A Flow Injection (FI) Biosensor System for Pentachlorophenol (PCP

A flow injection (FI) biosensor system has been designed for the analysis of pentachlorophenol (PCP) using a substrate recycling scheme comprising ...
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Environ. Sci. Technol. 2000, 34, 3291-3295

A Flow Injection (FI) Biosensor System for Pentachlorophenol (PCP) Using a Substrate Recycling Scheme MARIETTA LABRA-ESPINA,‡ KEITH B. MALE, AND JOHN H. T. LUONG* Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R2

A flow injection (FI) biosensor system has been designed for the analysis of pentachlorophenol (PCP) using a substrate recycling scheme comprising immobilized bilirubin oxidase (BOX) in the presence of excess of NADH. PCP was efficiently converted to tetrachloro-p-benzoquinone (1,4TCBQ) and then tetrachloro-p-hydroquinone (1,4-TCHQ) by bis(trifluoroacetoxy)iodobenzene (BTFAIB) and zinc powder, respectively. BOX immobilized on aminopropyl glass beads rapidly oxidized 1,4-TCHQ to 1,4-TCBQ, which in turn was readily reduced back to 1,4-TCHQ in the presence of excess NADH. This recycling scheme enabled one molecule of PCP to consume several NADH molecules leading to enhanced sensitivity. Under optimized conditions the rate of NADH uptake measured as the absorbance decrease at 340 nm yielded a detection limit for 1,4-TCHQ or oxidized PCP of 250 nM. The detection limit was improved to 25 nM for both analytes using a fluorescence detector with excitation and emission wavelengths of 345 and 450 nm, respectively. The PCP level in contaminated soil samples was measured using the FI biosensor system, and the results obtained compared well with capillary zone electrophoresis (CZE) analysis.

Introduction The large-scale use of pentachlorophenol (PCP) and other chlorinated phenols has led to the contamination of both aquatic and terrestrial ecosystems, and PCP, commonly used as a wood preservative, has been classified by the U.S. EPA as a priority pollutant (1). Due to its widespread use and toxicological properties, PCP regulations under the Resource Conservation and Recovery Act have been created specially for wood treatment facilities in the United States. Therefore, there is continuing and urgent interest in developing simple, sensitive, and accurate analytical procedures for measuring PCP in various environmental and biological samples for the investigation of animal and human exposure. The use of enzymes in colorimetric assays is one popular approach to achieve fast and simple measurement, and such a detection system can be miniaturized for online and field screening. However, enzymic procedures using tyrosinase (2-12), horseradish peroxidase (13-15), chloroperoxidase (16), and, to a lesser extent, laccase (17) are not applicable * Corresponding author phone: (514)496-6175; fax: (514)496-6265; e-mail: [email protected]. ‡ Current address: University of the Philippines, Diliman, Quezon City, Philippines. 10.1021/es0009370 CCC: $19.00 Published on Web 06/23/2000

Published 2000 by the Am. Chem. Soc.

for PCP assay. Such enzymes are not effective with highly chlorinated phenols such as PCP, and/or a multitude of products including polymers is often formed during the course of enzymatic reactions. A substrate recycling assay using bilirubin oxidase (BOX) together with NADH was recently reported for the determination of tetrachloro-pbenzoquinone (1,4-TCBQ), an oxidation product of PCP and 2,3,5,6-tetrachlorophenol (18). In such a procedure, 1,4-TCBQ could be easily reduced to its tetrachlorohydroquinone form (1,4-TCHQ) by NADH. This 1,4-TCHQ species was then reoxidized by BOX, a copper-containing enzyme, which has been also shown to be capable of oxidizing several organic compounds in the presence of oxygen (19, 20). Consequently, a recycle for the substrate (1,4-TCBQ) was established where one 1,4-TCBQ molecule could consume several NADH molecules. This substrate-recycling assay could detect 1,4TCBQ as low as 110 nM and 30 nM, respectively, using absorbance or fluorescence measurement (18).

This article describes a flow injection (FI) biosensor system for the detection and determination of PCP in contaminated soil. Bilirubin oxidase was covalently immobilized onto porous aminopropyl glass beads to form an immobilized enzyme column and the consumption rate of NADH was monitored by measuring the absorbency or fluorescence decrease during the course of the substrate recycling assay. The FI biosensor system was then demonstrated for analysis of PCP in contaminated soil by combining this substrate recycling scheme with a chemical reaction using bis(trifluoroacetoxy)iodobenzene (21, 22) for the oxidation of PCP to 1,4-TCBQ. This high yield reaction was recently reported for the efficient oxidation of PCP and other chlorophenols under mild reactions. The applicability of the FI biosensor system was also demonstrated by comparing the results obtained with those of certified values and/or capillary zone electrophoretic measurements.

Experimental Section Materials. Bilirubin oxidase, BOX (EC 1.3.3.5 from Myrothecium verrucaria, lyophilized powder, 15-65 U/mg protein), NADH, glutaraldehyde (25% w/v), and porous aminopropyl glass beads (pore size 70 nm) were purchased from Sigma (St. Louis, MO). Bis(trifluoroacetoxy)iodobenzene (BTFAIB), 1,4-tetrachlorobenzoquinone (1,4-TCBQ), 1,2-tetrachlorobenzoquinone (1,2-TCBQ), 2-chloro-1,4-benzoquinone (2CBQ), 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ), 2,6dichloro-1,4-benzoquinone (2,6-DCBQ), pentachlorophenol (PCP), and zinc powder were obtained from Aldrich (Milwaukee, WI). All other reagent grade chemicals were purchased from Anachemia (Montreal, QC, Canada). Certified PCP-contaminated soil samples were purchased from Resource Technology Corp. (Laramie, WY), whereas other PCPcontaminated soil samples were obtained from woodpreserving plants in the Montreal region. Immobilization of Bilirubin Oxidase. BOX was covalently immobilized onto aminopropyl glass beads by glutaraldehyde activation (23). In brief, aminopropyl glass beads (500 mg) were washed with phosphate-buffer saline (PBS, 20 mM VOL. 34, NO. 15, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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phosphate, pH 7 containing 9 g/L NaCl) and then activated with glutaraldehyde (3 mL, 2.5% w/v) in PBS for 3 h at room temperature. The resulting orangeish-pink beads were then washed to remove excess glutaraldehyde. BOX (50 units, 1.6 mg protein) in 20 mM phosphate buffer (pH 7) was slowly rotated with the activated beads in a capped tube overnight at 4 °C. The resulting beads were washed and then packed into a 3 mm microbore borosilicate glass chromatography column from Omnifit (Toms River, NJ). The 500 mg of immobilized bilirubin oxidase beads could be packed into two 10 cm columns. It should be noted that immobilized bilirubin oxidase beads are often stable for up to 1 year when stored at 4 °C. PCP Oxidation Using Bis(trifluroacetoxy)iodobenzene. Based on the method reported by Saby and Luong (21, 22), the oxidation of PCP was performed in 100 mM trichloroacetic acid, pH 1.0 containing 50 mM L-tartaric acid using PCP (10 mM in methanol) and freshly prepared BTFAIB (100 mM in methanol) stock solutions. The PCP reactions (20 mL, 25 nM-10 µM) containing 500 µM BTFAIB were carried out at room temperature, with light protection for 1 h. At the end of the reaction, 500 µM hydrogen peroxide was added to neutralize any unreacted BTFAIB, followed by the addition of zinc powder (100 mg) to reduce the 1,4-TCBQ to 1,4TCHQ. The use of tartrate in the buffer was necessary to maintain the pH below 4 upon zinc addition, otherwise the reduction reaction could not proceed to completion. The pH was altered to about 3 by the addition of 200 µL of 8 M NaOH. The 1,4-TCHQ sample was then passed through a 0.45 µm Millex-HV filter (Millipore, Bedford, MA) to remove the zinc powder. Directly after this filter was a Sep-Pak C18 cartridge which had been prewashed with methanol and then a 100 mM TCA solution, pH 3.0 containing 50 mM L-tartaric acid. Notice that if the cartridge was washed with TCA at pHs below 3, a precipitate would be released during the elution of 1,4-TCHQ. After loading of the 1,4-TCHQ sample, the cartridge was washed extensively with water to remove all excess TCA and hydrogen peroxide, which are known to inhibit the bilirubin oxidase reaction (18). The cartridge was submerged into a vial containing 10 mL of 50 mM phosphate pH 3.0. The absorbed 1,4-TCHQ was then eluted by passing 2 mL of methanol through the cartridge followed by 8 mL of water to arrive at a final concentration of 25 mM phosphate pH 3.0 containing 10% methanol. This protocol ensures that the running buffer and sample have the same ionic strength and solvent concentration. However without this precaution, injection artifacts were noticed which hampered the accuracy of 1,4-TCHQ determination at concentrations near the detection limit. It should be noted that the recovery of standard 1,4-TCHQ was always greater than 95% using this method. Preparation of Soil Extracts. Soil samples (1 g) were extracted with 50 mL of 10 mM NaOH for 4 h under light protection. After centrifugation, the supernatants were collected, and the PCP was partially purified and concentrated if necessary by using Sep-Pak tC18 cartridges (Waters Corp., Milford, MA) employing the method of Male et al. (24). In brief, cartridges were wetted with methanol and then washed with 50 mM phosphate (pH 7.5). The samples (20 mL) were neutralized (2 mL of 500 mM phosphate, pH 7.5) and passed through the cartridges. After washing with phosphate buffer, contaminants which interfered with the biosensor measurement using either absorbance or fluorescence were released from the cartridges employing 2 mL of a 50% methanol solution containing 50 mM phosphate (pH 7.5). The PCP was only eluted from the cartridges when the methanol concentration was increased to 100%. The samples (1 mL) were then diluted back to the original volume with 10 mM NaOH for analysis using capillary zone electrophoresis (CZE) or the biosensor system. The concentration of PCP was 3292

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measured directly by CZE. In brief, a P/ACE 5500 CE system (Beckman, Fullerton, CA) equipped with a UV detector module set at 214 nm was used for the detection of PCP. Separation was achieved using a polyimide-coated fusedsilica capillary (length to detector: 40 cm; total length: 47 cm; 50 µm ID) provided by Polymicro Technologies (Phoenix, AZ) and a separation buffer containing 20% acetonitrile and 80 mM phosphate pH 7.5. Samples were injected by pressure for 5 s, and the separation was conducted at 25 kV with a controlled temperature of 30 °C. PCP was detected at about 11-12 min, and the relationship between concentration and integrated area was linear between 5 and 200 µM. For FI biosensor analysis, the PCP partially purified extracts were diluted appropriately (10-100 fold) with the tartrate buffer containing TCA and treated as above with BTFAIB to oxidize any PCP present. The PCP concentration determined by the biosensor was then compared to the values obtained by CZE. For soil samples containing PCP below 80 ppm, the concentrated methanol extract could be used directly without dilution for CZE analysis, since the detection limit was much higher than that of the biosensor system. For the biosensor system, the methanol extract (1 mL) was diluted directly to 20 mL with the tartrate buffer containing TCA. The sample preparation technique using the Sep-Pak tC18 cartridges resulted in recoveries of PCP of greater than 95%. Apparatus. The FI biosensor system consisted of a peristaltic pump (FIA Pump 1000, Eppendorf North America, Madison, WI) that delivered the sample, buffer, and NADH solution at a preset flow rate (Figure 1). A 100 µL sample was injected into a 25 mM phosphate buffer (pH 3.0, containing 10% methanol) stream by a motorized injection valve (EVA injector, Eppendorf). The loading and injection times were controlled from the EVA injector. The resulting sample stream then merged at a T-joint with the NADH containing buffer (25 mM phosphate, pH 7.5), and homogeneous mixing (final pH of about 6.8) was effected by a mixing coil just before the sample entered the immobilized enzyme (BOX) column. The NADH consumption was monitored by following the absorbance at 340 nm (Waters LC spectrophotometer, model 481, Milford, MA) or fluorescence (Waters scanning fluorescence detector, model 747, with excitation at 345 nm, emission 450 nm and a gain setting of 100). Signals were digitized using an IBM-AT type computer equipped with a DAS-8 A/D card and custom software to obtain both peak height and peak area. The output of the detectors after conversion to voltage was also recorded on a strip chart recorder. Optimization of FI Biosensor. The effects of buffer strength, pH, flow rate, NADH concentration, and column length on the signal response were investigated. The response time, sensitivity, and reproducibility of the 1,4-TCHQ (1,4TCBQ reduced by zinc powder) signal were examined as well as the stability of the enzyme column. For fluorescence detection the NADH concentration needed to be reoptimized, whereas the other operating parameters remained the same. The optimized systems were then applied to samples of PCP, which had been oxidized by BTFAIB as described earlier.

Results and Discussion Optimization of FI Biosensor Detection for 1,4-TCHQ. The biosensor system used a 10 cm column of immobilized bilirubin oxidase and a T-joint to ensure a constant concentration of NADH (Figure 1). The signal responses for 1,4TCBQ and 1,4-TCHQ (1,4-TCBQ treated with zinc powder) were virtually identical; therefore, even if the reduction of 1,4-TCBQ by zinc powder was not 100% complete it would not effect any decrease in the signal response. All lines in contact with the sample were in stainless steel 316 to minimize adsorption to the lines that could result in a tailing effect on the peaks. Based on such considerations, 1,4-TCHQ

FIGURE 1. Schematic diagram of the flow-injection (FI) biosensor system for oxidized pentachlorophenol detection. which is less hydrophobic than 1,4-TCBQ was used for all subsequent experiments since it should cause less problems with adsorption to the various lines. The response for 1,4-TCHQ was maximal at pH 6-7 and decreased 30% at pH 8 (figure not shown). NADH remained stable for days if the pH was maintained above 7.5; however, at pH 6.5 the concentration dropped 40% in just 2 days. In contrast, 1,4-TCHQ was not very stable at pHs above 3, and even at pH 3 in the presence of methanol it was only stable for about 24-36 h. For these reasons, NADH was placed in a phosphate buffer at pH 7.5, whereas 1,4-TCHQ was placed in a pH 3 phosphate buffer. Methanol (10%) was also added to the pH 3 phosphate buffer to enhance the solubilization of the analyte as well as to prevent its adhesion to the various lines. Unlike acetonitrile, methanol at 5-10% did not inhibit bilirubin oxidase. After mixing at the T-joint the pH of the running buffer was about 6.8 and provided optimal results. The ionic strength of the phosphate buffer exhibited little effect on the response. Maximal response was achieved from 25 to 50 mM, while at 100 mM the signal dropped to 90% of the maximum. As a result, 25 mM phosphate was used in subsequent experiments. The response decreased 4-fold with an increase in the sample flow rate from 2.6 to 27.6 mL/h. Such a result was expected in accordance with the theoretical prediction for FI systems with negligible mass transfer in the bulk solution (25) as well as experimental observations (26, 23). Similarly, the response time decreased from 60 to 5 min over the same flow rate range. As a compromise between sensitivity of analysis and sample throughput (assays per hour), a flow rate of 5.6 mL/h (11.2 mL/h at the detector) was selected for all subsequent studies. The response time at this flow rate was about 30 min. The response for 1,4-TCHQ was also dependent upon the NADH concentration in the buffer. The maximum response was observed between 200 and 300 µM NADH (Figure 2), while the response dropped to 40% at 50 µM. Above 300 µM NADH, the background noise became very pronounced, and it was difficult to autozero the detector; therefore, 200 µM NADH (100 µM at the detector) was chosen for all subsequent

FIGURE 2. Effect of NADH concentration on the FI biosensor response to 1,4-TCHQ: (B) absorbance detector and (9) fluorescence detector. experiments using the UV detector. In the case of the fluorescence detector the optimal concentration for NADH was about 50 µM, while at 10 µM the response dropped to 50% (Figure 2). Above 60 µM NADH, the gain of the detector had to be set at 10 rather than 100 which greatly reduced the sensitivity to 1,4-TCHQ. Sensitivity could not be improved with a gain setting of 1000 owing to an extremely low NADH concentration (2 µM). The response increased as the column length increased with a maximum response obtained at 5 cm. The signal at 2 cm was 80% of the maximum, and as a result a 10 cm column length was chosen as a compromise between response time and the reusability of the immobilized BOX column. This series of experiments was performed in peak area to account for the difference in peak heights caused by changing dispersions due to varying column lengths. Response of the Biosensor System to 1,4-TCHQ. In peak area mode, there was an excellent linear relationship between the response of the FI biosensor and 1,4-TCHQ up to 10 µM (R 2 ) 0.999). The sensitivity of the biosensor (95% confidence interval, n ) 8) was determined to be 0.200 ( 0.005 AU‚ min/µM. The detection limit (signal/noise ) 3) was determined to be about 250 nM (125 nM at the detector), and no VOL. 34, NO. 15, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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response was noted in the absence of 1,4-TCHQ. The relationship in peak height mode was not linear, so as a result peak area mode was used in all subsequent experiments. Good reproducibility ((1.6% at 95% confidence interval) was obtained as reflected by the average response for 53 repeated analyses of 2 µM 1,4-TCHQ (0.437 ( 0.007 AU‚min). The experiment could not be performed for longer than 30 h since the 1,4-TCHQ standard solution became unstable and the signal began to drop. The enzyme column could be used at room temperature for 4-6 weeks without any noticeable decrease in the sensitivity to 1,4-TCHQ. However, the biosensor had to be calibrated each day (especially if the column is removed from the system and stored at 4 °C), since the absolute sensitivity from time to time with the same enzyme column could vary up to ( 10%, although the reproducibility and stability of the system remained unchanged. This could be due to different flow dynamics within the column since recycling systems would be very sensitive to the packing of the beads in the column causing different tailing effects from time to time. The sensitivity from column to column was noticed to vary (20%. PCP Detection Using the FI Biosensor System. PCP was oxidized by the reaction with BTFAIB as described earlier. Tartrate (50 mM) was added to the TCA solution (pH 1.0) to maintain pH below 3.0, otherwise the subsequent addition of zinc powder to the reaction mixture would increase pH above 5.0 to effect instability of the 1,4-TCBQ/1,4-TCHQ. The experimental data also confirmed that PCP was completely consumed after the reaction as verified by capillary zone electrophoresis. However, the reaction yield from PCP to 1,4-TCHQ was only 85-95% when compared by the biosensor to an equivalent 1,4-TCHQ standard. Such a noticeably lower reaction yield could be due to the instability of the 1,4-TCBQ product during the BTFAIB reaction as well as the conversion of PCP to 1,2-TCBQ which gave no response to the biosensor. An excellent linear relationship between the response of the FI biosensor system and 1,4-TCHQ, generated from oxidized PCP, up to 10 µM was obtained (R 2 ) 0.993). The sensitivity of the FI biosensor system (95% confidence interval, n ) 8) was determined to be 0.196 ( 0.015 AU‚ min/µM. The detection limit (signal/noise ) 3) was determined to be 250 nM. Good reproducibility (2.3% at 95% confidence interval) was obtained by the average response for 39 repeated analyses (figure not shown) of 2 µM 1,4TCHQ generated from oxidized PCP (0.426 ( 0.010 AU‚min). PCP Determination in Contaminated Soil. The concentration of PCP in the contaminated soil sample (Resource Technology Corp.) was determined by capillary electrophoresis and compared to the value obtained by the biosensor system for 1,4-TCHQ generated from oxidized PCP. The sample (reference value 1425 ppm, confidence interval 11091742 ppm) gave a value of 1270 ( 108 ppm with capillary electrophoresis (95% confidence interval, n ) 4) and 1380 ( 20 ppm with the biosensor system (95% confidence interval, n ) 4). Good reproducibility (2.8% at 95% confidence interval) was obtained by the average response for 67 repeated analyses (figure not shown) of a converted soil sample (0.216 ( 0.006 AU‚min), and the peak area signal did not decrease during this time indicating that the biosensor system was very stable. Detection of 1,4-TCHQ by Fluorescence. The decrease in fluorescence of NADH (50 µM) in the presence of 1,4TCHQ was monitored with an excitation wavelength of 345 nm and an emission wavelength of 450 nm. An excellent linear relationship between the response of the FI biosensor and 1,4-TCHQ up to 2 µM (R 2 ) 0.999) was achieved in peak area mode. The sensitivity of the biosensor (95% confidence interval, n ) 8) was determined to be 4.18 ( 0.12 V‚min/µM. The detection limit (signal/noise ) 3) was determined to be about 25 nM (12.5 nM at the detector), a 10-fold improvement 3294

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FIGURE 3. Response of the FI biosensor to PCP oxidized by BTFAIB using the fluorescence detector.

TABLE 1. Comparison of Determinations of Concentrations of Pentachlorophenol in Contaminated Soil sample no.

PCP concn (ppm) (CZE method)

PCP concn (ppm) FI biosensor methoda

differenceb (%age)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1.6 1.8 84 88 122 137 137 160 169 620 753 1124 1200 1318 1331 1335 1415 2157 2259 2285

1.5 2.1 94 97 133 142 156 147 191 720 874 1067 1300 1290 1553 1270 1411 2153 2315 2228

6 -17 -12 -10 -9 -4 -14 8 -13 -16 -16 5 -8 2 -17 5 0 0 -2 2

a The value for the FI biosensor method was determined from the average of duplicates. The reproducibility expressed as the coefficient of variation (standard deviation/mean) varied from 1.1% to 8.4%, and the overall coefficient of variation was determined to be 3.8 ( 1.0% (at 95% confidence interval). b Difference ) 100% * (CZE(value) - FI biosensor system(value))/CZE(value).

over the detection limit with the UV detector. Good reproducibility ((1.4% at 95% confidence interval) was also obtained by the average response for 38 repeated analyses (figure not shown) of 0.5 µM 1,4-TCHQ (2.09 ( 0.03 V‚min). As shown in Figure 3, an excellent linear relationship between the response of the FI biosensor system and 1,4TCHQ generated from oxidized PCP up to 2 µM was obtained (R 2 ) 0.998). The sensitivity of the FI biosensor system (95% confidence interval, n ) 8) was determined to be 3.65 ( 0.12 V‚min/µM with a detection limit (signal/noise ) 3) of 25 nM. Good reproducibility (2.1% at 95% confidence interval) was obtained by the average response of 38 repeated analyses (figure not shown) of 0.5 µM 1,4-TCHQ generated from oxidized PCP (2.10 ( 0.04 V‚min). The biosensor system was very substrate specific with respect to 1,4-TCBQ since 2-CBQ, 2,5-DCBQ, and 2,6-DCBQ only gave responses of 4%, 8%, and 6%, respectively, when compared to an equivalent amount of 1,4-TCBQ (1 µM). These values were much lower than those reported by Cybulski et al. (18) using soluble BOX, an indication that immobilization could alter the substrate specificity of the immobilized enzyme. The PCP concentration in contaminated soils was determined by CZE and compared to the values obtained by

based biosensor was very specific to 1,4-TCBQ, the oxidized product of PCP and 2,3,5,6-tetrachlorophenol in comparison to 2-CBQ, 2,5-DCBQ, and 2,6-DCBQ, the oxidized products of the other chlorophenols (22).

Literature Cited

FIGURE 4. Reproducibility of the response of the FI biosensor to PCP in a soil sample oxidized by BTFAIB using the fluorescence detector. the FI biosensor system for oxidized PCP. The PCP contamination level for the 20 soil samples tested ranged from 1.5 to 2300 ppm (µg of PCP/g of soil). Table 1 indicates good agreement between the two techniques with an overall discrepancy of 8.3 ( 2.7% at the 95% confidence interval. When the biosensor system values were plotted against those of CZE, a straight line resulted with a slope of 1.01 ( 0.03 and a correlation coefficient of 0.993 (95% confidence interval, n ) 20). Such good agreement thus validated the applicability of the BOX recycling FI biosensor system for measuring PCP in contaminated soil samples. It should be noted that without the PCP purification procedure with the tC18 cartridges, the biosensor values for PCP were lower due to contaminants which possessed a positive fluorescence and thus suppressed the NADH fluorescence decrease (up to 50% for samples contaminated with 100-200 ppm PCP). Good reproducibility (0.7% at 95% confidence interval) was obtained by the average response for 40 repeated analyses of a converted soil sample (approximately 100 µM, diluted 100-fold) (Figure 4). The peak area signal (4.4 ( 0.03 V‚min) remained constant during this time to demonstrate the stability of the biosensor system. In brief, oxidized PCP was integrated into a biosensor system such that contaminated soil samples containing PCP as low as 2 ppm (µg PCP/g of soil) could be monitored. The fluorescence detector could detect PCP in solution as low as 25 nM, which was superior to CZE (5 µM) or HPLC-UV detection (1 µM (24)). Although the FI electrochemical biosensor system using glucose oxidase (24) could detect about 10 nM PCP, this system detects all chlorophenols present in the samples. In contrast, the bilirubin oxidase

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Received for review January 31, 2000. Revised manuscript received May 1, 2000. Accepted May 8, 2000. ES0009370

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