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.

The authors have no conflicts of interests.

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, thickness, induced thermotropic-phase changes, kinetics of membrane-active species, and the energetics of adhesion of the bilayer interface37,38.
The protocol consists of three parts prior to use in a DIB study: 1) preparation and assembly of the heated stage fixture; 2) connecting the various instruments; and 3) confirming suitable temperature control performance with the chosen proportional and integral control gains. Most important in part 2, the user must make sure to avoid shared conduction paths between the output of power amplifier (&#62;mA currents) and the patch clamp headstage (pA-nA currents). An inadvertent short could cause permanent damage to the headstage. Additionally, ensuring that the PC and all instruments are connected to a common AC power ground, and the use of a grounded Faraday cage near the headstage and droplet electrodes helps to minimize noise in bilayer current measurements. After setup in part 2 is complete, the user must first assess the open-loop heating response of the oil reservoir by applying a fixed voltage to the heating elements and recording the subsequent rise in temperature (like shown in Figure 4a). This type of exponential response can be used to define and simulate a simple model of the closed-loop system for varying values of control gains (see Figure S4 for details). The control gains reported herein allow the system to heat to a desired temperature level quickly (~ 2 minutes) and with little overshoot and maintain set point value accurately. But the specific gains required will depend on the power level of the heating elements as well as the geometry of the fixture that supports the oil reservoir. Once suitable values of control gains are determined and the feedback control system operates as desired, the user may then begin to assemble and characterize a DIB.
The protocol does not change the process of DIB formation or characterization, however there are limitations and considerations. Raising the temperature of the oil can affect how droplets hang on the electrodes, due to reductions in monolayer tension and oil density that increase droplet sagging and convective currents in the oil that can move the droplets. Hence, the protocol suggests lowering the tips of the electrodes to near the bottom surface of the substrate such that droplets are supported and held still by the acrylic reservoir. The user should assess how much the substrate may be distorting the droplets (if lowered too far), and consider this distortion when calculating of the area of the bilayer from images of DIBs as discussed elsewhere22.
While the described system is limited to heating of the oil bath, a Peltier cooling device could be used in place of the resistive heating elements if testing at temperatures below RT is needed. In this case, however, the user will need to consider the freezing point of the oil phase. Many alkanes freeze at temperatures higher that 0 &#176;C; hexadecane described herein freezes at 18 &#176;C. If the oil freezes, droplets will no longer be moveable and a bilayer between droplets may become unstable or rupture.
For a previously untested lipid composition, key unknowns are the incubation time and temperature required to enable sufficient monolayer assembly at the surfaces of the droplets. The general rule is to heat the oil to a temperature above TM, where lipid mobility is enhanced allowing for faster lateral diffusion and tighter packing at the oil-water interface45, and wait long enough such that monolayer packing at the oil-water interface is high. The user may review published literature or consider their own complementary measurements to determine suitable time and temperature values: interfacial tension measurements on a pendant drop goniometer can be used to assess the time required for monolayer assembly46&#160;and differential scanning calorimetry is often used to identify thermotropic transitions of lipids38. Or an iterative approach may be pursued to identify suitable time and temperature where bilayer formation is consistent, the membrane is stable for more than a few minutes, and the resistance of the bilayer is &#62;1 G&#937;. In recent studies with E. coli total lipid extract (ETLE)37 and BTLE38,47&#160;a starting temperature &#62;50 &#176;C consistently leads to stable bilayer formation. Similarly, the minimum stable temperature after DIB for a given lipid type may also vary between lipid selections. For example ETLE DIBs can be cooled to 25 &#176;C37, whereas single component DPPC DIBs always coalesced below TM~40 &#176;C38. Observation has shown that BTLE DIBS show that 27 &#176;C is a safe minimum temperature for maintaining a stable bilayer.
Our representative results show that changes in temperature can greatly affect the properties of the resulting DIB. The data in Figure 5 shows that the nominal capacitance of the membrane decreases as temperature rises. Because capacitance, C, is directly proportional to bilayer area, A, and inversely proportional to thickness, d, as given by
<img alt="Equation 1" src="/files/ftp_upload/62362/62362eq.jpg" />, (1)
a decrease in C can be manifest by a decrease in A, an increase in d, or both (assuming a fixed dielectric permittivity, &#949;). These relationships motivate the use of capacitance measurements and DIB images to assess changes in C, A, and Cm&#160;versus temperature to determine which effects are significant. The data included in Figure 5 and Figure 6 for BTLE DIBs shows that both C and Cm (which represents the ratio &#949;/d) decrease by nearly 50% as temperature rises from 30 C to 60 C. Together, these indicate that higher temperature thickens the bilayer, due to an increased solubility of the acyl chains of the lipids in hexadecane48. The additional oil in the membrane can also affect the interfacial tension of the bilayer and the contact angle between droplets22,38. These effects can be quantified by analyzing images of a DIB at user-specified time intervals to monitor bilayer area and contact angle during heating and cooling.
The temperature favorability of oil in the membrane can also be used to assess thermotropic melting temperatures of the lipids and affect ion channel kinetics. The melting temperature for a lipid mixture can be defined by locating nonmonotonic changes in C versus T relationships as in Figure 6. The current measurements in Figure 7 further reveal that temperature-induced changes in phase (i.e. fluidity) and thickness can impact the threshold voltage for insertion of ionophores like Mz. These physical associations are important for understanding ion channel behaviors in model membranes, especially in scenarios aimed at replicating body temperature environments. However, they may also be useful for tuning the conductivity of the bilayer in applications such as neuromorphic computing devices47&#160;For example, increased channel kinetics are a desirable feature when fabricating devices exhibiting memory resistance that need to mimic the speed, functionality, and short-term plasticity of the brain.

Cellular membranes are selectively permeable barriers comprised of&#160;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&#8212;including lipid bilayers assembled from synthetic or naturally occurring lipids&#8212;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., &#34;painting&#34;) 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 &#176;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 &#176;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 &#176;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&#160;(37 &#176;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.

<table><tbody><tr><td>25 mm x 40 mm x 1 mm insulative rubber (x2)</td><td>Any</td><td></td><td>Insulates the bottom of the aluminum fixture from the stage of the microscope</td></tr><tr><td>25 mm x 40 mm x 6 mm insulative rubber (x2)</td><td>Any</td><td></td><td>Protects heating elements from being damaged by the microscope stage clips and insulates the top of the heating elements.</td></tr><tr><td>3-(N-morpholino) propanesulfonic acid&amp;nbsp;</td><td>Sigma Aldrich</td><td>M3183</td><td>Buffering agent for lipid solution</td></tr><tr><td>Acrylic substrate</td><td>Fabricated in house</td><td>HTD_STG_2</td><td>~1000 uL acrylic well with a poka-yoke exterior profile to fix orientation</td></tr><tr><td>Aluminum fixture</td><td>Fabricated in house</td><td>HTD_STG_1</td><td>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&amp;nbsp;</td></tr><tr><td>Brain Total Lipid Extract</td><td>Avanti</td><td>131101C-100mg</td><td>25 mg/mL porcine lipid extract&amp;nbsp;</td></tr><tr><td>Compact DAQ Chassis (cDAQ)</td><td>National Instruments&amp;nbsp;</td><td>cDAQ-9174&amp;nbsp;</td><td>Chassis to house multiple types of sensor measurement or output modules</td></tr><tr><td>Data Acquisition System (DAQ)</td><td>Molecular Devices&amp;nbsp;</td><td>Digidata 1440A&amp;nbsp;</td><td>High resolution analog to digital converter</td></tr><tr><td>Fixed gain amplifier/power supply</td><td>Hewlitt Packard</td><td>HP 6826A</td><td>Amplifies DC voltage output from the voltage output module</td></tr><tr><td>Glass Cover Slip</td><td>Corning</td><td>CLS284525</td><td>Seals bottom of aluminum base and allows for optical characterization of the bilayer</td></tr><tr><td>Heating element (x2)</td><td>Omega</td><td>KHLV-101/5</td><td>25 mm x 25 mm polymide film kapton heating element with a 5 watt power limit.&amp;nbsp;</td></tr><tr><td>M3 Stainless Steel Screw</td><td>McMaster Carr</td><td>90116A150</td><td>Secures thermocouple to aluminum fixture</td></tr><tr><td>Patch clamp amplifier</td><td>Molecular Devices&amp;nbsp;</td><td>AxoPatch 200B&amp;nbsp;</td><td>Measures current and outputs voltage to the headstage</td></tr><tr><td>Personal computer</td><td>Any</td><td></td><td>Computer with mulitiple high speed usb ports and a minimum of 6 Gb of ram</td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P3911</td><td>Electrolyte solution of dissociated ions</td></tr><tr><td>Temperature input module</td><td>National Instruments&amp;nbsp;</td><td>NI 9211</td><td>Enables open and cold junction thermocouple measurements for the cDAQ chassis</td></tr><tr><td>Thermocouple</td><td>Omega</td><td>JMTSS-020U-6&amp;nbsp;</td><td>U-type thermocouple with a diameter of 0.02 inches and 6 inches in length</td></tr><tr><td>UV Curable Adhesive</td><td>Loctite</td><td>19739</td><td>Secures glass coverslip to aluminum base fixture</td></tr><tr><td>Voltage output module</td><td>National Instruments&amp;nbsp;</td><td>NI 9263</td><td>Analog voltage output module for use with the cDAQ chassis</td></tr><tr><td>Waveform generator</td><td>Agilent</td><td>33210A&amp;nbsp;</td><td>Used to output a 10 mV 10 Hz sinusoidal waveform</td></tr></tbody></table>

temperature-controlled assembly and characterization of a droplet interface bilayer

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&#8212;and perhaps the biological relevance&#8212;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.

Figure 1&#160;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 outlines the components used to establish feedback temperature control and perform electrical measurements on a DIB: a PC, a fixed gain power amplifier, a patch clamp amplifier and a DAQ system (or equivalent instrument for applying voltage and measuring pA-nA level currents), a second DAQ with appropriate analog inputs and outputs, a waveform generator, and the assembled aluminum fixture with attached resistive heaters. DAQ-2 makes use of two modules (Figure 2b). A 4-channel, &#177;10 V, 16-bit analog voltage output module is used to initiate the independent voltages supplied to the input of the power amplifier (blue connection in Figure 2a) and an External Command input on the patch clamp amplifier (green connection). The voltage output module is limited by a maximum output current of 46 mA and a maximum output voltage of 10 V, whereas each heating element used herein consumes as much as 5 W of power (~180 mA max) at a maximum voltage of 28 V. For this reason, the power supply/amplifier was included to pre-amplify the output voltage and supplement the supplied current needed for powering the heating elements (wired in parallel) attached to the aluminum fixture. A 4-channel, 24-bit thermocouple input device is used to digitize temperature measurements from the oil reservoir near the DIB (yellow connection). Since the thermocouple input device module allows up to 4 thermocouples, the user may consider monitoring temperatures at other locations in the fixture. If done, they will also need to consider which signal or combination of signals is used for comparison to the desired set point temperature within the feedback loop.
These outputs and measured signals are controlled via two software: 1) the custom GUI for temperature control; and 2) electrophysiology measurement software. Figure 3 shows a screenshot of the GUI and includes annotations to corresponding steps in the protocol. The GUI is used to define key parameters (Set Point temperature, PI control gains, voltage limits), compare the measured temperature to the set point temperature and compute the control signal supplied to the amplifier and then the heating elements, and record data of the temperature and applied voltage versus time. This program also includes the ability to command the voltage applied to DIB electrodes (Figure 2c) via the patch clamp amplifier. Separately, measurement software is used to configure measurements of both the voltage applied to the DIB electrodes and the induced current through the lipid bilayer. A voltage proportional to DIB current is output by the patch clamp amplifier and sent via BNC cable to DAQ-1 (connection not shown).
Figure 4&#160;plots the change in temperature and absolute electrical power sent to the heaters versus time under both open-loop and closed-loop heating scenarios. For the former, an arbitrary input voltage corresponding to ~5.2 W of power was applied to the heaters, which resulted in an exponential rise in temperature with a time constant of ~125 s and a steady state&#160;&#916;T &#8776; 4.5 &#176;C/W after an initial delay of ~20 s. These characteristics of the open-loop system were used to construct a model of the closed-loop system in a simulation software (see Figure S4 for details) that can be used to determine values for the proportional and integral control gains. The closed-loop and simulated model responses in Figure 4 thus represent the measured and simulated responses of the tuned PI controller, with KP and KI values of 0.598 and 0.00445, respectively, to a set point temperature 20 &#176;C higher than RT. Compared to the open-loop case, both the simulation and the measurements confirm the increased speed of response in the closed-loop system (time constant ~63 s). The reduction in heating time comes at the expense of higher initial applied power. Still, the desired set point temperature and the measured oil temperature remained within 0.6 &#176;C at steady state, which was deemed suitable for use. Total supplied power is limited within the program during closed-loop control so as not to eclipse the 10 W total power limit for the two heaters.
The temperature control system was used to showcase the temperature-dependence of solvent in a DIB formed from BTLE lipids and its impact on membrane capacitance (Figure 5). BTLE lipids were chosen for this measurement because heating is required for DIB formation due to a lipid phase transition that occurs between 35-42 &#176;C38. The protocol described herein was performed to initiate bilayer formation at 60 &#176;C. Following membrane formation and equilibration, the temperature can be successively lowered or raised as needed to characterize the response of the membrane. For example, Figure 5a shows representative measurements of raw capacitive current (square-shaped waveform) and temperature versus time during a heating cycle from RT to ~60 &#176;C. Observe that the amplitude of the capacitive current waveform reduces by more than half as temperature rises, which is caused by the uptake of oil into the hydrophobic core of the membrane. This change thickens the interface and alters the lateral tension of the bilayer22,37,38.
The data in Figure 5b document changes in C (normalized by the capacitance at 27 &#176;C) versus T across one complete cooling-heating cycle after initial bilayer formation at 60 &#176;C. Just like in Figure 5a, as temperature rises, capacitance drops. However, what this presentation shows more clearly are the nonmonotonic changes that occur at temperatures between ~30-42 &#176;C, which represents the collective melting temperature, TM, during which the lipid mixture transitions between a liquid-ordered and a liquid-disordered thermotropic phase. The temperature where the nonmonotonic change in capacitance occurs corresponds to a change in bilayer thickness from exclusion of oil from the membrane38. Also, note that the hysteresis shown between the heating cycle and the cooling cycle is due to irreversible changes in bilayer area that occur between subsequent cycles, which were typically performed 10 min apart.
Similarly, Figure&#160;6a,b show how quasi-static measurements of Cm at different temperatures can be used to identify TM. Here, the area of the membrane is varied successively by manually increasing the distance between the droplet electrodes. During this experiment, the droplets are first pushed together to promote maximum membrane area prior to subsequent reductions in contact area with stepwise separations between the electrodes. At each level of contact, the nominal capacitance of the bilayer is assessed from the induced current and its area is determined through image analysis. Plotting C versus A allows for a linear regression, where the slope represents the value of Cm as shown in Figure 6a. Repeating this procedure across multiple temperatures (Figure 6b) shows that Cm decreases by nearly 50% at temperatures above TM, confirming an increase in the hydrophobic thickness of the membrane due to heating-induced hexadecane uptake (see Figure S5 for complete C versus A data). At higher temperatures, the additional solvent in the membrane also reduces the maximum contact area between the droplets, and thus maximum nominal capacitance. Reducing the temperature reverses these effects. The DIB image in Figure 6c shows that when the temperature (25 &#176;C) is well below TM, the membrane can stably adopt a highly adhesive state-even under the tension of stretched droplets caused by well-separated electrodes. This is the result of complete exclusion of hexadecane from the bilayer, which increases the adhesion energy of the droplets. In this state, the bilayer area cannot be reliably changed through manipulation of the electrodes and hinders the ability to accurately measure specific capacitance (see Figure S5 for more details).
Finally, the representative data in Figure 7 show how temperature changes can affect the behaviors of pore-forming species that create ion conducting channels through a DIB. Monazomycin (Mz), a positively-charged antibiotic that forms cation selective channels through the bilayer at sufficient transmembrane potentials37,44, was chosen to demonstrate this relationship. These measurements were conducted on a BTLE-based (2 mg/mL final concentration in both droplets) DIB doped with Mz (1 &#181;g/mL final concentration in both droplets). The current versus voltage traces shown in Figure 7a were obtained by applying sinusoidal membrane voltages and measuring the induced current at two different temperatures; the arrows and subsequent numbers in Figure 7a aid in visualizing the successive quarters of the sinusoidal voltage with respect to time. This type of measurement is often performed to examine the voltage-dependence of current through ion channels. The data here show that increasing the temperature of the DIB from 27 &#176;C to 45 &#176;C causes the threshold for channel formation to rise from ~|100 mV| to ~|110 mV|. This change, likely driven by the higher membrane thickness due to absorbed oil, shows that the energy barrier for insertion has risen. The hysteresis in these curves-which signifies memory resistance-can be caused by voltage-induced changes in either bilayer area or the kinetics of Mz channel formation and inactivation44.
To help separate these factors in DIBs, transient changes in ion current can be measured in response to a DC step voltage. Figure 7b shows the measured current density for the same Mz-doped BTLE membrane at the same voltage level (+90 mV) and two different temperatures (27 &#176;C and 45 &#176;C). The data show clearly that the kinetics of the channel responses are quite different. Notably, at 27 &#176;C, the membrane exhibits a faster, larger increase in current that is then followed by a transient decay (the latter is a result of Mz channels translocating across the bilayer to an inactive state44). The response is much more muted at 45 &#176;C, where the S-shaped rise in current is not proceeded by a subsequent drop. Differences such as these are helpful for assessing the kinetics of channel responses and understanding how these could contribute to the total dynamic resistance of the membrane.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62362/62362fig04.jpg" /> 
Figure 4: Open-loop versus closed-loop heating. Panel (a) compares the time responses for the measured and simulated (See SI) closed-loop system to a +20 &#176;C temperature step to the open-loop heating response under fixed applied power. Panel (b)&#160;displays the power dissipated by each system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/62362fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62362/62362fig05.jpg" /> 
Figure 5: Measuring capacitance and varying temperature. The typical square waveform current response to a 10 mV, 10 Hz triangular waveform input on a BTLE lipid membrane undergoing a phase transition is shown in (a). The phase transition of the lipids can also be seen in the area measurement data displayed above panel (a). Capacitance normalized by initial capacitance at 27 &#176;C is shown in panel (b) plotted as a function of temperature for a heating and cooling cycle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/62362fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62362/62362fig06.jpg" /> 
Figure 6: Specific capacitance measurements Panel (a) shows nominal capacitance versus bilayer area obtained at successive contact areas for two different temperatures. Linear regressions to each set are used to determine their respective values of CM. Panel (b) plots&#160;CM versus T, while panel (c) shows the stable capacitive current waveform (left) and contact area (right) under attempted droplet separation at 25 &#176;C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/62362fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62362/62362fig07.jpg" /> 
Figure 7: Voltage-dependent membrane resistance and Mz ion-channel kinetics versus temperature. Panel (a)&#160;shows how the current-voltage relationship changes with temperature for BTLE DIBs formed between droplets containing 1 &#181;g/ml Mz. The arrows and numbers represent the successive portions of the applied sine wave. The differences in these traces illustrate how temperature shifts the voltage threshold for Mz insertion, which is identified as the magnitude of the voltage where induced current increases sharply. Likewise, panel (b) shows the impact temperature has on the transient current response induced by a DC step voltage of 90 mV. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/62362fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure S1: Aluminum fixture. This drawing shows the necessary dimensions and features for fabrication of the aluminum fixture that is the base of the heated stage. The 25.2 mm X 26 mm flat spots adjacent to the oil well were designed to allow for a maximal amount of surface-area contact between the fixture and the heating elements for heat conduction. Likewise, aluminum was chosen for the base fixture material due to its high thermal conductivity. The M3 X 0.5 mm screw hole called out in the print is used to secure and position the thermocouple in the oil well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/FigureS1.docx" target="_blank">Please click here to download this File.</a>
Figure S2: Acrylic substrate. The acrylic substrate is relatively simple piece to fabricate, with no critical outstanding features, except for the profile. The exterior profile was designed with Poka -yoke in mind so the acrylic substrate can only be oriented in the fixture in such a way to allow ample room for the thermocouple to fit in the oil well.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/FigureS2.docx" target="_blank">Please click here to download this File.</a>
Figure S3: Heated stage assembly. An exploded view of the assembled heated stage has been provided to aid the experimenter during initial setup. Also, take note of the area highlighted by the dashed circle, as this is the ideal position to fill the aluminum fixture with oil during protocol step 1.8.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/FigureS3.docx" target="_blank">Please click here to download this File.</a>
Figure S4: Open loop data and Simulink modeling panel. (a) shows the open-loop temperature responses to varying dc power levels that were used to assess the delay time, td, the time constant, &#964;, and open-loop heating gain, &#945;, of the system. The delay time represents the time lag before temperature starts rising (~20 s). Each value of &#964; (marked by *, ~125 s) is defined as the time required for 63.2% of the total rise in temperature to occur. Panel (b) shows the steady state change in temperature (&#8710;T) versus the applied power. The slope of the data plotted in (b) was used to compute the &#945;, which represents the ratio of temperature change per supplied power. These parameters were used in the model shown in panel (c) and provided as a supplementary file to tune the PI controller to achieve a desired closed-loop temperature control response.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/FigureS4.docx" target="_blank">Please click here to download this File.</a>
Figure S5: Additional specific capacitance data. The plots shown in Figure 6a,b were compiled from this CM data set. This plot also showcases the inability to accurately measure capacitance at temperatures of 25 &#176;C and below, therefore this measurement was excluded from the dataset. The area changes necessary for an accurate Cm measurement require excessive force to be applied to the droplets from the micromanipulators, which causes sever distortion of the droplets shape and area of contact. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/FigureS5.docx" target="_blank">Please click here to download this File.</a>
Supplementary Coding Files. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62362/supplementary coding files.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer at JoVE.com

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

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 ...

temperature-controlled-assembly-characterization-droplet-interface

Nanyang Technological University

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3.6K Views.  University of Tennessee. 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.

Video: Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Chemistry

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Biochemistry

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 &#181;m in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

Neuroscience

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...