Achieving control over the switching of a gate between "on" and "off" states, either allowing or blocking transport, presents numerous possibilities for the separation, isolation, and release of targeted species. Nature takes advantage of this strategy using cell-membrane embedded transport proteins, such as nanochannels or pumps, to regulate signal transduction and nutrient transport across cell membranes as a response towards specific stimuli, such as electrostatic interactions, ligand-binding, concentration gradients, transmembrane potentials, or pH changes. 1 Subnanometer hydrophobic cavities play a crucial role as selective filters in biological constructs, such as transport proteins, that exhibit an empty, dewetted resting state, with a transition to a filled, wetted structure occurring when a channel-specific response is triggered, thereby opening the gate and turning on transmembrane transport. [2][3][4] Biological channels, however, fail to function outside their native bilayer and are difficult to render in arbitrary device structures. To circumvent these challenges, artificial solid-state biomimetic nanochannels have been developed and used in a number of applications, including energy conversion, nanofluidics, filtration, and biosensing. [5][6][7][8][9][10][11][12][13][14] External control over gating in artificial nanochannels can be achieved by decorating the interior of the channel with functionalized molecules that alter their conformation or physical properties in response to specific stimuli. For example, Smirnov and coworkers demonstrated pH-and lightresponsive gating in single hydrophobic nanopores with chemically functionalized interiors, 15,16 however, the reversible gating of these systems was limited by high kinetic barriers after activation or chemical cross-reactivity. Potential-induced gating provides an attractive way to circumvent this problem, as demonstrated by the same group that used an external bias applied to focused-ion beam (FIB) milled single hydrophobic nanopores to achieve reversible switching between lowand high-conductivity states. 17 Siwy and coworkers examined reversible gating in large aspect-ratio track-etched nanopores exhibiting a mixture of hydrophilic and hydrophobic domains on the interior nanopore surfaces. 18 More recently, the influence of electrolyte on voltage-gating has been studied, making it possible to fine-tune wetting/dewetting potential thresholds, as demonstrated in asymmetric hydrophilic/hydrophobic nanopores with sub-10 nm diameters. [19][20][21] Furthermore, hydrophobic gating was extended by Jiang and coworkers to dual-stimuli responsive systems, combining electric-field induced wetting of single nanopores with light-and pH-sensitive molecular agents bound to the interior pore wall. 22,23 The reversibility of hydrophobic gating depends on the dewetting mechanisms in the pore. Dewetting is thermodynamically favorable, but kinetically delayed due to the presence of a free energy barrier, which has been shown to depend on the hydrophobicity of the pore lining, the pore diameter, and the ionic nature of the utilized solution. 17,21,24 Thus, effective reversible gating employs an optimum interplay of stimulusresponsive wetting and spontaneous dewetting.
Single nanopore systems exhibit superior characteristics for examining structure-function relationships, however, a number of applications, e.g., sensing and filtration, benefit from membranes with multiple pores to maximize flux. Block copolymer (BCP) membranes have emerged as alternatives to conventionally fabricated multipore membranes, because they can undergo phase segregation and self-assemble into nanostructures, for example, nanocylindrical channels with nearly monodisperse size distribution, tunable feature sizes, and ultrahigh pore densities. 25 Pore diameters down to ~ 10 nm are routinely attainable, and further reduction of the feature size may be achieved by modifying the copolymer with polyelectrolyte brushes, resulting in diameters as small as 1 nm. 26,27 Additionally, judicious choice of the block composition can endow BCPs with stimulus-responsive gating properties enabling, for example, biomimetic transport. [28][29][30][31][32][33][34][35] Our laboratory has examined mass transport and electrochemical behavior in multi-electrode embedded nanopore electrode arrays (NEAs), where the potential of each electrode can be addressed individually. 36,37 Upon application of suitable potentials, redox cycling (RC) can be initiated, enabling ultrasensitive electrochemical measurements due to the highly efficient mass transport at the small length scales characteristic of nanopore-embedded electrodes. [38][39][40][41][42][43] Furthermore, additional embedded electrodes have been utilized to gate particle and ion access to the NEA pores, to achieve voltage-gated blockage of Ag nanoparticles diffusing into the vestibule of the NEAs and electrochemical transistor action in 3-electrode NEAs utilizing electrowettingmediated defects. 44,45 Going further, stimulus-responsive control over mass transport in a BCP can be combined with the highly sensitive detection in NEAs by fabricating hierarchically organized architectures, obtained by coating NEAs with functional layers, e.g., Nafion to achieve permselective ion transport, dual-stimuli responsive BCP membranes for pH-and potentialresponsive gating, and poly(dimethylsiloxane) (PDMS) to accomplish electrochemical rectification. 34,[46][47][48] Contrary to a completely closed electrode, where analyte confinement arises from an impermeable blocking layer, 36,48 the BCP@NEA architecture benefits from spatial and temporal control over analyte introduction, making it possible to integrate the architecture into micro-and nanofluidic devices. Additionally, multiple benefits arise from spatial confinement, such as altered modes of mass transport, double layer effects, single entity studies, and enhanced electrocatalytic activity. [49][50][51] In this work, we explore using hierarchically organized NEAs as transport-gated electrochemical biosensors by combining voltage-induced gating in hydrophobic self-assembled polystyrene-bpoly(4-vinyl)pyridine (PS-b-P4VP) BCP membranes with tyrosinase-modified dual-embedded NEAs. Figure 1(A) depicts the biosensing BCP@NEA architecture. The nanopores consist of a metal-insulator-metal (MIM) structure exhibiting a ring-disk dual electrode geometry, utilizing Au as the bottom and top electrodes (BE/TE), respectively, while a thin SiNx layer provides electrical insulation. By designing the device as an array, the electrochemical signal can be integrated across the entire array of pores, while simultaneously exploiting the small-volume characteristics of the individual nanopores, thus maintaining their beneficial properties and leading to enhanced sensitivity. A PS-b-P4VP gating layer, composed of vertically aligned nanocylindrical P4VP channels that support pH-dependent and voltage-responsive transport across the BCP membrane, conformally covers the NEA. The biomolecular recognition agent tyrosinase, a copper-containing enzyme characterized by monophenolase and oxidase activity, 52 is selectively immobilized to the BE to facilitate the amperometric detection of alkylphenols, of interest as environmental pollutants. 53 Figure 1(B) shows the enzyme-catalyzed conversion of non-electroactive 4-ethyl phenol, which was used as a model compound in this work, to redox-active 4-ethyl quinone. The reversible 2e -/2H + redox reaction to 4-ethyl catechol can be exploited for redox cycling in nanoscale two-electrode systems, [54][55][56] allowing for the sensitive detection of non-electroactive alkylphenols. To gain external control over transmembrane transport, a potential bias is applied to gate a transition of the P4VP nanochannels from a dewetted, "off" state to a wetted, "on" state, allowing passage of solutes into the vestibule of the NEA from the bulk, Figure 1(C). Upon removing the stimulus, the P4VP nanochannels return to their dewetted state, thereby trapping the analyte inside the interior of the NEA. To determine the amount of enzymatically generated 4ethyl quinone inside the NEA, the sensing architecture is operated in generator-collector (GC) mode, which supports redox cycling of 4-ethyl quinone between BE and TE to generate a sensitive amperometric readout as shown in Figure 1(D). The GC-enhanced amperometry was used to extract the sensitivity of the sensor, resulting in a limit-of-detection of ~120 nM for 4-ethyl phenol. This work demonstrates the feasibility of electrochemical biosensing in hierarchically organized BCP@NEA structures that can take advantage of spatial and temporal control of analyte placement in ultrasmall sample volumes. Furthermore, the molecular recognition element, i.e., the enzyme tyrosinase, can be changed to render the architecture amenable to a range of targets, providing a general route to ultrasensitive multiplex biosensing with externally controllable biomimetic gating layers.
BCP@NEA Fabrication and Characterization. The block copolymer (BCP) polystyrene-blockpoly(4-vinyl)pyridine (PS48400-b-P4VP21300) was used to spin-coat thin membranes for use as voltage-responsive gating layers. Under the processing conditions used here, the P4VP polymer segregates into vertically aligned nanocylinders embedded in the flexible PS matrix, providing a physical connection between cis and trans sides of the membrane that can be switched between dewetted, empty and wetted, filled states in response to a potential bias, Figure 1(C). Because spin-coating of BCP solution directly onto NEAs results in filling of the pores, constructing the BCP@NEA structure requires an alternative strategy. Previously, this issue was addressed using a thermal-release-tape to transfer thin BCP membranes with strong attachment. 34 However, this method is incompatible with architectures that host immobilized biomolecular recognition agents, such as enzymes, because they can lose activity at elevated temperatures. To circumvent this problem, we used a thin polyacrylic acid (PAA) layer as a water-soluble, sacrificial compound to enable biocompatible transfer under ambient conditions. The polar properties of PAA are ideal for non-preferential coating of the hydrophobic diblock copolymer, enabling the desired vertically oriented morphology rather than lamellar architectures. As shown schematically in Figure 2(A In addition, a plan-view SEM image of the BCP@NEA device was taken to investigate the P4VP pattern throughout device assembly, Figure 2(D). The phase segregation of multi-block BCPs into structural motifs is mostly dictated by the chemical properties of the predefined polymer moieties, although, deposition parameters, such as substrate polarity and the rate of evaporation, directly influence the segregation as well. 57 To avoid entrapment of P4VP structures in a poorly defined kinetic state upon fast solvent evaporation during spin-coating, the BCP was briefly immersed in ethanol, which acts as a P4VP-selective solvent, swelling the structures and increasing the mobility of the P4VP blocks, allowing them to assemble in a thermodynamically favorable state. The SEM image of the BCP surface reveals approximately hexagonally-packed nanochannels with an average diameter of ~14 nm and a pitch of ~41 nm with no visible cracks or defects, in agreement with previous reports, 28,35,58 demonstrating that the use of membrane swelling and a sacrificial PAA layer yields PS-b-P4VP membranes with well-defined vertical nanochannels on the surface. Here, a 0.2 M phosphate buffer solution (pH = 6.8) was used, because it is well above the pKa of P4VP (pKa ~ 4.8), thus yielding dewetted, hydrophobic P4VP channels. 34 The potential of the coated electrode was swept between +0.4 V ≥ Eappl ≥ -1.2 V vs. Ag/AgCl producing the voltammogram shown in Figure 3(A). Initially, the forward scan produces small capacitive currents for Eappl > -0.5 V, consistent with effective blockage of mass transport to the electrode.
At more negative potentials, a steep increase in current is observed at an onset potential of ca.
-1.1 V, consistent with a transition of the P4VP channels to a wetted state, allowing mass transport to the electrode. Furthermore, the voltammogram exhibits a large hysteresis -the current returning to intersect the forward scan near -0.4 V, which is significantly more positive than the threshold potential to initiate wetting. The larger currents observed in the reverse, anodic scan, suggest that the filled P4VP channels experience a kinetic barrier against returning to their original dewetted, empty state.
Next, potential-induced gating in hierarchically organized BCP@NEA architectures was investigated by stepping the potential of the TE to -1.2 V to induce transmembrane transport, producing the current-time response shown in Figure 3(B) (red trace). Intriguingly, a slow initial current decay is observed before reaching a quasi-steady state at around ~7 s. This non-canonical behavior contrasts with the current decay observed in open-NEA structures as shown in Figure 3(B) (black trace), in which rapid decay of the charging current is dictated by the inherent RC time constant of the cell. In general, the mechanism of potential-induced wetting in hydrophobic nanopores has been attributed to a combination of electrowetting, electroosmotic pressure, and the ionic environment, leading to dynamic water condensation and evaporation. 17,20,21 We hypothesize that the slower current decay observed for the potential step in BCP@NEA structures S1, yielding the membrane characteristic decay constant τ. Measurement of i(t) across five different BCP@NEA architectures and subsequent current fitting to the developed model produced an average decay constant of τ = 1.88 ± 0.36 s. Previous gating experiments have measured single nanopore transitions between wetted and dewetted states occurring over a vast range of timescales (~10 -7 s -~10 1 s), depending on the applied potential bias, the nature of the pore, and environmental conditions. 18,21,59,60 The wetting constant measured here, τ ~ 2 s, is intermediate between fast wetting and kinetically delayed wetting. Thus, the applied potential was held for 30 s to allow sufficient time for nanochannel wetting and subsequent mass transport from the bulk solution into the vestibule of the pores.
Next, the reversibility of potential-induced gating in BCP@NEA structures was investigated. A solution containing the model redox probe Fe(CN)6 3-was placed on the device, and the analyte was introduced into the vestibule of the nanopores by stepping ETE to -1.2 V vs. Ag/AgCl to induce transport across the membrane (vide supra). After releasing the applied potential, the BCP nanochannels return to their dewetted resting state, isolating the bulk solution from the nanopore volume. To examine how effectively redox species were introduced and captured in the attoliter pore volume during the wetting-dewetting sequence, cyclic voltammetry (CV) was conducted at varying scan rates using the BE as working electrode as shown in Figure 3(C). The CVs exhibit typical thin-layer-cell (TLC) behavior. First, they show a narrow peak separation of ~ 5 mV, which is near the ideal value of 0 mV for thin-layer behavior. Second, the charges obtained during cathodic and anodic scans are approximately equal and remain constant with varying scan rates as shown in Figure 3(D). Finally, the anodic and cathodic peak currents increase linearly with scan rate, as expected for thin-layer behavior. The observation of TLC characteristics is consistent with potential-induced dewetting of the BCP nanochannels, switching them back to the closed state, leading to the encapsulation of redox species to the attoliter volume of the NEA nanopores. The depth of a single nanopore (700 nm) is well below the diffusional boundary layer, δ < 2Dt 1/2 ~ 370 µm for the slowest scan rate used (10 mV/s), leading to full electrolysis of the pore content within the experimental timeframe, which is consistent with the observed current behavior. Additionally, the occurrence of thin-layer behavior after potential-induced wetting indicates that diffusive transport of the negatively charged analyte across the membrane overcomes repulsive electrostatic interactions, such as those responsible for electrophoretic forces.
Redox Cycling Behavior and Amperometry of 4-Ethyl Catechol. Closely spaced two-electrode systems can take advantage of redox cycling to produce enhanced currents for the sensitive detection of reversible redox analytes. To test this idea, the cycling behavior of the redox pair 4ethyl catechol/4-ethyl quinone (4-EC/4-EQ) was probed in open NEAs, i.e., without the BCP membrane, by operating the device in generator-collector (GC) mode. Here, BE and TE were both utilized as working electrodes, where the potential of BE (generator) was swept to generate the cycling species, while TE (collector) was poised at a complementary potential to regenerate the original redox species. CVs obtained in GC and non-GC modes are presented in Figure 4(A). A comparison of the two conditions shows a transition from a peak-shaped CV in non-GC mode to a sigmoidal-shaped current trace in GC mode, demonstrating efficient redox cycling of the redox pair. To extract the current amplification factor (AF), the cathodic limiting current, ilim = -684 nA, was divided by the cathodic peak current, ip = -28.1 nA, yielding AF~24 for redox cycling of 4-EC/4-EQ in the open NEA configuration. Subsequently, the effect of attoliter confinement on redox cycling was examined in BCP@NEA architectures. After utilizing a pair of potential steps to induce a wetting-dewetting cycle in the BCP membrane, the NEA device was operated in GC and non-GC modes, producing the CVs shown in Figure 4(B). While the non-GC CV shows no peak separation, consistent with thin-layer behavior and successful trapping of the analyte, the current traces obtained in GC mode show efficient redox cycling. Current enhancement with AF~104 (ilim = -310 nA, ip = -2.97 nA) was observed, outperforming the open NEAs. This increased cycling current enhancement relative to open NEAs is attributed to confinement of the redox species within the attoliter volume of the nanopore vestibule, with the dewetted BCP membrane effectively blocking diffusion of the redox species back into bulk solution. Finally, currents experienced a modest decrease with multiple consecutive scans, indicating an increase in charge transfer resistance, which likely arises from the polymerization of quinones and the adsorption of phenoxy radicals resulting in concentrationdependent electrode fouling, as has been previously reported. 55,61,62 Amperometric Sensing of 4-Ethyl Phenol in BCP@NEA Structures. After establishing suitable conditions for potential-induced gating and redox cycling of 4-ethyl catechol in confined NEAs, the functionality of BCP@NEA devices as electrochemical biosensors was examined. The enzyme tyrosinase was covalently immobilized onto the BE of the sensor utilizing sequential deposition of self-assembled monolayers of cysteamine followed by activation with glutaraldehyde, Figure S3.
To increase redox cycling efficiency, the enzyme was selectively immobilized on the BE by sweeping the potential of the TE to a reducing potential to induce desorption of the cysteamine number of immobilized enzymes is required per pore. For example, a single enzyme with a turnover number of 14 s -1 for 4-ethyl phenol 55 would suffice to generate up to ~840 product molecules min -1 , which would produce a concentration of ~28 µM for a nanopore volume of 50 aL. Because the geometry allows enzyme to be used so efficiently, low enzyme loadings (exposure of 10 µM for 30 min) were used in order to minimize the effect of increased electron transfer resistance upon enzyme immobilization. In addition, enzyme activity was tested after immobilization by incubating varying concentrations of 4-ethyl phenol on tyrosinase-modified planar Au electrodes. The generated amount of enzymatic product, i.e., 4-ethyl quinone, was detected utilizing CV, verifying that the enzyme follows Michaelis-Menten behavior, thus validating retention of enzyme activity after immobilization, Figure S6.
After covalently linking tyrosinase onto the BE of an NEA, a 350 nm thick PS-b-P4VP membrane was applied to the top surface of the NEA using sacrificial membrane transfer (vide supra).
Phosphate buffered analyte solution with varying 4-ethyl phenol concentrations was placed onto the sensor and after a 5 min incubation, but prior to potential-induced wetting of the BCP nanochannels, the device was operated in GC mode to test for solution leakage, Figure S7. Current traces obtained in these control experiments are dominated by capacitive current, demonstrating the effective isolation of the bulk solution from the interior of the nanopores. After potentialinduced wetting to enable introduction of analyte, Figure 5(A), shows the TE currents collected for different analyte concentrations when operating the device in GC mode, where the TE was poised at a constant potential of +0.4 V vs. Ag/AgCl. The current traces exhibit a sigmoidal shape and show an increase in current at larger analyte concentrations. An increase in background current also observed at higher analyte concentrations is attributed to the oxidation of adsorbed species on the TE. Furthermore, a working curve was generated by plotting the limiting current vs. 4-ethyl phenol concentration as shown in Figure 5(B). The working curve exhibits canonical behavior, with a linear dynamic range from 0.5 -5 mM, and a limit-of-detection (LOD) of 120 nM for 4ethyl phenol, was obtained, confirming the capabilities of these hierarchically organized potentialgated BCP@NEA devices as amperometric biosensors.
We have demonstrated the assembly and characterization of hierarchically organized BCP@NEA sensing architectures utilizing a novel sacrificial layer transfer strategy. These structures exhibit a useful hydrophobic gating capability based on potential-induced wetting-dewetting of PS-b-P4VP membranes that was exploited in these amperometric sensing experiments. Specifically, these structures exhibit: (a) a well-defined threshold potential for transitions from a dewetted, collapsed BCP@NEA structures after capture and trapping. The higher AFs result from confinement in attoliter-volume nanopores presenting two separately controllable nanoelectrodes, thus exploiting the superior detection capabilities of dual-electrode thin-layer cells. Finally, the BCP@NEA architecture was used for amperometric biosensing by immobilizing tyrosinase on the BE for sequential enzymatic conversion of the non-redox active analyte 4-ethyl phenol to redox-active 4ethyl quinone after which it is detected by redox cycling to 4-ethyl catechol. Spatial separation of the bulk analyte solution from the interior of the pores was demonstrated, and control over mass transport was achieved by potential-induced gating to first capture, then encapsulate, the analyte inside the NEA nanopores. 4-Ethyl phenol was detected utilizing ultrasensitive redox cycling of enzymatically generated quinones with a LOD of ~120 nM, demonstrating the biosensing potential of the hierarchically organized structures. Major advantages of the BCP@NEA sensor are reflected in short assay times, temporal and spatial control over transmembrane transport, and the redundancy of adding cofactors or redox mediators, while providing an ultrasensitive readout on a miniaturized solid-state chip. The BCP@NEA sensing architectures described here can serve as a versatile platform for the sensitive detection of a wide range of biomarkers by incorporating suitable enzymes for additional targets, while the spatial and temporal control over the composition of interior pore spaces afforded by hydrophobic gating should find use in a plethora of applications ranging from controlled release to nanoscale reactors. Furthermore, the BCP layer is expected to endow the sensor with additional anti-fouling properties based on its intrinsic filtration and size exclusion properties. The BCP is expected to block transmembrane transport of larger proteins through the vertically oriented P4VP nanochannels of the membrane, rendering the BCP@NEA architecture suitable for sensing biomarkers in complex biosamples.
Chemicals and Materials. 1,4-Dioxane, 4-ethyl catechol, 4-ethyl phenol, acetone, cysteamine, ethyl alcohol, glutaraldehyde solution (50% in H2O), poly(acrylic acid) solution (MW ~ 100,000, 35 wt.% in H2O), polystyrene latex beads (0.46 µm mean particle size), potassium ferricyanide(III) (K3Fe(CN)6), potassium phosphate dibasic, potassium phosphate monobasic (anhydrous), sulfuric acid (conc.), and tyrosinase from mushroom (>1000 units/mg, EC number: 1.14.18.1) were purchased from Sigma-Aldrich, USA. Nexterion glass slides D uncoated (cleanroom cleaned) were obtained from Applied Microarrays, Inc., USA. Poly(dimethylsiloxane) monomer and curing agent were purchased from Dow Corning, USA. Polystyrene-b-poly(4-vinyl)pyridine (PS48,400-b-P4VP21,300, MW/MN = 1.09) was purchased from Polymer Source Inc., Canada. Aqueous solutions were prepared using deionized (DI) water (ρ~18.2 MΩ cm) filtered by a Milli-Q water purification system (Millipore). Except where noted, all reagents and materials were used as received without further purification.
Fabrication and Characterization of NEA Devices. Two-electrode ring-disk NEAs were fabricated by adapting procedures reported previously, 43,63 using a combination of contact photolithography, nanosphere lithography, and reactive-ion etching. A 100 nm thick Au layer was sandwiched between 5 nm thin Ti layers on a cleanroom-clean glass slide using electron-beam evaporation (UNIVEX 450B, Oerlikon) to serve as the bottom electrode. Next, a 100 nm SiNx layer was deposited by plasma-enhanced chemical vapor deposition (PECVD, Unaxis 790, Plasma-Therm).
A similar procedure was utilized to deposit the Au top electrode, followed by PECVD deposition of 500 nm SiO2. Then, nanosphere lithography was conducted by transferring a self-assembled monolayer of polystyrene beads (0.46 mm mean particle size) onto the layered substrate, after which the mean particle size was reduced to 0.33 mm by using O2 plasma etching. A 70 nm thick Cr layer was deposited for use as a hard photomask via electron-beam deposition, and the beads were subsequently removed in acetone. The array size was defined to a 100 mm x 100 mm square using contact photolithography, and ring-disk nanopore electrodes were fabricated by sequential reactive-ion etching (RIE, Plasma-Therm 790) of SiO2, Au, and SiNx. A detailed fabrication scheme is shown in Figure S8. The nanoporous structures fabricated by this scheme were confirmed by scanning electron microscopy (FEI-Helios dual-beam focused ion beam). BCP membrane preparation. BCP membranes were prepared by one of two approaches: (1) direct spin-coating on bare Au electrodes, or (2) sacrificial membrane transfer, for preparation of BCP@NEA structures. In the former method, a 1,4-dioxane solution of 3 wt% PS48400-b-PS21300 was spin-coated at 3000 rpm onto the substrate. To execute the sacrificial membrane transfer method, a solution of 5 wt% polyacrylic acid in water was spin-coated at 4000 rpm onto an O2 plasma-cleaned Si wafer. Subsequently, a solution of 3 wt% PS48400-b-PS21300 in 1,4-dioxane was spin-coated onto the PAA layer at 3000 rpm and dried under N2. The layered substrate was immersed in pure ethanol for 30 s and dried under N2. Then, the PAA layer was dissolved in 0.2 M phosphate buffer until a free-standing BCP membrane emerged at the water-air interface. To transfer the BCP membrane onto an NEA device, the membrane was mechanically collected and placed onto the NEA device after emersion, and excess solution was removed with N2, yielding hierarchically organized BCP@NEA structures. The BCP membrane was characterized by scanning electron microscopy after sputter coating a 1.5 nm Ir film onto it.
Covalent Immobilization of Tyrosinase. An aqueous solution of cysteamine (100 mM) was placed onto the electrode for 60 min and thoroughly rinsed with DI water. For NEA devices, the monolayer formed on the top electrode was removed by reductive desorption in 50 mM KOH by sweeping the potential from -0.4 V to -1.0 V vs. Ag/AgCl for 20 consecutive scans. Next, the device was incubated in a solution of glutaraldehyde (5 % v/v, aq.) for 1 min after which it was rinsed with phosphate buffer solution. Finally, a 0.2 M phosphate buffer solution containing tyrosinase (10 µM) was drop-cast onto the structure for 30 min, followed by rinsing with phosphate buffer solution.
Electrochemical Measurements. Cyclic voltammetry and potential step amperometry were conducted on a dual-channel CHI842C electrochemical workstation (CH Instruments, USA). For all electrochemical measurements, a Ag/AgCl electrode and a Pt wire were used as reference and counter electrodes, respectively. They were immersed in a 100 µL solution inside a PDMS reservoir, covering the region of interest containing the nanoelectrodes. In non-GC mode measurements, the potential of the bottom electrode of the NEA was swept while the top electrode
was left at open circuit potential. For GC mode measurements, the bottom and the top electrode were both utilized as working electrodes, with the potential of the bottom electrode being swept while the potential of the TE was poised at a constant potential of +0.4 V vs. Ag/AgCl. All electrochemical experiments were conducted with a scan rate of 100 mV/s unless otherwise noted.
To induce potential gating, a potential step of -1.2 V vs. Ag/AgCl was applied for 30 s to the TE.