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Method Article
This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.
The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions—and perhaps the biological relevance—of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.
Cellular membranes are selectively permeable barriers comprised of thousands of lipid types1, proteins, carbohydrates, and sterols that encapsulate and subdivide all living cells. Understanding how their compositions affect their functions and revealing how natural and synthetic molecules interact with, adhere to, disrupt, and translocate cellular membranes are, therefore, important areas of research with wide-reaching implications in biology, medicine, chemistry, physics, and materials engineering.
These aims for discovery directly benefit from proven techniques for assembling, manipulating, and studying model membranes—including lipid bilayers assembled from synthetic or naturally occurring lipids—that mimic the composition, structure, and transport properties of their cellular counterparts. In recent years, the droplet interface bilayer (DIB) method2,3,4 for constructing a planar lipid bilayer between lipid-coated water droplets in oil has received significant attention5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23, and has demonstrated practical advantages over other approaches for model membrane formation: the DIB method is simple to perform, requires no sophisticated fabrication or preparation (e.g., "painting") of a substrate to support the membrane, consistently yields membranes with superior longevity, allows for standard electrophysiology measurements, and simplifies the formation of model membranes with asymmetric leaflet compositions3. Because the bilayer forms spontaneously between droplets and each droplet can be tailored in position and makeup, the DIB technique has also attracted considerable interest in developing cell-inspired material systems that build on the use of stimuli-responsive membranes18,24,25,26,27,28,29, balanced compartmentalization and transport14,30,31, and tissue-like materials17,23,32,33,34,35,36.
The majority of published experiments on model membranes, including those with DIBs, have been performed at room temperature (RT, ~20-25 °C) and with a handful of synthetic lipids (e.g., DOPC, DPhPC, etc.). This practice limits the scope of biophysical questions that can be studied in model membranes and, based on observation, it can also restrict the types of lipids that can be used to assemble DIBs. For example, a synthetic lipid such as DPPC, which has a melting temperature of 42 °C, does not assemble tightly-packed monolayers or form DIBs at RT37. DIB formation at room temperature has also proven difficult for natural extracts, such as those from mammals (e.g., brain total lipid extract, BTLE)38 or bacteria (e.g., Escherichia coli total lipid extract, ETLE)37, which contain many different types of lipids and originate from cells that reside at elevated temperatures (37 °C). Enabling study of diverse compositions thus provides opportunities to understand membrane-mediated processes in biologically relevant conditions.
Raising the temperature of the oil can serve two purposes: it increases the kinetics of monolayer assembly and it can cause lipids to undergo a melting transition to reach a liquid disordered phase. Both consequences aid in monolayer assembly39, a pre-requisite for a DIB. In addition to heating for bilayer formation, cooling the membrane after the formation can be used to identify thermotropic transitions in single lipid bilayers38, including those in natural lipid mixtures (e.g., BTLE) that can be difficult to detect using calorimetry. Aside from assessing thermotropic transitions of lipids, precisely varying the temperature of the DIB can be used to study temperature-induced changes in membrane structure38 and examine how lipid composition and fluidity affect the kinetics of membrane-active species (e.g., pore-forming peptides and transmembrane proteins37), including mammalian and bacterial model membranes at a physiologically relevant temperature (37 °C).
Herein, a description of how to assemble a modified DIB oil reservoir and operate a feedback-temperature controller to enable monolayer assembly and bilayer formation at temperatures higher than RT will be explained. Distinguished from a previous protocol40, explicit detail is included regarding the integration of instrumentation needed for measuring and controlling temperature in parallel to assembly and characterization of the DIB in the oil reservoir. The procedure will thus enable a user to apply this method for forming and studying DIBs across a range of temperatures in a variety of scientific contexts. Moreover, the representative results provide specific examples for the types of measurable changes in both membrane structure and ion transport that can occur as temperature is varied. These techniques are important additions to the many biophysical studies that can be designed and performed effectively in DIBs, including studying the kinetics of membrane-active species in different membrane compositions.
1. Heated fixture preparation
Figure 1: Heated stage assembly. Images show the assembly of the thermally conductive fixture and oil reservoir for DIB formation; numbers beneath each image identify the corresponding step of the protocol. Please click here to view a larger version of this figure.
2. Instrumentation for simultaneous feedback temperature control and electrical characterization of a DIB
NOTE: This protocol integrates the following instruments for enabling feedback temperature control and simultaneous electrical characterization of a DIB: a personal computer (PC) with two available universal serial bus (USB) connections, a patch clamp amplifier paired to a dedicated data acquisition (DAQ-1) system, a waveform generator, a second programable DAQ (DAQ-2) with voltage output and temperature input modules, and a power supply/amplifier. The following steps describe the necessary connections of these instruments (as illustrated in Figure 2a) needed for isolating the measurement and control of temperature from simultaneous electrophysiology of a DIB. Substitutions for equivalent instruments may be made as required.
Figure 2: System wiring connections. A schematic of the devices and wiring required for the system is shown in (a), while a detailed look at the DAQ-2 connections is provided in (b). The illustration in (c) shows aqueous droplets on hydrogel-coated electrodes submerged in oil for DIB formation. The two electrodes are connected to the grounded and ungrounded (V+) connections, respectively, on the headstage unit of the patch clamp amplifier. Please click here to view a larger version of this figure.
3. Feedback temperature control of droplet interface bilayers
NOTE: The following steps for operating the feedback temperature control system are based on a custom graphical user interface (GUI) created for implementing proportional-integral (PI) feedback temperature control40,41 (see Supplementary Coding Files). Other software and control algorithms may be used instead. A copy of this program is provided to the reader with the supplementary information for the paper, however the user is responsible to configure it for their own equipment and needs.
4. Characterization of temperature-dependent behaviors in DIBs
NOTE: Many physical processes can be studied in DIB-based model membranes, including how changes in temperature affect the structure and transport properties of the membrane. The following steps should be performed after successful bilayer formation at a desired temperature.
Figure 3: The temperature control GUI. This figure highlights and labels the critical steps required to use the program's GUI to control the temperature of the oil bath. Please click here to view a larger version of this figure.
Figure 1 shows how the aluminum fixture and acrylic oil reservoir are prepared on the microscope stage for DIB formation. Assembly steps 1.2-1.4 serve to thermally insulate the fixture from the stage for more efficient heating. Steps 1.5-1.7 show how to properly attach the thermocouple to the fixture and position the oil reservoir, and steps 1.8 -1.9 show recommended locations for dispensing oil into these pieces.
Figure 2 outlin...
The protocol described herein provides instructions for assembling and operating an experimental system to control the temperature of the oil and droplets used to form DIBs. It is especially beneficial for enabling DIB formation using lipids that have melting temperatures above RT. Moreover, by precisely varying the temperature of the oil reservoir, the bilayer temperature can be manipulated to study the effects of elevated temperatures on various membrane properties and characteristics, including capacitance, area, thic...
The authors have no conflicts of interests.
Financial support was provided by the National Science Foundation Grant CBET-1752197 and the Air Force Office of Scientific Research Grant FA9550-19-1-0213.
Name | Company | Catalog Number | Comments |
25 mm x 40 mm x 1 mm insulative rubber (x2) | Any | Insulates the bottom of the aluminum fixture from the stage of the microscope | |
25 mm x 40 mm x 6 mm insulative rubber (x2) | Any | Protects heating elements from being damaged by the microscope stage clips and insulates the top of the heating elements. | |
3-(N-morpholino) propanesulfonic acid | Sigma Aldrich | M3183 | Buffering agent for lipid solution |
Acrylic substrate | Fabricated in house | HTD_STG_2 | ~1000 uL acrylic well with a poka-yoke exterior profile to fix orientation |
Aluminum fixture | Fabricated in house | HTD_STG_1 | Base fixture with an oil well that holds the acylic fixture and includes two flat pads adjacent to the oil well for the heating elements |
Brain Total Lipid Extract | Avanti | 131101C-100mg | 25 mg/mL porcine lipid extract |
Compact DAQ Chassis (cDAQ) | National Instruments | cDAQ-9174 | Chassis to house multiple types of sensor measurement or output modules |
Data Acquisition System (DAQ) | Molecular Devices | Digidata 1440A | High resolution analog to digital converter |
Fixed gain amplifier/power supply | Hewlitt Packard | HP 6826A | Amplifies DC voltage output from the voltage output module |
Glass Cover Slip | Corning | CLS284525 | Seals bottom of aluminum base and allows for optical characterization of the bilayer |
Heating element (x2) | Omega | KHLV-101/5 | 25 mm x 25 mm polymide film kapton heating element with a 5 watt power limit. |
M3 Stainless Steel Screw | McMaster Carr | 90116A150 | Secures thermocouple to aluminum fixture |
Patch clamp amplifier | Molecular Devices | AxoPatch 200B | Measures current and outputs voltage to the headstage |
Personal computer | Any | Computer with mulitiple high speed usb ports and a minimum of 6 Gb of ram | |
Potassium Chloride | Sigma Aldrich | P3911 | Electrolyte solution of dissociated ions |
Temperature input module | National Instruments | NI 9211 | Enables open and cold junction thermocouple measurements for the cDAQ chassis |
Thermocouple | Omega | JMTSS-020U-6 | U-type thermocouple with a diameter of 0.02 inches and 6 inches in length |
UV Curable Adhesive | Loctite | 19739 | Secures glass coverslip to aluminum base fixture |
Voltage output module | National Instruments | NI 9263 | Analog voltage output module for use with the cDAQ chassis |
Waveform generator | Agilent | 33210A | Used to output a 10 mV 10 Hz sinusoidal waveform |
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