This work was supported by grants to S.H.O. from the National Institutes of Health (R01 GM092993), the National Science Foundation (NSF CAREER Award and DBI 0964216), the Office of Naval Research (ONR) Young Investigator Program and the Minnesota Partnership Award for Biotechnology and Medical Genomics. Device fabrication was performed at the University of Minnesota Nanofabrication Center (NFC), which receives support from the NSF through the National Nanotechnology Infrastructure Network. This work was also supported by grants to M.R. from the National Institutes of Health (NS048357, R21 NS073684), the National Multiple Sclerosis Society (CA1060A11), the Applebaum, Hilton, Peterson and Sanford Foundations and the McNeilus family. The authors wish to thank Hyungsoon Im for assistance with illustrations and Shailabh Kumar for assistance with scanning electron microscopy.

1. Microfabrication of Microwell Array Substrate
<ol>
	<li>Start with a 4 inch silicon wafer with 100 nm of thermally grown oxide.</li>
	<li>Spin SPR-955 0.7 photoresist on the wafer at 4,000 rpm for 30 sec.</li>
	<li>Bake on a hotplate at 115 &#176;C for 90 sec.</li>
	<li>Expose photoresist.
	<ol>
		<li>Use a mask that will create 1 &#181;m holes arranged in a hexagonal array with a 3 &#181;m period where the array covers a 2 mm x 2 mm area.</li>
		<li>Expose wafer in an i-line stepper using a step size of 6 mm and an exposure of 200 mJ/cm2.</li>
	</ol>
	</li>
	<li>Bake on a hotplate at 115 &#176;C for 90 sec.</li>
	<li>Develop using a spin developer to deposit 2 puddles of CD-26 on the wafer for a total development time of 90 sec.</li>
	<li>Rinse thoroughly with ultrapure DI water and dry with N2.</li>
	<li>Etch the oxide in a reactive-ion etcher for 6 min with 50 sccm of Ar, 25 sccm of CF4, and 50 sccm of CHF3 flowing at a pressure of 75 mTorr.</li>
	<li>Etch silicon in an ICP deep-trench reactive-ion etcher for 2 min with 63 sccm of C4F8, 27 sccm of SF6, 40 sccm of Ar, and 10 sccm of O2 flowing at a pressure of 14 mTorr.</li>
	<li>Rinse in acetone, methanol, and isopropanol to remove resist.</li>
	<li>Place in a bath of H2SO4:H2O2 1:1 for 10 min to clean the surface.</li>
	<li>Rinse thoroughly with ultrapure DI water and dry with N2.</li>
	<li>Deposit 1,080 &#197; of Al2O3 by atomic layer deposition at 250 &#176;C.</li>
</ol>
2. Preparation of Poly(dimethylsiloxane) Squeegee
<ol>
	<li>If possible, work in a cleanroom. Otherwise work in the cleanest environment possible.</li>
	<li>Obtain a plastic Petri dish (or other clean disposable container) with &#8805;13 mm sides.</li>
	<li>In that dish, pour out 5 g PDMS curing agent, followed by 50 g PDMS base agent (or anything with a 1:10 ratio that will mostly fill the Petri dish). Mix thoroughly with a disposable plastic pipette or rod.</li>
	<li>Remove bubbles by placing the mixed PDMS in a chamber with a vacuum line, applying vacuum until bubbles come out of mixture (approximately 30 min).</li>
	<li>If not already in the Petri dish, pour PDMS in Petri dish, avoiding creation of air bubbles.</li>
	<li>Cure overnight on a hotplate at &#62;50 &#176;C (higher heat for less time also works).</li>
	<li>With new/clean razor blade, carefully cut a rectangular piece approximately 2.5 x 2.5 cm, keeping top surface as clean as possible. Peel out with a clean forceps. Trim off one edge (down to 1.5 cm) by pressing down on razor blade in one motion, to make top edge as sharp as possible (this will be the edge used in the squeegee process).</li>
	<li>Clean with acetone, methanol, isopropyl alcohol, and ultrapure DI water, and then blow dry with clean nitrogen gas. Store in a clean Petri dish.</li>
</ol>
3. Preparation of Vesicles
<ol>
	<li>Collect lipid stock solutions of 25 mg/ml egg phosphatidylcholine (PC) and 1 mg/ml 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt (Rho-DPPE) in chloroform and a 0.5 mg/ml stock solution of monosialoganglioside GM1 in chloroform/methanol (2:1 v/v).</li>
	<li>In a small glass vial make a mixture that results in 97 mol% PC, 2 mol% GM1 and 1 mol% Rho-DPPE by adding 18.9 &#181;l of 25 mg/ml PC, 39.6 &#181;l of 0.5 mg/ml GM1 and 7.9 &#181;l of 1 mg/ml Rho-DPPE using glass syringes.</li>
	<li>Note: The supplementary material for Nair et al.25 contains an excellent customizable Excel spreadsheet for calculating the amounts of lipids required to create vesicles of any desired composition.</li>
	<li>Place vial in a vacuum desiccator and remove the solvent by leaving the vial under vacuum for 6 hr.</li>
	<li>Make an aqueous solution of 0.1 M NaCl. Add 0.5 ml of the 0.1 M NaCl solution to the vial containing the dried lipid film.</li>
	<li>Allow the aqueous lipid mixture to rest overnight.</li>
	<li>Briefly vortex mix to create a vesicle suspension, then sonicate for 20 min in a bath sonicator at room temperature.</li>
	<li>Extrude the vesicle suspension through a polycarbonate membrane with 100 nm pore size. Pass the vesicle suspension through the filter 17x.</li>
	<li>Store the extruded vesicles in a glass vial at 4 &#176;C. </li>
</ol>
4. Formation of Spherical Supported Lipid Bilayers
<ol>
	<li>Collect 700 nm diameter SiO2 beads. The beads are supplied in distilled water with a stock concentration of 1.4 x 1011 beads/ml.</li>
	<li>In a 1.5 ml Eppendorf tube prepare a 1 ml bead suspension with a concentration of 1.5 x 1010 beads/ml by adding 107 &#181;l of bead stock solution to 893 &#181;l of 0.1 M NaCl.</li>
	<li>Vortex the suspension to ensure it is well mixed.</li>
	<li>Centrifuge the suspension at 1,700 x g for 20 min then discard the supernatant. Resuspend the beads in 1 ml of 0.1 M NaCl. Repeat this step twice more to thoroughly wash the beads.</li>
	<li>To form the SSLBs, in a 1.5 ml Eppendorf tube add 25 &#181;l of the SiO2 bead suspension to 200 &#181;l of 1 mg/ml vesicle suspension. Vortex the mixture and let stand for 1 hr at room temperature. At this stage the SSLB concentration should be 1.7 x 109 SSLBs/ml.</li>
	<li>Centrifuge at 1,700 x g for 20 min to pellet the SSLBs. The pellet should appear pink due to the rupture of vesicles with Rho-DPPE on the beads.</li>
	<li>Discard the supernatant and resuspend in 225 &#181;l phosphate buffered saline (PBS), pH = 7.4. Repeat Steps 4.6-4.7 two more times to remove any unruptured vesicles from the SSLB suspension.</li>
</ol>
5. Assembly of SSLB Array
<ol>
	<li>Cleave wafer of microwell arrays resulting in rectangular pieces with 4-6 microwell arrays per piece.</li>
	<li>Vortex mix the SSLB suspension, then place 10 &#181;l of SSLB suspension on each microwell array. Lest rest for 1 hr to allow SSLBs to settle to the surface.</li>
	<li>Gently wash the microwell array chip with PBS from a wash bottle, then submerge in a PBS bath prepared in a shallow dish.</li>
	<li>While submerged, gently place the PDMS squeegee flush on the microwell array chip and gently slide it along the surface 5x to remove SSLBs that are not immobilized in microwells.</li>
	<li>Grip the microwell array chip with tweezers and gently shake in the PBS bath to remove any excess SSLBs.</li>
	<li>Quickly remove the chip from the squeegee bath and place in a fresh PBS bath until further use. Make sure the top surface of the chip remains wet to preserve the SSLBs.</li>
</ol>
Note: The assembly method described above can be used to create arrays of natural membrane particles, like myelin particles isolated from mouse brain, or phospholipid vesicles without the SiO2 bead supports. This work is described in detail in Wittenberg et al.21 and representative results are shown below.
6. Cholera Toxin Binding Assay
<ol>
	<li>Prepare a 2 mg/ml solution of bovine serum albumin (BSA) in PBS.</li>
	<li>Prepare a solution with desired concentration of Alexa 488-conjugated cholera toxin B-subunit in PBS with 2 mg/ml BSA.</li>
	<li>Remove the SSLB array chip from the PBS bath and wick away most of the PBS solution using a laboratory wipe. Leave just enough PBS on the chip to keep the SSLB array hydrated.</li>
	<li>To prevent nonspecific binding, add 200 &#181;l of 2 mg/ml BSA solution to the SSLB array chip. Let rest for 1 hr in a humidified box.</li>
	<li>With a micropipette, remove 200 &#181;l of solution from the top of the SSLB array chip.</li>
	<li>Add 200 &#181;l of cholera toxin solution to the SSLB array chip and let rest for 1 hr in a humidified box.</li>
	<li>Gently wash the SSLB array chip with PBS from a wash bottle to remove any unbound cholera toxin.</li>
	<li>Wick away excess PBS using a laboratory wipe. Before imaging cover chip with a 24 mm x 40 mm cover slip.</li>
	<li>Image arrays with an upright microscope using filter sets appropriate for the fluorophores of interest.</li>
	<li>Analyze the fluorescence intensity of individual SSLBs in an array using the automated particle analysis function of ImageJ software.</li>
	<li>Compile mean fluorescence intensities from individual SSLBs into histograms that summarize the intensities of SSLBs found on a given array.</li>
</ol>

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H., Castillo, J., Gether, U., Hedegard, P., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Curved+Membranes+Recruit+Amphipathic+Helices+and+Protein+Anchoring+Motifs.">How Curved Membranes Recruit Amphipathic Helices and Protein Anchoring Motifs.</a> Nat. Chem. Biol. 5 (11), 835-841 (2009).</li><li>Roizard, S., Danelon, C., Hassaine, G., Piguett, J., Schulze, K., Hovius, R., Tampe, R., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+G-Protein-Coupled+Receptors+in+Cell-Derived+Plasma+Membranes+Supported+on+Porous+Beads.">Activation of G-Protein-Coupled Receptors in Cell-Derived Plasma Membranes Supported on Porous Beads.</a> J. Am. Chem. Soc. 133 (42), 16868-16874 (2011).</li></ol>

The authors have no competing financial interests to disclose.

In this work we show that monodisperse SiO2 beads coated with supported lipid bilayers can be arrayed into microwell arrays without the need for targeting ligands on the lipid bilayers or the substrate surface, and the arrays can be used for characterizing toxin-lipid interactions. The dissociation constant we calculated for CTx/GM1 binding compares favorably, given the wide disparity of values in the literature, with a previous report by Winter et al., where colloidal assembly of lipid-coated beads w...

Lipid bilayer membranes are essential structures in nature. Cellular plasma membranes and organelle membranes are composed of lipid bilayers that incorporate a number of molecules that are necessary for life. Many life-sustaining processes occur on the surface of cells or are mediated by molecules associated with lipid-bilayer membranes. In fact, many pharmaceuticals target processes or molecules are found on or in membranes1,2. It is therefore necessary to analytically investigate processes, such as chemical reactions or noncovalent binding events that occur on membrane surfaces. Because natural membranes can be difficult to isolate and/or interface with sensors, many researchers employ simplified model membranes to carry out analytical studies. A number of model membrane systems are described in the literature, ranging from giant vesicles that can be tens to hundreds of microns in diameter to liposomes with nanoscale dimensions3,4. Alternatively, planar lipid bilayers deposited on solid supports, i.e., supported lipid bilayers (SLBs), can be formed on a number of different surfaces and have been widely used in biophysical, biochemical, and analytical applications5. Coupling SLBs with electrical or optical materials enables investigation of membrane biochemistry and biophysics through the use of different analytical techniques. Fluorescence microscopy6,&#160;electrochemistry7, optical spectroscopy8, scanning probe microscopy9, surface plasmon resonance10, and mass spectrometry11 have all been employed to study the structure and properties of SLBs.
SLB arrays offer additional versatility in the design of sensors for multiplex assays12,13. Other applications use SLB arrays to mimic the junction that forms between immune cells14. Preparation methods for SLB arrays have varied from microfluidic approaches15 to those that employ physical barriers between adjacent SLB patches.16 Other groups have used printing methods17, photochemical patterning18 and various nanoengineering approaches19 to create SLB arrays.
In this paper and accompanying video we demonstrate a method for forming SLB arrays by depositing SLB-coated SiO2 beads into ordered arrays of microwells20. We refer to the SLB-coated SiO2 beads as spherical supported lipid bilayers (SSLBs). This technique is an extension of earlier work that created arrays of phospholipid vesicles and biomembranes derived from natural sources21, from which we also show example results. Other methods for arraying biomembrane particles or vesicles have relied on patterns of specific targeting ligands on surfaces that associate with complementary ligands contained on the vesicle surface. Examples include biotin-avidin association22,23 and DNA hybridization schemes24. Our approach only requires a microwell array with no targeting or recognition moieties necessary. The size of SSLBs is defined by the diameter of the SiO2 bead supports, which have low poly-dispersity. By tuning the microwell diameter to just larger than the SSLB diameter, only a single SSLB settles into each microwell. A poly(dimethylsiloxane) (PDMS) squeegee then removes from the surface all SSLBs that are not immobilized in microwells. The microwells and resultant SSLB arrays have high density (~105 SSLBs/mm2) with 3 &#181;m center-to-center spacing and hexagonal periodicity. By serially depositing SSLBs with different lipid compositions, it is possible to create multicomponent arrays with randomly positioned SSLBs. To demonstrate the sensing capability of SSLB arrays, we used the interaction of cholera toxin (CTx) with a ganglioside (GM1) incorporated into the SSLBs. With natural membrane particles, we were able to detect cell-specific lipids in multicomponent arrays containing membrane material from two different cell types.&#160;

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			<td height="42" style="height:42px;width:239px;">4-inch silicon wafers</td>
			<td style="width:91px;">University Wafer</td>
			<td align="right" style="width:119px;">425</td>
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			<td height="42" style="height:42px;width:239px;">Shipley MEGAPOSIT SPR955-CM 0.7 photoresist</td>
			<td style="width:91px;">MicroChem</td>
			<td style="width:119px;">SPR955-CM</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Shipley MICROPOSIT CD-26 developer</td>
			<td style="width:91px;">MicroChem</td>
			<td style="width:119px;">CD-26</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">i-line stepper</td>
			<td style="width:91px;">Canon</td>
			<td style="width:119px;">2500 i3 stepper</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Vision 320 reactive ion etcher</td>
			<td style="width:91px;">Advanced Vacuum</td>
			<td style="width:119px;">Vision 320 RIE</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Deep trench reactive ion etcher</td>
			<td style="width:91px;">Plasma Therm</td>
			<td style="width:119px;">SLR-770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Atomic layer depostion system</td>
			<td style="width:91px;">Cambridge NanoTech</td>
			<td style="width:119px;">Savannah</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Dow Corning Sylgard 184 poly(dimethylsiloxane) kit</td>
			<td style="width:91px;">Ellsworth Adhesives</td>
			<td style="width:119px;">184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">egg phosphatidylcholine</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>840051C</td>
			<td></td>
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		<tr height="84">
			<td height="84" style="height:84px;width:239px;">1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>810158C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">monosialoganglioside GM1&#160;</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td style="width:119px;">860065P</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Silica beads</td>
			<td style="width:91px;">Bangs Laboratories</td>
			<td style="width:119px;">SS03N/4666</td>
			<td>Packaging on the bead container states the beads are 900 nm in diameter. However, after light-scattering and electron microscopy we determined the beads are roughly 700 nm in diameter.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Cholera toxin B-subunit, Alexa 488 conjugate</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">C-34775</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Anti-oligodentrocyte antibody IgM O4, NorthernLights 557 conjugate</td>
			<td style="width:91px;">R&#38;D Systems</td>
			<td style="width:119px;">NL1326R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">FM1-43</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">T-3136</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Eppendorf MiniSpin centrifuge</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td style="width:119px;">05-401-09</td>
			<td></td>
		</tr>
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formation of biomembrane microarrays with a squeegee-based assembly method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

When SiO2 beads are mixed with a solution of vesicles composed of phospholipids, fluorescent lipids and other lipids such as gangliosides, the vesicles rupture on the SiO2 bead surfaces to form SSLBs, as shown schematically in Figure 1a. After washing the SSLBs, a drop of SSLB solution is placed on a microwell array, and the beads are allowed to settle to the surface. (Figure 1b1) This can also be done with a suspension of phospholipid vesicles or natural membrane p...

Watch this Scientific Journal Video about Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method at JoVE.com

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

formation-biomembrane-microarrays-with-squeegee-based-assembly

Research

JoVE Journal

Bioengineering

13.6K vues.  University of Minnesota.  Bicouches lipidiques supportées et des particules de la membrane sont des systèmes physiques avantageuses qui peuvent rapprocher les propriétés des membranes des cellules et être incorporés dans une variété de stratégies d'analyse. Nous démontrons ici un procédé pour préparer des microréseaux composées de bicouches lipidiques supportées revêtu de SiO 2, des perles ou des particules des vésicules de phospholipides membranaires naturelles. ...

Vidéo: Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.