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New enzyme sensors for urea and creatinine analysis
195
Bioelectrochemistry and Bioenergetics, 23 (1990) 195-202 A section of J. Electroanal. Chem., and constituting Vol. 298 (1990) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
New enzyme sensors for urea and creatinine analysis * L. Campanella **, F. Mazzei, M.P. Sammartino Dipartimento (Received
di Chimica, Vniversrta ’
29 September
and M. Tomassetti
“LA Sapienza”, p.le A. More, 5, 00185 Roma (Italy)
1989; in revised form 28 December
1989)
ABSTRACT
New selective sensors sensitive to urea or creatinine have been developed and different geometries of the electroenzymatic systems investigated. The sensors were characterised and used to analyse clinical matrices (urines and control sera).
INTRODUCTION
In the program presently running in our laboratory, aimed at the development of new electrochemical potentiometric sensors and ion-selective electrodes [1,2], we recently developed both a new assembly procedure for ion-selective polymeric electrodes [3] and an efficient immobilisation method for the entrapment of enzymes [4]. The most recent results of these investigations concern the realisation of two new kinds of enzymatic sensor for the determination of urea and creatinine, respectively. By using an electrode selective to the ammonium ion, with a PVC-based membrane containing sebacate as plasticising agent and nonactine as ionophore, a probe sensitive to urea and an other one sensitive to creatinine were designed and built up, by immobilisation respectively of urease and creatinine deiminase by polyazetidine prepolymer [1,5]. The entrapment of one of these two enzymes, in cellulose triacetate membranes, by a procedure developed by us [4,6], avoiding any possible denaturation of the enzyme, was also carried out. Thus, we obtained two new biosensors for urea and creatinine, characterised by a completely different geometry. In this case a cellulose triacetate membrane, containing one of the two
* Presented at the 10th Bioelectrochemical Conference September 1989. * * To whom correspondence should be addressed. 0302-4598/90/%03.50
8 1990 - Elsevier Sequoia
S.A.
(BEC
X), Pont-a-Mousson
(France),
24-30
196
enzymes, was superimposed on the membrane of a commercially available ammonia gas sensor, resulting a modified response of the latter. A complete electroanalytical characterisation of all the prepared electrodes was carried out; they were also applied to biological fluids analysis. The main results are reported in this paper. EXPERIMENTAL
Chemicals and reagents Urease (E.C. 3.5.1.5., from jack bean, 25 U/mg) and lyophilised control sera were purchased from Boehringer Mannheim (F.R.G.); microbial creatinine deiminase (E.C. 3.5.4.21., 1.9 U/mg) and dialysis membrane cat. D-9777, were from Sigma St. Louis (U.S.A.), urea (G.R.) from Welka S.p.A. (Milano, Italy), creatinine (GR) from Merck (Darmstadt, F.R.G.); cellulose triacetate (TAC) was from Fluka (Buchs, Switzerland). The polyazetidine prepolymer (PAP) solution (Polycup 172, 12% in water) was from Hercules (Wilmington, DE, U.S.A.), the high molecular mass poly-vinylchloride (PVC), nonactin and bis(2_ethylhexyl)-sebacate were from Fluka (Buchs, Switzerland). All other reagents, of analytical grade, were from Farmitalia Carlo Erba (Milano, Italy). Measurements and apparatus The measurements were carried out in steady-state conditions, in a 25 ml glass cell, thermostatted by means of forced water circulation, under magnetic stirring. A simple apparatus, consisting of the enzyme membrane, superimposed on the gas ammonia, or ISE ammonium electrode, and secured to it by a rubber O-ring, was used. Alternatively, the enzyme was entrapped on the surface electrode by means of a dialysis membrane and a rubber O-ring. Finally the electrode as such was dipped into a homogeneous solution of the enzyme. The working conditions, optimised in previous investigations [7,8], were: (a) if using an ammonia gas sensor and enzyme free in solution, or entrapped on a dialysis membrane, or immobilised in a cellulose triacetate membrane, a 0.1 M tris buffer solution, at pH 8, was used, and the temperature was maintained at 25 or 35°C for urea or creatinine sensor, respectively; (b) using an ammonium ISE electrode and enzyme free in solution, or entrapped on a dialysis membrane, or immobilised with polyazetidine, a 0.01 M tris-phosphate buffer solution, at pH 7.5,was employed, while the temperature was maintained respectively at 25 or at 32°C when urease or creatinine deiminase enzyme was employed. The gas sensor for ammonia was supplied by Ingold (cod. 157401); the original gas-permeoselective membrane of the electrode was replaced by a Celgard 2400 polypropylene microporous film (Celanese Corp., Summit, NJ). The ammonium electrode was a PVC-ISE, described in a previous paper [7], consisting of a PVC-sebacate polymeric membrane containing nonactine as ionophore, glued to the bottom of a PVC tube, filled with a lo-* M solution of ammonium chloride and KC1 and an Ag/AgCl reference electrode, dipped into this reference solution. A saturated calomel electrode served as the external reference electrode.
197
An Orion research Ionalyzer 901 potentiometer, an Amel model 686 recorder and a Julabo W/3 thermostat were used for all the electrochemical measurements. Immobilisation of the enzymes For the immobilisation of the enzyme on the PVC membrane of the ammonium ISE [5], 10 mg of the enzyme (urease, or creatinine deiminase) was mixed with 200 ~1 of the prepolymer solution (PAP) as obtained. The solution was stratified on the polymeric ion-selective membrane of the ISE and left for 24 h at + 4 o C. The result is a block polymer, chemically bound to itself and with a good adhesion to the polymeric membrane of the ISE. Any enzyme loss can be prevented by covering the head of the sensor with a dialysis membrane. The immobilisation procedure of the enzymes on the gas diffusion ammonia electrode, by entrapment in cellulose triacetate membranes, is the same as described in detail in a previous paper [4]. In short, by dissolution, under magnetic stirring, of a weighted amount of TAC in formic acid/water 90/10 (v/v), we obtained a viscose that, stratified on a glass plate and dipped into distilled water, furnishes a gelled polymeric film. By cutting this film in the form of disks, after abundant washing to remove the formic acid completely, we obtained a membrane free of any toxic agent and porous enough to allow the enzyme to diffuse into it freely. After drying, the irreversible contraction of the fibers results in entrapment of the enzyme. RESULTS
As previously said, for the construction of the two enzyme sensors for urea and creatinine, we used in one case a commercially available gas diffusion ammonia
-70
- 70
- 50
-50
-4c
- 40
‘I
250 act,wty
500 i LJ cm-~
750
0 25 actlvLty/u
0.50
0.75
cm-3
Fig. 1. Response of the electroenzymatic system, as slopes of the calibration curves, vs. urease activity, (enzyme free in solution). Fig. 2. Response of the electroenzymatic system, as slopes of the calibration curves, vs. creatinine deiminase (enzyme free in solution).
198 TABLE
1
Comparison of linearity range, sensitivity, precision, response time and standard urea solutions by enzyme sensors obtained by coupling a polymeric ion, with urease enzyme, by different techniques
Response time/mm Slope, AE[A log(c/M)]-‘/mV Linearity range/M Correlation coefficient Precision/pooled standard Lifetime/days
deviation
4;
lifetime, in the analysis of ISE, selective to ammonium
PVC ammonium electrode and urease free in solution
PVC ammonium electrode and urease immobilised by dialysis membrane
PVC ammonium electrode and urease irmnobilised by polyazetidine prepolymer
61 36.0 1.1 x10-‘+4.0x10-* 0.9977 3.0
91 37.1 8.0~10-~1.5x10-2 0.9973 2.3 12-14
electrode, and in the other, an NH: ion-selective electrode developed by us. The investigations for constructing and characterising the sensors was carried out in three steps: (1) a study of the response to concentration variations with either indicator electrode dipped in the buffer solution containing free urease or creatinine deiminase. The optimisation of the working conditions, as regard pH, ionic strength, buffer nature and temperature, was already performed in previous research [7,8]. In Figs. 1 and 2 we show the influence of increasing the activity of the enzyme free in solution, on the system response. It is observed that, for concentrations greater than
TABLE
2
Comparison of linearity range, sensitivity, precision, response time and lifetime, in the analysis standard creatinine solutions by enzyme sensors obtained by coupling a polymeric ISE, selective ammonium ion, with creatinine deiminase enzyme, by different techniques
Response time/mm Slope, A E[ A log( c/M)] - i/mV Linearity range/M Correlation coefficient Precision/pooled Lifetime/days
standard
deviation%
PVC ammonium electrode and creatinine deiminase free in solution
PVC ammonium electrode and creatinine deiminase immobilised by dialysis membrane
PVC ammonium electrode and creatinine deiminase immobilised by polyazetidine prepolymer
63 33.7 9.9x10-43.9x10-2 0.9985
<3 37.2 2.0x 10-45.3x10-2 0.9972
<3 35.8 1.0x 10-‘3.0x10-2 0.9981
2.8
1.9 4-5
2.4 7-9
of to
199 TABLE
3
Comparison of linearity range, sensitivity, precision, response standard solutions by enzyme sensors, obtained by coupling urease enzyme, by different techniques
Response time/mm Slope, A E [ A log( c/M)] - ‘/mV Linearity range/M Correlation coefficient Precision/pooled standard Lifetime/days
deviation
%
time and lifetime, in the analysis of urea a gas diffusion ammonia electrode with
Gas ammonia electrode and urease free in solution
Gas ammonia electrode and urease immobilised by dialysis membrane
Gas ammonia electrode and urease immobilised in cellulose triacetate membrane
61 - 59.3 2.0x10-42.2x10-2 0.9985 7.0
=zl - 58.0 2.0x10-42.2x10-2 0.9997 6.2
41 - 54.1 5.0x10-41.9x10-2 0.9988 1.5 < 90
67
50 U/ml of urease, or 0.3 U/ml of creatinine deiminase, there is no further increase of the signal when the enzyme concentration is raised. (2) A study of the response of the two sensors when the enzyme is immobilised on the surface of each by means of a dialysis membrane. (3) A study of the behaviour of the sensors when the enzyme is permanently immobilised on the electrode: to this end we developed two different methods, namely entrapment in cellulose triacetate membranes, or chemical reaction with polyazetidine prepolymer. The main results obtained in the three steps of the
TABLE
4
Comparison of linearity range, sensitivity, precision, response time and lifetime, creatinine standard solutions by enzyme sensors, obtained by coupling a gas diffusion with creatinine deiminase enzyme, by different techniques
Response time/mine Slope, AE[A log(c/M)]-‘/mV Linearity
range/M
Correlation coefficient Precision/pooled standard Lifetime/days
deviation
%
in the analysis of ammonia electrode
Gas ammonia electrode and creatinine deiminase free in solution
Gas ammonia electrode and creatinine deiminase immobilised by dialysis membrane
Gas ammonia electrode and creatinine deiminase immobilised in cellulose triacetate membrane
20 - 59.1 2.0x10-42.1 x10-2 0.9950 7.5 _
61 - 45.4 7.9x10-42.1 x 10-2 0.9992 8.5
<7
200 TABLE
5
Substances enzyme
with possible
interference
activity
between
10m4 and
IO-’ M concentration,
on the urea
sensors
Species analysed
PVC ammonium electrode and urease immobilised by polyazetidine prepolymer. Relative activity/%
Gas ammonia electrode and urease immobilised in cellulose triacetate membrane. Relative activity/%
Urea Ammonia Creatinine Ethanolamine Diethanolamine Triethanolamine Alanine
100.0 140.0 15.0 8.5 7.8 3.1 1.1
100.0 104.6 29.0 11.3 11.5 3.1 0
Substances with possible interference deiminase enzyme sensors
activity
between
10m4 and 10F2 M concentration,
on the creatinine
Species analysed
PVC ammonium electrode and creatinine deiminase immobilised by polyazetidine prepolymer. Relative activity/%
Gas ammonia electrode and creatinine deiminase immobilised in cellulose triacetate membrane. Relative activity/%
Creatinine Ammonia Urea Ethanolamine Diethanolamine Triethanolamine Alanine
100.0 154.0 10.1 8.3 10.5 9.4 2.7
100.0 106.7 8.6 10.6 12.4 10.6
TABLE
6
Accuracy of urea and creatinine determinations in biological fluids and standard solutions with the urea enzyme sensor and the creatinine enzyme sensor, respectively, assembled with different techniques Accuracy with standard solution/% (by direct method)
Accuracy with control sera/% (by direct method)
Accuracy with urine/% (by standard addition method)
Urea a
96.3-102.1 (RSD% Q 4.5)
Q 103.4 (RSD% d 2.3)
98.1-105.7 (RSDX d 2.3)
Urea b
102.8 (RSDS
_ = 1.8)
103.8 (RSD% Q 2.2) 101.4-104.8 (RSD% < 2.3)
Creatinine
a
96.2-104.4 (RSD% < 2.9)
Creatinine
b
103.2 (RSD% = 3.1)
_
104.2 (RSD% Q 3.7)
a Ammonia gas sensor with enzyme immobilised in cellulose triacetate membrane. b PVC ammonium ellectrode with enzyme immobilised by means of polyazetidine
prepolymer.
201
program are summarised in Tables 1-4, showing all the electroanalytical data of the studied systems. In Table 5 interference data for the most commonly found compounds are reported for both electrodes. Lastly, in Table 6, accuracy data are given for both standard solutions and clinical real matrices. In the last case, a correction for the interference of free ammonia solution was performed for the ammonia electrode with or without enzyme immobilised and the latter value obtained was subtracted from that given by the enzyme sensor. DISCUSSION
On the basis of the reported results (Tables l-4) it is possible to draw the following conclusions: the response times of the sensors to urea are comparable to or shorter than those to creatinine, while the experimental temperature, in the latter case, is higher than in the former (32-35°C vs. 25 “C). For each of the systems studied the response time can be affected by the configuration of the electrode; thus, when the indicator electrode is sensitive to ammonium, the response time remains practically constant, irrespective of whether enzyme free in solution, entrapped in a dialysis membrane or immobilised with polyazetidine prepolymer is used. But on using as indicator the gas diffusion ammonia electrode and in the case of the creatinine sensor, the response time becomes longer when the enzyme creatinine deiminase is free in solution, while it is three times shorter, if the enzyme is entrapped or chemically immobilised. The slope value for the former range between 36 and 38.5 mV per concentration decade for urea and between 34 and 31 mV for creatinine, varying only modestly as a function of the system configuration. The same observation can be made for the correlation coefficients, while the width of the linearity intervals ranges from 1.5 to 2 concentration decades. Generally the maximum width, as the lowest limit of detection (above 10e4 M), corresponds to the case of the enzyme chemically bound by means of polyazetidine prepolymer. Lastly, the precision of the measurements (as pooled standard deviation %) is satisfying (2-3%). In the case of enzyme sensors having an ammonia electrode as indicator, it is observed that the slope values are nearest to Nernstian ones (between - 54 and - 59 mV per concentration decade and from -55.5 to -59 mV per concentration decade, respectively, for urea and creatinine), the correlation coefficients are as in the previous case and the useful concentration ranges are between 1.5 and 2.5 decades, reaching the maximum value when the enzyme is used free in solution. The minimum detection limit is of the same order as previously reported and the precision (as pooled standard deviation W) is satisfying, always less than 5% when the enzyme is immobilised; when the enzyme is free in solution, or immobilised in a dialysis membrane, the precision values are lower. The life times do not exceed one week, when the enzyme is entrapped in a dialysis membrane, while they are 1.5 or 2 times longer, when the enzyme is immobilised by reaction with polyazetidine prepolymer or in cellulose triacetate membrane; in the latter case the sensor for urea has a lifetime of more than three months. As regards possible interferences, these
202
are generally very low (Table 5), with the only exception of that from ammonia and, less heavily, but clearly, from creatinine for urea electrodes. Finally, in Table 6, accuracy data are reported, both with standard solutions and real matrices (urines, control sera): they are between 96.3 and 105.7%. In conclusion it can be said that both sensors developed can realise determinations of urea and creatinine, both in standard solutions and in bioclinical matrices, with comparable analytical characteristics, except for their sensitivities and lifetimes, which are generally better for the enzyme sensor employing immobilisation in cellulose triacetate membrane, than in polyazetidine prepolymer. ACKNOWLEDGEMENT
This work was supported by the National Research Council (CNR) of Italy. REFERENCES 1 L. Campanella and M. Tomassetti in G.G. Guilbault and M. Mascini (Eds.), Analytical Uses of Immobilized Biological Compounds for Detection, Medical and Industrial uses, Reidel, Dordrecht, 1988, p. 169. 2 L. Campanella and M. Tomassetti, Select. Electr. Rev., 11 (1989) 69. 3 L. Campanella, M. Tomassetti, F. Marxi and R. Sbrilli, J. Pharm. Biomed. Anal., 6 (1988) 717. 4 L. Campanella, M.P. Sammartino and M. Tomassetti, Sensors Actuators, 16 (1989) 235. 5 L. Campanella, F. Mazrei, C. Morgia, M.P. Sammartino, M. Tomassetti, V. Baroncelli, M. Battilotti, C. Colapicchioni, I. Giamrini and F. Porcelli, Analusis, 16 (suppl. 9-10) (1988) 120. 6 L. Campanella, M. Tomassetti and M.P. Sammartino, Analyst, 113 (1988) 77, 994. 7 L. Campanella, M.P. Sammartino and M. Tomassetti, Analyst, in press. 8 M. Battilotti, C. Colapicchioni, I. Giamiini, F. Porcelli, L. Campanella, M. Cordatore, F. Mazzei and M. Tomassetti, Anal. Chim. Acta, 221 (1989) 157.
Bioelectrochemistry and Bioenergetics, 23 (1990) 195-202 A section of J. Electroanal. Chem., and constituting Vol. 298 (1990) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
New enzyme sensors for urea and creatinine analysis * L. Campanella **, F. Mazzei, M.P. Sammartino Dipartimento (Received
di Chimica, Vniversrta ’
29 September
and M. Tomassetti
“LA Sapienza”, p.le A. More, 5, 00185 Roma (Italy)
1989; in revised form 28 December
1989)
ABSTRACT
New selective sensors sensitive to urea or creatinine have been developed and different geometries of the electroenzymatic systems investigated. The sensors were characterised and used to analyse clinical matrices (urines and control sera).
INTRODUCTION
In the program presently running in our laboratory, aimed at the development of new electrochemical potentiometric sensors and ion-selective electrodes [1,2], we recently developed both a new assembly procedure for ion-selective polymeric electrodes [3] and an efficient immobilisation method for the entrapment of enzymes [4]. The most recent results of these investigations concern the realisation of two new kinds of enzymatic sensor for the determination of urea and creatinine, respectively. By using an electrode selective to the ammonium ion, with a PVC-based membrane containing sebacate as plasticising agent and nonactine as ionophore, a probe sensitive to urea and an other one sensitive to creatinine were designed and built up, by immobilisation respectively of urease and creatinine deiminase by polyazetidine prepolymer [1,5]. The entrapment of one of these two enzymes, in cellulose triacetate membranes, by a procedure developed by us [4,6], avoiding any possible denaturation of the enzyme, was also carried out. Thus, we obtained two new biosensors for urea and creatinine, characterised by a completely different geometry. In this case a cellulose triacetate membrane, containing one of the two
* Presented at the 10th Bioelectrochemical Conference September 1989. * * To whom correspondence should be addressed. 0302-4598/90/%03.50
8 1990 - Elsevier Sequoia
S.A.
(BEC
X), Pont-a-Mousson
(France),
24-30
196
enzymes, was superimposed on the membrane of a commercially available ammonia gas sensor, resulting a modified response of the latter. A complete electroanalytical characterisation of all the prepared electrodes was carried out; they were also applied to biological fluids analysis. The main results are reported in this paper. EXPERIMENTAL
Chemicals and reagents Urease (E.C. 3.5.1.5., from jack bean, 25 U/mg) and lyophilised control sera were purchased from Boehringer Mannheim (F.R.G.); microbial creatinine deiminase (E.C. 3.5.4.21., 1.9 U/mg) and dialysis membrane cat. D-9777, were from Sigma St. Louis (U.S.A.), urea (G.R.) from Welka S.p.A. (Milano, Italy), creatinine (GR) from Merck (Darmstadt, F.R.G.); cellulose triacetate (TAC) was from Fluka (Buchs, Switzerland). The polyazetidine prepolymer (PAP) solution (Polycup 172, 12% in water) was from Hercules (Wilmington, DE, U.S.A.), the high molecular mass poly-vinylchloride (PVC), nonactin and bis(2_ethylhexyl)-sebacate were from Fluka (Buchs, Switzerland). All other reagents, of analytical grade, were from Farmitalia Carlo Erba (Milano, Italy). Measurements and apparatus The measurements were carried out in steady-state conditions, in a 25 ml glass cell, thermostatted by means of forced water circulation, under magnetic stirring. A simple apparatus, consisting of the enzyme membrane, superimposed on the gas ammonia, or ISE ammonium electrode, and secured to it by a rubber O-ring, was used. Alternatively, the enzyme was entrapped on the surface electrode by means of a dialysis membrane and a rubber O-ring. Finally the electrode as such was dipped into a homogeneous solution of the enzyme. The working conditions, optimised in previous investigations [7,8], were: (a) if using an ammonia gas sensor and enzyme free in solution, or entrapped on a dialysis membrane, or immobilised in a cellulose triacetate membrane, a 0.1 M tris buffer solution, at pH 8, was used, and the temperature was maintained at 25 or 35°C for urea or creatinine sensor, respectively; (b) using an ammonium ISE electrode and enzyme free in solution, or entrapped on a dialysis membrane, or immobilised with polyazetidine, a 0.01 M tris-phosphate buffer solution, at pH 7.5,was employed, while the temperature was maintained respectively at 25 or at 32°C when urease or creatinine deiminase enzyme was employed. The gas sensor for ammonia was supplied by Ingold (cod. 157401); the original gas-permeoselective membrane of the electrode was replaced by a Celgard 2400 polypropylene microporous film (Celanese Corp., Summit, NJ). The ammonium electrode was a PVC-ISE, described in a previous paper [7], consisting of a PVC-sebacate polymeric membrane containing nonactine as ionophore, glued to the bottom of a PVC tube, filled with a lo-* M solution of ammonium chloride and KC1 and an Ag/AgCl reference electrode, dipped into this reference solution. A saturated calomel electrode served as the external reference electrode.
197
An Orion research Ionalyzer 901 potentiometer, an Amel model 686 recorder and a Julabo W/3 thermostat were used for all the electrochemical measurements. Immobilisation of the enzymes For the immobilisation of the enzyme on the PVC membrane of the ammonium ISE [5], 10 mg of the enzyme (urease, or creatinine deiminase) was mixed with 200 ~1 of the prepolymer solution (PAP) as obtained. The solution was stratified on the polymeric ion-selective membrane of the ISE and left for 24 h at + 4 o C. The result is a block polymer, chemically bound to itself and with a good adhesion to the polymeric membrane of the ISE. Any enzyme loss can be prevented by covering the head of the sensor with a dialysis membrane. The immobilisation procedure of the enzymes on the gas diffusion ammonia electrode, by entrapment in cellulose triacetate membranes, is the same as described in detail in a previous paper [4]. In short, by dissolution, under magnetic stirring, of a weighted amount of TAC in formic acid/water 90/10 (v/v), we obtained a viscose that, stratified on a glass plate and dipped into distilled water, furnishes a gelled polymeric film. By cutting this film in the form of disks, after abundant washing to remove the formic acid completely, we obtained a membrane free of any toxic agent and porous enough to allow the enzyme to diffuse into it freely. After drying, the irreversible contraction of the fibers results in entrapment of the enzyme. RESULTS
As previously said, for the construction of the two enzyme sensors for urea and creatinine, we used in one case a commercially available gas diffusion ammonia
-70
- 70
- 50
-50
-4c
- 40
‘I
250 act,wty
500 i LJ cm-~
750
0 25 actlvLty/u
0.50
0.75
cm-3
Fig. 1. Response of the electroenzymatic system, as slopes of the calibration curves, vs. urease activity, (enzyme free in solution). Fig. 2. Response of the electroenzymatic system, as slopes of the calibration curves, vs. creatinine deiminase (enzyme free in solution).
198 TABLE
1
Comparison of linearity range, sensitivity, precision, response time and standard urea solutions by enzyme sensors obtained by coupling a polymeric ion, with urease enzyme, by different techniques
Response time/mm Slope, AE[A log(c/M)]-‘/mV Linearity range/M Correlation coefficient Precision/pooled standard Lifetime/days
deviation
4;
lifetime, in the analysis of ISE, selective to ammonium
PVC ammonium electrode and urease free in solution
PVC ammonium electrode and urease immobilised by dialysis membrane
PVC ammonium electrode and urease irmnobilised by polyazetidine prepolymer
61 36.0 1.1 x10-‘+4.0x10-* 0.9977 3.0
91 37.1 8.0~10-~1.5x10-2 0.9973 2.3 12-14
electrode, and in the other, an NH: ion-selective electrode developed by us. The investigations for constructing and characterising the sensors was carried out in three steps: (1) a study of the response to concentration variations with either indicator electrode dipped in the buffer solution containing free urease or creatinine deiminase. The optimisation of the working conditions, as regard pH, ionic strength, buffer nature and temperature, was already performed in previous research [7,8]. In Figs. 1 and 2 we show the influence of increasing the activity of the enzyme free in solution, on the system response. It is observed that, for concentrations greater than
TABLE
2
Comparison of linearity range, sensitivity, precision, response time and lifetime, in the analysis standard creatinine solutions by enzyme sensors obtained by coupling a polymeric ISE, selective ammonium ion, with creatinine deiminase enzyme, by different techniques
Response time/mm Slope, A E[ A log( c/M)] - i/mV Linearity range/M Correlation coefficient Precision/pooled Lifetime/days
standard
deviation%
PVC ammonium electrode and creatinine deiminase free in solution
PVC ammonium electrode and creatinine deiminase immobilised by dialysis membrane
PVC ammonium electrode and creatinine deiminase immobilised by polyazetidine prepolymer
63 33.7 9.9x10-43.9x10-2 0.9985
<3 37.2 2.0x 10-45.3x10-2 0.9972
<3 35.8 1.0x 10-‘3.0x10-2 0.9981
2.8
1.9 4-5
2.4 7-9
of to
199 TABLE
3
Comparison of linearity range, sensitivity, precision, response standard solutions by enzyme sensors, obtained by coupling urease enzyme, by different techniques
Response time/mm Slope, A E [ A log( c/M)] - ‘/mV Linearity range/M Correlation coefficient Precision/pooled standard Lifetime/days
deviation
%
time and lifetime, in the analysis of urea a gas diffusion ammonia electrode with
Gas ammonia electrode and urease free in solution
Gas ammonia electrode and urease immobilised by dialysis membrane
Gas ammonia electrode and urease immobilised in cellulose triacetate membrane
61 - 59.3 2.0x10-42.2x10-2 0.9985 7.0
=zl - 58.0 2.0x10-42.2x10-2 0.9997 6.2
41 - 54.1 5.0x10-41.9x10-2 0.9988 1.5 < 90
67
50 U/ml of urease, or 0.3 U/ml of creatinine deiminase, there is no further increase of the signal when the enzyme concentration is raised. (2) A study of the response of the two sensors when the enzyme is immobilised on the surface of each by means of a dialysis membrane. (3) A study of the behaviour of the sensors when the enzyme is permanently immobilised on the electrode: to this end we developed two different methods, namely entrapment in cellulose triacetate membranes, or chemical reaction with polyazetidine prepolymer. The main results obtained in the three steps of the
TABLE
4
Comparison of linearity range, sensitivity, precision, response time and lifetime, creatinine standard solutions by enzyme sensors, obtained by coupling a gas diffusion with creatinine deiminase enzyme, by different techniques
Response time/mine Slope, AE[A log(c/M)]-‘/mV Linearity
range/M
Correlation coefficient Precision/pooled standard Lifetime/days
deviation
%
in the analysis of ammonia electrode
Gas ammonia electrode and creatinine deiminase free in solution
Gas ammonia electrode and creatinine deiminase immobilised by dialysis membrane
Gas ammonia electrode and creatinine deiminase immobilised in cellulose triacetate membrane
20 - 59.1 2.0x10-42.1 x10-2 0.9950 7.5 _
61 - 45.4 7.9x10-42.1 x 10-2 0.9992 8.5
<7
200 TABLE
5
Substances enzyme
with possible
interference
activity
between
10m4 and
IO-’ M concentration,
on the urea
sensors
Species analysed
PVC ammonium electrode and urease immobilised by polyazetidine prepolymer. Relative activity/%
Gas ammonia electrode and urease immobilised in cellulose triacetate membrane. Relative activity/%
Urea Ammonia Creatinine Ethanolamine Diethanolamine Triethanolamine Alanine
100.0 140.0 15.0 8.5 7.8 3.1 1.1
100.0 104.6 29.0 11.3 11.5 3.1 0
Substances with possible interference deiminase enzyme sensors
activity
between
10m4 and 10F2 M concentration,
on the creatinine
Species analysed
PVC ammonium electrode and creatinine deiminase immobilised by polyazetidine prepolymer. Relative activity/%
Gas ammonia electrode and creatinine deiminase immobilised in cellulose triacetate membrane. Relative activity/%
Creatinine Ammonia Urea Ethanolamine Diethanolamine Triethanolamine Alanine
100.0 154.0 10.1 8.3 10.5 9.4 2.7
100.0 106.7 8.6 10.6 12.4 10.6
TABLE
6
Accuracy of urea and creatinine determinations in biological fluids and standard solutions with the urea enzyme sensor and the creatinine enzyme sensor, respectively, assembled with different techniques Accuracy with standard solution/% (by direct method)
Accuracy with control sera/% (by direct method)
Accuracy with urine/% (by standard addition method)
Urea a
96.3-102.1 (RSD% Q 4.5)
Q 103.4 (RSD% d 2.3)
98.1-105.7 (RSDX d 2.3)
Urea b
102.8 (RSDS
_ = 1.8)
103.8 (RSD% Q 2.2) 101.4-104.8 (RSD% < 2.3)
Creatinine
a
96.2-104.4 (RSD% < 2.9)
Creatinine
b
103.2 (RSD% = 3.1)
_
104.2 (RSD% Q 3.7)
a Ammonia gas sensor with enzyme immobilised in cellulose triacetate membrane. b PVC ammonium ellectrode with enzyme immobilised by means of polyazetidine
prepolymer.
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program are summarised in Tables 1-4, showing all the electroanalytical data of the studied systems. In Table 5 interference data for the most commonly found compounds are reported for both electrodes. Lastly, in Table 6, accuracy data are given for both standard solutions and clinical real matrices. In the last case, a correction for the interference of free ammonia solution was performed for the ammonia electrode with or without enzyme immobilised and the latter value obtained was subtracted from that given by the enzyme sensor. DISCUSSION
On the basis of the reported results (Tables l-4) it is possible to draw the following conclusions: the response times of the sensors to urea are comparable to or shorter than those to creatinine, while the experimental temperature, in the latter case, is higher than in the former (32-35°C vs. 25 “C). For each of the systems studied the response time can be affected by the configuration of the electrode; thus, when the indicator electrode is sensitive to ammonium, the response time remains practically constant, irrespective of whether enzyme free in solution, entrapped in a dialysis membrane or immobilised with polyazetidine prepolymer is used. But on using as indicator the gas diffusion ammonia electrode and in the case of the creatinine sensor, the response time becomes longer when the enzyme creatinine deiminase is free in solution, while it is three times shorter, if the enzyme is entrapped or chemically immobilised. The slope value for the former range between 36 and 38.5 mV per concentration decade for urea and between 34 and 31 mV for creatinine, varying only modestly as a function of the system configuration. The same observation can be made for the correlation coefficients, while the width of the linearity intervals ranges from 1.5 to 2 concentration decades. Generally the maximum width, as the lowest limit of detection (above 10e4 M), corresponds to the case of the enzyme chemically bound by means of polyazetidine prepolymer. Lastly, the precision of the measurements (as pooled standard deviation %) is satisfying (2-3%). In the case of enzyme sensors having an ammonia electrode as indicator, it is observed that the slope values are nearest to Nernstian ones (between - 54 and - 59 mV per concentration decade and from -55.5 to -59 mV per concentration decade, respectively, for urea and creatinine), the correlation coefficients are as in the previous case and the useful concentration ranges are between 1.5 and 2.5 decades, reaching the maximum value when the enzyme is used free in solution. The minimum detection limit is of the same order as previously reported and the precision (as pooled standard deviation W) is satisfying, always less than 5% when the enzyme is immobilised; when the enzyme is free in solution, or immobilised in a dialysis membrane, the precision values are lower. The life times do not exceed one week, when the enzyme is entrapped in a dialysis membrane, while they are 1.5 or 2 times longer, when the enzyme is immobilised by reaction with polyazetidine prepolymer or in cellulose triacetate membrane; in the latter case the sensor for urea has a lifetime of more than three months. As regards possible interferences, these
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are generally very low (Table 5), with the only exception of that from ammonia and, less heavily, but clearly, from creatinine for urea electrodes. Finally, in Table 6, accuracy data are reported, both with standard solutions and real matrices (urines, control sera): they are between 96.3 and 105.7%. In conclusion it can be said that both sensors developed can realise determinations of urea and creatinine, both in standard solutions and in bioclinical matrices, with comparable analytical characteristics, except for their sensitivities and lifetimes, which are generally better for the enzyme sensor employing immobilisation in cellulose triacetate membrane, than in polyazetidine prepolymer. ACKNOWLEDGEMENT
This work was supported by the National Research Council (CNR) of Italy. REFERENCES 1 L. Campanella and M. Tomassetti in G.G. Guilbault and M. Mascini (Eds.), Analytical Uses of Immobilized Biological Compounds for Detection, Medical and Industrial uses, Reidel, Dordrecht, 1988, p. 169. 2 L. Campanella and M. Tomassetti, Select. Electr. Rev., 11 (1989) 69. 3 L. Campanella, M. Tomassetti, F. Marxi and R. Sbrilli, J. Pharm. Biomed. Anal., 6 (1988) 717. 4 L. Campanella, M.P. Sammartino and M. Tomassetti, Sensors Actuators, 16 (1989) 235. 5 L. Campanella, F. Mazrei, C. Morgia, M.P. Sammartino, M. Tomassetti, V. Baroncelli, M. Battilotti, C. Colapicchioni, I. Giamrini and F. Porcelli, Analusis, 16 (suppl. 9-10) (1988) 120. 6 L. Campanella, M. Tomassetti and M.P. Sammartino, Analyst, 113 (1988) 77, 994. 7 L. Campanella, M.P. Sammartino and M. Tomassetti, Analyst, in press. 8 M. Battilotti, C. Colapicchioni, I. Giamiini, F. Porcelli, L. Campanella, M. Cordatore, F. Mazzei and M. Tomassetti, Anal. Chim. Acta, 221 (1989) 157.