- Email: [email protected]
Enzyme electrode system for oxalate determination utilizing oxalate decarboxylase immobilized on a carbon dioxide sensor
Analytica Chimica A&x. 121f1980) 111-118 63Blsevier Scientific Bubliihmg Company, Amsterdam -
Printed in-The Netherlands
ENZYME ELE~RODE SYSTEM FOR OXALATE D~TE~INATION UTILIZING OXALATE DECARBOXYLASE IMMOBILIZED ON A CARBON DIOXIDE SENSOR
R. K. KOBOS*
and T. A. RAMSEY
Department of Chemistry, (U.S.A.)
Virginia Commonwealth
University. Richmond,
VA 23284
(Received 2nd June 1980)
SUMMARY A highly selective enzyme electrode system for oxaiate is described in which the enzyme oxalate decarboxylase is immobilized on a carbon dioxide gas-sensing electrode. The response of the system is linear with the logarithm of the oxalate concentration between 2 x lo-* and 1 x 10m2M with a slope of 5’7-60 mV/decade. The oxalate detection limit is 4 x lo-’ M. Electrodes used with chemically immobilized enzyme are not affected by phosphate and sulfate at levels normahy found in urine and are very stable showing no decrease in response after one month of operation. The enzyme eketrade system functions well in urine, requiring minimal sample pretreatment. The recovery of oxalate added to five ahquots of a human control urine sample averaged 97.7% with an average relative standard deviation of 4.5%.
The determination of oxalate in urine is clinically important for the diagnosis of various forms of hyperoxaluria as well as in urinary stone research. In addition, a simple method for oxalate determination is needed in the analysis of foods and other nonbiological samples [l].Consequently, many different methods have been reported for the determination of oxalate El, 2] _ Most of these published me~odolo~es are complicated, requiring sample pretreatment as part of the determination. Enzymatic methods for oxalate have been reported using oxalate decarboxylase (E.C. 4.1.1.2) and more. recently oxalate oxidase (E.C. 1.2.3.4). Oxalate decarboxylase is commercially available and has been shown to be highly specific in catalyzing the reaction: oxalate + CO* i- formate. Methods have been developed, using the soluble enzyme, based either on the detection of CO, by means of manometric ]3,4], spectrophotometric [5,6], conductometric [‘I], and potentiometric techniques [6,8], or the spectrophotometric detection of formate using NAD requiring format-e reductase f9, lo] _ The problem with these techniques is that the enzyme is expensive and since the soluble enzyme is used, the methods are very costly. Furthermore, phosphate and sulfate, at levels normally found in urine, have been found to inhibit the enzyme [ 563, thereby complicating the determination.
112 Oxalate oxidase catalyzes the reaction: oxalate + O2 -+ X02 + HzOr. Methods based on this enzyme have used the detection of a pH change caused by the release of CO2 into an alkaline buffer using a pH electrode [Xl], and the spec~ophotome~c determination of the hydrogen peroxide produced by the immobilized enzyme [X2]. Oxalate oxidase is not available commercially and must be isolated from barley seedlings. In addition, several anions and cations, such as calcium, copper, and fluoride [11,13,143 in urine, inhibit the enzyme. Potentiometric enzyme electrode systems have been reported for substances such as urea [X5], uric acid [ 161, and lysine f 173 using decarboxylating enzymes immobilized on carbon dioxide gas-sensing electrodes. This configuration has the advantage that the enzyme can be reused for many assays and is therefore very cost-efficient. Furthermore, it has been reported that the effects of enzyme inhibitors can be greatly reduced or eliminated by using chemical immobilization techniques [X3, 191. The use of enzyme activators has also been described to increase enzyme activity and therefore the lifetime of enzyme electrode systems [ZO] . This paper describes the preparation and evaluation of oxaIate enzyme electrode systems based on commercially available oxalate decarboxylase. Chemical immobilization techniques were investigated as a means of reducing phosphate and sulfate inhibition as well as to increase the lifetime of the system. An enzyme activator, hydroquinone, which has been reported to increase the enzyme activity of oxalate decaxboxylase by 60-100% [21], was also used to improve the lifetime of the system. The resulting sensor provides a simple method for oxalate determination in complex samples such as urine, with minimal sample pretreatment. EXPERIMEN'i'AL Apparatus and reugents An Orion Model 95-02 carbon dioxide gas-sensor was used in the construction of the sensor systems. Potential measurements were made with a Corning Model 130 digital pEI meter in conjunction with a Houston Instruments Omniscribe recorder. All measurements were performed in a thermostated cell. Oxalate decarboxylase was obtained (Sigma Chemical Company) as a partially purified powder isolated from Cdybia velutipes. With some lots it was necessary to purify the enzyme further because of iow specific activity. An acetone fractionation procedure as described by Shimazono and Hayaishi 1211 was used, Standard sodium oxalate (Thorn Smith), amino acids, bovine serum albumin and glutaraldehyde (Sigma Chemical Company) and ultrapure urea (Schwarz and Mann) were used_ AlI other chemicals were analytical reagent grade_ Solutions were prepared with distilled-deionized water. Human control urine samples, Level I (normal), were obtained from Fisher Scientific.
113 Procedures
Activity determinations were made for each enzyme lot at 37 f 0.5”C in pH 3.0 citrate buffer by using the carbon dioxide sensor to monitor the rate of carbon dioxide production. A unit is defined as the amount of enzyme catalyzing the production of I pmol CO2 mine1 at 37°C and pH 3.0. The enzyme was immobilized either by entrapment within a dialysis membrane (Uni-Pore Polycarbonate Membrane, pore size 0.05 pm) or by covalent binding to the membrane of the COZ electrode using bovine serum albumin (SSA) and glu~dehyde f177 _ in the entrapment method, the desired amount of enzyme was dissolved in 30 r_tlof buffer directly on the gas-permeable membrane. The enzyme solution was covered with the dialysis membrane to hold the enzyme on the electrode surface. In the cov~ent-b~d~g procedure, the desired amount of enzyme was dissolved in 30 r.ll of buffer containing 15% BSA on the membrane of the COi sensor. Then, 3 ~1 of 25% glutaraldehyde was added. The solution was mixed thoroughly and allowed to cross-link for lo-15 min until complete solidification was achieved. A dialysis membrane was then placed over the immobilized enzyme. _ The enzyme electrode system was conditioned in 0.1 M, pH 3.0 citrate buffer for 1 h before use. All electrode studies were done at 30 & O.S’C. Calibration curves were obtained by making additions of 0.1 M sodium oxalate solution to 25 ml of buffer and recording the steady-state potentials. Recovery studies were performed using Urine Controls, reconstituted with distilled water according to the manufacturer’s instructions. The urine sample was then diluted (1 + 1) with double strength buffer. The carbon dioxide level of the diluted urine sample was first determined with the CO1 sensor and any endogenous carbon dioxide present was removed by passing nitrogen gently through the solution for 15 min. The oxalate sensor was then placed in the sample solution and the stable potential reading obtained. The initial oxalate concentration was determined from a priorcalibrationcurve. Additions of standard oxalate solution were made and the corresponding oxalate levels determined from the calibration curve. Rl3SULTS
AND
DISCUSSION
Preliminary studies, with a dialysis-membrane-entrapped enzyme electrode system containing 8 units of oxalate decarboxylase, were done in 0.1 M, pH 3.0 citrate buffer. This pH has been reported to be the optimum for the enzyme [Zl] and is highly compatible with the gas-sensing electrode, since essentially all of the carbon dioxide in solution is in the CO* form. Under these conditions, the enzyme electrode system exhibited a response that was linear with the logarithm of the oxalate concentration between 2 X 10d4 and 1 X low2 M with a slope of 57.6 + 2.1 mV/decade and with standard error and correlation coefficient of 1.5 and 0.9994, respectively. To determine the infhxence of pH on the enzyme electrode system, it
ws placed in a pH 6.0 citricacid buffer ccxxkdkng 1 X UT3 M ox&ate. Additions of concentrated hydrochloric acid were made, and the pH of the solution and the electrode potentiaI were recorded. The results of this study are shown in Fig. I_ The pH maximum response range was found to be 3.22.5 for this electrode system with the enzyme entrapped in the dialysis membrane. The chemically immobilized enzyme electrode system had a much broader optimum range, pH 45-2.5. AU subsequent studies were done at pR 3.9 for purposes of comparison, However, the chemically immobiized enzyme could be used at pH 4.5 with no loss of oxal&@ response. The response of the carbon dioxide gas-sensor to organic acids is fess at this pH [22], therefore the abiity to use the enzyme e&&rode system at pH 4.5 would diminish possible interferences in some samples. Enzyme electrode systems were prepared by using various amounts of enzyme ranging from 2 to 12 units. Et was found that a minimum of 8 uniti was required to produce maximum response. Therefore, 8-3.2 units of enzyme were used in all systems studied. The effect of hydruquinone, a reported activator of oxalate decarboxylase 1213) on the response of the enzyme electrode system was aIso investigated. Figure 2 shows the response of an eight-day old enzyme electrode system, the response of which had beguu to deteriorate {slope = 35.5 mV/decade), in the presence of 1 X 1 ON2M hydroquinone, The response slope under activated conditions was 49.3 mV/decade, indicating that the hydroquinone has a significant effect on the enzyme activity and therefore can be used Lo
Fig_ 1. Effect afpH on the potential of oxalateseusora Uxalate concentration is 1 X IO-” 3% (a) Dialysis-membrane-entrapped enzyme electrode system; (a) chemically immobilized enzyme electrode system. Fig_ 2. Response of an eight-day aid electrode system with enzyme membrane under aouactivated (a) andactivated(I) conditioa-l;.
e&a~ped in dialysis
115 incre;ise the, lifetime of the system. However, at this concentration level, hydroquinone inhibited the enzyme at low oxalate concentrations, i.e., 1 x lO-4 -3 X 10s4 M. The optimum amount of hydroquinone to be used was determined by ma&g additions of hydroquinone to a solution eontainin~ 1 X 10e4M oxah and recordkg the potential of the enzyme L electrode system. Figure 3 indicates that the concentration of the activator for maximum response is between ‘7 X low4 and 1.5 X 10W3M. Hydroquinone was added to the buffer solution at a concentration of 1 X 10m3M for all further studies. ‘I&pical calibration curves for the chemically immobilized enzyme electrode system under optimum conditions, i.e., pH 3.0 citrate buffer containing 1 X 10m3M hydroyuinone, are shown in Fig. 4. The response was linear with the logarithm of the oxdate concentration fkom 2 X lOF4 to 1 X lO-2 M with a slope of 59-5 I 2.1 m’lflcrecade (standard error 0.66; correlation coefficient 0.9998). Similar response was obtained with the dialysk-membrane-entrapped enzyme electrode system. The lower limit of detection, defined as the concentration at the point of intersection of the extrapolated linear segments of &he calibration curve [23], was 4 X low5 M, This range is &table for urinary ox&ate determinations, since ozkaiateis normahy present at concentrations of 1.6 X 10-4-5.5 X 10M4M (14-50 mg 1-l) [l]. If the urine sample is diluted (1 + 1) with double strength buffer, the normal ox&&e concentration range would skill be above the detection limit of the
Fig. 3. Effect of hydroquinoaeon the potential of the enzyme electrode system at an oxalate concentmtion of 1 x IO-’ M. Fig. 4. Calibration Day 1; f=) day 29.
curves of a cbemicalIy immabiIized
enzyme electrode system. fa)
116 electrode and could be determined by using the non-linear portion of the calibration curve. The response times of the enzyme electrode system ranged from 8 to 30 min depending on the thickness of the enzyme layer, Therefore, it is important to use an enzyme preparation with a high specific activity so that the required amount of enzyme results in a thin layer. By purification of the commercially available enzyme, the specific activity could be increased sufficiently to yield a thin enzyme layer, resulting in systems with response times of S-10 min. A specific activity of 1 unit mg-’ of solid was found to be adequate. The slowness of response is probably the most serious limitation of the sensor. However, when compared with other methods which require extensive sample pretreatment, the time of determination is favorable. The selectivity of the oxalate sensor was studied by testing the response to a number of substances. The Lisomers of the ammo acids lysine, glutamic acid, arginine, histidine, phenylahmine, methionine, alanine, asparagine, glutamine, leucine, aspartic acid, and tyrosine as well as urea produced no response at concentrations up to 3 X 10W3M, Similarly, the enzymeelectrode system did not respond to the following organic acids at these levels: acetic, formic, pyruvic, maleic, _malonic and suecinic acids. Some response was obtained to benzoic and sahcylic acids because of the carbon dioxide sensor itself_ The effects of phosphate and sulfate, which are known inhibitors of ox&to decarboxylase [5], were also investigated. The response of the enzyme electrode system based on a dialysis membrane was decreased in the presence of phosphate or sulfate at concentrations of 0.082 M, the highest concentration likely to be encountered in human urine [6]. This effect increased as the activity of the enzyme decreased over a period of one week_ Figure 5 shows the result of this level of inhibitors on a seven-day old oxalate sensor. The chemically immobilized enzyme electrode system was not affected by phosphate and sulfate at this same concentration level. Calibration curves obtained in the presence of phosphate and sulfate, each at a concentration of O-082 M, had a slope of 59-4 + 2.8 mV/decade_ This response is identical to that obtained in the absence of the inhibitors. The long-tt3rm stability of the two types of enzyme electrode systems differed markedly. The dialysis-membrane-entrapped enzyme electrode system began to show a decrease in response after 4 days when stored refrigerated in pH 4.5 citrate buffer_ This pH was chosen for the storage solution because it has been reported that it is optimal for enzyme stability fZ1] _ The response was analytitally useful for up to eight days_ The iifetime could be extended to two weeks by the use of hydroquinone to activate the enzyme. The chemically immobilized enzyme electrode system, as would be expected, had a much longer lifetime. When stored refrigerated in the storage sol&ion containing I X 10e3 M hydroquinone, the system showed no decrease in response after one month of daily use, as shown in F’ig. 4.
117
Fig. 5. Effect of phosphate and sulfate on the response of a seven-day old electrode system with enzyme entrapped in dialysis membrane. (e). No inhibitors present; (a) inhibitors present at a concentration of 0.082 M.
TABLE
1
Standard addition studies bf oxalate added to human control urine R.s.d. (%)
OxaIate (mg 1-l) Added= 0 18.0 35.9 53.7 89.1
Recovery (a)
Foundb 8.76 17.9 34-7 53.3 84.9
0.1 6.1 4.6 3.4 8.4
99.6 96.6 99.2 95.3
aAs anbydrous oxalic acid, bAverage of three determinations_
In order to demonstrate the possible future application of the oxalate sensor in clinical chemistry, a calibration curve was obtained in a control urine sample, The curve is essentially the same as that obtained in buffer, having a slope of 59.9 2 1.6 mV/decade with standard error of estimate of 0.76, and a correlation coefficient of 0.9997. Table 1 shows the recovery of oxalate added to control urine samples. The average recovery was 97.7%, indicating the potential usefulness of the enzyme electrode system for urinary oxalate determinations. We gratefully acknowledge the support of a grant from Research Corporation.
118
REFERENCES 1 A. Hodgkinson, Oxalic Acid in Biology and Medicine, Academic Prw, New York, 1977. 2 A. Hodgkinson, Clin. Chem., 16 (1970) 547. 3 G. G, Mayer, D. Markow and F. I&p, Ciin. Chem., 9 (1963) 334. 4 M. E. Ribeiro and J. S. Eiiiot, Invest. Ural., 2 (1964) 78. 5 C. F. Knowles and A. Hodgkinson, Analyst, 97 (1972) 474. 6 P_ C. Haiison and G. A, Rose, CIin. Chim. Acta, 55 (1974) 29. 7 J. D. Sailis, M. F. LumIey and J. E. Jordon, Biochem. Med., 18 (1977) 371. 8 S. J. Yao, S. K. Woifson and J. M. Tokarsky, Bioelectrochem. Bioenerg., 2 (1975) 348. 9 J. Costello, M. Hatch and E. Bourke, J. Lab. Ciin. Med., 87 (1976) 903. 10 M, Hatch., E. Bourke and J. Costello, Clin. Chem., 23 (1977) 76. 11 G. Kohlbecker, L. Richter and M_ Butz, J_ Clin. Chem. Ciin. Biochem., 17 (1979130912 R. Bais, N. Potezny, J. B. Edwards, A. M_ Rofe and R. A. J- Conyers, Anal. Chem., 52 (1980) 508. 13 J_ Chiriboga, Biochem. Biophys. R.es_ Commun., ll(lQ63) 277. 14 J_ Chiriboga, Arch_ Biochea Biophys., 116 (1966) 516. 15 G. G. Guiibault and F. R. Shu, Anai. Chea; 44 (1972) 2161. 16 T. Kawashima and G. A. Rechnitz, Anal. Chim. Acta, 83 (1976) 9. 17 W. C. White and G, G. Guiibault, AnaI. Chem, 50 (1978) 1481. 18 G. G_ Guiibauit, Handbook of Enzymatic Me&hods of Analysis, M. Dekker, New York, 1977, p- 497. 19 A. M. Rlibanov, Anal. Biochem., 93 (1979) 1. 20 M. E. Meyerhoff and G. A_ Rechnitz, Anal. Chim. Acta, 85 (1976) 277. 21 H. Shimazono and 0. Hayaishi, J. BioI. Chem., 227 (1957) 151. 22 Orion Carbon Dioxide Electrode Instruction Manual, Orion Research Inc., 1977, p. 19. 23 IUPAC Analytical Chemistry Division, Pure Appl, Chem., 48 (1976) 127.
Printed in-The Netherlands
ENZYME ELE~RODE SYSTEM FOR OXALATE D~TE~INATION UTILIZING OXALATE DECARBOXYLASE IMMOBILIZED ON A CARBON DIOXIDE SENSOR
R. K. KOBOS*
and T. A. RAMSEY
Department of Chemistry, (U.S.A.)
Virginia Commonwealth
University. Richmond,
VA 23284
(Received 2nd June 1980)
SUMMARY A highly selective enzyme electrode system for oxaiate is described in which the enzyme oxalate decarboxylase is immobilized on a carbon dioxide gas-sensing electrode. The response of the system is linear with the logarithm of the oxalate concentration between 2 x lo-* and 1 x 10m2M with a slope of 5’7-60 mV/decade. The oxalate detection limit is 4 x lo-’ M. Electrodes used with chemically immobilized enzyme are not affected by phosphate and sulfate at levels normahy found in urine and are very stable showing no decrease in response after one month of operation. The enzyme eketrade system functions well in urine, requiring minimal sample pretreatment. The recovery of oxalate added to five ahquots of a human control urine sample averaged 97.7% with an average relative standard deviation of 4.5%.
The determination of oxalate in urine is clinically important for the diagnosis of various forms of hyperoxaluria as well as in urinary stone research. In addition, a simple method for oxalate determination is needed in the analysis of foods and other nonbiological samples [l].Consequently, many different methods have been reported for the determination of oxalate El, 2] _ Most of these published me~odolo~es are complicated, requiring sample pretreatment as part of the determination. Enzymatic methods for oxalate have been reported using oxalate decarboxylase (E.C. 4.1.1.2) and more. recently oxalate oxidase (E.C. 1.2.3.4). Oxalate decarboxylase is commercially available and has been shown to be highly specific in catalyzing the reaction: oxalate + CO* i- formate. Methods have been developed, using the soluble enzyme, based either on the detection of CO, by means of manometric ]3,4], spectrophotometric [5,6], conductometric [‘I], and potentiometric techniques [6,8], or the spectrophotometric detection of formate using NAD requiring format-e reductase f9, lo] _ The problem with these techniques is that the enzyme is expensive and since the soluble enzyme is used, the methods are very costly. Furthermore, phosphate and sulfate, at levels normally found in urine, have been found to inhibit the enzyme [ 563, thereby complicating the determination.
112 Oxalate oxidase catalyzes the reaction: oxalate + O2 -+ X02 + HzOr. Methods based on this enzyme have used the detection of a pH change caused by the release of CO2 into an alkaline buffer using a pH electrode [Xl], and the spec~ophotome~c determination of the hydrogen peroxide produced by the immobilized enzyme [X2]. Oxalate oxidase is not available commercially and must be isolated from barley seedlings. In addition, several anions and cations, such as calcium, copper, and fluoride [11,13,143 in urine, inhibit the enzyme. Potentiometric enzyme electrode systems have been reported for substances such as urea [X5], uric acid [ 161, and lysine f 173 using decarboxylating enzymes immobilized on carbon dioxide gas-sensing electrodes. This configuration has the advantage that the enzyme can be reused for many assays and is therefore very cost-efficient. Furthermore, it has been reported that the effects of enzyme inhibitors can be greatly reduced or eliminated by using chemical immobilization techniques [X3, 191. The use of enzyme activators has also been described to increase enzyme activity and therefore the lifetime of enzyme electrode systems [ZO] . This paper describes the preparation and evaluation of oxaIate enzyme electrode systems based on commercially available oxalate decarboxylase. Chemical immobilization techniques were investigated as a means of reducing phosphate and sulfate inhibition as well as to increase the lifetime of the system. An enzyme activator, hydroquinone, which has been reported to increase the enzyme activity of oxalate decaxboxylase by 60-100% [21], was also used to improve the lifetime of the system. The resulting sensor provides a simple method for oxalate determination in complex samples such as urine, with minimal sample pretreatment. EXPERIMEN'i'AL Apparatus and reugents An Orion Model 95-02 carbon dioxide gas-sensor was used in the construction of the sensor systems. Potential measurements were made with a Corning Model 130 digital pEI meter in conjunction with a Houston Instruments Omniscribe recorder. All measurements were performed in a thermostated cell. Oxalate decarboxylase was obtained (Sigma Chemical Company) as a partially purified powder isolated from Cdybia velutipes. With some lots it was necessary to purify the enzyme further because of iow specific activity. An acetone fractionation procedure as described by Shimazono and Hayaishi 1211 was used, Standard sodium oxalate (Thorn Smith), amino acids, bovine serum albumin and glutaraldehyde (Sigma Chemical Company) and ultrapure urea (Schwarz and Mann) were used_ AlI other chemicals were analytical reagent grade_ Solutions were prepared with distilled-deionized water. Human control urine samples, Level I (normal), were obtained from Fisher Scientific.
113 Procedures
Activity determinations were made for each enzyme lot at 37 f 0.5”C in pH 3.0 citrate buffer by using the carbon dioxide sensor to monitor the rate of carbon dioxide production. A unit is defined as the amount of enzyme catalyzing the production of I pmol CO2 mine1 at 37°C and pH 3.0. The enzyme was immobilized either by entrapment within a dialysis membrane (Uni-Pore Polycarbonate Membrane, pore size 0.05 pm) or by covalent binding to the membrane of the COZ electrode using bovine serum albumin (SSA) and glu~dehyde f177 _ in the entrapment method, the desired amount of enzyme was dissolved in 30 r_tlof buffer directly on the gas-permeable membrane. The enzyme solution was covered with the dialysis membrane to hold the enzyme on the electrode surface. In the cov~ent-b~d~g procedure, the desired amount of enzyme was dissolved in 30 r.ll of buffer containing 15% BSA on the membrane of the COi sensor. Then, 3 ~1 of 25% glutaraldehyde was added. The solution was mixed thoroughly and allowed to cross-link for lo-15 min until complete solidification was achieved. A dialysis membrane was then placed over the immobilized enzyme. _ The enzyme electrode system was conditioned in 0.1 M, pH 3.0 citrate buffer for 1 h before use. All electrode studies were done at 30 & O.S’C. Calibration curves were obtained by making additions of 0.1 M sodium oxalate solution to 25 ml of buffer and recording the steady-state potentials. Recovery studies were performed using Urine Controls, reconstituted with distilled water according to the manufacturer’s instructions. The urine sample was then diluted (1 + 1) with double strength buffer. The carbon dioxide level of the diluted urine sample was first determined with the CO1 sensor and any endogenous carbon dioxide present was removed by passing nitrogen gently through the solution for 15 min. The oxalate sensor was then placed in the sample solution and the stable potential reading obtained. The initial oxalate concentration was determined from a priorcalibrationcurve. Additions of standard oxalate solution were made and the corresponding oxalate levels determined from the calibration curve. Rl3SULTS
AND
DISCUSSION
Preliminary studies, with a dialysis-membrane-entrapped enzyme electrode system containing 8 units of oxalate decarboxylase, were done in 0.1 M, pH 3.0 citrate buffer. This pH has been reported to be the optimum for the enzyme [Zl] and is highly compatible with the gas-sensing electrode, since essentially all of the carbon dioxide in solution is in the CO* form. Under these conditions, the enzyme electrode system exhibited a response that was linear with the logarithm of the oxalate concentration between 2 X 10d4 and 1 X low2 M with a slope of 57.6 + 2.1 mV/decade and with standard error and correlation coefficient of 1.5 and 0.9994, respectively. To determine the infhxence of pH on the enzyme electrode system, it
ws placed in a pH 6.0 citricacid buffer ccxxkdkng 1 X UT3 M ox&ate. Additions of concentrated hydrochloric acid were made, and the pH of the solution and the electrode potentiaI were recorded. The results of this study are shown in Fig. I_ The pH maximum response range was found to be 3.22.5 for this electrode system with the enzyme entrapped in the dialysis membrane. The chemically immobilized enzyme electrode system had a much broader optimum range, pH 45-2.5. AU subsequent studies were done at pR 3.9 for purposes of comparison, However, the chemically immobiized enzyme could be used at pH 4.5 with no loss of oxal&@ response. The response of the carbon dioxide gas-sensor to organic acids is fess at this pH [22], therefore the abiity to use the enzyme e&&rode system at pH 4.5 would diminish possible interferences in some samples. Enzyme electrode systems were prepared by using various amounts of enzyme ranging from 2 to 12 units. Et was found that a minimum of 8 uniti was required to produce maximum response. Therefore, 8-3.2 units of enzyme were used in all systems studied. The effect of hydruquinone, a reported activator of oxalate decarboxylase 1213) on the response of the enzyme electrode system was aIso investigated. Figure 2 shows the response of an eight-day old enzyme electrode system, the response of which had beguu to deteriorate {slope = 35.5 mV/decade), in the presence of 1 X 1 ON2M hydroquinone, The response slope under activated conditions was 49.3 mV/decade, indicating that the hydroquinone has a significant effect on the enzyme activity and therefore can be used Lo
Fig_ 1. Effect afpH on the potential of oxalateseusora Uxalate concentration is 1 X IO-” 3% (a) Dialysis-membrane-entrapped enzyme electrode system; (a) chemically immobilized enzyme electrode system. Fig_ 2. Response of an eight-day aid electrode system with enzyme membrane under aouactivated (a) andactivated(I) conditioa-l;.
e&a~ped in dialysis
115 incre;ise the, lifetime of the system. However, at this concentration level, hydroquinone inhibited the enzyme at low oxalate concentrations, i.e., 1 x lO-4 -3 X 10s4 M. The optimum amount of hydroquinone to be used was determined by ma&g additions of hydroquinone to a solution eontainin~ 1 X 10e4M oxah and recordkg the potential of the enzyme L electrode system. Figure 3 indicates that the concentration of the activator for maximum response is between ‘7 X low4 and 1.5 X 10W3M. Hydroquinone was added to the buffer solution at a concentration of 1 X 10m3M for all further studies. ‘I&pical calibration curves for the chemically immobilized enzyme electrode system under optimum conditions, i.e., pH 3.0 citrate buffer containing 1 X 10m3M hydroyuinone, are shown in Fig. 4. The response was linear with the logarithm of the oxdate concentration fkom 2 X lOF4 to 1 X lO-2 M with a slope of 59-5 I 2.1 m’lflcrecade (standard error 0.66; correlation coefficient 0.9998). Similar response was obtained with the dialysk-membrane-entrapped enzyme electrode system. The lower limit of detection, defined as the concentration at the point of intersection of the extrapolated linear segments of &he calibration curve [23], was 4 X low5 M, This range is &table for urinary ox&ate determinations, since ozkaiateis normahy present at concentrations of 1.6 X 10-4-5.5 X 10M4M (14-50 mg 1-l) [l]. If the urine sample is diluted (1 + 1) with double strength buffer, the normal ox&&e concentration range would skill be above the detection limit of the
Fig. 3. Effect of hydroquinoaeon the potential of the enzyme electrode system at an oxalate concentmtion of 1 x IO-’ M. Fig. 4. Calibration Day 1; f=) day 29.
curves of a cbemicalIy immabiIized
enzyme electrode system. fa)
116 electrode and could be determined by using the non-linear portion of the calibration curve. The response times of the enzyme electrode system ranged from 8 to 30 min depending on the thickness of the enzyme layer, Therefore, it is important to use an enzyme preparation with a high specific activity so that the required amount of enzyme results in a thin layer. By purification of the commercially available enzyme, the specific activity could be increased sufficiently to yield a thin enzyme layer, resulting in systems with response times of S-10 min. A specific activity of 1 unit mg-’ of solid was found to be adequate. The slowness of response is probably the most serious limitation of the sensor. However, when compared with other methods which require extensive sample pretreatment, the time of determination is favorable. The selectivity of the oxalate sensor was studied by testing the response to a number of substances. The Lisomers of the ammo acids lysine, glutamic acid, arginine, histidine, phenylahmine, methionine, alanine, asparagine, glutamine, leucine, aspartic acid, and tyrosine as well as urea produced no response at concentrations up to 3 X 10W3M, Similarly, the enzymeelectrode system did not respond to the following organic acids at these levels: acetic, formic, pyruvic, maleic, _malonic and suecinic acids. Some response was obtained to benzoic and sahcylic acids because of the carbon dioxide sensor itself_ The effects of phosphate and sulfate, which are known inhibitors of ox&to decarboxylase [5], were also investigated. The response of the enzyme electrode system based on a dialysis membrane was decreased in the presence of phosphate or sulfate at concentrations of 0.082 M, the highest concentration likely to be encountered in human urine [6]. This effect increased as the activity of the enzyme decreased over a period of one week_ Figure 5 shows the result of this level of inhibitors on a seven-day old oxalate sensor. The chemically immobilized enzyme electrode system was not affected by phosphate and sulfate at this same concentration level. Calibration curves obtained in the presence of phosphate and sulfate, each at a concentration of O-082 M, had a slope of 59-4 + 2.8 mV/decade_ This response is identical to that obtained in the absence of the inhibitors. The long-tt3rm stability of the two types of enzyme electrode systems differed markedly. The dialysis-membrane-entrapped enzyme electrode system began to show a decrease in response after 4 days when stored refrigerated in pH 4.5 citrate buffer_ This pH was chosen for the storage solution because it has been reported that it is optimal for enzyme stability fZ1] _ The response was analytitally useful for up to eight days_ The iifetime could be extended to two weeks by the use of hydroquinone to activate the enzyme. The chemically immobilized enzyme electrode system, as would be expected, had a much longer lifetime. When stored refrigerated in the storage sol&ion containing I X 10e3 M hydroquinone, the system showed no decrease in response after one month of daily use, as shown in F’ig. 4.
117
Fig. 5. Effect of phosphate and sulfate on the response of a seven-day old electrode system with enzyme entrapped in dialysis membrane. (e). No inhibitors present; (a) inhibitors present at a concentration of 0.082 M.
TABLE
1
Standard addition studies bf oxalate added to human control urine R.s.d. (%)
OxaIate (mg 1-l) Added= 0 18.0 35.9 53.7 89.1
Recovery (a)
Foundb 8.76 17.9 34-7 53.3 84.9
0.1 6.1 4.6 3.4 8.4
99.6 96.6 99.2 95.3
aAs anbydrous oxalic acid, bAverage of three determinations_
In order to demonstrate the possible future application of the oxalate sensor in clinical chemistry, a calibration curve was obtained in a control urine sample, The curve is essentially the same as that obtained in buffer, having a slope of 59.9 2 1.6 mV/decade with standard error of estimate of 0.76, and a correlation coefficient of 0.9997. Table 1 shows the recovery of oxalate added to control urine samples. The average recovery was 97.7%, indicating the potential usefulness of the enzyme electrode system for urinary oxalate determinations. We gratefully acknowledge the support of a grant from Research Corporation.
118
REFERENCES 1 A. Hodgkinson, Oxalic Acid in Biology and Medicine, Academic Prw, New York, 1977. 2 A. Hodgkinson, Clin. Chem., 16 (1970) 547. 3 G. G, Mayer, D. Markow and F. I&p, Ciin. Chem., 9 (1963) 334. 4 M. E. Ribeiro and J. S. Eiiiot, Invest. Ural., 2 (1964) 78. 5 C. F. Knowles and A. Hodgkinson, Analyst, 97 (1972) 474. 6 P_ C. Haiison and G. A, Rose, CIin. Chim. Acta, 55 (1974) 29. 7 J. D. Sailis, M. F. LumIey and J. E. Jordon, Biochem. Med., 18 (1977) 371. 8 S. J. Yao, S. K. Woifson and J. M. Tokarsky, Bioelectrochem. Bioenerg., 2 (1975) 348. 9 J. Costello, M. Hatch and E. Bourke, J. Lab. Ciin. Med., 87 (1976) 903. 10 M, Hatch., E. Bourke and J. Costello, Clin. Chem., 23 (1977) 76. 11 G. Kohlbecker, L. Richter and M_ Butz, J_ Clin. Chem. Ciin. Biochem., 17 (1979130912 R. Bais, N. Potezny, J. B. Edwards, A. M_ Rofe and R. A. J- Conyers, Anal. Chem., 52 (1980) 508. 13 J_ Chiriboga, Biochem. Biophys. R.es_ Commun., ll(lQ63) 277. 14 J_ Chiriboga, Arch_ Biochea Biophys., 116 (1966) 516. 15 G. G. Guiibault and F. R. Shu, Anai. Chea; 44 (1972) 2161. 16 T. Kawashima and G. A. Rechnitz, Anal. Chim. Acta, 83 (1976) 9. 17 W. C. White and G, G. Guiibault, AnaI. Chem, 50 (1978) 1481. 18 G. G_ Guiibauit, Handbook of Enzymatic Me&hods of Analysis, M. Dekker, New York, 1977, p- 497. 19 A. M. Rlibanov, Anal. Biochem., 93 (1979) 1. 20 M. E. Meyerhoff and G. A_ Rechnitz, Anal. Chim. Acta, 85 (1976) 277. 21 H. Shimazono and 0. Hayaishi, J. BioI. Chem., 227 (1957) 151. 22 Orion Carbon Dioxide Electrode Instruction Manual, Orion Research Inc., 1977, p. 19. 23 IUPAC Analytical Chemistry Division, Pure Appl, Chem., 48 (1976) 127.