JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Bioingegneria

温度控制组件和液滴接口比莱耶的特征

Published: April 19, 2021
doi:
10.3791/62362
,
1Department of Mechanical, Aerospace and Biomedical Engineering,University of Tennessee

Summary

该协议详细说明了使用反馈温度控制加热系统来促进脂质单层组件和液滴接口双层形成，用于熔化温度升高的脂质，以及电容测量，以描述膜中温度驱动的变化。

Abstract

用于组装脂质双层的液滴接口双层 （DIB） 方法 （即， 与其它方法相比，油中脂质涂层水滴之间的 DIB 具有关键益处：DIB 是稳定的，而且通常具有持久性，双层区域可以逆向调整，传单不对称很容易通过液滴成分控制，并且通过相邻的许多液滴可以获得类似组织的双层水滴网络。形成 DIB 需要在液滴表面自发地将脂质组装成高密度脂质单层。虽然这在室温下很容易发生，但足够的单层或稳定的双层纤维不能在类似条件下形成，其熔点高于室温，包括一些细胞脂质提取物。这种行为可能限制了模型膜研究中DIB的组成——或许还有生物学相关性。为了解决这个问题，提出了一个实验方案，以仔细加热油藏托管DIB液滴，并描述温度对脂质膜的影响。具体来说，此协议展示了如何使用反馈环控制的热导电铝夹具和电阻加热元件来规定温度升高，从而改善单层组件和双层形成，用于更广泛的脂质类型。通过测量 DIB 电容的变化，可以量化膜的结构特征以及由双层纤维组成的脂质的温相过渡。该程序有助于评估模型膜中不同温度的生物物理现象，包括确定多成分脂质混合物的有效熔化温度（TM）。因此，这种能力将允许在模型膜中更密切地复制自然相位转换，并鼓励从更广泛的膜成分（包括那些能够更好地捕获其细胞对应体异质性）形成和使用模型膜。

Introduction

细胞膜是选择性渗透屏障，由数千种脂质类型1、蛋白质、碳水化合物和固醇组成，这些屏障封装和细分所有活细胞。因此，了解其组成如何影响其功能，揭示自然分子和合成分子如何与细胞膜相互作用、粘附、破坏和转移，是生物学、医学、化学、物理学和材料工程领域具有广泛影响的重要研究领域。

这些发现的目的直接受益于经过验证的组装、操纵和研究模型膜的技术，包括从合成或自然产生的脂质中组装的脂质双层，这些双层细胞模仿其细胞对应体的组成、结构和传输特性。近年来，在油脂涂层水滴之间构建平面脂质双层的液滴接口双层（DIB）方法2、3、4受到重视，5、6、7、8、9、10、11、11、12、13、14、15、16、17、18、19、20、21、22、23，在模型膜形成的其他方法上显示出实用优势：DIB方法操作简单， 不需要复杂的制造或准备（例如， “绘画”）的基板，以支持膜，一贯产生膜与优越寿命长，允许标准电生理学测量，并简化模型膜的形成与不对称的传单成分3。因为双层在液滴之间自发形成，每个液滴都可以在位置和妆容中量身定做， DIB技术也吸引了相当的兴趣，开发细胞启发材料系统，建立在使用刺激反应膜18，24，25，26，27，28，29、平衡分割和运输14、30、31和组织状材料17、23、32、33、34、35、36。

大多数已公布的模型膜实验，包括使用 DIB 的实验，都是在室温下（RT，+20-25 °C）和少量合成脂质（如 DOPC、DPHPC 等）进行的。这种做法限制了可在模型膜中研究的生物物理问题的范围，基于观察，它还可以限制可用于组装DIB的脂质类型。例如，合成脂质，如 DPPC，其熔化温度为 42 °C，不会在 RT37中组装紧密包装的单层或形成 DIB。室温下的DIB形成也被证明是困难的自然提取物，如那些来自哺乳动物（如大脑总脂质提取物，BTLE）38或细菌（如大肠杆菌总脂质提取物，ETLE）37，其中含有许多不同类型的脂质，并来自细胞，居住在高温（37°C）。因此，能够研究不同的成分，为了解生物相关条件下膜介介的过程提供了机会。

提高油的温度可以达到两个目的：它增加单层组件的动力学，并可能导致脂质经历融化过渡，以达到液体紊乱阶段。这两种后果都有助于单层组件39，这是 DIB 的先决条件。除了为双层形成加热外，形成后冷却膜还可用于识别单脂双层38中的热向过渡，包括天然脂质混合物（如BTLE）中难以使用热量测量检测的热向过渡。除了评估脂质的温电性过渡外， 精确改变DIB的温度可用于研究膜结构38 的温度诱发变化，并检查脂质组成和流动性如何影响膜活性物种的动力学（例如，孔隙形成肽和转膜蛋白37），包括哺乳动物和细菌模型膜在生理相关温度（37°C）。

在此，将解释如何组装经过改进的 DIB 油藏并操作反馈温度控制器，以便在高于 RT 的温度下实现单层组件和双层地层的形成。与以前的第40号议定书不同，在油藏DIB的组装和定性的同时，对测量和控制温度所需的仪器进行了整合，其中包括了明确的细节。因此，该过程将使用户能够应用此方法在各种科学环境中在各种温度下形成和研究 DIB。此外，具有代表性的结果为膜结构和离子传输中可能因温度变化而发生的可测量变化类型提供了具体示例。这些技术是许多生物物理研究的重要补充，这些研究可以在DIB中有效设计和执行，包括研究不同膜成分中膜活性物种的动力学。

Protocol

1. 加热夹具准备 收集2块1毫米厚的绝缘橡胶修剪为25毫米×40毫米的宽度和长度，分别2件6毫米厚的橡胶，也是25毫米×40毫米，一个预制的铝基夹具组装，和一个丙烯酸油藏，适合在铝基夹具的观景窗口（见 图S1，S2和S3 的制造细节和爆炸的组装视图）。首先将铝夹具连接到夹具底部，用紫外线可固化胶粘剂将玻璃盖片查看窗口连接起来，并将 1 个电阻加热元件固定在每根 25 mm x …

Representative Results

图1显示了铝夹具和丙烯酸油储层是如何在显微镜阶段为DIB形成准备的。装配步骤 1.2-1.4 用于将固定装置与舞台隔热，以便更高效地加热。步骤 1.5-1.7 显示如何正确地将热电偶连接到夹具上并定位油藏，步骤 1.8 -1.9 显示将油分配到这些部件中的建议位置。 图 2 概述用于建立反馈温度控制并在 DIB 上执行电气测量的组件：PC、固定增?…

Discussion

此处描述的协议为组装和操作用于形成 DIB 的油和液滴的温度提供了组装和操作实验系统的说明。它特别有利于使用温度高于RT的脂质形成DIB。此外，通过精确改变油藏的温度，可以操纵双层温度来研究温度升高对各种膜特性和特征的影响，包括电容、面积、厚度、诱导的温带相变化、膜活性物种的动力学以及双层界面37、38的粘附能量。

<p class="jove_c…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

财政支助由国家科学基金会赠款CBET-1752197和空军科学研究办公室赠款FA9550-19-1-0213提供。

Materials

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
DOWNLOAD MATERIALS LIST

Riferimenti

  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).

Tags

Droplet Interface BilayerDIBTemperature-controlled AssemblyCharacterizationMembraneHeat TransferThermocoupleAluminum FixtureAcrylic ReservoirHexadecane OilInsulative RubberResistive Heating ElementsElectrical/optical CharacterizationTemperature-related EffectsCellular EnvironmentsTransport And Signaling PropertiesMembrane ConstituentsNatural Cellular Extracts
check_url/it/62362?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Ringley, J. D., Sarles, S. A. Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer. J. Vis. Exp. (170), e62362, doi:10.3791/62362 (2021).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image