexiglas flow cell. Fix the flow cell to ensure that the sample reflectivity is measured at the same spot during all the measurements.
2. Incubate the samples with E. coli K12 suspension (10⁴ cell/ml) for 30 min at room temperature. Then remove the bacteria suspension by flushing the cell with 0.85% w/v saline solution for 30 min.
3. Monitor the changes in the reflectivity data throughout the experiment. All optical measurements need to be carried out in an aqueous surrounding. Spectra should be collected using a CCD spectrometer and analyzed by applying fast Fourier transform (FFT), as previously described^25,26 .
4. Confirm the presence of the bacteria on the biosensor surface, by observation of the samples under an upright light microscope immediately after the biosensing experiment.

Results

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...

Discussion

A label-free optical immunosensor, based on a PSiO₂ nanostructure (a Fabry-Pérot 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...

Materials

Name	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