Chimica Actu, 83 (1976) 9-17 0 Elsevier Scientific Publishing Company, Amsterdam Analytica
POTEN~O~ETR~C
TAKUJI KAWASHIkIA* Lfeparfment
of Chemistry,
ENZYME ELECTRODE
Printed in The Netherlands
FOR URIC ACID
and G. A. RECHNITZ State
Lhiuersity
of New
York.
Buffalo, New
York
14.214
(U.S.A.) (Received 29tb September 1975)
SUMMARY An enzyme electrode for uric acid, with immobilized uricase on a pC0, membrane electrode, is described. Evaluation studies show that the enzyme electrode attains performance characteristics similar to homogeneous enzymatic conversion of uric acid under optimum solution conditions. Comparison of analyses carried out with the enzyme electrode and classical procedures on urine control samples demonstrates acceptable accuracy and precision for the electrode method.
Uric acid is an end-product of human purine metabolism and, as a result, is routinely measured in clinical laboratories as a diagnostic parameter [I]. Commonly used methods include spe~trophotometri~ determinations based on tungsten blue formation 12, 33, enzymatic oxidation by uricase enzyme [ 2, k-1], and other optical methods based on the use of colored or fluorescent reagents [8-H] . The enzyme-catalyzed oxidation of uric acid according to Uricase Uric acid -I- 0, A Allantoin -I-I-&O2 •!-CO2
(1)
has been recently utilized by Nanjo and Guilbault [13] as the basis for an amperometric electrode method where a urkase-coated p~at~um electrode is employed to monitor the disappearance of oxygen during.the reaction. The design and evaluation of a poten~~omet~~ enzyme electrode for uric acid is reported in this paper. The electrode employs immobilized uricase enzyme in conjunction with the Severinghaus-type [X4] gas-sensing pCOl membrane electrode. The properties and design characteristics of pC0, gas sensors have recently received renewed attention [15, 163 and have been used for enzyme electrodes for substrates such as urea and I/-tyrosine [IL?] _ In order to optimize reaction conditions and evaluate electrode performance, the results of enzyme electrode measurements were compared with studies on homogeneous reaction mixtures by both potentiometric and spectrophotometric techniques. It will be seen that the uricase enzyme *Visiting Research Scientist from Ragosbima University (Japan).
10
electrode, based on the pC02 sensor, yields results in excellent agreement with the homogeneous methods for both aqueous and urine control samples. EzXPERIhlENTAL
Reagents
Unless otherwise specified, all chemicals used were of reagent grade and all solutions were prepared with water which had been distilled, deionized and boiled. Stock solutions of uric acid (5 - low3 M) were prepared from the monosodium salt of uric acid (Sigma Chemical Co., St. Louis, MO.) and stored under refrigeration. Uricase enzyme (from Candida Utilis) was a gift from Toyobo Biochemicals, Osaka, Japan and had an activity of 3.5 units per mg. For the enzyme electrode studies, 3 mg of uricase were mixed with 20 mg of hydroxyethylcellulose (gift from Electronic Instruments, Ltd., Surrey, Englandj in 0.5 ml of phosphate buffer (0.1 M, pH 6.5); the resulting slurry could be stored under refrigeration for at least four months without significant loss of enzyme activity. Urine control samples representing a lyophilized preparation of pooled normal adult urine were obtained from Hycel, Inc., Houston, Texas (Assay Lot B120503) and contained 44 2 4 mg of uric acid per 100 ml as determined spectrophotometrically. Apparatus
A Corning pCOl electrode (Cat. No. 476107) was used as the principal component of the enzyme electrode assembly. Its internal filling solution was replaced with a solution 10m3M in NaHC03, 0.2 M in KCl, and saturated with AgCl in order to optimize response times at low CO, levels. The modified pCOl electrode was used for all homogeneous reaction measurements, and also for the automated analysis experiments after being fitted with a flow-through adapter_ Enzyme electrodes were constructed by coating the gas-permeable membrane of the pC0, electrode with the enzyme slurry and covering the sensing assembly with moistened cellophane. The resulting enzyme electrode was stored in buffer solution between use but could also be kept in air when not in use. Enzyme electrodes were also constructed by binding the uricase with albumin and glu’iaraldehyde, or directly to nylon nets, but the cellulose slurry technique was found to be best for the present purpose. All potentiometric measurements were done with a Beckman Research Model pH meter in conjunction with a Beckman lo-in Recorder. Automated analyses employed Technicon Type II automated samplers and proportioning pumps. All measurements were made in a thermostated cell which could be made gas-tight to prevent loss of CO2 or contamination from the room atmosnhere. r------
11
RESULTS
AND DISCUSSION
Uricase-catalyzed oxidation in homogeneous solution In order to establish optimum reaction conditions and to provide a valid basis for comparison among methods, the critical characteristics of the uricase-catalyzed oxidation of uric acid were first studied in homogeneous solution. For this purpose, the modified pCOZ electrode without immobilized enzyme was used to monitor the CO, produced under various reaction conditions. Preliminary experiments on purely inorganic calibration standards had shown that the electrode itself would not be limiting in terms of response time, sensitivity, or pH range required.
Figure 1 shows typical potential vs. time plots obtained when the production of CO, was monitored at various solution pH values for the oxidation of 1.43 - 10m3 M uric acid in the presence of 1 unit of uricase enzyme at 37 “C. Although it is apparent that best results are obtained at pH 6.5, the overall situation is considerably more complicated than can be shown in Fig. 1. First, the reaction consumes oxygen and the rate can be limited by the level of dissolved oxygen in the reaction mixture. The experiments of Fig. 1 were carried out in oxygen; parallel experiments made under atmospheres of air and of pure nitrogen showed considerable decreases of the reaction rate. however ) +Lr\ . ..-.+..LJ’“LS ..l#.C, +,1-n, :., ,:, P-w l__--..I.-a..-LA I^_.^ I^_A _^__^ ~..-.‘L1CI1.c lab= -cII 111 aLI. I”1 I”W ~U”bblal,e Ie”el> al.t! rrp,l”uucl”le
0
5
10 itme.
Fig.
1. Effect
of pH on CO:
formation
c, pH ‘7.0s; d, pH ‘7.38; e, pH S.00. 1.43 - lo-’ M uric acid.
15 min
from
20
25
uric acid. Curve a, pH 5.95;
All at 37 T,
1 unit of uricase enzyme
30
b, pH 6.50; per 2 ml of
12
and the final steady-state potentials are not appreciably different in air and pure oxygen on the time-scale shown_ At high uric acid levels, it may be necessary to bubble oxygen over the reaction mixture to prevent kinetic complications. The reaction rate for the enzyme-catalyzed oxidation of uric acid depends not only on the pH of the solution but on the buffer used to control the pH. For example, the pH-rate profile will be different in phosphate and in Tris buffers, because phosphate acts as an enzyme activator for uricase [ 181. For the sake of faster response and higher sensitivity, the phosphate buffer system at pH 6.5 was therefore selected for analytical purposes. A special situation arises in the ease of borate buffers. There is evidence to suggest that the stoichiometry of the uricase-catalyzed oxidation of uric acid is considerably altered from that of reaction (I) in the presence of borate ffZ+24] . As a result;, the production of CO2 is considerably lower than the consumption of uric acid or oxygen, and erroneous analytical results could be obtained in borate buffers by any method which monitors CO, production This effect was tested experimentally, and significant reductions in the amount of CO* produced in the presence of borate were indeed found. For example, only 73 % and 25 % of the expected amount of CO, were found when 1.4 - 10d3 M uric acid was reacted at pH 6.5 in the presence of low3 M and lo-’ M borate, respectively. Borate buffers must, therefore, be avoided in the present method_ Within limits, the reaction rate will depend on the enzyme catalyst concentration, This effect is seen in Fig, 2 where the enzyme concentration is varied from 0.1 to 2 units for the oxidation of 1.43 lop3 M uric acid (in a 2-ml reaction volume) under otherwise constant conditions. Although the initial reaction rate increases with increasing enzyme concentration over the range studied, the steady-state potentials obtained are very similar. In the case of the immobilized enzyme electrode (see below) the effective enzyme concentration in the micro-environment at the sensor surface will, of course, be quite high in comparison to the homogeneous studies. The effect of temperature on the enzyme-catalyzed reaction has been previously studied over the range 26-60 “C [19] _ The activation energy for the reaction changes from 12.4 kcal mol-’ at lower temperature to 4.5 kcal mol-’ at the highest temperature, suggesting a change in mechanism. The present study was restricted to the range 26-45 “C; the initial rates increased with increasing temperatures, but there was some decline in steady-state vatues in the upper temperature range_ (Fig, 3) On the basis of these considerations, 37 “C was selected as a convenient compromise between optimum rate and enzyme stability. The best conditions found in the above studies (pH 6.5 phosphate buffer, 37 OC, and 1 unit of enzyme per 2 ml of final reaction mixture) were used for the construction of analytical calibration curves for uric acid. From the typical calibration curve shown in Fig. 4, it can be seen that the method gives a linear response (slope 57 mV per concentration decade) over the l
13
-
160
> E _ Lu -
200
- 240 30
20 Time,
min
Fig. 2. Effect of enzyme concentration. Curve a, 2 units of uricase enzyme per 2 ml of 1.43 - UP M uric aicid; b, 1 unit; c, 0.5 unit; d, 0.3 unit; e, 0.1 unit. All at 37 “C and
pH 6.5 (0.1 M phosphate buffer).
Time,
Fig. 3_ Effect
min
of temperature_ Curve a, 45-8 OC!; b, 40-8 “C!; c, 37-O “C!; d, 30-O “C; 0.5 unit of uricase enzyme per 2 ml of 1.43 1tF M uric acid.
e, 26.0 “C, All at pH 6.5,
l
Fig. 4. Calibration curve for analysis in homogeneous solution_ buffer), 1 unit of uricase enzyme per 2 ml of reaction solution.
37 “C, pH 6.5
(phosphate
range 10 -4-2 . 5 - 10m3M uric acid. These results form the basis for comnarison mc ~~- -~ with the immobilized enzyme electrode to be discussed below. For purposes of practical, routine analysis using the homogeneous reaction method, it would be more convenient to automate the procedure. Figure 5 shows a schematic diagram of an automated system set up to determine uric acid by this method. A typical recording of the resulting tied-time analysis peaks over a range of uric acid concentrations is shown in Fig. 6. Although the automatic analysis method was not evaluated in full detail, it can be seen from the comparison in Table 1 that accuracy and precision are likely to be comparable to the manual method. Tmmnhilizd _.._.._ - ____ -_-
cwzvrne -‘--J ..__ &c&&g
The complete enzyme electrode, consisting of cellulose-trapped uricase held on the pC0, electrode by a cellophane film, was evaluated under the conditions found to be optimal by the normal measurements. Figure 7 shows a calibration curve obtained for the response of the enzyme electrode to uric acid at pH 6.5 (phosphate buffer) and 37 “C. In this case, the immobilized layer contained 3 mg of uricase per 20 mg of the cellulose matrix and was evenly spread over the ga+permeable membrane of the pCOZ electrode before being covered by the restraining cellophane film. Figure 7 should be compared with Fig. 4, the calibration curve for the homogeneous reaction under otherwise identical experimental conditions. It
15
incubation 37QC 25min I Buffer Water
pH 6.5 :,: ” I I ly\l LYJ
1.0
I
1.0
Proportioning P”“P
Fig. 5. Schematic diagram of automated 0.5 units enzyme per 0.1 ml.
flow
analysis system.
I
10
0
20
60
40
80
Uricase solution
contained
mv
100
Time,min
Fig. 6. mpical
output
peaks for analysis system.
Peak a, 4.76 - lo-’ M uric acid; b. 9.14 * M; f, 8.95 - lo-’ M; g. 1.30 - lo-’ M;
10-I M; c, 1.76 - IO* M; d, 3.97 - IO* M; e, 6.80 - 10“ h, 2.38
- 10-l
M. Other conditions
as in Fig. 5.
16 TABLE 1 Comparison analyses of uric acid in urine control= (uric acid found, mg/lOO ml) Method
Trial :
2
3
X+-S
Spectrophotometric
45.9
-
-
-
Manual enzyme Automated enzymeb Enzyme electrode=
44.4 45.4 42.4
43.4 43.4 44.4
44.4 41.3 42.4
44.1 + 0.6 42.1 + 1.5 43.1 + 1.2
=A.ll measurements at 37 OC, pH 6.5 (0.1 M phosphate buffer). bSample to wash ratio, 1:4. =0.2 ml of urine control sample.
Fig. 7_ Calibration curve fcr immobilized enzyme electrode. Enzyme layer contained 3 mg of uricase per 20 mg of cellulose. 37 OC, pH 6.5 (0.1 M phosphate buffer). can be seen that the two calibration
curves are not only very similar in range and slope (57 mV/decade), but also that the actual potential values obtained are essentially the same whether the pC0, electrode is used in the homogeneous reaction mixture or when its surface is covered with the trapped enzyme layer. This finding indicates that the enzyme electrode is operating efficiently in catalytic terms and that true steady-state potentials are being observed_ Over the concentration range studied, the enzyme electrode has a response time in the 5-lO-min range, which is similar to previously reported values for other enzyme electrodes of similar construction.
17
The reproducibility of the enzyme electrode was evaluated over a lo-day period by constructing calibration curves of the type shown in Fig. 7 on each day. Little change in operating characteristics could be found, except that the entire calibration curve shifted by ca. 2 mV over the 10-d period; the linear range and response slope were not changed. In order to evaluate the behavior of the enzyme electrode under more realistic clinical conditions, a comparison study of the manual, automated, and enzyme electrode methods was undertaken with urine control samples. These controls are lyophilized preparations of pooled normal adult urine and contain steroids, urea, creatinine, hydroxy acids, and inorganic materials in addition to uric acid. For further confidence, the control samples were also remeasured spectrophotometrically. Results of these studies are shown in Table 1. It can be seen that there is excellent agreement between the four methods employed and good reproducibility among the individual trials. For clinical purposes, a precision of -~5 % relative on uric acid determinations in this concentration range would be considered completely acceptable. We gratefully acknowledge support of a grant from the National Institutes of Health. T. Kawashima wishes to thank the Ministry of Education, Japan, frrr L"I
fin3nrsiQl IIIIc4IILIcLI
12tannnt-t clLLpp"I".
REFERENCES 1
J. D. Giorgio, in R. J. Henry, D. C. Cannon and J. W. Winkelman(Eds.), Clinical Chemistry;
2 3 4 5
6 C. k 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Principles
and Techniques,
2nd edn.,
Harper and Row
Publishers,
1974, p_ 526. R. J. Henry, C. Sobel and J. Kim, Am. J. Clin. Path., 28 (1957) 152. V. J. Pileggi, J. D. Giorgio and D. R. Wybenga, Clin. Chim. Acta, 37 (1972) E. Praetorius and H. Pouisen, Stand. J. Clin. Lab. Invest., 5 (1953) 273. T_ V. _ L Pn;,.r.tm‘x;r .,..rl II. u TI. xx,-rr.. t-l QCQ\ U”“_ Q-2-2 LaL.xC‘zIFAI41.U ,, 1-cU“) Am. d. C!in. Dn,L I cat.,..,cB= 2” (La”“, Dubbs,
F. W. Davis and W. S. Adams,
J_ Biol.
Chem.,
218
(1956)
N-Y.,
141.
497.
L. Liddle, J. E. Seegmiller and L. Laster, J. Lab. Clin. Med., 54 (1959) 93. K. Lorentz and W. Berndt, Anal. Biochem., 18 (1967) 58. ?. Hunzicker and H. Keller, 2. Klin. Chem. Klin. Biochem., 13 (1975) 89. N. Gochman and J. M. Schmitz, Clin. Chem.. 17 (1971) 1154. N. Kageyama, Clin. Chim. Acta, 30 (1971) 421. G. G. Guilbault and P. Hodapp, Anal. Lett., 1 (1968) 789. M. Nanjo and G. G. Guilbault, Anal. Chem., 46 (1974) 1769. J. W. Severinghaus and A. F. Bradley, J. Appl. Physiol.. 13 (1958) 515. J. W. Ross, J. H. Riseman and J. A. Krueger, Pure Appl. Chem., 36 (1973) 473. U. Fiedler, E. H. Hansen and J. Ruzicka, Anal. Chim. Acta, 74 (1975) 423. G. G. Guilbault and F. R. Shu, Anai. Chem., 44 (1972) 2161. K. Arima and K. Nose, Biochim. Biophys. Acta, 151 (1968) 54. H. Baum, G. Hiibscher and H. R. Mahler, Biochim. Biophys. Acta, 22 (1956) 514. F. Felix, F. Scheel and W. Schuler, 2. Physiol. Chem., 180 (1929) 90. W. Schuler, Z. Physiol. Chem., 208 (1932) 237. F. W. Kiemperer, J. Biol. Chem., 160 (1945) 111. E. S. Canellakis and P. P. Cohen, J_ Biol. Chem., 213 (1955) 385. G. Hiibscher, H. Baum and H. R. Mahler, Biochim. Biophys. Acta, 23 (1957) 43.
POTEN~O~ETR~C
TAKUJI KAWASHIkIA* Lfeparfment
of Chemistry,
ENZYME ELECTRODE
Printed in The Netherlands
FOR URIC ACID
and G. A. RECHNITZ State
Lhiuersity
of New
York.
Buffalo, New
York
14.214
(U.S.A.) (Received 29tb September 1975)
SUMMARY An enzyme electrode for uric acid, with immobilized uricase on a pC0, membrane electrode, is described. Evaluation studies show that the enzyme electrode attains performance characteristics similar to homogeneous enzymatic conversion of uric acid under optimum solution conditions. Comparison of analyses carried out with the enzyme electrode and classical procedures on urine control samples demonstrates acceptable accuracy and precision for the electrode method.
Uric acid is an end-product of human purine metabolism and, as a result, is routinely measured in clinical laboratories as a diagnostic parameter [I]. Commonly used methods include spe~trophotometri~ determinations based on tungsten blue formation 12, 33, enzymatic oxidation by uricase enzyme [ 2, k-1], and other optical methods based on the use of colored or fluorescent reagents [8-H] . The enzyme-catalyzed oxidation of uric acid according to Uricase Uric acid -I- 0, A Allantoin -I-I-&O2 •!-CO2
(1)
has been recently utilized by Nanjo and Guilbault [13] as the basis for an amperometric electrode method where a urkase-coated p~at~um electrode is employed to monitor the disappearance of oxygen during.the reaction. The design and evaluation of a poten~~omet~~ enzyme electrode for uric acid is reported in this paper. The electrode employs immobilized uricase enzyme in conjunction with the Severinghaus-type [X4] gas-sensing pCOl membrane electrode. The properties and design characteristics of pC0, gas sensors have recently received renewed attention [15, 163 and have been used for enzyme electrodes for substrates such as urea and I/-tyrosine [IL?] _ In order to optimize reaction conditions and evaluate electrode performance, the results of enzyme electrode measurements were compared with studies on homogeneous reaction mixtures by both potentiometric and spectrophotometric techniques. It will be seen that the uricase enzyme *Visiting Research Scientist from Ragosbima University (Japan).
10
electrode, based on the pC02 sensor, yields results in excellent agreement with the homogeneous methods for both aqueous and urine control samples. EzXPERIhlENTAL
Reagents
Unless otherwise specified, all chemicals used were of reagent grade and all solutions were prepared with water which had been distilled, deionized and boiled. Stock solutions of uric acid (5 - low3 M) were prepared from the monosodium salt of uric acid (Sigma Chemical Co., St. Louis, MO.) and stored under refrigeration. Uricase enzyme (from Candida Utilis) was a gift from Toyobo Biochemicals, Osaka, Japan and had an activity of 3.5 units per mg. For the enzyme electrode studies, 3 mg of uricase were mixed with 20 mg of hydroxyethylcellulose (gift from Electronic Instruments, Ltd., Surrey, Englandj in 0.5 ml of phosphate buffer (0.1 M, pH 6.5); the resulting slurry could be stored under refrigeration for at least four months without significant loss of enzyme activity. Urine control samples representing a lyophilized preparation of pooled normal adult urine were obtained from Hycel, Inc., Houston, Texas (Assay Lot B120503) and contained 44 2 4 mg of uric acid per 100 ml as determined spectrophotometrically. Apparatus
A Corning pCOl electrode (Cat. No. 476107) was used as the principal component of the enzyme electrode assembly. Its internal filling solution was replaced with a solution 10m3M in NaHC03, 0.2 M in KCl, and saturated with AgCl in order to optimize response times at low CO, levels. The modified pCOl electrode was used for all homogeneous reaction measurements, and also for the automated analysis experiments after being fitted with a flow-through adapter_ Enzyme electrodes were constructed by coating the gas-permeable membrane of the pC0, electrode with the enzyme slurry and covering the sensing assembly with moistened cellophane. The resulting enzyme electrode was stored in buffer solution between use but could also be kept in air when not in use. Enzyme electrodes were also constructed by binding the uricase with albumin and glu’iaraldehyde, or directly to nylon nets, but the cellulose slurry technique was found to be best for the present purpose. All potentiometric measurements were done with a Beckman Research Model pH meter in conjunction with a Beckman lo-in Recorder. Automated analyses employed Technicon Type II automated samplers and proportioning pumps. All measurements were made in a thermostated cell which could be made gas-tight to prevent loss of CO2 or contamination from the room atmosnhere. r------
11
RESULTS
AND DISCUSSION
Uricase-catalyzed oxidation in homogeneous solution In order to establish optimum reaction conditions and to provide a valid basis for comparison among methods, the critical characteristics of the uricase-catalyzed oxidation of uric acid were first studied in homogeneous solution. For this purpose, the modified pCOZ electrode without immobilized enzyme was used to monitor the CO, produced under various reaction conditions. Preliminary experiments on purely inorganic calibration standards had shown that the electrode itself would not be limiting in terms of response time, sensitivity, or pH range required.
Figure 1 shows typical potential vs. time plots obtained when the production of CO, was monitored at various solution pH values for the oxidation of 1.43 - 10m3 M uric acid in the presence of 1 unit of uricase enzyme at 37 “C. Although it is apparent that best results are obtained at pH 6.5, the overall situation is considerably more complicated than can be shown in Fig. 1. First, the reaction consumes oxygen and the rate can be limited by the level of dissolved oxygen in the reaction mixture. The experiments of Fig. 1 were carried out in oxygen; parallel experiments made under atmospheres of air and of pure nitrogen showed considerable decreases of the reaction rate. however ) +Lr\ . ..-.+..LJ’“LS ..l#.C, +,1-n, :., ,:, P-w l__--..I.-a..-LA I^_.^ I^_A _^__^ ~..-.‘L1CI1.c lab= -cII 111 aLI. I”1 I”W ~U”bblal,e Ie”el> al.t! rrp,l”uucl”le
0
5
10 itme.
Fig.
1. Effect
of pH on CO:
formation
c, pH ‘7.0s; d, pH ‘7.38; e, pH S.00. 1.43 - lo-’ M uric acid.
15 min
from
20
25
uric acid. Curve a, pH 5.95;
All at 37 T,
1 unit of uricase enzyme
30
b, pH 6.50; per 2 ml of
12
and the final steady-state potentials are not appreciably different in air and pure oxygen on the time-scale shown_ At high uric acid levels, it may be necessary to bubble oxygen over the reaction mixture to prevent kinetic complications. The reaction rate for the enzyme-catalyzed oxidation of uric acid depends not only on the pH of the solution but on the buffer used to control the pH. For example, the pH-rate profile will be different in phosphate and in Tris buffers, because phosphate acts as an enzyme activator for uricase [ 181. For the sake of faster response and higher sensitivity, the phosphate buffer system at pH 6.5 was therefore selected for analytical purposes. A special situation arises in the ease of borate buffers. There is evidence to suggest that the stoichiometry of the uricase-catalyzed oxidation of uric acid is considerably altered from that of reaction (I) in the presence of borate ffZ+24] . As a result;, the production of CO2 is considerably lower than the consumption of uric acid or oxygen, and erroneous analytical results could be obtained in borate buffers by any method which monitors CO, production This effect was tested experimentally, and significant reductions in the amount of CO* produced in the presence of borate were indeed found. For example, only 73 % and 25 % of the expected amount of CO, were found when 1.4 - 10d3 M uric acid was reacted at pH 6.5 in the presence of low3 M and lo-’ M borate, respectively. Borate buffers must, therefore, be avoided in the present method_ Within limits, the reaction rate will depend on the enzyme catalyst concentration, This effect is seen in Fig, 2 where the enzyme concentration is varied from 0.1 to 2 units for the oxidation of 1.43 lop3 M uric acid (in a 2-ml reaction volume) under otherwise constant conditions. Although the initial reaction rate increases with increasing enzyme concentration over the range studied, the steady-state potentials obtained are very similar. In the case of the immobilized enzyme electrode (see below) the effective enzyme concentration in the micro-environment at the sensor surface will, of course, be quite high in comparison to the homogeneous studies. The effect of temperature on the enzyme-catalyzed reaction has been previously studied over the range 26-60 “C [19] _ The activation energy for the reaction changes from 12.4 kcal mol-’ at lower temperature to 4.5 kcal mol-’ at the highest temperature, suggesting a change in mechanism. The present study was restricted to the range 26-45 “C; the initial rates increased with increasing temperatures, but there was some decline in steady-state vatues in the upper temperature range_ (Fig, 3) On the basis of these considerations, 37 “C was selected as a convenient compromise between optimum rate and enzyme stability. The best conditions found in the above studies (pH 6.5 phosphate buffer, 37 OC, and 1 unit of enzyme per 2 ml of final reaction mixture) were used for the construction of analytical calibration curves for uric acid. From the typical calibration curve shown in Fig. 4, it can be seen that the method gives a linear response (slope 57 mV per concentration decade) over the l
13
-
160
> E _ Lu -
200
- 240 30
20 Time,
min
Fig. 2. Effect of enzyme concentration. Curve a, 2 units of uricase enzyme per 2 ml of 1.43 - UP M uric aicid; b, 1 unit; c, 0.5 unit; d, 0.3 unit; e, 0.1 unit. All at 37 “C and
pH 6.5 (0.1 M phosphate buffer).
Time,
Fig. 3_ Effect
min
of temperature_ Curve a, 45-8 OC!; b, 40-8 “C!; c, 37-O “C!; d, 30-O “C; 0.5 unit of uricase enzyme per 2 ml of 1.43 1tF M uric acid.
e, 26.0 “C, All at pH 6.5,
l
Fig. 4. Calibration curve for analysis in homogeneous solution_ buffer), 1 unit of uricase enzyme per 2 ml of reaction solution.
37 “C, pH 6.5
(phosphate
range 10 -4-2 . 5 - 10m3M uric acid. These results form the basis for comnarison mc ~~- -~ with the immobilized enzyme electrode to be discussed below. For purposes of practical, routine analysis using the homogeneous reaction method, it would be more convenient to automate the procedure. Figure 5 shows a schematic diagram of an automated system set up to determine uric acid by this method. A typical recording of the resulting tied-time analysis peaks over a range of uric acid concentrations is shown in Fig. 6. Although the automatic analysis method was not evaluated in full detail, it can be seen from the comparison in Table 1 that accuracy and precision are likely to be comparable to the manual method. Tmmnhilizd _.._.._ - ____ -_-
cwzvrne -‘--J ..__ &c&&g
The complete enzyme electrode, consisting of cellulose-trapped uricase held on the pC0, electrode by a cellophane film, was evaluated under the conditions found to be optimal by the normal measurements. Figure 7 shows a calibration curve obtained for the response of the enzyme electrode to uric acid at pH 6.5 (phosphate buffer) and 37 “C. In this case, the immobilized layer contained 3 mg of uricase per 20 mg of the cellulose matrix and was evenly spread over the ga+permeable membrane of the pCOZ electrode before being covered by the restraining cellophane film. Figure 7 should be compared with Fig. 4, the calibration curve for the homogeneous reaction under otherwise identical experimental conditions. It
15
incubation 37QC 25min I Buffer Water
pH 6.5 :,: ” I I ly\l LYJ
1.0
I
1.0
Proportioning P”“P
Fig. 5. Schematic diagram of automated 0.5 units enzyme per 0.1 ml.
flow
analysis system.
I
10
0
20
60
40
80
Uricase solution
contained
mv
100
Time,min
Fig. 6. mpical
output
peaks for analysis system.
Peak a, 4.76 - lo-’ M uric acid; b. 9.14 * M; f, 8.95 - lo-’ M; g. 1.30 - lo-’ M;
10-I M; c, 1.76 - IO* M; d, 3.97 - IO* M; e, 6.80 - 10“ h, 2.38
- 10-l
M. Other conditions
as in Fig. 5.
16 TABLE 1 Comparison analyses of uric acid in urine control= (uric acid found, mg/lOO ml) Method
Trial :
2
3
X+-S
Spectrophotometric
45.9
-
-
-
Manual enzyme Automated enzymeb Enzyme electrode=
44.4 45.4 42.4
43.4 43.4 44.4
44.4 41.3 42.4
44.1 + 0.6 42.1 + 1.5 43.1 + 1.2
=A.ll measurements at 37 OC, pH 6.5 (0.1 M phosphate buffer). bSample to wash ratio, 1:4. =0.2 ml of urine control sample.
Fig. 7_ Calibration curve fcr immobilized enzyme electrode. Enzyme layer contained 3 mg of uricase per 20 mg of cellulose. 37 OC, pH 6.5 (0.1 M phosphate buffer). can be seen that the two calibration
curves are not only very similar in range and slope (57 mV/decade), but also that the actual potential values obtained are essentially the same whether the pC0, electrode is used in the homogeneous reaction mixture or when its surface is covered with the trapped enzyme layer. This finding indicates that the enzyme electrode is operating efficiently in catalytic terms and that true steady-state potentials are being observed_ Over the concentration range studied, the enzyme electrode has a response time in the 5-lO-min range, which is similar to previously reported values for other enzyme electrodes of similar construction.
17
The reproducibility of the enzyme electrode was evaluated over a lo-day period by constructing calibration curves of the type shown in Fig. 7 on each day. Little change in operating characteristics could be found, except that the entire calibration curve shifted by ca. 2 mV over the 10-d period; the linear range and response slope were not changed. In order to evaluate the behavior of the enzyme electrode under more realistic clinical conditions, a comparison study of the manual, automated, and enzyme electrode methods was undertaken with urine control samples. These controls are lyophilized preparations of pooled normal adult urine and contain steroids, urea, creatinine, hydroxy acids, and inorganic materials in addition to uric acid. For further confidence, the control samples were also remeasured spectrophotometrically. Results of these studies are shown in Table 1. It can be seen that there is excellent agreement between the four methods employed and good reproducibility among the individual trials. For clinical purposes, a precision of -~5 % relative on uric acid determinations in this concentration range would be considered completely acceptable. We gratefully acknowledge support of a grant from the National Institutes of Health. T. Kawashima wishes to thank the Ministry of Education, Japan, frrr L"I
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