Research Article www.acsami.org
Tuning Structural Changes in Glucose Oxidase for Enzyme Fuel Cell Applications Barbara Mecheri,*,†,‡ Diana De Porcellinis,‡ Patricia T. Campana,*,†,§ Alberto Rainer,⊥ Marcella Trombetta,⊥ Alexandre Marletta,∥ Osvaldo N. Oliveira, Jr.,# and Silvia Licoccia‡ ‡
Department of Chemical Science and Technologies, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 00133 Rome, Italy § School of Arts, Sciences and Humanities, University of São Paulo, Av. Arlindo Bettio, 1000, São Paulo CEP 03828-000, São Paulo, Brazil ⊥ Università Campus Bio-Medico di Roma, Via Á lvaro del Portillo 21, 00128 Rome, Italy ∥ Institute of Physics, Federal University of Uberlândia, Avenida João Naves de Á vila, 2121, Uberlândia, CEP 38408-100, Minas Gerais, Brazil # São Carlos Institute of Physics, University of São Paulo, CP 369, São Carlos 13560-970, São Paulo, Brazil S Supporting Information *
ABSTRACT: Stabilization and electrical contacting of redox enzymes with electrodes are fundamental requirements for bioelectronics devices, including biosensors and enzyme fuel cells (EFCs). In this study, we show increased glucose oxidase (GOx) stability by immobilization with Nafion. The immobilization process affected GOx conformation but was not detrimental to its activity, which was maintained for more than 120 days. The GOx/Nafion system was interfaced to a carbon cloth electrode and assembled in a prototypal EFC fed with glucose. Polarization and power density curves demonstrated that GOx/Nafion system was able to generate power, exploiting a Nafion-assisted electron transfer process to the electrode. Our findings are consistent with the onset of pHdependent conformational equilibrium for the enzyme secondary structure and its active site. Significantly, the protective effect exerted by Nafion on the enzyme structure may be tuned by varying parameters such as the pH to fabricate durable EFCs with good electrocatalytic performance. KEYWORDS: Nafion, glucose oxidase, enzyme activity, enzyme secondary structure, enzymatic fuel cells nanoparticles,8,9 mesoporous materials,10 graphene and graphene oxide sheets,11−13 and carbon nanotubes.14 In most of bioelectrocatalytic applications, enzymes are immobilized onto an electrode surface using a polymer matrix, which acts as a scaffold maintaining the enzyme conformation, while allowing for easy diffusion of molecules and ions for enzymatic conversion.15 A common mechanism of action of polymeric matrices includes creation of hydrophobic regions for encapsulating and stabilizing enzymes.16 Owing to its micellar structure with hydrophobic domains,17,18 Nafion has been investigated as a support matrix for enzyme immobilization. Nafion/oxidase systems have been developed to enhance the sensitivity of biosensors in amperometric detection of several components of body fluids, such as uric acid,19 ethanol,20 and glucose.21−24 Nafion/oxidase electrodes were
1. INTRODUCTION The unique catalytic activity accomplished by enzymes provides considerable opportunities for the industry to carry out sophisticated, efficient and economical biocatalytic conversion. Indeed, biocatalysis enables accelerated rates for difficult chemical transformations and guarantees extraordinary specificity toward substrates depending on enzyme stereochemistry.1 The integration of redox enzymes with an electrode support and the formation of an electrical contact between biocatalysts and the electrode surface offers an enormous potential for niche applications, such as bioelectronics and optobioelectronics.2 However, bioelectrocatalysis is still confronted with various challenges, such as understanding and tuning electron transfer within the redox enzymes and improving enzyme stability by preventing denaturation under external stress.3 To this end, enzyme immobilization and entrapment strategies have been pursued on a wide range of supports, including hydrophilic sol−gel matrices,4 ionic liquids,5,6 polymer carriers,7 and nanomaterials with different sizes and shapes, such as metal © XXXX American Chemical Society
Received: September 12, 2015 Accepted: December 7, 2015
A
DOI: 10.1021/acsami.5b08610 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
colorimetric probe according to Bateman.42 The method involved running a series of enzyme assays at varying substrate concentrations. Assay solutions contained GOx (0.025 U mL−1), HRP (1.25 U mL−1), ABTS (2.5 mM), and glucose (varied incrementally in the 0−0.25 M range) in PBA buffer for a total assay volume of 1 mL. The reaction was monitored by measuring absorbance at 414 nm as a function of time (2 readings per second) on an Infinite M200 Pro multimode microplate reader (Tecan, Männedorf, Switzerland) at 28 °C. The reaction rate was derived from the slope of the quasi-linear part of the curve, and reported as a function of substrate concentration. The activity of immobilized GOx was assayed on GOx-modified carbon cloth disks. GOx was dissolved in a 0.5 wt % Nafion solution in PBA at a concentration of 1500 IU/mL. Specimens were prepared by casting the GOx solution onto carbon cloth disks (6 mm diameter) at either 30 IU cm−2 (GOx_CC_30) or 100 IU cm−2 (GOx_CC_100), followed by drying overnight. They were then immersed in a working solution containing HRP (1.25 U mL−1), ABTS (2.5 mM), and glucose (varied incrementally in the 0−0.25 M range) in PBA buffer for a total assay volume of 1 mL. Spectroscopic determination of kinetic parameters was performed as described above. 2.3. Conformational Studies. Conformational studies of GOx in solution were performed by circular dichroism (CD) and fluorescence spectroscopy in PBA buffer at different pH values, both in the absence (GOx/PBA samples) and in the presence (GOx/PBA/Nafion samples) of Nafion. Far-UV (190−250 nm) CD spectra were recorded in a Jasco J810 spectropolarimeter (Jasco Inc., Japan), at 25 °C and in a 1 mm path length quartz cell. All spectra were recorded after accumulation of four runs, with subtraction of the buffer spectrum (for all pHs) and smoothed using a FFT (Fast Fourier Transform) filter to minimize background effects. The enzyme concentration varied between 3 and 5 μM for solution samples, and their spectra are shown in molar ellipticity [θ]. Quantitative prediction of the secondary structure was performed by deconvolution of the CD spectra using CONTINLL, CDSSTR, and SelCon programs.43−45 Optimized results were achieved from the calculations with a root-mean-square difference between the experimental and calculated curves (RMSDExp-Calc) lower than 6% for all deconvolutions. Fluorescence spectra of GOx in PBA deriving from tryptophan emission were obtained at 25 °C in a RF 5301 PC spectrofluorimeter (Shimadzu Corporation, Japan) using rectangular quartz cuvettes (1 cm path length) and at optical density (at 280 nm) less than 0.1 to avoid inner filter effects. The fluorescence spectra of buffer solutions were subtracted to eliminate scattering. The excitation wavelength was 295 nm, and the emission spectra were recorded in the 305−450 nm range, with bandwidths of 5 nm for excitation and emission. 2.4. Electrochemical Tests. The electrochemical cell used for acquiring polarization and power density curves, shown in Figure 1, was made of two plexiglass chambers (11 mL) separated by a Nafion 117 membrane (12 cm2 surface area). The anolyte chamber consisted of the GOx_CC_100 electrode in contact with a 100 mM glucose and 2 mM HQ solution in PBA (pH 6). The catholyte chamber consisted
also developed as anodes for generations of new classes of miniaturized enzymatic cells.25,26 Among oxidase enzymes, glucose oxidase (GOx) has received special attention as an “ideal” enzyme because of its high specific activity, low cost, and reliability.27 These features are responsible for the leading role of GOx in glucose biosensing28 and fuel cells.25,29−31 GOx is a globular dimeric protein extracted from the fungi Aspergillus and Penicillium species, which catalyzes the oxidation of glucose into Dglucono-1,5-lactone, using molecular oxygen as an electron acceptor. It contains two identical subunits, each of which folds in two domains: one binds to the substrate, whereas the other domain binds noncovalently to the flavin adenine dinucleotide (FAD) redox center. The active site of GOx contains important amino acids involved in catalysis: His516 and Glu412, which is hydrogen-bonded to His559, deeply buried below the protein surface.32,33 Hence, GOx is in principle incapable of direct electron transfer to an electrode, as the redox center is shielded from the conductive support by the electrically insulating GOx matrix. Redox mediators34,35 and plugging metal/carbonaceous connectors into GOx have allowed electrical contact between GOx and electrodes.36−38 However, enzyme confinement in a matrix does not necessarily stabilize its structure. The immobilization procedure inevitably alters the enzyme active site microenvironment, potentially leading to denaturation/ unfolding processes and reduction of catalytic activity, due to conformational restraints and mass transfer limitations.39 Nafion has been shown a suitable matrix for GOx immobilization, with its microstructure affecting enzyme activity and stability.40 The balance of hydrophobic/hydrophilic domains is a key feature to achieve optimal microenvironments for enzyme immobilization and to tailor charge transfer and mass transport effects. In a previous work, we immobilized GOx on a carbon cloth electrode modified by a Nafion matrix.41 A comprehensive spectroscopic and electrochemical characterization of the electrode indicated that the immobilization procedure of GOx strongly affected its catalytic activity. We have now extended our studies to the investigation of conformational equilibrium of GOx in different physicochemical environments, using circular dichroism spectroscopy to verify the correlation between secondary structure and activity, and steady-state fluorescence of tryptophan and FAD to understand changes at the active site. The bioelectrocatalytic activity of immobilized GOx toward glucose oxidation was also investigated, providing the basis for developing an electrode/ enzyme interface specifically adapted to EFC applications.
2. EXPERIMENTAL SECTION 2.1. Materials. Glucose oxidase (GOx, EC 1.1.3.4, type X-S from Aspergillus niger), Nafionperfluorinated resin (5 wt % solution in a mixture of lower aliphatic alcohols and water), 2,2′-azino-bis-3ethylbenzthiazoline-6-sulfonic acid (ABTS), horseradish peroxidase (HRP, EC 1.11.1.7, type II), β-D-glucose, poly(tetrafluoroethylene) (PTFE, 60 wt % dispersed in water), and hydroquinone (HQ) were purchased from Sigma-Aldrich (St. Louis, MO). Vulcan XC72R was purchased from Cabot Corporation (Boston, MA), Platinum 10% on Vulcan (C-10-Pt) and plain carbon cloth CC-G-5N with PTFE were purchased from QuinTech (Göppingen, Germany). Electrochemical and spectroscopic measurements were carried out in 50 mM sodium phosphate, sodium borate, sodium acetate buffer (PBA), in the pH range between 2 and 12. 2.2. GOx Activity Assay. The activity of free GOx in solution was measured by a coupled enzyme assay, in which GOx oxidizes beta-Dglucose with production of hydrogen peroxide that reacts with a
Figure 1. Schematic diagram of the enzyme fuel cell. B
DOI: 10.1021/acsami.5b08610 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces of a platinum-modified carbon cloth electrode (CC_Pt), prepared according to Iannaci,46 in contact with PBA pH 6 solution. Four cell configurations were used: (a) double chamber (DC) EFC with nitrogen-saturated anolyte and oxygen-saturated catholyte; (b) DC EFC with oxygen-saturated catholyte; (c) DC EFC without gas flow at the anolyte and catholyte sides; (d) single chamber EFC (obtained by removing the Nafion membrane) without gas flow. The electrodes were connected to a VMP3Multi-Channel potentiostat (Bio-Logic Science Instruments SAS, Claix, France) and the voltage under open circuit conditions (OCV) was recorded and allowed to reach stable values for at least 30 min. Polarization curves were obtained by linearly sweeping the voltage from OCV to 0.005 V at 1 mV·s−1. Electrochemical impedance spectroscopy (EIS) measurements were performed during the cell functioning, in potentiostatic mode (0.15 V). An AC sinusoidal perturbation (amplitude 10 mV) was introduced to the DC load, and impedance spectra were acquired by sweeping frequencies from 5 mHz to 100 kHz, with 10 points per decade being recorded. All measurements were performed at 28 °C.
3. RESULTS AND DISCUSSION 3.1. Enhanced Bioelectrocatalytic Activity of GOx Immobilized on Nafion. We previously demonstrated Nafion-induced stabilization of the secondary structure of GOx at pH 7.4 by means of a combined spectroscopic and electrochemical study. CD features indicated that the immobilization of GOx on an electrode surface through Nafion matrix allowed to preserve GOx active site while decreasing the content of unordered structural conformations vs free GOx.41 To investigate whether this effect also occurs under other pH conditions, here we performed a systematic study of GOx conformation either in the presence or in the absence of Nafion for the pH range between 2 and 11. The effect from Nafion addition is readily seen by comparing the CD spectra in Figure 2A (GOx/PBA) and B (GOx/PBA/Nafion). The data can be interpreted by assuming four GOx conformations depending on the pH, with the native structure being better preserved in the pH range from 5 to 8, both for GOx/PBA and GOx/PBA/ Nafion samples. The pH dependence was similar in the two cases, but the contents of regular structures (i.e., α-helices and β-sheets) increased in the presence of Nafion, as indicated in the percentages of each conformation extracted by deconvolution of the spectra (see below). Important differences to be noted between panels A and B in Figure 2, which point to increasing amounts of regular structures in the presence of Nafion, are as follows. At pH 5 (acidic-neutral region), the negative broad band at 210 nm for GOx/PBA in Figure 2A became better defined as two negative bands around 210 and 220 nm in the presence of Nafion, which also displayed a positive peak at 199 nm in Figure 2B. At pH 7, a positive peak appears upon Nafion addition, which is absent in Figure 2A. The two negative bands at 210 and 222 nm are more pronounced in Figure 2B, suggesting an increase in the number of helical structures. As expected, GOx loses part of its regular contents at pH 10, which is indicated by the disappearance of the positive peak and the shift in the negative band from 210 to 204 nm in Figure 2A. In the presence of Nafion, there appears to be more preservation of regular structures since the negative band is still seen at 210 nm. Considerable amounts of regular structures are observed at pH 2, in spite of this extreme acid condition, for both GOx/PBA and GOx/PBA/Nafion samples, though their spectra differ. A more quantitative analysis was carried out by comparing the percentages of the different structures, obtained by deconvolution of the CD spectra, as shown in Tables 1 and
Figure 2. (A) CD spectra of GOx in PBA at pH 2, 5, 6, 7, 10. (B) CD spectra of GOx in PBA buffer with Nafion addition at selected pH values.
2 for GOx/PBA and GOx/PBA/Nafion, respectively. A detailed description of the spectra, particularly with regard to the pH dependence, is given in the Supporting Information. Table 1. CD Spectra Deconvolution of GOx in PBA Buffer at Various pH Values, from 2 to 11 pH 2 3 4 5 6 7 8 9 10 11
helix (%)a β (%)b turn (%) 19 19 12 11 16 17 17 17 10 09
19 21 35 28 28 27 28 26 28 29
23 23 23 25 23 22 22 22 26 25
unordered (%)
RMSD (Exp-Calc)
39 39 30 36 33 34 33 33 36 36
2 5 5 2 4 2 2 2 3 5
a
The values for helix include regular and distorted helices. bThe values for β include parallel and antiparallel β sheets.
The normalized quantum efficiency for the fluorescence emission spectra of GOx in Figure 3 also indicates a pH dependence for the vicinity of the tryptophan (λexcitation = 295 nm) and FAD (λexcitation = 375 and 450 nm) fluorophores. Tryptophans are in average partially exposed to the solvent at pH values from 2 to 10, with λmax varying from 336 to 340 nm, consistent with their position in the crystallographic structure described by Wohlfahrt and co-workers.47 Only at extremely basic pH values (11−12) the tryptophans are considerably exposed to the solvent, as denoted by the red shift in emission to 344 nm, in comparison with λmax = 337 in the neutral region. This red shift is compatible with a partial or initial GOx C
DOI: 10.1021/acsami.5b08610 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces Table 2. CD Spectra Deconvolution of GOx in PBA Buffer with Nafion Addition at Different pH Values from 2 to 11 pH 2 3 4 5 6 7 8 9 10 11
helix (%)a β (%)b turn (%) 11 12 36 9 20 21 18 18 7 7
35 30 15 34 32 34 30 31 31 23
25 28 22 24 22 22 22 22 25 29
unordered (%)
RMSD (Exp-Calc)
29 30 27 33 26 22 30 30 37 41
