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A glucose electrode based on carbon paste chemically modified with a ferrocene-containing siloxane polymer and glucose oxidase, coated with a poly(ester-sulfonic acid) cation-exchanger
Analyt~ca Chimica Acta, 228 (1990) 23-30 Elsevier Science Publishers B.V., Amsterdam
23 - Printed
in The Netherlands
A glucose electrode based on carbon paste chemically modified with a ferrocene-containing siloxane polymer and glucose oxidase, coated with a poly(ester-sulfonic acid) cation-exchanger L. GORTON Materials
‘.*, H.I. KARAN
Scrence Division, Department
b, P.D. HALE,
T. INAGAKI,
of Applied Science, Brookhaven (Received
Y. OKAMOTO
’ and T.A. SKOTHEIM
National Laboratory,
Upton, NY 11973 (U.S.A.)
13th June 1989)
SUMMARY
A carbon-paste chemically modified with glucose oxidase and a ferrocene-containing siloxane polymer was further modified by coating the electrode surface with a poly(ester-sulfonic acid) cation-exchanger, Eastman AQ-29D. The polymer is obtained as a homogeneous aqueous dispersion at pH 5-6; when dried, the polymer coating is not water-soluble. The coating was shown not to be detrimental to the enzyme activity but to prevent electrochemically active anionic interferents such as ascorbate and urate from reaching the electrode surface. The polymer coating also prevented glucose oxidase from leaking out of the carbon paste into the contacting solution and protected the electrode surface from fouling agents present in urine and bovine serum albumin. Uncoated electrodes lost some lo-15% of their original response to glucose after storage in buffer for three weeks whereas the response of the coated electrodes remained constant. Calibration curves for glucose were strictly linear up to about 5 mM for uncoated and up to 20 mM for coated electrodes. The response current to glucose was not decreased after coating.
A new class of ferrocene-containing siloxane polymers was recently developed [l]. The ferrocene in the polymer proved to be electrochemically active and was shown to mediate the electron transfer from reduced glucose oxidase when mixed with carbon paste to form a chemically modified carbon-paste electrode, CMCPE [2]. In the formula shown for the ferrocene/siloxane polymer, the m/n ratio is 1 : 2; the subunits are ’ Permanent address: University of Lund, h Permanent address: Mathematics, Medgar New York, Brooklyn, ’ Permanent address: University, Brooklyn, 0003-2670/90/$0350
Department of Analytical Chemistry, P.O. Box 124. S-221 00 Lund, Sweden. Division of Natural Science and Evers College, The City University of NY 11225, U.S.A. Department of Chemistry, Polytechnic NY 11201, U.S.A. 0 1990 Elsevier Science
Publishers
B.V.
randomly distributed copolymer).
(i.e., it is a random
block
Fc
The reason for the successful mediation, compared to other ferrocene-containing polymers that fail [3] to mediate the electron transfer from glucose oxidase, is believed to be due to the flexible nature of the siloxane backbone [1,2]. Maximal catalytic currents for glucose oxidation occurred around + 300 mV vs. SCE. In this potential range, several common interferents in biological fluids,
24
e.g., ascorbic acid and uric acid, are either directly oxidized at the electrode [4,5] or catalytically oxidized by the ferrocene [6]. This additional contribution to the response current may make it difficult to obtain accurate quantification of the glucose concentration in the sample. Other interferents in biological fluids or in samples with a complex matrix may foul or alter the properties of the electrode surface. In a recent paper, Harrison et al. [7] described the protection of the active surface of a glucose oxidase-modified platinum electrode by coating the active surface with a layer of Nafion, a perfluorosulfonic acid polymer, which has been much studied as a modifier for making chemically modified electrodes [8,9]. Because of the anionic nature of the polymer, negatively charged molecules, such as hexacyanoferrate(II), were largely prevented from reaching the electrochemically active surface. The Nafion-coated glucose oxidase electrode was shown to have a much improved stability in fresh whole blood compared with an uncoated electrode. Other investigations [lO,ll] have also shown an improved stability for Nafion-coated electrodes in biological fluids. Nafion is, however, only soluble in at least partly organic solvents (1 : 1 2-propanel/water [7]). Much care was therefore needed to obtain proper dilution of the commercial product and to apply the appropriate amount of the Nafion solution to the enzyme layer of the electrode surface to prevent denaturation [7]. Moreover, the sulfonic acid groups of Nafion are protonated, so when the coated electrode is conditioned in an aqueous solution, a drastic pH change is expected to take place at the electrode surface. This may also have a negative influence on fhe remaining enzyme activity after coating [7]. In this paper, the use of a new class of anionic polymeric resin [12] is reported; the polymer, Eastman AQ-29D, is a poly(ester-sulfonic acid) cation-exchanger commercially available as a homogeneous aqueous dispersion with a pH of 5-6 (Eastman Chemical Products, Publ. no. GN-375, 1987). Once dried, the polymer is water-insoluble at room temperature. Thin films of this polymer were coated onto the surface of carbon-paste electrodes modified with glucose oxidase and the ferrocene/siloxane polymer. The coating was shown
L. GORTON
ET AL.
not to be detrimental to the enzyme activity but it excluded small anionic interferents and protected the electrode surface from fouling agents present in biological fluids.
EXPERIMENTAL
The chemically modified carbon paste (CMCP) was prepared by thorough mixing of 100 mg of graphite powder (Fluka, cat. no. 50870) and 20 ~1 of paraffin oil (Fluka, cat. no. 76235) with 10 mg of glucose oxidase (EC 1.1.3.4 from Aspergilh niger; Sigma cat. no. G-2133, 177 U mg-‘) and 1.5 mg of the ferrocene/siloxane polymer [2] [poly(methyl, /3-ferrocenylethylsiloxane dimethylsiloxane] to form a uniform paste. This paste was stored at 4°C until further use. Plastic syringe holders (l.O-ml syringe with a tip of 4.0 mm o.d. and 1.8 m i.d.) were packed with about 0.1 ml of carbon paste (100 mg of graphite powder, 20 ~1 of paraffin oil) in the end of the holder leaving about 3-4 mm in the syringe tip empty to be filled with the CMCP to produce the final electrode. Electrical contact was made by inserting a silver wire in the carbon paste. After the tip of the syringe had been filled with CMCP, the end was rubbed gently on fine smooth paper to produce a flat shining electrode surface with an area of about 0.024 cm2. The outer end-parts of the plastic syringe were polished with fine emery paper to promote adherence of the Eastman AQ-29D coating. This coating was obtained by dipping the enzyme electrode into a 0.5% (w/v) solution prepared by proper dilution in distilled water of the 30% stock solution (kindly obtained as a gift from Professor J. Wang, New Mexico State University, Las Cruces). The amount of the 0.5% solution that remained on the electrode was estimated to be about 5 ~1. After this dipping, the electrodes were allowed to dry for at least 1 h at room temperature with the electrode surfaces facing down. (Drying with the electrode surface facing up caused worse reproducibility.) The electrodes were then ready for use. Conditioning in buffer before experiments was found to be unnecessary. When not in use, all electrodes, coated and uncoated, were
CARBON
PASTE
GLUCOSE
25
ELECTRODE
stored in 0.1 M phosphate buffer (pH 7.0 with 0.1 M KCl). Constant-potential experiments were done by connecting the CMCPE to a three-electrode potentiostat (PAR model 173). A platinum wire and a saturated calomel electrode were used as the auxiliary and reference electrodes, respectively. All potentials are referred to this reference electrode. All constant-potential experiments were done at + 300 mV. The electrochemical cell used was a glass beaker with a volume of about 25 ml. Portions (10.0 ml) of the 0.1 M potassium phosphate/ 0.1 M KC1 buffer (pH 7.0) were thoroughly deoxygenated by purging with nitrogen for at least 15 min prior to each experiment. The purging also provided fast and thorough stirring of the solution during these experiments. Samples injected into the cell were also thoroughly deoxygenated. Stock solutions of glucose (P-D-glucose, Sigma cat. no. G-5250), L-ascorbic acid (Sigma cat. no. A-0278), uric acid (Sigma, cat. no. U-2625) and bovine serum albumin (BSA; Pel-Freez, cat. no. 27001-2) were prepared daily by dissolving appropriate amounts in the 0.1 M phosphate/O.1 M KC1 buffer, pH 7.0. Oxidation of dissolved ascorbic acid by molecular oxygen was prevented by purging the buffer with nitrogen before use. The glucose was allowed to reach mutarotational equilibrium before use (ca. 24 h). All other chemicals used were of reagent grade.
1.5
RESULTS
AND
DISCUSSION
Characteristics of the CMCPEs Glucose is not directly oxidizable at carbon or carbon-paste electrodes within a reasonable potential range. By the addition of glucose oxidase (GOD) and the ferrocene polymer to the carbon paste, an electron shuttle from glucose to the carbon can be obtained if the applied potential of the CMCPE is more positive than the formal potential of the polymer-bound ferricinium/ ferrocene redox couple, as shown below. ZH+
GOD-FAD (flavin adenine dinucleotide) and GOD-FADH, denote the oxidized and reduced forms of glucose oxidase and Fc+ and Fc the oxidized (ferricinium) and reduced forms of the ferrocene. Maximal response currents for glucose are found at applied potentials of at least + 300 mV [2]. In this study, the response to glucose was compared mainly to that of ascorbate because this
1.5
4.0
4.0
A
3.0
3.0
1.0
1.0
;; a ._
-* 2.0
2.0
0.5
;i 1
.-
0.5 1.0
1.0
,
-?!k$ 0
2
number
4
of coatings
0
6
number
of coatings
Fig. 1. Variation of the response to 7.0 mM glucose (0) and 0.68 mM ascorbate AQ-29D solution for three identically prepared CMCPEs, A, B and C.
2
number
(m) with the number
4
6
Of cOatlng*
of coatings
of a 0.5% Eastman
26
compound is directly oxidizable at the carbon paste. It is also a common substance in most biological fluids and its molecular weight and structure are similar to those of glucose, except it is negatively charged. Figure 1 shows the variation in the response to 7.0 mM glucose and 0.68 mM ascorbate with the number of dip-coatings of the AQ-29D polymer for three identically prepared CMCPEs, denoted A, B, and C. Compared with the current for the direct oxidation of ascorbate, the conversion efficiency for glucose oxidation is around 2%. The response to ascorbate decreases with each coating, as is clearly shown in Fig. 1. When the polymer dispersion applied to the electrode surface is dried, a thin membrane is formed on the surface of the CMCPE. The anionic nature of the membrane makes it more difficult for the ascorbate anion to reach the electrochemically active surface. After six or seven coatings, typically less than 10% of the initial response to ascorbate remains. The variation of the initial response to ascorbate between the electrodes may be due to inhomogeneous mixing during preparation of the CMCP. A variation is, however, expected even if perfect mixing is obtained. The electrochemical oxidation of ascorbate on carbon electrodes is strongly irreversible and a variation in the response of electrodes equally pretreated and prepared from the same carbon black is expected [4]. Figure 2 shows calibration curves for ascorbate for an uncoated and a coated CMCPE. At + 300 mV, maximal oxidation currents for ascorbate are not obtained. The kinetically controlled behaviour of the response for the uncoated electrode is obvious,
Fig. 2. Ascorbate response of an uncoated CMCPE (0) and a CMCPE coated six times with a 0.5% Eastman AQ-29D solution (m).
L. GORTON
ET AL.
as the response curve is not linear. The response curve for the coated electrode is more linear (and much lower), owing to the effect of the membrane. The linear response for ascorbate at the coated electrode makes a correction for this compound much easier. The response to ascorbate at the uncoated CMCPE indicates the strong interference of this compound at the applied potential, + 300 mV. The concentration of ascorbate in this experiment is about five times the typical value of a human blood serum, 0.125 PM. The response to such an ascorbate concentration is approximately equal to the response obtained for a glucose concentration of about 4 mM, the normal serum concentration. By coating the CMCPE with this anionic polymer, less than 10% of the original response to ascorbate will remain whereas the glucose response will be virtually the same. Figure 1 also shows that the exposure of the active surface of the CMCPE to the anionic polymer dispersion is by no means detrimental to the enzyme activity. Rather than a decreased glucose response, most coated electrodes showed a pronounced increase in the glucose response on the first two or three coatings. The reason for this is not fully clear but it could be explained by some initial dissolution of the water-soluble glucose oxidase from the hydrophobic CMCP into the polymer solution before the polymer has dried. It was observed that it took at least 20 min for the polymer drop to become visibly dry. An increased activity of the enzyme at the electrode surface is expected to result in an increase in the glucose response. In other experiments with soluble glucose oxidase in the contacting buffer, much larger response values for an equal glucose concentration were obtained with uncoated CMCPEs [13]. The variation in the initial glucose response of the uncoated electrodes (Fig. 1) is probably due to inhomogeneous mixing of glucose oxidase in the CMCP. The effect of coating the CMCPE on the characteristics of the calibration curves for glucose is shown in Fig. 3. The linear response range is extended to much higher concentrations for the coated CMCPEs, compared to the uncoated electrodes. Plotting the response current versus response current/ glucose concentration produced
CARBON
PASTE
GLUCOSE
ELECTRODE
Fig. 3. Glucose response of a freshly prepared uncoated CMCPE (A), an uncoated CMCPE subjected to 5% BSA (m), and a CMCPE coated six times with a 0.5% Eastman AQ-29D solution (0). For details see text.
straight lines (electrochemical Eadie-Hofstee plots [14]). From the slope of the plot, the apparent Michaelis-Menten constant, Kzp, can be evaluated [14]. An uncoated electrode typically gave a Kgp value of 50-70 mM, a value in close agreement with that obtained for other electrodes with glucose oxidase immobilized directly on the electrode surface [15-191. An electrode coated six times gave a much higher value of around 200 mM. The increase in Ksp clearly shows the expected additional resistance for diffusion of glucose to the active enzyme layer, obtained by coating the electrode with a membrane. That the response level remains at the same order of magnitude as that of the uncoated electrode can only be explained by an increased activity of glucose oxidase at the polymer CMCP interface after coating. With a Kzp value of 200 mM, the response to glucose for the coated electrode is expected to be strictly linear up to about 20 mM ([glucose] G 0.1 Kzp), which is well within the range of interest in biological fluids. For the uncoated electrode, however, the calibration curve will deviate from linearity for glucose concentration less than 10 mM (Fig. 3). The responses to glucose and ascorbate were measured every second day for three weeks for a set of four uncoated electrodes and four electrodes coated six times. These electrodes were not exposed to any solutions other than pure buffers and pure sample solutions (cf. below). The uncoated electrodes after this period had lost between 10 and 15% of their initial glucose response.
27
The response to ascorbate remained essentially constant, in contrast to what can be expected for graphite-based electrodes [4]. It was noticed that when uncoated CMCPEs were stored in buffer, the solution slowly turned slightly yellow probably because of dissolution of glucose oxidase from the carbon paste. Commercial preparations of glucose oxidase also contain unbound FAD [20]. Dissolution of this compound into the buffer during storage is more unlikely, as it is known that it strongly adsorbs on graphite [21]. However, no investigations were done to trace the nature of this yellow compound. The coated electrodes fully retained (_t 1%) their initial responses to both glucose and ascorbate over this three-week period. The slight variation is within experimental errors. This shows that storage does not change the basic characteristics of the electrodes, because the same amount of glucose and ascorbate can pass through the membrane when newly coated and after three weeks. The membrane also seems to prevent leakage of components from the CMCP into the contacting solution. No yellow colour could be seen in the buffer in which the coated electrodes were stored. The Kzp values for glucose for the uncoated and coated electrodes were also nearly constant (+ 10%) after three weeks. This also supports the suggestion that the loss in response to glucose of the uncoated electrodes is due to leakage of glucose oxidase rather than denaturation of the trapped enzyme at the electrode surface. A layer of denatured enzyme would serve as an additional resistance to diffusion of glucose to the undenatured enzyme, resulting in an increased Kzp value. No special precautions were taken to protect the coated electrodes. When stored in buffer, the coated surface was allowed to touch the bottom of the beaker. Between experiments, the electrodes were thoroughly washed with a jet of distilled water and the surface was gently wiped with a tissue. It was noticed, however, that the coated surface was somewhat sticky. Application to urine and BSA solutions Uric acid or urate is, like ascorbate, a common interfering substance in biological fluids; it is elec-
102 105
3’
4s 5s 102 108
108 162 145
Ab
Response ratio (W) after (A) and before (B) exposure. Immediately after exposure. Four days after exposure. Before exposure. 24 h after exposure. ’ Uncoated electrode. E Coated electrode.
a b ’ d ’
39 63 30
63 81 44
97 94
BC
Ab
To glucose
To ascorbate
100 103
108 141 102
BE
0.05 0.05
0.01 0.01 0.01
Cd
0.4 0.2
0.15 0.4 0.25
DC
0.08 0.08
0.1 0.25 0.15
B’
4.0 2.0
0.3 0.3 0.3
Cd
6 5
5 5 5
Ab
4 2
4 4 4
BC
Time to steady background (s)
Background current ( p A)
CME
Response a (W)
CMCPEs exposed to undiluted urine for 1 b
1
Responses to 27 mM glucose and 0.68 mM ascorbate and background currents for three uncoated and two six-times-coated
TABLE
CARBON
PASTE
GLUCOSE
29
ELECTRODE
trochemically active in the investigated potential range [ll]. The responses of the uncoated CMCPEs to both 0.25 and 0.5 mM urate were only about 30% of the responses to equal concentrations of ascorbate. Because of its anionic nature at pH 7.0, the response at the coated electrodes was very small, less than 10% of the response obtained at the uncoated electrodes. Neither the response to glucose nor that to ascorbate decreased after the CMCPEs had been subjected to urate oxidation. Other investigations have shown that urate oxidation causes fouling of the electrode surface [5]. It could not be found here. When the electrodes were subjected to fresh urine samples diluted a hundred times, the response of the coated electrode was about 20% of that of the uncoated electrode. From the results obtained with the small anionic molecules, ascorbate and ureate, the ratio of the responses of the coated and uncoated electrodes should be about 1 : 10. The probable reason that the response of the coated electrodes in urine is 20% of that of the uncoated electrodes is that urine also contains neutral and cationic species that can be directly oxidized electrochemically. Calibrating the uncoated and coated CMCPEs for glucose and ascorbate directly after the experiments with diluted urine samples did not result in any response changes. Coated and uncoated electrodes were stored at open circuit in undiluted fresh urine for 1 h. They were then thoroughly washed and recalibrated for glucose and ascorbate. A somewhat larger background current was noticed, as well as an increase in the time necessary to reach a steady background current level (Table 1). While the uncoated electrodes showed a noticeable and immediate decrease in the response to glucose and also an increase in the response to ascorbate, the coated electrodes seemed to be largely unaffected by the exposure to the urine. These response changes displayed by the uncoated electrodes remained after storage for a few days in buffer (see Table 1) with an even more pronounced decrease in glucose response and virtually no increase in the response to ascorbate. Uncoated and coated electrodes were also calibrated for ascorbate and glucose in 2.5%
TABLE
2
Responses to 27 mM glucose and 0.68 mM ascorbate uncoated and three 6-times-coated CMCPEs exposed CME
Response
a (W)
To glucose Ab
of three to BSA
To ascorbate B”
Ab
B’
3d
86 81 84
75 77 78
82 89 85
77 92 79
4e 5= 6e
98 99 101
98 99 100
100 100 98
100 102 101
a See footnote to Table 1. b After exposure to 2.5% BSA/buffer solution an applied potential of + 300 mV. ’ After storage in 5% BSA/buffer solution overnight at 4O C. d Uncoated electrode. e Coated electrode.
for 10 mm with at open
circuit
BSA/buffer solutions. No increase in background current level was observed compared to a pure buffer background. The uncoated electrodes immediately lost some 15-20% of the glucose response and 10-15s of the ascorbate response, which clearly reveals the effects of some components in the BSA solution. The coated electrodes, however, showed no decrease in response on exposure to the BSA solution. After the uncoated electrodes had been soaked in 5% BSA solution overnight, the response to glucose and to ascorbate was further lowered to about 70% of their original responses. The fact that both the glucose and the ascorbate responses decreased is probably due to precipitation of some BSA onto the electrode. The apparent Ksp value for glucose oxidase also increased to about 100 mM for the uncoated electrodes. Conclusion These studies demonstrate that the ion-exchange characteristics and transport properties of water-cast Eastman AQ-29D poly(ester-sulfonic acid) make it ideal for use as a protective membrane on enzyme-based glucose sensors. Because the polymer is cast from an aqueous dispersion, and not from a partially organic solvent, enzyme activity is easily maintained on coating. The
30
L. GORTON
material improves the performance of redox polymer/ glucose oxidase/ carbon-paste electrodes, through the exclusion of anionic interferents and the prevention of electrode fouling by biological agents. In addition, sensor lifetime is increased substantially, because glucose oxidase is no longer free to diffuse away from the electrode. The polymer membrane also improves the useful concentration range of the glucose sensor with no decrease in response current. Work is presently underway to extend these studies by investigating other Eastman polymers in similar electrode configurations. This investigation was financially supported by the Swedish Board for Technical Development (STUF), the Swedish National Energy Administration (STEV), and the U.S. Department of Energy, Division of Material Science, Office of Basic Energy Science. T.I. is the recipient of a research fellowship from UBE Industries. The gift of the Eastman AQ-29D from Professor J. Wang is gratefully acknowledged.
REFERENCES 1 T. Inagaki, H.S. Lee, T.A. Skotheim and Y. Okamoto, Chem. Sot., Chem. Commun., (1989) 1181.
J.
ET AL.
2 P.D. Hale, T. Inagaki, HI. Karan, Y. Okamoto and T.A. Skotheim, J. Am. Chem. Sot., 111 (1989) 3482. 3 J.A. Chambers and N.J. Walton, J. Electroanal. Chem., 250 (1988) 417. 4 G.N. Kamau, Anal. Chim. Acta, 207 (1988) 1. 5 J. Wang and M. Shan Lin, Anal. Chem., 60 (1988) 499 (and references cited therein). 6 M. Pettersson, Anal. Chim. Acta, 147 (1983) 359. 7 D.J. Harrison, R.F.B. Turner and H.P. Bates, Anal. Chem., 60 (1988) 2002. 8 R.W. Murray, A.G. Ewing and R.A. Durst, Anal. Chem., 59 (1987) 379A. 9 S. Dong and Y. Wang, Electroanalysis, 1 (1989) 99. 10 E.W. Kristensen, W.G. Kuhr and R.M. Wightman, Anal. Chem., 59 (1987) 1752. 11 H. Huiliang, D. Jagner and L. Renman, Anal. Chim. Acta, 207 (1988) 17. 12 J. Wang and T. Golden, Anal. Chem., 61 (1989) 1397. 13 P.D. Hale, T. Inagaki, H. Karan, H.S. Lee, Y. Okamoto and T.A. Skotheim, unpublished work. 14 R.A. Kamin and G.S. Wilson, Anal. Chem., 52 (1980) 1198. 15 V.J. Razumas, J.J. Jasaitis and J.J. Kulys, Bioelectrochem. Bioenerg., 12 (1984) 297. 16 G. Jiinsson and L. Gorton, Anal. Lett., 20 (1987) 839. 17 L. Gorton, F. Scheller and G. Johansson, Stud. Biophys., 109 (1985) 199. 18 R.M. Ianniello and A.M. Yacynych, Anal. Chem., 53 (1981) 2090. 19 J.C. Bourdillon, J.P. Bourgeois and D. Thomas, Biotechnol. Bioeng., 21 (1979) 1877. 20 R.E. Holt and T.M. Cotton, J. Am. Chem. Sot., 109 (1987) 1841. 21 L. Gorton and G. Johansson, J. Electroanal. Chem., 113 (1980) 151.
23 - Printed
in The Netherlands
A glucose electrode based on carbon paste chemically modified with a ferrocene-containing siloxane polymer and glucose oxidase, coated with a poly(ester-sulfonic acid) cation-exchanger L. GORTON Materials
‘.*, H.I. KARAN
Scrence Division, Department
b, P.D. HALE,
T. INAGAKI,
of Applied Science, Brookhaven (Received
Y. OKAMOTO
’ and T.A. SKOTHEIM
National Laboratory,
Upton, NY 11973 (U.S.A.)
13th June 1989)
SUMMARY
A carbon-paste chemically modified with glucose oxidase and a ferrocene-containing siloxane polymer was further modified by coating the electrode surface with a poly(ester-sulfonic acid) cation-exchanger, Eastman AQ-29D. The polymer is obtained as a homogeneous aqueous dispersion at pH 5-6; when dried, the polymer coating is not water-soluble. The coating was shown not to be detrimental to the enzyme activity but to prevent electrochemically active anionic interferents such as ascorbate and urate from reaching the electrode surface. The polymer coating also prevented glucose oxidase from leaking out of the carbon paste into the contacting solution and protected the electrode surface from fouling agents present in urine and bovine serum albumin. Uncoated electrodes lost some lo-15% of their original response to glucose after storage in buffer for three weeks whereas the response of the coated electrodes remained constant. Calibration curves for glucose were strictly linear up to about 5 mM for uncoated and up to 20 mM for coated electrodes. The response current to glucose was not decreased after coating.
A new class of ferrocene-containing siloxane polymers was recently developed [l]. The ferrocene in the polymer proved to be electrochemically active and was shown to mediate the electron transfer from reduced glucose oxidase when mixed with carbon paste to form a chemically modified carbon-paste electrode, CMCPE [2]. In the formula shown for the ferrocene/siloxane polymer, the m/n ratio is 1 : 2; the subunits are ’ Permanent address: University of Lund, h Permanent address: Mathematics, Medgar New York, Brooklyn, ’ Permanent address: University, Brooklyn, 0003-2670/90/$0350
Department of Analytical Chemistry, P.O. Box 124. S-221 00 Lund, Sweden. Division of Natural Science and Evers College, The City University of NY 11225, U.S.A. Department of Chemistry, Polytechnic NY 11201, U.S.A. 0 1990 Elsevier Science
Publishers
B.V.
randomly distributed copolymer).
(i.e., it is a random
block
Fc
The reason for the successful mediation, compared to other ferrocene-containing polymers that fail [3] to mediate the electron transfer from glucose oxidase, is believed to be due to the flexible nature of the siloxane backbone [1,2]. Maximal catalytic currents for glucose oxidation occurred around + 300 mV vs. SCE. In this potential range, several common interferents in biological fluids,
24
e.g., ascorbic acid and uric acid, are either directly oxidized at the electrode [4,5] or catalytically oxidized by the ferrocene [6]. This additional contribution to the response current may make it difficult to obtain accurate quantification of the glucose concentration in the sample. Other interferents in biological fluids or in samples with a complex matrix may foul or alter the properties of the electrode surface. In a recent paper, Harrison et al. [7] described the protection of the active surface of a glucose oxidase-modified platinum electrode by coating the active surface with a layer of Nafion, a perfluorosulfonic acid polymer, which has been much studied as a modifier for making chemically modified electrodes [8,9]. Because of the anionic nature of the polymer, negatively charged molecules, such as hexacyanoferrate(II), were largely prevented from reaching the electrochemically active surface. The Nafion-coated glucose oxidase electrode was shown to have a much improved stability in fresh whole blood compared with an uncoated electrode. Other investigations [lO,ll] have also shown an improved stability for Nafion-coated electrodes in biological fluids. Nafion is, however, only soluble in at least partly organic solvents (1 : 1 2-propanel/water [7]). Much care was therefore needed to obtain proper dilution of the commercial product and to apply the appropriate amount of the Nafion solution to the enzyme layer of the electrode surface to prevent denaturation [7]. Moreover, the sulfonic acid groups of Nafion are protonated, so when the coated electrode is conditioned in an aqueous solution, a drastic pH change is expected to take place at the electrode surface. This may also have a negative influence on fhe remaining enzyme activity after coating [7]. In this paper, the use of a new class of anionic polymeric resin [12] is reported; the polymer, Eastman AQ-29D, is a poly(ester-sulfonic acid) cation-exchanger commercially available as a homogeneous aqueous dispersion with a pH of 5-6 (Eastman Chemical Products, Publ. no. GN-375, 1987). Once dried, the polymer is water-insoluble at room temperature. Thin films of this polymer were coated onto the surface of carbon-paste electrodes modified with glucose oxidase and the ferrocene/siloxane polymer. The coating was shown
L. GORTON
ET AL.
not to be detrimental to the enzyme activity but it excluded small anionic interferents and protected the electrode surface from fouling agents present in biological fluids.
EXPERIMENTAL
The chemically modified carbon paste (CMCP) was prepared by thorough mixing of 100 mg of graphite powder (Fluka, cat. no. 50870) and 20 ~1 of paraffin oil (Fluka, cat. no. 76235) with 10 mg of glucose oxidase (EC 1.1.3.4 from Aspergilh niger; Sigma cat. no. G-2133, 177 U mg-‘) and 1.5 mg of the ferrocene/siloxane polymer [2] [poly(methyl, /3-ferrocenylethylsiloxane dimethylsiloxane] to form a uniform paste. This paste was stored at 4°C until further use. Plastic syringe holders (l.O-ml syringe with a tip of 4.0 mm o.d. and 1.8 m i.d.) were packed with about 0.1 ml of carbon paste (100 mg of graphite powder, 20 ~1 of paraffin oil) in the end of the holder leaving about 3-4 mm in the syringe tip empty to be filled with the CMCP to produce the final electrode. Electrical contact was made by inserting a silver wire in the carbon paste. After the tip of the syringe had been filled with CMCP, the end was rubbed gently on fine smooth paper to produce a flat shining electrode surface with an area of about 0.024 cm2. The outer end-parts of the plastic syringe were polished with fine emery paper to promote adherence of the Eastman AQ-29D coating. This coating was obtained by dipping the enzyme electrode into a 0.5% (w/v) solution prepared by proper dilution in distilled water of the 30% stock solution (kindly obtained as a gift from Professor J. Wang, New Mexico State University, Las Cruces). The amount of the 0.5% solution that remained on the electrode was estimated to be about 5 ~1. After this dipping, the electrodes were allowed to dry for at least 1 h at room temperature with the electrode surfaces facing down. (Drying with the electrode surface facing up caused worse reproducibility.) The electrodes were then ready for use. Conditioning in buffer before experiments was found to be unnecessary. When not in use, all electrodes, coated and uncoated, were
CARBON
PASTE
GLUCOSE
25
ELECTRODE
stored in 0.1 M phosphate buffer (pH 7.0 with 0.1 M KCl). Constant-potential experiments were done by connecting the CMCPE to a three-electrode potentiostat (PAR model 173). A platinum wire and a saturated calomel electrode were used as the auxiliary and reference electrodes, respectively. All potentials are referred to this reference electrode. All constant-potential experiments were done at + 300 mV. The electrochemical cell used was a glass beaker with a volume of about 25 ml. Portions (10.0 ml) of the 0.1 M potassium phosphate/ 0.1 M KC1 buffer (pH 7.0) were thoroughly deoxygenated by purging with nitrogen for at least 15 min prior to each experiment. The purging also provided fast and thorough stirring of the solution during these experiments. Samples injected into the cell were also thoroughly deoxygenated. Stock solutions of glucose (P-D-glucose, Sigma cat. no. G-5250), L-ascorbic acid (Sigma cat. no. A-0278), uric acid (Sigma, cat. no. U-2625) and bovine serum albumin (BSA; Pel-Freez, cat. no. 27001-2) were prepared daily by dissolving appropriate amounts in the 0.1 M phosphate/O.1 M KC1 buffer, pH 7.0. Oxidation of dissolved ascorbic acid by molecular oxygen was prevented by purging the buffer with nitrogen before use. The glucose was allowed to reach mutarotational equilibrium before use (ca. 24 h). All other chemicals used were of reagent grade.
1.5
RESULTS
AND
DISCUSSION
Characteristics of the CMCPEs Glucose is not directly oxidizable at carbon or carbon-paste electrodes within a reasonable potential range. By the addition of glucose oxidase (GOD) and the ferrocene polymer to the carbon paste, an electron shuttle from glucose to the carbon can be obtained if the applied potential of the CMCPE is more positive than the formal potential of the polymer-bound ferricinium/ ferrocene redox couple, as shown below. ZH+
GOD-FAD (flavin adenine dinucleotide) and GOD-FADH, denote the oxidized and reduced forms of glucose oxidase and Fc+ and Fc the oxidized (ferricinium) and reduced forms of the ferrocene. Maximal response currents for glucose are found at applied potentials of at least + 300 mV [2]. In this study, the response to glucose was compared mainly to that of ascorbate because this
1.5
4.0
4.0
A
3.0
3.0
1.0
1.0
;; a ._
-* 2.0
2.0
0.5
;i 1
.-
0.5 1.0
1.0
,
-?!k$ 0
2
number
4
of coatings
0
6
number
of coatings
Fig. 1. Variation of the response to 7.0 mM glucose (0) and 0.68 mM ascorbate AQ-29D solution for three identically prepared CMCPEs, A, B and C.
2
number
(m) with the number
4
6
Of cOatlng*
of coatings
of a 0.5% Eastman
26
compound is directly oxidizable at the carbon paste. It is also a common substance in most biological fluids and its molecular weight and structure are similar to those of glucose, except it is negatively charged. Figure 1 shows the variation in the response to 7.0 mM glucose and 0.68 mM ascorbate with the number of dip-coatings of the AQ-29D polymer for three identically prepared CMCPEs, denoted A, B, and C. Compared with the current for the direct oxidation of ascorbate, the conversion efficiency for glucose oxidation is around 2%. The response to ascorbate decreases with each coating, as is clearly shown in Fig. 1. When the polymer dispersion applied to the electrode surface is dried, a thin membrane is formed on the surface of the CMCPE. The anionic nature of the membrane makes it more difficult for the ascorbate anion to reach the electrochemically active surface. After six or seven coatings, typically less than 10% of the initial response to ascorbate remains. The variation of the initial response to ascorbate between the electrodes may be due to inhomogeneous mixing during preparation of the CMCP. A variation is, however, expected even if perfect mixing is obtained. The electrochemical oxidation of ascorbate on carbon electrodes is strongly irreversible and a variation in the response of electrodes equally pretreated and prepared from the same carbon black is expected [4]. Figure 2 shows calibration curves for ascorbate for an uncoated and a coated CMCPE. At + 300 mV, maximal oxidation currents for ascorbate are not obtained. The kinetically controlled behaviour of the response for the uncoated electrode is obvious,
Fig. 2. Ascorbate response of an uncoated CMCPE (0) and a CMCPE coated six times with a 0.5% Eastman AQ-29D solution (m).
L. GORTON
ET AL.
as the response curve is not linear. The response curve for the coated electrode is more linear (and much lower), owing to the effect of the membrane. The linear response for ascorbate at the coated electrode makes a correction for this compound much easier. The response to ascorbate at the uncoated CMCPE indicates the strong interference of this compound at the applied potential, + 300 mV. The concentration of ascorbate in this experiment is about five times the typical value of a human blood serum, 0.125 PM. The response to such an ascorbate concentration is approximately equal to the response obtained for a glucose concentration of about 4 mM, the normal serum concentration. By coating the CMCPE with this anionic polymer, less than 10% of the original response to ascorbate will remain whereas the glucose response will be virtually the same. Figure 1 also shows that the exposure of the active surface of the CMCPE to the anionic polymer dispersion is by no means detrimental to the enzyme activity. Rather than a decreased glucose response, most coated electrodes showed a pronounced increase in the glucose response on the first two or three coatings. The reason for this is not fully clear but it could be explained by some initial dissolution of the water-soluble glucose oxidase from the hydrophobic CMCP into the polymer solution before the polymer has dried. It was observed that it took at least 20 min for the polymer drop to become visibly dry. An increased activity of the enzyme at the electrode surface is expected to result in an increase in the glucose response. In other experiments with soluble glucose oxidase in the contacting buffer, much larger response values for an equal glucose concentration were obtained with uncoated CMCPEs [13]. The variation in the initial glucose response of the uncoated electrodes (Fig. 1) is probably due to inhomogeneous mixing of glucose oxidase in the CMCP. The effect of coating the CMCPE on the characteristics of the calibration curves for glucose is shown in Fig. 3. The linear response range is extended to much higher concentrations for the coated CMCPEs, compared to the uncoated electrodes. Plotting the response current versus response current/ glucose concentration produced
CARBON
PASTE
GLUCOSE
ELECTRODE
Fig. 3. Glucose response of a freshly prepared uncoated CMCPE (A), an uncoated CMCPE subjected to 5% BSA (m), and a CMCPE coated six times with a 0.5% Eastman AQ-29D solution (0). For details see text.
straight lines (electrochemical Eadie-Hofstee plots [14]). From the slope of the plot, the apparent Michaelis-Menten constant, Kzp, can be evaluated [14]. An uncoated electrode typically gave a Kgp value of 50-70 mM, a value in close agreement with that obtained for other electrodes with glucose oxidase immobilized directly on the electrode surface [15-191. An electrode coated six times gave a much higher value of around 200 mM. The increase in Ksp clearly shows the expected additional resistance for diffusion of glucose to the active enzyme layer, obtained by coating the electrode with a membrane. That the response level remains at the same order of magnitude as that of the uncoated electrode can only be explained by an increased activity of glucose oxidase at the polymer CMCP interface after coating. With a Kzp value of 200 mM, the response to glucose for the coated electrode is expected to be strictly linear up to about 20 mM ([glucose] G 0.1 Kzp), which is well within the range of interest in biological fluids. For the uncoated electrode, however, the calibration curve will deviate from linearity for glucose concentration less than 10 mM (Fig. 3). The responses to glucose and ascorbate were measured every second day for three weeks for a set of four uncoated electrodes and four electrodes coated six times. These electrodes were not exposed to any solutions other than pure buffers and pure sample solutions (cf. below). The uncoated electrodes after this period had lost between 10 and 15% of their initial glucose response.
27
The response to ascorbate remained essentially constant, in contrast to what can be expected for graphite-based electrodes [4]. It was noticed that when uncoated CMCPEs were stored in buffer, the solution slowly turned slightly yellow probably because of dissolution of glucose oxidase from the carbon paste. Commercial preparations of glucose oxidase also contain unbound FAD [20]. Dissolution of this compound into the buffer during storage is more unlikely, as it is known that it strongly adsorbs on graphite [21]. However, no investigations were done to trace the nature of this yellow compound. The coated electrodes fully retained (_t 1%) their initial responses to both glucose and ascorbate over this three-week period. The slight variation is within experimental errors. This shows that storage does not change the basic characteristics of the electrodes, because the same amount of glucose and ascorbate can pass through the membrane when newly coated and after three weeks. The membrane also seems to prevent leakage of components from the CMCP into the contacting solution. No yellow colour could be seen in the buffer in which the coated electrodes were stored. The Kzp values for glucose for the uncoated and coated electrodes were also nearly constant (+ 10%) after three weeks. This also supports the suggestion that the loss in response to glucose of the uncoated electrodes is due to leakage of glucose oxidase rather than denaturation of the trapped enzyme at the electrode surface. A layer of denatured enzyme would serve as an additional resistance to diffusion of glucose to the undenatured enzyme, resulting in an increased Kzp value. No special precautions were taken to protect the coated electrodes. When stored in buffer, the coated surface was allowed to touch the bottom of the beaker. Between experiments, the electrodes were thoroughly washed with a jet of distilled water and the surface was gently wiped with a tissue. It was noticed, however, that the coated surface was somewhat sticky. Application to urine and BSA solutions Uric acid or urate is, like ascorbate, a common interfering substance in biological fluids; it is elec-
102 105
3’
4s 5s 102 108
108 162 145
Ab
Response ratio (W) after (A) and before (B) exposure. Immediately after exposure. Four days after exposure. Before exposure. 24 h after exposure. ’ Uncoated electrode. E Coated electrode.
a b ’ d ’
39 63 30
63 81 44
97 94
BC
Ab
To glucose
To ascorbate
100 103
108 141 102
BE
0.05 0.05
0.01 0.01 0.01
Cd
0.4 0.2
0.15 0.4 0.25
DC
0.08 0.08
0.1 0.25 0.15
B’
4.0 2.0
0.3 0.3 0.3
Cd
6 5
5 5 5
Ab
4 2
4 4 4
BC
Time to steady background (s)
Background current ( p A)
CME
Response a (W)
CMCPEs exposed to undiluted urine for 1 b
1
Responses to 27 mM glucose and 0.68 mM ascorbate and background currents for three uncoated and two six-times-coated
TABLE
CARBON
PASTE
GLUCOSE
29
ELECTRODE
trochemically active in the investigated potential range [ll]. The responses of the uncoated CMCPEs to both 0.25 and 0.5 mM urate were only about 30% of the responses to equal concentrations of ascorbate. Because of its anionic nature at pH 7.0, the response at the coated electrodes was very small, less than 10% of the response obtained at the uncoated electrodes. Neither the response to glucose nor that to ascorbate decreased after the CMCPEs had been subjected to urate oxidation. Other investigations have shown that urate oxidation causes fouling of the electrode surface [5]. It could not be found here. When the electrodes were subjected to fresh urine samples diluted a hundred times, the response of the coated electrode was about 20% of that of the uncoated electrode. From the results obtained with the small anionic molecules, ascorbate and ureate, the ratio of the responses of the coated and uncoated electrodes should be about 1 : 10. The probable reason that the response of the coated electrodes in urine is 20% of that of the uncoated electrodes is that urine also contains neutral and cationic species that can be directly oxidized electrochemically. Calibrating the uncoated and coated CMCPEs for glucose and ascorbate directly after the experiments with diluted urine samples did not result in any response changes. Coated and uncoated electrodes were stored at open circuit in undiluted fresh urine for 1 h. They were then thoroughly washed and recalibrated for glucose and ascorbate. A somewhat larger background current was noticed, as well as an increase in the time necessary to reach a steady background current level (Table 1). While the uncoated electrodes showed a noticeable and immediate decrease in the response to glucose and also an increase in the response to ascorbate, the coated electrodes seemed to be largely unaffected by the exposure to the urine. These response changes displayed by the uncoated electrodes remained after storage for a few days in buffer (see Table 1) with an even more pronounced decrease in glucose response and virtually no increase in the response to ascorbate. Uncoated and coated electrodes were also calibrated for ascorbate and glucose in 2.5%
TABLE
2
Responses to 27 mM glucose and 0.68 mM ascorbate uncoated and three 6-times-coated CMCPEs exposed CME
Response
a (W)
To glucose Ab
of three to BSA
To ascorbate B”
Ab
B’
3d
86 81 84
75 77 78
82 89 85
77 92 79
4e 5= 6e
98 99 101
98 99 100
100 100 98
100 102 101
a See footnote to Table 1. b After exposure to 2.5% BSA/buffer solution an applied potential of + 300 mV. ’ After storage in 5% BSA/buffer solution overnight at 4O C. d Uncoated electrode. e Coated electrode.
for 10 mm with at open
circuit
BSA/buffer solutions. No increase in background current level was observed compared to a pure buffer background. The uncoated electrodes immediately lost some 15-20% of the glucose response and 10-15s of the ascorbate response, which clearly reveals the effects of some components in the BSA solution. The coated electrodes, however, showed no decrease in response on exposure to the BSA solution. After the uncoated electrodes had been soaked in 5% BSA solution overnight, the response to glucose and to ascorbate was further lowered to about 70% of their original responses. The fact that both the glucose and the ascorbate responses decreased is probably due to precipitation of some BSA onto the electrode. The apparent Ksp value for glucose oxidase also increased to about 100 mM for the uncoated electrodes. Conclusion These studies demonstrate that the ion-exchange characteristics and transport properties of water-cast Eastman AQ-29D poly(ester-sulfonic acid) make it ideal for use as a protective membrane on enzyme-based glucose sensors. Because the polymer is cast from an aqueous dispersion, and not from a partially organic solvent, enzyme activity is easily maintained on coating. The
30
L. GORTON
material improves the performance of redox polymer/ glucose oxidase/ carbon-paste electrodes, through the exclusion of anionic interferents and the prevention of electrode fouling by biological agents. In addition, sensor lifetime is increased substantially, because glucose oxidase is no longer free to diffuse away from the electrode. The polymer membrane also improves the useful concentration range of the glucose sensor with no decrease in response current. Work is presently underway to extend these studies by investigating other Eastman polymers in similar electrode configurations. This investigation was financially supported by the Swedish Board for Technical Development (STUF), the Swedish National Energy Administration (STEV), and the U.S. Department of Energy, Division of Material Science, Office of Basic Energy Science. T.I. is the recipient of a research fellowship from UBE Industries. The gift of the Eastman AQ-29D from Professor J. Wang is gratefully acknowledged.
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