Financial support was provided by the National Science Foundation Grant NSF ECCS-1631472. Research for G.J.T., C.D.S., A.B., and C.P.C. was partially sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U.S. Department of Energy. A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility.&#160;

<ol>
	<li>Thompson, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+learning+and+memory.">The neurobiology of learning and memory.</a> Science. 233 (4767), 941-947 (1986).</li><li>Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+systems+of+the+brain:+a+brief+history+and+current+perspective.">Memory systems of the brain: a brief history and current perspective.</a> Neurobiology of learning and memory. 82 (3), 171-177 (2004).</li><li>Benfenati, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+and+the+neurobiology+of+learning+and+memory.">Synaptic plasticity and the neurobiology of learning and memory.</a> Acta Bio Medica Atenei Parmensis. 78 (1Suppl), 58-66 (2007).</li><li>Marx, G., Gilon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+memory.">The molecular basis of memory.</a> ACS Chemical Neuroscience. 9 (8), 633-642 (2012).</li><li>Izquierdo, I., Medina, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+formation:+the+sequence+of+biochemical+events+in+the+hippocampus+and+its+connection+to+activity+in+other+brain+structures.">Memory formation: the sequence of biochemical events in the hippocampus and its connection to activity in other brain structures.</a> Neurobiology of learning and memory. 68 (3), 285-316 (1997).</li><li>Merolla, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+million+spiking-neuron+integrated+circuit+with+a+scalable+communication+network+and+interface.">A million spiking-neuron integrated circuit with a scalable communication network and interface.</a> Science. 345 (6197), 668-673 (2014).</li><li>Benjamin, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogrid:+A+mixed-analog-digital+multichip+system+for+large-scale+neural+simulations.">Neurogrid: A mixed-analog-digital multichip system for large-scale neural simulations.</a> Proceedings of the IEEE. 102 (5), 699-716 (2014).</li><li>Furber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+neuromorphic+computing+systems.">Large-scale neuromorphic computing systems.</a> Journal of neural engineering. 13 (5), 051001 (2016).</li><li>Di Ventra, M., Pershin, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parallel+approach.">The parallel approach.</a> Nature Physics. 9 (4), 200-202 (2013).</li><li>Chua, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memristor-the+missing+circuit+element.">Memristor-the missing circuit element.</a> IEEE Transactions on circuit theory. 18 (5), 507-519 (1971).</li><li>Di Ventra, M., Pershin, Y. V., Chua, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit+elements+with+memory:+memristors,+memcapacitors,+and+meminductors.">Circuit elements with memory: memristors, memcapacitors, and meminductors.</a> Proceedings of the IEEE. 97 (10), 1717-1724 (2009).</li><li>Najem, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memristive+Ion+Channel-Doped+Biomembranes+as+Synaptic+Mimics.">Memristive Ion Channel-Doped Biomembranes as Synaptic Mimics.</a> ACS Nano. , (2018).</li><li>Strukov, D. B., Snider, G. S., Stewart, D. R., Williams, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+missing+memristor+found.">The missing memristor found.</a> Nature. 453 (7191), 80-83 (2008).</li><li>Prezioso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Training+and+operation+of+an+integrated+neuromorphic+network+based+on+metal-oxide+memristors.">Training and operation of an integrated neuromorphic network based on metal-oxide memristors.</a> Nature. 521 (75550), 61-64 (2015).</li><li>Prodromakis, T., Toumazou, C., Chua, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+centuries+of+memristors.">Two centuries of memristors.</a> Nature Materials. 11 (6), 478 (2012).</li><li>Berzina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+an+organic+memristor+as+an+adaptive+memory+element.">Optimization of an organic memristor as an adaptive memory element.</a> Journal of Applied Physics. 105 (12), 124515 (2009).</li><li>van de Burgt, Y., Melianas, A., Keene, S. T., Malliaras, G., Salleo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+electronics+for+neuromorphic+computing.">Organic electronics for neuromorphic computing.</a> Nature Electronics. 1, (2018).</li><li>Dan, Y., Poo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spike+timing-dependent+plasticity:+from+synapse+to+perception.">Spike timing-dependent plasticity: from synapse to perception.</a> Physiological reviews. 86 (3), 1033-1048 (2006).</li><li>Zucker, R. S., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+synaptic+plasticity.">Short-term synaptic plasticity.</a> Annual Reviews of Physiology. 64 (1), 355-405 (2002).</li><li>Shepherd, J. D., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+synaptic+plasticity:+AMPA+receptor+trafficking.">The cell biology of synaptic plasticity: AMPA receptor trafficking.</a> Annual Review of Cell Developmental Biology. 23, 613-643 (2007).</li><li>Taylor, G. J., Venkatesan, G. A., Collier, C. P., Sarles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+in+situ+measurement+of+specific+capacitance,+monolayer+tension,+and+bilayer+tension+in+a+droplet+interface+bilayer.">Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer.</a> Soft Matter. 11 (38), 7592-7605 (2015).</li><li>Najem, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+bacterial+channel+MscL+in+mechanically+stimulated+droplet+interface+bilayers.">Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.</a> Scientific Reports. 5, 13726 (2015).</li><li>Taylor, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitive+Detection+of+Low-Enthalpy,+Higher-Order+Phase+Transitions+in+Synthetic+and+Natural+Composition+Lipid+Membranes.">Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes.</a> Langmuir. 33 (38), 10016-10026 (2017).</li><li>Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+interrogation+of+asymmetric+droplet+interface+bilayers+reveals+surface-bound+alamethicin+induces+lipid+flip-flop.">Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop.</a> Biochimica et biophysica acta (BBA)-Biomembranes. , (2018).</li><li>Sarles, S. A., Garrison, K. L., Young, T. T., Leo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+Encapsulation+of+Biomolecular+Arrays+for+Developing+Arrays+of+Membrane-Based+Artificial+Hair+Cell+Sensors.">Formation and Encapsulation of Biomolecular Arrays for Developing Arrays of Membrane-Based Artificial Hair Cell Sensors.</a> Proceedings of the Asme Conference on Smart Materials, Adaptive Structures and Intelligent Systems (Smasis 2011), Vol 2. , 663-671 (2011).</li><li>Sarles, S. A., Leo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-based+biomolecular+smart+materials.">Membrane-based biomolecular smart materials.</a> Smart Materials &amp; Structures. 20 (9), (2011).</li><li>Sarles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physical encapsulation of interface bilayers. , (2010).</li><li>JoVE Science Education Datatbase. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+Chemistry+II.+Cleaning+Glassware.">Organic Chemistry II. Cleaning Glassware.</a> Journal of Visualized Experiments. , (2018).</li><li>Taylor, G. J., Sarles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heating-enabled+formation+of+droplet+interface+bilayers+using+Escherichia+coli+total+lipid+extract.">Heating-enabled formation of droplet interface bilayers using Escherichia coli total lipid extract.</a> Langmuir. 31 (1), 325-337 (2014).</li><li>Shlyonsky, V., Dupuis, F., Gall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OpenPicoAmp:+an+open-source+planar+lipid+bilayer+amplifier+for+hands-on+learning+of+neuroscience.">The OpenPicoAmp: an open-source planar lipid bilayer amplifier for hands-on learning of neuroscience.</a> Plos One. 9 (9), e108097 (2014).</li><li>Najem, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropipette-based+Method+for+Incorporation+And+Stimulation+of+Bacterial+Mechanosensitive+Ion+Channels+in+Droplet+Interface+Bilayers.">Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers.</a> Journal of Visualized Experiments. (105), (2015).</li><li>Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+interface+bilayers.">Droplet interface bilayers.</a> Molecular Biosystems. 4 (12), 1191-1208 (2008).</li><li>Nguyen, M., Srijanto, B., Retterer, S., Collier, C. P., Sarles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+trapping+for+rapid+assembly+and+in+situ+electrical+characterization+of+droplet+interface+bilayer+arrays.">Hydrodynamic trapping for rapid assembly and in situ electrical characterization of droplet interface bilayer arrays.</a> Lab on a Chip. 16, 3576-3588 (2016).</li><li>Weiss, R., Najem, J. S., Hasan, M. S., Schuman, C. D., Belianinov, A., Collier, C. P., Sarles, S. A., Rose, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Soft-Matter+Biomolecular+Memristor+Synapse+for+Neuromorphic+Systems.">A Soft-Matter Biomolecular Memristor Synapse for Neuromorphic Systems.</a> , (2018).</li></ol>

This manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC0500OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for the United States Government purposes.

This paper presents a protocol for assembling and characterizing biomolecular memristors based on ion channel-doped synthetic biomembranes formed between two droplets of water in oil. The soft-matter, two-terminal device is designed and studied to: 1) overcome constraints that are associated with solid-state technology, such as high noise, high energy consumption, and high switching voltages, 2) more closely mimic the composition, structure, switching mechanisms of biological synapses, and 3) explore the mechanisms and f...

It is widely recognized that biological synapses are responsible for the high efficiency and enormous parallelism of the brain due to their ability to learn and process information in highly adaptive ways. This coordinated functionality emerges from multiple, highly complex molecular mechanisms that drive both short-term and long-term synaptic plasticity1,2,3,4,5. Neuromorphic computing systems aim to emulate synaptic functionalities at levels approaching the density, complexity, and energy efficiency of the brain, which are needed for the next generation of brain-like computers6,7,8. However, reproducing synaptic features using traditional electronic circuit elements is virtually impossible9, instead requiring the design and fabrication of new hardware elements that can adapt to incoming signals and remember information histories9. These types of synapse-inspired hardware are known as mem-elements9,10,11 (short for memory elements), which, according to Di Ventra et al.9,11, are passive, two-terminal devices whose resistance, capacitance, or inductance can be reconfigured in response to external stimuli, and which can remember prior states11. To achieve energy consumption levels approaching those in the brain, these elements should employ similar materials and mechanisms for synaptic plasticity12.
To date, two-terminal memristors13,14,15 have predominantly been built using complementary metal-oxide-semiconductor (CMOS) technology, characterized by high-switching voltages and high noise. This technology does not scale well due to high power consumption and low density. To address these limitations, multiple organic and polymeric memristors have been recently built. However, these devices exhibit significantly slower switching dynamics due to time-consuming ion diffusion through a conductive polymer matrix16,17. As a result, the mechanisms by which both CMOS-based and organic memristive devices emulate synapse-inspired functionalities are highly phenomenological, encompassing only a few synaptic functionalities such as Spike Timing Dependent Plasticity (STDP)18, while overlooking other key features that also play essential roles in making the brain a powerful and efficient computer, such as pre-synaptic, short-term plasticity19.&#160;
Recently, we introduced a new class of memristive devices12 featuring voltage-activated peptides incorporated in biomimetic lipid membranes that mimics the biomolecular composition, membrane structure, and ion channel triggered switching mechanisms of biological synapses20.&#160; Here, we describe how to assemble and electrically interrogate these two-terminal devices, with specific focus on how to evaluate short-term plasticity for implementation in online learning applications12. Device assembly is based on the droplet interface bilayer (DIB)21 method, which has been used extensively in recent years to study the biophysics of model membranes21 and membrane-bound ion channels22,23,24, and as building blocks for the development of stimuli-responsive materials25,26. We describe the membrane assembly and interrogation process in detail for those interested in neuromorphic applications but have limited experience in biomaterials or membrane biology. The protocol also includes a full description of the characterization procedure, which is as important as the assembly process, given the dynamic and reconfigurable electrical properties of the device27. The procedure and representative results described here are foundations for a new class of low-cost, low-power, soft mem-elements based on lipid interfaces and other biomolecules for applications in neuromorphic computing, autonomous structures and systems, and even adaptive brain-computer interfaces.

<table><tbody><tr><td>1,2-diphytanoy-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DPhPC)</td><td>Avanti Polar Lipids</td><td>850356P/850356C</td><td>Purchased as lyophilized powder (P) or in chloroform (C)&amp;nbsp;</td></tr><tr><td>Agarose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>Agarose (0.5g Agarose Tablets)</td><td>Benchmark</td><td>A2501</td><td>You can either use the powder form or the tablets&amp;nbsp;</td></tr><tr><td>Alamethicin&amp;nbsp;</td><td>AG Scientific</td><td>A-1286</td><td></td></tr><tr><td>Analytical balance&amp;nbsp;</td><td>Mettler Toledo</td><td>ME204TE/00</td><td></td></tr><tr><td>Axopatch 200B Amplifier&amp;nbsp;</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>BK Precision 4017B 10 MHz DDs Sweep/Function Generator</td><td>Digi-Key</td><td>BK4017B-ND</td><td></td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>Brain Total Lipid Extracts (Porcine)</td><td>Avanti Polar Lipids</td><td>131101</td><td></td></tr><tr><td>DigiData 1440A system</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>Extruder Set With Holder/Heating Block&amp;nbsp;</td><td>Avanti Polar Lipids</td><td>610000</td><td>This includes a mini-extruder, 2 syringes, 100 PC membranes, 100 filter supports, and 1 holder/heating block</td></tr><tr><td>Freezer (-20 &amp;deg;C)</td><td>VWR International</td><td>SCUCBI0420AD</td><td></td></tr><tr><td>Glassware</td><td>VWR International</td><td>-</td><td></td></tr><tr><td>Hexadecane, 99%</td><td>Sigma-Aldrich</td><td>544-76-3</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>VWR International</td><td>BDH1133-4LP</td><td></td></tr><tr><td>Microelectrode Holder&amp;nbsp;</td><td>World Precision Instruments</td><td>MEH1S</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>Nitrogen (N&lt;sub&gt;2&lt;/sub&gt;) Gas</td><td>Airgas</td><td>UN1066</td><td></td></tr><tr><td>Parafilm M All-Purpose Laboratory Film</td><td>Parafilm</td><td>PM999</td><td></td></tr><tr><td>Powder Free Soft Nitrile Examination Gloves&amp;nbsp;</td><td>VWR International</td><td>CA89-38-272</td><td></td></tr><tr><td>Precleaned Microscope Sildes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>22-267-013</td><td></td></tr><tr><td>Refrigirator (4 &amp;deg;C)</td><td>VWR International</td><td>SCUCFS-0504G</td><td></td></tr><tr><td>Silver wire</td><td>GoodFellow</td><td>147-346-94</td><td>Different diameters could be used depending on the application&amp;nbsp;</td></tr><tr><td>Sodium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Stirring Hot Plate</td><td>Thermo Scientific&amp;nbsp;</td><td>SP131325</td><td></td></tr><tr><td>VWR Light-Duty Tissue Wipers</td><td>VWR International</td><td>82003-820</td><td></td></tr><tr><td>VWR Scientific 50D Ultrasonic Cleaner</td><td>VWR International</td><td>13089</td><td></td></tr></tbody></table>

assembly and characterization of biomolecular memristors consisting of ion channel-doped lipid membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Figure 1 displays the experimental setup used to assemble and characterize the biomolecular memristor. Lowering the free ends of the electrodes to the bottom of the oil reservoir, as shown in Figure 1b, was found helpful to minimize vibrations of the electrodes and droplets that can result in variations in measured current and bilayer area, especially in cases where heating the oil can generate convective flow in the oil. Figure 2 s...

Watch this Scientific Journal Video about Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes at JoVE.com

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

assembly-characterization-biomolecular-memristors-consisting-ion

Research

Education

JoVE Core

JoVE Journal

Physics

Bioengineering

Unit 1

Alternating-Current Circuits

SCIENTISTS IN ACTION

7.7K Views.  Oak Ridge National Laboratory. Ons protocol beschrijft hoe te monteren en elektrisch karakteriseren een peptide-doped biomembrane die nauw nabootst de samenstelling, structuur, en transport eigenschappen van biologische synapsen en die tunable geheugenweerstand vertoont. Deze techniek stelt gebruikers in staat om activiteitsafhankelijke, geheugenbestendigheid en kortdurende plasticiteit in gemanipuleerde systemen te beoordelen op tijdschalen en excitatieniveaus die relevant zijn voor biologische synapsen en ionenkanalen. D...

Video: Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Biochemistry

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 ...

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 ...

Chemistry

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

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 ...

A Method for Growing Bio-memristors from Slime Mold

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and computing architectures based on biology. This specific paper focuses on harnessing the unicellular slime mold Physarum polycephalum to develop bio-memristors (or biological memristors) and bio-computing devices. The memristor is a resistor that possesses memory. It is the 4th fundamental passive&#160;circuit element (the other three are the resistor, the capacitor, and the inductor), which is paving the way for the design of new kinds of computing systems; e.g., computers that might relinquish the distinction between storage and a central processing unit. When applied with an AC voltage, the current vs. voltage characteristic of a memristor is a pinched hysteresis loop. It has been shown that P. polycephalum produces pinched hysteresis loops under AC voltages and displays adaptive behavior that is comparable with the functioning of a memristor. This paper presents the method that we developed for implementing bio-memristors with P. polycephalum and introduces the development of a receptacle to culture the organism, which facilitates its deployment as an electronic circuit component. Our method has proven to decrease growth time, increase component lifespan, and standardize electrical observations.

This paper introduces an improved method for growing bio-memristors out of the plasmodium of Physarum polycephalum. Such a method has proven to decrease growth time, increase component lifespan, standardize electrical observations, and create a protected environment that can be integrated into conventional circuitry.

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and ...

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.

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 ...

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 ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video