The authors wish to acknowledge support from the National Research Foundation (NRF -NRFF2011-01 and NRF2015NRF-POC0001-19), the National Medical Research Council (NMRC/CBRG/0005/2012), and Nanyang Technological University to N.J.C.

1. Formation of Supported Lipid Bilayer on a Hydrophilic Solid Support
<ol>
	<li>Prepare lipid stock solutions of 10 mg/ml 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1 mg/ml 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) by dissolving the respective lipid powders (appropriately weighed beforehand with an analytical mass balance) in isopropanol solution.</li>
	<li>Dilute and mix the stock solutions in isopropanol in order to prepare the desired lipid mixture at the final concentration. For fluorescence microscopy and FRAP experiments,0.5 wt% Rh-PE should be included in the lipid mixture.</li>
	<li>Inject the lipid mixture in isopropanol into the microfluidic channel until it is filled.</li>
	<li>Incubate the lipid mixture on the glass surface for about 10 min.</li>
	<li>Gradually replace the lipid solution with water or buffer solution using a peristaltic pump at a very low flow rate (10-50 &#181;l/min). Alternatively, replace the lipid mixture by repeated pipetting.</li>
	<li>Rinse the channel thoroughly with excess buffer in order to remove the residual isopropanol.</li>
</ol>
2. Formation of Cholesterol-enriched Supported Membranes
Note: The solid surface (SiO2) supports vesicle fusion, but the membrane composition (high cholesterol) inhibits vesicle fusion because cholesterol-enriched vesicles have high bending rigidity30.
<ol>
	<li>Prepare a stock solution containing 10 mg/ml DOPC lipid, 10 mg/ml cholesterol, and 1 mg/ml Rh-PE by first dissolving the respective powders in isopropanol.</li>
	<li>Repeat steps 1.2-1.6 using the stock solutions prepared in step 2.1.</li>
</ol>
3. Membrane Fluidity Assay
<ol>
	<li>Immerse the glass slide in sodium dodecyl sulfate (SDS) solution (1%) for 10 min.</li>
	<li>Wash the slides thoroughly with deionized water and rinse with ethanol.</li>
	<li>Blow-dry slides using a gentle stream of nitrogen.</li>
	<li>Expose the slides to oxygen plasma for 30 sec at maximum radiofrequency power in the oxygen plasma chamber.</li>
	<li>Remove the protective film coating of the bottomless commercial microfluidic chamber (Figure 1A) using a tweezer and attach the glass slide onto the sticky side of the chamber (Figure 1B).</li>
	<li>Assemble the connectors and tubing into the inlet and outlet positions of the chamber and place the microfluidic channel on the microscope stage (Figure 1C).</li>
	<li>Form a fluorescently-labeled supported lipid bilayer in the microfluidic channel [repeat step 1 using the desired lipid composition (e.g., 0.5 mg/ml DOPC) including 0.5 wt% Rh-PE in organic solvent].</li>
	<li>Locate the bilayer plane with a 60X oil immersion objective (NA 1.49) in order to capture images.</li>
	<li>Take two pre-bleach images, then photo-bleach a 30 &#181;m wide circular spot with a 532 nm, 100 mW laser beam. Before photobleaching, warm up the laser until its intensity has become stable. Immediately after photobleaching, capture a series of images every 1 sec for 2 min in order to follow the recovery in fluorescence intensity at the bleached spot.</li>
</ol>
4. Cholesterol Quantification Assay
<ol>
	<li>Expose the silicon dioxide-coated quartz crystal sensor chip to oxygen plasma for 30 sec at maximum radiofrequency power in the oxygen plasma chamber. Remove the chip from the chamber and immediately mount the chip in the measurement chamber.</li>
	<li>Before experiment, first acquire the frequency and dissipation of the sensor chip in air in order to ensure proper mounting.
	<ol>
		<li>For the commercial Q-Sense E1 or E4 QCM-D instrument, run the Q-Soft software program and click &#8220;Acquisition&#8221; and select &#8220;Setup Measurement&#8221;.</li>
		<li>In the new window which appears, check the 3, 5, 7, 9, 11 and 13 overtones and then click Find and Run in order to check the resonance spectra. Set the temperature at 24 &#176;C. If one or more resonance frequencies do not agree with the expected values, check the chip mounting and re-perform this step until an agreement is reached.</li>
	</ol>
	</li>
	<li>Start the peristaltic pump and flow buffer solution (10 mM Tris, 150 mM NaCl, pH 7.5) into the measurement chamber at a flow rate of 100 &#181;l/min.</li>
	<li>In the software program, click &#8220;Acquisition&#8221; and select &#8220;Restart Measurement&#8221; in order to record the resonance frequency and energy dissipation signals. Repeat this step until a stable baseline is obtained for the frequency and dissipation shifts. Note: For steps hereafter, there will be additional frequency and dissipation shifts which can be recorded as a function of time.</li>
	<li>Inject isopropanol (without lipid) for 10 min.</li>
	<li>Inject the mixture of DOPC lipid/cholesterol at the desired molar ratio with a total lipid concentration of 0.5 mg/ml in isopropanol for 10 min.</li>
	<li>Inject buffer for 20 min at a flow rate of 100 &#181;l/min.</li>
	<li>Inject a solution of 1 mM methyl-&#946;-cyclodextrin (M&#946;CD) prepared in buffer (flow rate 100 &#181;l/min) until the frequency signal reaches a stable value.</li>
	<li>Measure the relative positive frequency shifts that is caused by the M&#946;CD treatment step and calculate the mass of cholesterol and DOPC lipids by converting the &#916;fcho and &#916;fDOPC values to mass values by using the Sauerbrey equation [&#916;m = -(C/n)&#215;&#916;f, where C=17.7 ng/cm2, n:overtone].</li>
	<li>Calculate the mole fraction of cholesterol in the SLB, taking into account the molecular weights of DOPC lipid (786.1 g/mol) and cholesterol (386.6 g/mol).</li>
</ol>

The authors have no competing financial interests to disclose.

In this work, a solvent-exchange protocol is presented in which lipids in alcohol (isopropanol, ethanol, or n-propanol) are incubated with a solid support and then the alcohol is replaced gradually with an aqueous buffer solution in order to drive a series of phase transitions eventually producing lamellar-phase lipid bilayers24. It is shown that the method enables fabrication of supported lipid bilayers on surfaces such as gold, which is intractable to the vesicle fusion method.
An optimal lipid concentration range (0.1 - 0.5 mg/ml) has been determined for complete bilayer formation in standard experimental formats tested thus far. At lipid concentrations below 0.1 mg/ml, discrete, microscopic patches of bilayers formed. On the other hand, at concentrations above 0.1 mg/ml and lower than 0.5 mg/ml, a complete and uniform bilayer is formed. At lipid concentrations above this range, a fluid bilayer was still formed as verified by FRAP analysis, however, fluorescence microscopy reveals the presence of additional lipid structures on top of the bilayer. Strikingly, the morphology of these additional lipid structures, as determined by QCM-D analysis, depended on the alcohol which was used during the incubation step. In the case of ethanol, the relatively high &#916;f and &#916;D shifts resemble the QCM-D signature obtained for an adsorbed vesicle layer. When isopropanol or n-propanol was instead used, the &#916;f was slightly higher than the value expected for a bilayer (final &#916;f between -30 to -40 Hz), while the &#916;D was appreciably higher. Such QCM-D responses would be expected for extended lipid structures (e.g., worm-like micelles) protruding outward from the membrane surface (as visible by fluorescence microscopy in some cases).
The rate of solvent exchange is another important parameter which can be critical, especially when lower lipid concentrations (e.g., 0.1 mg/ml) are used. Rapid solvent exchange at low lipid concentration can lead to the formation of incomplete bilayers. In the standard measurement chamber used for QCM-D measurements in this paper (Q-Sense E4 measurement chamber), flow rates of around 100 &#181;l/min, were suitable for highly reproducible complete bilayer formation. For flow cells with other geometries and volume, the optimal flow rate may vary and must be empirically determined based on the steps suggested herein.
In addition to form supported lipid bilayers on surfaces which are intractable to vesicle fusion, the SALB can be employed to circumvent the need for lipid vesicles which can rupture, thereby opening the door to the fabrication of supported membranes with complex compositions. As an illustrative example composition, lipid mixtures with a high fraction of cholesterol were examined. Cholesterol is an important component of mammalian cell membranes, and its fraction can approach 45&#8722;50 mol% of the membrane lipid composition (e.g., in erythrocytes). Thus, even a simple model of a lipid bilayer representing a human cell membrane should include cholesterol.
While vesicle fusion could be used to fabricate fluidic lipid bilayers containing only 10-15% cholesterol, the SALB method enables formation of fluidic lipid bilayers containing high fractions of cholesterol (up to 57 mol%, as quantified by QCM-D measurements)36. However, when the level of cholesterol was further elevated (up to 63 mol%), stripe-shape domains were observed37. The co-existing domains were liquid, reminiscent of those observed in the &#946; region in the phase diagram of the cholesterol/phospholipids monolayer at the air-water interface.
Overall, the SALB method is shown to be a simple and efficient approach to form supported lipid bilayers, especially in cases beyond the scope of the conventional vesicle fusion method. Thus far, the QCM-D technique and fluorescence microscopy were mainly used to characterize the supported lipid bilayers formed by the SALB method. Looking forward, a broad range of surface-sensitive analytical measurements techniques, including surface plasmon resonance (SPR)38, atomic force microscopy (AFM)39,40, Fourier-transform infrared spectroscopy41, X-ray42 and neutron reflectivity43, can be used to further characterize and study simple and complex bilayer configurations prepare by the SALB method. These emerging capabilities open the door to a greater number of scientists who can explore artificial cell membranes by taking advantage of a simple and robust experimental protocol.

The solid-supported lipid bilayer1 (SLB) is a versatile platform which preserves the basic characteristics of biomembranes such as the bilayer thickness, two-dimensional lipid diffusivity, and the ability to host membrane-associated biomolecules. Due to the complexity of natural cell membranes, this simple platform has been shown to function as an efficient platform for in vitro studies of membrane-related processes such as raft formation2, protein binding3, virus and virus-like particle binding4,5, and cell signaling6. Formed in close proximity to a solid support, the SLB platform is compatible with a range of surface-sensitive measurements techniques such as total internal reflection microscopy (TIRF), quartz crystal microbalance-dissipation (QCM-D), and impedance spectroscopy.
Several methods have been developed to produce different types of SLBs, including air bubble collapse7 and dip-pen nanolithography8 for submicron-sized lipid spots, spin-coating9 for bilayer stacks and Langmuir&#8722;Blodgett (LB)10 and vesicle fusion (VF)11 for full-spanning, single lipid bilayer coatings. The VF method consists of the adsorption of small unilamellar vesicles to a solid support and subsequent spontaneous rupture and fusion to form a continuous lipid bilayer. However, under physiological conditions, spontaneous vesicle rupture is mainly limited to silicon-based materials such as silicon dioxide, glass, and mica. In addition, vesicle rupture does not occur spontaneously for vesicles of complex lipid compositions such as those containing high fractions of cholesterol or negatively charged lipids. Depending on the system, vesicle rupture may be induced by further tailoring the experimental conditions such as temperature12, solution pH13, and salinity14, osmotic shock15 or pressure16, or addition of divalent ions such as Ca2+ 17 . Alternatively, the membrane-active AH peptide can be introduced in order to destabilize a layer of adsorbed vesicles, leading to vesicle rupture and bilayer formation on a range of surfaces18-22.
Moreover, successful bilayer formation requires preparation of a well-controlled population of small unilamellar vesicles that can be time consuming and difficult to achieve for certain membrane compositions. Therefore, despite its high efficiency in optimal cases (e.g., after extensive freeze-thaw pretreatment of vesicles23), the general application of vesicle fusion is limited by the scope of suitable substrates and membrane compositions .
The solvent-assisted lipid bilayer (SALB) method24-28 is an alternative fabrication technique which does not require lipid vesicles. The method is based on the deposition of lipid molecules onto a solid surface in the presence of a water miscible organic solvent followed by gradual exchange of this solvent with an aqueous buffer solution in order to trigger SLB formation. During the solvent-exchange step, the ternary mixture of lipids, organic solvent, and water undergoes a series phase transitions with increasing water fraction, which leads to the formation of lamellar phase structures in the bulk solution and an SLB on the solid substrate. Importantly, this self-assembly route bypasses the need for vesicle rupture, which is usually the limiting step for the transformation of adsorbed vesicles into an SLB. The protocol is applicable to a wide variety of surfaces including silicon dioxide, aluminum oxide, chrome, indium tin oxide, and gold. In this paper and in the accompanying video, a comparison of lipid deposition by the SALB and vesicle fusion methods is presented. In particular, the influence of experimental parameters, including lipid concentration, flow rate, and the choice of water miscible organic solvent, on the quality of the bilayer formed by the SALB method are discussed. Analytical characterization of the fabricated SLBs is performed by the QCM-D, fluorescence microscopy, and fluorescence recovery after photobleaching (FRAP) techniques. QCM-D monitoring is a surface-sensitive mass measurement technique which, since the pioneering work conducted by Keller and Kasemo29, has been extensively used to quantitatively investigate bilayer formation. Fluorescence microscopy permits inspection of membrane homogeneity as well as the visualization of membrane domains. The FRAP technique is a standard tool to determine the lateral mobility of lipid molecules in an SLB, which is an essential property of fluidic membranes.
The first part of this study involves QCM-D analysis of the SALB and vesicle fusion methods applied to attempt bilayer formation on silicon dioxide and gold. In the second part, the preparation and characterization of supported membranes containing a range of cholesterol concentrations with the SALB method are demonstrated and the results are compared with those obtained by the vesicle fusion method.

<table border="0" cellpadding="0" cellspacing="0" width="1459">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">QCM-D silicon dioxide-coated substrates</td>
			<td style="width:356px;">QSense AB,&#160; Sweden</td>
			<td style="width:217px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QCM-D gold-coated substrates</td>
			<td style="width:356px;">QSense AB,&#160; Sweden</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q-Sense E4 module</td>
			<td>QSense AB,&#160; Sweden</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Plasma Cleaner, PDC-32G</td>
			<td>Harrick Plasma, Ithaca, NY</td>
			<td>PDC-001 (115V)&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850375P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE)</td>
			<td>Avanti Polar Lipids</td>
			<td>810150P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td style="width:217px;">700000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methyl-&#946;-cyclodextrin</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td style="width:217px;">C4555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right">673773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Ethanol</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right" style="width:217px;">459844</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">n-propanol</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right">279544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Sticky-Slide I 0.1 Luer</td>
			<td style="width:356px;">IBIDI</td>
			<td align="right" style="width:217px;">81128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Male elbow 1/8&#8221;</td>
			<td style="width:356px;">Cole-Parmer</td>
			<td style="width:217px;">30505-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Silicon tubing 1.6mm ID</td>
			<td style="width:356px;">IBIDI</td>
			<td align="right" style="width:217px;">10842</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Glass coverslip No. 1.5H, 25 mm x 75 mm</td>
			<td style="width:356px;">IBIDI</td>
			<td align="right" style="width:217px;">10812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Reglo Digital M2-2/12 Peristaltic Pump</td>
			<td style="width:356px;">Ismatec</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Sodium dodecyl sulfate</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right" style="width:217px;">71725</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method at JoVE.com

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.

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 ...

biomembrane-fabrication-solvent-assisted-lipid-bilayer-salb

Research

JoVE Journal

Bioengineering

14.5K visualizações.  Nanyang Technological University.  Apresenta-se um protocolo experimental para formar uma bicamada lipídica suportado sobre substratos sem a utilização de vesículas lipídicas. Nós demonstramos um método de um só passo para formar uma bicamada lipídica em dióxido de silício e ouro, bem como membranas suportadas com o domínio enriquecida em colesterol durante várias aplicações biológicas. 