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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

Published: April 19th, 2021

DOI:

10.3791/62362

1Department of Mechanical, Aerospace and Biomedical Engineering, 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.

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 ....

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1. Heated fixture preparation

  1. Gather 2 pieces of 1 mm thick insulative rubber trimmed to 25 mm x 40 mm in width and length, respectively, 2 pieces of a 6 mm-thick rubber that are also 25 mm x 40 mm, a prepared aluminum base fixture assembly, and an acrylic oil reservoir that fits in the viewing window of the aluminum base fixture (see Figures S1, S2, and S3 for details on fabrication and an exploded view of assembly). Prepare the aluminum fixture first by attaching to the bottom of the fixtur.......

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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.......

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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.......

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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.

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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

  1. van Meer, G., de Kroon, A. I. P. M. Lipid map of the mammalian cell. Journal of Cell Science. 124 (1), 5-8 (2011).
  2. Bayley, H., et al. Droplet interface bilayers. Molecular BioSystems. 4 (12), 1191-1208 (2008).
  3. Hwang, W. L., Chen, M., Cronin, B., Holden, M. A., Bayley, H. Asymmetric droplet interface bilayers. Journal of the American Chemical Society. 130 (18), 5878-5879 (2008).
  4. Holden, M. A., Needham, D., Bayley, H. Functional bionetworks from nanoliter water droplets. Journal of the American Chemical Society. 129 (27), 8650-8655 (2007).
  5. Sarles, S. A., Leo, D. J. Physical encapsulation of droplet interface bilayers for durable, portable biomolecular networks. Lab on a Chip. 10 (6), 710-717 (2010).
  6. Stanley, C. E., et al. A microfluidic approach for high-throughput droplet interface bilayer (DIB) formation. Chemical Communications. 46 (10), 1620-1622 (2010).
  7. Gross, L. C. M., Heron, A. J., Baca, S. C., Wallace, M. I. Determining membrane capacitance by dynamic control of droplet interface bilayer area. Langmuir. 27 (23), 14335-14342 (2011).
  8. Huang, J., Lein, M., Gunderson, C., Holden, M. A. Direct quantitation of peptide-mediated protein transport across a droplet, interface bilayer. Journal of the American Chemical Society. 133 (40), 15818-15821 (2011).
  9. Leptihn, S., Thompson, J. R., Ellory, J. C., Tucker, S. J., Wallace, M. I. In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers. Journal of the American Chemical Society. 133 (24), 9370-9375 (2011).
  10. Castell, O. K., Berridge, J., Wallace, M. I. Quantification of membrane protein inhibition by optical ion flux in a droplet interface bilayer array. Angewandte Chemie International Edition. 51 (13), 3134-3138 (2012).
  11. Dixit, S. S., Pincus, A., Guo, B., Faris, G. W. Droplet shape analysis and permeability studies in droplet lipid bilayers. Langmuir. 28 (19), 7442-7451 (2012).
  12. Elani, Y., deMello, A. J., Niu, X., Ces, O. Novel technologies for the formation of 2-D and 3-D droplet interface bilayer networks. Lab on a Chip. 12 (18), 3514-3520 (2012).
  13. Michalak, Z., Fartash, D., Haque, N., Lee, S. Tunable crystallization via osmosis-driven transport across a droplet interface bilayer. CrystEngComm. 14 (23), 7865-7868 (2012).
  14. Punnamaraju, S., You, H., Steckl, A. J. Triggered release of molecules across droplet interface bilayer lipid membranes using photopolymerizable lipids. Langmuir. 28 (20), 7657-7664 (2012).
  15. Boreyko, J. B., Mruetusatorn, P., Sarles, S. A., Retterer, S. T., Collier, C. P. Evaporation-induced buckling and fission of microscale droplet interface bilayers. Journal of the American Chemical Society. 135 (15), 5545-5548 (2013).
  16. Leptihn, S., et al. Constructing droplet interface bilayers from the contact of aqueous droplets in oil. Nature Protocols. 8 (6), 1048-1057 (2013).
  17. Villar, G., Graham, A. D., Bayley, H. A Tissue-like printed material. Science. 340 (6128), 48-52 (2013).
  18. Barriga, H. M. G., et al. Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli. Journal of The Royal Society Interface. 11 (98), (2014).
  19. Boreyko, J. B., Polizos, G., Datskos, P. G., Sarles, S. A., Collier, C. P. Air-stable droplet interface bilayers on oil-infused surfaces. Proceedings of the National Academy of Sciences. 111 (21), 7588-7593 (2014).
  20. Mruetusatorn, P., et al. Dynamic morphologies of microscale droplet interface bilayers. Soft Matter. 10 (15), 2530-2538 (2014).
  21. Najem, J., Dunlap, M., Sukharev, S., Leo, D. J. The gating mechanism of mechanosensitive channels in droplet interface bilayers. MRS Proceedings. , 1755 (2015).
  22. Taylor, G. J., Venkatesan, G. A., Collier, C. P., Sarles, S. A. Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer. Soft Matter. 11 (38), 7592-7605 (2015).
  23. Bayley, H., Cazimoglu, I., Hoskin, C. E. G. Synthetic tissues. Emerging Topics in Life Sciences. 3 (5), 615-622 (2019).
  24. Oliver, A. E., et al. Protecting, patterning, and scaffolding supported lipid membranes using carbohydrate glasses. Lab on a Chip. 8 (6), 892-897 (2008).
  25. Maglia, G., et al. Droplet networks with incorporated protein diodes show collective properties. Nature Nanotechnology. 4 (7), 437-440 (2009).
  26. Najem, J. S., et al. Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers. Scientific Reports. 5, 13726 (2015).
  27. Freeman, E. C., Najem, J. S., Sukharev, S., Philen, M. K., Leo, D. J. The mechanoelectrical response of droplet interface bilayer membranes. Soft Matter. 12 (12), 3021-3031 (2016).
  28. Tamaddoni, N., Sarles, S. A. Toward cell-inspired materials that feel: measurements and modeling of mechanotransduction in droplet-based, multi-membrane arrays. Bioinspiration & Biomimetics. 11 (3), 036008 (2016).
  29. Restrepo Schild, V., et al. Light-patterned current generation in a droplet bilayer array. Scientific Reports. 7, 46585 (2017).
  30. Milianta, P. J., Muzzio, M., Denver, J., Cawley, G., Lee, S. Water permeability across symmetric and asymmetric droplet interface bilayers: Interaction of cholesterol sulfate with DPhPC. Langmuir. 31 (44), 12187-12196 (2015).
  31. Mruetusatorn, P., et al. Control of membrane permeability in air-stable droplet interface bilayers. Langmuir. 31 (14), 4224-4231 (2015).
  32. Wauer, T., et al. Construction and manipulation of functional three-dimensional droplet networks. ACS Nano. 8 (1), 771-779 (2013).
  33. Bayley, H. Building blocks for cells and tissues: Beyond a game. Emerging Topics in Life Sciences. 3 (5), 433-434 (2019).
  34. Booth, M., Restrepo Schild, V., Downs, F., Bayley, J. Droplet network, from lipid bilayer to synthetic tissues. Encyclopedia of Biophysics. , (2019).
  35. Booth, M. J., Cazimoglu, I., Bayley, H. Controlled deprotection and release of a small molecule from a compartmented synthetic tissue module. Communications Chemistry. 2 (1), 142 (2019).
  36. Gobbo, P., et al. Programmed assembly of synthetic protocells into thermoresponsive prototissues. Nature Materials. 17 (12), 1145-1153 (2018).
  37. Taylor, G. J., Sarles, S. A. Heating-enabled formation of droplet interface bilayers using escherichia coli total lipid extract. Langmuir. 31 (1), 325-337 (2015).
  38. Taylor, G. J., et al. Capacitive detection of low-enthalpy, higher-order phase transitions in synthetic and natural composition lipid membranes. Langmuir. 33 (38), 10016-10026 (2017).
  39. Lee, S., Kim, D. H., Needham, D. Equilibrium and dynamic interfacial tension measurements at microscopic interfaces using a micropipet technique. 2. Dynamics of phospholipid monolayer formation and equilibrium tensions at the water-air interface. Langmuir. 17 (18), 5544-5550 (2001).
  40. Najem, J. S., et al. Assembly and characterization of biomolecular memristors consisting of ion channel-doped lipid membranes. Journal of Visualized Experiments. (145), e58998 (2019).
  41. Wang, Y. G., Shao, H. H. Optimal tuning for PI controller. Automatica. 36 (1), 147-152 (2000).
  42. Needham, D., Haydon, D. A. Tensions and free energies of formation of "solventless" lipid bilayers. Measurement of high contact angles. Biophysical Journal. 41 (3), 251-257 (1983).
  43. Sarles, S. A., Leo, D. J. Physical Encapsulation of Interface Bilayers for durable portable biolayer network. Lab on a Chip. 10 (6), 710-717 (2010).
  44. Muller, R. U., Peskin, C. S. The kinetics of monazomycin-induced voltage-dependent conductance. II. Theory and a demonstration of a form of memory. The Journal of General Physiology. 78 (2), 201-229 (1981).
  45. Nenninger, A., et al. Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli. Molecular Microbiology. 92 (5), 1142-1153 (2014).
  46. Venkatesan, G. A., et al. Adsorption kinetics dictate monolayer self-assembly for both lipid-in and lipid-out approaches to droplet interface bilayer formation. Langmuir. 31 (47), 12883-12893 (2015).
  47. Najem, J. S., et al. Memristive ion channel-doped biomembranes as synaptic mimics. ACS Nano. 12 (5), 4702-4711 (2018).
  48. Tamaddoni, N., Taylor, G., Hepburn, T., Michael Kilbey, S., Sarles, S. A. Reversible, voltage-activated formation of biomimetic membranes between triblock copolymer-coated aqueous droplets in good solvents. Soft Matter. 12, 5096-5109 (2016).

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