Biosensors& Bioelectmnics 6 (1991) 581-587
An FIA biosensor system for the determination of phosphate K. B. Male & J. H. T. Luong* Biotechnology
Research Institute, National Research Council of Canada. Montreal, Quebec, Canada H4P 2R2
(Received 24 September 1990; revised version received 5 December 1990, accepted 5 December 1990)
Ah&met: A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at O-7V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-l-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100~~ phosphate, with a minimum detectable concentration of 1*25@b1phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.
Keywords: FIA biosensor, phosphate, oxidase, amperometric electrode.
INTRODUCTION Determination of phosphate is of importance in most water management programs because it is one of the vital nutrients that stimulates or supports excessive and undesirable growth of aquatic life (Reup, 1968). Phosphate also occurs in several food products, and the adverse effect of the excess phosphate intake upon human health has been reported (Watanabe et al., 1988).The US recommended dietary allowance for phosphate is 800 mg day-’ for adults (Tie& 1987).Phosphate is used extensively in the treatment of boiler waters and in many fermentation processes to support the growth of microorganisms. Inorganic *To whom correspondence should be addressed.
nucleoside
phosphorylase,
xanthine
phosphate is involved in many metabolic reactions concerned with the generation of metabolic energy. The level of phosphate in plasma is an important determinant of bone mineral turnover (Tie& 1987).In view of this, it is of importance to develop a rapid, reliable and inexpensive technique for the determination of phosphate. The development of biosensors using oxidase enzymes coupled with amperometric electrodes is at present a growing area of research and development (Luong et al., 1988).The specificity and sensitivity of enzymes are complemented by the transducer, which electronically measures and computes the signal. The main advantage of enzyme electrodes is their inexpensiveness and generally simple preparation. Among several
0956-5463/91/$03.50 @ 1991 Elsevier Science Publishers Ltd.
581
Biosensors& Bioelectmnics 6 (1991) 581-587
K B. Male, .I. H i? Luong
enzymatic methods for the determination of phosphate (Schutz et al., 1967; Scopes, 1972; Fawaz & Tejirian, 1972;Guilbault h Nanjo, 1975; Schubert et al., 19&l), the coupled assay using oxidase (X0) and nucleoside xanthine phosphorylase (NP) is of interest (Hwang & Cha, 1973): inosine + Pi 5
+o* x0_
hypoxanthine ribose-l-phosphate uric acid + 2H202
(1)
Both NP and X0 are very stable and the determination of phosphate can be achieved by following the oxygen consumption or the production of hydrogen peroxide and/or uric acid during the enzymatic reactions. Based on the above reaction, Watanabe m al. (1988)developed an enzyme sensor system for the determination of phosphate ions in food. Both X0 and NP were immobilized simultaneously on a triacetyl cellulose membrane containing l&diamino4aminomethyloctane and attached to the sensing area of an oxygen electrode. This enzyme sensor system was able to withstand at least 70 assays with a linear range of 0.3-l mM. The detection of hydrogen peroxide has become an established approach to the development of amperometric biosensors using immobilized oxidases. The main advantage of hydrogen peroxide over the oxygen-electrode-based sensors is high sensitivity (Hendry el al., 1!490). Recently, flow-through techniques have been developed to improve the sensors’ capability (Kolev et al., 1990); for instance, its detection limit, stability and reproducibility. In these techniques, the system in equilibrium is perturbed by a pulse or a periodic signal and the relaxation processes leading to the new equilibrium can be analyzed and quantified. The main purpose of this study was to develop a flow injection analysis (FIA) biosensor incorporating immobilized enzymes and an phosphate electrode for amperometric determination in food products and plasma. The two enzymes, NP and X0, were coimmobilized on a preactivated nylon membrane and attached to the tip of the electrode. The applicability of the FIA biosensor system, in view of its accuracy, sensitivity, reliability, and reusability, are also presented and discussed in detail. 582
MATERIALS AND METHODS Materials
Hypoxanthine, inosine, glutaraldehyde, bovine serum albumin (BSA) (Fraction V), rabbit serum, human plasma, X0 and NP were purchased from Sigma Chemical Co. (St Louis, MO, USA). Food products containing phosphate were bought from a local supermarket. The preactivated Immunodyne”” nylon 66 membrane (pore size 3~m) was obtained from Pall BioSupport Division (Glen Cove, NY, USA). All of the materials were used as received except that NP and X0 were dialyzed against 4 liters of 20 mM phosphate buffer (pH 7) for 2 h to remove high levels of ammonium sulfate present in the enzyme preparations. Coimmobilization of NP aad X0
A prewetted Immunodyne membrane (1.5 cm X 1.5 cm) was stretched on the tip of a hollow plastic cylinder (1 cm diameter) and held in place by an O-ring. To a mixture containing dialyzed X0 and NP was added 2 ~1 of BSA (400 mg ml-‘) 18~1of phosphate buffer (20 mM, pH 7) and 4~1 of glutaraldehyde (25%w/v) to initiate the reaction. Experiments were performed at various concentrations of the two enzymes to establish the optimal immobilization conditions; 35~1 of the resulting solution (50~1) was immediately layered on the stretched membrane and the reaction was allowed to proceed for 30-45 min at room temperature (20-22°C). The membrane was then washed extensively with 100 mM Imidazole100 mM NaCl buffer (pH 7) to remove unreacted glutaraldehyde. The membranes loaded with enzymes (henceforth referred to as enzymic membrane) were stored at 4°C in 25 mM phosphate buffer (pH 7) containing 1 mM Mti+. The presence of magnesium was reported to stabilize both X0 and NP (Mulchandani et al., 1990). Measurement of enzyme activity The activity of NP bound on the membrane was
assayed by immersing the enzymic membrane in a test tube containing 1.5 ml of 10 mM phosphate buffer (pH 7) and 500 PM inosine for 2 min. The contents of the test tube were vortexed during this incubation period. The resulting solution (1 ml)
An FL4 biosensorfor phosphate detmination
Biosensors& Bioeiectrwdcs6 (1991) 58l-587
equipped with a preamplifier board which converts and amplifies the current output of the amperometric electrode to voltage (4-S mV nA-‘). The signal was then recorded on a chart recorder. The FIA detection system also displayed the response of the amperometric probe in relative units (RU), in which 1 RU is equal to 2.86pV at the detection output. The data acquisition was performed in peak height mode, i.e. the signal amplitude at which the peak occurs. The FIA biosensor system was calibrated using a standard phosphate solution which was spectrophotometrically calibrated a priori.
was transferred to a cuvette where soluble X0 (5~1) was then added to ensure that all hypoxanthine produced from inosine in the presence of NP was completely converted to uric acid. The reaction was followed by measuring the absorbance increase at 290 nm due to the production of uric acid (eqn (1)).
Figure 1 depicts a schematic diagram of the FIA biosensor system. A peristaltic pump (FL4 pump 1000,FIAtron Laboratory Systems, Oconomowoc, WI, USA) delivered the buffer at a preset flow rate. Unless otherwise indicated, this buffer contains 1 mM inosine in 100 mM Imidazole and 100 mM NaCl (pH 7) (referred to as P7). An 80 ~1 sample containing either phosphate or hypoxanthine in P7 was injected into this stream by a motorized injection valve (FL4 valve 2000, FIAtron Laboratory Systems). The resulting solution was delivered to the FIA detector system (FIA-zyme 500, FIAtron Laboratory Systems) consisting of an amperometric electrode (platinum versus silver/silver chloride at O-7V) which was attached to a temperature-controlled flow cell (22°C). As reported by Luong et al. (1989) under such conditions the electrode detects both uric acid and H202. The enzymic membrane was tightly attached to the tip of the electrode and held in place by an O-ring. The FIA-zyme 500 is
Measurement of phosphate by enzymatic assay
The enzymatic procedure was used to determine the phosphate levels of the standard solution and the samples of interest. To a sample containing phosphate, excess inosine, NP and X0 were added to convert inosine to uric acid. The concentration of uric acid produced was followed spectrophotometrically by measuring the absorbance at 290nm. The absorbance for O-1mMuricacidat290 nmwasreportedtobe 1.18 (Weast, 1974). Blanks were carried out in the absence of the phosphate-containing samples, to account for any endogenous phosphate. In the presence of excess inosine, NP and X0,1 mol of phosphate will react with inosine to produce
FIA-Zyme
Sample
l-xl
Infect valve
Waste 7 Pump
Waste
F&. 1. Schematic diagram of the FL4 biosensorsystem.The sample containing phosphate diluted in I nrM inosine, IW mu Imidazole and 1W mhi NaCl (pH 7) (&rt& to as P7) is injectedinto the buffer stream P7 and introducedinto the det&tion chamber equipped with an ampetvmemk electrode. 583
K. B. Male, J H. T. Luong
Biosensors & Bioelectmnics 6
(1991)581-587
1 mol of uric acid. This stoichiometry is used to determine the level of phosphate. SunpIe preparation
Liquid samples containing phosphate were diluted using P7 before analysis. Lyophilized human blood plasma and rabbit serum commercially prepared from 5 ml of plasma and rabbit serum, respectively, were reconstituted in 10 and 5 ml of the above buffer, respectively, and then diluted lOO-fold with P7. Solid samples containing phosphate (2.1 g for water-melon, honeydew and raisin, and 0.5 g for tea) were homogenized in 5 ml of the same buffer and after centrifugation the supematant obtained was diluted using P7.
(4
RESULTS AND DISCUSSION Effect of enzyme loading on tbe enzymic membrane
FIA biosensor incorporating an amperometric electrode and the membrane was evaluated for its response with respect to enzyme loading. As shown in Fig. 2, the activity of X0 in the enzyme layer increased with X0 amount used and reached a plateau at about 049 U of X0 or 69 pg of protein. Consequently, a two-fold excess of X0 (0.18 U or 138c(g of X0) was used to prepare the membrane. Similarly, the resulting NP activity on the membrane prepared with O-18U of X0 also increased as NP amount increased and levelled off at 064U of NP or 18Fg. As a result of this finding, 0.18 U of X0 and 2.56 U of NP (four-fold excess ofNP) was used for the preparation of the enzymic membrane. It should be noted that the cross-linking of the enzyme with BSA via glutaraldehyde will depend both glutaraldehyde and BSA upon concentrations. As previously reported (Mulchandani ef al., 1990), the resulting NP with glutaraldehyde increased activity concentrations up to l-2%. Above 2% the NP activity decreased drastically. The protein layer formed at 2% glutaraldehyde, however, was considerable stronger than that of 1% A concentration of 2% glutaraldehyde. glutaraldehyde was thus considered optimal in view of the enzyme activity and the mechanical strength of the protein layer. Although the membrane exhibited excellent responses to both The
(b) Fig. 2. (a) The e#zct of the amount of X0 used on the response of the FL4 biosensor system. The sample containing loO/ur hypoxanthine in P7 (80~0 k injected into the P7 bu#er stream. (b) The ej@t of the amount of NP used on the response of the FL4 biosensor system. The sample containing 1oOp~ phosphate in P7 (80~1) tk injected into the P7 bufler stream.
phosphate and hypoxanthine, the response to either phosphate or inosine was only 65% of that for an equimolar amount of hypoxanthine at the identical flow rate condition (16 ml h-l), an indication of a kinetic-controlled reaction. If NP and X0 were used as received, the enzyme layer obtained was thick and slightly yellow, an indication of pronounced diffusional limitations. Indeed, the experiment confirmed that the sensitivity of the enzymic membrane to both hypoxanthine and phosphate was decreased five-fold if the two enzymes were not dialyzed to remove ammonium sulfate before immobilization. This was why both X0 and NP were dialyzed before use as mentioned in ‘Materials and methods’. Effects of pH and buffer on the enzymic membrane
Based on the above findings, the enzymic membrane was prepared and used together with
Biosensors & Bioelectronics 6 (1991) 581-587
the FIA biosensor to evaluate the response of the system as a function of pH. The effect of pH on the activity of the enzymic membrane is illustrated in Fig. 3. Although the membrane exhibited a broad pH optimum (6-9), its response to lOO~(rvtphosphate decreased with repeated injections at pH below 6 or above 85 but not at pH 7. Consequently, pH 7 was used for subsequent experiments. Luong et al. (1989) also reported a broad pH optimum with a maximum at pH 7.5 for these two enzymes in the soluble state. Among several buffers tested, Imidazole was selected as it did not produce any background signal to the amperometric electrode. Experimental data also confirmed that this compound up to 100 mM exhibited no inhibitory effect on either NP or X0 (data not shown). Sodium chloride was added to the carrier buffer as it improves the sensitivity of the electrode, as shown by Luong er al. (1989). Responses of the FIA biosensor
The peristaltic pump was first set at its minimum flow rate (4 ml h-‘) to constantly deliver a solution containing either 100~~ phosphate or 100~~ hypoxanthine in P7. The steady-state responses obtained (157 000 and 273 000 RU, respectively) were used to normalize the FIA response. The pump was then set at different flow rates to deliver the P7 buffer to the detection chamber. To study the response of the FIA system, 80~1 of either 100~~ phosphate or 100FM hypoxanthine in P7 was injected into the P7 buffer stream. Figure 4 illustrates the normalized peak height FIA response (peak height obtained/steady-state response) as a function of the P7 buffer flow rate.
Fig. 3. The e&t of pH on the perJormance of the FL4 biosensorsystem. Thesamplecontaining 100 @f phosphate in p7 (SO@)is injected into the P7 bufler stream.
An FL-4biosensor for phosphate detetmination
0
*o
40
en
80
800
now
(Lo
uo
mlc
(mlh.‘)
160
Ia0
Fig. 4. The ej@t of theflow rate on the FL4 response. The sample containing either loopy phosphate or 100~~ hypoxanthine (SO@) in P7 is injected into the P7 buffer stream. The peak height response was normalized with respect to the steady-state responsefor a constantfrow rate of 4mlh-?
The response increased with a decrease in the sample flow rate, as expected, in accordance with the theoretical prediction for FIA systems with negligible mass transfer resistance in the bulk solution (Olsson et al., 1986) and experimental observations of Heider et al. (1990) and Olsson et al. (1986). As a compromise between the sensitivity of analysis and sample throughput (assays per hour), a flow rate of 16 ml h-’ was selected for all subsequent studies. A series of experiments was then conducted to examine the effect of inosine on the system’s response. As shown in Fig. 5, the response to 0.1 mM phosphate increased with increasing inosine concentrations up to 1 mM. Beyond this level, the response was insensitive to any further increase in inosine concentration. The maximum response for phosphate was observed at an inosine-to-phosphate ratio of 1O:l. As a result, this molar ratio was used to establish a calibration for the biosensor system to phosphate. Because the system’s response was linear up to 100MMphosphate (see below), 1 mM inosine concentration was used in all subsequent studies. The response of the FIA phosphate biosensor was linear up to 1OOpMphosphate and had a sensitivity of 682 + 15 RU FM-’ (95% confidence interval) with a correlation coefficient of one (n = 13).The minimum detectable concentration of phosphate was 1.25,!JMand the response time to obtain the peak after sample injection was 585
K B. Male, .I H T. Luong
Biosensorsd Bioekctronics 6
(1991)581-587
TABLE 1 Phosphate concentrations &termined by biosensor and spectrophotometer BWdUCts
Phosphate concentration (mg phosphate pw 100 mg product) Biosensor Spectrophotometw (C&J (C,)
Fig 5. The eJect of inosine on the response of the FL4 biosensor system. The sample containing various concentrationsof inosine in 100 pi phosphate, 100 mM Imidazoleand I00 mh#NaCl(pH 7) is injectedintoa buffw streamcontaining100 mMImidazole 100 mMNaC1(PH 7) and the same inosine concentrations as used in the sample.
Coca-Cola” Ekerb Soya sau&
0.030 036 019
0031 0.036 0.18
Rabbit serum Human blood plasma
0.022
0.023
Ox-t099 o-14 O-018 GO15 0097
O-0096 0.14 0.017 o-014 0.095
Dried raisinsb Honeydew melon” Water-melond Earl Grey teaC Dilution factors: ‘100X,
b200X,clOOOX,d25X,c50X.
2 min. The mean value for 14 repeated analyses of 1OOpM POT2 was 72 000 + 380 RU at 95%
confidence interval, i.e. the result is reproducible within only f 0.53% error. Each assay could be performed in 5-6 min, including washing and reconditioning the chamber and the electrode, giving a throughput of 10 assays h-‘. The response of the system to 100~~ phosphate showed little decrease in the sensitivity over the first 6 h of continuous use at 22”C, and sensitivity decreased by only 20% after 16 h of continuous operation, i.e. the system could be used for 160 analyses with periodic recalibrations. The loss in sensitivity was probably caused by inactivation of NP as the system showed no loss in sensitivity when operated continuously for 16 h using 100~~ hypoxanthine instead of phosphate. The performance of the FIA biosensor system thus compared favorably with that developed by Watanabe et al. (1988) in terms of sensitivity (0.3-l mM) and number of analyses (70). The membrane stored for 3 weeks in 25 mM phosphate buffer containing 1 mM M2’ lost only 30% of its original activity when measured spectrophotometrically (data not shown). However, this membrane still gave similar results to a freshly prepared membrane when used in the FIA phosphate biosensor run continuously for 16 h. Without phosphate, 50% of the activity was lost over the same time period, and if inosine buffer alone was used for storage more than 60% of the activity was lost. 586
Applicability of the FIA-phosphate biosensor
The FIA biosensor system was then applied to determine phosphate in various food products and in blood plasma. As shown in Table 1, the results obtained (average of duplicate experiments) by the biosensor (Cbio)agreed very well with those of the enzymatic assay (Csr&. With the exception of the tea sample, all samples tested did not exhibit any background when they were injected in the FIA-biosensor system containing a blank Immunodyne membrane. For the tea sample, the background reading was subtracted from the biosensor’s response to correct for non-specific response. At 95% contidence interval, a correlation between Cbio and C,,, was established as Cbio= (l-04 f 0033) Cspec- (0.001 f O-0026), with a correlation coefficient of 0999.
CONCLUSIONS Xanthine oxidase and nucleoside phosphorylase were cross-linked with bovine serum albumin and deposited on a commercially preactivated nylon membrane to form an enzymic membrane for phosphate. The membrane was used together with an FIA biosensor equipped with an amperometric electrode for the determination of phosphate in several food products and in
Biosensors 6 Bioelecttonics 6 (1991) 581-587
plasma. The FIA biosensor system appears attractive for the routine determination of phosphate ions. Both NP and X0 are very stable and commercially available, and the preparation of the enzymic membrane is simple and does not involve any expensive materials. The application of the FIA biosensor for the determination of phosphate in samples containing reducing agents such as ascorbic acid, glutathione or uric acid deserves some consideration, as such reducing agents interfere with the Hz02 response. One solution to this problem is the use of two identical electrodes: one with the enzymes and one without. Measurement is performed by subtracting the current from the electrode without enzymes from the current from the electrode with enzymes to eliminate the interfering current.
REFERENCES Fawaz, E. N. & Tejirian, A (1972). A new enzymatic method for the estimation of inorganic phosphate in native sera. Z. klin. Chem. klin. B&hem., 10, 215-19. Guilbault, G. G. & Nanjo, M. (1975). A phosphateselective electrode based on immobilized alkaline phosphatase and glucose ox&se. Anal. Chim. Acta, 78, 69-80. Heider, G. H., Sasso, S. V., Huang, K., Yacynych, A M. & Wieck, H. J. (1990). Electrochemical platinization of reticulated vitreous carbon electrodes to increase biosensor response. Anal. Chem., 62, 1106-10. Hendry, S. P., Higgins, I. J. & Bannister, J. V. (I*). Amperometric biosensors. J Biotechnol., IS, 229-38. Hwang, W. Z. & Cha, S. (1973). A new enzymatic method for the determination of inorganic
An FL4 biosensor for phosphate determination phosphate and its application to the nucleoside diphosphatase assay. Analyt. Biochem., 55,379-87. Keup, L. L. (1968). Phosphorus in flowing waters. Water Res., 2, 373-86. Kolev, S. D., Toth, K, Linder, E. & Pungor, E. (1990). Flow-injection approach for the determination of the dynamic response characteristics of ionselective electrodes, Part 1. Theoretical considerations. Anal. Chim. Acta, 223,49-56. Luong, J. H. T., Mulchandani, A. & Guilbault, G. G. (1988). Developments and applications of biosensors. Trends Biotechnok, 6, 310-16. Luong, J. H. T., Male, K. B. & Nguyen, A. L. (1989). Application of polarography for monitoring the fish postmortem metabolite transformation. Enzym. Microbial Technol., 11, 277-82. Mulchandani, A, Male, K B. & Luong, J. H. T. (1990). Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues. Biotechnol. Bioengng., 35, 739-45. Olsson, B., Lunback, H., Johansson, G., Scheller, F. & Nentwig, J. (1986). Theory and application of diffusion-limited amperometric enzyme electrode detection in flow injection analysis of glucose. Anal. Chem., 5% 1046-52. Schubert, F., Renneberg, R., Scheller, F. W. & I&stein, L. (1984). Plant tissue hybrid electrode for determination of phosphate and fluoride. Anal. Chem., 56, 1682-5. Schutz, D. W., Passoneua, J. V. & Lowry, 0. H. (1%7). An enzymic method for the measurement of inorganic phosphate. Anal. Chem.. 19, 300-14. Scopes, R K (1972). A new enzymatic method for inorganic phosphate determination. Anal. Biochem.. 49, 88-94. Tietz, N. W. (1987). Fundamentals of Clinical Chemistry, 3rd edn. W. B. Saunders, Philadelphia, PA, p. 7%. Watanabe, E., Endo, H. & Toyama, K (1988). Determination of phosphate ions with an enzyme sensor system. Biosensors, 3,297-306. Weast, R. C. (1974). Handbook of Chemistry and Physics, 54th edn. Chemical Rubber Co., Cleveland, OH, p. c-534.
587
An FIA biosensor system for the determination of phosphate K. B. Male & J. H. T. Luong* Biotechnology
Research Institute, National Research Council of Canada. Montreal, Quebec, Canada H4P 2R2
(Received 24 September 1990; revised version received 5 December 1990, accepted 5 December 1990)
Ah&met: A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at O-7V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-l-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100~~ phosphate, with a minimum detectable concentration of 1*25@b1phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.
Keywords: FIA biosensor, phosphate, oxidase, amperometric electrode.
INTRODUCTION Determination of phosphate is of importance in most water management programs because it is one of the vital nutrients that stimulates or supports excessive and undesirable growth of aquatic life (Reup, 1968). Phosphate also occurs in several food products, and the adverse effect of the excess phosphate intake upon human health has been reported (Watanabe et al., 1988).The US recommended dietary allowance for phosphate is 800 mg day-’ for adults (Tie& 1987).Phosphate is used extensively in the treatment of boiler waters and in many fermentation processes to support the growth of microorganisms. Inorganic *To whom correspondence should be addressed.
nucleoside
phosphorylase,
xanthine
phosphate is involved in many metabolic reactions concerned with the generation of metabolic energy. The level of phosphate in plasma is an important determinant of bone mineral turnover (Tie& 1987).In view of this, it is of importance to develop a rapid, reliable and inexpensive technique for the determination of phosphate. The development of biosensors using oxidase enzymes coupled with amperometric electrodes is at present a growing area of research and development (Luong et al., 1988).The specificity and sensitivity of enzymes are complemented by the transducer, which electronically measures and computes the signal. The main advantage of enzyme electrodes is their inexpensiveness and generally simple preparation. Among several
0956-5463/91/$03.50 @ 1991 Elsevier Science Publishers Ltd.
581
Biosensors& Bioelectmnics 6 (1991) 581-587
K B. Male, .I. H i? Luong
enzymatic methods for the determination of phosphate (Schutz et al., 1967; Scopes, 1972; Fawaz & Tejirian, 1972;Guilbault h Nanjo, 1975; Schubert et al., 19&l), the coupled assay using oxidase (X0) and nucleoside xanthine phosphorylase (NP) is of interest (Hwang & Cha, 1973): inosine + Pi 5
+o* x0_
hypoxanthine ribose-l-phosphate uric acid + 2H202
(1)
Both NP and X0 are very stable and the determination of phosphate can be achieved by following the oxygen consumption or the production of hydrogen peroxide and/or uric acid during the enzymatic reactions. Based on the above reaction, Watanabe m al. (1988)developed an enzyme sensor system for the determination of phosphate ions in food. Both X0 and NP were immobilized simultaneously on a triacetyl cellulose membrane containing l&diamino4aminomethyloctane and attached to the sensing area of an oxygen electrode. This enzyme sensor system was able to withstand at least 70 assays with a linear range of 0.3-l mM. The detection of hydrogen peroxide has become an established approach to the development of amperometric biosensors using immobilized oxidases. The main advantage of hydrogen peroxide over the oxygen-electrode-based sensors is high sensitivity (Hendry el al., 1!490). Recently, flow-through techniques have been developed to improve the sensors’ capability (Kolev et al., 1990); for instance, its detection limit, stability and reproducibility. In these techniques, the system in equilibrium is perturbed by a pulse or a periodic signal and the relaxation processes leading to the new equilibrium can be analyzed and quantified. The main purpose of this study was to develop a flow injection analysis (FIA) biosensor incorporating immobilized enzymes and an phosphate electrode for amperometric determination in food products and plasma. The two enzymes, NP and X0, were coimmobilized on a preactivated nylon membrane and attached to the tip of the electrode. The applicability of the FIA biosensor system, in view of its accuracy, sensitivity, reliability, and reusability, are also presented and discussed in detail. 582
MATERIALS AND METHODS Materials
Hypoxanthine, inosine, glutaraldehyde, bovine serum albumin (BSA) (Fraction V), rabbit serum, human plasma, X0 and NP were purchased from Sigma Chemical Co. (St Louis, MO, USA). Food products containing phosphate were bought from a local supermarket. The preactivated Immunodyne”” nylon 66 membrane (pore size 3~m) was obtained from Pall BioSupport Division (Glen Cove, NY, USA). All of the materials were used as received except that NP and X0 were dialyzed against 4 liters of 20 mM phosphate buffer (pH 7) for 2 h to remove high levels of ammonium sulfate present in the enzyme preparations. Coimmobilization of NP aad X0
A prewetted Immunodyne membrane (1.5 cm X 1.5 cm) was stretched on the tip of a hollow plastic cylinder (1 cm diameter) and held in place by an O-ring. To a mixture containing dialyzed X0 and NP was added 2 ~1 of BSA (400 mg ml-‘) 18~1of phosphate buffer (20 mM, pH 7) and 4~1 of glutaraldehyde (25%w/v) to initiate the reaction. Experiments were performed at various concentrations of the two enzymes to establish the optimal immobilization conditions; 35~1 of the resulting solution (50~1) was immediately layered on the stretched membrane and the reaction was allowed to proceed for 30-45 min at room temperature (20-22°C). The membrane was then washed extensively with 100 mM Imidazole100 mM NaCl buffer (pH 7) to remove unreacted glutaraldehyde. The membranes loaded with enzymes (henceforth referred to as enzymic membrane) were stored at 4°C in 25 mM phosphate buffer (pH 7) containing 1 mM Mti+. The presence of magnesium was reported to stabilize both X0 and NP (Mulchandani et al., 1990). Measurement of enzyme activity The activity of NP bound on the membrane was
assayed by immersing the enzymic membrane in a test tube containing 1.5 ml of 10 mM phosphate buffer (pH 7) and 500 PM inosine for 2 min. The contents of the test tube were vortexed during this incubation period. The resulting solution (1 ml)
An FL4 biosensorfor phosphate detmination
Biosensors& Bioeiectrwdcs6 (1991) 58l-587
equipped with a preamplifier board which converts and amplifies the current output of the amperometric electrode to voltage (4-S mV nA-‘). The signal was then recorded on a chart recorder. The FIA detection system also displayed the response of the amperometric probe in relative units (RU), in which 1 RU is equal to 2.86pV at the detection output. The data acquisition was performed in peak height mode, i.e. the signal amplitude at which the peak occurs. The FIA biosensor system was calibrated using a standard phosphate solution which was spectrophotometrically calibrated a priori.
was transferred to a cuvette where soluble X0 (5~1) was then added to ensure that all hypoxanthine produced from inosine in the presence of NP was completely converted to uric acid. The reaction was followed by measuring the absorbance increase at 290 nm due to the production of uric acid (eqn (1)).
Figure 1 depicts a schematic diagram of the FIA biosensor system. A peristaltic pump (FL4 pump 1000,FIAtron Laboratory Systems, Oconomowoc, WI, USA) delivered the buffer at a preset flow rate. Unless otherwise indicated, this buffer contains 1 mM inosine in 100 mM Imidazole and 100 mM NaCl (pH 7) (referred to as P7). An 80 ~1 sample containing either phosphate or hypoxanthine in P7 was injected into this stream by a motorized injection valve (FL4 valve 2000, FIAtron Laboratory Systems). The resulting solution was delivered to the FIA detector system (FIA-zyme 500, FIAtron Laboratory Systems) consisting of an amperometric electrode (platinum versus silver/silver chloride at O-7V) which was attached to a temperature-controlled flow cell (22°C). As reported by Luong et al. (1989) under such conditions the electrode detects both uric acid and H202. The enzymic membrane was tightly attached to the tip of the electrode and held in place by an O-ring. The FIA-zyme 500 is
Measurement of phosphate by enzymatic assay
The enzymatic procedure was used to determine the phosphate levels of the standard solution and the samples of interest. To a sample containing phosphate, excess inosine, NP and X0 were added to convert inosine to uric acid. The concentration of uric acid produced was followed spectrophotometrically by measuring the absorbance at 290nm. The absorbance for O-1mMuricacidat290 nmwasreportedtobe 1.18 (Weast, 1974). Blanks were carried out in the absence of the phosphate-containing samples, to account for any endogenous phosphate. In the presence of excess inosine, NP and X0,1 mol of phosphate will react with inosine to produce
FIA-Zyme
Sample
l-xl
Infect valve
Waste 7 Pump
Waste
F&. 1. Schematic diagram of the FL4 biosensorsystem.The sample containing phosphate diluted in I nrM inosine, IW mu Imidazole and 1W mhi NaCl (pH 7) (&rt& to as P7) is injectedinto the buffer stream P7 and introducedinto the det&tion chamber equipped with an ampetvmemk electrode. 583
K. B. Male, J H. T. Luong
Biosensors & Bioelectmnics 6
(1991)581-587
1 mol of uric acid. This stoichiometry is used to determine the level of phosphate. SunpIe preparation
Liquid samples containing phosphate were diluted using P7 before analysis. Lyophilized human blood plasma and rabbit serum commercially prepared from 5 ml of plasma and rabbit serum, respectively, were reconstituted in 10 and 5 ml of the above buffer, respectively, and then diluted lOO-fold with P7. Solid samples containing phosphate (2.1 g for water-melon, honeydew and raisin, and 0.5 g for tea) were homogenized in 5 ml of the same buffer and after centrifugation the supematant obtained was diluted using P7.
(4
RESULTS AND DISCUSSION Effect of enzyme loading on tbe enzymic membrane
FIA biosensor incorporating an amperometric electrode and the membrane was evaluated for its response with respect to enzyme loading. As shown in Fig. 2, the activity of X0 in the enzyme layer increased with X0 amount used and reached a plateau at about 049 U of X0 or 69 pg of protein. Consequently, a two-fold excess of X0 (0.18 U or 138c(g of X0) was used to prepare the membrane. Similarly, the resulting NP activity on the membrane prepared with O-18U of X0 also increased as NP amount increased and levelled off at 064U of NP or 18Fg. As a result of this finding, 0.18 U of X0 and 2.56 U of NP (four-fold excess ofNP) was used for the preparation of the enzymic membrane. It should be noted that the cross-linking of the enzyme with BSA via glutaraldehyde will depend both glutaraldehyde and BSA upon concentrations. As previously reported (Mulchandani ef al., 1990), the resulting NP with glutaraldehyde increased activity concentrations up to l-2%. Above 2% the NP activity decreased drastically. The protein layer formed at 2% glutaraldehyde, however, was considerable stronger than that of 1% A concentration of 2% glutaraldehyde. glutaraldehyde was thus considered optimal in view of the enzyme activity and the mechanical strength of the protein layer. Although the membrane exhibited excellent responses to both The
(b) Fig. 2. (a) The e#zct of the amount of X0 used on the response of the FL4 biosensor system. The sample containing loO/ur hypoxanthine in P7 (80~0 k injected into the P7 bu#er stream. (b) The ej@t of the amount of NP used on the response of the FL4 biosensor system. The sample containing 1oOp~ phosphate in P7 (80~1) tk injected into the P7 bufler stream.
phosphate and hypoxanthine, the response to either phosphate or inosine was only 65% of that for an equimolar amount of hypoxanthine at the identical flow rate condition (16 ml h-l), an indication of a kinetic-controlled reaction. If NP and X0 were used as received, the enzyme layer obtained was thick and slightly yellow, an indication of pronounced diffusional limitations. Indeed, the experiment confirmed that the sensitivity of the enzymic membrane to both hypoxanthine and phosphate was decreased five-fold if the two enzymes were not dialyzed to remove ammonium sulfate before immobilization. This was why both X0 and NP were dialyzed before use as mentioned in ‘Materials and methods’. Effects of pH and buffer on the enzymic membrane
Based on the above findings, the enzymic membrane was prepared and used together with
Biosensors & Bioelectronics 6 (1991) 581-587
the FIA biosensor to evaluate the response of the system as a function of pH. The effect of pH on the activity of the enzymic membrane is illustrated in Fig. 3. Although the membrane exhibited a broad pH optimum (6-9), its response to lOO~(rvtphosphate decreased with repeated injections at pH below 6 or above 85 but not at pH 7. Consequently, pH 7 was used for subsequent experiments. Luong et al. (1989) also reported a broad pH optimum with a maximum at pH 7.5 for these two enzymes in the soluble state. Among several buffers tested, Imidazole was selected as it did not produce any background signal to the amperometric electrode. Experimental data also confirmed that this compound up to 100 mM exhibited no inhibitory effect on either NP or X0 (data not shown). Sodium chloride was added to the carrier buffer as it improves the sensitivity of the electrode, as shown by Luong er al. (1989). Responses of the FIA biosensor
The peristaltic pump was first set at its minimum flow rate (4 ml h-‘) to constantly deliver a solution containing either 100~~ phosphate or 100~~ hypoxanthine in P7. The steady-state responses obtained (157 000 and 273 000 RU, respectively) were used to normalize the FIA response. The pump was then set at different flow rates to deliver the P7 buffer to the detection chamber. To study the response of the FIA system, 80~1 of either 100~~ phosphate or 100FM hypoxanthine in P7 was injected into the P7 buffer stream. Figure 4 illustrates the normalized peak height FIA response (peak height obtained/steady-state response) as a function of the P7 buffer flow rate.
Fig. 3. The e&t of pH on the perJormance of the FL4 biosensorsystem. Thesamplecontaining 100 @f phosphate in p7 (SO@)is injected into the P7 bufler stream.
An FL-4biosensor for phosphate detetmination
0
*o
40
en
80
800
now
(Lo
uo
mlc
(mlh.‘)
160
Ia0
Fig. 4. The ej@t of theflow rate on the FL4 response. The sample containing either loopy phosphate or 100~~ hypoxanthine (SO@) in P7 is injected into the P7 buffer stream. The peak height response was normalized with respect to the steady-state responsefor a constantfrow rate of 4mlh-?
The response increased with a decrease in the sample flow rate, as expected, in accordance with the theoretical prediction for FIA systems with negligible mass transfer resistance in the bulk solution (Olsson et al., 1986) and experimental observations of Heider et al. (1990) and Olsson et al. (1986). As a compromise between the sensitivity of analysis and sample throughput (assays per hour), a flow rate of 16 ml h-’ was selected for all subsequent studies. A series of experiments was then conducted to examine the effect of inosine on the system’s response. As shown in Fig. 5, the response to 0.1 mM phosphate increased with increasing inosine concentrations up to 1 mM. Beyond this level, the response was insensitive to any further increase in inosine concentration. The maximum response for phosphate was observed at an inosine-to-phosphate ratio of 1O:l. As a result, this molar ratio was used to establish a calibration for the biosensor system to phosphate. Because the system’s response was linear up to 100MMphosphate (see below), 1 mM inosine concentration was used in all subsequent studies. The response of the FIA phosphate biosensor was linear up to 1OOpMphosphate and had a sensitivity of 682 + 15 RU FM-’ (95% confidence interval) with a correlation coefficient of one (n = 13).The minimum detectable concentration of phosphate was 1.25,!JMand the response time to obtain the peak after sample injection was 585
K B. Male, .I H T. Luong
Biosensorsd Bioekctronics 6
(1991)581-587
TABLE 1 Phosphate concentrations &termined by biosensor and spectrophotometer BWdUCts
Phosphate concentration (mg phosphate pw 100 mg product) Biosensor Spectrophotometw (C&J (C,)
Fig 5. The eJect of inosine on the response of the FL4 biosensor system. The sample containing various concentrationsof inosine in 100 pi phosphate, 100 mM Imidazoleand I00 mh#NaCl(pH 7) is injectedintoa buffw streamcontaining100 mMImidazole 100 mMNaC1(PH 7) and the same inosine concentrations as used in the sample.
Coca-Cola” Ekerb Soya sau&
0.030 036 019
0031 0.036 0.18
Rabbit serum Human blood plasma
0.022
0.023
Ox-t099 o-14 O-018 GO15 0097
O-0096 0.14 0.017 o-014 0.095
Dried raisinsb Honeydew melon” Water-melond Earl Grey teaC Dilution factors: ‘100X,
b200X,clOOOX,d25X,c50X.
2 min. The mean value for 14 repeated analyses of 1OOpM POT2 was 72 000 + 380 RU at 95%
confidence interval, i.e. the result is reproducible within only f 0.53% error. Each assay could be performed in 5-6 min, including washing and reconditioning the chamber and the electrode, giving a throughput of 10 assays h-‘. The response of the system to 100~~ phosphate showed little decrease in the sensitivity over the first 6 h of continuous use at 22”C, and sensitivity decreased by only 20% after 16 h of continuous operation, i.e. the system could be used for 160 analyses with periodic recalibrations. The loss in sensitivity was probably caused by inactivation of NP as the system showed no loss in sensitivity when operated continuously for 16 h using 100~~ hypoxanthine instead of phosphate. The performance of the FIA biosensor system thus compared favorably with that developed by Watanabe et al. (1988) in terms of sensitivity (0.3-l mM) and number of analyses (70). The membrane stored for 3 weeks in 25 mM phosphate buffer containing 1 mM M2’ lost only 30% of its original activity when measured spectrophotometrically (data not shown). However, this membrane still gave similar results to a freshly prepared membrane when used in the FIA phosphate biosensor run continuously for 16 h. Without phosphate, 50% of the activity was lost over the same time period, and if inosine buffer alone was used for storage more than 60% of the activity was lost. 586
Applicability of the FIA-phosphate biosensor
The FIA biosensor system was then applied to determine phosphate in various food products and in blood plasma. As shown in Table 1, the results obtained (average of duplicate experiments) by the biosensor (Cbio)agreed very well with those of the enzymatic assay (Csr&. With the exception of the tea sample, all samples tested did not exhibit any background when they were injected in the FIA-biosensor system containing a blank Immunodyne membrane. For the tea sample, the background reading was subtracted from the biosensor’s response to correct for non-specific response. At 95% contidence interval, a correlation between Cbio and C,,, was established as Cbio= (l-04 f 0033) Cspec- (0.001 f O-0026), with a correlation coefficient of 0999.
CONCLUSIONS Xanthine oxidase and nucleoside phosphorylase were cross-linked with bovine serum albumin and deposited on a commercially preactivated nylon membrane to form an enzymic membrane for phosphate. The membrane was used together with an FIA biosensor equipped with an amperometric electrode for the determination of phosphate in several food products and in
Biosensors 6 Bioelecttonics 6 (1991) 581-587
plasma. The FIA biosensor system appears attractive for the routine determination of phosphate ions. Both NP and X0 are very stable and commercially available, and the preparation of the enzymic membrane is simple and does not involve any expensive materials. The application of the FIA biosensor for the determination of phosphate in samples containing reducing agents such as ascorbic acid, glutathione or uric acid deserves some consideration, as such reducing agents interfere with the Hz02 response. One solution to this problem is the use of two identical electrodes: one with the enzymes and one without. Measurement is performed by subtracting the current from the electrode without enzymes from the current from the electrode with enzymes to eliminate the interfering current.
REFERENCES Fawaz, E. N. & Tejirian, A (1972). A new enzymatic method for the estimation of inorganic phosphate in native sera. Z. klin. Chem. klin. B&hem., 10, 215-19. Guilbault, G. G. & Nanjo, M. (1975). A phosphateselective electrode based on immobilized alkaline phosphatase and glucose ox&se. Anal. Chim. Acta, 78, 69-80. Heider, G. H., Sasso, S. V., Huang, K., Yacynych, A M. & Wieck, H. J. (1990). Electrochemical platinization of reticulated vitreous carbon electrodes to increase biosensor response. Anal. Chem., 62, 1106-10. Hendry, S. P., Higgins, I. J. & Bannister, J. V. (I*). Amperometric biosensors. J Biotechnol., IS, 229-38. Hwang, W. Z. & Cha, S. (1973). A new enzymatic method for the determination of inorganic
An FL4 biosensor for phosphate determination phosphate and its application to the nucleoside diphosphatase assay. Analyt. Biochem., 55,379-87. Keup, L. L. (1968). Phosphorus in flowing waters. Water Res., 2, 373-86. Kolev, S. D., Toth, K, Linder, E. & Pungor, E. (1990). Flow-injection approach for the determination of the dynamic response characteristics of ionselective electrodes, Part 1. Theoretical considerations. Anal. Chim. Acta, 223,49-56. Luong, J. H. T., Mulchandani, A. & Guilbault, G. G. (1988). Developments and applications of biosensors. Trends Biotechnok, 6, 310-16. Luong, J. H. T., Male, K. B. & Nguyen, A. L. (1989). Application of polarography for monitoring the fish postmortem metabolite transformation. Enzym. Microbial Technol., 11, 277-82. Mulchandani, A, Male, K B. & Luong, J. H. T. (1990). Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues. Biotechnol. Bioengng., 35, 739-45. Olsson, B., Lunback, H., Johansson, G., Scheller, F. & Nentwig, J. (1986). Theory and application of diffusion-limited amperometric enzyme electrode detection in flow injection analysis of glucose. Anal. Chem., 5% 1046-52. Schubert, F., Renneberg, R., Scheller, F. W. & I&stein, L. (1984). Plant tissue hybrid electrode for determination of phosphate and fluoride. Anal. Chem., 56, 1682-5. Schutz, D. W., Passoneua, J. V. & Lowry, 0. H. (1%7). An enzymic method for the measurement of inorganic phosphate. Anal. Chem.. 19, 300-14. Scopes, R K (1972). A new enzymatic method for inorganic phosphate determination. Anal. Biochem.. 49, 88-94. Tietz, N. W. (1987). Fundamentals of Clinical Chemistry, 3rd edn. W. B. Saunders, Philadelphia, PA, p. 7%. Watanabe, E., Endo, H. & Toyama, K (1988). Determination of phosphate ions with an enzyme sensor system. Biosensors, 3,297-306. Weast, R. C. (1974). Handbook of Chemistry and Physics, 54th edn. Chemical Rubber Co., Cleveland, OH, p. c-534.
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