Integrating Ion Channels with Bioelectronics for Biotic–Abiotic Systems

Precise and highly regulated flow of biomolecules and ions through complex cellular networks is crucial for communication and information processing in living systems. In contrast, human‐made electronics rely on the flow of electrons and holes through well‐defined semiconductor networks for processing. Ion channels play vital roles in regulating the flow of ions and biomolecules across the cell membrane with a complexity unmatched in any semiconductor device. To enable biotic–abiotic communication and leverage this complexity, supported lipid bilayers (SLBs) create a planar cell membrane for integration with bioelectronics. This review discusses the integration of ion channels in bioelectronic devices for biotic–abiotic communication with enhanced functionality. This review begins with an introduction of natural and artificial ion channels across SLBs, continues with a description of bioelectronic devices integrating SLBs, and concludes with examples of functional ion channel bioelectronics.


Biological Membrane: SLB
Cell membranes serve as vital barriers between cells and their environment, and are primarily composed of amphiphilic phospholipids that self-assemble into a continuous lipid bilayer when in water. [2,39] Since their first demonstration in 1985 by Tamm and McConnell, SLBs have become a versatile tool for studying membrane proteins and biomolecule interactions. [40][41][42] The mechanical and electrical properties of SLBs have been characterized using various techniques, including fluorescence and surface plasmon resonance ( Figure S1a, Supporting Information), [43,44] atomic force microscopy, and quartz crystal microbalance ( Figure S1b, Supporting Information). [45][46][47] Electrochemical impedance spectroscopy (EIS) and FETs ( Figure S1c, Supporting Information) were mainly used for SLBs characterizations. [48]

Ion channels
Ion channels span the lipid bilayer and enable the regulated flow of ions and small molecules across the cell membrane. Ion channels can be divided into a) passive ion channels and b) active ion channels ( Figure 1). Passive ion channels, also known as leakage channels, allow the continuous passive transport of intracellular and extracellular ions by means of hydrophilic pores that are always open (Figure 1a). These channels can be selective for specific ions, allowing certain ions to pass through more easily than others. [49] Examples of passive ion channels include gramicidin A (gA), which forms a channel permeable to small monovalent cations, protons, and water molecules but not divalent cations and anions. [50] Active ion channels, in contrast, can open, close, or act as pumps depending on external factors such as chemistry, voltage, and light ( Figure 1b). [51][52][53] For example, bacteriorhodopsins are light-gated ion channels that change conformation upon absorbing a specific wavelength of light, transporting ions across the membrane. [54] Recently, many strategies have emerged to create artificial ion channels (Figure 1c). These strategies include carbon nanotube porins (CNT), [55] DNA nanopores, [56] and random heteropolymers (RHPs). [57] CNT porins consist of CNTs (%10 nm in length) that are coated with a lipid layer so that they position themselves across the cell membranes. CNT porins transport protons, small ions, and single-strand DNAs. [55] DNA nanopores are designed with programmable triggers and transport small ions and dye for real-time detecting systems. [56] RHPs can position themselves across the cell membrane and efficiently transport protons. [57]

Bioelectronic Platforms
Recent advancements in microfabrication technologies and materials have enabled the development of bioelectronic devices that can interface with biological systems through electronic and ionic signals across multiple length scales. [22,38] For the integration with SLBs, typically two types of devices are used. The first type is FET-based devices, [58][59][60][61] where the gate dielectric is functionalized with the SLB, and the change in conductivity upon the insertion of the ion channels affects the charge on the FET channel and, consequently, the conductivity. The second type of device comprises a single electrode in contact with the SLB to www.advancedsciencenews.com www.advintellsyst.com measure the change in ionic conductivity of the SLB upon the insertion of ion channels either indirectly with EIS [62] or directly for protons with PdH x proton-conducting contact. [23,63,64]

Field Effect Transistors
In conventional FETs, two electrodes, namely sources and drain, are connected with a semiconductor material called a channel. A gate dielectric separates a third electrode, called gate, from the channel (Figure 2a). A voltage applied to the gate electrode modulates the conductivity of the channel. In bioelectronic applications, the gate dielectric is typically partially replaced with a gate electrolyte, and the gate electrode is replaced with an Ag/AgCl electrode. [65,66] The voltage on the Ag/AgCl electrodes can module the conductivity of the channel, which can then be used to detect analytes ( Figure 2a). The presence of dangling bonds on the surface of the gate dielectric can be functionalized with the immobilizing binding technique, such as biomolecules, antibodies, enzymes, and DNA strands, creating binding sites for the analyte of interest. [59,[67][68][69][70] When the analyte of interest binds to the functionalized surface of the gate dielectric, the binding event leads to a change in the charge density at the surface of the gate dielectric. This change affects the gate potential, which is the voltage applied to the gate electrode and controls the electrostatic potential distribution in the semiconductor channel. As a result of this change in the gate potential, the conductivity of the semiconductor channels is modulated and recorded as the source-drain current.
The magnitude of the change in conductivity is proportional to the amount of bound analyte and can be used to quantify the concentration of the analyte. The use of 1D and 2D nanomaterials as channel offers enhanced sensitivity due to their higher surface-tovolume ratio, resulting in a larger active area for sensing. [38,59] OECTs are compatible with fast and inexpensive manufacturing of flexible bioelectronics. [71] In OECTs, the semiconducting channel is typically a porous polymer that has an affinity for ions and small molecules, allowing them to penetrate its structure upon changes in the electrostatic environment, either due to changes in gate voltage or binding of charges molecules ( Figure 2b). [17,18] The electrostatic environment is determined by the concentration of charged species and their distribution in the vicinity of the polymer channel. Applying a gate voltage creates an electric field that attracts or repels hydrated ions and small molecules toward or away from the polymer channel, leading to changes in their concentration near the polymer channel. These changes affect the oxidation state of the polymer, altering its conductivity. The conjugated segments of polymer can be oxidized or reduced, changing its electronic structure and conductivity. The changes in conductivity are recorded as a change in the source-drain current, allowing the detection of analytes that modulate the electrostatic environment around the polymer channel. Both electronic systems have their advantages, and this article will discuss SLB incorporated with ion channels and FET hybrid structures.

Electrodes for EIS and Proton-Conducting PdH x Electrodes
In addition to active field-effect devices, electrodes made of metal or polymer can be used to record signals from ion channels using EIS or PdH x electrodes that are able to transduce H þ signals into electronic signals. [72] EIS involves a working electrode, a counter electrode, and a reference electrode in Figure 3a. EIS measures the impedance between the working electrode and the solution electrolyte and uses the resulting data to fit an equivalent circuit, which includes both resistance and capacitance components. EIS can also be used for biosensing when the working electrode surface is functionalized with redox species, allowing for the detection and quantification of specific biological targets through changes in impedance and resistance. This method is able to analyze the concentration of ions/biomolecules, charge transfer, and resistance of electrolytes, making it a powerful tool for studying and understanding Figure 2. Schematics of basic structure and mechanism of FETs and OECTs. a) FETs rely on a semiconducting material in the channel (such as graphene, silicon, molybdenum oxide, or CNTs) to change its conductivity based on the charge on the surface of the gate dielectric, which can be functionalized with biosensing receptors. b) OECTs rely on hydrated ions entering the conductive polymer and affecting its conductivity. The polymer or gate electrode can be functionalized to provide selectivity to specific biomarkers.
www.advancedsciencenews.com www.advintellsyst.com interfacial properties such as lipid bilayers/ion channels, antigen-antibodies, aptamers, receptors, and enzymes. [73] Many ion channels, including ATPase and bacteriorhodopsin, are proton conductors. [74][75][76][77] Pd/PdH x contacts are useful for measuring the conductivity of these channels, taking advantage of the reversible redox reaction of H þ at the Pd/solution interface and subsequent physisorption to create PdH ( Figure 3b). For V < 0 on the Pd contact, an H þ takes an electron from the contact to reduce to H, and H subsequently absorbs onto the Pd surface to create PdH x . This transfer results in an increase in solution pH. For every H þ transferred from the solution to PdH x, an electron flows from the circuit into the Pd electrode. Therefore, measuring electronic current provides a one-to-one correlation with the H þ current flowing through the ion channel. [64] For V > 0, oxidization takes place at the Pd surface, releasing H þ from PdH ads , and increasing the concentration of H þ in the solution while decreasing the pH (Figure 3c). These reactions depend on the difference in protochemical potential between the Pd/PdH x contact and the solution and are pH dependent. [72] For example, at low pH, with high H þ in solution and high activity, H þ may transfer directly from the solution into the Pd even for V = 0. The ability to measure H þ current with PdH x contacts makes them a suitable alternative to EIS measurements when using proton-conducting ion channels. [74][75][76][77] 4. Bioelectronic Platforms with SLB and Ion channels 4

.1. 1D Nanomaterial FET-Based Bioelectronics
The 1D nanostructures are well suited for biosensing and diagnostics as they have comparable dimensions to biological molecules. [78,79] Researchers have combined silicon nanowirebased FETs (SiNW-FETs) with SLBs and incorporated two ion channels, gramicidin A (gA) and alamethicin (ALM) (Figure 4a,b). [80] With gA, the SiNW conductivity becomes pH-dependent because gA allows H þ ions to cross the lipid bilayer onto the SiNW surface. These H þ ions change the surface pH, affecting the charged state of the surface ÀOH groups, effectively gating the SiNW (Figure 4a). The addition of Ca 2þ ions in the electrolyte blocks the gA channel and reduces the current response of the SiNW. [80] Integration of ALM with SiNWs demonstrates voltage-dependent active gating from the ALM (Figure 4b). ALM has voltage-dependent barrel-shaped pores that allow the diffusion of monovalent cations when open for V g = 0.15 V. [81] For V g < 0.15 V, the ALM is closed and does not allow H þ transport. Thus, the SiNW-FET is only pH sensitive when V g = 0.15 V. [80] Functionalizing top-down silicon nanoribbons with SLBs also offers a platform for integration with CNT porins (Figure 4c). [82] These porins allow for the fast Figure 3. Schematics of bioelectronic platforms with conducting contacts. a) Supported lipid bilayers (SLBs) on single electrodes for electrochemical measurement. b) With an applied negative bias, H þ transports to the Pd/solution surface and is absorbed onto the surface, then onto the Pd subsurface layer, and finally diffuses into the bulk layer to acquire PdH bulk . c) With an applied positive bias, H þ is initially released from the surface PdH ads , then from the subsurface layer Pd subs to the surface PdH ads , and further diffuses from PdH bulk . Adapted with permission. [64] Copyright 2020, AIP Publishing.
www.advancedsciencenews.com www.advintellsyst.com transport of protons across the SLB and induce pH sensitivity of the silicon (Figure 4d). [82] The results indicate that the CNT porins transport small ions at high speeds to the devices and increase the resolutions of sensing limitations through the passive ion channels. [55,82] However, it is worth noting that while the use of 1D nanomaterial FETs with SLBs and ion channels offers several advantages for bioelectronics, including high sensitivity and specificity, there are also limitations to these devices. For example, the stability of the lipid bilayer can be a challenge, which may impact the accuracy and reliability of the biosensing. [10] Additionally, the scalability and reproducibility of these devices can be challenging, and further development is needed to optimize the performance and broaden the applicability of these bioelectronic platforms.

2D Nanomaterial FET-Based Bioelectronics
The 2D nanomaterials advantages for biosensing include low noise, scalability, high surface-to-volume ratio, and compatibility with biomaterials, making them effective transducers of biosensors. [59,68,69] Among 2D materials, molybdenum disulfide (MoS 2 ) is particularly advantageous for biosensing due to its bandgap, which allows for integration with standard FET architectures, unlike graphene. MoS 2 -based biosensors are capable of sensing pH, DNA hybridization, and proteins ( Figure 5a). [59,68,69] The sensing mechanism of MoS 2 FET also relies on the protonation state of the surface hydroxyl groups, similar to SiNWs FETs (Figure 5b). [59] However, since MoS 2 is an n-type semiconductor, protonation and positive charging of surface hydroxyl groups at www.advancedsciencenews.com www.advintellsyst.com low pH induce more electrons in the MoS 2 channel, leading to higher current. This is in contrast to SiNWs, which are p-type semiconductors and exhibit the opposite trend. [80] The pH sensing in MoS 2 FET is also gate voltage dependent, as expected, and can be tuned by the application of a top gate voltage (Figure 5c). [60] The planar nature of MoS 2 -FETs allows for the integration with SLBs, as demonstrated by the integration of MoS 2 FETs with ion channels (Figure 5d). [43] In this proof of concept, gA across the lipid bilayer caused pH dependence of the MoS 2 current, and the blocking behavior with Ca 2þ ions was similar to that observed in SiNW FETs. [43] In summary, the integration of SLBs and 2D nanomaterial FETs holds significant promise for the development of bioelectronics with improved sensitivity and compatibility. However, further research is needed to optimize their performance and explore potential applications in diagnostic and therapeutic including stability and reproducibility issues with SLBs on 2D nanomaterials.

Organic Electrochemical Transistor-Based Bioelectronics
OECTs have emerged as a versatile platform for translating ionic currents into electronic signals, making them highly promising for bioelectronic applications. [17] When integrated with an SLB containing α-hemolysins (α-HL), and bacterial pores, OECTs transconductance increases because the ion channels facilitate ion transport across the SLB into the OECT channel ( Figure 6a). [83] The integration of OECTs with SLBs can also create organic neuromorphic memory devices. High-frequency pulses trap ions into the poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and result in short-term depression (Figure 6b). [84,85] This approach can lead to organic neuromorphic devices with memory that can mimic the functionality of biological synapses and potentially lead to new applications in artificial intelligence and machine learning. Most semiconductive polymers used in OECTs are p-type (hole-conducting) and less sensitive to cations than n-type (electron transporting) materials. This limits the sensitivity of the device to cationic biomolecules and affects the overall performance of the bioelectronics. To address this limitation, researchers have developed an n-type OECT in accumulation mode using the n-type polymer poly(naphthalene diimide) with thiophene and alkyl side chains functionalized with lysine-based moieties (p(NDI-T2-L2)). [86] These side chains enable the integration of the p(NDI-T2-L2) OECTs with SLBs and gA ion channels, resulting in a pH-sensitive device that exhibits the expected blocking response to Ca 2þ ions (Figure 6c,d). [86] The device showed a significant increase in sensitivity to cationic biomolecules compared to p-type OECTs and the use of n-type polymers can also provide complementary OECTs that can enhance the selectivity and sensitivity of biosensors.
In summary, OECTs integrated with SLBs and ion channels show potential for highly sensitive and selective biosensors. Challenges include limited stability, the predominance of p-type Lower pH values lead to increased drain currents. Reproduced with permission. [59] Copyright 2016, American Chemical Society. d) Time-current plots of MoS 2 -FETs for the bare device (black line), the device after incorporation with gA (blue line), and the device current that the passages of gA blocked by Ca 2þ ions (purple line). Reproduced with permission. [43] Copyright 2019, Elsevier.
www.advancedsciencenews.com www.advintellsyst.com semiconductive polymers, and the negative effect of swelling of the polymer channels on device performance. [21] However, with further research and optimization, the integration of OECTs with SLBs and ion channels holds great promise for the advancement of bio-applications.

Electrochemical Impedance Spectroscopy-Based Bioelectronics
Integrating SLBs and ion channels with electronics can be achieved by forming SLBs on working electrodes to perform EIS measurements. [87][88][89] EIS is a powerful technique that can record ion transport and changes in redox species between the SLB and the electrode by measuring changes in the capacitance resistance of the SLB. [11,66,87,[89][90][91][92] Various electrode materials, including gold, [90,93] platinum, [94] and organic polymers, [17,87,89] can be used. This technique can improve the biocompatibility and signal-to-noise ratio of the electrode by coating it with PEDOT:PSS. [95,96] The incorporation of α-HL channels into the SLB can be monitored using PEDOT:PSS-and SLB-modified electrodes (Figure 7a,b). [89] Additionally, this platform can support naturally derived SLBs (from human embryonic kidney cells, HEK293) (Figure 7c,d). Compared to conventional artificial SLBs, naturally derived SLBs may contain more defects due to the loose lipid packing and ion channels of cells. The stability and electrochemical characteristics of SLBs were characterized by measuring their electrical resistance and capacitances using the highly sensitive label-free EIS technique, which allows for direct evaluation of the molecular level of mechanistic detail. [89,[97][98][99][100] EIS measurement with SLBs on single electrodes can provide improved sensitivity by directly measuring the capacitance resistance of SLB, which is related to ion transport and changes in redox species. However, there are still challenges associated with distinguishing between the signal from the electrode and the signal from the SLB which can lead to interference and lower sensitivity. Despite the challenges, further optimization and refinement are needed for their practical use in biosensing.

Bioprotonic Devices-Based Bioelectronics
Bioprotonic devices use Pd/PdH x contacts to translate H þ currents into electronic currents and vice versa, forming a versatile interface with SLBs given that many ion channels are proton www.advancedsciencenews.com www.advintellsyst.com conductors ( Figure 8). [23] When gramicidin A is integrated in the SLB, a voltage (V Hþ ) applied between the Pd/PdH x working electrode and reference causes a current (i Hþ ) to flow (Figure 9). With V Hþ is negative, protons transfer from the solution into the Pd/PdH x contact. When V Hþ is positive, protons transfer from the Pd/PdH x contact into the solution. We measure i Hþ as a function of V Hþ for the Pd/PdH x contact with SLB ( Figure 9a,d), SLB and gA (Figure 9b,d), and SLB and gA in the presence of Ca 2þ (Figure 9c,d). First, V Hþ is set to À200 mV to load the Pd with protons and form PdH x , followed by V Hþ at 0 mV to allow the protons into the PdH x to transfer back into the solution. This transfer can occur even for V Hþ = 0 mV because the protochemical potential of PdH x is higher than the protochemical potential of the solution at neutral pH. As expected, i Hþ is small with SLB alone (Figure 9a) and with the gA channel blocked by Ca 2þ (Figure 9b) compared to when gA is present (Figure 9b,d) because gA facilitates the transport of H þ across the SLB. When we repeated the same measurements with ALM ( Figure 10), we did not observe any i Hþ for V Hþ = 0 V despite a high i Hþ for V Hþ = À200 V, indicating the formation of PdH x and the presence of the channel in the SLB (Figure 10a,d). This is because ALM is voltage gated and is closed for V Hþ = 0 V (Figure 10b,d). In contrast, for V Hþ = 100 mV, the ALM channel is open and allows i Hþ to flow (Figure 10c,d). Pd/PdH x contacts functionalized with SLB can also accommodate active ion channels such as  Reproduced with permission. [23] Copyright 2016, Springer Nature. c) The bioprotonic device integrated with a light-activated ion channel consisting of deltarhodopsin with SLB and Pd/PdH x contacts enables control of H þ flow. Reproduced with permission. [63] Copyright 2016, Wiley-VCH. d) Pd/PdH x is used to measure the H þ conductivity of a synthetic ion channel, random heteropolymers (RHPs). Adapted with permission. [57] Copyright 2020, Springer Nature.
www.advancedsciencenews.com www.advintellsyst.com  www.advancedsciencenews.com www.advintellsyst.com Figure 11. Bioprotonic devices with ligand-gated ion channels illustrating protonic current. Reproduced with permission. [63] Copyright 2016, Wiley-VCH. a) Photograph of the bioprotonic microfluidic device. Multiple lithographically fabricated Pd microcontacts inside a microfluidic channel (inset) comprise each manufactured chip. An Ag/AgCl electrode immersed in the buffer solution completed the circuit as reference electrode and counter electrode. b) Schematics of a bioprotonic device. The applied voltage controlled the H þ flow across the ligand-gated proton channel. c) The photocurrent response of devices prepared with SLBs derived from 1,2-dioleoyl-sn-glycero-3-phosphocholine control liposomes (black trace), HtdR-His 6 (blue), or Pd4-HtdR-His 6 proteoliposomes (purple) was measured under an applied voltage of À50 mV to Pd contacts versus Ag/AgCl electrode and thereafter exposed to successive 10 s illumination cycles with a 523 nm light-emitting diode. d) A device made with a Pd4-HtdR-His 6 was illuminated for 10, 30, and 20 s to show the onset of the steady-state better. The photocurrent decreased to a constant state due to higher device resistance or lower protein density. www.advancedsciencenews.com www.advintellsyst.com Haloterrigena turkmenica detarhodhopsin (HtdR) [63] (Figure 11). HtdR is a light-activated proton pump that drives protons across the cell membrane when exposed to green light. To ensure that the HtdR positioned itself across the cell membrane correctly, the N-terminus of the HtdR was fused with a Pd-binding peptide. [63] Applying À50 mV (vs. Ag/AgCl) to the Pd electrode did not generate any current to flow in the dark or when the HtdR did not have the Pd binding motif (Figure 11c). In contrast, green light shining on the HtdR with the Pd-binding peptide produced an H þ current measured with the Pd contact (Figure 11c). The illuminated HdtR could pump protons for up to 30 s (Figure 11d). Furthermore, HdtR selected to respond to multiple wavelengths could be assembled into a multicolor photodetector. [101] The Pd/PdH x contacts with SLB can also measure artificial proton channels made with RHPs ( Figure 12). [57]

Conclusions and Outlook
The integration of SLBs with bioelectronics offers a versatile and powerful platform for fundamental studies of ion channel conductivity and potential applications in sensing. SLBs serve a dual role in this context, acting as passivating layers to isolate the devices from the external environment and as hosts for specific ion channels. The diverse range of bioelectronic platforms described in this review demonstrates the potential of SLB integration with different materials, including 1D and 2D materials, organic bioelectronics, and bioprotonic devices. There is room for improvement in many areas, such as the stability of the lipid bilayer, miniaturization, and optimization of the signal-to-noise ratio for more accurate measurements of individual ion channels.
With the continued improvements of SLB-integrated bioelectronics, it may be possible to move beyond current applications toward multielectrode multiplexed patch clamping, where individual ion channels can be exposed to different conditions in parallel. This would provide an unprecedented level of detail in the study of ion channel function, enabling the investigation of afflictions that affect ion channels or fast and efficient drug screening.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.