Published: April 19th, 2021
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 ....
1. Heated fixture preparation
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.......
|25 mm x 40 mm x 1 mm insulative rubber (x2)
|Insulates the bottom of the aluminum fixture from the stage of the microscope
|25 mm x 40 mm x 6 mm insulative rubber (x2)
|Protects heating elements from being damaged by the microscope stage clips and insulates the top of the heating elements.
|3-(N-morpholino) propanesulfonic acid
|Buffering agent for lipid solution
|Fabricated in house
|~1000 uL acrylic well with a poka-yoke exterior profile to fix orientation
|Fabricated in house
|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
|25 mg/mL porcine lipid extract
|Compact DAQ Chassis (cDAQ)
|Chassis to house multiple types of sensor measurement or output modules
|Data Acquisition System (DAQ)
|High resolution analog to digital converter
|Fixed gain amplifier/power supply
|Amplifies DC voltage output from the voltage output module
|Glass Cover Slip
|Seals bottom of aluminum base and allows for optical characterization of the bilayer
|Heating element (x2)
|25 mm x 25 mm polymide film kapton heating element with a 5 watt power limit.
|M3 Stainless Steel Screw
|Secures thermocouple to aluminum fixture
|Patch clamp amplifier
|Measures current and outputs voltage to the headstage
|Computer with mulitiple high speed usb ports and a minimum of 6 Gb of ram
|Electrolyte solution of dissociated ions
|Temperature input module
|Enables open and cold junction thermocouple measurements for the cDAQ chassis
|U-type thermocouple with a diameter of 0.02 inches and 6 inches in length
|UV Curable Adhesive
|Secures glass coverslip to aluminum base fixture
|Voltage output module
|Analog voltage output module for use with the cDAQ chassis
|Used to output a 10 mV 10 Hz sinusoidal waveform
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