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Immobilisation of glucose oxidase in electrodeposited copper
Electrochemistry Communications 8 (2006) 450–454 www.elsevier.com/locate/elecom
Immobilisation of glucose oxidase in electrodeposited copper Carlos Palma Remirez, Daren J. Caruana
*
Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK Received 2 December 2005; received in revised form 20 December 2005; accepted 20 December 2005 Available online 8 February 2006
Abstract A metallic enzyme electrode responsive to glucose based on the electrodeposition of copper in the presence of glucose oxidase (GOx) is described. Generally, enzymes are entrapped in ‘soft’ porous polymeric matrices which allow the penetration of substrate to, and product away from the enzyme layer. We have determined the conditions for the incorporation of GOx into a metallic copper film. The enzyme remains active within the metal film, as indicated by the amperometric measurement of hydrogen peroxide produced by the enzyme in the presence of glucose in aerobic conditions. The magnitude of the current response to glucose of the enzyme electrode was greatly improved by the presence of crystal violet in the deposition solution. It is proposed that the presence of crystal violet in the deposition solution changes the porosity of the bulk metal deposit, allowing the penetration of glucose and oxygen into the enzyme-loaded metallic film. It was found that the optimum concentration of crystal violet was exactly half that of Cu2+ in the deposition solution. The glucose response was dependent on the enzyme concentration in the deposition solution and the presence of oxygen. 2006 Published by Elsevier B.V. Keywords: Glucose oxidase; Enzyme electrodes; Biosensor; Co-electrodeposition; Crystal violet
1. Introduction A large number of matrices have been shown to be suitable for the entrapment of biological molecules. Electrochemical co-deposition of matrix and enzyme is a convenient single step method that is fast and well controlled [1]. There are many examples of electropolymerisation of conducting [2] and non-conducting [3] polymers in the presence of an enzyme. These methods produce ‘soft’ matrices which are delicate and easily degraded; however, in many cases they contribute to the stability of the enzyme. In this paper, we have focused on developing a method of entrapping an enzyme within the electrochemically deposited copper films, which retains the enzymatic activity. The co-deposition of metallic matrices and enzymes has been described by several groups. Kim and Oh [4] deposited platinum black from a platinum salt to produce a high
*
Corresponding author. Tel.: +44 207 679 4527; fax: +44 207 679 7463. E-mail address: [email protected] (D.J. Caruana).
1388-2481/$ - see front matter 2006 Published by Elsevier B.V. doi:10.1016/j.elecom.2005.12.016
surface area deposit with immobilised GOx. The electrodeposition of iridium on glassy carbon also in the presence of GOx was presented by Rodriguez and Rivas [5]. In both these reports, the amperometric response to glucose was the measured electrochemical response of the hydrogen peroxide produced by the enzyme. We describe the electrodeposition of copper on gold with preadsorbed glucose oxidase. We found that crystal violet (hexamethylpararosaniline chloride) was essential to measure a significant response to glucose. Dakkouri et al. [6] and Haiss et al. [7] showed that crystal violet had changed the morphology of the deposited copper. In the absence of crystal violet, the growth was three-dimensional with the formation of copper crystallites. In the presence of crystal violet the growth of crystallites perpendicular to the surface was reduced; the so called rim effect. The crystallites spread laterally, following a quasi-two-dimensional growth mechanism, and this leads to a relatively uniform coverage of the surface. The Dakkouri et al. study was concerned with thin layers of copper; however, in this study we are interested in changing the properties of the bulk deposit to produce a matrix capable
C.P. Remirez, D.J. Caruana / Electrochemistry Communications 8 (2006) 450–454
of entrapping and retaining GOx. We describe a study to investigate the deposition of glucose oxidase within solid copper films in the presence of crystal violet. The motivation of this work is to produce solid metallic films, which are selective to chemical species and may be regenerated by polishing. 2. Experimental 2.1. Materials and instrumentation All solutions were prepared using deionised water (Millipore Milli-Q gradient, <0.05 S cm2). Salts and organic materials were of the highest purity and used as received. GOx was from Biozyme Laboratories, San Diego. Crystal violet and all other inorganic salts were of the highest purity grade from Sigma-Aldrich Co Ltd. A 3-electrode system was employed in these studies. Throughout the experiment, a saturated Ag/AgCl reference electrode was used; all potentials quoted in this paper are against this. A large surface area platinum wire was used as counter electrode. A gold rotating disc working electrode (0.783 cm2, Oxford Electrodes Ltd.) was polished with sandpaper and with Alumina powder (1.0 and 0.3 lm, Micropolish II, Buehler, Lake Bluff, IL) to produce a mirror finish. The electrodes were then thoroughly rinsed with deionised water. The electrode were cycled in sulphuric acid between 0.3 and +1.5 V for 30 cycles or until a stable cyclic voltammogram was obtained. The electrode were stored in water and used within an hour of finishing the cleaning procedure. All electrochemical measurements were made with a lAutolab Type II potentiostat (Windsor Scientific) connected to a personal computer (Dell). A rotator and controller (Oxford electrodes Ltd.) were used to control the rotation rate of the gold electrode. Data were interpreted and presented using SigmaPlot2001 (Version 7.0) software provided by SPSS Inc. Solution pH measurements were made using a model EIL 7045/46 pH meter (ABB KentTaylor Ltd).
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electrode was placed in a freshly cleaned cell containing (separate counter and reference electrodes to those used for the deposition) 25 ml of phosphate buffer solution (0.1 M, pH 7.0), and rotated at 9.0 Hz. All experiments were carried out at room temperature (23 ± 2 C) with an applied potential of 0.3 V. The background current was allowed to stabilise (no longer than 30 min), aliquots of 50 ll of glucose (1.0 M in phosphate buffer prepared at least 6 h before use) were added to the test solution to produce sequential increases of 2 mM in the concentration of glucose. The reproducibility of these experiments was improved by cleaning the electrodes in between experiments and taking care to avoid long exposure of the electrode surface to air. This was achieved by storing the electrode in phosphate buffer solution at all times. 3. Results and discussion Fig. 1 shows the response to glucose for an electrode deposited with copper from a solution containing GOx (3 mg ml1) and copper sulphate (2.5 mmol dm3) with sodium sulphate as background electrolyte. The current response when glucose was added to the buffered test solution was barely measurable and showed a small and steady decrease in current (reduction of hydrogen peroxide). The test was under steady state conditions held at 0.3 V to measure hydrogen peroxide produced from the enzymatic reaction. The response observed was probably from very small amount of glucose oxidase strongly adsorbed to the outside of the copper deposit. 3.1. Effect of crystal violet in the deposition solution on the glucose response The response time and magnitude were dramatically affected by the presence of crystal violet in the deposition -22x10 -6
2.2. Procedures
-24x10 -6
1st
2nd
3rd
4th
5th
Current / A
6th
Copper electrodeposition was carried out by a potential step to 0.35 V for a period of 600 s unless stated otherwise. A copper solution of 2.5 mM was prepared with cupric sulphate in 0.1 M sodium sulphate solution. Variable amounts of GOx and crystal violet were used in the deposition solution. The deposition solution was deoxygenated by purging with nitrogen for 30 min prior to deposition. The cell was then sealed while passing nitrogen over the surface of the solution, and the electrode was left to stand in the solution to facilitate adsorption of enzyme onto the electrode surface. After electrodeposition, the electrode was thoroughly washed by rotating (9.0 Hz) the electrode in phosphate buffer solution (0.1 M, pH 7.0) for 10 min to avoid contamination of the test solution with GOx. Once rinsed the
7th
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8th
9th
10th
-28x10 -6 -30x10 -6 -32x10 -6 -34x10 -6
1700
1800
1900
2000
2100
2200
2300
Time /s
Fig. 1. Current vs time trace from an electrode with copper (2.5 mM CuSO4) deposited in the presence of 3.0 mg cm3 GOx. Successive additions of 2.0 mM glucose. Deposition was carried out at 0.35 V vs saturated Ag/AgCl for 300 s. The testing was carried out in air saturated pH 7.04 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz.
C.P. Remirez, D.J. Caruana / Electrochemistry Communications 8 (2006) 450–454
solution. In the presence of crystal violet, the response to each addition of glucose is clearly distinguished in the current–time trace shown in Fig. 2(A). The current response measured was from the electrochemical reduction of hydrogen peroxide produced by the enzymatic reaction. In the absence of oxygen in the test solution, there was a significant decrease in the current signal, as shown in Fig. 2(B). Furthermore, in the absence of GOx in the deposition solution, there was no measurable change in current at all when glucose was added (not shown). Chronoamperometry was used for co-deposition of copper and GOx at 0.35 V in the presence of crystal violet. The influence of the electrodeposition time and potential was evaluated in a series of optimisation experiments, which will not be discussed here. From these experiments we determined that a deposition potential of 0.35 V, for 600 s, was found to yield reproducible coatings. Deposition by cycling potential was not successful. A maximum of 5 depositions was found to be possible from a freshly made solution. The reason for this was probably due to leaching of chloride from the reference electrode into the test solution, which led to the precipitation of CuCl at the electrode during the electroreduction. Crystal violet is an organic additive known to change the growth of copper deposit, which is known from Dakkouri et al.’s previous studies [6]. The reason for the change in the observed response was probably due to the direct effect of the crystal violet on the deposition of copper. We do not have any microstructure information on the resultant deposit, but it is certain that the porosity of these films is affected by the crystal violet which is necessary for the substrates (glucose and oxygen) to penetrate the film and react with the enzyme. Crystal violet concentration had a large effect on the response to glucose of the resultant copper layer. Fig. 3 shows the response to 20 mM glucose vs crystal violet con-10x10-6 -12x10-6
Current / A
-14x10-6 -16x10-6
B (B)
-18x10-6 -20x10-6
(A)
-22x10-6
A -24x10-6 1600
1800
2000
2200
2400
Time / s
Fig. 2. (A) Steady state current response to glucose vs time of an electrode with copper (2.5 mM CuSO4) deposited in the presence of GOx (3.0 mg cm3) with crystal violet (1.25 mM). Testing conditions are as stated in legend to Fig. 1. (B) The same conditions as (A), except the test buffer solution was deoxygenated for 20 min with oxygen free nitrogen prior to testing.
10x10-6
8x10-6
6x10-6
ibk-i / A
452
4x10-6
2x10-6
0 0.0
0.5
1.0
1.5
2.0
2.5
[Crystal Violet] / x 10 -3 mol dm-3
Fig. 3. Change in steady state current response glucose (25 mM) for different electrodes prepared from deposition solutions containing GOx (3.0 mg cm3) with copper sulphate concentration either 1.0 mM ( ) and () with different crystal violet concentrations ranging from 0.2 to 2.25 mM. Testing was performed in air saturated buffer pH 7.0 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz, only one addition of glucose was made to the solution to obtain the desired concentration of glucose.
centration for two concentrations of copper sulphate. There is clearly a peak of glucose response when the crystal violet concentration was close to half the copper concentration. This suggests that the mode of action of crystal violet may be due to homogeneous complex formation with copper. Copper–crystal violet complexes are known to form and used for the decolourisation of synthetic dyes [8]. However, the precise effect of crystal violet on the bulk copper deposit is unclear at this point, but we speculate that a change in the response to glucose is a direct effect on the porosity of the layer. As the concentration of crystal violet increases, the porosity increases which may result in the leaching out of the enzyme. The effect of the enzyme loading on the response to glucose was investigated over the range of 1.5–4.5 mg cm3 in the deposition solutions. Fig. 4 shows the change in current for three different concentrations of enzymes in the deposition solutions containing 2.5 and 1.25 mM of Cu2+ and crystal violet, respectively. The curves were fitted to a Michaelis–Menten type equation to determine the apparent Michaelis–Menten constants K 0M and Imax, which are given in the figure legend, for each concentration of GOx that was used. The low K 0M and Imax calculated for the electrode made with 1.5 mg cm3 GOx suggests that the enzyme loading in this layer was probably low, which was expected. Electrodes prepared with deposition solutions containing 3.0 and 4.5 mg cm3 GOx have similar values of K 0M but different Imax values. Imax decreases with increasing GOx concentration. At concentrations of substrate much higher than KM, the Michaelis–Menten equation reduces to kcateR, where kcat is the rate of the enzyme reaction 800 s1, [9] and eR is the total concentration of enzyme in the layer. At steady state the current density, iss, for the enzyme electrode may be given by [10]
C.P. Remirez, D.J. Caruana / Electrochemistry Communications 8 (2006) 450–454
453
9x10-6
10x10-6 3 mg/mL
8x10-6
8x10-6 4.5 mg/mL
7x10-6
4x10-6
1.5 mg/mL
ibk-i / A
ibk-i / A
6x10-6
6x10-6
2x10-6
5x10-6
0
4x10-6
0
5
10
15
20
25
[Glucose] / mmol dm-3
3x10-6 0
5
10
15
20
Time / Days
Fig. 4. Change in steady state current vs glucose concentration for electrodes prepared from deposition solutions containing copper (2.5 mM CuSO4), crystal violet (1.25 mM) and GOx at 1.5 (d, K 0M ¼ 16:75 mM, and Imax = 5.95 · 106 A), 3.0 (j, K 0M ¼ 4:91 mM, and Imax = 1.04 · 105 A) and 4.5 (N, K 0M ¼ 4:85 mM, and Imax = 9.01 · 106 A) mg cm3. Deposition was carried out at 0.35 V vs saturated Ag/AgCl for 300 s. The testing was carried out in air saturated pH 7.0 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz.
iss ¼ nFDs
ds ¼ nFk cat eR l; dx x¼l
ð1Þ
where n is the number of electrons transferred, F is the Faraday constant, Ds is the diffusion coefficient of the substrate, and l is the layer thickness. At concentrations of enzymes in the deposition solution higher than 3.0 mg cm3, the response does not remain dependent on the enzyme concentration, but falls. At high GOx concentrations, the integrity of the copper film could be affected. This behaviour has been described for other enzyme electrodes [11]. 3.2. Enzyme electrode stability The stability of the enzyme electrodes was investigated by creating an enzyme layer using 2.5 mM copper, 1.25 mM crystal violet and 3.0 mg cm3 GOx. The current response to 25 mM glucose was measured periodically for 20 days. The electrode was stored in pH 7.0 phosphate buffer at 4 C, when not in use. The electrode was allowed to warm up to room temperature for 20 min before testing. The glucose response vs time is shown in Fig. 5. The response appeared to drop significantly after the first day, then drops at a slower rate between 2 and 20 days. The long term stability is relatively good and comparable with enzymes immobilised in electropolymerised films [12]. 3.3. Measurement of copper quantity on electrode The quantity of copper deposited on the electrode was measured by a potential step to oxidative potentials (+0.8 V), which was found to be a suitable potential for anodic stripping of copper. The current from the chrono-
Fig. 5. Change in steady state current vs time in days in the presence of glucose (25 mM) for one electrode prepared from deposition solution containing glucose oxidase (3 mg cm3), copper sulphate (2.5 mM) with crystal violet (1.25 mM). Testing was performed in air saturated pH 7.0 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz, only one addition of glucose was made to the test solution to obtain the desired concentration of glucose. The electrode was stored in pH 7.0 buffer at 4 C.
amperometric trace was integrated, and corrected for non-Faradaic charge contribution to determine the charge for copper oxidation. It is possible to calculate the number of moles of copper, which have been deposited onto the electrode surface. The total charge from the oxidation for a freshly made electrode was 4.12 ± 0.73 · 104 C corresponding to 2.14 ± 0.37 · 109 M of copper. Interestingly, after the electrodes were tested with one single addition of 25 mM of glucose the quantity of copper had reduced to 0.70 ± 0.21 · 109 moles. The loss of copper from the electrode under test was significant. This loss of material from the layer was probably due to poor adherence of copper to the electrode surface. 4. Conclusion The present work demonstrates that the use of an electrodeposited copper film provides an effective technique for the immobilisation of the enzyme at an electrode surface. Using this method of immobilisation, we believe that we have enzymes entrapped within a porous bulk copper film. There are several variables which have been studied in this paper namely; electrodeposition methods, electrodeposition time, concentration of copper, concentration of enzyme and the effect of crystal violet. With this organic additive, the resulting films gave an improved current response in the presence of glucose. The molar ratio of 2:1, copper:crystal violet was important to produce good responsive films. The stability of the enzyme electrode was not impressive. However, we believe that incorporation of polyelectrolytes within the enzyme layer by co-electrodeposition will probably improve the long term stability [12].
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C.P. Remirez, D.J. Caruana / Electrochemistry Communications 8 (2006) 450–454
In conclusion, we have developed electrochemically a procedure involving the deposition of copper and GOx on a working electrode promoted by the presence of crystal violet.
[3] [4] [5] [6]
Acknowledgement
[7]
C.P. thanks the Socrates exchange programme for its support.
[8]
References
[9] [10] [11]
[1] S. Cosnier, Appl. Biochem. Biotech. 89 (2000) 127. [2] W. Schuhmann, Method. Biotech. 6 (1998) 143.
[12]
P.N. Bartlett, D.J. Caruana, Analyst 119 (1994) 175. C.S. Kim, S.M. Oh, Electrochem. Acta. 4 (1996) 2433. M.C. Rodriguez, G.A. Rivas, Electroanal. 11 (1999) 558. A.S. Dakkouri, N. Batina, D.M. Kolb, Electrochimica. Acta. 38 (1993) 2467. W. Haiss, D. Lackey, J.K. Sass, H. Meyer, R.J. Nichols, Chem. Phys. Lett. 200 (1992) 343. P. Verma, P. Baldrian, J. Gabriel, T. Trnka, F. Nerud, Chemosphere 57 (2004) 1207. B.E.P. Swoboda, V. Massey, J. Biol. Chem. 240 (1965) 2209. P.N. Bartlett, C.-S. Toh, in: J. Copper, A. Cass (Eds.), Biosensors, Oxford Univeristy Press, 2003. S. Arjsiriwat, M. Tanticharoen, K. Kirtikara, K. Aoki, M. Somasundrum, Electrochem. Commun. 2 (2000) 441. N.A. Chaniotakis, Anal. Bioanal. Chem. 378 (2004) 89.
Immobilisation of glucose oxidase in electrodeposited copper Carlos Palma Remirez, Daren J. Caruana
*
Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK Received 2 December 2005; received in revised form 20 December 2005; accepted 20 December 2005 Available online 8 February 2006
Abstract A metallic enzyme electrode responsive to glucose based on the electrodeposition of copper in the presence of glucose oxidase (GOx) is described. Generally, enzymes are entrapped in ‘soft’ porous polymeric matrices which allow the penetration of substrate to, and product away from the enzyme layer. We have determined the conditions for the incorporation of GOx into a metallic copper film. The enzyme remains active within the metal film, as indicated by the amperometric measurement of hydrogen peroxide produced by the enzyme in the presence of glucose in aerobic conditions. The magnitude of the current response to glucose of the enzyme electrode was greatly improved by the presence of crystal violet in the deposition solution. It is proposed that the presence of crystal violet in the deposition solution changes the porosity of the bulk metal deposit, allowing the penetration of glucose and oxygen into the enzyme-loaded metallic film. It was found that the optimum concentration of crystal violet was exactly half that of Cu2+ in the deposition solution. The glucose response was dependent on the enzyme concentration in the deposition solution and the presence of oxygen. 2006 Published by Elsevier B.V. Keywords: Glucose oxidase; Enzyme electrodes; Biosensor; Co-electrodeposition; Crystal violet
1. Introduction A large number of matrices have been shown to be suitable for the entrapment of biological molecules. Electrochemical co-deposition of matrix and enzyme is a convenient single step method that is fast and well controlled [1]. There are many examples of electropolymerisation of conducting [2] and non-conducting [3] polymers in the presence of an enzyme. These methods produce ‘soft’ matrices which are delicate and easily degraded; however, in many cases they contribute to the stability of the enzyme. In this paper, we have focused on developing a method of entrapping an enzyme within the electrochemically deposited copper films, which retains the enzymatic activity. The co-deposition of metallic matrices and enzymes has been described by several groups. Kim and Oh [4] deposited platinum black from a platinum salt to produce a high
*
Corresponding author. Tel.: +44 207 679 4527; fax: +44 207 679 7463. E-mail address: [email protected] (D.J. Caruana).
1388-2481/$ - see front matter 2006 Published by Elsevier B.V. doi:10.1016/j.elecom.2005.12.016
surface area deposit with immobilised GOx. The electrodeposition of iridium on glassy carbon also in the presence of GOx was presented by Rodriguez and Rivas [5]. In both these reports, the amperometric response to glucose was the measured electrochemical response of the hydrogen peroxide produced by the enzyme. We describe the electrodeposition of copper on gold with preadsorbed glucose oxidase. We found that crystal violet (hexamethylpararosaniline chloride) was essential to measure a significant response to glucose. Dakkouri et al. [6] and Haiss et al. [7] showed that crystal violet had changed the morphology of the deposited copper. In the absence of crystal violet, the growth was three-dimensional with the formation of copper crystallites. In the presence of crystal violet the growth of crystallites perpendicular to the surface was reduced; the so called rim effect. The crystallites spread laterally, following a quasi-two-dimensional growth mechanism, and this leads to a relatively uniform coverage of the surface. The Dakkouri et al. study was concerned with thin layers of copper; however, in this study we are interested in changing the properties of the bulk deposit to produce a matrix capable
C.P. Remirez, D.J. Caruana / Electrochemistry Communications 8 (2006) 450–454
of entrapping and retaining GOx. We describe a study to investigate the deposition of glucose oxidase within solid copper films in the presence of crystal violet. The motivation of this work is to produce solid metallic films, which are selective to chemical species and may be regenerated by polishing. 2. Experimental 2.1. Materials and instrumentation All solutions were prepared using deionised water (Millipore Milli-Q gradient, <0.05 S cm2). Salts and organic materials were of the highest purity and used as received. GOx was from Biozyme Laboratories, San Diego. Crystal violet and all other inorganic salts were of the highest purity grade from Sigma-Aldrich Co Ltd. A 3-electrode system was employed in these studies. Throughout the experiment, a saturated Ag/AgCl reference electrode was used; all potentials quoted in this paper are against this. A large surface area platinum wire was used as counter electrode. A gold rotating disc working electrode (0.783 cm2, Oxford Electrodes Ltd.) was polished with sandpaper and with Alumina powder (1.0 and 0.3 lm, Micropolish II, Buehler, Lake Bluff, IL) to produce a mirror finish. The electrodes were then thoroughly rinsed with deionised water. The electrode were cycled in sulphuric acid between 0.3 and +1.5 V for 30 cycles or until a stable cyclic voltammogram was obtained. The electrode were stored in water and used within an hour of finishing the cleaning procedure. All electrochemical measurements were made with a lAutolab Type II potentiostat (Windsor Scientific) connected to a personal computer (Dell). A rotator and controller (Oxford electrodes Ltd.) were used to control the rotation rate of the gold electrode. Data were interpreted and presented using SigmaPlot2001 (Version 7.0) software provided by SPSS Inc. Solution pH measurements were made using a model EIL 7045/46 pH meter (ABB KentTaylor Ltd).
451
electrode was placed in a freshly cleaned cell containing (separate counter and reference electrodes to those used for the deposition) 25 ml of phosphate buffer solution (0.1 M, pH 7.0), and rotated at 9.0 Hz. All experiments were carried out at room temperature (23 ± 2 C) with an applied potential of 0.3 V. The background current was allowed to stabilise (no longer than 30 min), aliquots of 50 ll of glucose (1.0 M in phosphate buffer prepared at least 6 h before use) were added to the test solution to produce sequential increases of 2 mM in the concentration of glucose. The reproducibility of these experiments was improved by cleaning the electrodes in between experiments and taking care to avoid long exposure of the electrode surface to air. This was achieved by storing the electrode in phosphate buffer solution at all times. 3. Results and discussion Fig. 1 shows the response to glucose for an electrode deposited with copper from a solution containing GOx (3 mg ml1) and copper sulphate (2.5 mmol dm3) with sodium sulphate as background electrolyte. The current response when glucose was added to the buffered test solution was barely measurable and showed a small and steady decrease in current (reduction of hydrogen peroxide). The test was under steady state conditions held at 0.3 V to measure hydrogen peroxide produced from the enzymatic reaction. The response observed was probably from very small amount of glucose oxidase strongly adsorbed to the outside of the copper deposit. 3.1. Effect of crystal violet in the deposition solution on the glucose response The response time and magnitude were dramatically affected by the presence of crystal violet in the deposition -22x10 -6
2.2. Procedures
-24x10 -6
1st
2nd
3rd
4th
5th
Current / A
6th
Copper electrodeposition was carried out by a potential step to 0.35 V for a period of 600 s unless stated otherwise. A copper solution of 2.5 mM was prepared with cupric sulphate in 0.1 M sodium sulphate solution. Variable amounts of GOx and crystal violet were used in the deposition solution. The deposition solution was deoxygenated by purging with nitrogen for 30 min prior to deposition. The cell was then sealed while passing nitrogen over the surface of the solution, and the electrode was left to stand in the solution to facilitate adsorption of enzyme onto the electrode surface. After electrodeposition, the electrode was thoroughly washed by rotating (9.0 Hz) the electrode in phosphate buffer solution (0.1 M, pH 7.0) for 10 min to avoid contamination of the test solution with GOx. Once rinsed the
7th
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8th
9th
10th
-28x10 -6 -30x10 -6 -32x10 -6 -34x10 -6
1700
1800
1900
2000
2100
2200
2300
Time /s
Fig. 1. Current vs time trace from an electrode with copper (2.5 mM CuSO4) deposited in the presence of 3.0 mg cm3 GOx. Successive additions of 2.0 mM glucose. Deposition was carried out at 0.35 V vs saturated Ag/AgCl for 300 s. The testing was carried out in air saturated pH 7.04 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz.
C.P. Remirez, D.J. Caruana / Electrochemistry Communications 8 (2006) 450–454
solution. In the presence of crystal violet, the response to each addition of glucose is clearly distinguished in the current–time trace shown in Fig. 2(A). The current response measured was from the electrochemical reduction of hydrogen peroxide produced by the enzymatic reaction. In the absence of oxygen in the test solution, there was a significant decrease in the current signal, as shown in Fig. 2(B). Furthermore, in the absence of GOx in the deposition solution, there was no measurable change in current at all when glucose was added (not shown). Chronoamperometry was used for co-deposition of copper and GOx at 0.35 V in the presence of crystal violet. The influence of the electrodeposition time and potential was evaluated in a series of optimisation experiments, which will not be discussed here. From these experiments we determined that a deposition potential of 0.35 V, for 600 s, was found to yield reproducible coatings. Deposition by cycling potential was not successful. A maximum of 5 depositions was found to be possible from a freshly made solution. The reason for this was probably due to leaching of chloride from the reference electrode into the test solution, which led to the precipitation of CuCl at the electrode during the electroreduction. Crystal violet is an organic additive known to change the growth of copper deposit, which is known from Dakkouri et al.’s previous studies [6]. The reason for the change in the observed response was probably due to the direct effect of the crystal violet on the deposition of copper. We do not have any microstructure information on the resultant deposit, but it is certain that the porosity of these films is affected by the crystal violet which is necessary for the substrates (glucose and oxygen) to penetrate the film and react with the enzyme. Crystal violet concentration had a large effect on the response to glucose of the resultant copper layer. Fig. 3 shows the response to 20 mM glucose vs crystal violet con-10x10-6 -12x10-6
Current / A
-14x10-6 -16x10-6
B (B)
-18x10-6 -20x10-6
(A)
-22x10-6
A -24x10-6 1600
1800
2000
2200
2400
Time / s
Fig. 2. (A) Steady state current response to glucose vs time of an electrode with copper (2.5 mM CuSO4) deposited in the presence of GOx (3.0 mg cm3) with crystal violet (1.25 mM). Testing conditions are as stated in legend to Fig. 1. (B) The same conditions as (A), except the test buffer solution was deoxygenated for 20 min with oxygen free nitrogen prior to testing.
10x10-6
8x10-6
6x10-6
ibk-i / A
452
4x10-6
2x10-6
0 0.0
0.5
1.0
1.5
2.0
2.5
[Crystal Violet] / x 10 -3 mol dm-3
Fig. 3. Change in steady state current response glucose (25 mM) for different electrodes prepared from deposition solutions containing GOx (3.0 mg cm3) with copper sulphate concentration either 1.0 mM ( ) and () with different crystal violet concentrations ranging from 0.2 to 2.25 mM. Testing was performed in air saturated buffer pH 7.0 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz, only one addition of glucose was made to the solution to obtain the desired concentration of glucose.
centration for two concentrations of copper sulphate. There is clearly a peak of glucose response when the crystal violet concentration was close to half the copper concentration. This suggests that the mode of action of crystal violet may be due to homogeneous complex formation with copper. Copper–crystal violet complexes are known to form and used for the decolourisation of synthetic dyes [8]. However, the precise effect of crystal violet on the bulk copper deposit is unclear at this point, but we speculate that a change in the response to glucose is a direct effect on the porosity of the layer. As the concentration of crystal violet increases, the porosity increases which may result in the leaching out of the enzyme. The effect of the enzyme loading on the response to glucose was investigated over the range of 1.5–4.5 mg cm3 in the deposition solutions. Fig. 4 shows the change in current for three different concentrations of enzymes in the deposition solutions containing 2.5 and 1.25 mM of Cu2+ and crystal violet, respectively. The curves were fitted to a Michaelis–Menten type equation to determine the apparent Michaelis–Menten constants K 0M and Imax, which are given in the figure legend, for each concentration of GOx that was used. The low K 0M and Imax calculated for the electrode made with 1.5 mg cm3 GOx suggests that the enzyme loading in this layer was probably low, which was expected. Electrodes prepared with deposition solutions containing 3.0 and 4.5 mg cm3 GOx have similar values of K 0M but different Imax values. Imax decreases with increasing GOx concentration. At concentrations of substrate much higher than KM, the Michaelis–Menten equation reduces to kcateR, where kcat is the rate of the enzyme reaction 800 s1, [9] and eR is the total concentration of enzyme in the layer. At steady state the current density, iss, for the enzyme electrode may be given by [10]
C.P. Remirez, D.J. Caruana / Electrochemistry Communications 8 (2006) 450–454
453
9x10-6
10x10-6 3 mg/mL
8x10-6
8x10-6 4.5 mg/mL
7x10-6
4x10-6
1.5 mg/mL
ibk-i / A
ibk-i / A
6x10-6
6x10-6
2x10-6
5x10-6
0
4x10-6
0
5
10
15
20
25
[Glucose] / mmol dm-3
3x10-6 0
5
10
15
20
Time / Days
Fig. 4. Change in steady state current vs glucose concentration for electrodes prepared from deposition solutions containing copper (2.5 mM CuSO4), crystal violet (1.25 mM) and GOx at 1.5 (d, K 0M ¼ 16:75 mM, and Imax = 5.95 · 106 A), 3.0 (j, K 0M ¼ 4:91 mM, and Imax = 1.04 · 105 A) and 4.5 (N, K 0M ¼ 4:85 mM, and Imax = 9.01 · 106 A) mg cm3. Deposition was carried out at 0.35 V vs saturated Ag/AgCl for 300 s. The testing was carried out in air saturated pH 7.0 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz.
iss ¼ nFDs
ds ¼ nFk cat eR l; dx x¼l
ð1Þ
where n is the number of electrons transferred, F is the Faraday constant, Ds is the diffusion coefficient of the substrate, and l is the layer thickness. At concentrations of enzymes in the deposition solution higher than 3.0 mg cm3, the response does not remain dependent on the enzyme concentration, but falls. At high GOx concentrations, the integrity of the copper film could be affected. This behaviour has been described for other enzyme electrodes [11]. 3.2. Enzyme electrode stability The stability of the enzyme electrodes was investigated by creating an enzyme layer using 2.5 mM copper, 1.25 mM crystal violet and 3.0 mg cm3 GOx. The current response to 25 mM glucose was measured periodically for 20 days. The electrode was stored in pH 7.0 phosphate buffer at 4 C, when not in use. The electrode was allowed to warm up to room temperature for 20 min before testing. The glucose response vs time is shown in Fig. 5. The response appeared to drop significantly after the first day, then drops at a slower rate between 2 and 20 days. The long term stability is relatively good and comparable with enzymes immobilised in electropolymerised films [12]. 3.3. Measurement of copper quantity on electrode The quantity of copper deposited on the electrode was measured by a potential step to oxidative potentials (+0.8 V), which was found to be a suitable potential for anodic stripping of copper. The current from the chrono-
Fig. 5. Change in steady state current vs time in days in the presence of glucose (25 mM) for one electrode prepared from deposition solution containing glucose oxidase (3 mg cm3), copper sulphate (2.5 mM) with crystal violet (1.25 mM). Testing was performed in air saturated pH 7.0 phosphate buffer with an applied potential of 0.30 V and rotated at 9.0 Hz, only one addition of glucose was made to the test solution to obtain the desired concentration of glucose. The electrode was stored in pH 7.0 buffer at 4 C.
amperometric trace was integrated, and corrected for non-Faradaic charge contribution to determine the charge for copper oxidation. It is possible to calculate the number of moles of copper, which have been deposited onto the electrode surface. The total charge from the oxidation for a freshly made electrode was 4.12 ± 0.73 · 104 C corresponding to 2.14 ± 0.37 · 109 M of copper. Interestingly, after the electrodes were tested with one single addition of 25 mM of glucose the quantity of copper had reduced to 0.70 ± 0.21 · 109 moles. The loss of copper from the electrode under test was significant. This loss of material from the layer was probably due to poor adherence of copper to the electrode surface. 4. Conclusion The present work demonstrates that the use of an electrodeposited copper film provides an effective technique for the immobilisation of the enzyme at an electrode surface. Using this method of immobilisation, we believe that we have enzymes entrapped within a porous bulk copper film. There are several variables which have been studied in this paper namely; electrodeposition methods, electrodeposition time, concentration of copper, concentration of enzyme and the effect of crystal violet. With this organic additive, the resulting films gave an improved current response in the presence of glucose. The molar ratio of 2:1, copper:crystal violet was important to produce good responsive films. The stability of the enzyme electrode was not impressive. However, we believe that incorporation of polyelectrolytes within the enzyme layer by co-electrodeposition will probably improve the long term stability [12].
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In conclusion, we have developed electrochemically a procedure involving the deposition of copper and GOx on a working electrode promoted by the presence of crystal violet.
[3] [4] [5] [6]
Acknowledgement
[7]
C.P. thanks the Socrates exchange programme for its support.
[8]
References
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