Droplet polymer bilayers

Abstract

Droplet polymer bilayers (DPBs), DPB devices, methods of making the DPBs, and methods of using the DPBs are provided. The DPBs are formed by bringing together a monolayer of lipid adsorbed directly onto the surface of a conducting polymer and a lipid-coated aqueous droplet comprising a lipid monolayer surrounding an aqueous droplet in an oil solution. The polymer surface on which the lipid bilayer is formed is not a hydrogel and, therefore, in the devices described herein, the lipid bilayer makes direct contact with a non-hydrogel polymer surface.

Description

DROPLET POLYMER BILAYERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisional patent application number 63 / 565,639 that was filed March 15, 2024, the entire contents of which are incorporated herein by reference.REFERENCE TO GOVERNMENT RIGHTS

[0002] This invention was made with government support under grant number FA9550- 22-1-0426 awarded by the Air Force Office of Scientific Research. The government has certain rights in the invention.BACKGROUND

[0003] Model membranes interfaced with bioelectronics allow for the exploration of fundamental cell processes and the design of biomimetic sensors. Organic conducting polymers are an attractive surface on which to study the electrical properties of membranes because of their low impedance, high biocompatibility, and hygroscopic nature. However, establishing supported lipid bilayers (SLBs) on conducting polymers has lagged significantly behind other substrate materials due to challenges in membrane electrical sealing and stability-.

[0004] Probing the electrical properties of model membranes enables the study of cell processes that alter physical bilayer properties or transmembrane protein activity. These electrical signals created by cell communication and environmental interactions are transported through dynamic ionic fluxes. For this reason, electrodes have become an essential tool to investigate artificial membranes.

[0005] Conducting polymer electrodes have been used to interface with increasingly complex membranes to investigate ion channel activity, the impact of antimicrobials, and mechanisms of virus-host entry. (Bernards, D. A., et al., Appl. Phys. Lett. 2006, 89 (5), 053505; Pitsalidis, C. et al., Adv. Mater. 2018, 30 (39), 1-8; Bali, K. et al., ACS Biomater. Sci. Eng. 2022, 2022.12.13.520201; Tang, T. et a , ACS Nano 2021, 15 (11), 18142-18152.) However, investigations of model membranes with conducting polymers have nearly exclusively employed SLBs. While SLBs demonstrate notable advantages over freestandingblack lipid membranes, their formation is highly dependent on the surface of the substrate, introducing difficulties with reproducibility, membrane leakiness, and low membrane resistance because of interactions with the underlying substrate. Further, while soft, hygroscopic polymer substrates can cushion fluid membranes, their rough surfaces can dissuade SLB formation and generate new sources of membrane defects. Previous works creating SLBs on conducting polymers have used polymer surface treatments and membrane formation technique to overcome this barrier. Despite this, SLBs on conducting polymer electrodes have displayed particularly low electrical sealing to nonspecific ionic currents, with normalized membrane resistances < 1 kQ*cm2, suggesting incomplete surface coverage and the presence of significant membrane defects that may hinder future investigation of ion transport processes. (Bali. K. et al., 2022; Tang, T. et al., 2021; Manzer, Z. A. et al., 2023; Zhang, Y. et al., A<A. Fund. Mater. 2016, 26 (40), 7304-7313; Liu, H. et al., Langmuir 2020, 36 (26), 7325-7331; Pappa, A.-M. et a / . ACS Nano 2020; Bint E Naser, S. F. et al., ACS Appl. Bio Mater. 2021, 4 (11), 7942-7950; Bint E Naser, S. F. et al., Langmuir 2023, 39 (28), 9831-9840; Schafer. E. A. et al., ACS Appl. Mater. Interfaces 2023, 15 (20), 24638-24647; Uribe, J. et al.. ACS Biomater. Sei. Eng. 2021. 7 (12). 5585-5597.)

[0006] Other types of model membranes include droplet interface bilayers (DIBs) and droplet hydrogel bilayers (DHBs). DIBs are liquid-supported model membranes assembled between lipid monolayer-coated water droplets in oil that show high electrical resistances and good stability. (Bayley, H. et al., Mol. Biosyst. 2008, 4 (12), 1191-1208.) The high electrical sealing and inherent fluidity of DIBs has facilitated electrophysiological study of membranes and integral membrane proteins. (Stephenson. E. B. et al.. Nat. Chem. 2022, 14 (8), 862- 870.) Similarly, DHBs exploit the energetically favorable arrangement of phospholipids between oil and water by allowing lipids to adsorb at a hydrated hydrogel layer and bringing a monolayer on an aqueous droplet in contact with the hydrogel. (Leptihn, S. et al.. Nat. Protoc. 2013, 8 (6), 1048-1057.) DHBs therefore stabilize on a surface, but retain strong electrical properties capable of single-channel recordings, high fluidity comparable to living cells, and the ability to insert functional transmembrane proteins. However, neither DIBs nor DHBs are readily merged with conducting polymer electronics.SUMMARY

[0007] DPBs, methods of using the DPBs, and methods of making the DPBs are provided.

[0008] One example of a droplet polymer bilayer includes: a substrate comprising a nonhydrogel polymer surface; a first lipid monolayer adsorbed directly on the non-hydrogel polymer surface; an oil solution over the first lipid monolayer; and a lipid-coated aqueous droplet in the oil solution. The lipid-coated aqueous droplet comprises: an aqueous droplet comprising a dissolved salt; and a second lipid monolayer around the aqueous droplet at an oil-aqueous interface formed between the aqueous droplet and the oil solution. The first lipid monolayer and the second lipid monolayer form a lipid bilayer at a polymer-droplet interface formed between the non-hydrogel polymer surface and the aqueous droplet.

[0009] The droplet polymer bilayers can be used to measure a property of the lipid bilayer applying a voltage across the lipid bilayer and measuring one or more of: the current through the lipid bilayer; the impedance of the lipid bilayer; the specific capacitance of the lipid bilayer; the lipid bilayer resistance; and the ion conductance across the lipid bilayer.

[0010] The droplet polymer bilayers can be made by: forming a first lipid monolayer directly on a non-hydrogel polymer surface of a substrate in an oil solution; forming a lipid- coated aqueous droplet in the oil solution, the lipid-coated aqueous droplet comprising: an aqueous droplet; and a second lipid monolayer around the aqueous droplet at an oil-aqueous interface formed between the aqueous droplet and the oil solution; and bringing the lipid- coated aqueous droplet and the first lipid monolayer on the non-hydrogel polymer surface together, whereby the first lipid monolayer and the second lipid monolayer form a lipid bilayer at a polymer-droplet interface formed between the non-hydrogel polymer surface and the aqueous droplet.

[0011] Other principal features and advantages of the invention will become apparent to those skilled in the art upon review of the following drawings, the detailed description, and the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Illustrative embodiments of the invention will hereafter be described with reference to the accompanying drawings, wherein like numerals denote like elements.

[0013] FIGS. 1A-1B. FIG. 1A is an illustration of a droplet polymer bilayer (DPB) on a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). A lipid monolayer formed on the polymer and along an aqueous droplet, and a bilayer formed upon contact of the droplet with the polymer. The equivalent circuit of the system’s electrical impedance isshown with an electrolyte resistance (Re), membrane resistance (Rm), membrane capacitance (Cm), and polymer capacitance (CP). FIG. IB shows impedance (solid line) and phase (dotted line) of the PEDOTPSS film and DPB.

[0014] FIGS. 2A-2B. FIG 2A shows impedance (circles) and phase (triangles) spectra of a PEDOTPSS film with a 0.5 wt. % agarose hydrogel coating and droplet hydrogel bilayer. The corresponding fits to the spectra are shown as lines for the PEDOTPSS and agarose and droplet hydrogel bilayer. FIG. 2B shows impedance (circles) and phase (triangles) spectra of the PEDOTPSS film and droplet polymer bilayer. The corresponding fits to the spectra are shown as lines for the PEDOTPSS and droplet polymer bilayer. The impedance and phase spectra of the PEDOTPSS electrode was fit to an electrolyte resistance to ionic flux in series with the capacitance of the conducting polymer film. Note that deviations between the fit and raw impedance and phase data for the pre-bilayer conditions arose from the effects of the squalene: hexadecane oil on PEDOTPSS. The impedance and phase spectra of droplet bilayers were fit to an R(RC) circuit with an electrolyte resistance in series with a resistance and capacitance of the bilayer in parallel.

[0015] FIG. 3. Membrane capacitance (Cm) and resistance (Rm) extracted from impedance spectra of DPBs on PEDOTPSS of pure DPhPC, l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), and a 4:3:3 ratio of DPhPC. sphingomyelin (SM), and cholesterol (Choi). Box plot lines indicate quartiles of each dataset (n > 4).

[0016] FIGS. 4A-4B. FIG. 4A shows impedance (solid line) and phase (dotted line) spectra of a droplet polymer bilayer on PEDOTPSS over time during zipping from no bilay er-to-bilay er formation. Final impedance after bilayer equilibration shows the expected impedance of a resistor in series with a resistor and capacitor in parallel. FIG. 4B shows bilayer capacitance fitted to the impedance spectra and is shown to increase over time during formation and zipping.

[0017] FIGS. 5A-5D. FIG. 5A shows impedance (solid line) and phase (dotted line) spectra of a representative DPhPC DPB on PEDOTPSS before and after the application of a 300 mV pulse for 30 s. FIG. 5B shows bilayer capacitance increased significantly after application of the voltage pulse (p < 0.05). Bilayer area (FIG. 5C) and resistance (FIG. 5D), on the other hand, did not change significantly after the application of the voltage pulse (p > 0.05).

[0018] FIGS. 6A-6C. FIG. 6A shows the contact angle of a sessile droplet without lipids on a plasma cleaned-PEDOTPSS surface in 4: 1 squalene: hexadecane oil. The mean contact angle was found to be 43.6° (n = 4). FIG. 6B shows the potential difference between PEDOT:PSS and an aqueous droplet in 4: 1 squalene: hexadecane as measured by open circuit potentiometry. The mean potential difference was found to be 231 mV (n = 7). FIG. 6C shows average impedance and phase spectra of unpattemed PEDOT:PSS in contact with an aqueous droplet (100 mM KC1 / 10 mM MOPS) in 4: 1 squalene: hexadecane. Error bars represent standard deviation.

[0019] FIGS. 7A-7B. FIG. 7A shows illustration and impedance spectra before and after formation of a SLB on a PEDOT:PSS-coated microelectrode. FIG. 7B shows illustration and impedance spectra before and after formation of a DPB on a PEDOT:PSS-coated microelectrode. Both plots use a dashed arrow to indicate measured dynamic range. Each micro-electrode is circular with a radius of 350 pm.

[0020] FIGS. 8A-8C. FIG. 8A shows a top-view image of in-plane reference electrode design where the theoretical droplet area is indicated by the dashed circle and the solid line indicates the cross-sectional view depicted in FIG. 8B. Scale bar represents 100 pm. FIG. 8B shows illustration of a DPB on a PEDOT:PSS working electrode (WE) with an in-plane PEDOT:PSS reference electrode (RE). The corresponding equivalent circuit demonstrates that the impedance of the working electrode (Zw) is much greater than the impedance of the reference electrode (ZR). FIG. 8C shows impedance (solid) and phase (dotted) spectra of a DPB on a patterned 80 pm radius PEDOT:PSS working electrode with the in-plane PEDOT:PSS reference electrode.

[0021] FIGS. 9A-9B. FIG. 9A shows open circuit potential across a PEDOTPSS working electrode and PEDOTPSS reference electrode in IX PBS. FIG. 9B shows a comparison of impedance (solid line) and phase (dotted line) spectra of PEDOTPSS working electrode using an external Ag / AgCl reference wire electrode and using the planar PEDOTPSS reference electrode.

[0022] FIGS. 10A-10C. FIG. 10A shows an optical micrograph of the planar electrode design using an Ag / AgCl reference. Scale bar represents 500 pm. FIG. 10B shows open circuit potential across the PEDOTPSS working electrode and planar Ag / AgCl reference electrode in IX PBS. FIG. 10C shows a comparison of impedance (solid line) and phase (dotted line) spectra of PEDOTPSS working electrode without a droplet bilayer presentusing an external Ag / AgCl reference wire electrode and using the planar Ag / AgCl reference electrode, along with the impedance and phase spectra directly of the fabricated planar Ag / AgCl electrode.

[0023] FIG. 11 shows impedance (solid line) and phase (dotted line) spectra of a PEDOT:PSS working electrode before and after the addition of a droplet polymer bilayer, measured using the fabricated planar Ag / AgCl design. The deviation in expected impedance for a DPB for frequencies > 102Hz is hypothesized to arise from incomplete monolayer formation on the rough Ag / AgCl surface.DETAILED DESCRIPTION

[0024] DPBs, DPB devices, methods of making the DPB devices, and methods of using the DPB devices are provided. The DPBs are stable, easily fabricated, and can be made from biocompatible components. The DPBs are formed by bringing together a monolayer of lipid adsorbed directly onto the surface of a polymer and a lipid-coated aqueous droplet comprising a lipid monolayer surrounding an aqueous droplet in an oil solution. The polymer surface on which the lipid bilayer is formed is not a hydrogel and, therefore, in the DPBs described herein, the lipid bilayer makes direct contact with a non-hydrogel polymer surface. The DPB devices are DPBs in which the bilayer is formed on the surface of a conducting polymer that acts as an electrode and / or in which the lipid bilayer of the DPB in in electrical communication with an electrode.

[0025] In some embodiments of the DPB devices, the non-hydrogel polymer is an electrically conducting organic polymer. Compared to traditional metal electrodes, organic electrically conducting polymers can intimately interface with a biological environment by directly transducing ionic fluxes to an electronic current. In addition, their low impedance enables a high sensitivity to small and otherwise undetectable biological signals.

[0026] The DPBs on electrically conducting polymers are characterized by good membrane electrical sealing, reliability, and bilayer fluidity, and leverage the sensing capabilities of conducting polymer electronics. Unlike SLBs that are highly dependent on surface interactions, the DPBs leverage the energetically favorable organization of lipids at atomically smooth liquid interfaces to build high-integrity’ membranes. The improved membrane electrical sealing and stability of DPBs, relative to SLBs, results in higher electrical impedances over SLBs on conducting polymers, including bilayer capacitancesrepresentative of homogenous, oil-free, defect-free membranes. As a result, DPB devices based on the DPBs have a high dynamic range and sensitivity. DPBs on conducting polymers can be used as electronic devices to mimic and investigate membrane processes and properties in cells, to fabricate bio-inspired, bioelectronic sensors, and in neuromorphic computing.

[0027] For practical device applications, the polymer surface upon which the lipid bilayers are created is desirably a planar polymer surface. The polymer surface is in contact with an oil solution comprising an oil having lipid molecules dissolved therein. The lipids present in the oil solution spontaneously adsorb to the polymer surface to form an ordered, low defect or defect free monolayer. At least one aqueous droplet is suspended in the oil solution and a monolayer of the lipid from in the oil solution forms around the aqueous droplet at its oil-aqueous interface. The droplet can be incubated to facilitate the selfassembly of the monolayer. A lipid bilayer is formed by bringing the lipid monolayer-coated droplet and the polymer surface-adsorbed monolayer together at an interface between the polymer surface and the aqueous droplet. When the two lipid monolayers come together, the oil between them is expelled and a lipid bilayer is formed. A lipid bilayer formed between a lipid-coated aqueous droplet in an oil bath and a lipid monolayer-coated polymer surface is shown schematically in FIG. 1A. The lipid-coated aqueous droplet can be introduced into the oil solution using, for example, a pipette and can be moved to the polymer surface using, for example, a micromanipulator. An electrically conducting reference electrode, such as a metal wire, inserted into the aqueous droplet can be used to move the aqueous droplet within the oil solution.

[0028] The surface of the polymer in contact with the lipid bilayer does not comprise or consist of a hydrogel. As such, the DPBs are readily distinguishable from DHBs.

[0029] The electrically conducting polymers are conjugated organic polymers that support electronic charge transport and ionic charge transport. A variety of organic polymers can be used, including amphiphilic polymers that are commonly used in biomedical applications and / or electrically conducting organic polymers. Examples of electrically conducting polymers that can be used include, but are limited to, polythiophene polymers, such as poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). To facilitate efficient lipid bilayer formation, it can be advantageous to use hydrophilicpolymers. A hydrophilic surface property can be imparted using, for example, plasma cleaning. Optionally, the polymer can be hydrated prior to exposure to the oil.

[0030] The polymer surface may be the surface of a bulk polymer substrate or may be the surface of a polymer film on an underlying support substrate. In the latter embodiment, the support substrate may comprise an electrically insulating material, such as a glass or ceramic. However, electrically conducting or semiconducting support substrate materials can also be used. A polymer film can be formed on a support substrate using a variety of film-forming techniques, such as spin-coating, electrodeposition, blade-coating, and the like.

[0031] In embodiment of the DPBs in which the polymer is an electrically conducting polymer, the polymer can serve as an electrode in a DPB device. In some embodiments of the DPB devices the polymer is patterned such that one or more areas of the surface in contact with the lipid bilayer comprise or consist of an electrically conductive polymer and one or more areas of the surface in contact with the lipid bilayer comprise or consists of a material other than an electrically conducting polymer, such as an electrically insulating material.Such patterning enables the formation of multiple discrete, electrically isolated electrodes, such as a working electrode and a reference electrode, in contact \\ i th the lipid bilayer. FIG. 8A, which is discussed in more detail below, shows an example of a polymer layer that is patterned to form a separate working electrode (WE) and reference electrode (RE) that are electrically insulated from one another by an intervening non-electrically conductive material. Suitable electrically insulating materials include, but are not limited to, electrically insulating polymers, such as parylene-C and other organic polymers that are used as dielectrics in biomedical applications.

[0032] Multiple DPB devices can be formed by patterning a substrate comprising an electrically conducting polymer into a planar array of electrically isolated electrodes. Lipid- coated aqueous droplets can then be contacted with the lipid monolayer adsorbed on each electrode to form an array of DPBs in which individual electrically conducting polymer electrodes in the array confine the effective DPB area. The electrodes may be microelectrodes designed to have a size comparable to that of the aqueous droplets disposed thereon.

[0033] The oil of the oil solution is an oil in which the lipids can be dissolved. The oil should be immiscible with water and sufficiently non-polar (hydrophobic) to promote lipid monolayer formation.

[0034] Suitable oils include hydrocarbons, such as squalane, squalene, and hexadecane. Other non-limiting examples include silicone oil, decane, and tetradecane. The oil solution can contain mixtures of two or more of these oils and / or other oils.

[0035] The aqueous droplets comprise, at a minimum, water and lipids, and may optionally have salts dissolved therein. Including the lipids in the aqueous droplets and in the oil can increase the rate at which the lipid monolayers are formed. Examples of dissolved salts include metal halide salts, such as potassium chloride. The salts provide anions and cations that can enable the aqueous droplet to function as an electrolyte and / or can be used to facilitate bilayer formation or bilayer stability and / or to enable the ion channel behavior. The aqueous droplets typically have nanoliter scale volumes (e.g., volumes of 100 nL or smaller, including 50 nL or smaller). However, larger aqueous droplets can be used.

[0036] The lipids that can be used to form the bilayer in a DPB can be natural lipids and / or synthetic lipids and / or a hybrid of different lipids. The lipids may be chosen to mimic mammalian cell membranes. Examples of suitable lipids include, but are not limited to, phospholipids, such as l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), l,2-dimyristoyl-5n-glycero-3-phosphocholine (DMPC), 1 .2-distearoyl-s77-glycero-3-phosphocholine (DSPC), 1 ,2-dipalmitoyl-5M-glycero-3- phosphocholine (DPPC), cholesterol, and sphingomyelin. Other non-limiting examples include monoglycerides and naturally-derived lipid extracts obtained from bacteria, plants, and animals.

[0037] The DPBs on electrically conducting substrates can be used in a variety of devices, including transistors, logic circuits, and biomimetic sensors. In some embodiments of the DPB devices an electrode is patterned in the electrically conducting polymer surface on which the bilayer is disposed and a second electrode is placed in electrical communication with the aqueous droplet. The second electrode can be embedded in the aqueous droplet, as shown in FIG. 1A, or can be an in-plane electrode patterned into the electrically conducting polymer surface, as shown in FIG. 8A.

[0038] The elements of the DPB can be modeled as electrical circuits, wherein the lipid bilayer membrane is modeled as a capacitor (Cm) in parallel with a resistor (Rm), the electrolyte solution of the aqueous droplet is modeled as a resistor (Re). and the electrically conducting polymer in contact with the bilayer is modeled as a capacitor (CP). The equivalentcircuit for a DPB device having an electrode embedded in an aqueous electrolyte droplet is shown in FIG. 1A.

[0039] For a DPB device in which in-plane working and reference electrodes are patterned into electrically conducting polymer, the lipid bilayer covers both electrodes. The equivalent circuit for DPB device of this type is shown in FIG. 8A. In the embodiment shown in this figure, the central electrode is a circular reference electrode (RE) and the working electrode (WE) is an in-plane ring electrode surrounding the RE. However, other electrode geometries can be used. The use of an in-plane reference electrode eliminates the need for and external reference electrode in the aqueous droplet and the need for either careful micromanipulation of the aqueous droplets and / or a detailed microfluidic design, which are not convenient in many biosensing applications, but which are required by devices based on traditional DIBs and DHBs.

[0040] The DPB devices can be used to mimic and / or study the properties and behavior of lipid bilayers, including biomimetic lipid bilayers by applying a voltage across the bilayer using the electrodes that are in electrical communication with the bilayer and measuring a generated signal. For example, the current through the lipid bilayer, the impedance of the lipid bilayer, the specific capacitance of the lipid bilayer, the lipid bilayer resistance, and / or ion conductance across the lipid bilayer can be measured. The measured signals can be used in a wide range of studies. By way of illustration only, the DPB devices can be used for electrophysiological studies of lipid bilayer membranes, for studies on ionic channel / protein activity in a DPB that mimics a biological cell, and / or for studies of the insertion of transmembrane proteins in the lipid bilayers of the DPBs.EXAMPLE

[0041] For the first time, this Example reports the formation of DPBs between an aqueous droplet and the high-performing conducting polymer PEDOT:PSS.

[0042] Droplet bilayers were formed using a combined lipid-in, lipid-out approach in a squalene-based oil mixture, selected for its ability to form oil-free bilayers and low interfacial tension with water.

[0043] For comparison, DHBs were first formed on agarose-coated PEDOT:PSS using methods comparable to previous reports, resulting in bilayers with high electrical sealing as measured by electrochemical impedance spectroscopy (EIS). (Leptihn, S. et al., 2013.) Droplet membranes added a bilayer resistance and capacitance in parallel with the impedanceof the PEDOT:PSS electrode, confirmed by a high fit agreement to empirical data (FIG. 2A). DHBs on agarose-coated PEDOTPSS demonstrated a high electrical resistance > 105Q*cm2and a capacitance of 0.70 pF / cm2, which is in good agreement with the reported value for specific capacitance (Cm) of a defect-free l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) droplet bilayer. (Gross, L. C. M. et al., Langmuir 2011, 27 (23), 14335-14342.)

[0044] Droplet bilayers can next be formed directly on PEDOTPSS without an agarose intermediate layer (FIG. 1A). DPBs of DPhPC on PEDOTPSS exhibited similarly high electrical impedance, having an average membrane resistance of 4.9 MQ*cm2and a specific capacitance of 0.63 pF / cm2(FIB. IB, FIG. 3). DPB formation was electrically monitored over time to measure the rise in capacitance from bilayer thinning (FIGS. 4A-4B).Interestingly, it was found that applying a direct voltage pulse after bilayer formation further increased bilayer capacitance without a significant change to bilayer resistance (FIGS. 5A- 5D). It was posited that the applied voltage both accelerated DPB expansion and expelled trapped oil inside the bilayer to achieve this effect. The DPB technique w as found to deliver highly reliable results with a low' formation failure rate. 100% of DPBs tested on PEDOTPSS had a normalized bilayer resistance > 1 kQ*cm2and -50% had a bilayer resistance > 1 M *cm2(n = 11), while no prior work of SLBs on conducting polymers has exhibited reliable electrical resistances > 1 k *cm2. The success of DPBs directly on PEDOTPSS can be explained by its material properties and deliberate efforts to promote monolayer formation. While PEDOTPSS electrodes were not intentionally hydrated prior to oil exposure, PEDOTPSS is hygroscopic and known to absorb water from air in seconds. (Zhanshayeva. L. et al., ACS Omega 2019, 4 (26). 21883-21890.) It was hypothesized that this property, alongside the strong hydrophilicity incurred from plasma cleaning, allowed the polymer to maintain enough moisture for homogenous monolayer formation (FIG. 6A). Additionally, the high concentration of salts in PEDOTPSS films created an intramembrane potential difference > 200 mV across the two lipid monolayers that likely encouraged bilayer zipping (FIGS. 6B-6C). (Tamaddoni, N. et al.. Soft Matter 2016, 12 (23), 5096-5109; Najem, J. S. et al., Nat. Commun. 2019, 10 (1), 1-11; Basham, C. M. et al., ACS Appl. Mater.Interfaces 2022, 14 (49), 54558-54571.)

[0045] To further prove the utility7of DPBs to study model membranes, bilayers were formed on PEDOTPSS of numerous lipid compositions. Membrane lipid compositions are well-known to greatly influence important properties like fluidity, stability, and transmembrane protein functionality7. Membrane composition can be leveraged as a tool tomore closely mimic biological processes and tune sensing performance. Here, DPB resistance could vary between lipid types, with DPhPC exhibiting the strongest resistance to ionic flux, but always displaying resistances > 1 k *cm2(FIG. 3). The membrane capacitance also revealed expected variations with lipid composition. Bilayers containing brain sphingomyelin and cholesterol, designed to mimic mammalian cell membranes with lipid rafts, demonstrated an expected lower Cm because of the longer hydrophobic chain length of sphingomyelin tails and increased trapped oil in the bilayers. These changes in membrane impedance demonstrated not only the ability to form DPBs of diverse compositions, but also the ability of DPBs to sensitively quantity7and differentiate membrane physical properties.

[0046] DPBs can also be established on patterned PEDOT:PSS circular microelectrodes with an adjacent parylene-C insulation layer comparable to microfabricated devices used in bioelectronic interfaces. Using patterned electrodes confines the effective DPB area to simplify characterization and enables more complex sensing circuitry such as multi-droplet arrays. The electrodes were designed to have comparable size to DPBs on unpattemed PEDOT:PSS. On identical electrode geometries, DOPC bilayers formed as DPBs had a substantially higher electrochemical impedance than DOPC SLBs, meaning that they formed a far greater barrier against nonspecific ionic fluxes (FIGS. 7A-7B). DPBs demonstrated an electrical resistance two orders of magnitudes higher than SLBs (2.1 kQ*cm2vs. 37 Q*cm2for representative plots shown). Dynamic range, defined as the difference between the fitted resistance of the bilayer and electrolyte resistance, also exhibited a two order of magnitude improvement over SLBs. DPBs exhibited a lower specific capacitance than SLBs that more closely agreed with Cm values reported in bilayer literature (0.35 pF / cm2vs. 5.72 pF / cm2for representative plots). The higher Cm extracted from SLBs reflected the high defect density7of the membrane, and thus higher fraction of w ater in the membrane, while DPBs had a capacitance value that closely aligned with a defect-free, oil-free bilayer. (Gross. L. C. M et al., 2011.) The lower defect density in DPBs over traditional SLBs on PEDOT:PSS can then improve the sensitivity7of resulting devices to measuring ion channel / protein activity7.

[0047] DIBs and DHBs traditionally require either careful micromanipulation of droplets or a detailed microfluidic design, both of which are not convenient tools in many biosensing applications. Thus, an in-plane gate configuration was designed that does not require an external reference electrode embedded in the aqueous droplet. A planar PEDOT:PSS-coated reference electrode surrounded the PEDOT:PSS-coated circular working electrode so that aDPB may interface with both electrodes simultaneously (FIG. 8A). Although a DPB was expected to form a bilayer over both the working and reference electrodes, resulting in a new equivalent circuit (FIG. 8B), the working and reference electrode areas were designed such that the impedance of the PEDOT:PSS and bilayer of the reference electrode could be neglected during equivalent circuit fitting. Using PEDOTPSS planar gates allowed for faithful measurements of the impedance of the working electrodes (FIGS. 9A-9B). DPBs formed on these planar microelectrodes presented membrane impedances similar in magnitude to those on PEDOTPSS films with an external reference (FIG. 8C). The fitted membrane resistance and capacitance of a DPhPC DPB show n in FIGS. 8A-8C are 4.7 kQ*cm2and 0.34 pF / cm2, respectively, with a high dynamic range > 107Q. DPBs can also be established over PEDOTPSS working electrodes with a non-polarizable silver / silver chloride reference electrode (FIGS. 10A-10C, FIG. 11). These fabrication approaches illustrate the usefulness of the design for more sophisticated bioelectronic circuits.

[0048] Materials: l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), brain sphingomyelin (SM), and cholesterol (Choi) were obtained from Avanti Polar Lipids (Alabaster, AL). PEDOTPSS (Clevios PH-1000) was obtained from Heraeus Holding. Ethylene glycol (EG). 3- glycidyloxypropyl)trimethoxysilane (GOPS), chloroform (CHCh), squalene, n-hexadecane, and (3-(N-morpholino)propanesulfonic acid) (MOPS) were all purchased from Sigma Aldrich.

[0049] Substrate preparation: Glass slides were cleaned through sonication in a 50% acetone, 50% isopropanol mixture for 30 min and plasma cleaned (Harrick Plasma) for 5 min at 45 W before spin coating at 1500 rpm with a dispersion of filtered PEDOTPSS with 5 wt.% EG and 1 wt.% GOPS. After spin-coating, slides were baked at 140 °C for 45 min. A poly(dimethylsiloxane) (PDMS) well was placed on the slide surface to contain the liquid during all experiments. Slides were plasma cleaned for 2 min at 45 W at a pressure of 200 mTorr immediately prior to the addition of lipid solutions during experiments. For those experiments involving agarose to form droplet hydrogel bilayers, 50 pL of 0.5 wt.% agarose solution was drop casted onto the PEDOTPSS surface and allowed to gel before the addition of lipid solution.

[0050] Lipid preparation: For all droplet bilayer experiments, lipid solutions of the desired phospholipid (DPhPC or DOPC) w ere prepared by first aliquoting the requiredamounts of lipid in CHCI3 in glass vials to yield a final concentration of 8 mg / ml for solutions in oil and 3 mM for solutions in aqueous. For lipid mixtures (4:3:3 DPhPC:SM:Chol), the proper molar ratio of lipids was mixed in CHCh in glass vials to reach the same total concentrations. Solutions were allowed to evaporate under N2 flow for 30 min, then excess solvent was further evaporated for > 24 hours under vacuum. Lipid solutions in the oil phase were rehydrated in a 4: 1 squalene: hexadecane and heated at 50 °C for 60 min to promote dissolution. Lipid solutions in the aqueous phase were rehydrated in 100 mM KC1 with 10 mM MOPS buffer and extruded through a 100 nm pore filter 9 times (Avanti Polar Lipids). For solvent-assisted lipid bilayers (SALBs), lipid solutions were prepared and evaporated with an identical procedure. Solutions were rehydrated to a concentration of 0.75 mM in 50 v / v% IPA and sonicated for 1 hour immediately prior to use.

[0051] Droplet bilayer formation and characterization: After plasma cleaning ofPEDOT:PSS substrates, 200 pL of lipid solution in the 4: 1 squalene: hexadecane solution was incubated over the desired surface for 20 min while on an orbital shaker at 90 rotations per minute (Fisher Scientific). Droplet hydrogel bilayers on agarose-coated PEDOT:PSS were not plasma cleaned but allowed to soak in deionized water prior to use to ensure proper hydration. All electrical measurements were performed in a two-electrode configuration with the reference and counter shorted together, which is ty pi cal for droplet bilayer interfacing. An Ag / AgCl reference electrode was prepared by incubating a 100 pm diameter Ag wire (Sigma) in bleach for 20 minutes, rinsing with deionized water for 60 s, and dipping the electrode tip in 1 wt.% agarose to increase hydrophilicity. The reference electrode was attached to a motorized micromanipulator (Narishige International USA. Inc.) and wire connections were made to the substrate and reference electrode with a SP-200 potentiostat (Biologic). The reference electrode was dipped into the PDMS well containing the lipid-oil solution and a 25 nL droplet of lipid-aqueous solution was pipetted onto the electrode tip. The droplet was allowed to incubate for approximately 5 min or until visible sagging began. The electrode was lowered until the droplet rested on the PEDOT:PSS surface. A 200 mV DC bias was applied to encourage bilayer formation, which could take between 5 and 30 min to begin. Droplet bilayers were then allowed to equilibrate in size and electrochemical impedance, as determined optically and electrically. Images of all droplet bilayers were taken using an objective lens of an Olympus 1X50 inverted microscope and acquired with a QI Click CCD camera (Teledyne Qlmaging). DPBs of pure DPhPC were given a 300 mV DC pulse for 30 s prior to final characterization. Electrochemical Impedance Spectroscopy (EIS) measurementswere performed with the potentiostat operated with EC -Lab software over a frequency range from 500 kHz to 50 mHz with a DC voltage of 0 V and AC voltage of 10 mV. The area of each DHB and DPB was determined as the area of the white oval contained by the black outline of the droplet and calculated using ImageJ software. Equivalent circuit values fitted from impedance spectra were finally normalized to the droplet bilayer area to calculate Cm and Rm. Dynamic range was calculated by subtracting the fitted electrolyte resistance of the impedance spectra without a bilayer from the fitted membrane resistance of the bilayer.

[0052] Sessile drop measurements of contact angle: 2 pL droplets of IX PBS or 100 mM KC1 10 mM MOPS solution were deposited on a glass substrate coated with PEDO PSS under n-hexadecane oil. Images were captured using a Dataphysics OCA 15EC optical contact angle platform (Filderstadt, Germany). Contact angles were extracted using their SCA20 software and the results were analyzed using a custom Matlab script.

[0053] Solvent-assisted lipid bilayer formation: The procedure for S ALB formation on patterned PEDOT:PSS microelectrodes was adapted from a previous work. (Schafer. E. A. et al., ACSAppl. Mater. Interfaces 2023, 15 (20), 24638-24647.) 50 pL of lipid solution was incubated over the desired surface for 20 minutes, then the solution was manually exchanged by pipetting to IX PBS over the course of 30 minutes. Finally, the wells were fully flushed with IX PBS to remove any remaining unadsorbed lipids or polymer.

[0054] PEDOT.PSS electrode fabrication and testing: PEDOT:PSS microelectrodes with and without a planar, ring-shaped PEDOT:PSS gate followed the same fabrication process. Cleaned glass slides (1 in. x 3 in.) were spun with the negative photoresist AZ nLOF 2035 (MicroChemicals) at 3500 rpm for 30 s and baked at 110 °C for 120 s, followed by UV exposure using a SUSS MJB4 mask aligner and baked again at 110 °C for 120 s, then developed in AZ300MIF for 40 s. The gold contacts and interconnects of 5 nm titanium / 100 nm gold of the devices were deposited using an AJA E-beam system, and the subsequent liftoff was performed in acetone for 15 min. PEDOT:PSS was patterned using a parylene-C peel-off method as described previously. (Schafer, E. A, et al., Sources and Mechanism of Degradation in p - Type Thiophene-Based Organic Electrochemical Transistors. 2022.) Approximately 2 pm parylene-C was deposited on slides to serve as the insulation layer (LabCoater II) with A-174 silane (Silquest) to promote surface adhesion. The sacrificial layer of parylene-C was deposited after spin-coating slides with a 2% soap solution (Micro90).Openings for PEDOT:PSS were patterned using the positive photoresist AZP4620 spun at3000 rpm for 1 min and baked at 110 °C for 2 min. Substrates were then exposed to UV light using the mask aligner and developed in AZ400 K (1 :4 dilution) for 180 s. Etching with a RAMCO reactive ion etcher (RIE) was performed at 175 W with 50 seem of O2 and 10 seem of CHF3 for ~ 25 min until the desired openings were revealed. The PEDOT:PSS dispersion was spin-coated on slides at 1500 rpm, baked at 90 °C for 60 s, and the second sacrificial layer of parylene-C was mechanically peeled. Finally, the devices were baked at 140 °C for 60 min to cross-link remaining PEDOT:PSS before use. DPBs were formed on patterned electrodes using the same procedure as detailed above with an Ag / AgCl wire electrode, while electrodes with a planar PEDOT:PSS reference electrode did not use a wire electrode. For both types of devices, EIS measurements were performed with a PalmSens4 potentiostat (PalmSens. Netherlands) operated with PS-Trace software using a two-electrode configuration (reference and counter shorted) as in previous experiments. DPB experiments using planar reference gates were conducted using a VC A Optima XE goniometer (AST Products, Inc.) with a vertical motorized syringe pump to dispense 40 nL droplets above each electrode on chip arrays.

[0055] Planar PEDOT.PSS electrode mask design and fitting: The electrode design was created such that the size of the droplet could reliably cover the entirety of the electrode system and simplify analysis of EIS measurement data. Using an in-plane PEDOT:PSS reference electrode as a polarizable gate for impedance measurements presented a new equivalent circuit with two bilayers for modeling (FIG. 8A). However, altering the impedances of the PEDOT:PSS working and reference electrodes allowed for the assumption that the impedance of the reference electrode and reference electrode bilayer do not significantly contribute to the measured impedance and that efficient current modulation of the working electrode was achieved. (Koutsouras, D. A et al., Adv. Mater. Technol. 2021, 6 (12).) The total impedance of the system (ZT) can firstly be written in equation (1) as a combination of the impedances of the PEDOT:PSS reference (ZR), bilayer over the reference (ZB.R), electrolyte (Ze), bilayer over the working electrode (ZB,W), and PEDOT:PSS working (Zw), all in series with each other:(1) ZT= ZR4- ZB R+ Ze4- ZB W+ Zw

[0056] Each of the PEDOT:PSS electrodes is modeled as a capacitor (Cw and CR for the working and reference electrodes, respectively) and the impedance of the electrolyte solution is modeled as a resistor (Re). The bilayers present over the working and reference electrodes(ZB,W and ZB,R, respectively) are modeled as a resistor (RB,W and RB,R, respectively) and capacitor (CB,W and CB,R, respectively) in parallel. Substituting the impedance equations for capacitors and resistors yields equation (2) using the angular frequency ®:

[0057] It can be assumed that the resistance (Rm) and capacitance (Cm) of the droplet polymer bilayer normalized to its area (AR and Aw for the reference and working electrodes, respectively) is the same for each bilayer in the equivalent circuit. The capacitance and resistance of these individual terms is therefore dependent on area, as shown in equations (3) and (4) illustrated for the reference electrode:(3) CB,R = m * R(4) RB,R — Rm * AR

[0058] PEDOT:PSS also has a known scaling relationship with its volume. (Proctor, C.M. et al., J. Polym. Set. Part B Polym. Phys. 2016, 54. 1433-1436.) Using the assumption that both the working and reference electrodes have the same PEDOT:PSS thickness (they are spun-coat in the same fabrication step), the CR and Cw can be understood as dependent on its geometric area AR and Aw, as shown in equation (5) illustrated for the reference electrode:(5) CR= Cp* AR

[0059] Simplifying equation (2) using the relationships determined in equations (3), (4), and (5) demonstrates a total |ZT| shown in equation (6):

[0060] In order to draw the assumption that the voltage drop across ZR and ZB,R is negligible and that the impedances of ZR and ZB,R can be neglected during circuit fitting, it was found that the area of the reference electrode AR must be much larger than Aw such that as the ratio of Aw to AR approaches zero, ZR and ZB.R approach zero, as show n in equation(7):(7)

[0061] The ideal planar electrode design satisfies this geometric criterion while also existing within sensor fabrication limitations. Aqueous droplets can reliably be produced to a700 pm diameter, presenting the outer size limit of the working and reference electrode pair. Additionally, photolithography and alignment limitations within an academic cleanroom dictate a gap between working and reference electrodes > 5 pm for high yield processes. Finally, the area of the working electrode must be large enough that bilayers of the expected resistance and capacitance can be fully captured in the measurement frequency and impedance range capabilities of the potentiostat. To balance these needs, it was therefore dictated that in the electrode design AR must be at least one order of magnitude greater than Aw. Because the thickness and volumetric capacitance of the PEDOT:PSS on the reference electrode and working electrode were equal, this also means that the ZR will be at least one order of magnitude smaller than Zw. In the final design shown in the Example, the circular PEDOT:PSS working electrode had a radius of 100 pm with an approximate area of 0.0003 cm2, while the annular PEDOT:PSS reference electrode had an inner radius of 125 pm and an outer radius of 350 pm with an approximate area of 0.003 cm2(FIGS. 9A-9B).

[0062] Planar Ag / AgC 'I gate electrode fabrication: Ag was patterned using an identical parylene-C peel-off method. The insulating and first sacrificial layer of parylene-C were deposited serially, separated by a soap solution. Openings for Ag were patterned and etched with the same parameters. 300 nm of Ag was deposited using the AJA E-beam system and the sacrificial parylene-C layer was mechanically peeled to remove unwanted Ag. Ag electrodes were subsequently exposed to FeCh for 10 min to establish Ag / AgCl reference electrodes. After, a second sacrificial parylene-C layer was deposited, patterned, and etched for PEDOT PSS working electrodes. The final design had a circular PEDOT:PSS working electrode with a radius of 150 pm and an annular Ag / AgCl reference electrode with an outer radius of 600 pm and an inner radius of 190 pm so as to maximize droplet area with the reference (FIGS. 10A-10C).

[0063] The word "illustrative" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "illustrative" is not necessarily to be construed as preferred or advantageous over other aspects or designs. Further, for the purposes of this disclosure and unless otherwise specified, "a" or "an" means "one or more.”

[0064] The foregoing description of illustrative embodiments of the invention has been presented for purposes of illustration and of description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention.The embodiments were chosen and described in order to explain the principles of the invention and as practical applications of the invention to enable one skilled in the art to utilize the invention in various embodiments and with various modifications as suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

WHAT IS CLAIMED IS:1 . A droplet polymer bilayer comprising: a substrate comprising a non-hydrogel polymer surface; a first lipid monolayer adsorbed directly on the non-hydrogel polymer surface; an oil solution over the first lipid monolayer; and a hpid-coated aqueous droplet in the oil solution, the lipid-coated aqueous droplet comprising: an aqueous droplet comprising a dissolved salt; and a second lipid monolayer around the aqueous droplet at an oil-aqueous interface formed between the aqueous droplet and the oil solution, wherein the first lipid monolayer and the second lipid monolayer form a lipid bilayer at a polymer-droplet interface formed between the non-hydrogel polymer surface and the aqueous droplet.

2. The droplet polymer bilayer of claim 1, wherein the non-hydrogel polymer of the non-hydrogel polymer surface comprises an electrically conducting organic polymer.

3. The droplet polymer bilayer of claim 2, wherein the electrically conducting organic polymer is a poly thiophene polymer.

4. The droplet polymer bilayer of claim 3, wherein the polythiophene polymer is PEDOT:PSS.

5. The droplet polymer bilayer of claim 2, wherein the non-hydrogel polymer surface underlying the lipid bilayer is a patterned surface comprising a first electrode area comprising a non-hydrogel electrically conducting organic polymer and a second electrode area comprising a non-hydrogel electrically conducting organic polymer, wherein the first electrode area and the second electrode area are separated by a non-electrically conducting material.

6. The droplet polymer bilayer of claim 5, wherein the first electrode area is a circular area and the second electrode area is a ring-shaped area surrounding the first electrode area.

7. The droplet polymer bilayer of claim 2, further comprising an electrode embedded in the lipid-coated aqueous droplet.

8. The droplet polymer bilayer of claim 1, wherein the oil is a hydrocarbon oil.

9. The droplet polymer bilayer of claim 1, wherein the lipid is a phospholipid.

10. The droplet polymer bilayer of claim 4, wherein the lipid is a phospholipid and the oil comprises squalane, n-hexadecane, or a mixture thereof.

11. The droplet polymer bilayer of claim 1, wherein the lipid is a naturally occurring lipid.

12. The droplet polymer bilayer of claim 4, wherein the lipid is a naturally occurring lipid and the oil comprises squalane, n-hexadecane, or a mixture thereof.

13. The droplet polymer bilayer of claim 1, wherein the substrate comprises a film of a non-hydrogel polymer on an electrically insulating substrate.

14. A method of measuring a property of the lipid bilayer of the droplet polymer bilayer of claim 1, the method comprising applying a voltage across the lipid bilayer and measuring one or more of the cunent through the lipid bilayer; the impedance of the lipid bilayer; the specific capacitance of the lipid bilayer; the lipid bilayer resistance; and the ion conductance across the lipid bilayer.

15. A method of making a droplet polymer bilayer, the method comprising: forming a first lipid monolayer directly on a non-hydrogel polymer surface of a substrate in an oil solution; forming a lipid-coated aqueous droplet in the oil solution, the lipid-coated aqueous droplet comprising: an aqueous droplet; and a second lipid monolayer around the aqueous droplet at an oil-aqueous interface formed between the aqueous droplet and the oil solution; and bringing the lipid-coated aqueous droplet and the first lipid monolayer on the nonhydrogel polymer surface together, whereby the first lipid monolayer and the second lipidmonolayer form a lipid bilayer at a polymer-droplet interface formed between the nonhydrogel polymer surface and the aqueous droplet.

16. The method of claim 15, wherein the non-hydrogel polymer of the nonhydrogel polymer surface comprises an electrically conducting organic polymer.

17. The method of claim 16, wherein the electrically conducting organic polymer is a polythiophene polymer.

18. The method of claim 17, wherein the polythiophene polymer is PEDOT:PSS.

19. The method of claim 18, wherein the lipid is a phospholipid or a naturally occurring lipid.

20. The method of claim 19, wherein the oil is a hydrocarbon oil.

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