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An improved urease electrode
ANALYTICAL
38,
BIOCHEMISTRY
An
Division
of Polymer P.
357-363
Improved
(1970)
Urease
Electrode
JOSEPH
G. MONTALVO,
and
Chemistry, Gulf South Research New Orleans, Louisiana 70126
0. Box
Physical 26600,
Received February
JR. Institute,
27, 1970
The importance of enzymes can hardly be overemphasized. Simpler methods are needed to assay the body enzymes of diagnostic value and to measure enzyme activity in other biological species. A new type of electrochemical sensor has been developed in this laboratory for the assay of the enzyme urease (1). The electrode sensor was made by coupling the substrate urea to the active surface of a cationic electrode responsive to ammonium ion, a product of the ureaurease reaction. The electrode was covered with a layer of cellophane trapping a thin layer of urea solution between the glass sensing bulb and the membrane. When bhe electrode was dipped into a solution containing urease, the urea which diffused out of the cellophane membrane reacted with urease (which cannot diffuse through the membrane) to produce ammonium ion at the membrane surface. The buildup of an ammonium ion activity gradient caused diffusion of the ion back to the electrode, where it was sensed. The urea concentration over the electrode surface was maintained constant during the assay by gravity flow from a small reservoir of urea. This type of electrode has been called an enzyme (urease) electrode because it is used to measure enzyme concentration (1). All preliminary studies with the urease electrode had been carried out with gravity or pressure flow control of urea. This type of flow control did not permit accurate measurements of the extremely small amounts of urea solution consumed in a urease assay and, of more importance, did not allow other necessary studies to be performed. This report describes a more elegant urease electrode and shows how the electrode response depended on the urea concentration, its flow rate through the membrane, and urease activity. 357
358
JOSEPH
G.
MONTALVO,
JR,
METHODS
Apparatus Figure 1 shows a diagram of the urease sensing electrode and configuration of the urea flow system. The urease electrode was made as previously described (1) except for the following changes. A 150 p nylon net was placed on both sides of the thin cellophane film. The netting between the glass bulb and cellophane film controls the thickness of the urea layer. The other nylon net helps to hold the cellophane against the glass electrode. All coverings were sealed to the glass electrode with Cutex nail hardener. Four applications of the sealent were applied. The viscosity of the first coating was increased by evaporation of the volatile solvent prior to application. MICRO TUBING SOLUTION
I.D. O.D.
‘-r
ION
NYLON
FIG.
NETTlNG‘-=~-
0.034” 0.050”
SENSING
ELECTRODE
::LOPHANF
1. Urease sensing electrode.
The concentration of urea in the space between the electrode and cellophane membrane was controlled with one chamber of a micro bilateral roller pump (Holter Company) . Procedures Procedures have been described utilizing gravity flow of urea (1). Assays performed with the urea pump followed this general scheme except for the change outlined below. A syringe was attached to the inlet of a pumping tube. The outlet of the pumping tube was attached to one of the micro tubings of the urease electrode. The syringe was filled with 0.5 M urea in Tris buffer, 0.1 M, pH 7.3. The space between the electrode and membrane was flushed with the urea by applying positive pressure to the syringe while allowing excess urea to flow out the open end of the other micro tubing.
AN
IMPROVED
UREASE
353
ELECTRODE
The pumping tube was then placed on the rollers. The reference electrode (saturated calomel electrode) and urease sensing electrode were stored at room temperature in an automatic electrode washer (miniature pipet washer) with the wash solvent (water or buffer) shut off until the electrode was ready to be used. Measurements were carried out in 50 ml of nonstirred solution which was thermostated at 25 -t O.lO”C. Enzyme solutions were prepared in Tris buffer from Type IV urease (Sigma Chemical Co., 3.53 units/mg). One unit will produce 1 mg ammonia nitrogen from urea in 5 min at pH 7.0 at 30°C. Wash solvent was allowed to flow through the electrode washer at 8 ml/min. The urea pump was turned on (flow rate 10 pl/min) and exactly 50 ~1 of urea was passed through the cellophane trap over the urease sensing electrode. Excess solution in the cellophane trap flowed out the opened end of the other micro tubing attached to the urease electrode and was discarded. The urea pump was turned off and both electrodes placed in the quiescent sample solution. A peak potential was obtained in about 3-5 min. Both electrodes were then rinsed in the automatic electrode washer. RESULTS
Figure 2 shows typical response curves obtained the flow rate of urea in the cellophane trap during
FIG. 2. Dependence of 0.06 unit/ml: ( > peaked llrease solution stirred.
electrode potential;
response on ( ) 98%
urea flow. response;
with 0.5 M urea vs assay. With no flow
Urease (vertical
concentration dashed line)
360
JOSEPH G. MONTALVO,
JR.
+110 -
95y: " v; ; '; ,' 80 -
65
r' 8.0
5.0
FIG. 3. Variation enzyme/ml.
2
I.0 [Ure.a],
of peak potential
with
M
urea concentration.
About
0.25 unit
of urea across the cation sensitive electrode surface during the assay, a peak potential was reached in 3.8-5.0 min. Curves B and C show the effect of urea flow across the glass electrode surface during assay. With 5 &min of urea flowing across the electrode the electrode response increases relative to that in curve A, but the
A B
+
0.5
5 M M
Urea urea
IO -
(Enzyme
FIQ. 4. Dependence
Cm.,
of peak potential
Unltr/ml.)
on urease concentration.
AN
IMPROVED
0
UREASE
Volume
5.
361
4
2 TIME
FIG.
ELECTRODE
(MIN.)
of substrate layer over the electrode.
shape of the curve is different. A steady-state potential obtained, but no peak potential, in 9 min. At 21.3 &min urea flow, increased response is again obtained relative to curve A, and a steady-state potential is obtained in less time than that of B. Figure 3 shows the effect of urea concentration on electrode response. The response of the urease electrode showed a first-order response to urea in the range, 0.25-5.0M. Above 5.0M, a zero-order dependence on urea was obtained. Figure 4 shows typical calibration curves for assay of urease with 0.5 and 5.0 M urea. First-order response curves were obtained in the region 0.0006-0.1 unit of urease/ml. The responses were highly reproducible. For example, with 0.5 M urea and 0.05 unit/ml, the deviation from the mean of five determinations was k1.6 mV. A study was conducted to determine the volume of the substrate trap over the electrode (Fig. 5), and the minimum volume of urea required per urease assay (Fig. 6). Figure 5 was obtained with the urease electrode in the electrode washer (flow rate, 7.5 ml,/min of buffer) while pumping 0.5 M urea at a flow rate of 10 &‘min across the space between the surface of the glass electrode and cellophane film. After the pump is turned on, the potential rises from a lower to a higher (more positive) steady-state potential in 2 min. This rise in potential is due to the cationic electrode sensing monovalent cationic impurities
0
FIG.
6.
40
20 MICROLITERS
OF
“REA
Minimum substrate volume per enzyme assay.
362
JOSEPH
G.
MONTALVO,
JR.
in the urea. Thus by multiplying the rate of flow of urea and the transition time, between the initial and final steady states, the volume of the cellophane trap over the electrode measures about 20 ~1. Figure 6 was obtained by plotting the peak potential obtained with 0.05 unit urease/ml against ~1 0.5 M urea which flowed across the glass electrode prior to urease assay. As shown in curve B, the peak potential is independent of the urea volume/assay down to about 30 ~1 of urea. DISCUSSION
The large differences in assay response curves as a result of changes in urea flow during assay is noteworthy. The peak potential response obtained with no flow across the electrode surface is due to depletion of urea at the electrode surface and subsequent diffusion of NH,+ ion outward from the electrode surface. The effect is enhanced by stirring and is known in other types of electroanalytical techniques (2). As the Aow rate of urea is increased during the assay, depletion of urea in the cellophane trap no longer exists. This results in attainment of a more sensitive and steady-state response. As the urea flow rate is increased still further, the response drops due to product (NH,’ ion) removed from the electrode surface rates approaching the diffusion of the NH,+ ion through the membrane trap to the glass surface. The first-order dependence of the electrode (product) response on substrate concentration is predicted when the substrate concentration is below the Km for the enzyme (3). With a urea indicating electrode (4, 5) a first-order response was obtained up to 0.01 M urea. The firstorder dependence found at higher urea coucentration with the urease electrode is due to dilution of urea in the boundary layer surrounding the cellophane film. Thus, when the urea concentration in the cellophane trap is 5 M, the concentration in the reaction layer adjacent to the trap must be at least 0.01 M, which could account for the observed dependence of electrode response on urea concentration. The first-order variation of t,he electrode response with urease activity was also obtained with gravity flow control of urea (l), which indicates that the responseto m-easeis first order whether or not urea flows across the electrode surface during the enzyme assay. To show just how little urea substrate is required per urease assay, a urease determination could be performed with 30 ~1 of 0.5 M urea, which is only 0.9 mg substrate. Using t,he smaller commercially available 39047 Beckman cationic electrode, this same assay could be performed with only 67 pg substrate. The advantages this electrode has over the more conventional use of an NH,+ electrode for sensing NH,+ concentration change in a stirred
Al;
IMPROVED
UREASE
ELECTRODE
363
urease-urea mixture are: (1) the quantity of substrate used per assay is always minimized and is always independent of the volume of enzyme solution assayed, and (2) the time required to reach the steady state or maximum (peak) potential is less because opposing urea and NH,+ gradient are set up at the electrode surface. SUMMARY
An important sensing electrode has been developed for assay of the enzyme urease. It is based on the peak or steady-state potential obtained with the aid of a roller pump to control the concentration of urea against the surface of an ammonium ion electrode. The urease sensing electrode is comparable in simplicity and mode of action with pH measurements with a glass electrode. ACKNOWLEDGMENTS The financial assistance of the National Institutes of Health (Grant 1 SO1 1 FR05672-01) is gratefully acknowledged. We are grateful to Bert Myers, M. D., Touro Infirmary, for use of the roller pump. REFERENCES 1. MONTALVO, J. G., And. Chem., 41, 2093 (1969). 2. “Electroanalytical Principles” (R. W. Murray and C. N. Reilley, eds.), Interscience, New York, 1966. 3. ‘LEnzymes” (M. Dixon and E. Webb. cds.). Academic Press New York, 1964. 4. GUILBAULT, G. G.. AND MONTALVO. J. G., Anal. Letters 2, 283 (1969). 5. GUILBAULT, G. G., AND MONTALVO, J. G., J. Am. Chem. Sot. 92, 2533 (1970).
38,
BIOCHEMISTRY
An
Division
of Polymer P.
357-363
Improved
(1970)
Urease
Electrode
JOSEPH
G. MONTALVO,
and
Chemistry, Gulf South Research New Orleans, Louisiana 70126
0. Box
Physical 26600,
Received February
JR. Institute,
27, 1970
The importance of enzymes can hardly be overemphasized. Simpler methods are needed to assay the body enzymes of diagnostic value and to measure enzyme activity in other biological species. A new type of electrochemical sensor has been developed in this laboratory for the assay of the enzyme urease (1). The electrode sensor was made by coupling the substrate urea to the active surface of a cationic electrode responsive to ammonium ion, a product of the ureaurease reaction. The electrode was covered with a layer of cellophane trapping a thin layer of urea solution between the glass sensing bulb and the membrane. When bhe electrode was dipped into a solution containing urease, the urea which diffused out of the cellophane membrane reacted with urease (which cannot diffuse through the membrane) to produce ammonium ion at the membrane surface. The buildup of an ammonium ion activity gradient caused diffusion of the ion back to the electrode, where it was sensed. The urea concentration over the electrode surface was maintained constant during the assay by gravity flow from a small reservoir of urea. This type of electrode has been called an enzyme (urease) electrode because it is used to measure enzyme concentration (1). All preliminary studies with the urease electrode had been carried out with gravity or pressure flow control of urea. This type of flow control did not permit accurate measurements of the extremely small amounts of urea solution consumed in a urease assay and, of more importance, did not allow other necessary studies to be performed. This report describes a more elegant urease electrode and shows how the electrode response depended on the urea concentration, its flow rate through the membrane, and urease activity. 357
358
JOSEPH
G.
MONTALVO,
JR,
METHODS
Apparatus Figure 1 shows a diagram of the urease sensing electrode and configuration of the urea flow system. The urease electrode was made as previously described (1) except for the following changes. A 150 p nylon net was placed on both sides of the thin cellophane film. The netting between the glass bulb and cellophane film controls the thickness of the urea layer. The other nylon net helps to hold the cellophane against the glass electrode. All coverings were sealed to the glass electrode with Cutex nail hardener. Four applications of the sealent were applied. The viscosity of the first coating was increased by evaporation of the volatile solvent prior to application. MICRO TUBING SOLUTION
I.D. O.D.
‘-r
ION
NYLON
FIG.
NETTlNG‘-=~-
0.034” 0.050”
SENSING
ELECTRODE
::LOPHANF
1. Urease sensing electrode.
The concentration of urea in the space between the electrode and cellophane membrane was controlled with one chamber of a micro bilateral roller pump (Holter Company) . Procedures Procedures have been described utilizing gravity flow of urea (1). Assays performed with the urea pump followed this general scheme except for the change outlined below. A syringe was attached to the inlet of a pumping tube. The outlet of the pumping tube was attached to one of the micro tubings of the urease electrode. The syringe was filled with 0.5 M urea in Tris buffer, 0.1 M, pH 7.3. The space between the electrode and membrane was flushed with the urea by applying positive pressure to the syringe while allowing excess urea to flow out the open end of the other micro tubing.
AN
IMPROVED
UREASE
353
ELECTRODE
The pumping tube was then placed on the rollers. The reference electrode (saturated calomel electrode) and urease sensing electrode were stored at room temperature in an automatic electrode washer (miniature pipet washer) with the wash solvent (water or buffer) shut off until the electrode was ready to be used. Measurements were carried out in 50 ml of nonstirred solution which was thermostated at 25 -t O.lO”C. Enzyme solutions were prepared in Tris buffer from Type IV urease (Sigma Chemical Co., 3.53 units/mg). One unit will produce 1 mg ammonia nitrogen from urea in 5 min at pH 7.0 at 30°C. Wash solvent was allowed to flow through the electrode washer at 8 ml/min. The urea pump was turned on (flow rate 10 pl/min) and exactly 50 ~1 of urea was passed through the cellophane trap over the urease sensing electrode. Excess solution in the cellophane trap flowed out the opened end of the other micro tubing attached to the urease electrode and was discarded. The urea pump was turned off and both electrodes placed in the quiescent sample solution. A peak potential was obtained in about 3-5 min. Both electrodes were then rinsed in the automatic electrode washer. RESULTS
Figure 2 shows typical response curves obtained the flow rate of urea in the cellophane trap during
FIG. 2. Dependence of 0.06 unit/ml: ( > peaked llrease solution stirred.
electrode potential;
response on ( ) 98%
urea flow. response;
with 0.5 M urea vs assay. With no flow
Urease (vertical
concentration dashed line)
360
JOSEPH G. MONTALVO,
JR.
+110 -
95y: " v; ; '; ,' 80 -
65
r' 8.0
5.0
FIG. 3. Variation enzyme/ml.
2
I.0 [Ure.a],
of peak potential
with
M
urea concentration.
About
0.25 unit
of urea across the cation sensitive electrode surface during the assay, a peak potential was reached in 3.8-5.0 min. Curves B and C show the effect of urea flow across the glass electrode surface during assay. With 5 &min of urea flowing across the electrode the electrode response increases relative to that in curve A, but the
A B
+
0.5
5 M M
Urea urea
IO -
(Enzyme
FIQ. 4. Dependence
Cm.,
of peak potential
Unltr/ml.)
on urease concentration.
AN
IMPROVED
0
UREASE
Volume
5.
361
4
2 TIME
FIG.
ELECTRODE
(MIN.)
of substrate layer over the electrode.
shape of the curve is different. A steady-state potential obtained, but no peak potential, in 9 min. At 21.3 &min urea flow, increased response is again obtained relative to curve A, and a steady-state potential is obtained in less time than that of B. Figure 3 shows the effect of urea concentration on electrode response. The response of the urease electrode showed a first-order response to urea in the range, 0.25-5.0M. Above 5.0M, a zero-order dependence on urea was obtained. Figure 4 shows typical calibration curves for assay of urease with 0.5 and 5.0 M urea. First-order response curves were obtained in the region 0.0006-0.1 unit of urease/ml. The responses were highly reproducible. For example, with 0.5 M urea and 0.05 unit/ml, the deviation from the mean of five determinations was k1.6 mV. A study was conducted to determine the volume of the substrate trap over the electrode (Fig. 5), and the minimum volume of urea required per urease assay (Fig. 6). Figure 5 was obtained with the urease electrode in the electrode washer (flow rate, 7.5 ml,/min of buffer) while pumping 0.5 M urea at a flow rate of 10 &‘min across the space between the surface of the glass electrode and cellophane film. After the pump is turned on, the potential rises from a lower to a higher (more positive) steady-state potential in 2 min. This rise in potential is due to the cationic electrode sensing monovalent cationic impurities
0
FIG.
6.
40
20 MICROLITERS
OF
“REA
Minimum substrate volume per enzyme assay.
362
JOSEPH
G.
MONTALVO,
JR.
in the urea. Thus by multiplying the rate of flow of urea and the transition time, between the initial and final steady states, the volume of the cellophane trap over the electrode measures about 20 ~1. Figure 6 was obtained by plotting the peak potential obtained with 0.05 unit urease/ml against ~1 0.5 M urea which flowed across the glass electrode prior to urease assay. As shown in curve B, the peak potential is independent of the urea volume/assay down to about 30 ~1 of urea. DISCUSSION
The large differences in assay response curves as a result of changes in urea flow during assay is noteworthy. The peak potential response obtained with no flow across the electrode surface is due to depletion of urea at the electrode surface and subsequent diffusion of NH,+ ion outward from the electrode surface. The effect is enhanced by stirring and is known in other types of electroanalytical techniques (2). As the Aow rate of urea is increased during the assay, depletion of urea in the cellophane trap no longer exists. This results in attainment of a more sensitive and steady-state response. As the urea flow rate is increased still further, the response drops due to product (NH,’ ion) removed from the electrode surface rates approaching the diffusion of the NH,+ ion through the membrane trap to the glass surface. The first-order dependence of the electrode (product) response on substrate concentration is predicted when the substrate concentration is below the Km for the enzyme (3). With a urea indicating electrode (4, 5) a first-order response was obtained up to 0.01 M urea. The firstorder dependence found at higher urea coucentration with the urease electrode is due to dilution of urea in the boundary layer surrounding the cellophane film. Thus, when the urea concentration in the cellophane trap is 5 M, the concentration in the reaction layer adjacent to the trap must be at least 0.01 M, which could account for the observed dependence of electrode response on urea concentration. The first-order variation of t,he electrode response with urease activity was also obtained with gravity flow control of urea (l), which indicates that the responseto m-easeis first order whether or not urea flows across the electrode surface during the enzyme assay. To show just how little urea substrate is required per urease assay, a urease determination could be performed with 30 ~1 of 0.5 M urea, which is only 0.9 mg substrate. Using t,he smaller commercially available 39047 Beckman cationic electrode, this same assay could be performed with only 67 pg substrate. The advantages this electrode has over the more conventional use of an NH,+ electrode for sensing NH,+ concentration change in a stirred
Al;
IMPROVED
UREASE
ELECTRODE
363
urease-urea mixture are: (1) the quantity of substrate used per assay is always minimized and is always independent of the volume of enzyme solution assayed, and (2) the time required to reach the steady state or maximum (peak) potential is less because opposing urea and NH,+ gradient are set up at the electrode surface. SUMMARY
An important sensing electrode has been developed for assay of the enzyme urease. It is based on the peak or steady-state potential obtained with the aid of a roller pump to control the concentration of urea against the surface of an ammonium ion electrode. The urease sensing electrode is comparable in simplicity and mode of action with pH measurements with a glass electrode. ACKNOWLEDGMENTS The financial assistance of the National Institutes of Health (Grant 1 SO1 1 FR05672-01) is gratefully acknowledged. We are grateful to Bert Myers, M. D., Touro Infirmary, for use of the roller pump. REFERENCES 1. MONTALVO, J. G., And. Chem., 41, 2093 (1969). 2. “Electroanalytical Principles” (R. W. Murray and C. N. Reilley, eds.), Interscience, New York, 1966. 3. ‘LEnzymes” (M. Dixon and E. Webb. cds.). Academic Press New York, 1964. 4. GUILBAULT, G. G.. AND MONTALVO. J. G., Anal. Letters 2, 283 (1969). 5. GUILBAULT, G. G., AND MONTALVO, J. G., J. Am. Chem. Sot. 92, 2533 (1970).