Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in

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Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in Supported Lipid Bilayer Using a Single Nanopore Chip Muhammad Shuja Khan, Noura Sayed Dosoky, Ghulam Mustafa, Darayas Patel, Bakhrom K. Berdiev, and John Dalton Williams Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02404 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in Supported Lipid Bilayer Using a Single Nanopore Chip Muhammad Shuja Khan1*, Noura Sayed Dosoky2, Ghulam Mustafa3, Darayas Patel4, Bakhrom Berdiev5, John Dalton Williams1 1

Electrical and Computer Engineering Department, University of Alabama in Huntsville,

Huntsville AL 35899 USA 2

Biotechnology Science and Engineering Program, University of Alabama in Huntsville,

Huntsville AL 35899 USA 3

Department of Nuclear Medicine, The State University of New York at Buffalo, Buffalo NY

14214 USA 4

Department of Mathematics and Computer Science, Oakwood University, Huntsville, AL 35896

USA 5

Department of Biomedical Sciences, Nazarbayev University School of Medicine, Astana,

010000, Kazakhstan *Email: [email protected]

1

ACS Paragon Plus Environment

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ABSTRACT Nanopore-based technologies are highly adaptable supports for developing label-free sensor chips to characterize lipid bilayers, membrane proteins and nucleotides. We utilized a single nanopore chip to study electrophysiology of epithelial Na+ channel (ENaC) incorporated in supported lipid membrane (SLM). An isolated nanopore was developed inside the silicon cavity followed by fusing large unilamellar vesicles (LUVs) of DPPS (1,2-dipalmitoyl-sn-glycero-3phosphoserine) and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) to produce a solvent-free SLM with giga-ohm (GΩ) sealed impedance. The presence and thickness of SLM on the nanopore chip were confirmed using atomic force spectroscopy. The functionality of SLM with and without ENaC was verified in terms of electrical impedance and capacitance by sweeping the frequency from 0.01 Hz to 100 kHz using electrochemical impedance spectroscopy. Nanopore chip exhibits long-term stability for lipid bilayer before (144 h) and after (16 h) incorporation of ENaC. Amiloride, an inhibitor of ENaC, was utilized at different concentrations to test the integrity of fused ENaC in lipid bilayer supported on single nanopore chip. The developed model presents excellent electrical properties and improved mechanical stability of SLM, making this technology a reliable platform to study ion channel electrophysiology. Keywords: Nanopore chip; supported lipid bilayer; epithelial sodium channel (ENaC); amiloride, electrochemical impedance spectroscopy; atomic force microscopy.

2


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1.

INTRODUCTION Several important biophysical processes can be studied by mimicking the cellular

environment across an artificial cell membrane. These processes are in great interest for medical applications such as drug screening, medical diagnostics and characterization of protein channels 1. Such transmembrane proteins are vital components of lipid bilayer for signalling, solute transport and immune recognition

2

. The traditional black lipid

membranes (BLMs) have been produced across the nano and micro-meter sized apertures in Teflon

3,4

, plastic septa 5, glass cover slip 6, Teflon coated silicon apertures

and other silicon based materials

8

by using painting and/or folding methods

7

9,10

.

3,5,6,11–16

However, these artificial biomembranes exhibit a short life

. To increase the

quality and stability of BLMs, a solid substrate was also considered

17

. Despite having a

solid platform to increase the BLM stability, most of the membranes could not tolerate the solution exchange. It is always desired to minimize the organic solvent in 6

membranes, even at the expense of membrane stability

because the remaining

residuals of organic solvent in the hydrophobic area of BLM could possibly disturb the embedded protein channels

18,19

.

Supported lipid membranes (SLMs)

14,20–25

developing biosensors with long-term stability

have been introduced and reported for

14,26

. The most commonly used techniques

to produce SLMs on hydrophilic substrates include Langmuir Blodgett followed by Langmuir Schaefer (LB/LS) and liposome fusion

34

27–30

, the vesicle adsorption and rupture

and droplet-interface bilayer

23,31–33

, combined LB

35–38

. SLMs based on these

techniques have shown more stability as compared with BLMs

39,40

. However, the

usability of these membranes is constrained by their close surface proximity, which substantially averts the fusion of integral proteins. Lipid bilayer produced on a solid support may not be consider as a significant apparatus to explore the electrophysiology of transmembrane proteins because of excessive ions present near the substrate and may denature the protein channels embedded in SLM

14

. To overcome this problem,

several other groups have explored methods to increase spacing using either a single or double hydrated polymer cushions between the membrane and the support

41–47

. One

interesting technique was to create a tethered-lipid bilayer membrane (t-LBM) directly on substrate for incorporating integral membrane proteins

48

. The increased distance,

however, still does not allow for a free ion movement and limit the possibility of monitoring single channel events. 3


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To overcome the above highlighted challenges, many other groups have been focused on preparing BLMs and SLMs on porous membranes (alumina, silicon and silicon nitride)

12,13,16,29,33,49

. However, sometimes such porous templates produce pores

with uneven distribution. A substrate with a single nanopore coated with an artificial membrane could be utilized to inspect the behavior of a lipid bilayer membrane with and without proteins 33,53–57

, Al2O3

17,50–52

. A single micro/nano pore or an array of pores produced in Si3N4

58

, and Si

29,59

utilized a complex fabrication procedure with high cost.

Nanospot GmbH’ developed an array of nanopores utilizing reactive ion etching techniques to investigate fusion process and membrane transport

60

. In another

approach, an array of nanopores was fabricated in pre-pattern arranged silicon cavities 61–63

. Hybrid biological solid-state nanopore has tremendous applications to study

sequencing of DNA

64

and bacteriophage phi29 connector channel fused in bilayer has

revealed the dynamics of dsDNA translocation 65,66. In this work, we present a label-free and highly robust nanopore chip supported with lipid bilayer to study the fusion procedure, stability and integrity of embedded Epithelial Na+ Channel (ENaC). A single pore was formed with size 167±12 nm inside the cavity of a Si surface. Lipid bilayer membrane was produced with thickness 4.7±0.4 nm on nanopore surface as verified using atomic force spectroscopy. The membrane coated on nanopore confirmed long term stability for up to 144 h and 16 h with impedance of >1 GΩ before and after incorporation of transmembrane protein ENaC respectively. EIS was employed within frequency domain of 0.01 Hz to 100 kHz for both SLM and the fused ENaC to determine the impedance and capacitance. Stability of embedded protein channels with varying concentrations in SLM was further monitored over several hours. Finally, integrity of the embedded channels formed across SLM was studied using amiloride blocker with different concentrations.

2. EXPERIMENTAL SECTION 2.1. Fabrication of Nanopore. Fabrication of a single nanopore chip has been reported previously

67

. Briefly, SOI wafer purchased from Silicon Valley Microelectronics,

Inc. was coated with Si3N4 layer (2 µm) on both sides using LPCVD technique followed by a lithographically transferred pattern of size 1x1 mm2 using SPR220. RIE was used to expose the Si3N4. The cavity was developed anisotropically in p-type Si with 35% KOH at 4


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60 °C followed by removal of a SiO2 layer (2 µm) using 5% HF for 2 min. Si3N4 layer (200 nm) was deposited on the n-type surface of Si followed by patterning the dimension of 20x20 µm2 window at the center of thin Si membrane. Another cavity in Si membrane was obtained with 8% KOH at RT for 20 min. Two Teflon supports were used to sandwich the chemically processed Si wafer using O-rings and photo-electrochemical etching was applied using an LED to create a single nanopore inside the Si cavity. 2.2. Supported Lipid Membrane Preparation and Protein Channel Fusion. Lipid bilayer membrane was prepared by spreading and fusing LUVs onto solid-state nanopore surface. Stock solution was prepared in chloroform with lipids’ ampoules (Avanti Polar Lipids Inc.) and stored in vials at -20 °C. Concentrations of 300 µL for DPPE (0.75 µg) and 300 µL for DPPS (0.375 µg) were prepared from stock solution. Nitrogen spray gun was used to evaporate the solvent. After desiccating for 1.5 h, the lipids were then rehydrated in buffer solution. LUVs were prepared with sonicator bath (Branson Sonicator 1510) to clarity at RT for 30 min followed by incubation on the hydrophilic surface of nanopore substrate at 65 °C for 20 min. Finally, with rate of 1 °C/min, the sample was allowed to cool at RT followed by spanning the lipid bilayer membrane on the nanopore surface. Optimized values for the formation of a robust and stable membrane were found by controlling different parameters such as incubation temperature, time and cooling rate as detailed in Table S1. The sample was gently rinsed with buffer to remove the unattached vesicles. The procedure to reconstitute ENaC proteins into proteoliposomes is detailed previously

68–70

.

2.3. Characterization with AFM and SEM. The nanopore chip grafted with supported lipid bilayer was assembled carefully inside the Teflon based cell and experiments were carried out using imaging buffer in the non-contact mode of Pico Plus AFM 1550 from Molecular Imaging. A cantilever with k=0.3 N/m (Bruker Inc.) was utilized in during imaging with scan rate set in the range 8-18 Hz for area size less than 2 µm². Further SEM experiments were taken on a LEO1550 SEM instrument. 2.4. Electrochemical Impedance Spectroscopy. EIS from VersaSTAT MC by Princeton Applied Research, AMETEK was used at frequency domain from 0.01 Hz to 100 kHz with a signal amplitude of 0.1 V. Fabricated nanopore chip with supported lipid bilayer was assembled using two Teflon chambers. In order to prevent electrolyte leakage across openings between the nanopore surface and the Teflon chamber, Orings were used. ENaC was used as a Na+ ion channel protein. Therefore, both Teflon 5


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chambers (cis and trans) were filled up with 10 mM NaCl as an electrolytic solution. To provide electrical current, EIS with three electrodes were utilized. Platinum electrode along with Ag/AgCl was immersed in electrolyte of one chamber (cis). The other platinum electrode was assembled with the secondary chamber (trans). Electrochemical characterization was performed for SLM coated on nanopore chip before and after incorporation of ENaC and data were presented in Nyquist and Bode plots.

3.

RESULTS AND DISCUSSION 3.1. Nanopore Chip with SLM. A SOI wafer was used to produce a single nanopore

inside the cavity of silicon. A process schematic is shown in Fig. 1a utilizing two steps chemical etching procedure KOH (i) and HF (ii). Fig. 1b shows the top view of the anisotropically etched cavities in p-type Si and after the removal of thin SiO2 layer using 5% HF, another small cavity was finally created. Fig. 1c reveals the SEM image of the fabricated groove cavity with a pore at high resolution and insets show the front and rear image of the nanopore. Results presented in Fig. 1d reveal AFM imaging of the nanopore surface followed by line profile to estimate the size of the fabricated pore. After production of supported lipid membrane using the method described in the experimental section, AFM imaging was performed in an aqueous environment to evaluate the membrane thickness on the nanopore surface. As shown in Fig. 2a, AFM tip directly implies stress on the membrane during the non-contact mode. The green line profile (1) reveals the visual illustration of the tip while exerting a force on membrane surface over the nanopore area at close proximity. Though the tip exhibits a reasonable force to rupture the membranes’ surface on the nanopore, the lipid bilayer confirms high strength with a thickness of 4.7±0.3 nm. An additional 1.2±0.3 nm contributes the surface roughness with 1–2 nm representing a thin hydrated layer on thin silicon membrane. A thermal drift of about 0.3–0.5 nm was observed during AFM measurements and it was most likely due to the phase transition temperature difference between two lipids (DPPS (54°C) and DPPE (63°C)). Moreover, these experiments were not conducted on a regular mica or glass instead we used chemically etched Si membrane as a substrate. The blue (2) and red (3) profiles show the presence of the SLM on Si surface near the nanopore area. The focus of this work was to investigate the long-term stability of the established

SLM

on

nanopore;

therefore

force

spectroscopy


experiment

was 6

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demonstrated to examine the rupture and the actual thickness of the membrane on the nanopore. Fig. 2b (left) shows the force-displacement relation for the lipid bilayer with AFM tip. The tip initially interacted with the top monolayer of SLM at a distance (Zad) of 6.1 nm with respect to the nanopore. The cantilever was then deflected to point ‘b’ from ‘a’ and the rupture was observed with force (FA) 1.33 nN at a distance (Zbd) of 4.7 nm. Fig. 2b (right) reveals the complete cycles for a single tip interaction with SLB and Gaussian distribution was used to fit the data. The obtained values for rupture force and the bilayer thickness are 1.41±0.12 nN and 4.7±0.4 nm respectively. The bilayer depth (Zbd) is comparable with previously estimated membrane thickness in the literature

15,65–

67

. 3.2. ElS Measurements of Nanopore Spanned SLM. Single nanopore chip was

assembled in Teflon based chambers as shown in Fig. 2c. Electrical recordings were demonstrated with a range of frequency at 0.01 Hz to 100 kHz as described in experimental section. Fig. 3a shows the electrical equivalent models for the bare nanopore and the SLM coated on nanopore chip. Details of both models and the role of each element present in these models are described in the electronic supplementary information. Bare nanopore impedance was previously calculated to be 5.4 MΩ

67

. The

impedance of SLM on nanopore was increased significantly from MΩ to GΩ as illustrated in Fig. 3b. Since the lipid bilayer prevents the flow of ions through the nanopore, the system changes its behavior from resistive to capacitive as depicted in the phase spectra of Fig. 3c. The phase shift of 80–90° reveals the capacitive behavior which confirms the presence of lipid bilayer membrane on the nanopore area. The impedance and capacitance of nanopore before (i) and after (ii) coating lipid bilayer was calculated with electrical models presented in Fig. 3a. After data fitting, the membrane capacitance was obtained to be 0.63±0.11 µF/cm2. The measured capacitance is in match with published literature capacitance of 0.7±0.3 µF/cm2 2 57

0.40 µF/cm

16

, 0.63 µF/cm2

29,30

, 0.65±0.2 µF/cm2

13

and

for lipid bilayer formed on porous surface with an average pore diameter 7

µm, ~2 µm, 280 nm and 200 nm respectively. Some other groups reported larger membrane capacitance with more stability than those produced using organic solvents 12,23,33,58,71–74

. Nevertheless, sometimes the larger capacitance occurs with incomplete

adsorption of vesicles. The membrane with capacitance ~0.62 µF/cm2

75,76

was reported

using the phosphatidylcholine lipids and our results are close to them. Sometimes, thin Au layer deposited in substrate could be the reason for high value of membrane 7


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capacitance (0.9–1.3 µF/cm2)

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12,77

. However, Au coated membrane accounts low

impedance and exhibits high defects in the bilayer production

72

.

Membrane impedance is dependent on its stability on the nanopore chip. In this work, SLM exhibited >1 GΩ sealed resistance (n=37). Equivalent circuit Fig. 3a (ii) was modelled to fit the data and membrane impedance was estimated to be 23.4±2.14 GΩ with a normalized value of 4.68 Ω.cm². This data is in agreement with SLM produced with GΩ impedance prepared using SUVs on the pore diameter of 800 nm fabricated in a thin layer of Si/Si3N4

74

. However, the success rate of their membrane formation was

30%, which is almost two times less as compared to the membrane formed using LUVs in this study on the nanopore with a diameter of ~167 nm. In their work

74

, they reported

the normalized resistivity of 5.0 Ω.cm² which is close to the data obtained in this work (4.68 Ω.cm²). Our data (23.4±2.14 GΩ) is in close agreement with membrane produced using aqueous droplet technique (26.3 GΩ)

37

. However, their normalized resistivity (15

Ω.cm²) is almost three times higher as compared to our data (4.68 Ω.cm²). One of the major reasons for this high value was the large surface contact area of the membrane. In spite of having high normalized resistivity, the solid surface hinders the application of a single channel activity and stops the actual current flow across the membrane channel. 3.3. Stability of SLM on Nanopore Chip. Membrane stability is verified in terms of impedance data obtained via Nyquist and phase plots as shown in Fig. 3d and 3e respectively. Prior to membrane formation, bare nanopore exhibits the impedance of ~5.4 MΩ and a phase < 5° as shown in Fig. 3c. The initial impedance was recorded to be 23.4 GΩ after the successful formation of the membrane. SLM impedance was then gradually decreased to 17.3, 15.6, 12.4, 8.05 and 4.51 GΩ at the time of 1, 2, 3, 4 and 5 days respectively. Finally, lipid bilayer exhibited the impedance of ~1.25 GΩ (n=11) at the time of ~144 h (sixth day) as shown in Fig. 3d. Bode plot of Fig. 3e shows the decrease of the profiles for 0.01–0.1 Hz. The decrease in the SLM resistance and increase in capacitance are calculated with respect to time after fitting the data as shown in Fig. 3f. Previously, SLM produced using small unilamellar vesicles on a single nanopore with the size of 200 nm showed the stability of up to 50 h 58. In another work

78

,

lipid bilayers with stability of up to 36 h and 21 h were created on triangle and beak apertures with size 60–80 µm respectively. In this work, we established SLM on single nanopore chip and validated its stability of up to 144 h. The other important parameters

8


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include an electric double layer with resistance (Redl=1.8 KΩ) and capacitance (Qedl=0.855 nF-sα-1 with α=0.709) as tabulating in Table 1. 3.4. ENaC Fused within SLM. The suitability of the membrane on the nanopore surface for the development of a highly robust biosensor was further investigated by observing the electrical resistance and capacitance of SLM after insertion of a transmembrane protein, ENaC. The procedure to reconstitute ENaC proteins into proteoliposomes is detailed previously

68–70

. ENaC reconstituted in proteoliposomes 20

ng/µL was spread near the SLM surface coated on nanopore chip. Transmembrane membrane

proteins

are

self-incorporated

in

membranes.

Therefore

real-time

experiments were demonstrated to study the incorporation of ENaC in SLM as shown in Fig. 4. Impedance change depicts the fusion mechanism of ENaC. Complete incorporation was evaluated by observing the dramatic decrease in the membrane impedance from 23.4 to 3.52 GΩ over the period of time. A similar trend of decreasing membrane impedance was also observed during the investigation of ENaC functionality in lipid bilayers supported on porous silicon support

29,30

. Data were recorded

continuously over the period of time for 5, 12, 20, 40min and presented using Nyquist plot in Fig. 4a. Capacitance change behavior of SLM after ENaC insertion is represented using phase spectra as shown in Fig. 4b. The double peaks in phase angle spectra confirm the successful incorporation of ENaC protein channels in SLM. The complete ENaC fusion in SLM was obtained for 20 min and the recorded time is compatible with data reported previously

70

. However, experimenting demonstrated in this work took an

additional 3–4 min to accomplish complete fusion of ENaC and the reason could be the variation in ENaC concentration during reconstitution of proteins into proteoliposomes. The obtained results were fit using Fig. 4c and electrical resistance and capacitance were then calculated. The capacitance of the membrane-embedded protein (ENaC) was finally estimated to be 0.77 µF/cm2 which is in close agreement with the published literature

79,80

. In another study, with the porous membrane, the ENaC capacitance was

found to be 0.537 µF/cm2

30

. Since porous membrane produces multiple channels

distributed throughout the support, this could be the potential reason for this slight change in capacitance in their work 30. 3.5. Stability of ENaC Embedded in SLM. ENaC with concentrations 20, 30 and 40 ng/µL reconstituted in proteoliposomes were used to validate the stability of SLM with embedded ENaC. Fig. 4e shows the impedance spectra of SLM fused with different 9


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concentrations of ENaC followed by data fitting using the electrical equivalent circuit of Fig. 4c to estimate the electrical impedance and capacitance. The Nyquist plot (Fig. 4e) reveals that the membrane embedded with ENaC protein exhibited good stability with the total impedance of more than 1 GΩ. The impedance of SLM with ENaC 40 ng/µL was low (1.52±0.12 GΩ) as compared with SLM having ENaC 30 ng/µL (1.71±0.37 GΩ) and 20 ng/µL (1.86±0.24 GΩ). This change in impedance could be due to the fact that high concentration of ENaC refers the presence of more channels as compared with low concentration in SLM. This causes more current across SLM via fused channels and finally reduction in impedance. Therefore, the difference in impedance is prominent at t=4 h due to the presence of more channels for 40 ng/µL ENaC. However, at t=16 h, Nyquist plot data show less difference between 20, 30 and 40 ng/µL. With passing time, channels starting to lose their stability within SLM and contributed in the reduction of SLM impedance. Table 2 summarizes the data obtained after fitting the curve for the individual concentration of ENaC. Further, the outcome of each element involved in the electrical equivalent circuit of Fig. 5c for bare nanopore, SLM coated on nanopore and ENaC fused in SLM is presented in Table 1. 3.6. Integrity of Embedded ENaC Channel with Amiloride. To validate the integrity of ENaC channels within SLM coated on single nanopore chip, we demonstrated the current across ENaC fused in SLM with amiloride concentrations, a specific inhibitor of ENaC, at different concentrations as shown in Fig. 5. Amiloride is high-affinity blockers of ENaC and it reasonably binds with the pore of Na+ channel

81

. Fig. 5a illustrates the

operating behavior of fused pores in SLM and switching time for these pores was exceptionally small. The slope of the straight line from current-voltage relation was used to determine the ENaC conductance as shown in Fig. 5b. ENaC conductance before treating with amiloride was 18±2.1 pS. It was reported previously that dwell time for ENaC channel incorporated in lipid bilayer occurs in the range from 10-6 to 10-3 sec

81

.

Even after the addition of amiloride, the dwell time duration is the same as the channel closed states and this makes the kinetics of ENaC blocked by amiloride more complex

81

.

Therefore, experiments were carried out using different concentrations of amiloride (30, 250, 800 and 1000 nM) to demonstrate the variations in blocked states of the channel. Initially, Fig. 5a shows data for ENaC channel activity in the absence of amiloride. Fig. 5c, 5d, 5e, and 5f reveal ENaC functionality in the presence of amiloride concentration of 30, 250, 800 and 1000 nM respectively. It can be seen that with the addition of amiloride, 10


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non-conducting states show different current signature as compared to previously recorded pattern (Fig. 5a) with longer duration depending on the concentration of the amiloride. It can be seen in Fig. 5g that the channel blocked states (τb) are not dependent on the concentration of amiloride. The slope and the constant were calculated based on the data presented in Fig. 5g to be 0.45 µM-1s-1 and 4.45 s-1 respectively. Here, the constant represents the dissociation rate, koff according to Eq. S15. On the other hand, channel open states (τo) is dependent on amiloride concentration as shown in Fig. 5h and is decreased with the addition of amiloride concentration as anticipated in Eq. S14. Current signatures with respect to time in Fig. 5c, 5d, 5e, and 5f represent qualitative information while the association rate constant (kon) reveals quantitative information in Fig. 5h. The slope and constant information were calculated to be 86.25 µM-1s-1 and 0.53 s-1; representing the association rate, kon according to Eq. S14 and the closing rate β respectively. Finally, Eq. S16 was used to determine the equilibrium dissociation constant (kD, 0.052 µM) with pre-obtained values of koff and kon. The kon for amiloride determined from these experiments is close to previously reported values

81–83

.

However, the kon is higher as compared with the value determined to close Na+ current in frog skin

84

. This slight difference could be due to the utilization of different techniques in

their work.

4.

CONCLUSION In this work, a solid-state nanopore chip, SSNP was developed and explored to

investigate SLM before and after incorporation of ENaC. Solvent-free SLM was prepared on SSNP substrate using LUVs of DPPE and DPPS. AFM was utilized in an aqueous environment to study the nanopore surface morphology after the production of SLM on nanopore chip with thickness 4.7±04 nm. The rupture force and the thickness of the established lipid bilayer were determined using atomic force spectroscopy. Giga-ohm (~23.4 GΩ at t=1 h) sealed membrane exhibited excellent stability up to 144 h with a minimum impedance of ~1.25 GΩ. Effect of ENaC protein concentrations (20, 30 and 40 ng/µL) in pre-formed SLM was investigated and SLM showed long-term stability of up to 16 h. Finally, pharmacological blocker (amiloride) at different concentrations (30, 250, 800 and 1000 nM) was utilized to test the integrity of fused ENaC in SLM supported on SSNP. It was found that channel open state (τo) correspond linearly with increasing 11


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concentration of amiloride and channel blocked state (τb) was independent of the amiloride concentration. Acknowledgement This work was supported by Office of the Vice President for Research and Economic Development, University of Alabama in Huntsville, Huntsville, AL. Dr. Berdiev was supported, in whole or in part, by National Institutes of Health Grant R21HL085112, the University of Alabama at Birmingham Health Services Foundation General Endowment Fund, and Nazarbayev University Social Policy Grant. Conflict of Interest The authors declare no conflict of interest.

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Table 1. Summary of each element involved in electrical equivalent circuits for bare Si nanopore, supported lipid membrane coated on nanopore and embedded transmembrane protein in SLM. System

RNP MΩ

CNP µF/cm²

RLBM GΩ

CLBM µF/cm²

RTP KΩ

CTP µF/cm²

REDL KΩ

QEDL nF/cm²

Nanopore

5.6±0.24

1.52±0.72

SLM

5.6±0.24

1.52±0.72

SLM with ENaC

5.6±0.24

1.52±0.72

3.25±0.24

α

-

-

-

-

2.36±0.02

87.8±3.12

0.22

23.4±2.14

0.63±0.11

-

-

1.82±0.04

0.85±0.07

0.71

0.76±0.04

1.11±0.07

1.19±0.08

1.11±0.07

0.64±0.04

0.62

Table 2. Resistance and capacitance of supported lipid membrane embedded with different ENaC concentrations (20, 30 and 40ng/µL). Time (h)

Resistance (GΩ)

Capacitance (µF/cm²)

20ng/µL

30ng/µL

40ng/µL

20ng/µL

30ng/µL

40ng/µL

4

1.86±0.24

1.71±0.37

1.52±0.12

0.76±0.04

0.77±0.02

0.79±0.05

16

1.21±0.17

1.11±0.81

1.05±0.23

0.86±0.03

0.87±0.04

0.91±0.06

24

0.81±0.10

0.75±0.68

0.73±0.11

1.11±0.07

1.13±0.05

1.19±0.08

20


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Fig. 1. Development of a single nanopore chip. (A) Cross section schematic of multistep chemical etching procedure with KOH (top) and HF (bottom). (B) SEM image of anisotropically etched cavities with the depth of ~500 µm in Si (p-type) using KOH. (C) Electrochemically fabricated nanopore in V-shaped groove cavity of Si (n-type) using HF. Insets reveal high-resolution SEM image of the front and rear sides of the fabricated single nanopore. (D) AFM imaging of the nanopore in silicon (top). Line profile confirms the diameter of the pore (bottom).

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Fig. 2. AFM representation of lipid bilayer coated on nanopore surface and assembly of nanopore chip coated with SLM in between two Teflon chambers. (A) AFM data (left) confirms the presence of the SLM on nanopore surface and line profile (right) reveals different segments of the sensor chip to estimate the thickness of the SLM spanned on nanopore chip. (B, left) A force-distance measurement for SLM produced on nanopore surface using AFM. (B, right) SLM rupture force (FR=1.33±0.18 nN) and thickness (Zbd=4.7±0.4nm). (C) Assembly schematic of a nanopore sensor in between two Teflon chambers using O-rings (left). Schematic of SLM coated on nanopore surface before and after fusion of transmembrane protein channel ENaC (right).

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Langmuir

Fig. 3.Electrical equivalent model and long-term stability of lipid bilayer membrane on a single nanopore chip. (A) Bare nanopore chip and nanopore coated with lipid bilayer membrane. (B) Magnitude plot reveals estimated impedance of nanopore (5.4 MΩ) and lipid bilayer (~23.3 GΩ). (C) Phase plot shows the resistive and capacitive behaviour of nanopore chip before and after deposition of lipid bilayer membrane. Brown circles represent experimental data for nanopore and green circles represent results for electrical insulation layer of lipid membrane across nanopore surface. Data fitting curves (solid black lines) are the results of electrical models presented in (A). (D) Stability of giga-ohm sealed membrane spanned on nanopore chip. Nyquist plot shows the GΩ impedance of SLM over continuous six days (144 h) with a minimum impedance of ~1.25 GΩ (E) Phase plot shows the capacitive behaviour with variations at different time intervals at lower frequency range, f < 1 Hz. (F) Membrane resistance and capacitance information obtained after employing data fitting models (A) as a function of time.

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Fig. 4. Incorporation process of ENaC transmembrane protein (20 ng/µL) into lipid bilayer over time. (A) Change in SLM impedance (A) and conductance (B) spectra over the period of time depict fusion process of ENaC in the membrane. (C) Electrical equivalent model of ENaC embedded in SLM on a single nanopore chip. (D) Time-dependent information of impedance and capacitance for ENaC channel in the membrane. Resistance and capacitance values were derived by applying data fitting electrical model (C) on experimental results (A, B). (E) Effect of ENaC concentrations on membrane stability. Data fitting lines are extracted and fit using electrical equivalent model (B). The Nyquist plot shows a negligible difference at 16 h in comparison with 4 h for 1, 1.5 and 2 µL.

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Fig. 5. Electrical recording of fused ENaC channel in SLM with varying amiloride (30, 250, 800 and 1000 nM). (A) Electrical data represents the behavior of embedded ENaC in lipid bilayer supported on a single nanopore. (B) Current-voltage relationship of ENaC in lipid bilayer reveals conductance of 18.2 pS, derived using the slope of the straight line. (C, D, E, F) ENaC channel activity following addition of amiloride at different concentrations of 30, 250, 800 and 1000 nM. (G) The channel blocked state (τb) is shown with respect to concentration of amiloride. The slope and the constant were calculated to be 0.45 µM-1s-1 and 4.45 s-1 respectively. (H) The channel open state (τo) is shown with varying amiloride concentration. The slope and constant information were calculated to be 86.25 µM-1s-1 and 0.53 s-1; representing the association rate, kon according to Eq. S14 and the closing rate β respectively.

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TOC

26


Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in

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