Development of Highly Selective Enzymatic Devices Based on

J. Phys. Chem. C 2009, 113, 6037–6041

6037

Development of Highly Selective Enzymatic Devices Based on Deposition of Permselective Membranes on Aligned Nanowires Frank N. Crespilho,*,† Marcos C. Esteves,‡ Paulo T. A. Sumodjo,‡ Elizabeth J. Podlaha,§ and Valtencir Zucolotto| Centro de Cieˆncias Naturais e Humanas, UniVersidade Federal do ABC, Santo Andre´, 09210-170, Brazil, Instituto de Quı´mica, UniVersidade de Sa˜o Paulo, Sa˜o Paulo, 05508-900, Brazil, Department of Chemical Engineering, Louisiana State UniVersity, Baton Rouge, Louisiana 70802, Instituto de Fı´sica de Sa˜o Carlos, UniVersidade de Sa˜o Paulo, Sa˜o Carlos, 13560-970, Brazil ReceiVed: December 17, 2008; ReVised Manuscript ReceiVed: January 28, 2009

We describe a simple and efficient strategy to fabricate enzymatic devices based on the deposition of glucose oxidase on aligned and highly oriented CoNiMo metallic nanowires. CoNiMo nanowires with an average diameter of 200 nm and length of 50 µm were electrodeposited on Au-covered alumina substrates via electrodeposition, using alumina membranes as templates. Enzyme-modified electrodes were fabricated via enzyme immobilization using a cross-linker. To minimize nonspecific reactions in the presence of interfering agents, a permselective membrane composed of poly(vinylsulfonic acid) and polyamidoamine dendrimer was deposited via electrostatic interaction. The formation of hydrogen peroxide as a product of the enzymatic reaction was monitored at low overpotential, 0.0 V (vs Ag/AgCl). The detection limit was estimated at 22 µM under an applied potential of 0.0 V. The apparent Michaelis-Menten constant determined from the Lineweaver-Burke plot was 2 mM. Introduction Immobilization of biological molecules in conjunction with nanostructured materials, the so-called nanobiocomposites, have been widely explored in electrochemical biodevices.1-7 In the case of enzymatic devices, including biosensors and biofuel cells, the control over the interface between the nanomaterial and enzyme is essential for developing analytical systems with high sensitivity and stability. Such interfacial control is crucial to overcome problems such as the nonspecific electrochemical reactions from interfering agents, a major drawback related to operation of amperometric devices. An example is the recent report from Wilners et al., who proposed a new concept on fabrication of nanoastructured biofuel cells based upon the chemical-to-electrochemical energy transformation.1,2 Another interesting approach has been proposed by Martin et al., who reported a protein biosensor based on a single conically shaped Au nanotube embedded in a polymeric membrane.8 In this case, the conical geometry exhibited a series of advantages, including the enhanced response signal under normal operation conditions. The use of dendrimer-based composites as nanoplatforms for enzyme immobilization has also been proved to be advantageous in terms of biocatalytic activity and sensitivity.3,10,11 We have recently reported the use of modified electrodes based on dendrimer-incorporated Au nanoparticles covered with redox mediators3,10,11 capable of promoting fast charge transfer between glucose oxidase (GOx) and the electrode surface.3 Different nanoscaled 1D materials, including nanotubes12 and nanowires,13 have been employed in conjunction with biomolecules in electrochemical devices, taking advantage of the high specific area and fast charge transport exhibited by these * Corresponding author. E-mail: [email protected]. † Universidade Federal do ABC. ‡ Instituto de Quı´mica, Universidade de Sa˜o Paulo. § Louisiana State University. | Instituto de Fı´sica de Sa˜o Carlos, Universidade de Sa˜o Paulo.

systems. One of the open challenges regarding the manipulation of nanotubes and nanowires for modified electrodes is the positioning of the materials perpendicularly to the electrode surface, in a way that both enzyme activity and fast electron transfer can be achieved. In this paper, we describe an efficient strategy to produce enzymatic devices based upon the deposition of GOx atop aligned CoNiMo nanowires. Electrode fabrication comprised the electrodeposition of the CoNiMo nanowires perpendicuarly to the gold electrode, followed by immobilization of the enzymatic layer using glutaraldehyde (GA) as the cross-linker. To minimize the nonspecific reaction in the presence of interfering agents, a block layer composed of poly(vinylsulfonic acid) (PVS) was deposited via electrostatic interaction over the enzyme layer. We used CoNiMo nanowires because they are strategic materials for the development and improvement of biosensors, in drug release devices or in medical diagnosis. In addition, CoNiMo nanowires are magnetic material, and a new approach can be used to build biological devices with magnetic properties. For example, the functioning of such devices requires cycles of enzyme recovery, and the regeneration can be done by the application of an external magnetic field if the enzymes are immobilized on magnetic particles. Experimental Section Deposition of Aligned Nanowires and Electrode Fabrication. CoNiMo nanowires (CoNiMo-NW) were deposited on Aucovered glass substrates via electrodeposition at room temperature (22 °C) using anodic alumina membranes from Whatman (0.2 µm pore size and 50 µm thickness) as scaffolds, as schematically represented in Figure 1. First, a gold layer was sputtered on one side of the membrane to provide the electrical contact. The membrane was placed inside a plastic electrode holder containing a copper wire used

10.1021/jp811153z CCC: $40.75  2009 American Chemical Society Published on Web 03/19/2009

6038 J. Phys. Chem. C, Vol. 113, No. 15, 2009

Crespilho et al.

Figure 2. Current as a function of CoNiMo NWs deposition time, under an applying potential of -1.2 V vs SCE.

Figure 1. Schematic illustration depicting the perpendicularly aligned deposition of nanowires on the Au-covered alumina electrodes.

as the electrical contact. This holder limited the exposed area to 1.24 cm2. Potentiostatic electrodeposition was performed using a BAS-Zahner model IM6e potentiostat with a threeelectrode cell in which the alumina membrane was the working electrode, a Pt mesh was the counter electrode and a saturated calomel electrode (SCE) was used as reference. An electrolytic solution of 0.24 mol L-1 NiSO4, 0.06 mol L-1 CoSO4, 0.006 mol L-1 Na2MoO4, 0.31 mol L-1 glycine (C2H5O2N), and 0.5 mol L-1 Na2SO4 was employed. All chemicals were provided by Fischer. The pH was set to 7 with NH3(aq), and the solutions were degassed with N2 prior to the deposition. Cyclic voltamograms were obtained using a Pt microelectrode to determine the optimal potential range for NW deposition. The optimized potential of -1.2 V vs SCE was selected for deposition of NWs. The surface morphology of the modified electrodes was investigated using scanning electron microscopy using both a JEOL JSM-840A and a JSM-7401F microscopes. To prevent charging and enhance the images quality, the alumina membranes were fractured and dissolved in a 1.0 mol L-1 KOH solution. The resulting material was rinsed with distilled water and dried using N2 gas. The chemical composition of the NWs was verified using a Noran EDS set at 15 kV coupled to the microscope. Enzyme Immobilization. A mixed layer of GOx and bovine serum albumin (BSA) was covalently attached to the CoNiMo NWs by drop-coating using glutaraldehyde as the cross-linker. A total amount of 680 µL of solution was prepared using 200 µL of GA (2.5% v/v in water) and 480 µL of enzyme solution. The enzyme solution was previously prepared upon adding 40 mg of BSA and 100 mg of GOx in 2 mL of 0.1 mol L-1 phosphate buffer at pH 7.0. The CoNiMo-NW/GOx electrodes were prepared by drop-coating of a mixture of proteic and crosslinker solutions (2 µL/2 µL). The electrode was dried for 1 h at 22 °C. Amperometric Measurements. Electrochemical experiments were performed in a three-electrode cell system. An Ag/AgCl electrode was used as reference, a Pt wire as the counter electrode, and CoNiMo-NW or CoNiMo-NW/glucose oxidase (CoNiMo-NW/GOx) electrodes (area of 0.70 cm2) as the working electrode. The electrolytic solution was a phosphate buffer solution, 0.1 mol L -1, pH 7.0. Amperometric measurements were carried out using an EG&G PAR M280 electrochemical analyzer at 0.0 V (Ag/AgCl). All measurements were performed at 22 ( 1 °C.

Figure 3. (a) SEM image showing the aligned CoNiMo NWs deposited on Au-covered alumina substrates. (b) Zoomed image of the nanowires.

Results and Discussion CoNiMo nanowires with an average diameter of 200 nm and length of 50 µm were electrodeposited from glycine-ammonia electrolytes using anodic alumina membranes as scaffolds (Figure 1). To determine the time required to fill out the membrane pores with the NWs, it was necessary to overgrow the wires and analyze the current transient in the process. Figure 2 shows the resulting current transient collected during the CoNiMo NWs deposition upon applying -1.2 V vs SCE during 110 min. Phase 1 is related to the filling of the pores, driven by ionic diffusion. After an initial decay, the current increased as the wires grew. Since the diffusion path shortened, the diffusioncontrolled reaction was enhanced. As the wires reached the top of the pores, the current increased as a result of mushroomlike deposit, and the diffusion became radial (phase 2). After coalescence of the mushroomlike aggregates, ionic diffusion becomes linear, and the current remained constant (phase 3). In this case, no more changes in the electrode area were observed. A similar behavior was reported by Martin and collaborators.18 SEM images of the samples confirmed the formation of the film over the nanowires. In this work, a deposition time of 35 min (required for phase 1 to be completed) was set for CoNiMo NWs deposition. The SEM images of the CoNiMo NWs are depicted in Figure 3. Nanowires with a diameter of 200 nm and 50 µm long (250 aspect ratio) are clearly seen. To determine the chemical composition of the CONiMo NWs, three regions along the wires were analyzed by EDS. The results indicated that the NWs are composed by 39% Co, 52% Ni, and 9% Mo (wt %). The electrocatalytic ability of CoNiMo-NWs toward hydrogen peroxide was investigated by cyclic voltammetry and crono-

Development of Highly Selective Enzymatic Devices

J. Phys. Chem. C, Vol. 113, No. 15, 2009 6039

Figure 4. Cyclic voltammograms of the CoNiMo-NWs in 0.1 mol L1 phosphate buffer (pH 7.0) with addition of 0.5 mM hydrogen peroxide. Scan rate 50 mV s-1. Inset: Zoomed region around 0.0 V (Ag/AgCl).

amperometry. Voltammograms in Figure 4 displayed a linear dependence of the Faradic current on the hydrogen peroxide concentration. Interestingly, the anodic and cathodic faradic currents increased at overpotential near to 0.0 V (Ag/AgCl), indicating a possibility of applying lower overpotentials for monitoring the redox process involving hydrogen peroxide (see inset of Figure 4). This effect is similar to that found for other electrodes based on nanostructured materials.3 For example, cobalt hexacianoferrate compounds can be used as a redox mediator to hydrogen peroxide electrocatalysis at 0.0 V (Ag/AgCl).3 In the latter case, however, metallic cobalt is present in the nanowires. In our system, we cannot unequivocally conclude that the presence of cobalt is the principal cause of the electrocatalytic effect. Nonetheless, the hydrogen peroxide electrocatalysis at 0.0 V (Ag/AgCl) was also verified in amperometric measurements. Figure 5 shows a typical cronoamperometry curve taken at 0.0 V (Ag/AgCl) in which the increase in the hydrogen peroxide concentration (0.6 µmol L-1 per aliquot) was monitored as a function of time. In the absence of hydrogen peroxide, a constant quasistationary current was observed during the first 15 min and remained constant for a long time, indicating a stable baseline shape, when equilibrium current was reached after 2 min. The cathodic current appeared upon the first addition of the hydrogen peroxide aliquot, leading to the well-known amperometric curve profile. After electrode characterizations, GOx was immobilized atop the CoNiMo NW-modified electrodes in conjunction with BSA using glutaraldehide as the cross-linker. As we have recently reported,3,10 the use of BSA in conjunction with the active enzyme may be advantageous in terms of preventing bioactivity losses, since a friendly environment for enzyme immobilization is created. The response of the CoNiMo-NW/Gox electrodes toward glucose oxidation was investigated using amperometry. The amperometric measurements revealed a Michaelis-Menten profile, which might be indicative of electrocatalytic reduction

Figure 5. (a) Cronoamperometry of the CoNiMo-NWs in 0.1 mol L1 phosphate buffer (pH 7.0) with addition of 0.5 mM hydrogen peroxide. Applied potential: 0.0 V (Ag/AgCl). (b) Linear fit of cathodic current (Ic) versus hydrogen peroxide concentration.

Figure 6. Glucose response curves at CoNiMo-NWs/GOx electrode in 0.1 mol L1 phosphate buffer (pH 7.0). Applied potential: 0.0 V (Ag/ AgCl).

of hydrogen peroxide at the CoNiMo layer. Figure 6 shows the faradic current (I - I0) as a function of the glucose concentration, in which a linear range up to 2 mM and a sensitivity of 64 nA mM-1 cm-2 was found. The detection limit (estimated using three times the signalto-noise ratio) was 22 µM under an applied potential of 0.0 V. The apparent Michaelis-Menten constant determined from the Lineweaver-Burke plot was 2 mM. The operational stability of CoNiMo-NW/GOx-modified electrodes was investigated upon monitoring the electrode response for a glucose concentration of 0.5 mM. After 50 cycles, the current response dropped by 20%. Also important to mention is that after 1 month of storage at 2 °C in phosphate buffer solution, the electrode maintained 91% of its initial current response. The selectivity of the CoNiMo-NW/GOx electrode was assessed by checking the influence of possible interfering agents, including chemical

6040 J. Phys. Chem. C, Vol. 113, No. 15, 2009

Figure 7. FTIR spectra in the transmission mode for cast films of PVS, PAMAM, and PVS/PAMAM LbL films.

Crespilho et al. -NH3+ terminal groups from PAMAM and -SO3- groups from PVS. The latter was inferred from the FTIR data, in which the bands assigned to SO3- at 1200 cm-1 and 1049 cm-1 from cast PVS were shifted to 1180 cm-1 and 1102 cm-1, respectively, in the PAMAM/PVS film, as shown in Figure 7. A schematic illustration showing the membrane architecture and the enzymatic reaction is shown in Figure 8a. After formation of the PAMAM/PVS membrane, the enzyme electrode containing a PAMAM/PVS layer was subject to the glucose biocatalysis in the presence of ascorbic acid. Using this new electrode configuration, no cronoamperometric current of ascorbic acid was detected (Figures 8b, c). We measured glucose without interfering agent currents in the presence of high ascorbic acid concentration, up to 0.5 mM. Due to the outermost negative PVS layer, an effective barrier for ascorbic acid was created, similarly to what has been reported for other systems.17 It is worth mentioning that other systems18,19 and nanomaterials20,21 have been proposed to prepare enzymatic devices. Similarly to CoNiMo-NW/GOx (without LbL membrane)modified electrodes, the long-term stability of the PVScontaining electrodes was also investigated using three electrodes stored in phosphoric buffer (pH 7.0) at 4.0 °C. After 15 days, the current response toward glucose oxidation decreased ∼8 ( 2%, whereas for 30 days of storage, the electrodes retained ∼75 ( 6% of their initial response. Conclusions

Figure 8. (a) Schematic illustration of the glucose oxidation at CoNiMo-NWs/GOx electrode after the PAMAM/PVS membrane deposition. (b) Cronoamperometry of the CoNiMo-NWs/GOx electrode after the PAMAM/PVS membrane deposition in 0.1 mol L1 phosphate buffer (pH 7.0) with addition of 0.1 mM glucose. Note that the enzyme electrode containing a PAMAM/PVS layer was subject to the glucose biocatalysis in the presence of ascorbic acid (0.5 mmol L1), and no current from ascorbic acid addition was observed. (c) Michaelis-Menten response curves of GOx using the same system described in part b. Applied potential: 0.0 V (Ag/AgCl).

species normally present in real and natural samples. The interfering agents analyzed were fructose, ethanol, and several organic acids (viz., acetic, ascorbic, citric, lactic, and oxalic acids). Among the interfering agents investigated, only ascorbic acid could be detected, at concentrations higher than 0.50 mM. To reduce a possible interference signal, as in the case of ascorbic acid, a permselective membrane of poly(vinyl sulfonic acid) was deposited via electrostatic attraction on the top of the CoNiMo-NW/GOx electrode. The rationale behind this approach was to establish a negatively charged membrane, capable of preventing diffusion of ascorbic anions through the electrode, due to electrostatic hindrance. We exploit the molecular engineering ability of the electrostatic layer-by-layer (LbL) technique16 to immobilize a bilayer of polycationic G4 polyamidoamine dendrimer (PAMAM) and anionic PVS over the CoNiMo-NW/GOx electrodes (see Figure 7). The deposition of the PAMAM/PVS bilayer was carried out by immersing the CoNiMo-NW/GOx electrode alternately into the 0.1 mol L-1 PAMAM (cationic) and 0.1 mol L-1 PVS (anionic) solutions for 10 min. The formation of a PAMAM/ PVS bilayer is a consequence of the strong interactions between

We developed an enzymatic device containing glucose oxidase immobilized at aligned CoNiMo nanowires, in which hydrogen peroxide was electrochemically reduced at 0.0 V (vs Ag/AgCl). This applied potential ensures minimization of interference effects when the CoNiMo-NW/GOx electrodes are used in real and complex matrices, such as biological substrates and natural environments. Furthermore, the PAMAM/PVS layer deposited on the CoNiMo NW/Gox electrodes was effective for blocking ascorbic acid interference. Acknowledgment. Financial support from FAPESP, CAPES, CNPq is gratefully acknowledged. References and Notes (1) Willner, I; Baron, R.; Willner, B. Biosens. Bioelectron. 2007, 22, 1841. (2) Willner, I.; Basnar, B.; Willner, B. FEBS 2007, 274, 302. (3) Crespilho, F. N.; Ghica, M. E.; Florescu, M.; Nart, F. C.; Oliveira, O. N., Jr.; Brett, C. M. A. Electrochem. Commun. 2006, 8, 1665. (4) Merkoc¸i, A. Electroanalysis 2007, 19, 739. (5) Huguenin, F.; Zucolotto, V.; Carvalho, A. J. F.; Gonzalez, E. R.; Oliveira, O. N., Jr. Chem. Mater. 2005, 17, 6739. (6) Zucolotto, V.; Pinto, A. P. A.; Tumolo, T.; Moraes, M. L.; Baptista, M. S.; Riul, A., Jr.; Arau´jo, A. P. U.; Oliveira Jr., O. N. Biosens. Bioelectron. 2006, 21 (7), 1320. (7) Ariga, K.; Hill, J. P.; Ji, Q. Phys. Chem. Chem. Phys. 2007, 9, 2319. (8) Sexton, L. T.; Horne, L. P.; Martin, C. R. Mol. BioSyst. 2007, 3, 667. (9) Zhang, H.; Hu, N. F. J. Phys. Chem. B 2007, 111, 10583. (10) Crespilho, F. N.; Ghica, M. E.; Gouveia-Caridade, C.; Oliveira, O. N., Jr.; Brett, C. M. A. Talanta 2008, 76, 922. (11) Crespilho, F. N.; Zucolotto, V.; Brett, C. M. A.; Oliveira, O. N., Jr.; Nart, F. C. J. Phys. Chem. B 2006, 110, 17478. (12) Merkoc, A.; Pumera, M.; Llopis, X.; Pe´rez, B.; del Valle, M.; Alegret, S. Trends Anal. Chem. 2005, 24, 9. (13) Wanekaya, A. K.; Chen, W.; Myung, N. V.; Mulchandani, A. Electroanalysis 2006, 18 (6), 533. (14) Esteves, M. C.; Sumodjo, P. T. A. J. Electrochem. Soc. 2006, 153 (8), 540. (15) Schuchert, U.; Molares, M. E. T.; Dobrev, D.; Vetter, J.; Neumann, R.; Martin, M. J. Electrochem. Soc. 2003, 150, 189. (16) Decher, G. Science 1997, 277, 1232.

Development of Highly Selective Enzymatic Devices (17) Hammonds, P. T. AdV. Mater. 2004, 16, 1271. (18) Okahata, Y.; Tsuruta, T.; Ijiro, K.; Ariga, K. Langmuir 1998, 4, 1373. (19) Okahata, Y.; Tsuruta, T.; Ijiro, K.; Ariga, K. Thin Solid Films 1989, 180, 65. (20) Ariga, K.; Hill, J. P.; Lee, M. V.; Vinu, A.; Charvet, R. Sci. Technol. AdV. Mater. 2008, 9, 014109.

J. Phys. Chem. C, Vol. 113, No. 15, 2009 6041 (21) Crespilho, F. N.; Borges, T.F.C.C.; Zucolotto, V.; Nart, F. C.; Oliveira, O. N., Jr. J. Nanosci. Nanotechnol. 2006, 6, 2588. (22) Caseli, L.; Crespilho, F. N.; Nobre, T. M.; Zaniquelli, M. E. D.; Zucolotto, V.; Oliveira, O. N., Jr. J. Colloid Interface Sci. 2008, 319, 100.

JP811153Z