Articles Anal. Chem. 1994,66, 2451-2457
“Wired” Enzyme Electrodes for Amperometric Determination of Glucose or Lactate in the Presence of Interfering Substances Tlmothy J. Ohara, Ravl RaJagopalan, and Adam Heller’ Department of Chemical Engineering, Universiw of Texas at Austin, Austin, Texas 78712- 1062
Glucose oxidase (COX) or lactate oxidase (LOX) were immobilized in an osmium-based three-dimensional redox hydrogel that electrically connected the enzyme’s redox centers to electrodes. The enzyme “wiring”hydrogel was formed by cross-linkingpoly( 1-vinylimidazole)(PVI) complexedwith Os(4,4‘-dimethylbpy)2CI (termed PVIls-dmeOs) with poly(ethylene glycol) diglycidyl ether (peg). Glucose and lactate sensors exhibited typical limiting current densities of 250 and 500 pAlcm2, respectively. When the electrodes were poised at 200 mV (SCE),the currents resulting from electrooxidation of ascorbate, urate, acetaminophen, and L-cysteine were negligible. When a Nafion film was employed, the linear range was extended from 6 to 30 mM glucose and from 4 to 7 mM lactate. The redox potential of the gel-forming polymer was 95 mV (Sa). Glucose and lactate measurements performed in bovine phosphate calf serum buffer.correlated well with a substrate calibration Electrooxidizable constituents of blood such as ascorbate, urate, and cysteine often interfere with glucose and lactate assays. Membranes that are permeable to the analyte but not to interferants are commonly used to improve the selectivity of enzyme electrodes. It has been suggested that NafionlJ excludes negatively charged interferants such as ascorbate and urate, but not neutrals such as acetaminophen. Membranes having very small pores that are transparent to H202 but not to electrooxidizable interferants have also been used in biosensors that electrooxidize H ~ O Z . ~Interferants -~ have also been preoxidized, e.g., on a gold grid: a precolumn containing an oxidizing cupric complex,’ or on a cross-linked layer of horseradish peroxidase.8 The problem of interferant electrooxidation can also be alleviated by using a mediator with a sufficiently low redox potential to allow its reoxidation at potentials where interferant oxidation is slow both on the (1) Harrison, D. J.; Turner, R. F. B.; Baltcs, H.P. Anal. Chem. 1988,60,20022007. (2) Bindra, D. S.;Wilson, G. S.Anal. Chem. 1989, 61, 2566-2570. (3) Sasso, S.V.; Pierce, R. J.; Walla, R.;Yacynych, A. M. Anal. Chem. 1990,
62,
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electrode and by the oxidized redox mediator. Examples of such mediators are polymeric viologens9 and 1,l’-dimethylferrocene.10 Recently, we showed that electrodes modified with a crosslinked mixture formed of glucose oxidase (GOX), poly(1vinylimidazole) (PVI) complexed with Os(bpy)2Cl (termed PV1,-Os), and poly(ethy1ene glycol) diglycidyl ether (peg) did not electrooxidize at their operating potential of +0.4 V (SCE) ascorbate or acetaminophen at appreciable rates at high glucose concentration.’ Although the potential of PV1,Os was lower than that of the earlier used poly(4-vinylpyri-
1111-1117.
(4) Lobcl, E.; Rishpn, J. Anal. Chem. 1981,53, 51-53. ( 5 ) Palleschi, G.; Rahni, M. A. N.; Lubrano, G. J.; Ngwainbi, J. N.; Guilbault,
G. G. Anal. Biochem. 1986, 159, 114121. (6) Iktda, T.; Katasho, I.; Senda, M. Anal. Sci. 1985, 1 , 455-457. (7)Yao, T.; Kobayashi, Y.; Sato, M. Anal. Chim. Acru 1983, 153, 337-340. (8) Maidan, R.; Hcller, A. Anal. Chem. 1992, 64, 2889-2896.
0003-2700/94/0366-2451$04.50/0 @ 1994 American Chemical Society
(9) Hale, P.D.; Boguslavsky, L. I.; Skotheim, T. A. Mol. Crysr. Liq. Crysr. 1990, 190, 259-264. (10) Cass, A. E. G.; Davis, G.; Francis,G. D.; Hill, H. A. 0.; Aston, W. J.; Higgins, I. J.; Plotkh, E.V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984.56,
661-671.
(11) Ohara, T. J.; Rajagopalan, R.; Hcller, A. Anal. Chem. 1993,24,3512-3517.
Analytical Chemistry, Vol. 66, No. 15, August 1, 1994 2451
Electrodes. Rotating disk electrodes were prepared by embedding vitreous carbon rods (3-mm diameter, V-1 0, Atomergic) in a Teflon shroud using a low-viscosity epoxy (Polysciences, Catalog No. 01916). Stationary electrodes for use in a flow cell were prepared by embedding the vitreous carbon rod in a glass rod, using a two-component epoxy (Armstrong Adhesives, Kit A12). Unless stated otherwise, glucose electrodes were prepared by syringing a 4-pL aliquot of 10 mg/mL PVIls-dmeOs solution onto the electrode surface (0.07 1 cm2). Next, 0.8 pL of a 10 mg/mL (10 mM HEPES, pH = 8.1) solution of GOX was added onto the electrode and stirred with a syringe needle. Note that all enzyme concentrations are based on the total weight of the solid constituents. In the final step, 1.2 pL of a 2.5 mg/mL solution of peg was added to the electrode and stirred. The electrode was allowed to cure for at least 20 h under air. Similarly, a typical lactate electrode was prepared by using the same amount of PVI15-dmeOs with 1.6 pL of a 20 mg/mL (10 mM HEPES, pH = 8.1) solution of LOX and 2 pL of a 2.5 mg/mL solution of peg. After the solutions applied to the electrodes dried, a relatively smooth dark purple film formed containing small dispersed aggregates. For glucose electrodes coated with Nafion, a 5-pL aliquot of a 0.5% Nafion solution in 95% ethanol was syringed onto the electrode, and after allowing the film to air dry for about 10 min, an additional 5-pL aliquot of 0.5% Nafion was added. After drying an additional 10 min, the electrode was soaked overnight at 4 “ C in a 20 mM phosphate buffer with 0.1 M EXPERIMENTAL SECTION NaCl at pH 7.1. Lactate electrodes were prepared similarly, Chemicals. 1-Vinylimidazole (Aldrich), K2OsC16 (Johnson except that they were not soaked in phosphate buffer prior to Matthey), 4,4’-dimethyl-2,2’-dipyridyl (Aldrich, 4,4’-ditesting. Unlike the glucose electrodes, lactate electrodes were methylbpy), sodium HEPES (sodium 4-(2-hydroxyethyl)- 1prepared and tested after being coated with either one or two piperazineethanesulfonate) (Aldrich), o-dianisidine (Sigma), 5-pL aliquots of the Nafion solution. Nafion (Aldrich, 5% by weight in 90% lower aliphaticalcohols Measurements. Electrochemical measurements were perand 10% water), poly(ethy1ene glycol) diglycidyl ether formed with a Princeton Applied Research 175 universal (Polysciences, peg, Catalog No. 082 lo), bovine calf serum programmer, a Model 173 potentiostat and a Model 179digital (Sigma, Catalog No. S6648), lactate oxidase (Genzyme, EC coulometer or with a Princeton Applied Research Model 400 1.1.3.2) from Pediococcus species (40 units/mg of solid, 3 1.5% bipotentiostat. The signal was recorded on a Kipp and Zonen protein), peroxidase (Sigma, EC 1.11.1.7) from horseradish X-Y-Y’ recorder. Rotating disk electrode experiments were (type VI-A, 1100 units/mg), and glucose oxidase (Sigma, EC performed with a Pine Instruments AFMSRX rotator with 1.1.3.4) from Aspergillus niger (type X-S, 198 units/mg of an MSRS speed controller. All electrochemical measurements solid, 75% protein) were used as received. Os(4,4’-dimethwere performed using a 20 mM phosphate buffer (pH = 7.1) ylbpy)zC12 and poly( 1-vinylimidazole) (PVI) were prepared containing 0.1 M NaCl except for experiments in which the as described.l1,l3 dependence of the steady-state electrooxidation current on PVIls-dmeOs. The osmium-derivatized polymer was pH or on NaCl concentration was determined. In the prepared by a procedure similar to that of Forster and V O S , ~ ~ experiments where the pH was changed, dilute solutions where Os(4,4’-dimethylbpy)2Cl2(132 mg, 0.21 mmol) was (approximately 1 M) of HC1 or NaOH were added to 10 mM refluxed with PVI (200 mg, 2.1 mmol) in 200 mL of absolute phosphate (pH = 7.1, 0.1 M NaC1). In experiments where ethanol for 3 days under N2. After filtering the solution, the the NaCl concentration was varied, the solution had a pH of product was precipitated by adding the solution to 1.5 L of 7.1 and contained 10 mM phosphate. The experiments were rapidly stirred diethyl ether and separated by decanting. The performed either in a three-electrode cell with a rotating glassy osmium complex-derivatized polymer is referred to as PVI15carbon working electrode or in a flow cell. The rotating disk dmeOs, the subscript representing the number of vinylimiexperiments were run at room temperature in 125 mL of dazole units per Os(4,4’-dimethylbpy)2Cl.Calcd for PVII5phosphate buffer. This cell had a rotating glassy carbon : 54.86; C1, 3.27; dmeOs.7H20, C&l2H1 1 4 N 3 4 0 ~ 7 H 2 0C, working electrode, a saturated calomel reference electrode H, 5.95; N, 21.97; Os, 8.78. Found: C, 56.21; C1, 3.14; H, (SCE), and a platinum counter electrode, isolated from the 6.42; N, 20.96; Os, 8.66. bulk solution with a vycor frit (EG & G, Princeton Applied Research). All rotating disk experiments were performed at (12) Gregg, B.; Heller, A. J . Phys. Chem. 1991, 95, 597G5975. (13) Kober, E. M.; Caspar, J. V.; Sullivan, B. P.;Meyer, T.J . Inorg. Chem. 1988, 1000 rpm with N2-purged solutions unless noted otherwise. 27,4587498. All flow cell electrochemical measurements were performed (14) Forster, R. J.; Vos, J. G.Macromolecules 1990, 23, 4372-4377. dine)-based osmium redox polymer (POs-EA),I2the ascorbate electrooxidation current was still high at low glucose concentrations, and urate rapidly degraded the sensor’s response, possibly through its electrooxidation to a reactiveintermediate. In order to further lower the operating potential, the ligand 2,2’-bipyridyl of PV1,-Os was substituted by 4,4’-dimethyl2,2’-bipyridyl (Figure 1). This reduced the redox potential of the polymer by 105 mV, from 200 to 95 mV (SCE). As will be shown here, the active site of GOX and lactate oxidase (LOX) are well “wired” by the lower potential redox polymer, PVI complexed with 0~(4,4’-dimethylbpy)~Cl, termed PVI15-dmeOs. Upon peg cross-linking of a mixture of PVI15-dmeOs and GOX or LOX, a film that adhered well to vitreous carbon was formed. The film conducted electrons and was permeable to the substrates and products of the enzyme-catalyzed reactions. Glucose and lactate electrodes constructed with PVI15-dmeOs, peg, and GOX or LOX were selective for their respective analytes in the presence of physiological concentrations of ascorbate, urate, acetaminophen, and cysteine. When overcoated with a Nafion film, the already adequate selectivity did not further improve, but because the film reduced the mass transport of glucose and lactate, the linear sensing range was extended from 6 to 30 mM for glucose and from 4 to 7 mM for lactate. The Nafioncoated glucose and lactate electrodes performed satisfactorily in serum.
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Analj4~calChemistt-y,Vol. 66,No. 15, August 1, 1994
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under air at a flow rate of 1.4 mL/min. The solution was pumped with a peristaltic pump (Eppendorf, EVA-pump). The cell had a void volume of 1 mL, and the linear flow velocity was 2.5 cm/min. The flow cell consisted of four glassy carbon working electrodes, a stainless steel counter electrode, and a Ag/AgCl reference electrode (3 M KC1saturated with AgC1). Serum samples were independently analyzed for glucose using a Yellow Springs Instrument Model 23A glucose analyzer. Serum lactate levels were measured spectrophotometrically, using a reagent solution containing 400 units/L LOX, 2400 units/L peroxidase, and 0.1 mg/mL o-dianisidine in a 20 mM phosphate buffer (0.1 M NaCl, pH = 7.1). A total of 10 pL of the sample solution was added to 1000 pL of the reagent solution, and after 5 min the absorbance was measured at 440 nm.
RESULTS AND DISCUSSION Cyclic Voltammetry of Cross-Linked PVIls-dmeOs Films Containing COX or LOX. The cyclic voltammogram of the film-coated rotating disk electrodes showed a half-wave potential of 95 mV (SCE) at 1 mV/s scan rate (Figure 2a). The peak separation for both cyclic voltammograms was 30 mV at a 1 mV/s scan rate. Upon the adding of substrate for electrodes rotated at 1000 rpm under Nz, the usual catalytic electrooxidation wave was observed (Figure 2b). Steady-State Amperometric Response of Cross-Linked PVIls-dmeOsFilms to Substrate. Figure 3 shows the potential dependence of the glucose and lactate electrooxidation current for rotating disk electrodes under Nz. The curves had a sigmoidal shape with an inflection point coinciding with the redox potential of PVIls-dmeOs. The glucose and lactate electrooxidation currents reached a plateau at 200 mV and 250 mV (SCE), respectively. In order to reach high glucose and lactate electrooxidation currents and at the same time minimize the electrooxidation of interferants, the electrodes were poised at 200 mV (SCE) in all experiments. A series of enzyme electrodes was prepared with a fixed amount of PVIls-dmeOs (40 pg), a constant percentage of peg (6% of the total weight), and varying enzyme amounts ranging from 0.6 to 180 pg. The limiting current densities for these glucoseor lactate electrodes measured with a rotating disk electrode under Nz at 48 mM glucose or at 10mM lactate
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concentrations increased initially with the weight fraction of the enzyme and then declined (Figure 4). In the rising part of the curve, the current was controlled by the enzyme activity in the film. In the declining part, it became limited by electron transfer to or through the polymer. Apparently, the glucose or lactate concentration in the redox hydrogel did not control the current. Both glucose and lactate are water soluble and expected to permeate through the film at high rates. Earlier measurements of benzoquinone/ hydroquinone permeation in related films showed that mass transport did not limit the current. 1 1312 The highest limiting current density was observed for glucose and lactate electrodes at 12 and 19 wt % of enzyme, respectively. The maximum limiting currents observed for glucose and lactate electrodes, observed for the non-methylated Os(bpy)zCl complex of PVI polymers, were at 32 and 22 wt % of enzyme p r ~ t e i n . ~ *The J ~ enzyme wt % represents the amount of protein in the enzyme preparation. The GOX and LOX sample preparations used contained 75% and 3 1.5% of protein, respectively. The optimal enzyme weight fraction was the same in the case of the lactate electrode for both the (15) Ohara, T.J. Unpublished results.
Anal~lcalChemlstry,Vol. 66, No. 15, August I, 1994
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methylated and non-methylated PVI-based osmium polymers. Note the major difference between the enzyme weight fractions where the highest glucose electrooxidation currents were observed at 12 wt % for PVIlsdme-Os and 32 wt % for PV1,Os. The maximum current density for lactate was twice that for glucose, which was surprising considering that the specific activity of the LOX preparation used was only half that of the GOX preparation. This difference suggests a more favorable electron-transferring interaction of LOX with PVI15-dmeOs. Figure 5a shows a typical calibration curve measured in a flow system under air for electrodes prepared with PVI15dmeOs, peg, and either GOX or LOX. The response to substrate is nearly linear up to 6 mM glucose and to 4 mM lactate at a flow rate of 1.4 mL/min. In order to augment the linear range, a mass transport controlling Nafion film was cast onto the electrode from a 0.5% solution in 95% ethanol. The film extended the linear range to 30 mM for glucose and to 7 mM for lactate (Figure 5b). The glucose electrode was prepared with two 5-pL aliquots of Nafion ( O S % ) , whereas the lactate electrode was prepared with only one 5-pL aliquot. Treating the electrodes with 95% ethanol without Nafion did not change the linear range nor did it greatly decrease the current. Glucoseelectrodes treated with Nafion that were tested 10 min after being overcoated showed an increase in linear range from 6 to 15 mM glucose. Upon soaking overnight in phosphate buffer, their linear range was further extended to 30 mM glucose. The linear range then remained steady. In contrast, the linear range of the Nafion-overcoated lactate electrodes did not change when they were soaked in phosphate buffer overnight. Therefore, the lactate electrodes were used immediately after their coating with Nafion. OxygenCompetition. 0 2 , the natural co-substrate for GOX and LOX, competes for electrons from the substrate-reduced active site causing the electrooxidation current to drop. Figure 6 shows the decline in the glucose and lactate electrooxidation currents with rotating disk electrodes at 1000 rpm when the N2 atmosphere was replaced by air. The catalytic current increased by a factor of 2 at low substrate concentration (i.e., 2454
AnalyticalChemistry, Vol. 66, No. 15, August 1, 1994
2 mM glucose or 1mM lactate) when the solution was switched from air to N2, but the change was smaller at higher substrate concentrations and became negligible at very high substrate concentrations. Current Density Dependence on the Osmium Loading. Figure 7 shows the dependence of the substrate electrooxidation current for rotating disk electrodes (1000 rpm, under N2) on the osmium loading at 48 mM glucose and 10 mM lactate in a series of electrodes in which the enzyme weight fraction (GOX or LOX) and peg were held constant with respect to the amount of osmium polymer. Assuming a density of 1 g/cm3, the thickness of the films on the electrodes ranged from approximately 0.3 to 10 pm. The catalytic current increased initially with osmium loading, then leveled off, in a manner similar to that found in the higher redox potential PV1,-Os based electrodes.” pH and NaCl Concentration Dependence of Substrate ElectrooxidationCurrents. Figure 8 shows the pH dependence of thecatalytic currents for both glucose and lactate electrodes rotated at 1000 rpm under Nz. The curves exhibit a plateau extending from pH 6 to pH 10 for both electrodes. Current was lost irreversibly at pH > 10. The change in current was, however, reversible between pH 5 and pH 7. In 02-requiring glucose or lactate assays involving GOX or LOX, the activity peaks at pH 5.5 and 7.5, respectively, and unlike in the present system, the response drops rapidly on both sides of the peaks. The glucose and lactate electrooxidation limiting currents that were measured with rotating disk electrodes at 1000rpm under N2 decreased with increasing NaCl concentration (Figure 9). As discussed previously, the anions screened the charges on the polycationic redox polymer.l1J6 Consequently, the polycation, which was extended at low ionic strength, coiled (“balled up”) when the repulsion between its positively charged centers was reduced by screening. Both the rate of electron transfer to the polycation from the reduced enzyme and the rate of electron diffusion through the cross-linked polyelectrolyte are reduced when the polymer undergoes transformation from its extended chain configuration to its coiled form. Effect of Interferants. Electrodes constructed with PVIISdmeOs, peg, and GOX or LOX showed the best analyte selectivity in the presence of interferants among all the wired enzyme electrodes built thus far in our laboratory.” The electrooxidation currents for glucose and lactate electrodes without Nafion overcoating were measured sequentially in a flow cell (1.4 mL/min) for solutions under air in the following order: first, for the solution of the interferant itself; second, for the interferant with either 6 mM glucose or 2 mM lactate; third, for either 6 mM glucose or 2 mM lactate without any interferant (Tables 1 and 2). The electrooxidation currents were not appreciable (
