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 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 dimensions^3,4. 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, 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 assays^12,13. 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 association^22,23 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 µm 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. 

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 deposit 2 puddles of CD-26 on the wafer for a total development time of 90 sec.
7. Rinse thoroughly with ultrapure DI water and dry with N₂.
8. 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.
9. 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.
10. Rinse in acetone, methanol, and isopropanol to remove resist.
11. Place in a bath of H₂SO₄:H₂O₂ 1:1 for 10 min to clean the surface.
12. Rinse thoroughly with ultrapure DI water and dry with N₂.
13. Deposit 1,080 Å of Al₂O₃ by atomic layer deposition at 250 °C.

2. Preparation of Poly(dimethylsiloxane) Squeegee

1. If possible, work in a cleanroom. Otherwise work in the cleanest environment possible.
2. Obtain a plastic Petri dish (or other clean disposable container) with ≥13 mm sides.
3. 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.
4. 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).
5. If not already in the Petri dish, pour PDMS in Petri dish, avoiding creation of air bubbles.
6. Cure overnight on a hotplate at >50 °C (higher heat for less time also works).
7. 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).
8. Clean with acetone, methanol, isopropyl alcohol, and ultrapure DI water, and then blow dry with clean nitrogen gas. Store in a clean Petri dish.

3. Preparation of Vesicles

1. 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).
2. 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 µl of 25 mg/ml PC, 39.6 µl of 0.5 mg/ml GM1 and 7.9 µl of 1 mg/ml Rho-DPPE using glass syringes.
3. 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.
4. Place vial in a vacuum desiccator and remove the solvent by leaving the vial under vacuum for 6 hr.
5. 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.
6. Allow the aqueous lipid mixture to rest overnight.
7. Briefly vortex mix to create a vesicle suspension, then sonicate for 20 min in a bath sonicator at room temperature.
8. Extrude the vesicle suspension through a polycarbonate membrane with 100 nm pore size. Pass the vesicle suspension through the filter 17x.
9. Store the extruded vesicles in a glass vial at 4 °C.

4. Formation of Spherical Supported Lipid Bilayers

1. Collect 700 nm diameter SiO₂ beads. The beads are supplied in distilled water with a stock concentration of 1.4 x 10¹¹ beads/ml.
2. 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 µl of bead stock solution to 893 µl of 0.1 M NaCl.
3. Vortex the suspension to ensure it is well mixed.
4. 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.
5. To form the SSLBs, in a 1.5 ml Eppendorf tube add 25 µl of the SiO₂ bead suspension to 200 µl 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.
6. 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.
7. Discard the supernatant and resuspend in 225 µl 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.

5. Assembly of SSLB Array

1. Cleave wafer of microwell arrays resulting in rectangular pieces with 4-6 microwell arrays per piece.
2. Vortex mix the SSLB suspension, then place 10 µl of SSLB suspension on each microwell array. Lest rest for 1 hr to allow SSLBs to settle to the surface.
3. Gently wash the microwell array chip with PBS from a wash bottle, then submerge in a PBS bath prepared in a shallow dish.
4. 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.
5. Grip the microwell array chip with tweezers and gently shake in the PBS bath to remove any excess SSLBs.
6. 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.

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

1. Prepare a 2 mg/ml solution of bovine serum albumin (BSA) in PBS.
2. Prepare a solution with desired concentration of Alexa 488-conjugated cholera toxin B-subunit in PBS with 2 mg/ml BSA.
3. 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.
4. To prevent nonspecific binding, add 200 µl of 2 mg/ml BSA solution to the SSLB array chip. Let rest for 1 hr in a humidified box.
5. With a micropipette, remove 200 µl of solution from the top of the SSLB array chip.
6. Add 200 µl of cholera toxin solution to the SSLB array chip and let rest for 1 hr in a humidified box.
7. Gently wash the SSLB array chip with PBS from a wash bottle to remove any unbound cholera toxin.
8. Wick away excess PBS using a laboratory wipe. Before imaging cover chip with a 24 mm x 40 mm cover slip.
9. Image arrays with an upright microscope using filter sets appropriate for the fluorophores of interest.
10. Analyze the fluorescence intensity of individual SSLBs in an array using the automated particle analysis function of ImageJ software.
11. Compile mean fluorescence intensities from individual SSLBs into histograms that summarize the intensities of SSLBs found on a given array.

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