Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Summary

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.

Abstract

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.

Introduction

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 membranes^1,2. 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 s....

Protocol

1. Microfabrication of Microwell Array Substrate

1. Start with a 4 inch silicon wafer with 100 nm of thermally grown oxide.
2. Spin SPR-955 0.7 photoresist on the wafer at 4,000 rpm for 30 sec.
3. Bake on a hotplate at 115 °C for 90 sec.
4. Expose photoresist.
   1. Use a mask that will create 1 µm holes arranged in a hexagonal array with a 3 µm period where the array covers a 2 mm x 2 mm area.
   2. Expose wafer in an i-line stepper using a step size of 6 mm and an exposure of 200 mJ/cm².
5. Bake on a hotplate at 115 °C for 90 sec.
6. Develop using a spin developer to ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4 inch silicon wafers	University Wafer	425
Shipley MEGAPOSIT SPR955-CM 0.7 photoresist	MicroChem	SPR955-CM
Shipley MICROPOSIT CD-26 developer	MicroChem	CD-26
i-line stepper	Canon	2500 i3 stepper
Vision 320 reactive ion etcher	Advanced Vacuum	Vision 320 RIE
Deep trench reactive ion etcher	Plasma Therm	SLR-770
Atomic layer depostion system	Cambridge NanoTech	Savannah
Dow Corning Sylgard 184 poly(dimethylsiloxane) kit	Ellsworth Adhesives	184 SIL ELAST KIT 0.5KG
Egg phosphatidylcholine	Avanti Polar Lipids	840051C
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt	Avanti Polar Lipids	810158C
Monosialoganglioside GM1	Avanti Polar Lipids	860065P
Silica beads	Bangs Laboratories	SS03N/4666	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.
Cholera toxin B-subunit, Alexa 488-conjugate	Molecular Probes	C-34775
Anti-oligodentrocyte antibody IgM O4, NorthernLights 557 conjugate	R&D Systems	NL1326R
FM1-43	Molecular Probes	T-3136
Eppendorf MiniSpin centrifuge	Fisher Scientific	05-401-09