Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving urea or urease
Reexamination Certificate
2001-04-19
2004-05-11
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving urea or urease
Reexamination Certificate
active
06733984
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK.
FIELD OF THE INVENTION
The present invention generally relates to sensors for the analysis of fluids, and more particularly relates to a sensor that uses liquid samples from fluids such as blood, urine and milk and measures the partial pressure of carbon dioxide as vapor in fluid communication with the liquid sample and generated by an enzymatic reaction, which pressure is related to concentration of a component, such as urea, in the fluid.
BACKGROUND OF THE INVENTION
Because urea is the primary waste carrier of nitrogen for mammals, measurement of dissolved urea is of interest to biomedical, agricultural and environmental professionals. Many techniques for measurement of urea have been developed in the biomedical industry for analyzing biological fluids such as blood or urine so as to monitor renal function and for control of artificial dialysis. For example, U.S. Pat. No. 5,008,078, issued Apr. 16, 1991, inventors Yaginuma et al., describes an analysis element in which gaseous ammonia may be analyzed from liquid samples such as blood, urine, lymph and the like biological fluids. U.S. Pat. No. 5,858,186, issued Jan. 12, 1999, inventor Glass, describes a urea biosensor for hemodialysis monitoring which uses a solid state pH electrode coated with the enzyme urease and is based upon measuring pH change produced by the reaction products of enzyme-catalyzed hydrolysis of urea.
Milk urea is well correlated to urea in the blood and urine, and thus some of the urea measurement techniques used in those fields have been adapted by the dairy industry for measurement of milk urea in order to balance feed rations for optimal nitrogen efficiency. This optimization often leads to considerable savings in feed costs because protein is the most costly feed supplement. In many locations, reduction of nitrogenous waste from the dairy is an even greater consideration than feed costs. Finally, it has been suggested that high systemic urea levels in dairy cows are associated with poor reproductive performance, which is a serious economic concern on dairy farms.
Most existing sensors for urea use the enzyme urease (EC# 3.5.1.5) to hydrolyze urea to ammonium and carbonate. Of these, it is most common to measure changes in the ionic composition of the solution with a pH or other ion selective electrode or by using a conductimetric electrode. These delicate electrodes, however, are susceptible to fouling with the high lipid and protein concentration of milk, thus limiting their use without expensive and complicated filtering or dialysis systems. Furthermore, these sensors are all dependent on the sample pH and buffering capacity.
Two colorimetric assays for urea are commonly used. One involves the reaction of urea with diacetyl monoxime in acid solution to give a pink complex, and another involves the reaction of ammonia from hydrolyzed urea with phenol to produce the blue dye indophenol. For the reaction with diacetyl monoxime, the milk must first be dialyzed to eliminate interferences due to peptides and other amide bonded molecules. Phenol and the catalyst for its reaction with ammonia are highly toxic. For these reasons, these assays are not well suited for farm applications.
Near infrared spectrographic instruments have also been used to provide analysis of materials, such as to determine the urea content of milk. For example, U.S. Pat. No. 5,912,730, issued Jun. 15, 1999, inventors Dahm et al. describes a spectrographic analysis instrument that is said to result in more accurate measurements.
SUMMARY OF THE INVENTION
In one aspect of the present invention, a sensor that is useful for assaying a component of a fluid is provided. The sensor comprises a chamber having an inlet adapted to admit a liquid sample, and further has a liquid containing portion and a vapor containing portion, with the two portions in fluid communication. A pressure monitor is in communication with the vapor containing portion, and is of a construction sufficient to measure pressure changes within the vapor containing portion. The measured pressure changes may be related to concentration of the component of the fluid being assayed.
In one preferred embodiment of the invention, the sample is taken from a biological fluid and is admitted into the liquid containing portion of the chamber. This sample is exposed to an enzyme for which the component of interest is a substrate. The enzyme exposure may be before the sample enters the chamber or during residence of the liquid sample within the chamber. A preferred enzyme is urease, which may be used to assay urea in biological fluids such as milk, urine and blood.
A particularly preferred embodiment of the sensor is where the pressure monitor is calibrated to provide urea (or milk urea nitrogen) concentration and may have a prediction error for milk urea nitrogen of not greater than about +/−1 mg/dl (over a physiological range of from about 6 ml/dl to about 24 ml/dl).
Another aspect of the present invention is a method of analyzing a component in a biological fluid. The analysis method includes the steps of providing a liquid sample of the biological fluid, contacting the sample with an enzyme for which the component is a substrate so as to form carbon dioxide as a reaction product, and detecting the amount of carbon dioxide so formed. One preferred embodiment of the present inventive method is in analyzing milk urea nitrogen (MUN) in dairy milk. In practicing this embodiment, the method comprises providing a dairy milk sample and contacting the sample with urease to yield carbonate and ammonium ions. The equilibrium is shifted towards carbon dioxide by adjusting pH, and carbon dioxide vapor is detected. This detected carbon dioxide may then be related to the concentration of MUN in the dairy milk sample.
Feed costs constitute the largest single expense of the dairy industry. Because of this and the increasing premium placed on milk protein content, there is considerable interest in optimizing nutritional input for the highest milk protein to feed cost ratio. In many localities, there is also concern about the environmental effects of excess nitrogen in dairy waste. Excessive levels of nitrogen in feed are believed to cause high systemic urea nitrogen levels without corresponding increase in milk protein. Use of the sensor and method in accordance with this invention can improve the nitrogen balance in dairy herds for economic benefit to the farmer and environmental benefit to the public.
Sensors of the invention may be used to automatically measure MUN during milking, and can thus be automated to run during an already automated milking process. The inventive sensors can complete one measurement cycle faster than the turn-around time for cows in the parlor (10 min), and are able to repeatably measure MUN to within 1 mg/dl in the physiological range from about 6 to 24 mg/dl.
Other aspects and advantages of this invention may be understood by reading the specification and claims.
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Oehler et al., “Detection of gases produced by biological systems with an enzyme-photoacoustic sensor”, Infrared Physics 25 (2):319-21 (1985).*
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Fuhrmann et al., “Enzymic assays based on the coulometric microflow titration of ammonia and carbon dioxide”, Biosensors & Bioelectronics 7(
Bondurant Robert H.
Delwiche Michael J.
Depeters Edward J.
Jenkins Daniel M.
Saucier Sandra E.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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