The authors gratefully acknowledge Prof. Andrea Armani at the University of Southern California for support during the time this protocol was developed. Funding for the initial development of this work was provided by the National Science Foundation [085281 and 1028440] and the National Institute of Health through NIH Director's New Innovator Award Program [1DP2OD007391-01]. Additional information is available at <a href="http://web.missouri.edu/~hunthk/" target="_blank">http://web.missouri.edu/~hunthk/</a>.

1. Background
The biotin is attached to the surface of these devices through a simple, three-step process (Figure 1). First, we clean the surface and populate it with hydroxyl groups by exposing the devices to either oxygen plasma or piranha solution. Second, we use vapor deposition to attach the silane coupling agent terminated with a primary amine to the hydroxyl groups through hydrolysis and condensation reaction. Third, we attach biotin to the surface via N-hydroxysuccinimide (NHS) ester chemistry. We direct the interested reader to our previous work for more information about the development of these techniques, as well as an explanation of our motivation for choosing these techniques.8
The success of these reactions can be evaluated through optical and fluorescence microscopy after the addition of the primary amine, as well as after the addition of the biotin. While optical microscopy can be used to determine if the surface functionalization protocols resulted in damage to the microsphere surface, or even contamination, fluorescence microscopy is used to verify the quality and uniformity of surface coverage of the biotin molecule, as well as the capability of the biotin on the surface to bind with (strept)avidin. To evaluate the coverage of amines on the surface, we used fluorescein isothiocyanate (FITC) fluorescent dye, which reacts with primary amines to form stable, covalent thiourea linkage to the microsphere. Texas Red fluorescent dye that has been conjugated to avidin is used to label the biotin groups on the surface through the biotin-avidin interaction. In both cases, dyes were chosen that can interact (through either receptor-ligand interactions or covalent bonding) with the functional groups on the surface.
Here, the silane coupling agent, which has three leaving groups and one functional group, provides the bridge between the inorganic surface and organic probe molecule. The primary amine functionality reacts quantitatively with NHS esters to form stable amide bonds. In this case, we use a biotin probe molecule whose valeric side chain has been modified with an NHS ester group. Realistically, any probe molecule to which an NHS ester group can be added to the surface using the following protocols. Additionally, these protocols are general, and can be used to functionalize any silica device surface with biotin.
The primary challenge with these protocols is not actually in the chemistry itself, but rather in the handling of the silica microspheres. Please note that, throughout the protocols, the microspheres should be handled by grapsing their stems lightly with sharp-tipped tweezers. This keeps the tweezers well away from the microspheres themselves, and allows for easy transport between steps. Many steps in the protocol below are specifically designed to address this factor.
2. Microsphere Fabrication
<ol>
	<li>Build the storage housing for the microspheres.
	<ol>
		<li>Using an exacto knife, cut a &frac14; in thick piece of cardboard into a 1 in x 1 in square; tape this onto a regular-sized glass slide, and attach a 1 in section of scotch tape, with its ends meeting to form a roll, to the cardboard.</li>
		<li>This creates a platform on which the microsphere can be elevated and isolated from the surrounding environment, and prevents damage to their surface.</li>
	</ol>
	</li>
	<li>Using scissors, cut a 3 inch section of optical fiber from a spool of optical fiber.</li>
	<li>Using a No-Nik fiber-stripper, strip the protective polymeric coating from the last 0.5 inches of the end of the cut piece of fiber, leaving just the silica core. Clean the surface of any remaining polymer with a Kimwipe dampened with methanol by gently wiping the fiber with the Kimwipe.</li>
	<li>Using a bare fiber cleaver, trim the stripped end so that only about 1 millimeter of stripped fiber remains on the end of the fiber.</li>
	<li>Place the stripped end of the optical fiber into the path of a CO2 laser, taking care to vertically align the fiber such that its stripped end is facing down.</li>
	<li>Turn on the CO2 laser, and direct it to the surface of the stripped end of the optical fiber. The laser will melt the stripped end of the fiber into a sphere using approximately 3.5% power in 2 seconds.</li>
	<li>Using tweezers, carefully grasp the stem (the non-stripped portion of the optical fiber) of the microsphere. Attach the stem to the tape roll on the microsphere housing. The glass slide may be itself stored in a petri dish. This allows for safe storage of the microsphere.</li>
</ol>
3. Populating the Surface with Hydroxyl Groups
<ol>
	<li>Using piranha solution:
	<ol>
		<li>In a 60 mL polypropylene vial with a hinged cap, prepare a piranha solution (70:30 by volume fuming H2SO4:H2O2 (30 wt%)) by adding 5 ml of hydrogen peroxide to the vial, followed by 11.6 mL of fuming sulfuric acid. CAUTION: wear acid-resistant gloves.</li>
		<li>Transfer the glass slide, holding at least 1 microsphere, to the vial. The microsphere should be in contact with the liquid, but the liquid should not touch the cardboard. Adjust the volume of the solution if needed.</li>
		<li>Gently remove the glass slide from the vial, and insert it into another plastic vial containing DDI H2O. Let the samples sit in the solution for 5 minutes.</li>
		<li>Gently remove the glass slide from the vial, and place it an oven at 80 &deg;C for 10 minutes to dry the surface.</li>
	</ol>
	</li>
	<li>Using oxygen plasma treatment:
	<ol>
		<li>Transfer the glass slide containing at least one microsphere to the oxygen plasma chamber.</li>
		<li>Set the oxygen pressure to 200 mTorr, and the power to 120 W. Expose the sample to the oxygen plasma for 2 minutes.</li>
		<li>Remove the glass slide from the plasma chamber.</li>
	</ol>
	</li>
</ol>
4. Attaching Silane Coupling Agents to the Surface
<ol>
	<li>Place the glass slide, containing at least one microsphere, into a vacuum desiccator in a fume hood.</li>
	<li>Also in the fume hood, open the silane coupling agent bottle (in this case, aminopropyltrimethoxysilane (APTMS) was used), and place the opened bottle in the desiccator.</li>
	<li>Replace the lid on the vacuum desiccator, and attach the outlet port to an aspirator or house vacuum line.</li>
	<li>Turn on the house vacuum or the water line, and evacuate the vacuum desiccator. Once a vacuum seal is formed between the lid and the base, begin timing the reaction. This will deposit the silane coupling agent onto the surface as a thin film.</li>
	<li>After 15 minutes, turn off the vacuum, and slowly open the port to let air into the desiccator. For this coupling agent, 15 minutes is sufficient to form a uniform monolayer on the surface. For other coupling agents, the time may need to be adjusted.</li>
</ol>
5. Attaching Biotin to the Surface
<ol>
	<li>Approximately one hour before attaching the biotin, prepare a 10 mM solution of N-hydroxylsuccinimide biotin (NHS-biotin) in anhydrous dimethylsulfoxide (DMSO) in a 60 mL polypropylene vial with a hinged cap.
	<ol>
		<li>If the NHS-biotin has been stored cold, allow it to equilibrate to room temperature before massing it out.</li>
		<li>Sonicate the solution for 1 hour to fully dissolve the NHS-biotin powder in the solvent.</li>
	</ol>
	</li>
	<li>Transfer the glass slide containing the microsphere into another plastic vial, with the microspheres at the bottom, and stems at the top of the vial.</li>
	<li>Transfer, using a plastic pipet, an appropriate volume of the NHS-biotin solution down the side of the vial, behind the glass slide (so the solution does not touch the microsphere as it is being added to the vial). Add enough solution to cover the microsphere surface.</li>
	<li>Place the vials now containing the microspheres and NHS-biotin in DMSO solution onto a rocking incubator (tilt tray) for 30 minutes at room temperature, with the speed and angle of tilt at 5 rpm and 5 degrees, respectively.</li>
	<li>Gently remove the glass slide from the vial, and gently slide it into another vial filled with DDI H2O. Place the vial on the tilt tray for another 10 minutes at the same speed and tilt angle as before. Repeat this step twice with fresh water each time. This helps remove excess DMSO from the surface, and removes any physically-adsorbed biotin that did not actually graft to the surface.</li>
	<li>Remove the glass slide from the vial, and place it in an oven at 80 &deg;C for 10 minutes, or until all water droplets are removed from the surface.</li>
</ol>
6. Fluorescent labeling of amine-terminated silica
<ol>
	<li>To label the amine groups present after treatment with APTMS, prepare the FITC solution in a darkened room by dissolving 1 mg FITC in 1 mL anhydrous DMSO.</li>
	<li>Dilute the solution in 6.1 by adding 50 &mu;L of the FITC solution to 1 mL of 0.1 M sodium bicarbonate buffer.</li>
	<li>Place the solution in a 60 mL polypropylene vial with a hinged cap, and gently slide the amine-terminated microspheres, housed again on the glass slide, into the vial.</li>
	<li>Place the vial in an ice bath, and let the sample react with the FITC solution for a minimum of 4 hours in the dark.</li>
	<li>Remove excess fluorophore through two, 10 minute rinses of the microspheres in sodium carbonate buffer. As before, fill a 60 mL polypropylene vial with a hinged cap with the buffer, and gently slide the glass slide into the solution, taking care that the solution only covers the microsphere, and not the cardboard housing. Cover the vial with aluminum foil, and place the vial on a tilt tray set at 5 degrees and 5 rpm, as before.</li>
	<li>Gently remove the glass slide from the vial, and gently slide it into another vial filled with DDI H2O and covered with aluminum foil. Place the vial on the tilt tray for another 10 minutes at the same speed and tilt angle as before. Repeat this step twice with fresh water each time. This helps remove excess dye from the surface.</li>
	<li>Gently remove the glass slide from the vial, and dry in an oven set at 80 C for 10 minutes before imaging.</li>
</ol>
7. Fluorescent labeling of biotin-terminated silica
<ol>
	<li>Prepare a 10 &mu;g/mL solution of Texas-Red avidin in phosphate buffered saline.</li>
	<li>Add the solution to a 60 mL polypropylene vial with a hinged cap, and gently slide the glass slide containing a biotin-terminated microsphere into the solution, so that the microsphere is just covered by the solution.</li>
	<li>React for 30 minutes at room temperature in the dark.</li>
	<li>Remove excess fluorophore through two, 10 minute rinses of the microspheres in PBS buffer.
	<ol>
		<li>As before, fill a 60 mL polypropylene vial with a hinged cap with the buffer, and gently slide the glass slide into the solution, taking care that the solution only covers the microsphere, and not the cardboard housing.</li>
		<li>Cover the vial with aluminum foil, and place the vial on a tilt tray set at 5 degrees and 5 rpm, as before.</li>
	</ol>
	</li>
	<li>Gently remove the glass slide from the vial, and gently slide it into another vial filled with DDI H2O and covered with aluminum foil. Place the vial on the tilt tray for another 10 minutes at the same speed and tilt angle as before. Repeat this step twice with fresh water each time. This helps remove excess dye from the surface.</li>
	<li>Gently remove the glass slide from the vial, and dry in an oven set at 80 &deg;C for 10 minutes before imaging.</li>
</ol>
8. Representative Results
Correctly functionalized microspheres can be identified through optical and fluorescence microscopy. If the surface functionalization is done correctly, it should result in a uniformly dense coverage of biotin molecules on the surface, and the surface should remain defect- and contaminant-free after functionalization in order to maintain their high sensitivities during detection experiments. Optical microscopy can be used to probe the latter, while fluorescent microscopy can probe the quality and uniformity of surface coverage. In Figure 2, we show examples of correctly functionalized microspheres. These images show that there is no surface damage or contamination due to functionalization (Fig. 2a), and that the microspheres show a uniform, consistent coverage of either amine groups (Fig. 2b) or biotin groups (Fig. 2c) on the surface.
If the microspheres have not been functionalized correctly, the optical microsphere images will exhibit surface contamination, clumping or non-uniform coverage, and obvious defects in the surface, like surface cracks (Figure 3). Here, we see a common example of surface contamination, resulting from clumping of reagents on the surface.
<img alt="Figure 1" src="/files/ftp_upload/3866/3866fig1.jpg" /> 
Figure 1. 3-step reaction scheme for attaching probe molecules to the surface of silica microspheres. <a href="https://www.jove.com/files/ftp_upload/3866/3866fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3866/3866fig2.jpg" /> 
Figure 2. Silica microspheres. a) Optical micrograph of silica microsphere populated with hydroxyl groups via exposure to oxygen plasma; b) Fluorescent micrograph of silica microsphere populated with primary amines and labeled with FITC dye; c) Fluorescent micrograph of silica microsphere populated with biotin and labeled with Texas Red-avidin conjugate. Reprinted with permission from Soteropulos, C. E., Hunt, H. K. &amp; Armani, A. M. Determination of binding kinetics using whispering gallery mode microcavities. Appl. Phys. Lett. 99, 103703-103703 (2011). Copyright 2011, American Institute of Physics.17
<img alt="Figure 3" src="/files/ftp_upload/3866/3866fig3.jpg" /> 
Figure 3. Optical micrograph of improperly functionalized sphere. Here, you can see dust on the top right surface, as well as contamination extending off the surface. Additionally, the right side of the optical microsphere shows a small divot on the surface.

<ol>
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No conflicts of interest declared.

As described in the protocols, we created a housing platform by which to transport the silica microspheres by their stems throughout the functionalization process. This housing platform was created as a solution to the surface contamination and damage that resulted from the microsphere coming into contact with the walls of the various containers used throughout the functionalization process. We realized the main difficulty arose from constantly attaching and detaching individual microspheres to different containers durin...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>67-56-1</td>
			<td>ACS grade</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>Fisher</td>
			<td>8014-95-7</td>
			<td>Fuming</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Fisher</td>
			<td>7722-84-1</td>
			<td>30 wt %</td>
		</tr>
		<tr>
			<td>Aminopropyltrimethoxysilane</td>
			<td>Fisher</td>
			<td>13822-56-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NHS-biotin EZ linker</td>
			<td>Pierce</td>
			<td>20217</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Fisher</td>
			<td>67-68-5</td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Fluorescein Isothiocyanate</td>
			<td>Pierce</td>
			<td>46425</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher</td>
			<td>7647-14-5</td>
			<td>Powder concentrate</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate Buffer</td>
			<td>Fisher</td>
			<td>NC0099321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Texas Red - Avidin Conjugate</td>
			<td>Pierce</td>
			<td>A820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optical Fiber</td>
			<td>Newport</td>
			<td>F-SC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Stripper</td>
			<td>Fiber Instrument Sales</td>
			<td>NN-175</td>
			<td>No-Nik 175 um stripper</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher</td>
			<td>06666A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bare Fiber Cleaver</td>
			<td>Ilsintech</td>
			<td>Cl-03A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene Vials</td>
			<td>Fisher</td>
			<td>03-341-75A</td>
			<td>60 mL, hinged cap</td>
		</tr>
		<tr>
			<td>Incubating Rocker</td>
			<td>VWR</td>
			<td>12620-910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>Fisher</td>
			<td>08-594-15B</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents at JoVE.com

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

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) ...

attaching-biological-probes-to-silica-optical-biosensors-using-silane

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JoVE Journal

Bioengineering

12.8K vistas.  University of Missouri. Biosensores interactuar con entornos complejos, biológicos y realizar una detección selectiva mediante la combinación de sensores altamente sensibles con sondas muy específicas que figuran en el sensor a través de modificación de la superficie. Aquí, se demuestra la funcionalización de la superficie de los sensores ópticos de sílice con biotina utilizando agentes de acoplamiento de silano para reducir el sensor y el entorno biológico.

Video: Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

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.

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).

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 ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

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 ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

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 ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.