Control of pH in bioelectronics and applications

Ions and biomolecules dominate intra- and inter-cell communication in biological processes.^1,2 Bioelectronics bridges biology and electronics with sensors and actuators with many potential applications ranging from therapeutics to synthetic biology.^3,4 Hybrid materials that conduct both ions and electrons can create a seamless interface between bioelectronic devices and biological systems.^5,6 This interface involves more efficient ion-to-electron conversion with low impedance^3,7,8 in applications such as drug delivery,^5,9 tissue regeneration,¹⁰ and neural recording and stimulation.^11,12 Iontronic devices deliver and sense chemical signals to directly control physiological processes.^13,14 Examples include organic electronic ion pumps (OEIPs) that deliver ions and biomolecules via electrophoresis to treat neuropathic pain in animal models¹⁵ and organic electrochemical transistors (OECTs) that sense biophysical signals.¹⁶ Among all the ionic species in physiological environment, protons (H⁺) play a critical role. Proton concentration (pH) affects oxidative phosphorylation of adenosine triphosphate (ATP),¹⁷ enzyme activity,¹⁸ gene expression,¹⁹ muscle contraction,²⁰ and neuronal excitability.^21,22 The regulation of pH has been used as therapy in cancer treatment²³ and epileptic seizure control.²⁴ Many biomaterials are proton conductors including chitosan,^25,26 proteins,^27,28 and glycosaminoglycans.²⁹ In these materials, protons hop along the hydrogen bonded network formed by the hydrated polymer chains following the Grotthuss mechanism.³⁰ To measure proton currents in these conducting materials, palladium/palladium hydride (Pd/PdH_x) used as contact converts a H⁺ current into an electrical current, and vice versa.^31–35 Here, we discuss recent results from using Pd/PdH_x as a proton conducting bioelectronic interface with solution for artificial membranes with ion channels, pH triggered delivery of biochemicals, pH enabled glucose sensing, and closed-loop control of pH actuation and sensing using a self-adaptive machine learning (ML) based feedback controller (Fig. 1).³⁶ 

Most metals are excellent contacts for e⁻ but poor for H⁺, so most investigations on proton conduction use alternating current to avoid ion accumulation at the interface. The couple Pd/PdH_x can act as a H⁺ to electron transducer, resulting in a proton-transparent contact for DC measurements of proton conducting materials.^25,26 Pd has a strong affinity to hydrogen and forms palladium hydride (PdH_x) either by absorbing hydrogen from H₂ gas or from H⁺ in solution as a reversible hydrogen electrode in electrochemistry. The incorporation of H⁺ from the solution into Pd is potential dependent and follows the reversible reduction, H⁺ + e⁻ ↔ H, at the Pd/PdH_x–solution interface and the subsequent physisorption of H onto Pd to form PdH_x.³⁷ An electrical potential applied (V) on the Pd/PdH_x contact shifts the reaction equilibrium and induces the transfer of H⁺ to or from the solution, which effectively controls [H⁺] in the solution (Fig. 2). In brief, when V < 0 and the contact is at a lower potential than the solution, an H⁺ in solution is adsorbed to the Pd/solution interface where it is reduced to PdH_ads by an incoming e⁻. PdH_ads subsequently absorbs onto the Pd subsurface to form PdH_subs and finally diffuses into the bulk Pd to form PdH_bulk following the concentration gradient.³⁸ The net result is the removal of an H⁺ from the solution for every e⁻ provided by the leads. This, in turn, lowers [H⁺] and increases pH. When V > 0, some of the H that is part of PdH_ads oxidizes at the Pd surface to form H⁺ and dissolves into solution with the e⁻ going into the electronic leads. The additional H⁺ in solution increases [H⁺] and consequently decreases pH.

A more quantitative way to describe the H⁺ transfer across the interface uses the protochemical potential (μ) because H⁺ transfer does not only follow V but is also affected by pH of the solution.³² We can define the difference in the protochemical potential between PdH_x (μ_PdHx) and the solution (μ_pH) using the following equation:

where $aH+$ is the activity of H⁺ in solution with pH = −log $aH+$⁠, $pH2$ being the hydrogen partial pressure in Pd, and V being the potential difference between Pd and solution.

When Eq. (1) leads to a positive value, H⁺ will transfer from the contact to the solution until equilibrium is reached. When Eq. (1) leads to a negative value, H⁺ will transfer from the solution to the contact until equilibrium is reached. In general, for solutions with low pH (high [H⁺]), the transfer of H⁺ into Pd will be favored vs transferring H⁺ from the contact into the solution. The opposite is true for solutions with high pH (low [H⁺]).

In a biological system, most intercellular communication is mediated by membrane proteins and ion channels that passively allow or actively control the flow of ions and small molecules across the cell membrane.³⁹ Thus, a bioelectronic device with ion channels that control ionic flow across a supported lipid bilayer (SLB) is ideal for interfacing with biological systems. Our group has demonstrated a bio-protonic device with Pd/PdH_x contacts that control H⁺ currents and modulate pH gradients across phospholipid membranes, as shown in Fig. 3(a).⁴⁰ Here, the SLB mimics the function of a cell membrane, acting as a barrier to divide the solution into bulk and isolation layers, and a self-healing support for the insertion of ion channels, gramicidin A (gA) and alamethicin (ALM). gA is an ion channel that allows the passage of small cations, while remaining impermeable to anions [Fig. 3(a), left].⁴¹ A voltage between the Pd/PdH_x contacts and AgCl electrode in the bulk solution controls the H⁺ flow (⁠$iH+$⁠) across the SLB. When gA is present, $iH+$ is much larger compared to that of only SLB. ALM is a voltage-gated ion channel that selectively transports cations in the direction of V applied and closes for V = 0 V [Fig. 3(a), right], which functions as a bidirectional voltage-gated channel analogous to an ON–OFF switch.⁴¹ When ALM is open, $iH+$ has a similar response to $iH+$ for gA. When ALM is closed for lower voltages, $iH+$ is much smaller as expected because ALM is in the closed state.

In addition to gramicidin and alamethacin, we demonstrated optical control of H⁺ flow with a similar design by incorporating detarhodhopsin (HtdR), a light-activated proton pump, into the SLB.⁴² In Fig. 3(b), H⁺ flow is recorded by using the Pd/PdH_x contact when HtdR is illuminated with the correct wavelength of light, while no flow is recorded in the dark. The responsive behavior is highly reproducible in shape and magnitude over repeated cycles and much faster than that made from a transistor.⁴⁴ Recently, our collaborators have engineered different peptides into this platform and developed a photodetector that responds to different wavelengths of light.⁴⁵ In detail, we integrated Pd/PdH_x contacts with blue absorbing proteorhodopsin (BPR) and green absorbing proteorhodopsin (GPR); thus, the devices exhibit wavelength dependent photocurrent production when illuminated between 450 nm and 600 nm. This can be considered as a primary step toward the construction of biological cameras with potential applications in artificial vision and retinal prostheses. In Fig. 3(c), we used carbon nanotube porins (CNTPs)^46,47 as synthesized ion channels inserted in the SLB and integrated them with Pd/PdH_x contacts to control H⁺ flow across the SLB through CNTPs by electrical means, which is a fully synthetic platform.⁴³ Moreover, since functionalized CNTs have the capability to penetrate plasma membranes, we suggest that CNTPs may be able to connect the bioprotonic devices directly with cells to modulate intracellular pH.

More recently, in collaboration with the Xu group, we used this Pd/PdH_x platform to integrate four-monomer-based random heteropolymers (RHPs) that mimic membrane proteins.³³ The bioprotonic device described above was used here to verify the selective proton transport across the SLB through the RHP [Fig. 3(d)]. Incorporating RHP1 into the SLB increased the membrane permeability from 8.1 × 10⁻⁵ s⁻¹ to 1.6 × 10⁻² s⁻¹.

The pH of a solution does not only affect the physiological environment but also electrochemical reactions at metal oxide surfaces. For example, cobalt oxide is a non-enzymatic glucose sensor that is only efficient at high pH.³⁵ The electrochemical redox reaction that involves glucose oxidation has much higher efficiency with CoO₂ (pH > 10) than with Co₃O₄ that is found in neutral conditions. As such, the use of cobalt oxide as a glucose sensor is limited in physiological conditions and fluids such as sweat and tears whose pH is close to 7. To overcome this limitation, our group built a cobalt oxide glucose sensor that exploits localized and transient alkaline conditions (high pH) from a Pd/PdH_x contact (Fig. 4).³⁵ In Fig. 4(a), for V_pH = −1 V, Pd absorbs H⁺ from the electrolyte and increases the local pH that coverts Co₃O₄ into CoO₂ for sensing. In Fig. 4(b), for V_pH = 0 V, the device is off restoring neutral local conditions and thus allowing for the integration into wearables without creating long-term localized alkalosis, which would damage the skin.^48,49 Figures 4(c) and 4(d) show glucose sensing current and device calibration at pH 7 and pH 11 cycled by applying V_pH, respectively. At pH 7, the sensing current, I_g, cannot be distinguished below 1 mM glucose, and I_g is overall smaller than I_g at pH 11 for all glucose concentrations. This restriction of working only at high pH does not only exist in cobalt oxide sensors but also occurs in many metal oxide and inorganic material-based sensors that oxidize target molecules.⁵⁰ Thus, the localized and transient pH control can be broadly applied to the detection of various biological relevant analytes, such as dopamine and ascorbic acid,⁵¹ and enables continuous sensing in neutral biological fluid.

The intracellular pH levels of the cytosol and different organelles are critical for many cell functions. The regulation of pH has significance in the modulation of cell function, metabolism, as well as bioelectronic therapies.^52,53 We have demonstrated that the pH control using Pd/PdH_x contacts can affect the rate of enzyme reactions and bioluminescence.⁵⁴ The local pH is also a parameter that can trigger delivery of drugs from vesicles for precise localized delivery.⁵⁵ We demonstrated Pd/PdH_x contacts to control pH in physiological relevant media and trigger the delivery of a fluorescent label in cardiac fibroblast cells [Fig. 5(a)].⁵⁶ A positive potential applied on Pd/PdH_x increases [H⁺] in solution and decreased the pH, triggering the disruption of acid sensitive microparticles containing fluorescein diacetate (FDA). The FDA released into solution is uptaken by the cardiac fibroblast where it turned into fluorescein. The capability of pH control with Pd/PdH_x contacts depends on the capacity of the electrodes to absorb and release H⁺, and it is usually limited to pH values close to the original pH of the solution. To expand the pH modulation range in the physiological environment, we integrated Pd/PdH_x contacts with an organic electronic ion pump (OEIP), which is able to modulate pH in a larger range [Fig. 5(b)].³⁴ Here, Pd/PdH_x is used as the electrode contacts in both the reservoir (right) and the target (left) electrolyte. A mixture of Poly(vinyl alcohol):Polystyrene Sulfonate (PVA:PSS) works as the H⁺ conducting material in which H⁺ is driven by V_d. The innovative design of a Donnan, Polyethylenimine (PEI), later alleviates the passive diffusion from the reservoir to the target. By applying positive V_d, H⁺ are transferred from the reservoir to the target solution, as illustrated by the arrow in Fig. 5(b), and pH in the buffer decreases. When V_d is reversed, H⁺ are transferred from the buffer solution back to the reservoir and increase pH. Here, we put the pH sensitive microparticles encapsulated with FDA in the buffer solution and switch the environment to the acidic condition by applying V_d = 1 V for 180 s. After incubation for 24 h, we induce a basic condition to hydrolyze FDA into fluorescein and take fluorescence images of the sample kept at pH 7.4 [Fig. 5(c)] and at pH 6 and 8 [Fig. 5(d)]. This platform uses a H⁺ reservoir to provide a large supply of H⁺ to the Pd/PdH_x contact in the target solution and shows the capability to modulate pH in buffer conditions, which has the potential to control a variety of biochemical reactions that require a larger dynamic range.

Life revolves around closed-loop feedback control in regulatory systems and organisms.⁵⁷ Closing the loop of actuation and sensing using bioelectronic devices can provide new opportunities for bioelectronic interfacing. This closed-loop control is also challenging due to the complexity of biological systems and the risk of affecting their innate self-regulation. We have recently introduced closed-loop control of cell membrane voltage (V_mem) using bioelectronic devices controlled by using a self-adaptive machine learning (ML) based feedback controller to close the loop between sensing and actuation [Fig. 6(a)].^36,58 In detail, the H⁺ pump is able to change pH with an applied voltage, and the pH change is monitored with a fluorescence dye in real time. The fluorescence intensity is read by using the ML controller, which compares it with the desired value and decides whether further actuation (the applied voltage on H⁺ pump) is required. Figure 6(b) shows that a specific [H⁺] value can be regulated precisely in the buffered fluids in this automated fashion, such as triangle, sine, and square waveforms. By regulating extracellular pH of human-induced pluripotent stem cells (hiPSCs), we showed a proof-of-concept prolonged control of membrane voltage (V_mem) toward a desired set-point over 10 h [Fig. 6(c)]. This is important because biological systems are adaptive and will typical react with their own feedback to localized changes in pH and the ML controller can adapt to the cellular self-regulation mechanisms.

Besides bioelectronic devices with Pd/PdH_x, organic polymers have also been used to control pH. Berggren et al. used PEDOT:PSS as the active material in an organic electronic ion pump to electronically control protons’ transport between two electrolytes and change pH from 7 to 3 in a few minutes.⁵⁹ More recently, Miyake and co-workers used a high H⁺-coupling conductive polymer, sulfonated polyaniline (SPA), to modulate pH in the solution.⁶⁰ In neutral pH, negatively charged sulfonate groups are covalently attached to the backbone of SPA acting as dopant anions, compensating positive charges at protonated nitrogen atoms. By applying a negative voltage to the contact, protons are absorbed in SPA and coupled with sulfonate groups and equivalent amount of e⁻ flows into SPA microelectrodes to neutralize the charge at nitrogen atoms on the SPA backbone. In Fig. 7(a), this strategy is used to control ATP synthesis by pH modulation in mitochondria. Figure 7(b) shows that ATP synthesis is more efficient at pH 7.4 compared to pH 8.3 because the proton translocation across the inner membrane at pH 7.4 is faster. The authors measured the rate of ATP synthesis in mitochondria by switching the transducer between the ON and OFF states [Fig. 7(c)], indicating an electronically controlled biological reaction. Katz’s group also reported pH control using biochemical logic gates with enzymes, such as alcohol dehydrogenase, glucose dehydrogenase, and glucose oxidase, which can change pH in solution by adjusting the chemical inputs, such as NADH, acetaldehyde, glucose, and oxygen.^61,62 Different from electronically controlled pH, chemical signals used here allow bioelectronic devices to respond to physiological changes and provide the possibility for autonomous actuation.

In this Research Update, we have summarized bioelectronic devices that are able to control [H⁺] and pH in solution by transferring H⁺ using Pd/PdH_x contacts. The ability to control pH with an electronic stimulus opens up many opportunities for interfacing bioelectronics with artificial membranes with ion channels, biochemical delivery, transient biosensors, and closed-loop control of the biological environment. By adding machine learning to our devices, we are able to precisely tune pH in biological systems that, in turn, allows for unprecedented control of pH dependent biological processes such as cellular response. The expansion of these types of devices to other ions such as Ca⁺⁺, Cl⁻,⁶³ K⁺, and Na⁺ will further expand the reach of bioelectronics into biology.

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

This research was sponsored by the Defense Advanced Research Projects Agency (DARPA), Army Research Office, under Cooperative Agreement No. W911NF-18-2-0104, and the Department of the Interior, Award No. D20AC00003.