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...

<ol>
	<li>Drews, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery:+A+Historical+Perspective.">Drug discovery: A Historical Perspective.</a> Science. 287 (5460), 1960-1964 (1126).</li><li>Cooper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Membrane+Receptor+Screening+and+Analysis.">Advances in Membrane Receptor Screening and Analysis.</a> J. Mol. Recognit. 17 (4), 286-315 (2004).</li><li>Voskuhl, J., Ravoo, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Recognition+of+Bilayer+Besicles.">Molecular Recognition of Bilayer Besicles.</a> Chem. Soc. Rev. 38 (2), 495-505 (2009).</li><li>Walde, P., Cosentino, K., Engel, H., Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+Vesicles:+Preparations+and+Applications.">Giant Vesicles: Preparations and Applications.</a> Chembiochem. 11 (7), 848-865 (1002).</li><li>Castellana, E. T., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+Supported+Lipid+Bilayers:+From+Biophysical+Studies+to+Sensor+Design.">Solid Supported Lipid Bilayers: From Biophysical Studies to Sensor Design.</a> Surf. Sci. Rep. 61 (10), 429-444 (2006).</li><li>Johnson, J. M., Ha, T., Chu, S., Boxer, S. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+Planar+Bilayers+in+Studies+on+Immune+Cell+Adhesion+and+Communication.">Supported Planar Bilayers in Studies on Immune Cell Adhesion and Communication.</a> J. Immunol. Methods. 278 (1-2), 19-32 .</li><li>Yang, T. L., Jung, S. Y., Mao, H. B., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Phospholipid+Bilayer-Coated+Microchannels+for+On-Chip+Immunoassays.">Fabrication of Phospholipid Bilayer-Coated Microchannels for On-Chip Immunoassays.</a> Anal. Chem. 73 (2), 165-169 (2001).</li><li>Groves, J. T., Ulman, N., Boxer, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Density+Arrays+of+Submicron+Spherical+Supported+Lipid+Bilayers.">High-Density Arrays of Submicron Spherical Supported Lipid Bilayers.</a> Anal. Chem. 84 (19), 8207-8213 (2012).</li><li>Wittenberg, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Assembly+of+Micro-+and+Nanoarrays+for+Sensing+with+Natural+Cell+Membranes.">Facile Assembly of Micro- and Nanoarrays for Sensing with Natural Cell Membranes.</a> ACS Nano. 5 (9), 7555-7564 (2011).</li><li>Stamou, D., Duschl, C., Delamarche, E., 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=Self-Assembled+Microarrays+of+Attoliter+Molecular+Vessels.">Self-Assembled Microarrays of Attoliter Molecular Vessels.</a> Angew. Chem. Int. Ed. 42 (45), 5580-5583 (2003).</li><li>Kalyankar, N. 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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;

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="42">
			<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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
	</tbody>
</table>

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 visualizações.  University of Minnesota.  Bicamadas lipídicas suportadas e partículas de membranas naturais são sistemas convenientes que podem aproximar as propriedades das membranas celulares e ser incorporadas em uma variedade de estratégias de análise. Aqui demonstramos um método para a preparação de compostos de microarrays de suporte revestidas de lípidos em bicamada-SiO 2 grânulos, vesículas de fosfolípidos ou partículas de membranas naturais. 