Electrochemistry and Current Control in Surface Films Based on Silica

Dec 17, 2012 - chitosan. The second film (F2) contained also added enzyme glucose ... tion, and sensing.1 For such applications, the nanoparticles hav...
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Electrochemistry and Current Control in Surface Films Based on Silica-Azure Redox Nanoparticles, Carbon Nanotubes, Enzymes, and Polyelectrolytes Sushma Karra, Maogen Zhang, and Waldemar Gorski* Department of Chemistry, University of Texas at San Antonio, San Antonio, Texas 78249-0698, United States ABSTRACT: The redox active nanoparticles were developed by covalently attaching redox dye Azure C (AZU) to commercial silica nanoparticles (SN) via the silylated amine and glutaric dialdehyde links. The SN-AZU nanoparticles were studied as redox mediators for the oxidation of reduced β-nicotinamide adenine dinucleotide (NADH) in two polymeric films. The first film (F1) was composed of SN-AZU, carbon nanotubes, and cationic polyelectrolyte chitosan. The second film (F2) contained also added enzyme glucose dehydrogenase and its cofactor β-nicotinamide adenine dinucleotide (NAD+). The films F1 and F2 were cast on the glassy carbon electrodes, covered with an anionic polyelectrolyte Nafion, and their electrochemical properties were probed with NADH and glucose, respectively, using voltammetry, amperometry, and potentiometry. The Nafion overcoat reduced the sensitivity of F1/Nafion film electrodes to NADH by >98%. In contrast, depending on the concentration of Nafion, the sensitivity of the F2/Nafion film electrodes (reagentless biosensors) to glucose increased by up to 340%. The amplification of glucose signal was ascribed to the Donnan exclusion and ensuing Nafion-gated ionic fluxes, which enhanced enzyme activity in films F2. The proposed model predicts that such signal amplification should be also feasible in the case of other enzyme-based biosensors. chitosan films and Nafion-induced signal amplification in enzyme-based biosensors. We also demonstrate the usefulness of iridium oxide (IrOx) films in monitoring local pH changes in polymeric surface films.

T

he silica nanoparticles (SN) have been used in a variety of applications including optical imaging, labeling, separation, and sensing.1 For such applications, the nanoparticles have been modified with optically1−3 and electrochemically active compounds.4−16 Recently, the redox active silica nanoparticles have been prepared by encapsulating or covalently attaching small redox moieties. The ferrocene-functionalized silica nanoparticles have been investigated as potential charge storage systems,4 catalysts,5,6 molecular recognition interfaces,7 and biosensors.8 The silica nanoparticles doped with ruthenium9 and iridium10 complexes and luminol11 have been studied as electrochemiluminescent immunosensors and sensors. The amperometric sensing systems have also been developed on the basis of the silica nanoparticles that were doped with redox dyes such as thionine,12 methylene blue,13,14 toluidine blue,15 and neutral red.16 The present paper describes the redox active silica nanoparticles that were prepared by the covalent attachment of phenothiazine dye Azure C (AZU). The covalent bonding rather than doping was chosen in order to avoid dye leaching from the particles. The SN-AZU nanoparticles were studied as the signal transducers in surface films of electrochemical nicotinamide adenine dinucleotide (NADH) sensors and reagentless biosensors based on a NAD-dependent dehydrogenase enzyme. The focus of this research was on the synergistic effects between the SN-AZU nanoparticles and carbon nanotubes in the electro-oxidation of NADH in © 2012 American Chemical Society

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EXPERIMENTAL SECTION Reagents. Chitosan (MW ∼1 × 10 6 Da; ∼80% deacetylation), spherical silica nanoparticles (5−15 nm diameter, 590−690 m2 g−1, density 2.0 g cm−3), nicotinamide adenine dinucleotide (NAD+, λ = 259 nm), dihydronicotinamide adenine dinucleotide (NADH), iodoacetic acid, Nafion (5.0 wt %, acid form), methanol, 3-aminopropyl triethoxysilane (APS), glutaric dialdehyde (25 wt % solution), Azure C (AZU), D(+)-glucose, glucose dehydrogenase (from Pseudomonas sp., EC 1.1.1.47, 275 units mg−1), and KNO3 were purchased from Sigma-Aldrich. Multiwalled carbon nanotubes (CNT, ∼95% nominal purity) were purchased from Nanolab (Brighton, MA). The NaH2PO4·H2O, Na2HPO4, HCl, NaOH, and tris(hydroxymethyl) aminomethane (Tris) were from Fisher. All solutions were prepared using deionized water that was purified with a Barnstead NANOpure cartridge system. The chitosan solutions (0.10 wt %) were prepared by dissolving chitosan flakes in a hot 0.10 M HCl solution (90 Received: November 1, 2012 Accepted: December 17, 2012 Published: December 17, 2012 1208

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°C), cooling to room temperature, and adjusting their pH to 5.0 with a NaOH solution.17 The colorless solutions were filtered using a 0.45 μm Millex-HA syringe filter unit (Millipore) and stored in a refrigerator (4 °C) when not in use. The suspensions of carbon nanotubes in chitosan solutions (1.0 mg mL−1) were prepared by a 15 min sonication. Electrochemical Measurements. The electrochemical data were collected using the CHI 832B workstation (CH Instruments, Inc.). The experiments were performed at room temperature (20 ± 1 °C) in a conventional three-electrode system with a 3.0 mm diameter glassy carbon (GC) disk working electrode (Bioanalytical Systems, Inc.), platinum wire auxiliary electrode, and Ag/AgCl/3 M NaCl (BAS) reference electrode. Prior to use, the GC electrodes were wet polished on an Alpha A polishing cloth (Mark V Lab) with successively smaller particles (0.3 and 0.05 μm diameter) of alumina. The slurry that accumulated on the electrode surface was removed by ultrasonication for 30 s in deionized water and methanol. Experiments were repeated at least three times, and results are presented with the relative standard deviations (RSD). Covalent Modification of Silica Nanoparticles with Azure C. The SN were activated at 550 °C for 6 h in air and modified with AZU in three synthetic steps. First, 500 mg of activated SN was refluxed in ethanolic solution of APS (3.68 mmol, 200 mL) for 24 h in order to obtain the SN-APS nanoparticles. The reaction mixture was cooled to room temperature and centrifuged to separate the nanoparticles, which were subsequently washed with three 20 mL aliquots of methanol in order to remove the excess of APS. Second, the aqueous suspension of SN-APS nanoparticles (10.0 mL, 45 mg mL−1) was slowly (15 min) added to a vigorously stirred solution of GDI (25 vol %) and stirred overnight at room temperature to synthesize the SN-APS-GDI nanoparticles. The reaction mixture was centrifuged, and the nanoparticles were washed with water (4 × 15 mL) in order to remove the unreacted GDI. Third, the aqueous suspension of SN-APS-GDI nanoparticles (10.0 mL, 45 mg mL−1) was reacted with 30.0 mL of aqueous solution of Azure C (2.0 mmol, 554 mg) at 80 ± 5 °C for 24 h to obtain the SN-APS-GDI-AZU nanoparticles (called SN-AZU thereafter). The SN-AZU nanoparticles were separated by centrifugation, washed with seven 40 mL aliquots of water, and stirred in fresh aliquots of water for 2 days in order to remove any unreacted or adsorbed AZU molecules. They were dried at 90 °C for 5 h and stored dry in a closed container at room temperature when not used. Synthesis of CHIT-GDI and CHIT-NAD+. The chemically modified chitosans were prepared following our previous synthetic protocols.18,19 Here, we used the CHIT-GDI and CHIT-NAD+ solutions that were prepared in 2005 and 2006, respectively. Preparation of Film Electrodes. Two types of films were prepared and optimized in order to maximize their sensitivity toward the NADH (first film (F1)) and glucose (second film (F2)). The films F1 were composed of SN-AZU nanoparticles, carbon nanotubes, and polysaccharide chitosan (SN-AZU/ CNT/CHIT films). They were prepared by mixing 10.0 μL of aqueous suspension of SN-AZU nanoparticles (1.0 mg mL−1) and 10.0 μL of CNT suspension in 0.10 wt % CHIT solution (1.0 mg mL−1) on the surface of glassy carbon electrode. After the evaporation of water at room temperature (3 h), the F1 film electrodes were soaked in a stirred pH 7.40 phosphate buffer

solution for 30 min to remove any weakly attached components. The wet F1 films were coated with 20 μL aliquots of 1.0, 3.0, or 5.0 wt % Nafion solutions and dried at room temperature for 1 h. The films F2 contained also the enzyme glucose dehydrogenase (GDH) and its cofactor β-nicotinamide adenine dinucleotide, NAD+ (SN-AZU/CNT/CHIT/GDH/NAD+ films). They were prepared by the sequence of mixing steps on the surface of glassy carbon electrode using the following solutions: (A) 30.0 μL of CHIT-NAD+ solution (0.094 wt % CHIT, 1.5 mM immobilized NAD+), (B) 5.0 μL of GDH enzyme solution (12.0 mg mL−1), (C) 20.0 μL of 0.10 wt % CHIT-GDI solution, and (D) a mixture of 10.0 μL of aqueous suspension of SN-AZU nanoparticles (1.0 mg mL−1) and 10.0 μL of CNT suspension in 0.10 wt % CHIT solution (1.0 mg mL−1). First, the solutions A and B were mixed and equilibrated for 30 min to establish the CHIT-NAD+-GDH interactions. A 20 μL aliquot of water was added, if necessary, to prevent drying. Second, the solution C was added to cross-link the system and secure the affinity interactions between the enzyme and its cofactor. The mixture was kept liquid while equilibrating for 30 min. Finally, the suspension D was added in order to provide a redox mediator for the enzymatic reaction and improve the electronic conductivity of the surface film. The water was allowed to evaporate at room temperature (3 h) in order to form well adhering surface films, which were then soaked in a stirred pH 7.40 phosphate buffer solution for 1 h in order to remove any weakly attached components. The wet F2 films were coated with 20 μL aliquots of 1.0, 3.0, or 5.0 wt % Nafion solutions and dried at room temperature for 1 h. All of the film electrodes were soaked in a background electrolyte solution for 30 min before their first use. Determination of pH inside Surface Films. The pHsensitive IrOx films were electrodeposited on the surface of glassy carbon electrodes from a 0.20 mM solution of Na3IrCl6 that was aged for 2 h at 80 °C. The films were made by a 15 min amperometric deposition at 1.20 V.20 The CHIT-CNT films were prepared by casting 20.0 μL of CNT suspension in 0.10 wt % CHIT solution (1.0 mg mL−1) on top of the IrOx films and drying at room temperature for 2 h. The pH changes in the CHIT-CNT and CHIT-CNT/Nafion films were determined by following a four-step protocol, which involved the (1) preparation of a calibration plot by measuring the open circuit potential EOCP at a IrOx/CHIT-CNT film electrode in a stirred phosphate buffer solution in the pH range of 6.0−8.0 (adjusted with 1.0 M HCl solution), (2) collection of ten 5 min EOCP vs time traces at the IrOx/CHIT-CNT film electrode in a stirred pH 7.40 phosphate buffer solution (0.050 M), (3) covering the IrOx/CHIT-CNT wet film electrode with a 20.0 μL aliquot of 5.0 wt % Nafion solution and drying for 2 h at room temperature, and (4) collecting again ten 5 min EOCP vs time traces at the IrOx/CHIT-CNT/Nafion film electrode in a stirred pH 7.40 phosphate buffer solution. The control experiments were performed by repeating steps 1−4 with a Tris buffer (pH range of 6.8−8.8, 0.050 M) and 0.10 M KNO3 solutions. In the case of KNO3 solution, the calibration plot was collected in Tris buffer solution and the IrOx/CHIT-CNT film electrode was soaked afterward in a KNO3 solution for 20 min before proceeding with the collection of ten EOCP vs time traces in a KNO3 solution. 1209

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RESULTS AND DISCUSSION Covalent Attachment of Azure C to Silica Nanoparticles. The modification of commercial silica nanoparticles (SN) with Azure dye (AZU) started with the hydrolysis and condensation of 3-aminopropyl triethoxysilane (APS) on the surface of SN. This yielded the SN that were covalently modified with terminal primary amines. The attached amines were subsequently reacted with glutaric dialdehyde (GDI) to form Schiff bases. The concentrated GDI solution was used in order to react only one aldehyde group of GDI with a primary amine. Finally, the unreacted aldehyde group of the attached GDI was reacted with the amino group of AZU to form the modified silica nanoparticles SN-APS-GDI-AZU, which will be referenced to as SN-AZU for the sake of brevity (Figure 1).

line c). The spectrum of SN-APS-GDI suspensions displayed only a minor band at 278 nm that was due to the absorption by the GDI, which further confirmed our synthetic scenario. The hypsochromic shift in the absorption bands upon dye immobilization from 612 to 594 nm could be ascribed to the transition dipole interactions between chromophore regions of AZU molecules arranged parallel to each other (Haggregates)21−24 on the silica surface. Apparently, the covalent confinement of AZU molecules on SN resulted in a close packing of dye molecules leading to dye aggregation.25 The intensity of the 594 nm absorption band of SN-AZU suspension indicated that 1 g of SN-AZU contained 1.4 mmol of AZU based on the Beer−Lambert’s law. This translated into ∼880 molecules of AZU attached to one nanoparticle when 2.0 g mL−1 and 10 nm as the silica density and average nanoparticle diameter, respectively, were used. The comparable number of ferrocenes has been attached to silica nanoparticles recently.4 The covalent immobilization of AZU on SN was further corroborated by the electrochemical studies (vide infra). Redox Properties of SN-AZU/CNT/CHIT Films (Films F1). The redox properties of the films composed of SN-AZU nanoparticles, CNT, and CHIT (films F1) were probed with NADH molecules. The role of individual film components was investigated by recording the cyclic voltammograms at the SNAZU, SN-APS-GDI, and F1 film electrodes in the presence and absence of NADH in a solution (Figure 3). In the initial control experiments, cyclic voltammograms of the free AZU dissolved in a solution were recorded at a bare glassy carbon electrode. The voltammograms displayed a pair of current peaks at a midpeak potential of −0.21 V (Figure 3, black line a1). They were generated by the oxidation and reduction of AZU species at the electrode surface

Figure 1. Silica nanoparticle (SN) with covalently attached phenothiazine dye Azure C.

This synthetic scenario was supported by the UV−visible spectra of AZU solution and SN-AZU suspension in water. Figure 2 (line a) shows the electronic spectrum of AZU

AZU (red) ⇔ AZU + (ox) + H+ + 2e−

(1)

The addition of NADH to the AZU solution resulted in the appearance of an anodic wave with a half-wave potential E1/2 = +0.26 V in the cyclic voltammogram (Figure 3, red line a). The new anodic wave was due to the AZU-mediated oxidation of NADH to NAD+ NADH + AZU + (ox) ⇒ NAD+ + AZU (red)

(2)

The new wave could not be ascribed to the direct oxidation of NADH at a bare glassy carbon electrode NADH ⇒ NAD+ + H+ + 2e−

(3)

because such an electrode process required much more positive potentials (>0.6 V). The large separation between the current peaks of AZU couple (−0.21 V) and the anodic mediation wave (+0.26 V) indicated that the mediation process was slow on the experimental time scale. The alternative explanation for the shift in the oxidation of NADH from >0.6 to +0.26 V may also involve the catalysis due to the complexation between NAD+ and AZU dye. The SN-AZU film electrodes displayed a different voltammetric behavior in a background electrolyte solution. The current peaks at −0.21 V due to the redox of silica-bound AZU molecules substantially decreased and broadened (Figure 3, black line b1) when compared to those of freely diffusing AZU molecules (Figure 3, black line a1). The lowering and broadening of the current peaks could be ascribed to the hindered charge transport in the surface film and heterogeneity of surface redox sites with distributed formal potentials and electron tunneling distances.26−28 The site heterogeneity could

Figure 2. UV−visible absorbance spectra of (a) 5.3 × 10−4 M Azure C solution, (b) 0.33 mg mL−1 suspension of SN-APS-GDI-Azure C (i.e., SN-AZU) nanoparticles, and (c) 0.278 mg mL−1 suspension of SNAPS-GDI nanoparticles in deionized water. In order to provide internal consistency, samples a and b underwent the same treatment, including heating at 80 °C for 24 h, before taking the spectra.

solution with two main absorption bands at 285 and 612 nm, which is characteristic for the monomeric AZU species. The shoulder at 580 nm indicated the presence of molecular aggregates of AZU in a solution.21 The suspension of SN-AZU nanoparticles in water yielded a similar spectrum with two main absorption bands at 282 and 594 nm (Figure 2, line b), which confirmed the attachment of AZU molecules to silica surface. No such absorption bands were observed in the control experiments with SN-APS-GDI suspensions in water (Figure 2, 1210

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tion matrix for other components of the surface film. The F1 film electrodes displayed cyclic voltammograms with large capacitive background currents due to the large surface area of CNT. The voltammograms displayed two distinct pairs of current peaks at the midpeak potentials of −0.26 and −0.13 V due to the redox of AZU species (Figure 3, black line d1). Considering the UV−visible evidence of dye aggregation, one can assign these pairs of current peaks to AZU monomers (−0.26 V) and aggregates (−0.13 V). In the presence of NADH in a solution, the voltammograms displayed an anodic mediation wave at E1/2 = −0.03 V, which had an inception point that coincided with the anodic peak for the AZU aggregate (Figure 3, red line d). This large decrease in E1/2 could not be explained by the direct oxidation of NADH at CNT because such process required much more positive potentials (>0.3 V). Apparently, the CNT and CHIT had a profound effect on the capacity of SN-AZU to mediate the oxidation of NADH. The small and broad overlapping peaks due to the redox of SNAZU (black line b1) increased and transformed into two pairs of current peaks in the presence of CNT and CHIT (black line d1). Each pair displayed a small peak separation (40 mV) indicating faster kinetics of the electrode process. The E1/2 of the anodic mediation wave shifted from +0.22 V (red line b) to −0.03 V (red line d) upon mixing the SN-AZU nanoparticles with the CNT and CHIT. This indicated a positive synergy between the three film components that resulted in the facilitation of SN-AZU-mediated oxidation of NADH. These positive changes could be ascribed to a better charge transport in films F1, which could be modeled as the collection of SNAZU redox sites embedded in the interpenetrating network of electronic (CNT) and ionic (CHIT) conductors. Indeed, the CNT are known to form well-dispersed and stable colloidal suspensions in CHIT solutions.18,32 Such intimate dispersion of CNT throughout the films F1 shortened the electron tunneling distances for all subsets of redox sites including those distant from the surface of glassy carbon. The shorter distances for electron hopping between the silica-immobilized AZU species reduced the demand for charge-compensating ion transport in films F1. This facilitated the mediation reaction 2 leading to a lower E1/2 for the anodic wave (red line d). Nafion-Induced Changes in Sensitivity of Films F1 and F2 to NADH and Glucose. Nafion33 has been used extensively in the design of electrochemical sensors and biosensors to provide a charge-based selectivity and protect them from fouling. Here, the influence of Nafion on the sensitivity of films F1 and F2 to NADH and glucose, respectively, was investigated. The experiments were conducted in stirred pH 7.40 phosphate buffer solutions using amperometry at 0.40 V. Figure 4 shows a drastic decrease in the sensitivity of the F1 film electrodes to NADH after they were covered with a Nafion film. For example, the current due to the oxidation of 10.0 mM NADH decreased by 98, 99, and 99.6% after the F1 film electrodes were coated with 1.0, 3.0, and 5.0 wt % Nafion films, respectively. The current decreased because the NADH species are negatively charged due the presence of the phosphate groups and were electrostatically rejected by negatively charged pores of Nafion. The Nafion effect on the sensitivity of F2 film electrodes to glucose was opposite (Figure 5). For example, the sensitivity to 10.0 mM glucose increased by 260, 320, and 340% after F2 film electrodes were coated with a 1.0, 3.0, and 5.0 wt % Nafion films, respectively. The analyte preconcentration is an unlikely

Figure 3. Cyclic voltammograms recorded in 1.0 mM NADH solutions at (a) bare GC electrode, and (b) SN-AZU, (c) SN-APSGDI, and (d) F1 film electrodes. Traces a and a1 were recorded in 1.0 mM NADH + 0.25 mM AZU and 0.25 mM AZU solutions, respectively. Background traces b1, c1, and d1 were recorded without AZU in a solution. Background electrolyte, pH 7.40 phosphate buffer solution (0.050 M). Scan rate, 50 mV s−1.

be due to a number of factors including the dye aggregation (vide supra) and nonuniform size distribution of nanoparticles, which frequently contribute to the complex charge/mass transport patterns in such films. Nevertheless, the SN-AZU films facilitated the mediated oxidation of NADH (reaction 2) as indicated by the shift in the anodic mediation wave from E1/2 = +0.26 V (Figure 3, red line a) to E1/2 = +0.22 V (Figure 3, red line b). This shift could be attributed to the presence of stronger oxidants such as the AZU aggregates in the SN-AZU film. Indeed, it has been shown that the dimerization of phenothiazine dyes shifts their standard potential to higher values.29−31 The anodic mediation wave displayed an additional feature, which was a sharply increasing current at potentials more positive than +0.45 V (Figure 3, red line b). In the control experiments with SN-APS-GDI film electrodes, a similar sharp increase in current at > +0.45 V was observed in the presence of NADH in a solution (Figure 3, red line c). The lack of such current increase in the absence of NADH in a solution (Figure 3, black line c1) indicated that the SN-APSGDI structures promoted the oxidation of NADH at > +0.45 V. The mechanism of this promotion is not clear at present. In order to improve the charge transport, the films F1 were formed by dispersing the SN-AZU nanoparticles with the electron conductive CNT in an ion-conductive matrix of CHIT. It should be pointed out that the CHIT was not electroactive and played the role of good ionic conductor and immobiliza1211

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fluxes of chemical species exchanging between the film F2 and a solution. We hypothesize that the Nafion-gated ionic fluxes created a chemical environment (i.e., ionic composition and pH) inside the film F2 that was conducive to the enhanced enzymatic activity of GDH. This led to the faster enzymatic oxidation of glucose and, thus, larger glucose currents at F2/ Nafion film electrodes. In order to test this hypothesis, two sets of experiments were performed. Initially, the influence of ionic composition inside films F2 on the glucose current at F2/Nafion film electrodes was investigated. This was done by equilibrating films F2 with different solutions and trapping the exchanged species by covering the films F2 with Nafion overcoat. The following experimental protocol was used. First, a freshly prepared F2 film electrode was equilibrated in a pH 7.40 phosphate buffer solution for 30 min and its current response (IF2) to 10.0 mM glucose aliquot was recorded. Second, the electrode was removed from the solution, rinsed, and equilibrated with a pH 7.40 phosphate buffer, deionized water, pH 7.40 Tris buffer, or KNO3 solution for 30 min before coating it with a 5.0 wt % Nafion film. The current response of the F2/Nafion film electrode (IF2/Nafion) to 10.0 mM glucose aliquot was recorded again in pH 7.40 phosphate buffer solution. Figure 6A shows

Figure 4. Amperometric response (E = 0.40 V) of (a) F1 film electrode and F1 film electrode that was coated with (b) 1.0, (c) 3.0, and (d) 5.0 wt % Nafion film to additions of NADH aliquots (0.025, 0.050, 0.075, 0.15, 0.30, 0.60, 1.0, 3.0, 5.0, and 10.0 mM) into a stirred solution of pH 7.40 phosphate buffer. Inset: the relevant calibration plots. The right y-axis is for traces b, c, and d.

Figure 5. Amperometric response (E = 0.40 V) of (a) F2 film electrode and F2 film electrode that was coated with (b) 1.0, (c) 3.0, and (d) 5.0 wt % Nafion film to additions of glucose aliquots (0.025, 0.050, 0.075, 0.15, 0.30, 0.60, 1.0, 3.0, 5.0, and 10.0 mM) into a stirred solution of pH 7.40 phosphate buffer. Inset: the relevant calibration plots.

explanation for the increased sensitivity because Nafion is not known for the preconcentration of neutral and hydrophilic molecules such as glucose. In order to explain the Nafioninduced increase in glucose current at F2/Nafion film electrodes (reagentless biosensors), the following aspects of the system were considered. First, the glucose current was generated by the sequence of reactions starting with the enzymatic reaction

Figure 6. (A) Nafion effect on glucose current measured in a pH 7.40 phosphate buffer solution at F2 film electrodes that were equilibrated with (a, e) pH 7.40 phosphate buffer, (b) deionized water, (c) pH 7.40 Tris buffer, and (d) 0.10 M KNO3 solutions. Bar e: glucose current measured in a pH 7.40 Tris buffer solution. R = [(IF2/Nafion − IF2)/IF2] × 100% where IF2/Nafion and IF2 are glucose currents measured at F2/ Nafion and F2 film electrodes, respectively, at 0.40 V. (B) pH effect on the enzymatic activity of enzyme glucose dehydrogenase (GDH). Background electrolyte, 0.050 M Britton Robinson buffer.

ß‐D‐glucose + GDH(NAD+) ⇒ GDH(NADH) + D‐glucono‐1,5‐lactone + H+

the effect of equilibration of films F2 in different media on the sensitivity of F2/Nafion film electrodes to glucose. The effect was quantified as a ratio R = [(IF2/Nafion − IF2)/IF2] × 100%. The casting of Nafion film over the F2 film that was equilibrated with a phosphate buffer resulted in the large amplification of glucose current (Figure 6A, bar a, R = 340%), which was consistent with the data in Figure 5. The amplification of glucose current was an order of magnitude smaller when films F2 were equilibrated with deionized water (Figure 6A, bar b, R = 35%) and practically nonexistent in the case of the films F2 that were equilibrated with Tris buffer (Figure 6A, bar c, R=-5%). In contrast, the soaking of films F2 in KNO3 solution practically eliminated the sensitivity of F2/ Nafion film electrodes to glucose (Figure 6A, bar d, R = −99.9%). This was ascribed to the deactivation of enzyme GDH by nitrate ions in unbuffered media. Indeed, our

(4)

which was followed by the AZU-mediated reoxidation of NADH to the enzymatically active NAD+ (reaction 2), reoxidation of AZU (reaction 1), and hydrolysis of lactone D‐glucono‐1,5‐lactone

⇒ D‐gluconate + H+

(5)

Second, all these reactions were taking place inside the film F2 because the enzyme GDH, enzyme cofactor NAD+, and redox mediator AZU were covalently immobilized either on the CHIT chains or SN in the film F2. The hydrolysis reaction could eventually spread outside the film F2 at longer experimental times. Third, the polymeric chains of Nafion were cast on the top of a solid film F2 and could not penetrate the cross-linked film F2. Under these conditions, one can argue that Nafion played the role of ionic gate that controlled the 1212

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electrochemical enzyme assays34 revealed that GDH lost on average 97% of its enzymatic activity in a 0.10 M KNO3 solution when compared to that in a pH 7.40 phosphate buffer solution (not shown). One can hypothesize that the increase in glucose current shown in Figure 6A (bar a) was due to the enhanced enzymatic activity of GDH in the presence of phosphate anions, which were trapped within a film F2 under Nafion. The phosphate ions are known to augment the activity of GDH.35−38 For example, we determined that the enzymatic activity of GDH increased by ∼100% when 10.0 mM Na2HPO4 was added to a pH 7.40 Tris buffer solution (not shown). Such behavior was also confirmed in the case of films F2 by recording the glucose current at a F2 film electrode in a pH 7.40 Tris buffer solution, equilibrating the F2 film electrode with a pH 7.40 phosphate buffer solution, and recording the glucose current again in a Tris buffer solution. The phosphate ions trapped in the F2 film under Nafion amplified the glucose current by R = 270% (Figure 6A, bar e). In contrast, there was no amplification of glucose current when Tris species were trapped within a film F2 under Nafion (Figure 6A, bar c). Thus, the signal amplification was unique to phosphate ions. After documenting the importance of ionic composition in signal amplification, the second set of experiments focused on the Nafion effect on pH inside films F2 in the context of pHdependent enzymatic activity of GDH. The amperometric enzyme assays showed that the enzymatic activity of GDH improved as pH increased and reached maximum at pH 8.0 (Figure 6B). The potentiometric measurements of Nafion effect on pH inside films F2 required more judicious selection of experimental conditions. First, the CHIT-CNT matrix of films F2 was chosen instead of whole films. This was done in order to avoid interferences from mixed potentials generated by the H+-sensitive AZU+/AZU and NAD+/NADH redox systems that were present in films F2. Second, the pH-sensitive IrOx film was used to measure the difference in pH before and after coating the CHIT-CNT films with Nafion. The calibration plots of the open circuit potential EOCP vs pH for the IrOx/ CHIT-CNT film electrodes in phosphate buffer solutions had a negative slope of −64 ± 5 mV pH−1 (R2 = 0.999). In a pH 7.40 phosphate buffer solution (0.050 M), the EOCP of IrOx/CHITCNT film electrodes shifted toward a less positive value by 37 ± 4 mV after they were coated with Nafion (Figure 7, red lines a1 and a2). Such shift in potential indicated that the Nafion overcoat caused an increase in pH inside the CHIT-CNT films by ∼0.60 units considering the slope of the calibration plot. The increase in pH could be explained by the preferential retention of a basic component of buffer (HPO42−) in CHITCNT films due to the more efficient Donnan exclusion of divalent ions (HPO42−) than monovalent ones (H2PO4−) by the Nafion overcoat. The control experiments with pH 7.40 Tris buffer solutions (0.050 M) revealed a slow drift of EOCP and practically no potential shift (