Electrochemical behavior of ascorbate oxidase immobilized on graphite electrode modified with Au-nanoparticles

Materials Science and Engineering B 178 (2013) 1497–1502

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Electrochemical behavior of ascorbate oxidase immobilized on graphite electrode modified with Au-nanoparticles Totka Dodevska a,∗ , Elena Horozova b , Nina Dimcheva b a b

Department Inorganic and Physical Chemistry, University of Food Technologies, 26, Maritsa Boulevard, Plovdiv 4002, Bulgaria Department Physical Chemistry, Plovdiv University, 24, Tsar Assen Street, Plovdiv 4000, Bulgaria

a r t i c l e

i n f o

Article history: Received 24 April 2013 Received in revised form 17 August 2013 Accepted 20 August 2013 Available online 3 September 2013 Keywords: Ascorbate oxidase Direct electron transfer (DET) Modified graphite electrodes l-ascorbic acid

a b s t r a c t Direct electrochemistry of ascorbate oxidase was observed when immobilized on graphite modified with nano-sized gold structures. Au-structures were electrodeposited onto the graphite surface by means of cyclic voltammetry, then the enzyme was chemisorbed onto their surface. The electron transfer between the enzyme active center and the modified electrode surface was probed by square wave voltammetry (SWV) and cyclic voltammetry (CV). The dependence of the current maxima on the scan rate was found linear, suggesting that the redox process is controlled by surface chemistry. Bioelectrocatalytic oxidation of the enzyme substrate l-ascorbic acid was explored by constant potential amperometry over the potential range from 200 to 350 mV (vs. Ag/AgCl, 3 M KCl) at the рНs 5.6 and 7.0. At a potential as low as 200 mV, pH 7.0 and temperature 25 ◦ C following operational parameters were determined for the enzyme electrode: a sensitivity: 1.54 ␮A mM−1 mm−2 (r2 = 0.995 ), linear dynamic range up to 3.3 mМ, detection limit of 1.5 ␮М, response time up to 20 s. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The phenomenon of direct electron transfer (DET) is on the focus of research in recent years in the field of biosensor technology, since DET between the immobilized redox-active biomolecules and the electrode surface is a prerequisite for the development of third generation biosensors [1–3]. Direct electron transfer between the active site and the electrode surface has, so far, been proven for several oxidoreductases. With the participation of the ascorbate oxidase (AOx), a representative of the group of multicopper oxidases, biosensor systems for ascorbic acid of first generation have been developed [4–11]. At the same time, however, there are a limited number of publications for achieved effective DET with this enzyme [12–17]. The first publication describing DET-reactions of AOx dates back to 1996 [12]. Voltammetric studies of AOx at a gold electrode modified with thiols showed values of the formal redox potential, which the authors assigned to T1 copper site of the enzyme. Despite the presence of three redox copper centers (T1, T2 and T3) in the structure of this enzyme, and in subsequent reports only a single redox response, related to T1 copper site was detected in the cathodic and anodic waves of AOx [14,15]. The presence of three clearly defined pairs of anodic and cathodic peaks, relating to T1 and the trinuclear

∗ Corresponding author. Tel.: +359 32 603 679; fax: +359 32 644 102. E-mail address: [email protected] (T. Dodevska). 0921-5107/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mseb.2013.08.012

copper centers (T2, T3) of the AOx were observed by Ivnitski et al. [16] on a screen-printed carbon electrode. Effective enzyme-electrode communication was achieved by including AOx in different types of polymeric films, covering the surface of the basic Au-electrode – [13–15]; or binding AOx with a bifunctional reagent such as glutaraldehyde to the surface of screen-printed carbon electrode [16]; as well as entrapment of the enzyme in a polymer–MWCNTs composite deposited on the surface of a Pt disk electrode [17]. In designing third generation biosensor systems, both the method of enzyme immobilization and the nature and surface condition of the electrode are of key importance. The electrode should provide a stable surface for the immobilization of the enzyme molecules while retaining their activity, as well as provide an electron-conducting pathway for transferring electrons between the biomolecules’ prosthetic groups of and the electrode surface. In this connection, the present work deals with the studies on the electrochemical behavior of AOx immobilized on graphite electrode modified with Au-nanodeposits; and the characterization of the produced enzyme electrode with respect to quantitative amperometric determination of l-ascorbic acid at low applied potentials. Electrodeposition was chosen as an attractive, cheap method for modifying electrode materials, which does not require expensive equipment, and which allows experimental control of the size and the surface morphology of the deposited metal particles.

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2. Experimental 2.1. Materials Ascorbate oxidase (AOx) (E.C. 1.10.3.3) from Cucurbita sp. (Fluka) with homogeneous activity 268 U mg−1 (1 U oxidizes 1.0 ␮mol of l-ascorbate to dehydroascorbate per min at pH 5.6 at 25 ◦ C). l-ascorbic acid (Fluka), Na2 HPO4 and NaH2 PO4 (Sigma-Aldrich), HAuCl4 .H2 O (Acros) were of analytical grade and used as received. 0.1 M buffer solutions were prepared with monobasic and dibasic sodium phosphates dissolved in double distilled water with pHs 5.6 and 7.0, adjusted with a pH meter pH 211 (Hanna Instruments, USA). The working electrode was disk from spectroscopic graphite with diameter of the working surface d = 5.6 mm and visible surface area of ca. 25 mm2 (RWO, Ringsdorf, Germany). 2.2. Apparatus and measurements All electrochemical experiments were performed using computer controlled electrochemical workstation PalmSens (Palm Instruments BV, The Nederland) with a non-compartmentalized three-electrode cell (working volume 10–15 mL). A Ag/AgCl (3 M KCl) was used as a reference electrode, and a platinum wire as an auxiliary electrode. Cyclic voltammograms (CV) were recorded at scan rates from 1 to 10 mV s−1 . Parameters used in square wave voltammetry (SWV) were as follows: step potential of 5 mV, amplitude of 30 mV, at a frequency 1 Hz. Peak intensities of SWV and CVs were reported with baseline correction. The amperometric experiments (calibrations) were performed by successive addition of aliquots of 10−2 M ascorbic acid solution to 0.1 M sodium phosphate buffer (pH 5.6 or 7.0) in the cell (10 mL initial volume) with simultaneous registration of the current at a constant potential. The solution was stirred at 460 rpm during the measurements. The 10−2 M ascorbic acid stock solution was freshly prepared before each measurement. All of the results reported here were obtained as an average of at least three independent measurements. The surface morphology of the modified electrode was examined with scanning electron microscope JEOL JSM-5500. No conductive coatings or other treatment were performed on the sample prior to SEM observations. 2.3. Preparation of the modified electrode Before modification, the graphite electrode was carefully polished to mirror-like finish with emery paper with decreasing particle size (P800, P1200 and P2000), rinsed with double distilled water, sonicated in water for 3 min. Several different procedures for electrodeposition of Aunanostructures on polished and cleaned graphite were tested. The metal particles were grown onto the graphite electrode surface by electroreduction of tetrachloroaurate ion electrolyte containing 2% HAuCl4 , dissolved in 0.1 M HCl, using following electrochemical techniques: electrodepositon at constant potential with durations from 1 to 10 s [18] and electrodeposition under potentiodynamic conditions [19]. Each one of the so obtained modified electrodes was examined by SEM and the images indicate that the surfaces of the electrodes are populated with Au-nanostructures, but they drastically differ both in shape and the size of the deposits. Their sizes, shapes, structural features and population on the graphite surface are strictly specific for each method of electrodeposition. Only one particular type of graphite modification yielded electrochemically and enzymatically active AOx, when immobilized on the surface of the modified graphite, namely: the electrodeposition

of gold structures onto the clean working surface of the graphite electrode by means of CV at a scan rate 0.1 V s−1 from electrolyte containing 2% HAuCl4 , dissolved in 0.1 M HCl. The electrode surface was seeded with gold when starting the cycle at −0.6 V, which is below the potential where the electroreduction of Au3+ on the graphite starts (i.e. −0.35 V, determined by CV) then the scan goes up to 0 and then back to −0.6 V. 2.4. Enzyme immobilization The electrochemical treatment, preceding the immobilization of AOx consisted of continuous cycling (CV, scan rate 0.1 V s−1 ) over the potential range from 0 to 1.7 V (vs. Ag/AgCl, 3 M KCl) in 2 M H2 SO4 for at least 10–20 cycles, followed by water-rinsing of the electrode. The adsorption of AOx was carried out under static conditions by immersing the modified electrode in solution containing 5 mg mL−1 AOx dissolved in 0.1 M sodium phosphate buffer, pH 7.0. The duration of the adsorption process was 1 or 12 hours at temperature 20 or 4 ◦ C. After completing the measurements the enzyme electrode was carefully washed with doubly distilled water and stored in 0.1 M sodium phosphate buffer pH 7.0 at temperature 4 ◦ C until next measurements. 3. Results and discussion 3.1. Electrochemical behavior of AOx immobilized on modified graphite The morphology of the gold deposits on the surface of modified graphite electrode was examined by scanning electron microscopy (SEM; Fig. 1). The deposited metal phase is not evenly distributed on the carrier. The shape and dimensions of the deposits vary to a large extent; the surface is chaotically scattered with oval formations which size reaches up to 700 nm. A large number of single fine ‘pearls’, with a size of 20–50 nm are also visible on the SEM image, and they cover a large part of the electrode surface. The presence of differentiated, separate massive, solid metal islands, with sharpened edges, is also visible. In the periphery of the bigger islands, granular groups are also identified, which evidences a complicated, multilayer deposition of the metal phase in those areas of the matrix. The presence of different forms of Au deposited on the graphite carrier is confirmed by the cyclic voltammogram, recorded in 2 M H2 SO4 (Fig. 2). Both anodic and cathodic maxima are observable on the voltammetric curves, however they result not from redox transformations taking place on the electrode surface, but are rather due to the adsorption/desorption of surface bond oxygen. Most probably, the three separate peaks on the cathodic course of the curve (at ca. 1.42 V, 0.96 V and 0.43 V), result from the oxygen desorption of three distinct Au–O types, differing in binding energies: the first two being assigned to the strongly chemisorbed O2 , while the third one relates to the loosely-bond oxygen. Two enzyme electrodes, produced on the basis of the modified graphite by 1 h’s adsorption of AOx at ambient temperature and temperature of 4 ◦ C, respectively, were screened for electrochemical activity by square wave voltammetry (Fig. 3). The immobilized enzyme has been found electrochemically active – a current peak is registered at potential 355 mV. Moreover the peak intensity is much higher when the adsorption of AOx is carried out at the temperature of 4 ◦ C (Fig. 3, curve 2). This fact evidences that on the surface of the modified electrode there is adsorption of protein macromolecules oriented in such a way as to be suitable for accomplishing electron communication between the redox center and the electrode surface.

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1499

270 240

Current (µA)

210 180

2

150 120

1

90 -0,25

0,00

0,25

0,50

0,75

1,00

Potential (V) Fig. 3. Square wave voltammograms of AOx adsorbed on modified with Au-deposits graphite. Supporting electrolyte: 0.1 M sodium phosphate buffer pH 5.6; adsorption of AOx 1 h: at ambient temperature (curve 1), at 4 ◦ C (curve 2); reference electrode Ag/AgCl, 3 M KCl.

Fig. 1. SEM images of spectroscopic graphite modified with Au-deposits by cyclic voltammetry (1 cycle from −0.6 to 0 V at a rate of 0.1 V s−1 ) at a different magnification: (a) 5000× and (b) 20,000×.

For the same enzyme electrode on the cyclic voltammogram, at scan rate of 5 mV s−1 a pair of clearly defined redox-peaks are established (Fig. 4). The anodic (Ep,a ) and the cathodic (Ep,c ) peaks are located at 430 and 240 mV, respectively. The value of the formal redox potential Eо  = (Ep,a + Ep,c )/2 = 335 mV is in very good agreement with Ep , obtained by SWV. The observed independent electrochemical activity of the redox co-factor, integrated into the active center of the enzyme molecule, in the absence of enzyme substrate, is a direct proof that an

electron exchange with the electrode surface takes place [2]. I–E curve is registered reproducibly, which evidences that AOx is irreversibly adsorbed on the Au-modified graphite electrode. About this process contributes the strong chemical bond between superficial sulphur-containing groups of the enzyme protein shell and the gold nanoparticles, which takes place during the immobilization of the enzyme on the gold plated graphite electrode. By varying the duration of the adsorption of AOx the optimal conditions for enzyme immobilization were determined. In all further studies the process was carried out for 12 hours in refrigerator at 4 ◦ C – conditions not only facilitating the sorption process, but also assuring the retention of the catalytic activity of the enzyme. The direct electrochemistry of AOx, immobilized on modified electrode was explored by cyclic voltammetry. Fig. 5 shows the CVs of the enzyme electrode recorded at different scan rates. When the scan rate increases from 1 to 5 mV s−1 , a shift to the anodic peak potential in a positive direction is observed, and of the cathodic peak – in a negative direction, respectively. The dependence of the peak currents on the scan rate is linear (Fig. 5b), which evidences a redox process controlled by surface chemistry. The peak-to-peak

200 100 Current (µA)

Current (mA)

15

10

5

0 -100 -200

0

-300

-5 0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

Potential (V) Fig. 2. Cyclic voltammogram of modified with Au-deposits graphite; supporting electrolyte: 2 M H2SO4; scan rate 0.1 V s−1 ; reference electrode Ag/AgCl, 3 M KCl.

0,0

0,2

0,4

0,6

0,8

Potential (V) Fig. 4. Cyclic voltammograms of AOx adsorbed on modified graphite electrode; 1 h adsorption of AOx at 4 ◦ C; 0.1 M sodium phosphate buffer pH 5.6; scan rate 5 mV s−1 ; reference electrode Ag/AgCl, 3 M KCl.

1500

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(a)

400

Current (µA)

200

0

-200

-400 -0,2

0,0

0,2

0,4

0,6

0,8

1,0

Potential (V)

(b)

100

Fig. 6. Steady-state response of the enzyme electrode based on Au-modified graphite as a function of l-ascorbic acid concentration at applied potential of 200 mV (vs. Ag/AgCl, 3 M KCl); stirring rate: 460 rpm; initial working volume: 10 ml; supporting electrolyte: 0.1 M sodium phosphate buffer, pH 7.0; temperature 25 ◦ C. Inset: authentic record of the current change upon addition of l-ascorbic acid aliquots.

i p,a (µA)

80

60

40

20

0

2

4

6

8

10

-1

Scan rate (mV s ) Fig. 5. (a) Cyclic voltammograms of AOx adsorbed on modified graphite; 0.1 M sodium phosphate buffer pH 7.0 at scan rates from 1 to 5 mV s−1 (from inner to outer). (b) Plot of the anodic peak currents vs. scan rate.

separation E < 200 mV suggests a quasi-reversible electrochemistry of the enzyme. 3.2. Bioelectrocatalytic oxidation of l-ascorbic acid The enzyme electrode based on the modified with Au nanoparticles graphite electrode was tested for amperometric detection of l-ascorbic acid at low working potentials (200–350 mV). The concentration dependence of the amperometric response of the enzyme electrode was investigated with constant potential amperometry over the potential range from 200 to 350 mV in 0.1 M phosphate buffer solution at the pH-values 5.6 and 7.0. The chronoamperometric records indicate that the oxidative current increased stepwise upon introducing in the buffer aliquots of the substrate stock solution, rapidly reaching a steady-state current in less than 20 s (Fig. 6). Calibration plots of the enzyme electrode build on the basis of chronoamperometric measurements at different constant potentials (Fig. 7a) clearly show that at pH 7.0, as the applied potential increases to 300 mV, the electrode sensitivity (determined as the slope of the linear section of the calibration graph) also grows. At the same time, the portion of the strict linear concentration dependence of the signal remains unchanged – up to 3.3 mM for the studied potential range. The calibration plots follow a hyperbolic trend; the ‘plateau’ observed in the dependence Is = f (C) at high substrate concentrations is typical for the kinetic model of Michaelis–Menten. The apparent Michaelis constant was

Fig. 7. (a) Steady-state response of the enzyme electrode based on modified electrode as a function of l-ascorbic acid concentration at different applied potentials (vs. Ag/AgCl, 3 M KCl); stirring rate: 460 rpm; initial working volume: 10 ml; supporting electrolyte: 0.1 M sodium phosphate buffer, pH 7.0; temperature 25 ◦ C. (b) Lineweaver–Burk plot based on the data of panel (a); potential 300 mV.

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Table 1 Operational parameters of ascorbate oxidase enzyme electrode, based on modified with Au-deposits spectroscopic graphite; potential range 200–350 mV (vs. Ag/AgCl, 3 M KCl); background electrolyte 0.1 M sodium phosphate buffer; temperature 25 ◦ C. Potential (mV)

pH

Sensitivitya (␮A mM−1 mm−2 )

r2

Linearityb (mM)

200

5.6 7.0 5.6 7.0 5.6 7.0 7.0

1.38 1.54 1.73 1.84 1.74 1.97 1.96

0.993 0.995 0.990 0.999 0.996 0.997 0.999

3.94 3.33 3.94 3.33 4.44 3.33 4

250 300 350 a b

app

KM

(mM)

2.74 2 16.8 18.8 22.5

The electrode sensitivity was determined as the slope of the linear portion of the calibration graph. The linear portion of the calibration graph (in mM).

Table 2 Comparison of the analytical characteristics of amperometric biosensors for ascorbic acid reported in the literature with the achieved in the present work. Support for immobilization a

c-MWCNT/PANI Egg shell membrane PEDOT/MWCNTsb PEDOT/SWCNTs Polyaniline film Au/graphite a b

Working potential 0.6 V (vs. Ag/AgCl) 0.6 V (vs. Ag/AgCl) 0.4 V (vs. SCE) 0.4 V (vs. SCE) 0.4 V (vs. SCE) 0.2 V (vs. Ag/AgCl)

Linearity range (M) −6

−4

2 × 10 –2.06 × 10 1 × 10−5 –4 × 10−4 0.5 × 10−4 –2 × 10−2 1 × 10−6 –1.8 × 10−4 1 × 10−5 –1 × 10−4 2 × 10−6 –3.3 × 10−3

Detection limit (␮M)

Response time (s)

Ref.

0.9 10 15 0.7 – 1.5

2 10 20 10 120 20

[5] [6] [17] [7] [16] Present work

Carboxylated multiwalled carbon nanotubes/polyaniline (c-MWCNT/PANI). Poly(3,4-ethylenedioxythiophene) (PEDOT)/multiwalled carbon nanotubes (MWCNTs) composite film.

calculated in accordance with the electrochemical version of the equation of Lineweaver–Burk: 1 = Is



app

KM

Imax

  1 C

+

I , Imax

where Is is the steady-state current after the substrate is added; C is the concentration of l-ascorbic acid in the working volume; Imax is the maximum enzyme electrode current (in conditions of substrate saturation). The calculated operational parameters for bioelectrocalatytic oxidation of l-ascorbic acid (electrode sensitivity, linear range, apparent Michaelis constant) are presented in Table 1. In the studied potential range, the apparent Michaelis constant has values higher than the one corresponding to the native enzyme (KM = 0.25 mM). This effect is expected in view of the conformation changes occurring during the adsorption of the protein molecule. Thus, at a potential of 300 mV, at which the highest electrode sensitivity is registered (1.97 ␮A mM−1 mm−2 ), the apparent Michaelis app constant KM = 18.8 mM (estimated from Fig. 7b); detection limit of the substrate–1.5 ␮M at a signal/noise ratio 3:1. At pH 5.6, the enzyme electrode operated with a sensitivity which is by 6–10% lower, but with a linearity of the signal spanning over a considerably longer concentration interval. The linearity range expands by 1/3 as the applied potential rises from 200 to 250 mV. A further increase of the potential to 300 mV, does not increase significantly the electrode sensitivity, and its value is very close to the one determined at 250 mV. Under the discussed experimental conditions, an assessment of the reproducibility of the signal was also made – the electrode response give a RSD of 4.7% in 4 consecutive tests in the presence of 100 ␮M ascorbic acid at 250 mV, pH 5.6. The operational stability of the enzyme electrode was tested over a time interval of 8 hours by calibrating it 6 consecutive times. No apparent decrease of the electrode sensitivity was noticed. However after 24 h storage in the refrigerator (4 ◦ C, in 0.1 M buffer, pH = 7.0) less than 50% of the initially registered electrode response to 100 ␮M l-ascorbate was achieved. Constant-potential amperometric measurements of the enzyme electrode response upon addition of the substrate l-ascorbic acid have shown that a temperature increase from 20 to 25 ◦ C gives a rise

of the electrode sensitivity by 10%, however a further temperature increase above 30–35 ◦ C did not result in a substantial improvement of the sensitivity. The last finding could be due to the higher rate of a purely chemical oxidation of the l-ascorbic acid, but also could be a result from the decreased oxygen solubility in the buffer at the elevated temperature. The advantages of the enzyme electrode here reported can be appreciated and assessed by comparative study with other amperometric biosensors for ascorbic acid (presented in Table 2). The analytical system is characterized by a relatively simple preparation, low working potential, fast response, wide linear dynamic range and very low detection limit of the analyte. The sensitivity of detection (1.54 ␮A mM−1 mm−2 ), is also a significant advantage, as it is about 5 times as high as the one achieved by Liu et al. by means of two types of electrodes with a complex sandwichtype architecture: PEDOT/MWCNTs/AscOx – 23.95 mA M−1 cm−2 [17] and PEDOT/SWCNTs/AscOx/Nafion – 28.5 mA M−1 cm−2 [10], obtained at a much higher operating potential (+400 mV vs. SCE). 4. Conclusions With the here reported method for electrodeposition of gold by cyclic voltammetry (1 cycle from −0.6 to 0 V (vs. Ag/AgCl, 3 M KCl) at a rate of 100 mV s−1 ) are obtained gold nano- and microstructures on the graphite, which provides a favorable for DET orientation of the enzyme AOx upon its chemisorption. The observed efficient DET between active site of immobilized AOx and the surface of modified graphite electrode is controlled by surface chemistry. For the bioelectrocatalytic oxidation of the enzyme substrate l-ascorbic acid, carried out at pH 7.0, 25 ◦ C and at a potential of 200 mV (vs. Ag/AgCl, 3 M KCl) following operational parameters were determined for the enzyme electrode: • • • •

sensitivity: 1.54 ␮A mM−1 mm−2 (r2 = 0.995 ); linear dynamic range: up to 3.3 mМ; detection limit: 1.5 ␮М (signal/noise 3:1); and response time: up to 20 s.

The results presented in this study, including the original approach for obtaining effectively structured gold deposition on

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the graphite surface and the operational parameters of the enzyme electrode, would be of interest for the biosensor technologies and the other aspects of applied bioelectrochemistry. Acknowledgements Authors acknowledge the support from the Bulgarian National Science Fund (grant DDVU-02/38), University of Food Technologies Research Fund (grant 5/12 – N) and the help of the student Boris Dzhuvinov (Computer Science in Plovdiv University). References [1] W. Zhang, G. Li, Analytical Science 20 (2004) 603–609. [2] S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A. Yaropolov, J. Whittaker, L. Gorton, Biosensors and Bioelectronics 20 (2005) 2517–2554. [3] Y. Wu, S. Hu, Microchimica Acta 159 (2007) 1–17. [4] E. Akyilmaz, E. Dinc¸kaya, Talanta 50 (1999) 87–93. [5] E. Turkusic, V. Milicevic, H. Tahmiscija, M. Vehabovic, S. Basic, V. Amidzic, Fresenius Journal of Analytical Chemistry 368 (2000) 466–470.

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