This work has been supported by Western Kentucky University&#8217;s Ogden College of Science and Engineering, including internal support from the Department of Chemistry and from the Office of Research (RCAP 13-8032). The assistance of Dr. John Andersland at the WKU Microscopy Facility (SEM images) and Associate Professor Yan Cao of the WKU Institute for Combustion Science and Engineering (BET analysis) has been central to conducting this work.

1. Preparation of Emulsion
<ol>
	<li>Emulsion Contents
	<ol>
		<li>Mass an appropriate amount of salt to produce 10 ml of 0.03-M solution. For platinum(IV) chloride measure 0.101 g, for zinc(II) chloride (ZnCl2) measure 0.032 g, and sodium chloride (NaCl) measure 0.018 g.</li>
		<li>In individual test tubes, dissolve each salt into 10 ml of DI water. Set aside for later use.</li>
		<li>Use a 20-ml, sealable glass vial for the contents throughout this procedure. Tare a balance to the glass vial.</li>
		<li>Weigh vinyl-terminated polydimethylsiloxane by slowly pouring it over a stir rod and into the glass vial resting on the zeroed scale. Weigh out 1.02 g (equivalent to 1.080 ml). 
		NOTE: The high viscosity of this polymer makes pipetting impractical.</li>
		<li>Pipette 1.02 ml of n-Heptane to the vial. Add 2 drops of non-ionic surfactant (sorbitan monoleate) to the vial. Pipette 0.3 ml of salt solution and 0.45 ml of DI-water to the vial.</li>
		<li>Seal the glass vial by screwing the lid on tightly. Shake vigorously for 60 sec to initiate the emulsion before beginning sonication.</li>
	</ol>
	</li>
	<li>Construction of Water-bath Sonicator Apparatus
	<ol>
		<li>Fill sonicator with water up to the minimum fill line. Add 250 ml of tap water to a 400-ml beaker. Fill the 400-ml beaker with ice so that the water level is just at the rim.</li>
		<li>Place this beaker inside the water-bath sonicator. Check the fill-line on the sonicator, adjusting if needed. Place a ring stand directly beside the water-bath sonicator.</li>
		<li>Using two ring stand clamps, position them so that an arm is extended out, perpendicular to the ring stand and another one is extended towards the water bath so that it is pointing downwards into the beaker filled with ice water. Attach another clamp to the ring stand with a thermometer down in the 400-ml beaker so that the temperature can be monitored throughout sonication.</li>
	</ol>
	</li>
	<li>Emulsification Procedure
	<ol>
		<li>Place the emulsion-containing vial so that it is fully submerged in the 400-ml beaker by securing it into the clamp protruding down into the beaker.</li>
		<li>Ensure that the glass vial containing the PDMS mixture is not touching the sides of the beaker to eliminate heat caused by friction.</li>
		<li>Turn on the sonicator and set sonication time for 7 min. Because the emulsion is very heat sensitive, ensure that the temperature inside the beaker is between 0 and 5 &#176;C throughout sonication. Start sonication once the temperature inside the beaker is desirable.</li>
		<li>After 7 min of sonication, remove the vial and gently shake/swirl for 1 min, holding the top of the vial to eliminate any clumps that might form in the emulsion.</li>
		<li>Dispose of the contents of the beaker. Refill with 250 ml of water and add ice to the fill to within 1 cm the top of the beaker.</li>
		<li>Place the vial back in the clamp. Submerge it under the ice water to resume sonication for 7 min.</li>
		<li>Repeat steps 1.3.3 to 1.3.6 for a total of eight, 7-min sonication periods, or until the sonication appears to be homogenous and no clumps are present. Store at RT. 
		NOTE: The emulsification should be stable for several days, but can be recovered by sonication as above in 7-min intervals until it appears homogenous. Store at RT.</li>
	</ol>
	</li>
</ol>
2. Cross-linking
<ol>
	<li>Setup for Addition of Triethoxysilane/Surfactant Solution
	<ol>
		<li>Pipette 5.4 ml triethoxysilane into a test tube. Place in a test tube rack under the hood for later use.</li>
		<li>Fill a 400-ml beaker with ice water and place it under the hood. Next to the 400-ml beaker place a ring stand with a clamp attached, extended directly over the opening of the beaker. This will be the ice-bath for the addition of triethoxysilane.</li>
		<li>Place a hot plate on the other side of the ring stand. Fill an 800-ml beaker with approximately 700 ml of tap water. Place it on the hot plate.</li>
		<li>Turn on the hot plate and maintain a temperature of 75 to 85 &#176;C inside the 800-ml beaker. Attach a clamp with a thermometer to the ring stand so the temperature of the water inside the 800-ml beaker can be monitored.</li>
		<li>Produce the surfactant solution by dissolving 0.5 g of sodium dodecyl sulfate to 375 ml of water (4.62 mM). Add approximately 10 ml of surfactant solution to a clean, empty test tube.</li>
		<li>Attach another clamp to the ring stand under the hood with the surfactant test tube secured such that its liquid level is below the surface of the water inside the 800-ml beaker. Allow 10 min for thermal equilibration.</li>
		<li>Wet a piece of filter paper and place it in the top of a small funnel. Place the stem of the funnel inside a 250-ml Erlenmeyer flask and place the flask under the hood.</li>
	</ol>
	</li>
	<li>Addition of Triethoxysilane
	<ol>
		<li>Place the emulsion vial in the clamp over the ice-water beaker inside the hood. Position the PDMS mixture in the clamp so that the vial contents are below the surface of the water. The addition of triethoxysilane causes an exothermic reaction, so the emulsion must be kept cold in order to maintain its structure.</li>
		<li>Remove the lid from the glass vial to avoid gaseous build up.</li>
		<li>Slowly pour the test tube containing triethoxysilane into the glass vial in a continuous stream over a period of approximately 10 sec (roughly 0.5 ml/sec). 
		CAUTION: The addition of triethoxysilane initiates an exothermic reaction and the release of caustic hydrogen chloride (HCl) gas. The vial will become extremely hot and a toxic gas will evolve from the mixture. DO NOT STIR the contents while adding the triethoxysilane.</li>
		<li>After adding triethoxysilane completely, gently stir the contents with a glass stir rod while wearing a heat protective glove. Wait for 2 min or until gas stops evolving from the vial.</li>
		<li>Following cross-linking, there is no phase separation visible in the sample. If clumps are present, seal the vial and shake rigorously for 20 sec while holding the vial by the lid.</li>
	</ol>
	</li>
	<li>Bead production
	<ol>
		<li>Use a clean, glass Pasteur pipette to draw the cross-linked emulsion from the glass vial. Add the cross-linked emulsion drop-wise to the surfactant solution (which should be maintained between 75 and 85 &#176;C) into the test tube taking as little time as possible in between drops.</li>
		<li>30 sec to 1 min after the addition of the emulsion, the surfactant solution will slowly begin to evolve gas as solids begin to form inside the test tube.</li>
		<li>While wearing heat protective gloves, take the test tube out of the clamp and pour its entire contents into the filtration apparatus under the hood. Filter for 5 min. Remove the filter paper from the filter.</li>
		<li>Transfer the filtered solids onto a watch glass and separate beads for O/N drying under the hood. Cleaning of the beads can be postponed indefinitely. Store dried beads at RT in a sealed glass vial until needed, and clean immediately prior to use.</li>
	</ol>
	</li>
	<li>Bead Cleaning
	<ol>
		<li>Create another filtration apparatus under the hood and place the dried beads inside the funnel on top of the filter paper.</li>
		<li>Use a plastic wash bottle filled with DI-water to rinse the beads gently, moving them around slightly to ensure all the beads are rinsed.</li>
		<li>Let the beads dry for 1 hr by placing them on a watch glass under the hood. Use a wash bottle filled with hexanes to rinse the beads using the same method for rinsing with water.</li>
		<li>Place the beads on a watch glass. Place the watch glass and the beads under the hood to dry.</li>
		<li>After the beads are completely dry, place them in a small sealable glass vial and store at RT for future use.</li>
	</ol>
	</li>
	<li>Mounting the Beads for SEM Analysis and SEM Settings
	<ol>
		<li>Place a strip of double-sided carbon conductive tape on top of the stub onto which the beads will be mounted. Using scissors, trim around the stub to ensure no tape hangs over the edges.</li>
		<li>Place a piece of filter paper under the stub on a flat surface. Remove the top layer from the tape so that the adhesive underside is exposed.</li>
		<li>Gently pour the beads over the stub. Some beads will stick to the tape, but most will bounce off and land on the filter paper. Pour these back into the vial if they stayed on the filter paper. Repeat if necessary, washing any beads (according to 2.4.2 to 2.4.5) that become contaminated.</li>
		<li>To ensure the beads are secure on the stub, use a bulb syringe and lightly blow very closely to the stub surface. Pour more beads over the stub if only a few adhered to the tape. Ensure that all beads are secure before placing the stub into the SEM chamber and evacuating it.</li>
		<li>Once the samples have been mounted properly they are now ready to undergo SEM analysis15. Collect images in LOW VAC mode at 15 keV to optimize the resolution of the bead&#8217;s surface features.</li>
	</ol>
	</li>
</ol>

<ol>
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C., Marois, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemocompatibility,+biocompatibility,+inflammatory+and+in+vivo+studies+of+primary+reference+materials+low-density+polyethylene+and+polydimethylsiloxane:+A+review.">Hemocompatibility, biocompatibility, inflammatory and in vivo studies of primary reference materials low-density polyethylene and polydimethylsiloxane: A review.</a> J. Biomed. 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The authors have nothing to disclose.

The beads produced using this protocol (and by adjusting the electrolyte concentration and identity) are fundamentally different from those produced with a low-ionic strength emulsion, as seen by comparison of Figure 1A to the other SEM images in Figure 1. Our initial report used PtCl4 with the intention of further catalyzing the polymerization cross-linking at the aqueous-aliphatic interface14. In that report, large concave indentations were seen. Since that report...

Polydimethylsiloxane (PDMS) is one of the most widely used silicone compounds. Its biocompatibility has led to widespread use in implant and other biomedical engineering structures1,2. It is trivially cross-linked into elastic structures using an organosilyl compound (such as triethoxysilane), a simple and reliable procedure which has made it useful for cast polymer applications where some flexibility is required3. Once cross-linked, PDMS is largely inert, particularly in biological conditions, and is therefore useful for a variety of food and medical applications4,5. Ease of casting, chemical inertness, and hydrophobicity have made it a natural choice for microfluidic devices6,7. Its affinity for non-halogenated, non-polar organic compounds has made it a popular stationary phase in separations chemistry8-10. 
Recently, microbubble fabrications have been used to generate porous beads for use as catalyst structural substrates or in chemical separations11,12. In both applications, ideal materials will have a maximized surface-area-to-volume (SAV) ratio for best efficiency. In a microbubble fabrication process, microstructuring of materials is typically accomplished by isolating the polymer in aliphatic &#8220;microbubbles&#8221; by emulsification in an aqueous continuous phase. The initial report of microporous PDMS beads produced them by mechanical emulsification of two phases (aliphatic and aqueous)13. The stock PDMS liquid (and its cross-linking agent) is dissolved into the aliphatic phase, which is structured into microscopic beads by being forced to cavitate within the (continuous) aqueous phase. The emulsification is stabilized by the addition of a non-ionic surfactant. When the emulsion is added dropwise to a heated bath, solid beads form by agglomeration of the microbubbles into clusters of tiny spheres of cross-linked PDMS. Our goal in this protocol is to modify this procedure to develop beads with an inverted porosity to improve the SAV ratio of the material.
As reported previously, control of the beads can be directed to some extent by the aliphatic:aqueous ratios in the emulsion. However, we have reported recently that addition of platinum(IV) chloride (PtCl4) inverts the porosity: materials are formed in which the PDMS is riddled with concave pores14. This indicates that the aqueous layer cavitates inside the aliphatic one, despite having similar aliphatic:aqueous ratios to those published in the original work13. The primary advantage of our method is that this concave porosity should naturally result in an increased SAV ratio, and thus, improved efficiency for analytical chemistry applications. While we are continuing to explore the specific effects of the addition of the platinum compound, we show here that the same effect can be accomplished using any aqueously soluble ionic compound, though perhaps to a reduced extent. Because our techniques also differ in some key aspects from what has been previously reported, we present our protocols here as a video to encourage others to extend our methods. Most notably, we use a common bath sonicator of the type used to clean glassware or other equipment, rather than the (considerably more expensive) probe sonicator often used in microbubble fabrication. This adjusted approach to the microbubble fabrication procedure could potentially be extended for the production of large quantities of bulk materials as well, creating porous sheets or slabs which could have applications for biomedical devices, aerospace and automotive industry, or substrates for chemical catalysis. Users seeking to generate high-SAV-ratio, microstructured materials using other similar polymers for such analyses may find that our protocols can be extended to any polymer for which the microbubble emulsion technique can be applied.

<table border="0">
	<tbody>
		<tr>
			<td>Poly(dimethylsiloxane), vinyl terminated</td>
			<td>Sigma-Aldrich</td>
			<td>68083-19-2</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Heptane</td>
			<td>Sigma-Aldrich</td>
			<td>142-82-5</td>
			<td>Flammable</td>
		</tr>
		<tr>
			<td>Triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>998-30-1</td>
			<td>Flammable, Accutely Toxic</td>
		</tr>
		<tr>
			<td>Sorbitan Monoleate (Span-80)</td>
			<td>Fluker</td>
			<td>1338-43-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum (IV) Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>13454-96-1</td>
			<td>Accutely Toxic</td>
		</tr>
		<tr>
			<td>Zinc (II) Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7646-85-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>2.8L Water Bath Sonicator</td>
			<td>VWR</td>
			<td>97043-964</td>
			<td></td>
		</tr>
	</tbody>
</table>

microbubble fabrication of concave-porosity pdms beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Representative SEM images of beads arising from emulsions with different electrolyte conditions are shown in Figure 1. Figure 1A shows a bead similar to those obtained by DuFaud, et al.13, produced using our procedures, without the addition of any electrolyte. Beads shown in Figure 1B-D, resulting in different morphologies for each metal ion. For all images shown, 300 &#956;l of 0.03-M electrolyte solutions were used in place of 300 &#956;l of the DI ...

Watch this Scientific Journal Video about Microbubble Fabrication of Concave-porosity PDMS Beads at JoVE.com

Microbubble Fabrication of Concave-porosity PDMS Beads

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

microbubble-fabrication-of-concave-porosity-pdms-beads

Research

JoVE Journal

Chemistry

8.2K vistas.  Western Kentucky University. Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized. 

Video: Microbubble Fabrication of Concave-porosity PDMS Beads

Fabrication of Large-area Free-standing Ultrathin Polymer Films

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films. Typically, ultrathin films are prepared using either sacrificial layers, which may damage the film or affect its mechanical properties, or they are made on freshly cleaved mica, a substrate that is difficult to scale. Further, the size of ultrathin film is typically limited to a few square millimeters. In this method, we modify a surface with a polyelectrolyte that alters the strength of adhesion between polymer and deposition substrate. The polyelectrolyte can be shown to remain on the wafer using spectroscopy, and a treated wafer can be used to produce multiple films, indicating that at best minimal amounts of the polyelectrolyte are added to the film. The process has thus far been shown to be limited in scalability only by the size of the coating equipment, and is expected to be readily scalable to industrial processes. In this study, the protocol for making the solutions, preparing the deposition surface, and producing the films is described.

We describe a method for the fabrication of large-area (up to 13 cm diameter) and ultrathin (as thin as 8 nm) polymer films. Instead of using a sacrificial interlayer to delaminate the film from its substrate, we use a self-limiting surface treatment suitable for arbitrarily large areas.

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films.  Typically, ultrathin films are prepared ...

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Influence of Hybrid Perovskite Fabrication Methods on Film Formation, Electronic Structure, and Solar Cell Performance

Hybrid organic/inorganic halide perovskites have lately been a topic of great interest in the field of solar cell applications, with the potential to achieve device efficiencies exceeding other thin film device technologies. Yet, large variations in device efficiency and basic physical properties are reported. This is due to unintentional variations during film processing, which have not been sufficiently investigated so far. We therefore conducted an extensive study of the morphology and electronic structure of a large number of CH3NH3PbI3 perovskite where we show how the preparation method as well as the mixing ratio of educts methylammonium iodide and lead(II) iodide impact properties like film formation, crystal structure, density of states, energy levels, and ultimately the solar cell performance.

We present an extensive study on the effects of different fabrication methods for organic/inorganic perovskite thin films by comparing crystal structures, density of states, energy levels, and ultimately the solar cell performance.

Hybrid organic/inorganic halide perovskites have lately been a topic of great interest in the field of solar cell applications, with the potential to ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

Fabrication of Thin Film Silver/Silver Chloride Electrodes with Finely Controlled Single Layer Silver Chloride

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin film silver electrodes. Thin film silver electrodes sized 80 &#181;m x 80 &#181;m and 160 &#181;m x 160 &#181;m were sputtered on quartz wafers with a chromium/gold (Cr/Au) layer for adhesion. After passivation, polishing and cathodic cleaning processes, the electrodes underwent galvanostatic oxidation with consideration of Faraday&#39;s Law of Electrolysis to form smooth layers of AgCl with a designated degree of coverage on top of the silver electrode. This protocol is validated by inspection of scanning electron microscope (SEM) images of the surface of the fabricated Ag/AgCl thin film electrodes, which highlights the functionality and performance of the protocol. Sub-optimally fabricated electrodes are fabricated as well for comparison. This protocol can be widely used to fabricate Ag/AgCl electrodes with specific impedance requirements (e.g., probing electrodes for impedance sensing applications like impedance flow cytometry and interdigitated electrode arrays).

This paper aims to present a method to form smooth and well-controlled films of silver chloride (AgCl) with designated coverage on top of thin film silver electrodes.

This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin ...

Porphyrin-Modified Beads in Flow Cytometry

Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. 
Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

Author Spotlight: Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry  

The protocol describes how porphyrin-based compensation beads for flow cytometry are prepared by the reaction of amine-functionalized polystyrene beads with the porphyrin TCPP and the amide coupling reagent EDC. A filtration procedure is used to reduce the particulate byproducts.

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or ...

Combining Amine-Functionalized Polystyrene Beads with TCPP Solution

Begin by adding 4.3 milliliters of 0.1 molar MES buffer solution to a 15 milliliter polypropylene tube. Then add 288 microliters of the vortexed amine functionalized polystyrene bead suspension to the 15 milliliter polypropylene tube containing MES buffer. Vortex the MES bead solution for 15 seconds at maximum speed.Next, vortex the one milligram per milliliter TCPP solution for 60 seconds at maximum speed. Then add 1.2 milliliters of this solution to the MES bead suspension. After vortexing the resultant suspension for 15 seconds, cover the tube with foil.