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 nitrogen gas.</li>
	<li>Oxidize the freshly-etched PSi samples in a tube furnace at 800 &#176;C for 1 hr in ambient air (place the sample in the furnace at room temperature, heat the furnace to 800 &#176;C, leave in furnace for 1 hr, turn off the furnace and remove the samples from the furnace only at room temperature).</li>
</ol>
2. Biofunctionalization of PSiO2 Scaffolds
<ol>
	<li>Incubate an oxidized PSi (PSiO2) sample for 1 hr in mercaptopropyl(trimethoxysilane-3) 95% (MPTS) solution (108 mM in toluene).</li>
	<li>Rinse the silane-treated PSiO2 sample with toluene, methanol, and acetone; dry under a dry nitrogen gas.</li>
	<li>Incubate the silane-modified PSiO2 sample for 30 min in 1 ml of 100 mM PEO-iodoacetyl biotin solution.</li>
	<li>Rinse the biotin-treated PSiO2 sample with 0.1 M phosphate&#160;buffered saline (PBS) solution several times.</li>
	<li>Incubate the biotin-modified PSiO2 sample for 30 min in 1 ml of 100 &#956;g/ml streptavidin (SA) solution.</li>
	<li>Rinse the SA-treated PSiO2 sample with 0.1 M PBS solution several times.</li>
	<li>Incubate the resulting SA-modified PSiO2 sample for 30 min in 1 ml of 100 &#956;g/ml biotinylated E. coli monoclonal antibody (Immunoglobulin G, IgG) solution or with 100 &#956;g/ml biotinylated-rabbit IgG (as a model for a monoclonal antibody).</li>
</ol>
3. Fluorescent Labeling and Fluorescence Microscopy
<ol>
	<li>Incubate the IgG-modified surfaces with 15 &#956;g/ml fluorescein (FITC) tagged anti rabbit IgG for 40 min and with 15 &#956;g/ml fluorescein (FITC) tagged anti mouse IgG as a control.</li>
	<li>Rinse the modified samples with 0.1 M PBS solution several times.</li>
	<li>Examine the samples under a fluorescence microscope.</li>
</ol>
4. Bacteria Culture
<ol>
	<li>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 &#176;C.</li>
	<li>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).</li>
</ol>
5. Bacteria Sensing
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 described25,26 .</li>
	<li>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.</li>
</ol>

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The authors declare no competing financial interests.

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 biosensor sensitivity 31,32. To minimize this issue, conjugation of the antibodies to the PSiO2 surface is accomplished through biotin-SA bridges. SA and biotin are commonly used in many biotechnological applications due to their high binding affinity (Kd ~10-13 M). The biotin-SA link is one of the most common chemistries for receptor immobilization practiced in biosensor development10,32,33,34. The accessibility of the immobilized antibodies can be regulated by the biotin-SA reaction that provides a &#64258;exible linkage between the solid surface and the IgGs31,35. Thus, the PSiO2 is first biofunctionalized with biotin/SA to allow the conjugation of biotinylated antibodies onto the nanostructure.
Limitations of the technique
The main limitations of this biosensing scheme are attributed to the use antibodies as the bacteria capture element. As is with other immunosensors, we are limited to the detection of targets that have available antibodies, the specificity of the used antibodies and their stability36. The type of antibodies to be used depends on the specific application and whenever possible monoclonal antibodies are preferred. Yet, despite the high degree of specificity that monoclonal antibodies provide, the detection of whole bacteria cells below 1,000 cells/ml is still a challenge35. Additionally, the high cost involved in the production of these antibodies should be taken into consideration.
Another limitation is associated with chemical stability of the oxidized PSi transducer in corrosive/aqueous environments. Although, the PSiO2 stability is superior in comparison to PSi thin films, still when conducting long (several hours) flow experiments baseline drifts may be observed14,15,21,24. Moreover, it is favored to work around neutral pH (in the range of 6-8) in order to prevent mechanical instability of the nanostructure.
Of course that when dealing with real food or water samples, which are contaminated with bacteria, an appropriate pretreatment process has to be considered prior to the sensing step. The pretreatment should be designed in accordance to the specific characteristics of studied sample. For example, preparation procedures such as dispersion, filtration, pH balance, etc., could be considered. However, these pretreatment techniques are beyond the scope of this work.
Significance with respect to existing methods
To date, detection and identification of bacterial contaminations rely mainly on classical microbiology culturing techniques or on rapid techniques in microbiology, such as biochemical kits, ELISA (enzyme-linked immunosorbent assay), and PCR (polymerase chain reaction) assays2,3. These methods are laborious, time-consuming and require several days to obtain results, thus &#39;&#39;real-time&#39;&#39; assessment of water and food safety remain unfeasible2,4,5. In the present work, we demonstrate the capability of PSi-based biosensors to overcome the drawbacks of these techniques. The present biosensor allows for a simple and relatively sensitive detection of bacteria via a &#39;&#39;direct cell capture&#39;&#39; approach, by monitoring changes in its optical interference spectrum. Our preliminary results display a low detection limit of 104 cell/ml E. coli and a rapid response time of several minutes. For comparison, the detection limit of current state-of-the-art surface Plasmon resonance (SPR) biosensors is in the range of 102-106 cell/ml37-40. In terms of response time, our biosensors response to bacteria exposure is comparable to that of SPR techniques. Thus, the major strengths of this biosensing scheme are the simplicity of the assay and rapid detection allowing for &#34;real-time&#34; identification of the target bacteria.
Future applications
Several approaches are currently being explored to improve the detection limit and sensitivity of the biosensor, including optimization of the antibody concentration and orientation, use antibody fragments instead of whole antibodies. Additionally, we are studying the potential of aptamers as alternative capture probes. In conclusion, the work presented here provides a generic biosensing platform that can be translated to many biosensing applications of a variety of microorganisms.
Critical steps within the protocol
In order to achieve optimal performance of the biosensor, it is highly important to confirm the biofunctionalization of the PSiO2 scaffolds (see section 3 in the protocol text, Fluorescent Labeling and Fluorescence Microscopy). The fluorescence studies allow us to confirm the attachment of the antibodies to the transducer (PSiO2) surface and to characterize the activity and antigenic specificity of the attached antibodies, by binding of fluorescently tagged anti-rabbit IgG and anti-mouse IgG as a control.
Moreover, in order to eliminate false results in the optical readout, it is essential to properly rinse the surface of the biosensor following exposure to the target bacteria (for the removal of bacteria cells that are nonspecifically bound or reside on the surface) and to conduct control experiments with neat PSiO2. The importance of the control experiment is for the elimination of unspecific bacteria adsorption on the biosensor surface (see section 5 in the protocol text, Bacteria Sensing).

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 biotin-SA system. The detailed synthesis scheme is outlined in Figure 2. First, the thermally oxidized surface is silanized by MPTS, resulting in a thiol-silanized surface (Figure 2c). In the following step, the activated surface is incubated with a SH-reactive biotin linker molecules (Figure 2d), followed by attachment of the SA proteins to the biotin-modified surface via biotin-SA bridges 30 (Figure 2e). The final step in the functionalization of the biosensor surface involves the bioconjugation of biotinylated monoclonal antibodies to the SA (Figure 2f).
The attachment of the antibodies to the PSiO2 surface is confirmed by fluorescent labeling followed by observation of the surface under a fluorescence microscope. In addition, fluorescence studies allow us to characterize the activity and antigenic specificity of the surface-immobilized antibodies (rabbit IgG) by binding of fluorescently tagged anti-rabbit IgG and anti-mouse IgG as a control. Figure 3 summarizes the results of these experiments.
In order to demonstrate bacteria biosensing we have used a specific E. coli IgG instead of the model IgG (rabbit IgG). Again, the approach is to monitor changes in the amplitude (intensity) of the light reflected from the PSiO2 nanostructure. During the sensing experiments, the biosensors are fixed in a flow cell in order to assure that the sample reflectivity is measured at the same spot during all measurements. The entire experiment is carried out in wet environment and the sample reflectivity spectrum is continuously monitored. The biosensors are exposed to E. coli K12 suspension (104 cells/ml) for 30 min, after which a buffer solution is used for washing the biosensor surface (for the removal of unattached bacteria). A FFT spectrum of the biosensor before and after the introduction of the E. coli bacteria (104 cells/ml) is shown in Figure 4a (top). Thus, following exposure to E. coli bacteria (and subsequent rinsing step) an intensity decrease of 7&#177;1% is recorded, while insignificant changes (less than 0.5% decrease in the FFT intensity) are observed for the unmodified PSiO2 surface (Figure 4b, top). Moreover, in order to confirm the presence of captured cells onto the PSiO2 surface, the biosensors are observed under a light microscope immediately after the completion of the biosensing experiment. Figure 4a (bottom) displays immobilized bacteria cells on the biosensor surface, while no cells are observed on the unmodified surfaces (control); see Figure 4b (bottom).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50805/50805fig1highres.jpg" src="/files/ftp_upload/50805/50805fig1.jpg" /> 
Figure 1. (A) Typical reflectivity spectrum of a typical Fabry-P&#233;rot PSiO2 nanostructure (left) and the corresponding spectrum following a fast Fourier transform (right). The position and magnitude of the resulting single peak are monitored. (B) A cross-sectional high-resolution scanning electron micrograph (secondary electrons) of a PSiO2 layer (etched for 30 sEC at 385 mA/cm2).
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50805/50805fig2highres.jpg" src="/files/ftp_upload/50805/50805fig2.jpg" /> 
Figure 2. Schematic representation of the synthesis steps followed to biofunctionalize the PSiO2 surface with antibodies. (a) An anodic electrochemical etch is used to prepare a PSi layer from a single-crystal Si wafer. (b) The freshly-etched sample is then thermally oxidized at 800 &#176;C. (c) The PSiO2 surface is initially reacted with MPTS to create free thiol group surface (activated PSiO2 film). (d) Incubation of the activated film with PEO-iodoacetyl biotin to generate biotinylated surface. (e) Incubation of the biotinylated surface with SA. (f) Incubation of the modified surface with biotinylated antibodies. <a href="https://www.jove.com/files/ftp_upload/50805/50805fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/50805/50805fig3.jpg" /> 
Figure 3. Results of fluorescence labeling experiments to confirm the activity and antigenic specificity of the immobilized IgG. The samples are observed under a fluorescent microscope at a constant exposure time. (A) Complete bioconjugation, PSiO2 + MPTS + biotin + SA + biotinylated-rabbit-IgG and FITC-anti rabbit IgG; (B) control experiment with FITC-anti mouse IgG; (C) control experiment, no IgG, PSiO2 + MPTS + biotin + SA and FITC-anti rabbit IgG.
Note: These schematics are for illustration purposes only as conjugation of IgG also occurs inside the pores.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50805/50805fig4highres.jpg" src="/files/ftp_upload/50805/50805fig4.jpg" /> 
Figure 4. E. coli biosensing experiments and corresponding optical microscope images of the biosensors immediately after the experiments. The biosensors are incubated with 104 cells/ml E. coli suspensions. Key: (a) IgG-modified PSiO2, (b) neat (unmodified) PSiO2.

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 ...

optical-detection-of-e-coli-bacteria-by-mesoporous-silicon-biosensors

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

16.8K Views.  Technion - Israel Institute of Technology. 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...

Video: Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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 skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

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

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

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) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

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 antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

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) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

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

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

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 membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

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 increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

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 studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

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 multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

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

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.