Phosphate sensing system using pyruvate oxidase and chemiluminescence detection

Biosensors & Bioelectronics Vol. 11, No. 10, pp. 959-965, 1996

© 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0956--5663/96/$15.00

ELSEVIER ADVANCED TECHNOLOGY

Phosphate sensing system using pyruvate oxidase and chemiluminescence detection Kazunori Ikebukuro, Hideaki Wakamura & Isao Karube* Research Centre for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153, Japan Tel: 81-3-3481-442 Fax: 81-3-3481-4581

Izumi Kubo Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangicho, Hachioji, Tokyo 192, Japan

Masako Inagawa, Takako Sugawara & Yoshiko Arikawa Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112, Japan

Masayasu Suzuki Department of Biochemical Engineering Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawatsu, Iizuka-shi, Fukuoka 820, Japan

& Toshifumi Takeuchi Faculty of Information Science, Hiroshima City University, 151-50tsuka, Numata-cho, Asanami-ku, Hiroshima 731-13, Japan (Received 9 September 1994; revised version received 8 June 1995; accepted 24 October 1995)

Abstract: A flow injection phosphate analysis system based on an enzymic

reaction and a subsequent luminol chemiluminescence reaction has been developed. The system consists of an immobilized pyruvate oxidase column, mixing chamber for the chemiluminescent reaction and a photomultiplier. The H202 generated by the reaction of phosphate and pyruvate oxidase then reacts with luminol and horseradish peroxidase and the consequent chemiluminescence is detected using a photomultiplier. This system is capable of the rapid determination of phosphate, the time required for one measurement cycle being approximately 3 min. A linear response was observed from 4.8 to 160/zM phosphate. © 1996 Elsevier Science Limited Keywords: phosphate, luminol, chemiluminescence, pyruvate oxidase, flow injection analysis 959

Kazunori lkebukuro et al.

INTRODUCTION Phosphate ion concentration is commonly used as an index for the estimation of water enrichment. When water is enriched with phosphate, the amount of phytoplankton in the water increases (Krebs, 1972; APHA AWWA WPCF, 1976). Therefore, determination of phosphate in water is important in the monitoring of water quality. Previously, phosphate concentration has generally been determined by spectrophotometric methods such as that based on molybdenum blue (Lowry & Lopez, 1946). This method is time consuming and is inappropriate for on-site monitoring. Currently, there is no simple sensing system available for this purpose and thus the development of a sensitive method for the determination of phosphate ions is of great importance for river water monitoring. Several enzymatic methods for the determination of phosphate have been reported. One method is based on the inhibition of alkaline phosphatase in the presence of phosphate (Weetall & Jacobson, 1972; Guilbault & Nanjo, 1975). A method using a combination of nucleoside phosphatase and xanthine oxidase was also reported (Watanabe et al., 1988; D'Urso & Coulet, 1990). In this method, the consumption of oxygen or the production of hydrogen peroxide that occurs as a result of two enzyme reactions is measured. We have reported a biosensor system suitable for on-site phosphate monitoring (Kubo et al., 1991). This system involves phosphate-dependent pyruvate oxidation mediated by pyruvate oxidase (EC 1.2.3.3.) derived from pediococcus sp. (Ngo, 1986) as shown in Eq. (1): pyruvate oxidase pyruvate + phosphate + H20 + 02 (1) acetylphosphate + H 2 0 2 + C O 2 In the presence of phosphate and oxygen, pyruvate is oxidized and converted to acetylphosphate with concomitant formation of hydrogen peroxide and carbon dioxide. Flavin adenine dinucleotide (FAD) and thiamine pyrophosphate (TPP) are necessary cofactors of the enzyme reaction. In the previous paper, the consumption of dissolved oxygen due to the reaction was detected amperometrically with a dissolved oxygen electrode.

* To whom correspondence should be addressed.

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Biosensors & Bioelectronics

Although this system was simple and easy to handle, it was not satisfactory for water quality monitoring because of its insufficiently low detection limit (12/~M phosphate). In order to improve the sensitivity of this system the present study employs a chemiluminescence system using horseradish peroxidase (EC 1.11.1.7) and luminol for the determination of the hydrogen peroxide produced by the pyruvate oxidation process described above. The basis of this system concerns two consecutive reactions: the above-mentioned phosphate acetylation reaction catalyzed by pyruvate oxidase and the subsequent luminol chemiluminescence reaction: peroxidase luminol + H202 + 2OH----) N 2 + 4H20 + hv

(2)

The maximum intensity of luminescence occurs at 420 nm and is detected by a photomultiplier tube. The method is expected to be highly sensitive because the background luminescence is essentially zero (Kricka & Thorpe, 1983). Optimum conditions for the pyruvate oxidation and peroxidase-catalyzed chemiluminescence reactions were determined and the system was evaluated for the determination of phosphate.

EXPERIMENTAL Materials

Pyruvate oxidase (POP) from Pediococcus sp. (EC 1.2.3.3.) was kindly donated by Toyojozo (Tokyo, Japan). Peroxidase (type VI) from horseradish (EC 1.11.1.7) was purchased from Sigma Chemical Co. (St. Louis, USA). Pyruvate, thiamine pyrophosphate chloride (TPP), 3-aminophthalic hydrazine (luminol), monopotassium phosphate, pyruvic acid and 4-iodophenol were purchased from Wako Pure Chemicals (Osaka, Japan). All chemicals used were laboratory grade. The pyruvate oxidase reaction mixture solution was similar to that used in the previous work (Kubo et al., 1991): 0.02 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (pH 7.0) consisting of TPP (0.6 mM), FAD (10~M), MgC12 (5 mM) and pyruvic acid (0.5 mM). TPP, a cofactor of pyruvate oxidase, was

Biosensors & Bioelectronics

purified by ion exchange column chromatography according to a previously reported method (Rindi & Gluseppe, 1961; Yusa, 1959). The ion exchanger resin employed was Dowex-1- X8 acetate form (200/400 mesh). The maximum absorbance of TPP occurs at 245 nm and fractions exhibiting this were collected. Phosphate concentrations of the TPP solutions before and after purification were determined by the molybdenum blue method (Lowry & Lopez, 1946). Sodium carbonate buffer (0.2 M, pH 9.5)or Tris-HCl buffer (0.2 M, pH 8.5) was employed for the chemiluminescent reaction. Luminol was dissolved in dimethylsulfoxide at a concentration of 20 mM and the solution was diluted to 12.5/~M with buffer solution. For the chemiluminescent reaction mixture, a buffer solution containing luminol, 4-iodophenol (10/~M) and horseradish peroxidase (2.8 U/ml) was used. Immobilization of pyruvate oxidase

Pyruvate oxidase (POP) was immobilized on aminoalkylated controlled pore glass (CPG, 80/ 120 mesh, pore size 500 A) by crosslinking with glutaraldehyde (Weetall & Hersh, 1969). Aminoalkylated CPG (500 mg), which had been degassed in vacuo in advance, was treated with 2 ml of 2.5% glutaraldehyde solution and rotated at room temperature. The resulting CPG was washed with water at least three times and then with 20 mM HEPES buffer (pH 7.0) containing 5/zM 2-mercaptoethanol once. The washed CPG was suspended in 12.5 ml of the same buffer. The same buffer (2.5 ml) containing pyruvate oxidase (700 U/ml) was mixed with the CPG suspension and treated at 4°C for 20 h and rotated for immobilization. It was washed well with 1 M NaC1 and stored in 20 mM HEPES buffer at 4°C. Measurement and apparatus

The phosphate sensing system is illustrated in Fig. 1. The system is composed of the immobilized pyruvate oxidase column, two peristaltic pumps (EYELA MP-3, Tokyo), a sample injector with a 25/xl sample loop (Model 7125, Rheodyne, Cotati, CA), a mixing joint (GL Science, Tokyo) and a luminometer (UPP2000, Meidensha, Tokyo) which consisted of a photomultiplier tube with a cell and a data processing unit. One pump delivered the pyruvate and cofactor solution for

Phosphate sensing system

the POP reaction to the POP column. When a sample solution containing phosphate was injected, it reacted with pyruvate on the POP column. The other pump delivered the chemiluminescence reagent containing luminol, peroxidase and 4-iodophenol to the mixing point. H202 from the POP column reacted with luminol at the mixing point and the resulting luminescence was detected with the photomultiplier tube. All experiments were conducted at ambient temperature.

RESULTS AND DISCUSSION Buffer for chemiluminescent reaction

Prior to phosphate determination, the optimum conditions of the luminol chemiluminescence were established. In this experiment the POP column was not incorporated in the system, and the 0.02 M HEPES buffer (pH 7.0) was pumped through the tube and H202 was injected directly to the mixing joint. It has been reported that the optimum pH for the chemiluminescent reaction is around pH 9.0 (Kricka & Thorpe, 1983). Therefore, 0.1 M Tris-HCl buffer (pH 8.5) and 0.2 M Na2CO3-NaHCO3 buffer (pH 9.5) were compared. The chemiluminescent reagents and buffer solutions were pumped to the mixing point with a flow rate of 1.5 ml/min. H202 solutions of various concentrations were injected and the resulting chemiluminescence (photon counts per second, CPS) were plotted as shown in Fig. 2. The chemiluminescence in sodium carbonate buffer showed a linear relationship to the H202 concentration from approximately 10-5 to 10 -7 M, but the chemiluminescence in Tris-HCl buffer did not. No stable chemiluminescence was obtained in Tris-HC1 buffer. Consequently, sodium carbonate buffer was employed in the subsequent experiments. Effect of enhancer on chemiluminescence

It was reported that 4-iodophenol was effective as an enhancer of the luminol chemiluminescence system (Kricka & Thorpe, 1983; Thorpe et al., 1985), so the efficacy of this compound was examined in our system. The detection was carried out as described above. Luminol chemiluminescence was measured with and without 4961

Kazunori Ikebukuro et al.

Biosensors & Bioelectronics

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Fig, 1. Schematic diagram of the phosphate sensing system based on immobilized pyruvate oxidase and chemiluminescent detection.

Response to hydrogen peroxide

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In these experiments the H 2 0 2 solution was passed through the immobilized POP column and then combined with the chemiluminescent reaction mixture at the mixing point. The flow rates of both of the solutions were 1.1 ml/min. The resulting chemiluminescence is plotted versus the concentration of H202 in Fig. 3. A linear relationship between the concentration and luminescence was observed above 10-9 M H 2 0 2 .

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Time dependence of detection [H202], M Fig. 2. Effect of pH and buffer on chemiluminescence. POP column reagent (2 mM pyruvic acid, 0.01 mM FAD, 0.6 mM TPP, 0.02 M HEPES pH 7.0), flow rate 1.5 ml/min, room temperature. ((3) Sodium carbonate buffer (0.2 M, pH 9.5); (0) Tris-HCl buffer (0.2 M,

p a 8.5). iodophenol (10/zM) at various concentrations of H202. The enhancement was apparently observed in our system above 10-7 M (data not shown). Thus 4-iodophenol was used in subsequent experiments. 962

In these experiments phosphate solutions of known concentration were injected into the system. Figure 4 illustrates a typical response curve of the phosphate. The reagents and flow rates were the same as for the detection of H202 . Before injection of the sample solution, the background chemiluminescence was measured. Injection of the sample solution brought about an increase in the luminescence which reached a maximum approximately 70-80 s after injection and subsided to the initial level within 150 s. No response was observed upon injection of water. Thus the time required for one measurement

Biosensors & Bioelectronics

Phosphate sensing system

Effect of flow rate

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The effect of the flow rate of the chemiluminescence reagents was examined using standard 80/zM phosphate, which corresponds to 2.5 ppm of phosphate. The flow rate of both the POPreagent solution and the chemiluminescence reagent was altered from 0.34 to 2.5 ml/min. As shown in Fig. 5, luminescence increased linearly with the flow rates up to 1.1 ml/min, plateauing at higher rates. Therefore a flow rate of 1.1 ml/ min is optimal for the analysis.

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The luminescent intensity is known to be greater at high pH (Kricka & Thorpe, 1983). The concentration of the buffer should be higher than that of POP reactions to ensure alkaline conditions for the chemiluminescent reaction. Effect of pH on the chemiluminescent reaction was examined using a phosphate standard (160/~M) and sodium carbonate buffers of pH9.0, 9.5, 10.0 and 10.5. The maximum response was observed at pH 9.5 where the CPS value was 10 times higher than those observed at pH 9.0 and 10.0 and 100 times higher than that at pH 10.5 (data not shown). Therefore carbonate buffer pH 9.5 was employed in this system.

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Fig. 4. Time dependence of the phosphate sensing system. POP column reagent (2 mM pyruvic acid, 0.01 nM FAD, 0.6 mM TPP, 0.02 M HEPES pH 7.0), chemiluminescence reagent (12.5 IzM luminol, 10 IxM 4-iodophenol, 10 mg/l horseradish peroxidase, 0.2 M Na2CO3--NaHC03, pH 9.5), flow rate 1.1 ml/min, room temperature. (--) 0.17 mM phosphate; (__) 0.33 mM phosphate. cycle was 120-150 s, 5 min shorter than that of

the electrode system which we had reported (Kubo et al., 1991).

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Fig. 5. Effect of flow rate on chemiluminescence. POP column reagent (2 mM pyruvic acid, 0.01 mM FAD, 0.6 mM TPP, 0.02 M HEPES pH 7.0), chemiluminescence reagent (12.5 FM luminol, 10 IxM 4-iodophenol, 10 mg/l horseradish peroxidase, 0.2 M Na2CO3--NaHC03, pH 9.5), room temperature.

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Biosensors & Bioelectronics

Determination of phosphate

Using our experimentally determined optimal conditions, various standard solutions of phosphate concentrations were analyzed. Figure 6 shows a typical calibration graph. As can be seen, a linear relationship was observed from 4.8 to 160/zM phosphate. The dynamic range is wider than that of the electrode system, which was 12-80/zM phosphate (Kubo et al., 1991). However, no response was observed below 3.2/~M of phosphate. The selectivity of this sensor system was also examined. When 10 mg/l sulfuric, nitric or hydrochloric acid was added to the standard solution of phosphate (160/zM, which corresponds to 5 mg/l phosphate), the response of the sensor was not affected (data not shown). The reproducibility of the response was also examined. Solutions of 80/.~M phosphate were measured five times every day using the same buffer. After five measurements over the period of 1 day, the POP column was filled with distilled water and stored at 4°C. The response was found to be constant over 4 days, after which it rapidly decreased (data not shown). The variances of these values were approximately 5%. We also examined the response of this sensor to an actual river water sample and did not observe any difference from a standard prepared with buffer. Thus it is possible to use this sensor

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[Phosphate], ~M Fig. 6. Response of system to phosphate concentration. POP column reagent (2 mM pyruvic acid, 0.01 mM FAD, 0.6 mM TPP, 0.02 M HEPES pH 7.0), chemiluminescence reagent (12.5 t~M luminol, 10 i~M 4iodophenol, 10 mg/l horseradish peroxidase, 0.2 M Na2CO3-NaHC03, pH 9.5), flow rate 1.1 ml/min, room temperature.

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system for the analysis of real river water. Whitehead et al. (1993) reported the use of this chemiluminescent reaction for the assessment of biological oxygen demand (BOD) in contaminated raw waters. In our sensor system no interference with the sensor response was observed due to the BOD. This is probably due to the dilution of the specimen resulting from the use of a flow injection system. In our previous paper (Kubo et al., 1991), the time was 7 min, and the dynamic range was 12-80/~M. In this system, the time was shortened to 3 min and the range expanded to 4.8-160/~M. The sensitivity of this system is thus better than the previously described sensor based on the oxygen detection. However, it is still inadequate for the detection of phosphate in river water for drinking. A lower detection limit should in principle be possible with further improvements. One factor is the purity of TPP. TPP is indispensable to pyruvate oxidase reaction, but phosphate contamination in TPP is a serious problem. In this study, phosphate contamination was reduced to 2.9/~M (0.09/~g/ml phosphate) after purification. The detection limit of this system is considered to be determined mainly by the contaminating phosphate. Therefore further purification is necessary to achieve lower detection limits. The sensitivity can also be improved by integrating the photon counting of chemiluminescence. In the electrode system (Kubo et al., 1991), the enzyme reaction was performed at an optimum temperature of 30°C, while in this system all the measurements were performed at room temperature and thus some improvement may be realized. In addition to this the chemiluminescence detection system should be optimized. In this system, only chemiluminescence that occurs directly in front of the photomultiplier can be detected. The lifetime of chemiluminescence is so short that the reaction should ideally occur immediately in front of the photomultiplier rather than be pumped from the mixing chamber. In this study, a commercial luminometer was used without any attempts at customization. In order to improve the system the time of flow from the mixing point to photomultiplier should be minimized. These improvements are being implemented and the detection limit of this system improved. In conclusion, a phosphate detection system based on enzymic conversion to H202 and a

diosensors & Bioelectronics subsequent chemiluminescent reaction has been developed for the rapid determination of phosphate. The system displays a linear response to phosphate concentrations ranging from 4.8 to 160/~M and is capable of directly analyzing unprepared river water samples.

ACKNOWLEDGEMENT We would like to thank Dr. S. McNiven for his helpful advice and discussions.

REFERENCES APHA AWWA WPCF (1976). Standard Methods for the Examination of Water and Wastewater, 14th edn., p. 466. D'Urso, E. M. & Coulet, P. R. (1990). Phosphatesensitive enzyme electrode: a potential sensor for environmental control. Anal. Chim. Acta, 239, 1-5. Guilbault, G. G. & Nanjo, M. (1975). A phosphate selective electrode based on immobilized alkaline phosphatase and glucose oxidase. Anal. Chim. Acta, 78, 69-74. Krebs, C. J. (1972). Ecology, The Experimental Analysis of Distribution and Abundance. Harper & Row, New York, 454--456. Kricka, L. J. & Thorpe, G. H. G. (1983). Chemiluminescent and biochemiluminescent methods in analytical chemistry. Analyst, 108, 1274-1296. Kubo, I., Inagawa, M., Sugawara, T., Arikawa, Y. & Karube, I. (1991). Phosphate sensor composed from immobilized pyruvate oxidase and an oxygen electrode. Anal. Lett., 24, 1711-1727.

Phosphate sensing system Lowry, O. H. & Lopez, J. A. (1946). The determination of inorganic phosphate in the presence of labile phosphate esters. J. Biol. Chem., 162, 421-428. Ngo, T. T. (1986). Single-enzyme-basedamperometric assay for phosphate ion. Appl. Biochem., 13, 127-131. Rindi, G. & Gluseppe, L. (1961). New chromatographic method for the determination of thiamine and its mono- di- tri- phosphate in animal tissue. Biochem. J., 78, 602. Thorpe, G. H. G., Kricka, L. J., Moseley, S. B. & Whitehead, T. P. (1985). Phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: application in luminescence-monitored enzyme immunoassays. Clin. Chem. 31/8, 1335-1341. Watanabe, E., Endo, H. & Toyama, K. (1988). Determination of phosphate ions with an enzyme sensor system. Biosensors, 3, 297-306. Weetall, H. H. & Hersh, L. S. (1969). Urease covalently coupled to porous glass. Biochim. Biophys. Acta, 85, 464. Weetall, H. H. & Jacobson, M. A. (1972). Studies on phosphate inhibition and quantitation using immobilized bacterial alkaline phosphatase. In: Fermentation Technology Today, G. Terui (ed.), Society of Fermentation Technology of Japan, p. 361. Whitehead, T. P., Thorpe, G., Lane, M., Watson, A. & Billings, C. (1993). A rapid and simple chemiluminescent assay for water quality monitoring. In: Biologie Prospective Comples Rendus du 8th Colloque de Pont a Mousson, M. M. Galteau, F. Siest & J. Henry (eds.). John Ubbey Eurotext, Paris, pp. 377-381. Yusa, T. (1959). Crystalline thiamine pyrophosphate (TPP); the preparation and characterization of authentic specimen. J. Biochem., 46, 391-395.

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