US3776819A - Urea determination and electrode therefor - Google Patents

Urea determination and electrode therefor Download PDF

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US3776819A
US3776819A US00887200A US3776819DA US3776819A US 3776819 A US3776819 A US 3776819A US 00887200 A US00887200 A US 00887200A US 3776819D A US3776819D A US 3776819DA US 3776819 A US3776819 A US 3776819A
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/333—Ion-selective electrodes or membranes
    • G01N27/3335—Ion-selective electrodes or membranes the membrane containing at least one organic component

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  • BACKGROUND OF THE INVENTION quired the isolation or removal of various blood components prior to the determination. It has previously been known that urease activity in bulk solution can be determined potentiometrically from the amount of ammonia formed by action of the enzyme upon urea in a buffered solution (Katz, Anal. Chem., vol. 36, p. 2500 (1964).
  • a layer of urease solution or film is held against a cation-sensitive electrode, and this enzyme containing electrode is then placed in a solution containing urea.
  • Ammonium ion is generated in the enzyme layer by action of the urea, and the logarithm of the ammonium ion concentration is measured versus a reference electrode. After corrections for the presence of other ions, the urea concentration is calculated.
  • the electrod utilizes only a small quantity of enzyme and is reusable. The procedure makes it possible to determine the concentration of urea in whole blood without prior removal of other blood components, and without using excessive amounts of enzyme.
  • the present invention utilizes the known reaction:
  • the electrode of the present invention has the urease bound or contained close to the surface of the cationsensitive electrode.
  • the urease can be incorporated into a medium or film material which is held against the electrode. Such material can comprise or be coated with a dialysis medium to retard diffusion loss of the urease while permitting entry of the urea.
  • a cation-sensitive electrode is employed as the base electrode component to which the urease is bound, such electrodes being known and avaliable.
  • a glass electrode can be used, such as the common glass electrodes with an Ag/AgCl internal reference.
  • the figure is an illustration of the electrodes as employed in determining potentials in accordance with the invention.
  • the potentiometric measurements in the present invention employ a cell with a cation-sensitive electrode and a standard reference electrode, such as a saturated calomel electrode.
  • the measurements are made on an electrometer connected across the electrodes, employing a bucking voltage from a reference to maintain a low millivolt full scale reading on the electrometer if desired.
  • the figure is an illustration of the electrodes, test solu tion and potentiometric set up employed in the present invention.
  • the electrode 1 has encompassing layers 2 and 3 shown in cross-section, 2 comprising urease, and 3 a covering membrane material.
  • the lead wire 4 from electrode 1 is connected through an electrometer 5 and a precision reference voltage 6 and lead wire 7 to a saturated calomel electrode 8, and both electrode 1 and electrode 8 are in contact with test solution 9.
  • the urease layer 2 around the electrode may be a coating of urease medium firmly adherent to the surface of the electrode, possibly comprising the urease in admixture with some resinous or gelatinous material or other film forming material, or it may be a solution or gel of the urease which is contained around the electrode by the membrane material 3.
  • the membrane material can be adherent to the urease layer and the electrode, or it can be a physically discrete film which is mechanically afiixed to the electrode to contain the urease layer. If the urease layer 2 is sufiiciently adherent to the electrode surface and comprising material sufficiently resistant to attack by the test solution to substantially immobilize or prevent leaching out of the urease, the membrane layer 3 is then not essential.
  • such layer it is generally preferred to employ such layer and generally that it be of some semi-permeable material which will permit migration of urea to the urease layer, but will substantially prevent migration of the urease into the body of the test solution.
  • Materials which permit the passage of molecules of molecular weight up to say 8000 are suitable, although normally those passing molecules up to 1000 or so molecular weight will be used.
  • Any membrane or porous diaphragm materials used in dialysis or electrolysis processes can be used.
  • the dimensions of the urease and membrane layers can vary greatly without impairing operability, but for good results it is desirable to employ relatively thin layers in order to have a quick potentiometric response to the test concentrations. Diffusion of the urea is apparently a ratedetermining step.
  • the urease layer is preferably of the order of ten-thousandths of an inch but is often of greater thickness; and appropriate range is about 0.0005 to about 0.005 inch.
  • the thickness of the outer layer will depend to some extent on the material employed, particularly with the permeability and mechanical strength of such material.
  • EXAMPLE 1 An electrode was prepared by coating with a ureasegelatin film and then spraying with flexible collodion. To form the film, a solution of 50 mg. urease in 50 ml. Water was added to fifty ml. aqueous solution containing 5% gelatin and 0.5% glycerol at about 40 C. The solution was coated on the electrode by alternately dipping and cooling. A flexible collodion was then sprayed on the electrode.
  • the electrode was then used in a cell containing bovine plasma to measure the urea content thereof.
  • a saturated calomel electrode was used as a reference electrode.
  • a Keithley model 610A electrometer was hooked across the electrodes to make the potentiometric measurements.
  • An Emcee Electronics Precision Reference Voltage, Model 1118D was used as a bucking voltage to maintain a 10 mv. full scale reading on the electrometer.
  • a Corning Patented Dec. 4, 1973 Glass Co. monovalent cation glass electrode with an Ag/AgCl internal reference was the basic electrode component, such electrode being sensitive to NH but relatively insensitive to Na+.
  • E the potential of the blood media
  • E the potential of the blood media after the enzyme has converted some percentage of the urea to NH
  • E The last measurement is unnecessary if the value has been previously established.
  • the electrode response, E in the bovine plasma was 6.6 mv.
  • a 0.0312 molar amount of urea was added and E was determined as 14.1 mv.
  • the potential of the blood media, E, as determined by the same electrode minus the enzyme film was 5.2 mv.
  • EXAMPLE 2 An electrode with fibrin-urease and collodion coatings was prepared as follows. A 26.4 mg. amount of urease was added to 2.5 ml. of a solution containing 5 mg. fibrinogin per ml. water. A solution was prepared containing 5000 units thrombin per ml. water buffered to pH 7.4 with a phosphate buffer, and 0.02 ml. of the solution was added to the fibrinogin solution. A fibrous mat formed within seconds and was removed from the container and placed on the tip of a glass electrode. The mat and electrode were sprayed with collodion. The electrode was then connected in electrical circuit as described in Example 1 with a saturated calomel electrode and used to measure the potential of whole blood.
  • the potential was 2.4 millivolts. A 0.15 gram amount of urea was added (-0.025 mol/liter) and the potential determined as 10.4 millivolts. The potential of 100 ml. whole blood with the untreated glass electrode was 0.6 millivolt. Utilizing the equations described herein, the B.U.N. was calculated as 13.1 mg./100 ml. The percentage of the urea converted by the urease was 25.6%.
  • EXAMPLE 3 A small amount of filter paper pulp was dried on a 3 cm. square piece of cellophane dialysis membrane. Five drops of Water containing 14 mg. urease was dropped on and absorbed by the filter paper pulp. The piece of cellophane was then fitted to a glass electrode, being tightly stretched about the tip, with the urease-pulp between the cellophane and glass surface. The membrane was afiixed to the electrode by wrapping with polytetrafiuoroethylene tape at its upper edge. The electrode was used to determine the potential of a buffered solution with various amounts of added urea. The potential was found to be essentially a straight line function of the log of the cation concentration at concentrations tested in the range of 0.013 to 0.077 mole/liter.
  • the potential should be measured at some standard time interval sufficient for diffusion to come to a fairly steady state with the electrode employed. This time will vary with the thickness of the coatings on the electrode, particularly the urese coating. Ideally it will be relatively short, as a matter of seconds up to 30 seconds, but for thicker coatings can be five minutes or more, even hours, although the latter has limited practicality. Ordinarily a stable state will have been achieved within five minutes.
  • the percent conversions herein are recognized as being a measure of how much ammonium ion reaches the electrode surface while true conversion of the urea may be much higher if only a particular portion of the ammonium ion is reaching the electrode surface.
  • the basic electrode structure utilized in forming the electrodes of the present invention can be any electrode capable of determining the ammonium ion potentiometrically.
  • cation sensitive electrodes are suitable, with those having a strong response to the ammonium ion in the presence of other cations being preferred. Those electrodes more sensitive to monovalent cations than other cations are preferred.
  • the pH type electrodes have some sensitivity to monovalent cations other than H+ and can therefore be used although more sensitive electrodes are preferred.
  • the electrodes used generally have an internal reference standard, for example silver/silver chloride, in contact with a liquid and a glass surface separating the test solution from the internal standard. The physical characteristics and composition of the glass has an influence on the selectivity of the electrode.
  • some electrodes utilize a porous membrane in conjunction with a liquid ion exchange layer between the test solution and the inner solution.
  • Other electrodes utilize a synthetic crystal as a membrane to separate the inner liquid from the test liquid.
  • a monovalent cation sensitive electrode manufactured by Corning Glass Works can be used.
  • Other monovalent cation electrodes can be used, for example Beckman Instrument Co., Catalog No. 39137. Whatever the base electrode which is utilized, it must be provided with an urease layer in accord with the present invention in order to constitute the electrode of the present invention.
  • the present invention is useful for the determination of urea in various bodily fluids such as blood, urine, etc. It is also useful in other applications where the concentration of urea in a solution is to be determined.
  • An electrode for measuring urea concentrations comprising a cation sensitive electrode with an encompassing urease layer around and in direct contact with the electrode and confined thereto.
  • the cation sensitive electrode is a glass electrode and the urease layer is in direct contact with the glass.
  • the electrode of claim 1 in which the urease is covered by a film which is substantially impermeable to urease molecules but permeable to urea molecules.
  • the method of measuring urea content of blood which comprises providing a first electrode having an encompassing urease coating, said electrode being in electric circuit with a reference electrode, placing said first electrode and said reference electrode in direct contact with a blood sample, measuring the electric potential between the electrodes, providing a corresponding electric potential determined by measuring the electric potential between the electrodes in direct contact with blood without the urease coating on the electrode, determining the difference between such electric potentials, comparing such difference with that for known urea concentrations and determining the concentration of the urea from such comparison.
  • the first electrode is a cation sensitive glass electrode and the blood sample is whole blood and it serves as the electrolyte between the electrodes.

Abstract

THE INVENTION CONCERNS A POTENTIOMETRIC METHOD OF DETERMINING THE UREA CONTENT OF FLUIDS BY USE OF A CATION SENSITIVE ELECTRODE HAVING A UREASE LAYER ON ITS SURFACE.

Description

Dec. 4, 1973 L). L WILLIAMS 3,776,819
UREA DETERMINATION AND ELECTRODE THEREFOR Filed Dec. 22, 1969 INVENTOR DAVID L.W|LL\AMS ATTO R NEY Tlnited States Patent 6 3,776,819 UREA DETERMINATION AND ELECTRODE THEREFOR David L. Williams, Reading, Mass., assignor to Monsanto Company, St. Louis, Mo. Filed Dec. 22, 1969, Ser. No. 887,200 Int. Cl. G01n 27/46 US. Cl. 204-1 T 13 Claims ABSTRACT OF THE DISCLOSURE The invention concerns a potentiometric method of determining the urea content of fluids by use of a cation sensitive electrode having a urease layer on its surface.
BACKGROUND OF THE INVENTION quired the isolation or removal of various blood components prior to the determination. It has previously been known that urease activity in bulk solution can be determined potentiometrically from the amount of ammonia formed by action of the enzyme upon urea in a buffered solution (Katz, Anal. Chem., vol. 36, p. 2500 (1964).
SUMMARY OF THE INVENTION In the present invention a layer of urease solution or film is held against a cation-sensitive electrode, and this enzyme containing electrode is then placed in a solution containing urea. Ammonium ion is generated in the enzyme layer by action of the urea, and the logarithm of the ammonium ion concentration is measured versus a reference electrode. After corrections for the presence of other ions, the urea concentration is calculated. The electrod utilizes only a small quantity of enzyme and is reusable. The procedure makes it possible to determine the concentration of urea in whole blood without prior removal of other blood components, and without using excessive amounts of enzyme.
The present invention utilizes the known reaction:
The electrode of the present invention has the urease bound or contained close to the surface of the cationsensitive electrode. The urease can be incorporated into a medium or film material which is held against the electrode. Such material can comprise or be coated with a dialysis medium to retard diffusion loss of the urease while permitting entry of the urea. A cation-sensitive electrode is employed as the base electrode component to which the urease is bound, such electrodes being known and avaliable. For example a glass electrode can be used, such as the common glass electrodes with an Ag/AgCl internal reference.
The figure is an illustration of the electrodes as employed in determining potentials in accordance with the invention.
The potentiometric measurements in the present invention employ a cell with a cation-sensitive electrode and a standard reference electrode, such as a saturated calomel electrode. The measurements are made on an electrometer connected across the electrodes, employing a bucking voltage from a reference to maintain a low millivolt full scale reading on the electrometer if desired.
The figure is an illustration of the electrodes, test solu tion and potentiometric set up employed in the present invention. The electrode 1 has encompassing layers 2 and 3 shown in cross-section, 2 comprising urease, and 3 a covering membrane material. The lead wire 4 from electrode 1 is connected through an electrometer 5 and a precision reference voltage 6 and lead wire 7 to a saturated calomel electrode 8, and both electrode 1 and electrode 8 are in contact with test solution 9. The urease layer 2 around the electrode may be a coating of urease medium firmly adherent to the surface of the electrode, possibly comprising the urease in admixture with some resinous or gelatinous material or other film forming material, or it may be a solution or gel of the urease which is contained around the electrode by the membrane material 3. The membrane material can be adherent to the urease layer and the electrode, or it can be a physically discrete film which is mechanically afiixed to the electrode to contain the urease layer. If the urease layer 2 is sufiiciently adherent to the electrode surface and comprising material sufficiently resistant to attack by the test solution to substantially immobilize or prevent leaching out of the urease, the membrane layer 3 is then not essential. However it is generally preferred to employ such layer and generally that it be of some semi-permeable material which will permit migration of urea to the urease layer, but will substantially prevent migration of the urease into the body of the test solution. Materials which permit the passage of molecules of molecular weight up to say 8000 are suitable, although normally those passing molecules up to 1000 or so molecular weight will be used. Any membrane or porous diaphragm materials used in dialysis or electrolysis processes can be used.
The dimensions of the urease and membrane layers can vary greatly without impairing operability, but for good results it is desirable to employ relatively thin layers in order to have a quick potentiometric response to the test concentrations. Diffusion of the urea is apparently a ratedetermining step. The urease layer is preferably of the order of ten-thousandths of an inch but is often of greater thickness; and appropriate range is about 0.0005 to about 0.005 inch. The thickness of the outer layer will depend to some extent on the material employed, particularly with the permeability and mechanical strength of such material. It may commonly be of the order of a few mills with collodion and such materials, say about 1 to 3 mils, although lesser thickness will be preferable if sufiicient to prevent migration of the urease. With high- 1y permeable. materials the thickness can even be 0.01 inch or more.
The following examples are illustrative of the invention but the invention is not limited thereto.
EXAMPLE 1 An electrode was prepared by coating with a ureasegelatin film and then spraying with flexible collodion. To form the film, a solution of 50 mg. urease in 50 ml. Water was added to fifty ml. aqueous solution containing 5% gelatin and 0.5% glycerol at about 40 C. The solution was coated on the electrode by alternately dipping and cooling. A flexible collodion was then sprayed on the electrode.
The electrode was then used in a cell containing bovine plasma to measure the urea content thereof. A saturated calomel electrode was used as a reference electrode. A Keithley model 610A electrometer was hooked across the electrodes to make the potentiometric measurements. An Emcee Electronics Precision Reference Voltage, Model 1118D was used as a bucking voltage to maintain a 10 mv. full scale reading on the electrometer. A Corning Patented Dec. 4, 1973 Glass Co. monovalent cation glass electrode with an Ag/AgCl internal reference was the basic electrode component, such electrode being sensitive to NH but relatively insensitive to Na+. For the determination of the urea content, three potential measurements are necessary: the potential of the blood media (E the potential of the blood media after the enzyme has converted some percentage of the urea to NH (E and the potential of the system after the addition of a known amount of urea to determine the percentage of urea converted by an electrode covered by a particular type urease film (E The last measurement is unnecessary if the value has been previously established. The electrode response, E in the bovine plasma was 6.6 mv. A 0.0312 molar amount of urea was added and E was determined as 14.1 mv. The potential of the blood media, E, as determined by the same electrode minus the enzyme film was 5.2 mv. The response of the cation sensitive electrode was found to be E=54.5 log [NH +]+83.9 where E is millivolts vs. saturated calomel electrode at 25. Conversion of this relationship to B.U.N. by subtraction of the background electrolyte and multiplication by a sensitivity factor yields the following equation from which the blood urea nitrogen was calculated:
E' 83.9 3 B.U.N. 1.4x [antllog 54-5 E 83.9 -ant1log K where K 2 [urea added] ant-ilog 25-48539 B.U.N.= 12.9 mg./100 cc.
The percent conversion of the urea was 23%. The above equation can be converted into another form:
B.U.N.=2800 [urea added] antilog E E 1 2) antllog 1 antilog (%)-1 and when the urea added is 0.0312, this becomes B.U.N.=87.36 antilog E.E. antilog 54.5 1 54.5 3" 2) antllog 54.5 1
[N]=2 [urea added] antilog ntilo )1 (E E a g A A antilog )1 As reported above, the potential showed conversion of 23% of the urea in the solution. This value will vary with the time of the measurement and factors affecting the stability state achieved, but may often characteristically be in the range of to 30% conversion,
4 EXAMPLE 2 An electrode with fibrin-urease and collodion coatings was prepared as follows. A 26.4 mg. amount of urease was added to 2.5 ml. of a solution containing 5 mg. fibrinogin per ml. water. A solution was prepared containing 5000 units thrombin per ml. water buffered to pH 7.4 with a phosphate buffer, and 0.02 ml. of the solution was added to the fibrinogin solution. A fibrous mat formed within seconds and was removed from the container and placed on the tip of a glass electrode. The mat and electrode were sprayed with collodion. The electrode Was then connected in electrical circuit as described in Example 1 with a saturated calomel electrode and used to measure the potential of whole blood. The potential was 2.4 millivolts. A 0.15 gram amount of urea was added (-0.025 mol/liter) and the potential determined as 10.4 millivolts. The potential of 100 ml. whole blood with the untreated glass electrode was 0.6 millivolt. Utilizing the equations described herein, the B.U.N. was calculated as 13.1 mg./100 ml. The percentage of the urea converted by the urease was 25.6%.
EXAMPLE 3 A small amount of filter paper pulp was dried on a 3 cm. square piece of cellophane dialysis membrane. Five drops of Water containing 14 mg. urease was dropped on and absorbed by the filter paper pulp. The piece of cellophane was then fitted to a glass electrode, being tightly stretched about the tip, with the urease-pulp between the cellophane and glass surface. The membrane was afiixed to the electrode by wrapping with polytetrafiuoroethylene tape at its upper edge. The electrode was used to determine the potential of a buffered solution with various amounts of added urea. The potential was found to be essentially a straight line function of the log of the cation concentration at concentrations tested in the range of 0.013 to 0.077 mole/liter.
The equations to determine nitrogen content are developed from the expression:
E=A log [X] +B where [X] =the concentration of cations in moles per liter.
In blood potansium and sodium ions are ordinarily present, so it is necessary to make allowances for these or other extraneous ions when determining urea nitrogen through the ammonium ion produced by urease, and this is done by measuring the potential in the absence of urease and utilizing the value in the equations as illustrated.
A series of solutions from 5X10 to 5 l0- molar concentrations of (NH SO in distilled water were prepared and a plot of the log [NHJ] versus the observed potential was substantially a straight line of slope 54 mv./ decade and conforming to the equation A can be determined by A AE A log [X] If E is determined on a plasma, and E" on a half-fold dilution thereof, the applicable equation is:
and
From values 2.4 mv. for the plasma, 14.3 mv. for halffold dilution and 30.3 for quarter fold dilution, A was calculated as having an average value of 54.5 mv./ decade. These values and further values obtained with known amounts of NH added conformed to the equation:
Thus the values of A and B which were employed in the calculations of B.U.N. herein were established as 54.5 and 83.9 respectively. It will be recognized that these values could be established with greater precision, although considered adequate for ordinary purposes. The value for A will vary somewhat with the concentration range, possible complexing agents in the system and other factors, and will in practice ordinarily be determined by careful calibration of the potentiometric system with the type of solution employed.
In the potentiometric determinations, the potential should be measured at some standard time interval sufficient for diffusion to come to a fairly steady state with the electrode employed. This time will vary with the thickness of the coatings on the electrode, particularly the urese coating. Ideally it will be relatively short, as a matter of seconds up to 30 seconds, but for thicker coatings can be five minutes or more, even hours, although the latter has limited practicality. Ordinarily a stable state will have been achieved within five minutes. The percent conversions herein are recognized as being a measure of how much ammonium ion reaches the electrode surface while true conversion of the urea may be much higher if only a particular portion of the ammonium ion is reaching the electrode surface.
The basic electrode structure utilized in forming the electrodes of the present invention can be any electrode capable of determining the ammonium ion potentiometrically. In general cation sensitive electrodes are suitable, with those having a strong response to the ammonium ion in the presence of other cations being preferred. Those electrodes more sensitive to monovalent cations than other cations are preferred. The pH type electrodes have some sensitivity to monovalent cations other than H+ and can therefore be used although more sensitive electrodes are preferred. The electrodes used generally have an internal reference standard, for example silver/silver chloride, in contact with a liquid and a glass surface separating the test solution from the internal standard. The physical characteristics and composition of the glass has an influence on the selectivity of the electrode. Rather than thus using glass as a membrane, some electrodes utilize a porous membrane in conjunction with a liquid ion exchange layer between the test solution and the inner solution. Other electrodes utilize a synthetic crystal as a membrane to separate the inner liquid from the test liquid. A monovalent cation sensitive electrode manufactured by Corning Glass Works can be used. Other monovalent cation electrodes can be used, for example Beckman Instrument Co., Catalog No. 39137. Whatever the base electrode which is utilized, it must be provided with an urease layer in accord with the present invention in order to constitute the electrode of the present invention.
The present invention is useful for the determination of urea in various bodily fluids such as blood, urine, etc. It is also useful in other applications where the concentration of urea in a solution is to be determined.
What is claimed is:
1. An electrode for measuring urea concentrations comprising a cation sensitive electrode with an encompassing urease layer around and in direct contact with the electrode and confined thereto.
2. The electrode of claim 1 in which the cation sensitive electrode is a glass electrode and the urease layer is in direct contact with the glass.
3. The electrode of claim 1 in which the urease is in a gelatinous film.
4. The electrode of claim 1 in which the urease is covered with a flexible collodion film.
5. The electrode of claim 1 in which the urease is covered by a film which is substantially impermeable to urease molecules but permeable to urea molecules.
6. The electrode of claim 1 in which the urease is embedded in a coating adhering to the electrode surface.
7. The electrode of claim 1 in which the urease is confined by a separate film.
8. The method of measuring urea content of blood which comprises providing a first electrode having an encompassing urease coating, said electrode being in electric circuit with a reference electrode, placing said first electrode and said reference electrode in direct contact with a blood sample, measuring the electric potential between the electrodes, providing a corresponding electric potential determined by measuring the electric potential between the electrodes in direct contact with blood without the urease coating on the electrode, determining the difference between such electric potentials, comparing such difference with that for known urea concentrations and determining the concentration of the urea from such comparison.
9.. The method of claim 8 in which the urease is confined to the electrode by a semi-permeable membrane.
10. The method of claim 8 in which the first electrode is a cation sensitive glass electrode and the blood sample is whole blood and it serves as the electrolyte between the electrodes.
11. The method of claim 8 in which 15 to 30% of the urea is converted to the ammonium ion.
12. The method of claim 8 in which the urease is in a gelatinous film.
13. The electrode of claim 1 in which the electrode is a monovalent cation sensitive electrode.
References Cited UNITED STATES PATENTS 3,421,982 1/1969 Schultz et a1 204-495 3,403,081 9/ 1968 Rohrback et a1 204-1 T 3,476,670 11/ 1969 Weiner 204 3,479,255 11/1969 Arthur 204-1 T 3,539,455 11/1970 Clark 204-195 3,542,662 11/ 1970 Hicks et a1 204-195 OTHER REFERENCES Clark et al.: Annals New York Academy of Sciences, vol. 102 (Art. 1), Oct. 31, 1962, pp. 29-45.
Analytical Chemistry, vol. 36, December 1964, pp. 2500 and 2501.
Guilbault et al.: JACS, vol. 91, April 1969, pp. 2164 and 2165.
Montalvo et al.: Analytical Chemistry, vol. 41, November 1969, pp. 1897-1899.
Riesel et al.: J. of Biological Chemistry, vol. 239, May 1964, pp. 1521-1524.
Hicks et al.: Analytical Chemistry, vol. 38, May 1966, pp. 726-730.
Guilbault et al.: JACS, vol. 91, April 1969, pp. 2164 1969, pp. 600-605.
TA-HSUNG TUNG, Primary Examiner U.S. Cl. X.R.
204-195 B, 195 G, 195 M
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US3869354A (en) * 1973-04-06 1975-03-04 Us Navy Ammonium ion specific electrode and method therewith
US3878049A (en) * 1973-04-03 1975-04-15 Massachusetts Inst Technology Biochemical temperature-sensitive probe and method for measuring reactant concentrations thereof
US3919052A (en) * 1973-01-10 1975-11-11 Battelle Memorial Institute Method and apparatus for continuously controlling an enzymatic reaction
US3979274A (en) * 1975-09-24 1976-09-07 The Yellow Springs Instrument Company, Inc. Membrane for enzyme electrodes
FR2319897A1 (en) * 1975-08-01 1977-02-25 Kyowa Hakko Kogyo Kk ENZYME MEMBRANE FOR CHEMICAL OR ELECTROCHEMICAL TRANSDUCER
US4016044A (en) * 1973-01-10 1977-04-05 Battelle Memorial Institute Method and apparatus for governing the reaction rate of enzymatic reactions
US4020830A (en) * 1975-03-12 1977-05-03 The University Of Utah Selective chemical sensitive FET transducers
US4216065A (en) * 1979-06-18 1980-08-05 University Of Delaware Bio-selective electrode probes using tissue slices
US4244787A (en) * 1979-06-11 1981-01-13 The United States Of America As Represented By The Secretary Of The Department Of Health, Education & Welfare Apparatus and method for determining serum concentrates of metabolites by monitoring dialysate fluid
US4476005A (en) * 1982-10-04 1984-10-09 Hitachi, Ltd. Urease-immobilized urea electrode and process for preparing the same
US4505784A (en) * 1981-07-29 1985-03-19 Siemens Aktiengesellschaft Method for urea analysis
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US3869354A (en) * 1973-04-06 1975-03-04 Us Navy Ammonium ion specific electrode and method therewith
US4020830A (en) * 1975-03-12 1977-05-03 The University Of Utah Selective chemical sensitive FET transducers
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US4244787A (en) * 1979-06-11 1981-01-13 The United States Of America As Represented By The Secretary Of The Department Of Health, Education & Welfare Apparatus and method for determining serum concentrates of metabolites by monitoring dialysate fluid
US4216065A (en) * 1979-06-18 1980-08-05 University Of Delaware Bio-selective electrode probes using tissue slices
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EP0142130A2 (en) * 1983-11-08 1985-05-22 Hitachi, Ltd. Electrochemical sensor having an immobilized enzyme membrane
EP0142130A3 (en) * 1983-11-08 1988-06-01 Hitachi, Ltd. Electrochemical sensor having an immobilized enzyme membrane
US4935106A (en) * 1985-11-15 1990-06-19 Smithkline Diagnostics, Inc. Ion selective/enzymatic electrode medical analyzer device and method of use
US4946651A (en) * 1985-11-15 1990-08-07 Smithkline Diagnostics, Inc. Sample holder for a body fluid analyzer
US4812220A (en) * 1986-08-14 1989-03-14 Unitika, Ltd. Enzyme sensor for determining a concentration of glutamate
US4950379A (en) * 1987-04-09 1990-08-21 Nova Biomedical Corporation Polarographic cell
US4891104A (en) * 1987-04-24 1990-01-02 Smithkline Diagnostics, Inc. Enzymatic electrode and electrode module and method of use
US5037527A (en) * 1987-08-28 1991-08-06 Kanzaki Paper Mfg. Co., Ltd. Reference electrode and a measuring apparatus using the same
EP0352717A2 (en) * 1988-07-25 1990-01-31 Nova Biomedical Corporation Method, analyzer and sensor for measuring urea concentration
EP0352717A3 (en) * 1988-07-25 1990-12-05 Nova Biomedical Corporation Method, analyzer and sensor for measuring urea concentration
US5063081A (en) * 1988-11-14 1991-11-05 I-Stat Corporation Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor
US5200051A (en) * 1988-11-14 1993-04-06 I-Stat Corporation Wholly microfabricated biosensors and process for the manufacture and use thereof
US5837446A (en) * 1988-11-14 1998-11-17 I-Stat Corporation Process for the manufacture of wholly microfabricated biosensors
US6306594B1 (en) 1988-11-14 2001-10-23 I-Stat Corporation Methods for microdispensing patterened layers
US20020090738A1 (en) * 1988-11-14 2002-07-11 I-Stat Corporation System and method of microdispensing and arrays of biolayers provided by same
US7074610B2 (en) 1988-11-14 2006-07-11 I-Stat Corporation System and method of microdispensing and arrays of biolayers provided by same
DE4027728A1 (en) * 1990-08-31 1992-03-05 Bayer Ag IMMOBILIZATION OF ORGANIC MACROMOLECULES OR BIOPOLYMERS IN A POLYMER MEMBRANE
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US10695481B2 (en) 2011-08-02 2020-06-30 Medtronic, Inc. Hemodialysis system having a flow path with a controlled compliant volume
US9707330B2 (en) 2011-08-22 2017-07-18 Medtronic, Inc. Dual flow sorbent cartridge
US9713668B2 (en) 2012-01-04 2017-07-25 Medtronic, Inc. Multi-staged filtration system for blood fluid removal
US9713666B2 (en) 2013-01-09 2017-07-25 Medtronic, Inc. Recirculating dialysate fluid circuit for blood measurement
US9707328B2 (en) 2013-01-09 2017-07-18 Medtronic, Inc. Sorbent cartridge to measure solute concentrations
US10881777B2 (en) 2013-01-09 2021-01-05 Medtronic, Inc. Recirculating dialysate fluid circuit for blood measurement
US11857712B2 (en) 2013-01-09 2024-01-02 Mozarc Medical Us Llc Recirculating dialysate fluid circuit for measurement of blood solute species
US11154648B2 (en) 2013-01-09 2021-10-26 Medtronic, Inc. Fluid circuits for sorbent cartridge with sensors
US11565029B2 (en) 2013-01-09 2023-01-31 Medtronic, Inc. Sorbent cartridge with electrodes
US10532141B2 (en) 2013-02-01 2020-01-14 Medtronic, Inc. Systems and methods for multifunctional volumetric fluid control
US11786645B2 (en) 2013-02-01 2023-10-17 Mozarc Medical Us Llc Fluid circuit for delivery of renal replacement therapies
US9623164B2 (en) 2013-02-01 2017-04-18 Medtronic, Inc. Systems and methods for multifunctional volumetric fluid control
US10010663B2 (en) 2013-02-01 2018-07-03 Medtronic, Inc. Fluid circuit for delivery of renal replacement therapies
US10850016B2 (en) 2013-02-01 2020-12-01 Medtronic, Inc. Modular fluid therapy system having jumpered flow paths and systems and methods for cleaning and disinfection
US9855379B2 (en) 2013-02-02 2018-01-02 Medtronic, Inc. Sorbent cartridge configurations for improved dialysate regeneration
US9827361B2 (en) 2013-02-02 2017-11-28 Medtronic, Inc. pH buffer measurement system for hemodialysis systems
US9144640B2 (en) 2013-02-02 2015-09-29 Medtronic, Inc. Sorbent cartridge configurations for improved dialysate regeneration
US9895477B2 (en) 2013-11-26 2018-02-20 Medtronic, Inc. Detachable module for recharging sorbent materials with optional bypass
US10004839B2 (en) 2013-11-26 2018-06-26 Medtronic, Inc. Multi-use sorbent cartridge
US9884145B2 (en) 2013-11-26 2018-02-06 Medtronic, Inc. Parallel modules for in-line recharging of sorbents using alternate duty cycles
US9943780B2 (en) 2013-11-26 2018-04-17 Medtronic, Inc. Module for in-line recharging of sorbent materials with optional bypass
US11219880B2 (en) 2013-11-26 2022-01-11 Medtronic, Inc System for precision recharging of sorbent materials using patient and session data
US10071323B2 (en) 2013-11-26 2018-09-11 Medtronic, Inc Module for in-line recharging of sorbent materials with optional bypass
US10343145B2 (en) 2013-11-26 2019-07-09 Medtronic, Inc. Zirconium phosphate recharging method and apparatus
US10537875B2 (en) 2013-11-26 2020-01-21 Medtronic, Inc. Precision recharging of sorbent materials using patient and session data
US10478545B2 (en) 2013-11-26 2019-11-19 Medtronic, Inc. Parallel modules for in-line recharging of sorbents using alternate duty cycles
US10172991B2 (en) 2014-06-24 2019-01-08 Medtronic, Inc. Modular dialysate regeneration assembly
US10357757B2 (en) 2014-06-24 2019-07-23 Medtronic, Inc. Stacked sorbent assembly
US10888800B2 (en) 2014-06-24 2021-01-12 Medtronic, Inc Replenishing urease in dialysis systems using urease pouches
US10926017B2 (en) 2014-06-24 2021-02-23 Medtronic, Inc. Modular dialysate regeneration assembly
US10807068B2 (en) 2014-06-24 2020-10-20 Medtronic, Inc. Sorbent pouch
US10821214B2 (en) 2014-06-24 2020-11-03 Medtronic, Inc. Urease introduction system for replenishing urease in a sorbent cartridge
US10016553B2 (en) 2014-06-24 2018-07-10 Medtronic, Inc. Replenishing urease in dialysis systems using a urease introducer
US10272363B2 (en) 2014-06-24 2019-04-30 Medtronic, Inc. Urease introduction system for replenishing urease in a sorbent cartridge
US10245365B2 (en) 2014-06-24 2019-04-02 Medtronic, Inc. Replenisihing urease in dialysis systems using a urease introducer
US10124274B2 (en) 2014-06-24 2018-11-13 Medtronic, Inc. Replenishing urease in dialysis systems using urease pouches
US10286380B2 (en) 2014-06-24 2019-05-14 Medtronic, Inc. Sorbent pouch
US11673118B2 (en) 2014-06-24 2023-06-13 Mozarc Medical Us Llc Stacked sorbent assembly
US11045790B2 (en) 2014-06-24 2021-06-29 Medtronic, Inc. Stacked sorbent assembly
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US10000784B2 (en) 2014-09-08 2018-06-19 Siemens Healthcare Diagnostics Inc. Creatinine biosensor and method of using the same
US10195327B2 (en) 2014-12-10 2019-02-05 Medtronic, Inc. Sensing and storage system for fluid balance
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US11167070B2 (en) 2017-01-30 2021-11-09 Medtronic, Inc. Ganged modular recharging system
US10960381B2 (en) 2017-06-15 2021-03-30 Medtronic, Inc. Zirconium phosphate disinfection recharging and conditioning
US11883794B2 (en) 2017-06-15 2024-01-30 Mozarc Medical Us Llc Zirconium phosphate disinfection recharging and conditioning
US11278654B2 (en) 2017-12-07 2022-03-22 Medtronic, Inc. Pneumatic manifold for a dialysis system
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