Anal. Chem. 1990, 62, 1106-1110
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Electrochemical Platinization of Reticulated Vitreous Carbon Electrodes To Increase Biosensor Response George
H.Heider,' Sylvia V. Sasso,2 Ke-ming Huang? and Alexander M.Yacynych*
Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 Henry J. Wieck2
Department of Chemistry and Physics, Kean College, Union, New Jersey 07083
Platlnlzed reHcukhd vltnaffi carbon (RVC) was used In the construction of an lmmobluzrd eneyme, chemkaUy moWed electrode sultable for flow Inanalyds (FIA) appllcatlons. The platlnlzed surface ylelds an increased current response due to the oxldatlon of hydrogen peroxlde as compared with nonplatlnlzed RVC. It also allows the electrode to operate at a much lower potential (+0.6 vs 4-03 V vs saturatedcakmel deck0de)ttWrs rewlhghl lncrsared !3lgnai to nolse. Platlnwn Is electrodeposited on only part of the electrode, allowing enzyme bMnobUlzation directly to the carbonaceous swfece. lhls system #em the advanteges of a platinum surface for electrochemical detection and a oarbon surface for enzyme ImmoMHzatlon, on the same electrode. The actlvity of the glucose oxidase immobiked on the plstMzed eleottode is very almller to that of the nonplatkrlzed electrode. The Unear range of the electrode is 0-50 mM glucose using a 5-pL sample volume.
INTRODUCTION The purpose of this paper to explore the possibilities of using electrode modification techniques for the construction of biosensors. This type of biosensor would incorporate various microcomponents on the surface of an electrode that would act, in concert, for the determination of a desired analyte. The various components are added chemically, creating a new microenvironment at the electrode surface. This microenvironment is engineered to optimize the electrochemical characteristics of the base electrode. By appropriate control of the microenvironment of the immobilized enzyme, more desirable properties, such as increased stability, high sensitivity, fast response, expanded linear range, high sampling rate (for applications involving flow injection analysis), great flexibility in the choice of electrode dimensions and shape (allowing easy miniaturization or use of complex surfaces, such as reticulated vitreous carbon), prevention of electrode fouling, and interferences from other species in solution, can be achieved. An early use of reticulated vitreous carbon (RVC), as a working electrode for electrochemical analysis, employed a cylinder of the material to electrolyze several analytes in a flow injection analysis (FIA) system (1). A linear relationship existed between the current and the concentration of ascorbic acid in the ranges from 1 to 1000 MM. The electrochemical efficiency of detection, for 2 X 1W6M ascorbic acid, was 100%
* Author to whom correspondenceshould be addressed.
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Present address: IC1 Americas, Inc., Analytical and Physical Chemistry Section, Corporate Research Department, Wilmington, DE 19897. *Present address: I-Stat Corp., 303 College Farm Rd. East, Princeton, NJ 08540. Present address: Chemistry Teaching Group, Nanjing Agricultural College, Nanjing, Jiangsu, People's Republic of China. 0003-2700/90/0362-1106$02.50/0
at flow rates less than 2 mL/min. Blaedel and Wang applied theoretical aspects of flow electrolysis to RVC electrodes (2). Strohl and Curran (3)used a RVC working electrode as both a coulometric and amperometric detector in FIA. To achieve 100% electrochemical efficiency from their coulometric detector, slow flow rates were necessary, which limited the sampling rate to about 24 samples per hour. However, they showed that the detector could operate in an amperometric mode (without 100% electrolysis), providing a much greater sampling rate. In a review, Wang ( 4 ) summarizes the uses of RVC as an electrode as well as its physical, chemical, and electrochemical characteristics. Wang and Dewald developed a coulometric flow cell to remove interfering ions for on-line stripping analysis (5). Wieck, Shea, and Yacynych used RVC as a support for the immobilization d glucose oxidase (6). Later Wieck, Heider, and Yacynych (7)incorporated the electrode into a detector for flow injection determination of glucose. Most of the enzyme electrodes employed for the determination of glucose use a platinum surface for the electrochemical oxidation of hydrogen peroxide. Lobe1 and Rishpon employed a platinum wire mesh, positioned behind a membrane containing immobilized glucose oxidase, as an amperometric sensor to monitor glucose concentration (8). Yao used glucose oxidase, cross-linked with bovine serum albumin, immobilized onto a silanized platinum sheet as an amperometric detector for glucose in a flow system (9). Srinivasan et al. have reviewed the use of immobilized enzymes in analytical amperometric sensors (10). Hydrogen peroxide is not easily detected by oxidation at carbon electrodes. Ianniello and Yacynych showed that the linear sweep voltammetry oxidation wave produced by hydrogen peroxide, at a spectrwcopic grade graphite rod, is very dependent upon the pH of the solution (11). At low pH, the oxidation of hydrogen peroxide does not produce any type of distinguishable current plateau, this is attributed to its anodic shift into the region of solvent breakdown. The potential at which the plateau appears, in the pH range from 7.0 to 10.0, is +0.9 V versus a saturated calomel electrode (SCE). The main advantage realized by using a platinum surface instead of a carbon surface is that a lower applied potential can be used for the electrochemical oxidation of hydrogen peroxide. Lowering the background current enhances the signal-to-noise ratio for detection of glucose. Another advantage of a system operating at a lower applied potential is less susceptibility to electrochemical interferents. Recently, a combination of platinization and enzyme attachment has been used on carbon fibers (12),glassy carbon (13),and platinum (14). Also, zinc metal has been electrodeposited on RVC surfaces (15), and platinum can be deposited onto RVC similarly. Partially platinized RVC provides a catalytic surface for the electrochemical oxidation of hydrogen peroxide without losing the carbonaceous sites for covalent attachment of glucose oxidase. The superior elec0 1990 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1999
trochemical properties of the platinum-plated surface allows detection of hydrogen peroxide a t much lower applied potentials and provides an increased current response over a wider range of substrate concentrations. Glucose oxidase immobilized onto the surface of a partially platinized RVC electrode is the reactor/detector in a FIA system for glucose determinations. The activity of the enzyme immobilized on the platinized RVC is similar to that immobilized on a nonplatinized RVC electrode. EXPERIMENTAL SECTION Apparatus. The flow cell design was shown and previously described (16,17). The potentiostat used to characterize the electrode was either a PARC Model 364 or 264A polarographic analyzer (Princeton Applied Research, Princeton, NJ). All potentials are referenced to a SCE. The output was recorded on a Omnigraph Model 2000 X-Y recorder (Houston Instruments, Austin, TX). For flow injection experiments, the current output from the potentiostat was also directed to a Supergrator I1 integrator (Columbia Scientific Industries, Austin, TX), which measured both peak heights and peak areas. A Varian Model 8500 syringe pump (Varian Instruments, Palo Alto,CA) was used in a FIA system shown previously (18). Samples were introduced into the carrier stream through a Rheodyne Model 5020 or 7125 injection valve (Rheodyne, Inc., Cotati, CA) having either a 5-ctL or 10O-kL sample loop. The open structure of the RVC electrodes does not impose any back pressure on the syringe pump. To ensure the reproducibility of the flow rate and to eliminate slight pulsing from the syringe pump, it was necessary to add a packed silica column, between the pump and the RVC electrode, to increase the back pressure on the syringe pump. This also provided a means of forcing small air bubbles from the RVC when first filled with solution. Pressure was allowed to build up by interposing the silica column and increasing the flow rate of the pump to a maximum, and when sufficient pressure was attained, a tee valve was turned allowing the pressurized buffer to purge the air from the RVC electrode. With the air bubbles purged from the RVC, the surface area for electrolysis was more reproducible and at a maximum. The platinum was electrodeposited with an ECO Model 549 potenticetat/galvanostat (ECO Instruments, Inc., Cambridge, MA) and was characterized electrochemically by hydrodynamic voltammetry. The platinum coating on the RVC was further characterized by using a JEOL Model JSM-25S scanning electron microscope equipped with a Kevex Model 7000 Si(Li) energydispersive X-ray detector. A Rabbit peristaltic pump (Rainin Instruments, Woburn, MA) circulated the solutions through the RVC electrodes for the platinum plating, the enzyme attachment procedure, and the determination of the immobilized enzyme activity. Enzymatic assays were done by using a Perkin-Elmer Model 101 spectrophotometer (Perkin-Elmer Corp., Norwalk, CT) equipped with a flow cell (1cm path length). The absorbance was recorded on a Recordall strip-chart recorder (Fisher Scientific Supply, Springfield,NJ). Temperature studies were done in a water bath thermostated with a Lauda constant temperature immersion circulator, Model MT (Fisher). Reagents. Hydrogen hexachloroplatinate was obtained as a hydrate salt, reagent grade (Aldrich Chemical Co., Milwaukee, WI).Hydrogen peroxide was obtained as a 30% solution (Fisher). The exact concentration of the hydrogen peroxide solution was determined by titrating an aliquot with potassium permanganate, previously standardized with primary standard grade arsenic trioxide. The carbodiimide used in the immobilization procedure was 1-cyclohexyl-3-(2-morpholinoethyl)carbometho-p-toluenesulfonate obtained from Fluka Chemical Co. (Hauppage, NY) and was biochemical grade. The glucose oxidase (EC 1.1.3.4, type 11, from Aspergillus niger), peroxidase (EC 1.11.1.7, type 11, from horseradish), and o-dianisidinewere obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals were reagent grade, and distilled/deionized water was used for all experiments. Procedure. RVC electrodes (RVC-8OS, The Eledrosynthesis Co., Inc., East Amherst, NY) were pretreated electrochemically by cycling the potential between +1.5 and -1.5 V dowing a 10-min
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equilibration at the potential extremes. This pretreatment was done twice on each electrode before use to ensure reproducibility of the electrochemical response of the RVC (2). The composition of the plating solution was developed from one suggested by Blum and Hogaboom (19).A 0.025 M solution of hydrogen hexachloroplatinate(1V)hydrate was prepared in 50 mL of water. This was placed in a 500-mL beaker into which 100 mL of a 0.67 M solution of Na2HP04and 100 mL of a 0.33 M solution of (NH4)2HP04were added. The solution was heated at just below full boil until it turned a pale yellow. After the solution had cooled to room temperature, it was placed in a 250-mL volumetric flask and diluted to the mark with water. The final solution concentration of platinum was 5.3 mM, and the amount of solution used for each plating was 50 mL. The electrodeposition on RVC was done galvanostatidy using two elecrodea. The flow cell was connected to the peristaltic pump, which circulated the solution through the electrode at a flow rate of 5.0 mL/min. The surface area of each electrode was calculated by using the previously reported value of 0.17 m2/g for RVC (6). Due to the geometry of the flow cell, the platinum plating on the RVC was always greater on the end closest to the counter electrode. Thus the electrode is always mounted with this end downstream to maxi.mize response. If the RVC cylinder is reversed, the response is typically halved. The Plexiglas cartridges containing the RVC electrodes were then connected to a four-channel peristaltic pump for the immobilization procedure. Two platinized electrodes and two without platinum were used in each attachment procedure to determine if the platinum plating had any effect on the enzyme immobilization. The carbodiimide attachment scheme used to immobilize the glucose oxidase to the RVC surface has been previously reported (7). After completion of the attachment scheme, the activity of the immobilized enzyme was determined by using a modification of the standard assay procedure for glucose oxidase provided by Sigma. The o-dianisidine solution was prepared by first dissolving 13.2 mg of the solid in 2 mL of water, then taking 1mL of the resulting solution and diluting further to 100 mL with pH 5.1 sodium acetate buffer (0.05 M). A glucose standard was prepared by dissolving 1 g in 10 mL of water. The peroxidase solution containing approximately 60 Purpurogallin units/mL of water was also prepared. The Plexiglas cartridge was attached to the peristaltic pump at the solution exit side. The inlet side of the pump was attached to the outlet of the flow cell installed in the spectrophotometer. A 10-mL beaker was used to hold the assay solutions and contained a magnetic stirbar to assure adequate mixing. The blank solution was prepared by pipetting 4.80 mL of the o-dianisidine solution and 0.20 mL of the peroxidase solutions into the 10-mL beaker. The peristaltic pump was set for maximum flow rate (approximately 8 mL/min), and the blank solution was circulated through the flow cell and electrode. After the spectrophotometer was calibrated for zero absorbance, 1.00 mL of the glucose standard was added to the beaker. As the enzymatic reaction takes place, the hydrogen peroxide produced oxidizes the o-dianisidine to a red product that is detected by the spectrophotometer. The change in absorbance after 4 min is used to calculate the enzymatic activity on the electrode. Note that the value obtained for the assay does not provide any information on the total amount of enzyme immobilized on the electrode but only represents the active enzyme bound to the electrode. It is only useful as a relative comparison of enzymatic activities on electrodes of the same size. The electrodes were evaluated in the FIA system after the enzyme assays were completed. The applied potential at the RVC working electrode was +0.6 V vs SCE. RESULTS AND DISCUSSION The electrochemical and hydrodynamic properties of RVC have made the material well suited for use as a flow-through electrode ( 4 ) . Coulometric response has been obtained by using various flow cell designs incorporating this material as the working electrode (2,20). The cell design and electrode size were optimized by using potassium ferricyanide as a test analyte (21). A variety of electrode sizes were tested, and the best size was a length of 2 cm and a diameter of 3 mm. This size was a compromise
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ANALYTICAL CHEMISTRY. VOL. &?'NO. 11. JUNE 1. 1990
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Figure 2. Scanning electron micrograph of an RVC electrode plated the m e buffer used for the carrier stream, were (njected into for 18 h at 0.1 mA/cm2; maqnification 30X. the PIA svstem. The current resnonses ioeak heiphts) dne -. to the sample injections were recorded as'the potential was stepped from 0.0 to +1.0 V in +O.l-V increments. The largest enhancement of the current signal was obtained with the platinum coating applied at a current density of 0.1 mA/cm2 for 18 h. On completion of the plating, the end of the RVC electrode closest to the auxiliary electrode had a visible band of platinum. The peak current, subtracted from the base line, at +0.9 V was amxoximatelv 10 times ereater than that oh~. tained without i h e platinum plating. -Typical background currents were less than 2 rA. At +0.6 V the platinum-plated electrode had a current response of approximately 600 r A for 100 pL of 10 mM hydrogen peroxide, while the nonplatinized electrode did not respond under the same conditions. In fact, the amount of current obtained with the plated electrode a t +0.6 V is about 6 times greater than the response of an untreated electrode a t +0.9 V. SEM/XRF Analysis. The platinum coating on the RVC surface was characterized by using scanning electron microscopy (SEM) and X-ray fluorescence analvsis iXRF). The SEM was operated a t a-25-kV accelerating voltage'and a 150-rm aperture. The samples were mounted a t a working distance of 10 m m and a tilt angle of On. Figure 2 shows the micrograph obtained for the platinum-plated RVC electrode. The surface of the electrode closest to the auxiliary electrode during platinum plating is shown (magnification 30X). The picture s h o w crystalline platinum at the top of the electrode (the portion closest to the auxiliary electrode), a smoother coating near the center of the picture, and some carbon surface appears near the bottom. As expected, more platinum is plated ... at the end of the electrode positioned closest to the ~~~~~
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exhibited by platinum at 2.08,9.i2, and 11.14 eV were used to obtain the X-ray map. Window were set up at these peaks so that the X-ray detector would display a white dot on the SEM when a platinum count was detected. A higher platinum concentration is noticed at the top of the picture, which de-
creases toward the bottom. A high-magnification (450X) picture of the platinum plating on the RVC is shown in Figure 4. It appears that the surface coverage of the platinum is not uniform, but varies from high to minimal coverage; this was confirmed with X-ray maps. The heterogeneous surface
ANALYTICAL CHEMISTRY. VOL. 62. NO. 11. JUNE 1. 1990
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GLUCOSE CONCENTRATION (mM1 Cabation curve l a @ w x eusing lhe Plinired. mmobiliied enzyme. RVC eleckode. Row rate. 2.0 M m l n : applied poten6al. +0.6 V vs SCE. 100 pL sample volume. Figwe 5.
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Scanning electron micrograph of the electrOde shown In
Figure 2 . magnilication 450X
I provides sites for enzyme immobilization and a platinum surface to facilitate the oxidation of hydrogen peroxide produced by the enzymatic reaction. Enzymatic Activity. The activity of the immobilized glucose oxidase on the platinized RVC electrodes was similar to that of the enzyme attached to electrodes without a platinum coating. Actually, the activity of the enzyme on the platinized electrodes was approximately 33% higher than that of the nonplatinized counterparts. The average activity on the nonplatinized electrodes was 0.0706 (i0.0161) pM unit per electrode, while the platinized electrodes had an activity of 0.0936 (*0.0152) pM unit per electrode. Notice that the enzyme activity is somewhat more reproducible on the platinum-plated electrodes than on the nonplatinized electrodes. It is possible that the surface of the RVC electrode subjected to the plating procedure is rougher than an electrode that was not electroplated. This increase in the surface area of the electrode could allow for more binding sites for enzyme immobilization, as well as making the RVC surface more reproducible, and compensate for the loss in binding sites due to platinization. Response. Figure 5 shows a calibration curve for a platinized RVC electrode with immobilized glucose oxidase. The flow rate for this experiment was 2.0 mL/min and the applied potential was +0.6 V. The data in this figure are the average current responses of five sample injections, with an average relative standard deviation of 0.63% over all the concentrations tested. The sample volume was 100 pL for each concentration tested. The working curve in Figure 5 ranges from 0 to 30 mM glucose, hut the linear portion ranged from 0 to 10 mM, which brackets the clinical region for glucose (3.5-6.5 mM). producing a linear current response with a slope of 1.56, intercept of 2.31, correlation coefficient of 0.993, and standard error of estimate of 0.221, The nonzero y intercept, a t zero concentration of glucose. is due to a response that results from changes in the carrier stream flow when moving the injection valve. The slope of the calibration curve for the platinized
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FLOWRATE ( M L / M I N l Figm 6 Sampling rate versus Row rate la llm plallnired. mmobluzed enzyme. RVC elechcde. Injected sampltts were 100 ML 01 2 mM glucose.
electrode proved to be greatly improved over the nonplatinid counterpart. The platinized immobilized enzyme RVC elec. trode had a slope of 1.56 rrA/mM in the range from 0 to 10 mM glucose. while the slope of the nonplatinized HVC electrude was only 021 pA/mM under the same ronditions. The correlation coefficient was also less for the nonplatinized electrode (0.983 versus 0.999 for the platinized electrode). Thus the platinization provides an increase in linearity and sensitivity. The flow rate of the carrier stream affects the current response of the electrode and the sampling rate for glucose determinations. The sampling rate is determined from the peak width at the base line and represents an upper limit for the numher of samples that can he run per unit time. The sampling rate for glucose determinations increases as the fluw rate of the FIA system is increased, as shown in Figure 6. A maximum sampling rate of approximately 230 samples per hour can he achieved at flow rates more than 8.0 mL/min. At 2.0 mL/min, the flow rate used in determining the calibration curve for glucose. the sampling rate is approximately I18 samples per hour. As expected. the current response decreases with increasing flow rate, as shown in Figure 7. At the flow rate where the sampling rate reaches a plateau, 8.0 mL/min. the current response exhibited by the electrode is 1.8 MA. This is the current response for a sample injection of 100 pL of 2.0 mM glucose, a concentration below the lower limit of the clinical range. The lowest detectable quantity of an analyte determined hy a particular method is useful for comparing different methods of analysis. The lowest de. tectable amount, in this work. is represented hy a signal that is twice the noise of the system. as measured peak-to-peak.
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
the electrode. The enhanced detection for hydrogen peroxide provided by the platinum coating has greatly improved the detection capabilities of the system for glucose. The advantages realized by using the platinized RVC immobilized enzyme electrode are in the precision, increased sensitivity, better signal-to-noise ratio, and increased sampling rate.
ACKNOWLEDGMENT The authors thank Maureen McCourt for all her help.
LITERATURE CITED
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FLOWRATE IML/MINI Figure 7. Peak current versus flow rate for the platinized, immobilized enzyme, RVC electrode, same conditions as for Figure 6.
For the electrodes used in this work, the lowest concentration of glucose detected ranged from 0.025 mM, for a freshly prepared electrode, to 0.1 mM, for an electrode that was used daily for 2 weeks. During this time, the linear range and slope decreases slightly, but still encompass the clinical range for glucose determinations. The electrode has a useful lifetime of about 1.5 months when stored at 2 "C in buffer between weekly uses. The calibration range increases to 50 mM by reducing the injection volume of glucose to 5 pL. As expected, the lower limit of detection increases (to 0.25 mM). This minimum value is still considerably below the clinical value for glucose in serum. Reducing the amount of glucose reaching the electrode prevents saturation of the glucose oxidase and minimizes oxygen demand, thereby extending the calibration range. The linear portion of the calibration curve has a slope, y intercept, correlation coefficient, and standard error of estimate of 1.64, 0.299, 0.998, and 0.178 respectively. Decreasing the sample volume to 5 p L also increases the sampling rate to 180 samples per hour at a flow rate of 2 mL/min. The electrodeposition of platinum on the RVC has not adversely affected the immobilization of glucose oxidase on
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) 15) 16) 17)
(21)
Strohl, A.; Curran, D. Anal. Chem. 1979, 51, 353. Bleedel, W.; Wang, J. Anal. Chem. 1979, 51, 799. Strohl, A.; Curran, D. Anal. Chem. 1979, 57, 1045. Wang. J. E h 9 c M m . Acta 1981, 26, 1721. Wang, J.; Dewald, H. Anal. Chem. 1983, 55. 933. Wieck, H.; Shea, C.; Yacynych, A. M. Anal. Chim. Acta 1982, 142, 277. Wieck, H.; HeMer, G.: Yacynych, A. M. Anal. Chim. Acta 1984, 158, 137. Lobel, E.; Rishpon, J. Anal. Chem. 1981, 53, 51. Yao. T. Anal. Chim. Acta 1983, 153, 175. Sdnivasan, V.; Povsic, T.; Huntington, J. Am. Lab. 1983, October,57. Iannielb, R. M.; Yacynych, A. M. Anal. Chem. 1981, 53, 2090-2095. Wang, J.; Li, R.; Lin, M. €lectraana/ys& 1989, I , 151-154. Gunasingham, H.; Tan, C. Ektroma/ysis 1989, 7 , 223-227. Ikariyama, Y.; Yamauchi, S.; Yukiashi, T.; Ushioda, H. J. Elecfrochem. Soc. 1989, 136. 702-706. Will, F.; Iacovangeb, C. J . Electrochem. SOC.1984, 131. 590. Sasso, S. V. W.D. Dissertation, Rutgers, The State University of New Jersey, New Brunswick, NJ, 1986. Yacynych, A. M.; Sasso, S. V.; Hekler, G. H.; Wieck. H. J. In proceedlngs of the Symposium on Sensor Science and Technobgy; Schumm, B., Jr., Liu, C. C.. Powers, R. A.. Yeaaer. E. B., Eds.; The Electrochemical Society, Inc.: Pennington, NJ, f987; p 85. Geise. R. J.; Yacynych. A. M. In Chemhi Sensors and M l c r h t r u mentation; Murray, R. W., Dessy, R. E., Heineman, W. R.; Janata, J.. Seitz. W. R., Eds.; ACS Symposium Series 4 0 3 American Chemical Society: Washington, DC, 1989; Chapter 4. Blum, W.; Hogaboom, G. Principles of €lectrop&ting end Electroforming; Mceaw-Hili: New York. 1930; p 372. Strohi. A.; Curran, D. Anal. Chem. 197% 51, 1050. Heider, G. H. Ph.D. Dissertation, Rutgers, The State University of New Jersey, New Brunswick, NJ, 1984.
RECEIVED for review February 16,1990. Accepted February 23, 1990. A.M.Y. thanks the Rutgers University Research Council and the Charles and Johanna Busch Memorial Fund for research support.
