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

Summary

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.

Abstract

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.

Introduction

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 plasticity^1,2,3,4,5. 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 computers^6,7,8. 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-elements^9,10,11 (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 memristors^13,14,15 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 matrix^16,17. 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. 

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.  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 channels^22,23,24, and as building blocks for the development of stimuli-responsive materials^25,26. 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.

Protocol

1. General Instructions and Precautions

1. Select suitable, undamaged measuring/mixing glassware (flasks, beakers, etc.) and other labware (spatulas, scoops, etc.) for use.
2. Handle glassware carefully to avoid damaging, and wear latex or nitrile gloves to avoid contaminating the glassware/labware with residues from fingertips and to protect your skin.
3. Clean chosen glassware/labware thoroughly using detergent solution and water by scrubbing with a soft bottle brush until clean and all residues are removed.
4. Rinse thoroughly with tap water and then with deionized (DI) water. Place on drying rack to air dry.
5. Optional: Rinse the cleaned glassware/labware with isopropyl (IPA, 99.5%) and place under vacuum to evaporate all residual IPA to ensure they are free of any contaminants (~2 h). Remove from vacuum chamber and place in clean environment.
   NOTE: Use lint-free wipes for wiping glassware and labware. Purchase and use sterile small glass vials and safe-lock tubes for materials preparation and sample storage. For further details on glassware cleaning and other laboratory standard operating procedures, refer to the JoVE Science Education Database²⁸.

2. Preparation of Aqueous Buffer Solution

1. Wearing latex or nitrile gloves, select an appropriate and clean glass container to prepare 50 mL of aqueous buffer (500 mM sodium chloride (KCl), 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0).
2. Using a digital, high precision mass balance and a clean spatula, dispense 1.86378 g of KCl onto clean weighing paper and then add to the glass container.
   NOTE: The amounts of KCl and MOPS should vary depending on the desired volume and desired final concentrations.
3. Weigh 0.10463 g of MOPS and add to the glass container. Then, add 50 mL of DI water to the glass container and vortex thoroughly until KCl and MOPS are completely dissolved.
4. Store the buffer solution at room temperature and use when needed.
   NOTE: While buffer solutions can be stored for relatively long periods of time, it is recommended to use freshly prepared buffer solutions for better and more consistent results.

3. Preparation of Liposomes

NOTE: Step 3.1 only applies if phospholipids are acquired as lyophilized powders, and therefore, may be skipped if the phospholipids are purchased in chloroform.

1. Dissolve 5 mg of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or Brain Total Lipid Extracts (BTLE) lipids in 1 mL of chloroform in a 5 mL sterile glass vial.
2. While gently swirling, evaporate the chloroform under a gentle stream of dry nitrogen until a lipid film remains at the bottom of the vial.
3. Place the vial containing the lipid film under vacuum for 10-12 h to allow for complete removal of residual chloroform.  
4. Remove the vial from the vacuum chamber and rehydrate the lipid film by adding 10 mL of the aqueous buffer solution prepared in Step 2 to achieve a final lipid concentration of 2 mg/mL.
5. Freeze (-20 °C) and completely thaw the lipid suspension six times to facilitate multilamellar liposome assembly.
   NOTE: Let the mixture thaw at room temperature, never in a heated environment.
6. Using a commercially available extruder, extrude the liposome solution by forcing the complete lipid suspension through a 0.1 μm pore diameter track-etched membrane. Extrude the suspension 11 times in immediate succession to obtain unilamellar liposomes with diameters of c.a. 100 nm needed for proper lipid monolayer formation. Store the liposome suspension at 4 °C and use within 1 week of preparation. For simplicity, refer to the resulting liposome solution as “A”.
   NOTE: For the extrusion of BTLE liposomes, the researcher is encouraged to warm up the extruder to 45-50 °C, higher than the phase transition temperature of BTLE lipids (~ 37 °C)^23,29, to enable easier extrusion. Hydrated BTLE liposome suspensions can also be directly prepared (in place of freeze-thaw and extrusion) by placing the closed suspension vial into in a bath sonicator at 55 °C for ~15 min.

4. Reconstitution of Alamethicin Peptides

NOTE: This procedure describes the process of alamethicin reconstitution in liposomes to a final concentration of 1 μM. This concentration is sufficient to induce nA-level currents similar to those previously published¹². Increasing the peptide concentration will reduce the switching threshold and increase the amplitudes of currents induced by applied voltage²⁹.

1. Dissolve alamethicin peptides in ethanol to a final concentration of 2.5 mg/mL, vortex briefly to mix well, and store the stock solution in freezer (-20 °C).
   ​NOTE: Alamethicin peptides are usually purchased in powder form.
2. In a 1.5 mL safe-lock tube, mix 99 μL of solution “A” with 1 μL of alamethicin stock solution to achieve a final alamethicin concentration of 13 μM in the liposome suspension.  Vortex to mix well. Refer to the resulting peptide-liposome solution as “B”.
3. Mix 117 μL of solution “A” with 10 μL of solution B to achieve a final alamethicin concentration of 1 μM, and then vortex to mix well. Refer to the resulting solution as “C”.
4. Store the solutions “B” and “C” at 4 °C and use as needed.

5. Preparation of Agarose gel

1. Using a digital, high precision mass balance and a clean spatula, add 0.5 g of agarose powder to a clean weighing paper.
2. Transfer weighed agarose to a 100-mL clean glass beaker and add 50 mL of DI water to the agarose.
   NOTE: This will yield a 1% (wt/vol) agarose gel solution.
3. Place a clean stirring magnet inside the glass beaker and place the beaker on a stirring hot plate.
4. While stirring, bring the mixture to a boil until agarose is completely dissolved.
5. Remove the beaker from the hot plate to let the mixture cool to room temperature. Store at 4 °C and use when needed.
6. Before using again, re-melt the agarose by heating with a hot plate or microwave.

6. Fabrication of the Oil Reservoir

NOTE: The procedure described below is just one of many ways that an oil reservoir can be fabricated. The reader is encouraged to design and fabricate a reservoir based on available materials, machining capabilities, and specific needs.

1. Using a band saw, cut a 12 x 12 x 12 mm acrylic cube from a larger 12 mm thick acrylic sheet.
2. Mill a 12 mm diameter hole to a depth of 8-12 mm in the acrylic tube (Figure 1a).

7. Preparation of Electrodes

1. Using scissors, cut two pieces (75 mm) of silver wires (125 μm-diameter).
2. Using an open-flame lighter, melt one end of each silver wire to form small spherical balls (around 250 μm in diameter).
3. Immerse the ball ends in bleach for 1-2 h to create a silver silver-chloride (Ag/AgCl) coating. A dark gray color indicates that the Ag/AgCl coating has formed (Figure 2a).
4. Remove both wires from bleach, rinse thoroughly with DI water and place aside on a clean lint-free wipe.
5. Dip the ball ends into molten agarose gel to create a thin layer. This gel coating helps to anchor the aqueous droplets onto the wires under oil.  
6. Using a glass cutter, split a 10-cm long, 1/0.58 OD/ID mm borosilicate glass capillary into two 5 cm capillaries.
7. Insert one of the glass capillaries into an electrode holder (Figure 2b, c), and then feed one of the Ag/AgCl wire into the glass capillary (Figure 2d). Feed the other Ag/AgCl wire into the second glass capillary.
8. Mount the second glass capillary to a glass micropipette holder (Figure 2e, f).

8. Setting Up the Experiment

1. Place a 1 mm thick, 25 x 75 mm glass slide on the stage of an inverted microscope (Figure 1a).
2. Dispense a few drops of hexadecane oil onto the center of the glass slide, and then place the oil reservoir directly onto the oil on the glass slide.
   NOTE: Adding oil between the glass slide and oil reservoir is used to match the refractive index of the substrate to provide clearer and sharper images.
3. Completely fill the oil reservoir with hexadecane oil. Make sure the reservoir is positioned above the objective lens.
   NOTE: Other hydrophobic oils may be used as well.
4. Connect the electrode holder to the headstage of a current amplifier. The headstage must be mounted on a micromanipulator (Figure 1a) to minimize electrode length and electrical noise.
5. Mount the glass micropipette holder with the second Ag/AgCl wire onto another micromanipulator (Figure 1a).
6. Using the manipulators, position the electrodes such that the agarose coated tips of the Ag/AgCl wires are fully submerged into the oil reservoir at a similar vertical plane.
7. Align the two electrodes and separate them by a few millimeters (Figure 1a, b).
   NOTE: After adding the droplets (described in Step 13), the wires must be brought all the way down until the electrode tips are touching the bottom of the oil reservoir. This step will ensure that the wires do not oscillate, and thus, will minimize unnecessary fluctuations in the measured current.

9. Proper Grounding to Reduce Electrical Noise

1. Create a Ground bus by threading a screw into the anti-vibration table on which the microscope is placed (Figure 3a).
   NOTE: Using an anti-vibration table is required to minimize vibrations from the surrounding, which might cause undesired fluctuations in measured current.
2. Using a conductive wire, connect the screw to an earth ground (Figure 3a), and then connect the microscope stage to the Ground bus.
3. Place a Faraday cage over the experimental setup to reduce noise and then electrically connect it to the Ground bus (Figure 3b).
   NOTE: It is always recommended to avoid unnecessary ground loops, as they may lead to an increase in measurement noise level.

10. Feedback-Controlled Heating

1. Machine an aluminum heating shell in which the oil reservoir can snugly fit²⁹.
2. Make sure to leave an opening at the bottom of the shell to be able to view through the shell via the inverted microscope.
3. Place a 30 x 30 mm resistive polyimide flexible heating element underneath the aluminum shell.
4. Place an insulating polydimethylsiloxane (PDMS) wafer beneath the heater to reduce heat loss in the downward direction and protect the microscope stage.
5. Insert a thermocouple into the oil phase. After making sure the thermocouple does not touch either Ag/AgCl wire, connect the thermocouple wires to a thermocouple data acquisition board and record temperature using custom programming software.
   NOTE: Write an On-Off, proportional integral (PI) feedback temperature control to enable heating and passive cooling of the oil temperature to a desired value. Codes can be provided to readers upon request.

11. Setting Up the Software and Equipment

1. Prepare the data acquisition software by powering on computer(s), microscope, function generator, current amplifier, and low-noise data acquisition systems.
   NOTE: While any current sensing equipment may be used, the following instructions are specifically for the one listed in Table of Materials. Researchers who wish to build their own current amplifier can refer to Shlyonsky et al.³⁰.
2. On the front panel of the patch clamp current amplifier, set the front panel display and source-measurement Mode dials to VHOLD/IHOLD and V-CLAMP, respectively.
3. On the front panel, set the Lowpass Bessel Filter to 1 kHz and Output Gain to 0.5.
   NOTE: Choosing a low output gain enables recording larger higher current amplitudes, whereas increasing the gain sacrifices measurement range for reduce measurement noise.
4. Set the Configuration to WHOLE CELL β = 1. This value may be later switched to 0.1 to allow recording of larger amplitude currents.
5. Set all other control dials to zero or in a neutral position.
6. Initialize the software by double-clicking on the icon of the desktop.
7. Click Configuration | Digitizer to open the Digitizer dialog, and then click the Change button.
8. In the Change Digitizer dialog, select the appropriate digitizer from the Digitizer Type list.
9. Click the Scan button to detect the digitizer.
10. Click OK to exit the Change Digitizer dialog, and then click OK to exit the Digitizer dialog.
11. Click Configure | Lab Bench.
12. In the Input Signals tab of the Lab Bench, set the scale factor to 0.0005 V/pA.
    NOTE: This value must be updated if the gain or β values are changed.

12. Pipette Offset

NOTE: The procedure described below applies only to current amplifier mentioned in Table of Materials.

1. Using a micropipette, deposit 200 nL of the aqueous lipid solution “A” onto the ends of each Ag/AgCl wire under oil.
2. Bring the droplets into contact and press the ZAP button on the front panel of the amplifier to coalesce the droplets into one volume spanning both electrodes. This should induce a short-circuit response.
3. Set source-measurement mode dial to TRACK.
4. Change the front panel display dial to V_TRACK.
5. Turn the PIPETTER OFFSET dial (clockwise or counterclockwise) until meter reads 0 mV and is stable. 
6. Return the source-measurement mode dial to V-CLAMP and the front panel display dial to V_HOLD/I_HOLD.

13. Formation of the Lipid Bilayer

1. Release the droplets that were previously deposited by moving the electrodes vertically out of the oil phase. This causes the droplets to fall from the electrodes into the oil. Re-submerge and position the electrodes in oil.
2. Use the micropipette to deposit 200 nL of lipid solution “A” on each of the wires.
3. Wait for 3-5 min to allow for spontaneous lipid monolayer assembly to occur at each water/oil interface.
   NOTE: As the lipid monolayer forms, the water/lipid/oil interface surface tension decreases, which can cause the droplets to sag if the surrounding oil is sufficiently less dense²¹.
4. Lower the electrodes (and droplets) until the ends of both electrodes barely touch the bottom of the oil reservoir (Figure 1b), and then move them horizontally to bring the droplets into contact.
   NOTE: The lipid bilayer will spontaneously thin by excluding excess oil from between the contacting droplets. Typically, this process occurs within 1 min.

14. Electrical Characterization of the Biomolecular Memristor

1. Lipid Bilayer Formation
   1. To record the lipid bilayer formation, which corresponds to an increase in electrical capacitance between droplets, apply a 10 Hz, 10 mV triangular waveform voltage using a function generator (Figure 4) connected to the external input of the patch clamp amplifier.
      NOTE: Due to the capacitive nature of the lipid membrane, the resulting current response should be a square waveform (Figure 4). During the lipid bilayer formation, Step 11.6, the researcher should see a growth in the peak-to-peak current amplitude and also observe a visual change between connected droplets (Figure 4).
2. Current-voltage measurements
   NOTE: The biomolecular memristor is modeled as a resistor and a capacitor in parallel^12,21. Therefore, the current response of the device can contain both resistive and capacitive components depending on the frequency of the applied voltage. To study the memristive nature of the device, and to obtain the pinched hysteretic current-voltage relationship¹², it may be necessary to subtract capacitive current from the total current. The protocol below describes this procedure.
   1. Using a function generator, apply a voltage waveform (triangular or sinusoidal) to an alamethicin-free lipid membrane assembled with droplets of solution “A”.
   2. Record the induced current response across multiple frequencies.
      NOTE: Capacitive currents are minimized at frequencies below 10 mHz.
   3. Record the size of the interfacial lipid bilayer by either measuring the diameter of the lipid membrane on computer, or by recording the peak-to-peak current amplitude resulting from the 10 Hz, 10 mV triangular wave. The current amplitude is proportional to the membrane capacitance, which in turn is proportional to the area of the membrane.
   4. Remove the droplets that contain no alamethicin.
   5. Add new aqueous droplets using solution “C” and form a lipid bilayer.
   6. Use the micromanipulators to adjust contact between droplets such that the bilayer has a similar area (diameter or square-wave current amplitude) as the one formed earlier.
   7. Repeat steps 14.2.1 and 14.2.2.
   8. Subtract current recorded in step 14.2.2 from current recorded in step 14.2.7.
   9. Plot the induced current versus applied voltage for each frequency and waveform to obtain the “pinched hysteresis” memristive response.
3. Pulse experiments
   1. Using a custom programming software and analog voltage source, generate voltage pulses with specific high and low amplitudes, ON time, and OFF time.
      NOTE: This is not needed if the voltage pulses could be generated using a commercial function generator.
   2. Record the current in response to applied pulses.  
   3. Due to the capacitive nature of the memristor, capacitive spikes will be recorded. Remove spikes by applying a low-pass filter with appropriate passband.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-diphytanoy-sn-glycero-3-phosphocholine (DPhPC)	Avanti Polar Lipids	850356P/850356C	Purchased as lyophilized powder (P) or in chloroform (C)
Agarose	Sigma-Aldrich	A9539
Agarose (0.5g Agarose Tablets)	Benchmark	A2501	You can either use the powder form or the tablets
Alamethicin	AG Scientific	A-1286
Analytical balance	Mettler Toledo	ME204TE/00
Axopatch 200B Amplifier	Molecular Devices	-
BK Precision 4017B 10 MHz DDs Sweep/Function Generator	Digi-Key	BK4017B-ND
Borosilicate Glass Capillaries	World Precision Instruments	1B100F-4
Brain Total Lipid Extracts (Porcine)	Avanti Polar Lipids	131101
DigiData 1440A system	Molecular Devices	-
Extruder Set With Holder/Heating Block	Avanti Polar Lipids	610000	This includes a mini-extruder, 2 syringes, 100 PC membranes, 100 filter supports, and 1 holder/heating block
Freezer (-20 °C)	VWR International	SCUCBI0420AD
Glassware	VWR International	-
Hexadecane, 99%	Sigma-Aldrich	544-76-3
Isopropyl Alcohol	VWR International	BDH1133-4LP
Microelectrode Holder	World Precision Instruments	MEH1S
MOPS	Sigma-Aldrich	M1254
Nitrogen (N2) Gas	Airgas	UN1066
Parafilm M All-Purpose Laboratory Film	Parafilm	PM999
Powder Free Soft Nitrile Examination Gloves	VWR International	CA89-38-272
Precleaned Microscope Sildes	Fisher Scientific	22-267-013
Refrigirator (4 °C)	VWR International	SCUCFS-0504G
Silver wire	GoodFellow	147-346-94	Different diameters could be used depending on the application
Sodium Chloride (KCl)	Sigma-Aldrich	P3911
Stirring Hot Plate	Thermo Scientific	SP131325
VWR Light-Duty Tissue Wipers	VWR International	82003-820
VWR Scientific 50D Ultrasonic Cleaner	VWR International	13089