Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-10-13
2003-07-15
Tung, T. (Department: 1753)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403010, C204S415000, C205S783000
Reexamination Certificate
active
06592746
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a hydrogen peroxide sensor for fluids, and applications therefore. In particular, the present invention relates to a sensor which permits continuous monitoring of hydrogen peroxide in fluids and use of the data from the sensor for various purposes, including predicting hypertension and other oxidative stress-related physical conditions.
BACKGROUND OF THE INVENTION
Hydrogen peroxide is formed in several biological and environmental processes. Hydrogen peroxide can be found in natural water (e.g., sea water, rain water), where it is an important species in redox reactions, in industrial processes, including drinking water purification, where it is used as a disinfectant, and in biological tissues, including blood, as a result of enzymatic reactions. Direct detection of hydrogen peroxide is an important analytical task, and numerous techniques have been devised for measurement of hydrogen peroxide levels in fluids as indications of, for example, medical conditions, environmental quality, or the presence of pathogens in cells of both animals and plants. Superoxide radicals (O
2
−
) in living tissue can be derived from many sources, such as activated granulocytes, endothelial cells, xanthine oxidase-catalyzed reactions, mitochondrial metabolism, and transition metal reactions with oxygen. Hydrogen peroxide (H
2
O
2
) can be produced from the dismutation of superoxide radicals catalyzed by the enzyme superoxide dimutase (SOD), from transition metal reactions with superoxide radicals, and from enzymes (e.g., glycollate oxidase and urate oxidase) which produce peroxide directly without first producing superoxide. The presence of antioxidants, including certain enzymes such as SOD and catalase, serves to limit the concentration of the reactive oxygen species in plasma and tissues. Therefore, either an increase in the production of free radicals and/or a decrease in antioxidants can cause oxidative stress, contributing to possible cardiovascular complications in animals. Similarly, oxygen free radicals may affect vascular resistance by inactivating nitric oxide (NO), thereby causing arteriolar vasoconstriction and elevation of peripheral hemodynamic resistance. Other conditions have also been associated with oxidative stress, including arthritis, acceleration of the progression of HIV to full-blown AIDS, and neurological diseases such as ALS.
The mortality of individuals with hypertension has been found to be more than double that of the normotensive population, with most of the deaths occurring suddenly. Untreated hypertension also predisposes individuals to end organ damage or failure, including cerebrovascular accident (e.g., intracranial hemorrhage, encephalopathy), myocardial infarction, renal failure, and retinal hemorrhage. The mechanisms that predispose individuals with elevated arterial pressure to develop vascular organ injury are only partially understood. Oxygen free radicals and related intermediates have been implicated in hypertension and may play a role by affecting vascular smooth muscle contraction and resistance to blood flow. In individuals with histories of conditions such as atherosclerosis, stroke and myocardial infarction, hypertension constitutes a risk factor.
Studies have shown that in persons with essential hypertension there exists not only reduced antioxidant enzyme and nitric oxide levels, but also an increase in the NADPH oxidase activity on neutrophil membranes. An increase in NADPH oxidase activity results in production of oxygen free radicals. Consequently, hypertensives (individuals experiencing increased systolic and diastolic blood pressures) have higher superoxide and hydrogen peroxide production by neutrophils than normotensive (individuals with normal blood pressure) controls. It has also been shown that hypertensive patients revert to normal free radical, antioxidant and nitric oxide levels after effective antihypertensive treatment.
A number of different techniques are known for measurement of oxygen free radicals and their intermediates. These methods include the use of electrodes, chemiluminescence, and fluorescence. All of the aforementioned methods are limited to measuring oxygen free radicals from stimulated neutrophils or deproteinized whole blood.
A new hydrogen peroxide sensing system for measuring hydrogen peroxide in plasma is disclosed in international PCT patent application PCT/US 98/19013 (filed Sep. 14, 1998). In this system the test sample of plasma from a fluid or fluid-containing material which is to be analyzed for hydrogen peroxide content is divided into two equal portions and a hydrogen peroxide oxidation sensor is inserted into each portion. An inhibitor for the enzyme catalase, such as sodium azide, is added to one of the portions to stabilize the hydrogen peroxide present. A quantity of catalase is added to the other portion to deplete any hydrogen peroxide present by catalyzing it to oxygen. Hydrogen peroxide oxidation of each portion at the respective sensor is then measured, along with background oxidation of any other oxidizable species in the sample. The signal from the sensor in the depleted hydrogen peroxide sample is subtracted from the signal from the stabilized hydrogen peroxide sample to eliminate the signals' contributions from background oxidation, thus yielding a resultant signal which is representative of the amount of hydrogen peroxide production in the subject fluid or material.
While the system described in that PCT application is quite useful, it requires two separate portions of the plasma from the sample fluid or material as well as chemical treatment of each of the portions. Such a system is useful primarily in a laboratory where there are facilities for chemically treating the portions, and where supplies of the treating chemicals can be made available. It is not, however, particularly useful for analysis of samples in the field or where the treating chemicals are not conveniently available. It also does not account for the fact that either or both of the treating agents may affect other components of the samples so that the two samples may end up being different from each other with respect to more than just the hydrogen peroxide component. Further, since the treating chemicals or enzymes must be added to each sample, the device must be recalibrated for each run.
SUMMARY OF THE INVENTION
We have now developed a new sensor probe which can measure hydrogen peroxide content of a single sample of a fluid or fluid-containing material (such as blood, tissue, environmental water steams or industrial water streams) using two oxygen sensors whose electrodes are encased in specified membranes. Each sensor has an oxygen sensing electrode group and both groups are inserted into the single sample of fluid or fluid-containing material, so that both measure from a homogeneous source, preventing testing errors due to differential reactions with treating chemicals.
The electrode end of each sensor is surrounded by a hydrophobic membrane which prevents the transport of electrochemical poisons or interferents and isolates the electrodes and an electrolyte fluid surrounding the electrodes from the sample fluid. The hydrophobic membrane is permeable to oxygen but not to hydrogen peroxide. The hydrogen-peroxide-generated-oxygen (HPGO) sensor also is encased in a hydrophilic membrane which contains an enzyme such as catalase, peroxidase or other enzymes of a family which catalyzes the reaction of hydrogen peroxide to oxygen and water, namely:
This hydrophilic membrane is permeable to both hydrogen peroxide and oxygen. It is positioned in series with the hydrophobic membrane, with the hydrophobic membrane disposed between the hydrophilic membrane and the electrodes of the first sensor.
Disposed within the contained volume or space formed by the inner (proximal) surface of each hydrophobic membrane and the electrodes of its respective sensor, is an electrolyte solution to provide for migration of oxygen or electric charge to the appropriate electrodes. T
Baker Dale A.
Gough David
Schmid-Schoenbein Geert W.
Brown Martin Haller & McClain LLP
The Regents of the University of California
Tung T.
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