Self-Assembly of Peptide Nanostructures onto an Electrode Surface

Article pubs.acs.org/JPCC

Self-Assembly of Peptide Nanostructures onto an Electrode Surface for Nonenzymatic Oxygen Sensing Camila P. Sousa,† Mauricio D. Coutinho-Neto,† Michelle S. Liberato,† Lauro T. Kubota,‡ and Wendel A. Alves*,† †

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-170, Santo André, SP, Brazil Instituto de Química, Universidade Estadual de Campinas, 13084-971, Campinas, SP, Brazil

‡

S Supporting Information *

ABSTRACT: We report here the fabrication of a biomimetic sensor for direct oxygen reduction; the sensor consists of multicopper oxidases derived from cyclic-tetrameric copper(II) species containing the ligand (4imidazolyl)ethylene-2-amino-1-ethylpyridine (apyhist) that are self-assembled with L-diphenylalanine micro/nanostructures (FF-MNTs). The [Cu4(apyhist)4]4+/FF-MNT complex was immobilized onto the surface of a glassy carbon (GC) electrode by poly ion complex formation with a Nafion film. This hybrid membrane allows regular proton transport to a Cu-based molecular oxygen reduction reaction catalyst, and the imidazole group in the imine ligand (apyhist) acts as a local buffer in the vicinity of the O2 reducing center, thus aiding the catalyst in retaining its selectivity for 4e−/ 4H+ oxygen reduction reaction. This nanocomposite provided improved sensing characteristics in the electrode interface with respect to the electroactive surface area, the diffusion coefficient, and the electron transfer kinetics. In addition, the hybrid film [Cu4(apyhist)4]4+/FF-MNT-coated GC electrode was successfully used as an enzymeless electrochemical sensor for the detection of dissolved oxygen in aqueous media at two concentration intervals, viz., 0.2−3.0 mg L−1 and greater than 3.0 mg L−1, with sensitivities of 25.0 and 80.2 μA L mg−1 cm−2, respectively, and a detection limit of 0.1 mg L−1. Evaluated in terms of relative standard deviation, the repeatability of the proposed sensor was less than 9.0% for ten measurements of a solution of 6.5 mg L−1 oxygen. Experimental efforts were conducted to use this proposed platform for O2 determination with real samples. Results from theoretical investigations using density functional theory support the hypothesis that the [Cu4(apyhist)4]4+ complex can act as the sole source of protons and electrons in the O2 reduction reaction.

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INTRODUCTION Copper proteins involved in natural redox processes exhibit highly different features and are distributed across all biota (fungus, plants, animals, and humans).1 Some of these materials exhibit a mononuclear active site, i.e., the so-called blue-copper proteins plastocyanin and stellacyanin, whereas others have a dinuclear active center, i.e., the tyrosinases and catecholases, and still others have multinuclear metal sites, i.e., laccases, ascorbate oxidases, nitrite reductases, cuprous oxidases, ceruloplasmin, and bilirubin oxidases. 2 Many of these substances are responsible for the oxidation of specific substrates, i.e., galactose oxidase, amine oxidase, or ascorbate oxidase, and others play an antioxidant role against free radicals, i.e., superoxide dismutase. The majority of multicopper oxidases (MCOs) contain four copper active sites (per monomer of a protein molecule) consisting of one copper ion at the T1 or blue site and three copper ions at the trinuclear cluster or T2/T3 sites.2 This family of enzymes reduces oxygen to water with simultaneous oxidation. Many research studies have been devoted to understanding the mechanism of oxygen reduction by MCOs in solution. The reduction to water occurs at the T2/T3 site, © 2014 American Chemical Society

and no free hydrogen peroxide has been registered as an evolving product.2 Many of the reported studies have been focused on simulation of the structural and spectral properties of these centers 3 as well as on their O 2 -activation mechanisms.4,5 Knowledge of the mechanisms that nature uses to synthesize materials with exceptional functional properties facilitates the use of molecular biomimetics to execute artificial functions at the molecular level and to apply biological principles to solve problems in human-made systems. Systems such as biomimetic lipid bilayer membranes6 and molecularly imprinted polymers7 can be used as versatile platforms for biosensors, whereas biomimetic enzymes,8,9 the so-called “artificial enzymes”, also offer excellent bioanalytical performance and long-term stability. Mimicking compounds are critical for the development of biosensors because they provide more resistant material for electron transfer with respect to the ease of their own oxidation Received: September 5, 2014 Revised: December 16, 2014 Published: December 16, 2014 1038

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The Journal of Physical Chemistry C or degradation than natural proteins. The manufacture of new sensors can be based on the biological characteristics of highly efficient and selective active sites in sophisticated proteins. However, when incorporated into supramolecular systems, these materials should be more robust, less expensive, and more easily obtained. No limitations exist for mimicking a determined structure or presenting a specific mechanism of action to better understand the phenomena that occur in a biological medium.10 In this context, novel biosensors constitute a challenge for researchers, particularly for bioanalytical chemists, with respect to the creation of new structures with new and useful properties. Over the past decade, the use of peptides has gained increasing attention from many materials scientists, primarily because of the biocompatibility and molecular recognition of peptides with other (bio)molecules or cells, resulting in the use of many novel hybrid materials as surface modifiers for implants or tissue engineering applications.11 Supramolecular selfassembly of nanofibers of amphiphilic oligopeptides12 or small molecules,13 i.e., the skeletons of artificial enzymes, has yet to be explored. Similar to the peptide chains that form active sites in enzymes, the self-assembled nanofibers of peptide moieties can act as matrices of artificial enzymes to aid in the function of the active site and as immobilization carriers to facilitate applications of the peptides.14−18 Peptide micro/nanostructures self-assembled from L-diphenylalanine (FF-MNTs) building blocks have been applied in the field of biosensors.15,19−23 A new electrochemical biosensing platform was fabricated by deposition of FFMNTs onto the surface of graphite electrodes.22 Cyclic voltammetric and time-based amperometric studies demonstrated that the presence of FF-MNTs can significantly improve the sensitivity of electrodes. In a similar study, thiol-modified FF-MNTs were applied to a gold electrode, rendering it sensitive to enzymes such as glucose oxidase and ethanol dehydrogenase.24 These modified electrodes exhibit improved sensitivity and reproducibility for the detection of glucose and ethanol based on enzyme-related electrocatalytic reactions. In addition, FF-MNT-modified electrodes offer other advantages, e.g., nonmediated electron transfer, short detection time, large current density, and comparatively high stability.17 Therefore, these findings demonstrate that FF-MNTs are an attractive alternative for the fabrication of sensors and biosensors with promising electrochemical performance. Recently, the construction of a biomimetic sensor for multinuclear copper(II) enzymes was reported.19,25 The architecture is based on the use of copper(II) complexes containing a tridentate ligand from a Schiff base, [Cu4(apyhist)4]4+, in which apyhist = (4-imidazolyl)ethylene2-amino-1-ethylpyridine was immobilized onto a GC electrode using a Nafion membrane. In this work, the [Cu4(apyhist)4]4+ complex is incorporated into micro/nanostructures constructed via the self-assembly of FF compounds. Specifically, we aimed to improve the electrochemical response and the stability of the complexes in a phosphate buffer solution at pH 7.0 for fabrication of a novel biomimetic oxygen sensor. We investigated the efficiency of the resulting modified electrodes in the electrocatalysis of O2 reduction via cyclic voltammetry and rotating-disk voltammetry and compared the results with those from the same electrode in the absence of FF-MNTs. This novel approach might provide important guidance for the development of artificial enzymes.

Article

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EXPERIMENTAL SECTION

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RESULTS AND DISCUSSION

Materials and Instrumentation. Sodium hydroxide, hydrochloric acid, potassium chloride, hydrogen peroxide (29%), absolute ethyl alcohol, and methyl alcohol were purchased from Synth. Histamine dihydrochloride, 1,1,1,3,3,3hexafluoride-2-propanol (HFP), diphenylalanine peptide (FF), 2-acetylpyridine, and potassium phosphate monobasic were purchased from Sigma-Aldrich. Potassium phosphate dibasic trihydrate was purchased from Carlo Erba Reagents. Copper(II) perchlorate was purchased from Acros Organics, Nafion (5% solution) was purchased from DuPont, and 0.5 and 0.03 μm alumina suspensions were purchased from Panambra Zwick. Cyclic voltammetry was performed using a μAutolab Fra 2 Type III potentiostat/galvanostat. Rotating-disk voltammetry was performed with an ASR2 rotator from Pine Instrument Corp. The dioxygen concentration in the electrolyte was measured with a Clark electrode connected to an oxymeter from Digimed (model DM-4). pH measurements were performed with a Metrohm-Pensalab model 827 lab pH meter using combined glass electrodes. The electrodes were cleaned in a Sanders model SW2000F ultrasonic cleaner equipped with a heating bath. All solutions were prepared using pure water with a resistivity of 18.2 MΩ cm at 25 °C produced using a Direct-Q system (Millipore). Functionalization of FF-MNTs with a Copper(II) Complex. The copper(II) complex [Cu4(apyhist)4]4+ was prepared according to the methodology described in the literature.25 The FF-MNTs were prepared by dissolving the lyophilized form of the peptide FF compound in HFP at a concentration of 100 mg mL−1 and diluting it to 5 mg mL−1 in ultrapure water.19 The diluted mixture was centrifuged for 15 min in a MPW centrifuge at 1200 rpm. The supernatant was removed; the precipitate was placed in a solution of [Cu4(apyhist)4]4+ (8 mg) previously dissolved in 1 mL of ultrapure water; and 5% Nafion solution (200 μL) was added. This solution was ultrasonicated for at least 15 min at room temperature to obtain a homogeneous blue solution. Fabrication of the Modified Electrode. A glassy carbon electrode (0.071 cm2) was carefully polished with 0.5 and 0.03 μm alumina and was ultrasonicated in ethanol and twicedistilled water. The modified GC electrode was prepared by adding a drop (4.5 μL) of the previously described mixture solution directly onto the top of the electrode. The solvent was allowed to evaporate at room temperature. Microscopy. Scanning electron microscope (SEM) images were obtained using a JEOL FEG-SEM JSM 6330 F and a JEOL LV-SEM microscope at the Laboratory of Electron Microscopy of the Nanotechnology National Laboratory (LNNano), Campinas, Brazil. Electrochemical Measurements. An electrochemical cell containing 10.0 mL of 0.1 mol L−1 phosphate buffer solution (pH 7.0) was used for all measurements, with a saturated calomel electrode (SCE) as reference, a Pt wire as the auxiliary electrode, and the modified GC electrode as the working electrode.

Electrochemical Behavior of the Modified GC Electrodes. Figure 1A shows the cyclic voltammograms of [Cu4(apyhist)4]4+/Nafion- and [Cu4(apyhist)4]4+/FF-MNT/ Nafion-modified GC electrodes in 0.1 mol L−1 phosphate 1039

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Figure 2. (A) Reduction of O2 on a rotating GC electrode coated with [Cu4(apyhist)4]4+/FF-MNT/Nafion in 0.1 mol L−1 phosphate buffer solution at pH 7.0 with different rotation rates under an O2-saturated atmosphere. (B) Koutecky−Levich plot at −0.7 V vs SCE obtained for [Cu 4 (apyhist) 4 ] 4+ /FF-MNTs/Nafion-modified (- ○ - ○ - ○ -) and [Cu4(apyhist)4]4+/Nafion-modified (-●-●-●-) GC electrodes.

buffer solution at pH 7.0. The peak currents at approximately −0.40 V (vs SCE) are assigned to the CuIIL/CuIL redox couple19 and are directly proportional to the square root of the scan rate, which is a characteristic of the diffusion of counterions into the hybrid film to maintain electroneutrality.26,27 Therefore, during the redox reactions of the copper complex, the charge is balanced via the insertion/ejection of electrolyte ions at the coating layer of the modified electrode, which facilitates the cyclic electrochemical reactivity of the hybrid film on the electrode surface. The amounts of metal complex in the modified electrodes were estimated using atomic absorption spectroscopy, i.e., [Cu4(apyhist)4]4+/Nafion = (0.82 ± 0.02) mg/L and [Cu4(apyhist)4]4+/FF-MNT/Nafion = (1.08 ± 0.04) mg/L, and the voltammograms were normalized to their respective values. Thus, cyclic voltammetry showed that the peak current is much higher for the electrode containing FF-MNTs, which indicates that the presence of the peptide nanostructure enhanced the electrochemical response toward redox reactions between the copper ion and the modified electrode. This behavior was previously demonstrated by Gazit et al. and by Matsui et al., who reported that peptide nanostructure-modified electrodes significantly improved the surface area of the electrode and induced electron transfer during the chemical reaction.17,28 To evaluate the contribution of the cyclic-tetrameric copper(II) complex and the effect of the FF-MNT film on the electrode surface, we fabricated different sensors and compared them using cyclic voltammetry. Figure 1B shows the

Figure 1. (A) Cyclic voltammograms of [Cu4(apyhist)4]4+/FF-MNT/ Nafion-modified (____), [Cu4(apyhist)4]4+/Nafion-modified (----) GC electrodes, and a bare electrode (····) recorded at a scan rate of 50 mV s−1 in phosphate buffer solution at pH 7.0 (0.1 mol L−1), 10th cycle, N2(atm). The inset shows the dependence of the redox peak currents on the potential sweep rates for the [Cu4(apyhist)4]4+/FF-MNTs/Nafionmodified (- ○ - ○ - ○ -) and [Cu 4 (apyhist) 4 ] 4+ /Nafion-modified (-●-●-●-) GC electrodes. (B) First cyclic voltammograms of [Cu 4 (apyhist) 4 ] 4 + /FF-MNT/Nafion-modified ( _ _ _ _ ) and [Cu4(apyhist)4]4+/Nafion-modified (----) GC electrodes and a bare electrode (····) at a scan rate of 50 mV s−1 in phosphate buffer solution at pH 7.0 (0.1 mol L−1), O2(atm). The inset shows the influence of the pH on the peak potentials in phosphate buffer solution (0.1 mol L−1), O2(atm). (C) Model scheme showing the change in chemical potential with changing pH for both reduced and oxidized [Cu4(apyhist)4] species. Protonated species are represented by red lines, and unprotonated species are represented by blue lines; Δμx represents the change in chemical potential due to reduction at different pH levels. 1040

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Figure 3. (A) Structure of the oxidized and (B) reduced [Cu4(apyhist)4] complex under vacuum, as obtained from DFT calculations. Copper atoms are represented in gold. (C) Reduced (blue) and oxidized (red) structures superimposed; hydrogen atoms are omitted for clarity.

electrode in solution at pH 7.0 (Figure 1B); this result indicates a faster electron transfer reaction between the oxygen molecules and the copper complex in the peptide nanostructures compared with that with the electrode in the absence of FF-MNTs. The catalysis can be associated with the larger diffused channels and hydrophobicity of the surfaces of the peptide micro/nanostructures, thus allowing rapid mass transfer of the O2 molecules and the product onto the modified electrode surface. In addition, we observed that FF-MNTs protected the cyclic tetranuclear imidazolate-bridged copper(II) complex [Cu4(apyhist)4]4+ by preventing dissociation and degradation in solution at pH 7.0, as the pKa for the corresponding dissociation of the imidazole group in the imine ligand was 8.3.25 The absence of dissociation and degradation facilitated the catalytic reaction by providing nanoporous diffusion channels, which possess unique flexibility to allow transport of the substrates. The influence of the solution pH on the electrochemical response for oxygen was investigated in 0.1 mol L−1 phosphate buffer solutions at varying pH values. The peak current reached a maximum at pH 7.0, as shown in the inset of Figure 1B. The peak potential was also dependent on the pH. The dependence of the peak potential on pH can be rationalized by considering the nonequilibrium processes that occur due to changes in the complex protonation. The scheme depicted in Figure 1C shows the change in chemical potential with pH approximated by straight lines for both the reduced (upper lines) and oxidized (lower lines) [Cu4(apyhist)4] species. In other words, it is assumed that the [Cu4(apyhist)4] complex has two regimes for the four electron redox reaction. Under equilibrium (reversible) conditions and high pH, reduction occurs between unprotonated species (blue curves), and at a low pH, this process involves protonated species (red curves). These processes are represented by green arrows in Figure 1C and correspond to the high-pH and low-pH reduction potentials and should remain valid as long as the integrity of the complex is maintained. However, given sufficient energy, nonequilibrium processes can occur. These processes are designated by black arrows in Figure 1C and represent proton-coupled electron transfer reactions (PCET) given below

Figure 4. (A) Cyclic voltammograms for [Cu4(apyhist)4]4+/FF-MNT/ Nafion-modified GC electrodes in various dissolved oxygen concentrations; the voltammograms were collected at a scan rate of 50 mVs−1 in phosphate buffer solution at pH 7.0 (0.1 mol L−1). (B) Plot of the cathodic peak currents as functions of the O2 concentration for [Cu 4 (apyhist) 4 ] 4+ /FF-MNT/Nafion-modified (- ○ - ○ - ○ -) and [Cu4(apyhist)4]4+/Nafion-modified (-●-●-●-) GC electrodes at 50 mV s−1 in phosphate buffer solution at pH 7.0 (0.1 mol L−1).

electrochemical performance in the absence and presence of O2 dissolved in phosphate buffer solution at pH 7.0 as a function of solution pH. As shown in the figure, O2 reduction on the [Cu4(apyhist)4]4+/FF-MNTs/Nafion hybrid film occurred at a potential that was ∼0.1 V more positive than that observed for the [Cu4(apyhist)4]4+/Nafion film and for bare electrodes. The current for the [Cu4(apyhist)4]4+/FF-MNT/Nafion-modified electrode in the presence of O2 was 2.3-fold higher than that observed for the [Cu4(apyhist)4]4+/Nafion-modified carbon

[Cu4(apyhist‐H)4 ]8 + + 4e− → [Cu4(apyhist)4 ]0 + 4H+ Low pH

(1)

[Cu4(apyhist)4 ]4 + + 4e− + 4H+ → [Cu4(apyhist‐H)4 ]4 + High pH 1041

(2) DOI: 10.1021/jp509020x J. Phys. Chem. C 2015, 119, 1038−1046

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The Journal of Physical Chemistry C Table 1. Comparison of Different Oxygen Sensorsa sensor

detection limit (mg L−1)

linear range (mg L−1)

refs

HOOC-2-AQ/AMWCNTs modified GC electrode cobalt tetrasulfonated pthalocyanine/poly-L-lysine FeTsPc/FeT4MPyP-modified electrode poly(nile blue)-modified GC βCDSH/FeTMPyP/CDAuNP PdNp/MWCNTs/GCE CPE−Si7.5Nb SiSb/CoTmPyP CdS/SPCEs hybrid fluorinated xerogels /[Ru(bpy)3]3+ Ru(II)/fluorinated xerogel Ag nanodendrites/GCE GNP/MWNTs-FeTMAPP SiO2/SnO2/MnPc [Cu4(apyhist)4]4+/FF-MNTs/Nafion/GCE

0.02 0.09 0.06 0.01 0.02 0.64 0.02 0.05 0.04 1.4 × 10−3 0.01 0.02 0.1

32 47 48 49 50 51 52 53 54 55 56 57 58 59 this work

[Cu4(apyhist)4]4+/Nafion/GCE

0.1

0.2−6.8 0.2−8.0 0.2−6.4 0.02−0.4 0.2−6.5 2.56−30.08 1.0−13.6 1.0−12.8 1.7−33.0 0.0−40.0 0.0−40.0 32 × 10−3−2.1 0.01−5.8 − 0.2−3.0 3.0-up 0.2-up

this work

a

HOOC-2-AQ/AMWCNTs - Nanowires of (anthraquinone-2-carboxylic acid/amino functionalized) multiwalled carbon nanotubes; FeTsPc iron(II) tetrasulfonated phthalocyanine; FeT4MPyP - iron(III) tetra-(N-methyl-pyridyl)-porphyrin; βCDSH - mono-(6-deoxy-6-mercapto)-βcyclodextrin; FeTMPyP - iron(III) tetra-(N-methyl-4-pyridyl)-porphyrin; CDAuNP - cyclodextrin-functionalized gold nanoparticles; PdNp palladium nanoparticles; MWCNTs - multiwalled carbon nanotubes; CPE - carbon paste electrode; SPCEs - screen-printed carbon electrodes; bpy 2,2′-bipyridine; GNP - gold nanoparticles; FeTMAPP - iron picket-fence porphyrin; MnPc - manganese(II) phthalocyanine.

activity, with a slope close to 0.059 V/pH at 25 °C, which is typical of Nernstian systems. The experimental behavior of the peak reduction potential closely follows the proposed mechanism; i.e., it rises with a slope of 0.059 V/pH when pH < 7 and exhibits the reverse trend when pH > 7, where the peak current potential decreases with an average slope of −0.070 V/pH (insert in Figure 1B). Moreover, the proposed interpretation predicts that the differences between peak potential and equilibrium reduction potential should be smallest at a pH close to the pKa of the oxidized species. Not coincidentally, the experimental curve for the peak current vs pH reaches a maximum at a pH close to (but slightly lower than) the pKa of the imidazole ring (Figure 1B). In this context, we believe that the Nafion film plays a fundamental role in providing charge stabilization and an exchange medium for protons, thus possibly enabling low-barrier proton transfer to the complex. In addition, at a pH close to the pKa of the oxidized species, reaction 2 becomes an equilibrium process and, as the pKa of the oxidized species is lower than the reduced one, can produce a protonated reduced adduct (golden arrow in Figure 1C). At this pH the overpotential should be minimal, allowing for an equilibrium PCET process. Mechanistic Studies of Oxygen Reduction. To investigate the electrocatalytic process involving the cathodic reduction of oxygen at the [Cu4(apyhist)4]4+/Nafion and [Cu4(apyhist)4]4+/FF-MNT/Nafion-modified GC electrodes in additional detail, we performed rotating-disk electrode voltammetric experiments in 0.1 mol L−1 phosphate buffer solution saturated with oxygen at pH 7.0 at different electrode rotation rates using a potential scan rate of 25 mV s−1. The limiting currents obtained for the modified electrode increased with increasing rotation speed. The linearity of the Levich plots observed at lower rates indicates that the reaction is controlled by mass transport (see Figure 2). However, a deviation from linearity in the Levich plots at higher rotation rates suggests a kinetic limitation. At higher rotation rates, the thickness of the

Figure 5. Stability test of [Cu4(apyhist)4]4+/FF-MNTs/Nafionmodified (- ○ - ○ - ○ -) and [Cu 4 (apyhist) 4 ] 4+ /Nafion-modified (-●-●-●-) GC electrodes at 50 mV s−1 in phosphate buffer solution at pH 7.0 (0.1 mol L−1) [O2] = 5.5 mg L−1.

Table 2. Results of Dissolved Oxygen Determination Using a Voltammetric Procedure Compared with Those Obtained Using a Commercial O2 Sensor (Triplicate Determinations) dissolved oxygen/mg L−1 proposed method samples

with FF-MNTs

without FF-MNTs

commercial sensor

crystal with gas crystal without gas

7.7 (±0.2) 1.1 (±0.1)

7.9 (±0.1) 1.0 (±0.1)

7.6 1.2

In both cases the protonated [Cu4(apyhist)4] complex can act as the sole source of electrons and protons for O2 reduction. Provided that the protonation/deprotonation barrier is low, the change in (nonequilibrium) reduction free energy for reactions 1 and 2 with changing pH is related to changes in the H+ 1042

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as well as the explicit treatment of direct coordination with the FF-MNT/Nafion matrix will be considered in a future investigation. In other words, the low pH regime, which should be valid for the pKa−14 pH range, is being computed. The optimized geometries for the [Cu4(apyhist)4] complex in both Cu(I) and Cu(II) oxidation states obtained using the LANL2DZ basis set are presented in Figure 3. The relatively small difference between their geometries points to a small reorganization energy for the redox process. Moreover, the small geometric reorganization might allow the complex to function efficiently while maintaining tight coordination to the FF-MNT/Nafion matrix, thus avoiding material loss. The computed value for the four-electron oxidation potential in water for the [Cu4(apyhist)4] complex was −0.24 V vs SCE under high-pH conditions. To obtain this value, ΔG0 for reaction 4 was computed using DFT-B3LYP and the 6311+G(d) basis set; the Nernst equation was applied; and the high pH reduction potential of O2 to H2O (0.401 V vs SHE at a pH 14) was subtracted from the overall reaction E0. This value compares favorably with the experimental E1/2 value of −0.28 V for [Cu4(apyhist)4]4+/FF-MNT/Nafion measured at pH 9 and with the E1/2 of −0.26 V measured in DMSO, where the nature of the tetranuclear imidazolate-bridged copper(II) complex was determined (data not shown). The agreement between the theoretically computed four-electron reduction potential and the E1/2 measured at pH 9 and in DMSO adds to the data that the integrity of the tetranuclear imidazolate-bridged copper complex is maintained under these conditions. It is also consistent with previously exposed arguments that suggest that the reduction potential should be pH independent over a wide range of pHs as long as the integrity of the complex is ensured. Dissolved Oxygen Detection. Investigation of the sensitivity of the composite electrode to varying dissolved oxygen concentrations was carried out in phosphate buffer solution at pH 7.0. A clear oxygen reduction peak was observed at approximately −0.40 V, showing a strong response to the dissolved oxygen concentration. A plot of peak currents vs dissolved oxygen concentration, as determined using a Clark electrode, is shown in Figure 4. The electrocatalytic current gradually increased with increasing dissolved oxygen concentration from 0.20 to 13 mg L−1. In particular, for concentrations greater than 3.0 mg L−1, the [Cu4(apyhist)4]4+/FF-MNT/ Nafion composite film exhibited high catalytic activity toward oxygen reduction compared to the [Cu4(apyhist)4]4+/Nafion system. In the [Cu4(apyhist)4]4+/Nafion system, the abundance of hydrophobic sites at the peptide interface appears to favor the creation of a nanoreactor in which nonpolar species such as the copper complex and dioxygen are brought together in a confined geometry. As shown in Figure 4B, the [Cu4(apyhist)4]4+/FF-MNT/Nafion/GC electrode produced two linear response ranges for O2 concentrations, viz., 0.2− 3.0 mg L−1 and greater than 3.0 mg L−1, with a detection limit (3σ/slope) of 0.1 mg L−1 and good sensitivities of 25.0 and 80.2 μA L mg−1 cm−2, respectively; the [Cu4(apyhist)4]4+/ Nafion/GC electrode exhibited a detection limit of 0.1 mg L−1 and a sensitivity of 25.0 μA L mg−1 cm−2. The high sensitivity of the current O2 sensor based on the [Cu4(apyhist)4]4+/FFMNT/Nafion/GC electrode for O2 concentrations up to 3.0 mg L−1 is attributed to the hydrophobic properties of surfaces treated with peptide nanostructures as well as to the electrocatalytic effect of FF-MNTs.41−43 A comparison of the performance of our sensor with that of other nonenzymatic O2 sensors is summarized in Table 1. The results show that the

Levich layer decreases, and the magnitude of the current begins to be controlled by the rate of the reaction between O2 and [Cu4(apyhist)4]4+ molecules immobilized within the supramolecular peptide assembly. In this case, the polarization curves are more conveniently analyzed using Koutecky−Levich plots (Figure 2B), in which the currents for oxygen reduction on the rotating-disk electrode are analyzed in potential regions for which the mass transport and kinetic contributions are related as follows29 1 Ilim

=

1 1 + 2/3 −1/6 nFA′k′ME cs 0.62nFAD ν csω1/2

(3)

where k′ME is the heterogeneous rate constant for the reaction between the catalyst and the substrate; D is the diffusion coefficient of oxygen (1.9 × 10−5 cm2 s); υ is the kinematic viscosity (0.009 cm2 s−1); cs is the solubility of oxygen (1.26 × 10−6 mol cm−3); and the other symbols have their usual meanings.25 Notably, the term A in the mass transport component (0.62nFAD2/3v−1/6csϖ1/2)−1 in the Koutecky− Levich equation refers to the geometric area, whereas the term A′ in the kinetic component (nFA′k′MEcs)−1 refers to the electroactive area, which itself depends on the film thickness. In this paper, we assume that A is equal to A′ for further calculations. The Koutecky−Levich plot of the inverse current at −0.7 V vs the inverse angular rotation rate is plotted in Figure 2, indicating that the current-limiting reaction is first order with respect to the O2 reduction, as previously reported for other complexes described in the literature.30−32 On the basis of the slope of the Koutecky−Levich plot in Figure 2B, we determined that the numbers of electrons involved in the catalytic reduction of O2 to H2O were 3.73 and 4.17 for the [Cu4(apyhist)4]4+/Nafion-modified and [Cu4(apyhist)4]4+/FFMNT/Nafion-modified GC electrodes, respectively, in phosphate buffer solution at pH 7.0. The kinetic currents (k′ME) given by the y-intercept in the Koutecky−Levich plot were determined to be 14.5 × 10−3 and 12.5 × 10−3 cm s−1 for the respective modified electrodes, similar to previously reported results.25 The reciprocal of the intercept of the Koutecky−Levich plot indicates that the limiting currents are controlled by a chemical step that precedes the electron transfer. The coordination between the O2 and Cu(I) of the catalyst present on the [Cu4(apyhist)4]4+-modified electrode surface most likely assumes this role. To support this interpretation, we computed the four-electron oxidation potential for [Cu4(apyhist)4] using density functional theory (DFT) and the IEF-PCM33 implicit solvent formalism. The B3LYP34−36 functional was employed along the 6-311+G(d)37,38 and LANL2DZ39 basis sets to compute the ΔG for the coupled oxygen reduction [Cu4(apyhist)4] oxidation reaction. All calculations were performed using the Gaussian09 code.40 The theoretical treatment assumed that no change occurred in the protonation state of the complexes in either oxidation state, which represents a low-pH experimental condition and leads to the corresponding reaction [Cu4(apyhist)4 ]0 + O2 + 2H 2O → [Cu4(apyhist)4 ]4 + + 4OH−

(4)

This choice was made to avoid complications due to changes in protonation and geometry of the ligands that might occur under low-pH conditions in solution. Inclusion of such effects 1043

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described in the literature, thereby demonstrating the advantages of FF-MNTs for O2 determination in practical applications.

incorporation of the copper(II) complex into the net-like FFMNT film was effective in improving the electrocatalytic activity of the modified electrode. Moreover, the [Cu4(apyhist)4]4+/FF-MNTs/Nafion-modified GC electrode exhibited better repeatability for oxygen determination compared with that of the electrode in the absence of FF-MNTs. The stability of the modified electrodes was checked by recording ten successive measurements in 0.1 mol L−1 phosphate buffer solution containing 6.5 mg L−1 of oxygen solution. Little change was observed for the electrode with the FF-MNT matrix, whereas the RSD of the peak current for [Cu4(apyhist)4]4+/Nafion/GC electrode was greater than 30%, as shown in Figure 5. In this case, the FF-MNTs provided a favorable microenvironment for the tetranuclear copper(II) complex to perform direct electron transfer to the electrode surface. Similar behavior has been observed for a hydrogen peroxide sensor based on microperoxidase-11/FF-MNTs deposited onto an ITO electrode surface. In this case, the microperoxidase-11/FF-MNTs were deposited via layer-bylayer deposition using poly(allylamine hydrochloride) in positively charged polyelectrolyte layers20 as well as in a supramolecular-hydrogel-encapsulated Hemin, which is an artificial enzyme that mimics peroxidase.44−46 We determined the dissolved oxygen concentration in commercial drinking water samples via cyclic voltammetry by adding sufficient KCl to the samples to bring the KCl concentration to 0.1 mol L−1; the results obtained agreed well with those obtained using a commercial sensor, as shown in Table 2. The results confirm the suitability of the modified electrode as a sensor and for further commercial application.

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ASSOCIATED CONTENT

S Supporting Information *

SEM images of [Cu4(apyhist)4]4+/Nafion and [Cu4(apyhist)4]4+/FF-MNT/Nafion-modified GC electrodes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +55 11 4996 0193. Fax: +55 11 4996 3166. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by FAPESP (grant no. 2013/129970), INCT in Bioanalytics (FAPESP, grant no. 08/57805-2 and CNPq, grant no. 573672/2008-3), and CNPq (grant no. 472197/2012-6). C.P.S. acknowledges FAPESP (proc. no. 2011/02346-6) for a fellowship grant. M. D. Coutinho-Neto thanks Prof. Pablo Alejandro Fiorito for insightful discussions. We are thankful to LNNano-CNPEM for the use of SEM facilities.

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REFERENCES

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CONCLUSIONS Biomimetic sensors built from an architecture based on a selfassembly [Cu4(apyhist)4]4+/FF-MNT complex immobilized onto a GC electrode by using a Nafion membrane produced improved response characteristics when compared to a previously developed sensor without FF-MNTs. The FFMNTs, with larger diffused channels and hydrophobic surfaces of the peptide micro/nanostructures, provided a novel host matrix on which biomimetic systems of multicopper(II) oxidase enzymes could be attached. This modification enhanced the stability of the catalyst in aqueous solution at pH 7.0 leading to a decrease in the overpotential toward O2 reduction when compared to a FF-MNT less sensor, most likely because of a change in the local environment of the catalyst from an aqueous medium to the hydrophobic peptide interior. By changing the pH of the bulk solution, we reversibly controlled the ability of the imidazole group in the imine ligand (apyhist) to act as a proton carrier inside a hybrid membrane. The resulting shift in peak potential with pH has been rationalized in terms of equilibrium and nonequilibrium PCET redox processes where the adduct, the reduced [Cu4(apyhist)4] complex, can act as the sole donor or electrons and protons for oxygen. Theoretical results obtained using density functional theory agree with results for pH = 9 supporting the idea that the role of the nanocomposite is to stabilize the complex at lower pHs, to provide an exchange media for protons, and to improve the O2 availability. This result confirmed the suitability of FF-MNTs as an electrocatalytic interface upon which a high density of copper complexes can be irreversibly linked. Optimization of the experimental conditions yielded a detection limit and sensitivity for O2 in aqueous solution that were much better than those 1044

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