Anal. Chem. 1990, 62, 1498-1501
1498
(9) Kuhr, R. J.; Dorough, H. W. Carbemate Insectkkjes: Chemistry, Biochemistry and Toxlcobgy; CRC Press: Cleveland, OH, 1976; Chapters 3 and 5. (10) Vukmanic, D.; Chiba. M. J . Chromatogr. 1989, 483, 189.
RECEIVED for review October 16,1989. Accepted April 2,1990.
The authors thank the Research and Technology Branch of of fie vir^^^^^ for financial support, the ontario Use of the equipment herein described does not represent endorsement by the Ontario Ministry of the Environment or by Agriculture Canada.
Microhole Array Electrode as a Glucose Sensor Yoshihiro Shimizu* a n d Ken-ichi Morital
Basic Research Laboratories, Toray Industries, Inc., 11 11 Tebiro, Kamakura 248, Japan
Mkrohole array enzyme electrodes (1000 mkrohdes of 7 pm diameter and 50-450 pm depth) were fabricated for the measurement of glucose. Enzyme was Immobilized on the surface of the platlnlzed mlcrohole array electrode. The enzyme electrode of hydrogen peroxide detection type was sensltlve to glucose concentration over a wide range. The sensitivity of the 50-450 pm depth microhole array enzyme electrode was hlgher than that of a nonetched one. The linearity of the electrode current response with glucose concentration was extended by lncreaslng the depth of the mlcrohoies. Increaslng the hole depth also increased the stabillty of the enzyme electrodes.
INTRODUCTION Microhole array electrodes can be fabricated by the electrochemical etching of carbon fibers embedded in an epoxy resin. They have proved to be promising as oxygen sensors ( I ) . The linear concentration gradient of the electroactive species in the diffusion layer is stably established in the steady state. The thickness of the diffusion layer for the reduction of dissolved oxygen is found to be the s u m of the depth of the microhole and the thickness of the solution boundary layer (1). Cyclic voltammograms obtained with the microhole array electrode are quite similar in shape to those for rotating disk or ultramicrodisk electrodes. In other words, the voltammograms obtained at slow potential sweep rates are sigmoidal (2). Other features of the microhole array electrode include its small size and being moderately flow-rate-insensitive and being discriminative against certain poisons ( I ) . In this paper we report on an amperometric biosensor utilizing a 0.5 mm diameter microhole array electrode. Amperometric glucose electrodes are based on the use of the enzyme glucose oxidase D-glucose H 2 0 + O2 D-gluconic acid H202 (1) This reaction has been followed by the amperometric detection of hydrogen peroxide at an electrode polarized a t +0.6 V (vs SCE)
-
+
-
Hz02
+
O2 + 2H+ + 2e-
(2) We have also pursued the current response for the oxygen reduction a t -0.6 V since oxygen is consumed by the reaction (1)
O2 + 2Hz0+ 4e-
-
40H-
(3) Glucose oxidase was immobilized on platinized carbon fibers Present address: Tbin University of Yokohama, 1614 Kuroganecho, Midori-ku, Yokohama 227, Japan 0003-2700/90/0362-1498$02.50/0
inside the microholes. I t was found that the hydrogen peroxide detection electrode (H202electrode) responded better than the oxygen detection electrode (02 electrode) and the sensitivity of the microhole array enzyme electrode for the hydrogen peroxide detection was higher than that of a nonetched array enzyme electrode. EXPERIMENTAL SECTION Chemicals and Reagents. The glucose oxidase used (100 X lo4 units/g) was obtained from Nagase Biochemicals, Ltd. The l-cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-ptoluenesulfonate and glucose (P-D-glUCOSe) were purchased from Tokyo-Kasei Japan. Phosphate buffer solutions (Na2HP02(0.033 mol/dm3) + NaH2P0, (0.033mol/dm3)) were used in all experiments. Preparation of Microhole Array Enzyme Electrode. Microhole array electrodes with a diameter of 0.5 mm were prepared from high-strength carbon fibers (TORAYCA T-300, the number of fibers in a single electrode, 1000, 6.93 pm in diameter) using fabrication techniques described in the previous paper ( I ) . The platinized electrode was immersed in a phosphate buffer solution for a few minutes and then placed in 1 mL of a slowly stirred phosphate buffer solution containing 5 mg of glucose oxidase and 25 mg of l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate at room temperature for 15 h. The microhole array enzyme electrodes thus prepared were stored in a phosphate buffer solution. Electrochemical Measurement. Amperometric measurements were carried out with a Nikko Keisoku potentiostat (Model N-POT 2501). The electrochemical cell (three-electrode system) consisted of a 1WmLglass beaker with a silicone rubber iid having four holes. Three of these holes were used for the electrodes and the other was used for the injection of concentrated glucose solution into the buffer solution. A saturated calomel electrode (SCE) and a platinum wire served as reference and the auxiliary electrodes. Potential values are referred to SCE. The electrodes were placed in air-saturated buffer solutions (100mL), stirred and thermostated at 37 "C,and then poised at +0.6 V for the hydrogen peroxide measurement or -0.6 V for the oxygen measurement. A solution of low oxygen concentration was prepared by purging one volume of air and three volumes of nitrogen gas into the solution. After background current decayed to a steady-state value, aliquots of a concentrated glucose solution were stepwise added. The steady-state current was recorded for each addition of aliquots. Each point shown in Figures 2, 4, 5, 6,and 7 corresponds to a single measurement. Definitions. Response time (tW)was defined as the time it took to reach 90% of the steady-state current after the glucose injection. Sensitivity was defined as an absolute value of the slope in the linear range of the calibration curve divided by the total cross-sectional area of the microholes. RESULTS AND DISCUSSION Immobilization. Glucose oxidase enzyme was immobilized by immersing the platinized microhole array electrode in the solution containing glucose oxidase and the soluble carbo0 1990 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 62. NO. 14. JULY 15. 1990
1499
Figure 1. Scanning electron micrographs of a microhole array enzyme electrode and a microhole array electrode: (a) a platinized carbon fiber covered with enzyme layer; (b) a platinized carbon fiber electrode.
diimide. The scanning electron micrograph (SEM) of the microhole array enzyme electrode (Figure l a ) shows that the enzyme mainly adhered to the surface of the platinum as a layer about 0.5 jLm thick. Ikariyama e t al. reported that the glucose oxidase was incorporated within the micropores of the platinized platinum (3). In their case, glucose oxidase could not be observed by SEM. A 30% decrease in the current response was observed after 100 h of operation for the 200-j~m-depthenzyme electrode, while the same decrease occurred after only 60 h for the enzyme electrode prepared without the carbodiimide. The addition of the soluble carbodiimide ( 4 ) to the enzyme immobilization solution increased the stability of the microhole array enzyme electrode, probably as a result of increasing the amount of glucose oxidase immobilized on the electrode surface by cross linking of each enzyme by the carhodiimide. In order to check whether enough amount of enzyme was immobilized, six enzyme electrodes with 400-j~mdepth were prepared and a mean current of 151 f 13 nA in 16 mM glucose solution was obtained. This finding indicates that the preparation is reproducible. Hz02a n d 0, Electrodes. Steady-state current values for oxygen reduction and hydrogen peroxide oxidation as a function of the concentration of glucose in a phosphate buffer solution were measured with a microhole array enzyme electrode with 200-j~m-depthmicroholes (Figure 2). The response for the H202 electrode was proportional to the glucose concentration between 0 and 11mM. On the other hand the response for the 0,electrode was proportional to the concentration between 0 and 0.8 mM and the current response was almost constant for higher glucose concentration. The maximum output current (40 nA a t zero glucose concentration) was also observed with an electrode of the same depth without enzyme. The fact shows that the enzyme layer does not
200-,
,
,
"
0
2
4
6 8 1 0
Glucose concenlrationlrnhn
Figure 2. Steady-state current values for the oxygen reduction (0) and hydrogen peroxide oxidation (0)as function Of the glucose cancentration. A microhole array enzyme electrode with ZOO-pmdepth microholes was used disturb the transport of oxygen. The current (20 nA) at the glucose concentration higher than 1.1 mM results from the reduction of hydrogen peroxide produced by the enzyme reaction. When the enzyme is fixed near the electrochemical hydrogen peroxide oxidation site, the enzyme activity can be controlled by the electrochemical oxygen regenerated on the electrode surface (5). The fact that there was a large difference in the linear range of the calibration curves between the 0% electrode and the H,O, electrode was substantiated by the concentration profile for the mass transfer in the microholes. Figure 3 shows the schematic illustration of the microhole array enzyme electrode and the schematic distribution of the concentration of oxygen and hydrogen peroxide as a function of the distance from the electrode surface inside the microhole before and after the injection of glucose. For the H,O2 electrode (I) before the injection of glucose, the concentration
1500
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
a b e
Table I. Sensitivity and Linearity
f
sensitivity: A M-I cm-2
depth of
microhole, Wm 0 50 100 200 300 400 440
linearity, mM
