An enzyme logic bioprotonic transducer

Ionic species dominate signaling in natural systems, so the conversion of biochemical ionic signals into electronic signals is an essential part of bioelectronics.^1–6 Examples include ionic transistors that deliver neurotransmitters,⁷ electrochemical transistors,⁸ membrane protein gated nanowires,¹ and squid-protein based protonic transistors.⁹ We have previously developed bioprotonic devices that control the flow of H⁺ currents such as complementary transistors, rectifying junctions, and memristive memories.^10–12 These protonic devices have a proton conducting polymer-based channel and use PdH_x protodes to convert a H⁺ current in the polymer channel into an electronic current in the contacts according to PdH↔Pd + H⁺ + e⁻.^10–12 Here, we extend the functionality of these devices to the conversion of a H⁺ current from solution, which is a common media for biological systems. To this end, we exploit the electrochemical properties of the formation of PdH_x and its dependence on the voltage applied on the contact (Fig. 1).^13–15 As a proof of concept, we measure with these devices the pH of a solution modulated by enzymatic reactions, such as the reaction catalyzed by glucose dehydrogenase (GDH) to produce gluconic acid from oxidized nicotinamide adenine dinucleotide (NAD⁺) and glucose.

To calibrate the protonic transducer, we first investigate the dependence of the formation and depletion of PdH_x on the potential applied to the metal (V) (Fig. 2). For this measurement, we prepare Pd films (100 nm thickness) on glass slides by electron beam deposition, and measure the electrochemical current at the Pd electrode (I) (0.2 cm² surface area) in a 0.5 M Na₂SO₄ unbuffered solution (pH 6.7). We use the standard three-electrode system with Ag/AgCl as the reference electrode and platinum as the counter electrode (0.5 cm² surface area) (Fig. 2(a)). First, we form PdH_x with a negative V applied to the PdH_x contact, which we refer to as V_F. A V_F applied to the Pd induces a reduction current of e⁻ to flow into the Pd, which we refer to as formation current (I_F). These e⁻ reduce the H⁺ from solution, which are absorbed as H into the Pd to form PdH_x. Second, once the Pd is fully converted to PdH_x with x = 0.6, we switch V to a positive depletion voltage (V_D) at which PdH_x is expected to return to Pd. For this specific measurement, V_D = 0 V. The difference in chemical potential of H⁺, or protochemical potential (μ) between the PdH_x (μ_PdH) and the solution (μ_pH), determines whether the H⁺ will transfer back to the solution. This difference is defined as¹⁶ 

where a_H+ = activity of H⁺ in solution with pH = − loga_H+, pH₂ = hydrogen partial pressure in the Pd.

From (1), the protochemical potential of H⁺ in the PdH_x (μ_PdH) for x = 0.6 and V = 0 V is the same as the protochemical potential for a solution at pH = 0 (μ_pH=0), and higher than the protochemical potential for a solution at pH = 6.7 (μ_pH=6.7).¹⁴ While hydrogen transport through the PdH_x electrode causes overpotential in protochemical potential, this overpotential is negligibly small.¹⁷ As a result, for V_D = 0 V, H⁺ flow from PdH_x into the solution and induce an oxidation current of e⁻ to flow from the electrode. This current is a product of the oxidation of H from the PdH_x into H⁺ (Ref. 18) (Fig. 2(b)) and we refer to it as depletion current (I_d). Since I_F is affected by dielectric charging of the solution, we use the magnitude of I_d to measure the extent of PdH_x formation for a given V_F assuming that for a specific V_D all of the H⁺ loaded onto the Pd for V_F returns to the solution. During the formation of PdH_x, we also observe a color change in the electrode from metallic (Pd, Fig. 2(c)) to white (PdH_x, Fig. 2(d)), while after depletion the electrode returns to its original metallic color (Pd, Fig. 2(e)).¹² The formation of PdH_x does not occur for every value of V_F. At pH 6.7, I_d at V_d = 0 V drops by an order of magnitude between V_F = − 1.0 V and V_F = − 0.9 V (see supplementary material Fig. S1¹⁹) indicating that formation of PdH_x did not occur for V_F > − 1.0 V. We assume that the threshold voltage for formation (at pH 6.7) is V_F = − 1.0 V. Similar to formation, the depletion of PdH_x is also governed by differences between μ_PdH and μ_pH and exhibits a threshold behavior. For fully loaded PdH_x, I_d is close to 0 for V_D < − 0.3 V (in a pH 6.7 solution, see supplementary material Fig. S2¹⁹). During depletion of the contact, we calculate the total charge transferred from the PdH_x to the solution by integrating I_d as function of time.¹² As we observed for the PdH_x-Nafion interface in memory devices,¹² the total charge transferred is constant (45 mC) as a function of V_D (for V_d = − 0.3, −0.2, −0.1, and 0 V). This observation suggests that as long as formation of PdH_x occurs and is allowed to go to completion, the PdH_x is loaded with a constant amount of H each time.

Second, we investigate the dependence of the PdH_x formation and depletion on the solution pH (Fig. 3(a)). We determine the threshold values for V_F and V_D for a range of pH values (0.0, 2.0, 3.0, 4.5, 6.7, and 11.0) (Fig. 3(b)). As solution pH increases, the threshold values for V_F and V_D decrease, as expected due to the influence of pH on the solution protochemical potential (1).^14,20 It is interesting to note that there is a range of pH and V (area in white) in which the solution and the PdH_x are in equilibrium and no exchange of H⁺ across the interface occurs. To further elucidate the depletion dynamics, we measure I_D as a function of solution pH by pre-loading the Pd with H₂ gas rather than loading electrochemically from solution. When exposed to H₂ gas, Pd absorbs H₂ to form PdH_x with x = 0.6 for 0.1 atm < H₂ pressure < 1 atm.^11,21,22 As expected, at V_D = 0 V, I_D for a solution with a low proton concentration (e.g., pH 11) is larger than I_D from a solution with a high proton concentration (e.g., pH 2), and no depletion occurs at pH 0 because μ_pH=0=μ_PdH. This is because an increase in proton concentration at the protode surface results in a smaller protochemical potential difference between the loaded PdH_x contact and the solution. The opposite is true for the formation of PdH_x. As the proton concentration at the protode surface is increased (lower pH), the favorability for the reduction is also increased. Importantly, we find that the threshold potentials for formation (V_F) and depletion (V _D) vary as the solution pH varies. When the reaction is proceeding very slowly, the calculated formation threshold voltage depends on the reaction rate in addition to activation energy, which probably causes the non-linear results for threshold V_F.

As proof of concept, we integrate this protonic transducer with enzyme logic (Fig. 4). We use GDH (EC 1.1.1.47, 250 U mg⁻¹) donated from TOYOBO.^23,24 Two input signals activate GDH; input A is NAD⁺ and input B is glucose (Fig. 4(a)). GDH requires the presence of both NAD⁺ and glucose to function, effectively serving as an enzymatic and logic.²⁵ The absence of glucose or NAD⁺ is defined as a logic “0,” while their presence is defined as logic “1.” We use the formation of PdH_x at V_D = − 0.85 V and the resulting I_D measured at V_D = 0 V as the logic output. I_D>0.1 mA corresponds to a logic “1,” while I_D<0.05 mA corresponds to a logic “0.” In the absence of either substrate (logic input “0,0,” “0,1,” or “1,0”), GDH is not active and the solution pH remains the same. At pH 6, a V_F = − 0.85 V does not form PdH_x and the resulting I_D<0.05 mA. The presence of both NAD⁺ and glucose (logic “1,1”) activates the biocatalytic reaction of GDH (Fig. 4(b)), which produces gluconic acid and lowers the solution pH from 6.0 to 4.0. At pH 4.0, V_F = − 0.85 V results in the formation of PdH_x. The resulting I_D = 0.164 mA at V_D = 0 V (Fig. 4(c)) corresponds to a logic output 1. These results demonstrate that the Pd/PdH_x system can transduce proton signals from biochemical inputs into readable electronic currents.

In conclusion, we have characterized the behavior of a Pd/PdH_x bioprotonic transducer in response to electrical and biochemical stimuli by measuring the redox currents for proton absorption (I_F) and depletion (I_D). These currents are controlled with both voltages applied on the transducer (V) and the solution pH. At the depletion voltage (V_D = 0 V), oxidation currents increase as the solution pH increases. In contrast, formation currents increase with decreasing pH. By measuring the current at V in different pH solutions, we have produced a process map for PdH_x formation and depletion. This map reflects the differences in protochemical potential between the solution and the PdH_x as function of pH and H₂ concentration in the PdH_x. The ability to prevent either process from occurring may be of interest in proton accumulation based devices.¹² Furthermore, we demonstrate the integration of this transducer with an enzyme logic system, which is activated by two biochemical input signals (glucose and NAD⁺). Our bioprotonic transducer is useful with many other dehydrogenase enzymes which produce H⁺ as the output signal, including alcohol dehydrogenase and lactate dehydrogenase, and it can serve as the proton source/sink in enzymatic type bioelectronic applications.

This work was supported by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES), under Award No. < DE-SC001044> (PdH_x formation and enzyme logic), and the National Science Foundation CAREER Award (DMR 1150630) (PdH_x depletion). T.M. acknowledges a fellowship from the Japan Society for the Promotion of Science. Part of this work was conducted at the Washington Nanofabrication Facility/Molecular Analysis Facility, a member of the NSF National Nanotechnology Infrastructure Network.