Anal. Chem. 1994,66, 1007-1011
Enzyme Microelectrode Array Strips for Glucose and Lactate Joseph Wang' and Qiang Chen Department of Chemistv and Biochemistty, New Mexico State Universi?y, Las Cruces, New Mexico 88003 Microdisk arrays, prepared by combination of thick-film technology and laser micromachining procedures, serve as hosts for glucose oxidase and lactate oxidase, to yield effective amperometrictest stripsfor glucose and lactate. The enzymes are localized electrochemically within the pores of the 15-pm disks via codeposition with rhodium or platinum. Scanning electron microscopy elucidates the unique growth patterns of the metalized enzyme layer within the individual laser-drilled holes. The strong and preferential catalytic action of the rhodium particles toward the oxidation of the enzymatically liberated hydrogen peroxide offers highly selective monitoring of the corresponding substrate and eliminates the need for membrane barriers. The high selectivity is coupled to high sensitivity accrued from the array character and large microscopic area of the individual disks. Such a simple one-step electrochemical immobilization onto microelectrode arrays offers fast and sensitive measurements from small sample volumes and can be used to mass produce reliable enzymebased diagnostic strips. The coupling of the inherent specificity of biocatalytic recognitionprocesses with the high sensitivityof amperometric transducers makes amperometric enzyme electrodespractical devices for routine clinical, environmental, or industrial applications. Particularly attractive for many decentralized testings are "one-shot" disposable enzyme electrodes. For example,commercial dry-reagent sensor strip are widely used by diabetic patients for self-monitoringof their blood glucose 1evels.lJ The fabrication of these and similar sensors is commonly accomplished by microelectronictechnologies, such as screen-printing or photolithographic schemes, which are suitable for large-scale mass p r o d ~ c t i o n . The ~ ~ ~ enzyme immobilization is accomplished by various means, including covalent binding, cross-linking, and entrapment in a gel or polymer. Innovative routes for preparing disposable enzyme electrodes are highly desired. The present article describes an effective and attractive approach for fabricating amperometric enzyme strips. Such strategy relies on the entrapment of the enzyme within the microdisk pores of newly introduced screen-printed composite electrodes. These disposable composite strips (from Ecosse Sensors Ltd., Edinburgh, UK) are fabricated by combination of thick-film printing and laser machining techniques to produce highly reproducible microdisk arrays.' For this purpose, the electrodematerial is first printed onto the substrate (1) (2) (3) (4) (5)
Matthews, D. Lancet 1987, 4, 778. Lewis, B. D. Clin. Chem. 1992, 38, 2093. Alvarez-Icaza, M.; Bilitewski, U. Anal. Chem. 1993, 65, 525A. Rudenziati, M.; Morten, B. Sens. Actuators 1986, 10, 65. Microdlsc Array Electrode; Ecosse Sensors: Edinburgh, U. K., 1992.
0003-2700/94/0368-1007$04.50/0 0 1994 Amerlcan Chemical Society
(via vapor deposition), then covered with a coating of a dielectric layer, and exposed to laser photoablation (in connection with projection masking) to 'drill" the desired array pattern through the insulator film. The resulting 15pm-diameter circular working electrode holes of the composite strips are used in the present work as templates for the immobilized enzyme. Model enzymes are thus electrochemically localized within the microholes via codeposition with rhodium or platinum or by entrapment in an electropolymerized film. The procedure for preparing the enzyme microelectrode array strips is summarized schematically in Figure 1 and discussed in detail in the following sections. The incorporationof enzymes within a growing metal layer has been used before for the localization of enzymes onto miniaturized surfaces (such as carbon fiber microcylinders).6*7 The use of rhodium is shown in the present study to offer not only efficient enzyme retention but also an efficient (and preferential) electrocatalytic action toward the anodic detection of the enzymatically liberated hydrogen peroxide. A highly selective glucose and lactate sensing is thus accomplished, without the need for permselective layer@)or redox mediators. The characterization and optimization of rhodinized enzyme composite electrode strips that couple the attractive properties of microelectrode arrays, disposable devices, and efficient electrocatalytic action are described in the following sections.
EXPER I MENTAL SECT1ON Apparatus. Amperometric and chronoamperometric experiments were performed with a BAS CV27 voltammetric analyzer (Bioanalytical Systems (BAS), West Lafayette, IN) in connectionwith a BAS X-Y-t recorder. Thescreen-printed carbon microdisk arrays electrodes were purchased from Ecosse Sensors (Edinburgh, U.K.). These strips consist of planar working (array) and reference (Ag/AgCl) electrodes on a 20 X 50 mm2plastic support. The semicircular reference electrode surrounds the disk-shaped array, with electrical contacts provided on the opposite side. Chronoamperometric experiments were performed by placing 200-pL sample drops on the strip (to cover the working and reference electrodes) and measuring the current 30 s following the potential step. Cyclic voltammetric and amperometric data were obtained using a 10-mL cell (Model VC-2, BAS), and a three-electrode system. Scanning electron micrographs were obtained with a Phillips Model 501B microscope, using an accelerating voltage of 7.2 kV. (6) Ihriyama, Y.; Yamauchi, S.;Yukiashi, T.; Ushioda, H. J . Electrochem. Soc. 1989, 136, 702. ( 7 ) Wang, J.; Angnes, L. Anal. Chem. 1992, 64,456.
Analytical Chemistry, Vol. 66,No. 7, April 1, 1994
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ielectric layer
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Flgure 1. Schematlc diagram of the procedure used to prepare the metalized enzyme microe-ode array strip.
Reagents. All solutions were prepared with deionized water. Glucose oxidase (EC 1.1.3.4, 166 100 units/g, from Aspergillus niger) and lactateoxidase (EC 1.1.3.2,40 000units/g from Pediococcus species) were received from Sigma. Hydrogen peroxide, uric acid, L-ascorbic acid, glucose, urea, tyrosine, glutathione, salicylic acid, lactic acid (lithium salt), acetaminophen,and o-phenylenediaminewere also purchased from Sigma. Rhodium and platinum solutions (atomic absorptionstandards, 1000ppm) were obtained from Aldrich. The supporting electrolyte solution was a 0.05 M phosphate buffer (pH 7.4) containing 0.03 M NaCl. Electrode Modification. The enzyme/metal deposition was accomplished by placing a 200-pL drop, containing 100 ppm of the metal (Rh or Pt), 332 units (2 mg) of glucose oxidase, or 50 units (1.3 mg) of lactate oxidase, as well as 0.03 M NaCl, on the sensor strip. The solution pH was adjusted to 5.2 or 4.8 (for Rh and Pt, respectively), with sodium hydroxide (before adding the enzyme). The potentiostatic codeposition proceeded for 15 min at a potential of -0.8 V. The resulting enzyme strips were rinsed with deionized water and stored in phosphate buffer (at 4 “C). The Rh- and Ptcoated arrays were prepared in a similar fashion, but in the absence of an enzyme. Glucose oxidase/poly(o-phen ylenediamine) deposits were electrochemicallygrown within the pores of the array using 200-pL drops, containing 5 X M o-phenylenediamine, 332 units of glucose oxidase, 0.05 M acetate buffer (pH 5.2), and 0.03 M NaCl. Electropolymerization proceeded for 20 min at +0.65 V (vs Ag/AgCl). The electrode was then rinsed with deionized water and stored in phosphate buffer (pH 7.4). RESULTS AND DISCUSSION Scanning Electron Microscopy. The growth patterns of the metalized and metal/enzyme deposits within the working electrode microholes of the printed compositestrips have been elucidated by scanning electron microscopy. Figure 2 shows typical micrographs, obtained with a 2500X magnification and 7.2-kV accelerating voltage, for one of the array microdisks, before any deposition (A) and after depositing the rhodium (B) and rhodium/glucose oxidase (Rh/GOx) (C). The micrograph for the naked microdisk illustrates a nearly circular hole of ca. 16-pm diameter in the dielectric layer (resulting from the laser “drilling”), which exposes the 1008 Am&thlChem&iry, Vol. 66, No. 7, April 1, 1994
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C
Flgure2. Scanning electron micrographs of a typical microdisk before (A) and after depositing the rhodium (B)and rhodium/glucoseoxidase (C). Magnification, 2500X
printed carbon layer. Both the rhodium and the rhodium/ enzymeare deposited within this microhole, with their growth occurring primarily over the inner walls. Apparently, the cavity walls support the metal/enzyme layer growing on the carbon substrate. Note, however, the significantly different morphologies of the deposits emerging from the microhole (B vs C). While the rhodium-coated surface appears as a ring consisting of small closely packed metal microparticles ( 0.51.O-pm diameter), the rhodium/enzyme deposit is composed of larger aggregates (-2-5-pm diameter). Such structural differencesare attributed to the effect of the enzyme upon the electrodeposition of rhodium. Apparently,an initial adsorption of the enzyme on the carbon substrate reduced the availability of rhodium nucleation sites and consequently yielded larger rhodium particles. Such explanation is supported by the deposition current vs time profiles shown in Figure 3. These indicate significant depression of the rhodinization process during the initial stage (-2 min) of thedeposition (solid line),
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TIME (min) Flgure 3. 7me course of the rhodization process in the presenceand absence of GOx (solid and dotted lines, respectively). The deposition conditions are ghren in the Experimental Section. Flgure5. Effect of the operatingpotentlaluponthechronoamperomebjc responsefor 2 X lo-* M glucose at the PpD/GOx (H), Pt/GOx (A),and Rh/GOx (0)screenprinted array electrodes. Solution, 200-pL drop of 0.05 M phosphate buffer (pH 7.4) containing 0.03 M sodium chloride.
1
Flgwe4. Scanning electronmicrographof a microdisk after depositing platinum/glucose oxidase. Magnification, 2500X
as compared to a large initial current spike observed without theenzyme (dotted line). In both cases, the deposition current subsequently increases, indicating a gradual increase in the surface area. Despite the different profiles, the total charge involved in the rhodinization process is very similar in the presence and absence of GOx (25.26 vs 25.57 mC, respectively). Different morphologies were reported for the electrodeposition of rhodium and rhodium/enzyme onto carbon fiber micro cylinder^.^ It is not fully clear from the scanningelectron micrograph whether the enzyme is incorporated/entrapped within the rhodium matrix or simply adsorbed on the surface of the microparticles. The porous rhodium microstructure results in a huge increase in the microscopic area (over the geometric area of the naked carbon disk) and hence yields largeanalytical signals (see discussion below). A SEM image of a larger (250 X 250 pm2) area illustrated a regular pattern of the enzyme microdisks, each located 10 diameters from its nearest neighbor (not shown). Scanning electron microscopy was used also to image the platinized glucose oxidase microdisk array (Figure 4). While the Pt/GOx also grows (and supported) on the inner walls of the microcavity, it has a morphology different from the Rh/GOx one (i.e., a layered structure vs the large aggregates of Figure 2C). Amperometric Biosensing. Electropolymerization represents another useful avenue for depositing enzymes within the microcavitiesof the screen-printed array. Figure 5 compares hydrodynamic voltammograms(HDVs) for 2 X 1O-*M glucose at the platinized (A),rhodinized (O), and electropolymerized
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(m) enzyme strips. As expected from the absence of electrocatalyticactivity, the electropolymerizedsensor array offers detection of the liberated hydrogen peroxideonly at potentials higher than + O S V. In contrast, glucose can be detected at substantially lower potentials at the metalized enzyme strips. With the rhodinized and platinized surfaces, such detection starts at +0.05 and +O. 15 V, respectively, and reaches a plateau at +0.46 and +0.60 V. Note also the greatly enhanced sensitivity in comparison to the electropolymerized enzyme layer (e.g., current enhancements of 12 (Rh) and 14 (Pt) at +0.80 V), as expected from the large microscopic area of the metalized surfaces. Such surfaces were thus employed in all subsequent analytical work. In addition to greatly enhanced sensitivity, the significant lowering of the overvoltage-accrued from the use of rhodinized surfaces-results in very high specificity toward the glucose substrate. Such selectivity improvements can be understood from cyclic voltammograms for several relevant compoundsat an “enzyme-free” rhodinized screen-printedstrip (Figure 6). While the oxidation of hydrogen peroxide at the rhodium sensor starts at +0.05 V (a), an anodic response for ascorbic acid (b), acetaminophen (c), or uric acid (d) is observed only above +O. 10, +0.25, and +0.30 V, respectively. With the naked (unmodified) carbon microelectrode array, the oxidation of hydrogen peroxide, ascorbic acid, acetaminophen, and uric acid starts at 0.65, 0.12, 0.30, and 0.31, respectively(not shown). Hence, the rhodium-coated surface preferentially catalyzesthe redox activity of hydrogen peroxide, with -600 mV lowering of the overvoltage, as compared to 20-50-mV decreases for the otherwise common electroactive interferences. (Notice also that the cathodic response for hydrogen peroxide also starts around 0.05 V.) Such catalytic action is of great significance, sincethe platinized array yielded a lower oxidation potential for ascorbic acid than that of hydrogen peroxide (not shown). It should be pointed out also that no differencein the catalytic properties towards hydrogen peroxide was observed at the two rhodium structures; similar peroxide voltammogramswere obtained at the rhodinized strip in the absenceand presence of GOx (not shown). Denaturation of the enzyme (by dipping the strip into boiling water) also did not influence the cyclic voltammetric behavior. Also noteworthy is the fact that direct oxidation of glucose is not Ana&tkalChemisby, Vol. 66, No. 7, April 1, 1994
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observed at the rhodinized surface (Figure 6e). Hence, the reported analytical data are attributed to the enzyme-catalyzed reaction, and not to a mixed enzymatic/nonenzymatic response,as is common for platinized glucose biosensors.* Cyclic voltammograms were recorded for additional relevant (electroactive endogeneous) compounds, including glutathione, salicylic acid, tyrosine, and urea (not shown). With the exception of tyrosine, which displayed oxidation at potentials higher than +0.30 V, these compounds did not exhibit any redox activity over the -0.2 to +0.5 V potential range. The biosensing implications of the electrocatalytic action of a rhodium-coated strip are illustrated in the selectivity experiments of Figure 7. This figure shows the effect of electroactive compounds such as ascorbic acid ( 2 ) , acetaminophen (3), and uric acid (4) upon the response to glucose (1) at Pt/GOx (A) and Rh/GOx (B) screen-printed array electrodes poised at different operating potentials (0.20-0.50 and 0.1 5-0.30 V, respectively). In the case of the platinized biosensor, the response for the substrate is accompanied by large contributions of the three oxidizable compounds. While the acetaminophen and uric acid currents are minimized at potentials lower than 0.40 V (Figure 7Ac-e), the glucose response is also compromised, and a large ascorbic acid interference is maintained. Indeed, at +0.20 V (Figure 7Ae), the signal of glucose nearly disappears while ascorbic acid yields a large response. Such interference is in agreement with the strong catalytic action of the platinized surface towards both ascorbic acid and hydrogen peroxide, with a lower overvoltage of ascorbic acid. In contrast, and as expected from the current/potential profiles of Figures 5 and 6, a careful tuning of the potential applied to the Rh/GOx strip (to the rising portion of the HDV, Le., in the 0.20-0.25-V region, Figure 7Bb, c) offers a well-defined glucose response, with negligible contributions from the oxidizable species. Overall, (8) Ikariyama, Y.; Yamauchi, S.:Yukiashi, T.; Ushioda, H. J . Elecrrounal. Chem. 1988, 251, 267.
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TIME Flgure 7. Currenthime recordlngs at the Pt/GOx (A) and Rh/GOx (B) screen-printed array electrodes for addltlons of 5 X lo4 M glucose (l), 1 X lo-' M ascorbic acid (2), 1 X lo4 acetaminophen (3), and 1 X lo-' M uric acid (4). Operating potential: (A) 0.50 (a), 0.40 (b), 0.30 (c), 0.25 (d), and 0.20 (e) V; (e) 0.30 (a), 0.25 (b), 0.20 (c), and 0.15 (d) V. A 10-mL stirred solution was employed.
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Flgure 6. Chronoamperometric response of the Rh/GOx (A) and Pt/ GOx (B) screen-printed array electrodes to successive concentration M glucose. Potential step to 4-0.45 (A) and increments of 5 X 4-0.50(8) V. Other conditions as in Figure 5.
thedata of Figures 6 and 7 indicate that the rhodiumdeposition greatly enhances the selectivity of screen-printed biosensors. The preferential electrocatalytic action of the rhodium coating should greatly simplify the large-scale fabrication of oxidasebased bioprobes, as it obviates the needs for permselective coatings or redox mediators (used otherwise for alleviating electroactive interferences). Figure 8 displays the effect of the substrate concentration upon the chronoamperometricresponse of the rhodinized (A) and platinized (B) enzyme composite strips. Both biosensors M increments in the glucose respond favorably to the 5 X level. Also shown in Figure 8 (right) are the resulting calibration plots. As expected for biocatalytic reactions, and from the absence of supporting membranes, both bioelectrodes exhibit a restricted linear range, with a curvature in the M (Rh) and response for concentrations higher than 5 X lom3 1X M (Pt). Such difference reflects the corresponding apparent Michaelis-Menten constants and indicates differences in the entrapment of GOx within the growing rhodium and platinum matrices. Additional diffusional limitations
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Flgure 0. Short-term (A) and long-term (8)stabilities of the Rh/GOx strip chronoamperometricresponse to 1 X lo-*(A) and 2 X lo-*M (B) glucose at 4-0.30and 4-0.45V, respecthrely.Other Conditions as In Figure 5.
could be introduced (e.g., via polymeric coatings) to further extend the linear range. The short-term and long-term stabilities of the Rh/GOx microelectrode array are illustrated in Figure 9A and B, respectively. The former is evaluated in a seriesof 40 repetitive measurements of 1 X 10-2 M glucose. Such a prolonged experiment yielded a mean peak current of 115 nA, a range of 108-120 nA, and a relative standard deviation of 3.0%. The glucose strip exhibits a highly stable response for -40 days (with intermittent usage and storageat 4 OC in phosphate buffer). A sharp decrease in the sensitivity is observed during the 40-50-day period. Overall, the good stability indicates that the enzyme is entrapped within the metal deposit (and not adsorbed onto its surface). The concept of enzyme microelectrode array strips is not limited to monitoring of glucose. Other enzymes can be hosted within the defined pores of dielectriclayer, with oxidasesbeing particularly promising in connection with the rhodiumimmobilization scheme. For example, Figure 10 displays calibration (A) and reproducibility (B) data for lactate at the lactate-oxidase rhodinized sensor strip. A well-defined chronoamperometric response is observed (at +0.45 V) for the 5 X M increments in the substrate concentration; the estimated detection limit is 1.5 X le5M (S/N = 3). The
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Figure 10. Response to lactate at the RhlLOx screen-prlnted array electrode. (A)ChronoampemtrIc response to successh/eIncrements of 5 X M lactate (a+: (8)stabillty of the response for 2 X 10-4 M lactate. Potentlal step to 4-0.45V. Other conditions as In Figure 5.
response is also highly reproducible; a relative standard deviation of 1.8% characterizes the series of 25 repetitive measurements of 5 X 1 V M lactate. Only small contributions (of less than 5%) to the 2 X M lactate response were observed in selectivity experiments in the presence of 1 X 1 V M ascorbic acid, uric acid, or acetaminophen (potential step to +0.25 V; not shown). Considering the physiologicalranges of lactate and ascorbic acid (3 X 10-4-1.5 X lo-) and 3 X 10-5-1.1 X 10-4 M,respectively), the error due to ascorbic acid would be very small. In conclusion,the microcavities of screen-printed electrode arrays were used as templates for electrochemically grown enzyme microdeposits. The rhodium codeposition scheme is particularly attractive for this task, as it offers not only a useful retention of the enzyme within the micropores but also a preferential electrocatalytic detection of liberated peroxide speciesand, hence, highly selectiveglucose sensing. Additional work is required to understand the mechanism of this catalytic action. The array character and large microscopic area also offer high sensitivity. The resulting strips thus hold great promise for diagnostic self-testing of glucose and lactate. Recelved for review October 5, 1993. Accepted January 18,
1994.' Abstract published in Aduonce ACS Abstracu. February 15, 1994.
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