CA1254117A - Enzymatic ethanol test - Google Patents
Enzymatic ethanol testInfo
- Publication number
- CA1254117A CA1254117A CA000480608A CA480608A CA1254117A CA 1254117 A CA1254117 A CA 1254117A CA 000480608 A CA000480608 A CA 000480608A CA 480608 A CA480608 A CA 480608A CA 1254117 A CA1254117 A CA 1254117A
- Authority
- CA
- Canada
- Prior art keywords
- test device
- mixture
- ethanol
- aqueous
- alcohol oxidase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
Abstract
ABSTRACT OF THE DISCLOSURE
The present invention provides a stable, conven-ient solid state test device for the determination of ethanol in an aqueous test sample, a method for its pre-paration and a method for its use. The use of carrier matrix incorporated with alcohol oxidase, a peroxida-tively active substance and a suitable chromogenic indicator for the determination of ethanol in aqueous samples such as body fluid samples (e.g., serum, urine or saliva) is part of the invention. The invention provides a method of incorporating the enzyme alcohol oxidase and a peroxidatively active substance into a carrier matrix with a chromogenic indicator system capable of providing a detectable response to the presence of at least 100 mg/dL ethanol in less than about 5 minutes. The method involves either a) the use of a specialized incorporation procedure or b) the addition of an azide to the test device. Either method overcomes the "false positive" problem seen when solu-tion assay reagents are incorporated at the increased concentrations necessary to provide a test device sensitive to the at least 100 mg/dL ethanol.
The present invention provides a stable, conven-ient solid state test device for the determination of ethanol in an aqueous test sample, a method for its pre-paration and a method for its use. The use of carrier matrix incorporated with alcohol oxidase, a peroxida-tively active substance and a suitable chromogenic indicator for the determination of ethanol in aqueous samples such as body fluid samples (e.g., serum, urine or saliva) is part of the invention. The invention provides a method of incorporating the enzyme alcohol oxidase and a peroxidatively active substance into a carrier matrix with a chromogenic indicator system capable of providing a detectable response to the presence of at least 100 mg/dL ethanol in less than about 5 minutes. The method involves either a) the use of a specialized incorporation procedure or b) the addition of an azide to the test device. Either method overcomes the "false positive" problem seen when solu-tion assay reagents are incorporated at the increased concentrations necessary to provide a test device sensitive to the at least 100 mg/dL ethanol.
Description
ENZYMATIC ETHANOL TEST
FIELD OF T~iE INVENTION
The invention relates generally to the deter-mination of an analyte in an aqueous test sample with a solid state reagent strip (test device). In par-ticular, the invention relates to a solid state test device useful for the determination of ethanol in an aqueous test sample. Of particular interest are body fluid samples such as blood, urine or saliva.
UTILI TY
Ethanol testing is useful industrially, medically and for law enforcement. Industrially, the level of ethanol present can be used to determine the progress of fermentation or of solvent purification. In addition, alcoholic beverages and medicinals must be tested to determine if the desired level of ethanol is present. Medically, the presence and level of ethanol in the blood stream can be used to aid differential diagnosis among possible origins of loss of motor function or life threatening coma. The determination pf blood ethanol could also be used as an aid in compliance programs for problem drinkers and for those diagnosed as alcoholics. Legally, the level of ethanol in the blood stream is used as an objective indicium Of fitness to operate machinary or to drive an auto-mobile or other vehicles. A simple, fast, convenient method of determining the blood alcohol is particu-larly important for law enforcement.
~S
1'7 INPORMATION DISCLOSURE
1. Ethanol Assays Given the importance and wide ranging utility for ethanol testing, it is not surprising that many assay methods are available. Ethanol testing can be accomp-lished instrumentally by potentiometric measurement, by infrared spectroscopy or by gas chromatography. In addition, both enzymatic and nonenzymatic solution assays are available. Nonenzymatic assays use strong oxidizing agents such as permanganate and dichromate which change color when they react with ethanol. Un-fortunately, due to their nonspecific nature they also react with many other oxidizable substances causing erroneously high results. Enzymatic assays are gen-erally based on the use of alcohol dehydrogenase or alcohol oxidase. These assays often also utilize a competitive inhibitor of the enzyme to facilitate quantitative determination of an analyte. (See, for example, U.S. Patent No. 3,977,944.) The enzymatic action of alcohol dehydrogenase (ADH) on ethanol proceeds as follows:
Ethanol Acetaldehyde ~ + ADH +
Nicotine Adenine < ~ Reduced NAD
Dinucleotide (NADH) (NAD) The reduced nicotine adenine dinucleotide (NADH)can then be determined directly by ultraviolet spec-troscopy or the above reaction can be coupled with asecond enzymatic reaction which allows the conversion 11'7 of ethanol to NADH to be followed in the visible region of the spectrum.
Since the equilibrium of the above reaction lies st9rongly toward ethanol, an acetaldehyde-trapping agent is often used as a means to drive the reaction toward the production of NADH. ~See, for example, U.S. Patent No. 3,926,736.) "Alcohol Analysis:
Clinical Laboratory Aspects, Part II", K.M.Dubowski, L~boratory Menageme~t, April 1982, p. 33, provides a table on the major features of enzymatic (ADH) oxi-dation methods for blood-alcohol determination. The table discloses that assays based on the foregoing reaction can be facilitated by the use of semicar-bizide as an acetaldehyde-trapping agent. The NADH
produced can then be determined by the use of dia-phorase and iodonitrotetrazolium chloride to form a colored end product (formazan).
Other methods have been found to obviate the need for an acetaldehyde-trapping agent. G.B. Patent No.
1,351,547 describes a method which comprises testing a sample with an aqueous solution of alcohol dehydro-genase and a specified tetrazolium salt in amounts which provide a quantitative colorimetric response when in contact with the sample, NAD, diaphorase, and a buffer. The patent discloses a stable lyophilized composition of alcohol dehydrogenase and tetrazolium salt and indicates that additional components, such as albumin or gelatin and an antioxidant (e.g., a rGduced mercaptan), will further stabilize the reaction so that an acetaldehyde-trapping agent is not required.
The other enzymatic pathway utilizes alcohol oxidase (AOD~ and proceeds as follows:
Ethanol ~ 2 AOD ~ Acetaldehyde ~ H2O2 '7 This reaction can be coupled with a second enzymatic reaction which allows the determination of hydrogen peroxide in the visible region of the spectrum by the addition of peroxidase and a chromogenic indicator.
This series of liquid reactions, used for the assay of alcohol oxidase, has been published by Phillips Chemical Co. in a Biochemicals Technical Information Bulletin on Alcohol Oxidase ~23785E).
In U.S. Patent No. 4,430,427 the sodium azide inhibition of the rate of methanol conversion by alcohol oxidase was measured by following the oxygen consumption in solution with a dissolved oxygen probe.
The patent describes the formation of a red absorbing combination when an azide is added to an alcohol oxidase preparation.
FIELD OF T~iE INVENTION
The invention relates generally to the deter-mination of an analyte in an aqueous test sample with a solid state reagent strip (test device). In par-ticular, the invention relates to a solid state test device useful for the determination of ethanol in an aqueous test sample. Of particular interest are body fluid samples such as blood, urine or saliva.
UTILI TY
Ethanol testing is useful industrially, medically and for law enforcement. Industrially, the level of ethanol present can be used to determine the progress of fermentation or of solvent purification. In addition, alcoholic beverages and medicinals must be tested to determine if the desired level of ethanol is present. Medically, the presence and level of ethanol in the blood stream can be used to aid differential diagnosis among possible origins of loss of motor function or life threatening coma. The determination pf blood ethanol could also be used as an aid in compliance programs for problem drinkers and for those diagnosed as alcoholics. Legally, the level of ethanol in the blood stream is used as an objective indicium Of fitness to operate machinary or to drive an auto-mobile or other vehicles. A simple, fast, convenient method of determining the blood alcohol is particu-larly important for law enforcement.
~S
1'7 INPORMATION DISCLOSURE
1. Ethanol Assays Given the importance and wide ranging utility for ethanol testing, it is not surprising that many assay methods are available. Ethanol testing can be accomp-lished instrumentally by potentiometric measurement, by infrared spectroscopy or by gas chromatography. In addition, both enzymatic and nonenzymatic solution assays are available. Nonenzymatic assays use strong oxidizing agents such as permanganate and dichromate which change color when they react with ethanol. Un-fortunately, due to their nonspecific nature they also react with many other oxidizable substances causing erroneously high results. Enzymatic assays are gen-erally based on the use of alcohol dehydrogenase or alcohol oxidase. These assays often also utilize a competitive inhibitor of the enzyme to facilitate quantitative determination of an analyte. (See, for example, U.S. Patent No. 3,977,944.) The enzymatic action of alcohol dehydrogenase (ADH) on ethanol proceeds as follows:
Ethanol Acetaldehyde ~ + ADH +
Nicotine Adenine < ~ Reduced NAD
Dinucleotide (NADH) (NAD) The reduced nicotine adenine dinucleotide (NADH)can then be determined directly by ultraviolet spec-troscopy or the above reaction can be coupled with asecond enzymatic reaction which allows the conversion 11'7 of ethanol to NADH to be followed in the visible region of the spectrum.
Since the equilibrium of the above reaction lies st9rongly toward ethanol, an acetaldehyde-trapping agent is often used as a means to drive the reaction toward the production of NADH. ~See, for example, U.S. Patent No. 3,926,736.) "Alcohol Analysis:
Clinical Laboratory Aspects, Part II", K.M.Dubowski, L~boratory Menageme~t, April 1982, p. 33, provides a table on the major features of enzymatic (ADH) oxi-dation methods for blood-alcohol determination. The table discloses that assays based on the foregoing reaction can be facilitated by the use of semicar-bizide as an acetaldehyde-trapping agent. The NADH
produced can then be determined by the use of dia-phorase and iodonitrotetrazolium chloride to form a colored end product (formazan).
Other methods have been found to obviate the need for an acetaldehyde-trapping agent. G.B. Patent No.
1,351,547 describes a method which comprises testing a sample with an aqueous solution of alcohol dehydro-genase and a specified tetrazolium salt in amounts which provide a quantitative colorimetric response when in contact with the sample, NAD, diaphorase, and a buffer. The patent discloses a stable lyophilized composition of alcohol dehydrogenase and tetrazolium salt and indicates that additional components, such as albumin or gelatin and an antioxidant (e.g., a rGduced mercaptan), will further stabilize the reaction so that an acetaldehyde-trapping agent is not required.
The other enzymatic pathway utilizes alcohol oxidase (AOD~ and proceeds as follows:
Ethanol ~ 2 AOD ~ Acetaldehyde ~ H2O2 '7 This reaction can be coupled with a second enzymatic reaction which allows the determination of hydrogen peroxide in the visible region of the spectrum by the addition of peroxidase and a chromogenic indicator.
This series of liquid reactions, used for the assay of alcohol oxidase, has been published by Phillips Chemical Co. in a Biochemicals Technical Information Bulletin on Alcohol Oxidase ~23785E).
In U.S. Patent No. 4,430,427 the sodium azide inhibition of the rate of methanol conversion by alcohol oxidase was measured by following the oxygen consumption in solution with a dissolved oxygen probe.
The patent describes the formation of a red absorbing combination when an azide is added to an alcohol oxidase preparation.
2. Reagent Strip Format It has been suggested that the solution assay composition for the determination of alcohol dehydro-genase can be incorporated onto solid state test devices. See, for example, U.S. Patent No. 4,394,444 commonly assigned to the present assignee, which describes an analyte determination wherein the analyte reacts with a dehydrogenase, such as alcohol dehydro-genase, to produce NADH. The NADH is then determined with an uncoupler and peroxidase to produce color.
~he patent suggests that such a system can be in-corporated into a solid state test device. However, there is no published information of which the in-ventors are aware on how to incorporate an alcohol oxidase solution assay into a test device.
'7 SUMMAR~ OF THE INVENTION
The present invention provides a test device for the determination of ethanol in an aqueous test sam-ple, a method for its preparation and a method for its use. The test device is composed of a carrier matrix incorporated with alcohol oxidase, a peroxidatively active substance and a chromogenic indicator system capable of providing a detectable colorimetric res-ponse, wherein the chromogenic indicator system is substantially in the reduced (uncolored) form and the alcohol oxidase is present in a quantity sufficient to provide a colorimetric response to at least 100 mg/dL
ethanol in less than 5 minutes. When azide is ad-ditionally incorporated, the device can be used to quantitatively determine ethanol content. The use of alcohol oxidase, a peroxidatively active substance and a suitable chromogenic indicator incorporated into a carrier matrix for determining ethanol in an aqueous test sample is also considered to be part of the invention.
In use, the aqueous test sample is contacted with the test device. The presence of 100 mg/dL ethanol and/or concentration of ethanol in the test sample is then determined by observing any detectable colori-metric response produced in less than about 5 minutes.
The test device of the present invention over-comes the false positive problem seen when increased concentrations of solution assay components, used to provide sufficient sensltivity to detect at least 100 mg/dL ethanol in an aqueous test sample, are simply incorporated into a carrier matrix. The test device provides rapid results, sufficient detectable response forming in less than 5 minutes. The device ~5~ 7 can be prepared with ordinary drying techniques and does not require lyophilization.
DETAILED DESCRIPTIO~ 0~ THE I~rVEZl1TIOlV
Solution assay reagent concentrations are usually too low to provide a sensitive assay when incorporated into a solid state test device. Therefore, the rea-gent concentrations incorporated into a carrier matrix must be increased and of course the concentra-tions in the final dry solid state test device are even higher. This concentration change itself often leads to peculiar problems related to differences in interactions and stabilities of the components in the solid state test device. These problems are particu-larly egregious when enzymes are involved.
In the case of an alcohol oxidase based ethanol test, direct incorporation of the solution assay rea-gent concentrations into a carrier matrix does not produce a test device with satisfactory sensitivity.
Required sensitivity can vary with the intended use of the test device. For compliance programs, very low levels of blood ethanol - 25 to 50 mg/dL (milligrams per deciliter) - would indicate noncompliance. For law enforcement, tests must be sensitive to the legally set limit, which in the United States is usually about lO0 mg/dL blood ethanol. The experimental protocols used herein required sensitivity to at least 100 mg/dL
ethanol in an aqueous fluid sample with a colorimetric response in less than about 5 minutes. A preferred test device can indicate the presence of lO0 mg/dL
ethanol in about l to 2 minutes, most preferably 1 minute o. less.
~Z~ JL'7 When solution assay reagent concentrations are increased to provide the requisite sensitivity as defined above, incorporated into a carrier matrix and dried, the indicator is substantially in the oxidized form (i.e., colored) even prior to contact with an aqueous test sample containing ethanol. In other words, a test device so prepared gives an instantaneous and ubiquitous false positive test for ethanol. While false positives are always undesirable, they are particularly detrimental in ethanol testing. False indications of noncompliance could seriously damage a program's ability to rehabilitate problem drinkers.
of course, a false positive test indicating a blood alcohol level over the legal limit would have par-ticularly serious consequences for a driver.
The false positive problem is either not seen inthe dilute solution assay or is perhaps negated by the use of a blank (i.e., comparison with an unreacted portion of the test composition). The problem also appears to be unique to alcohol oxidase since other oxidase er.zymes such as glucose oxidase, cholesterol oxidase, uricase and galactose oxidase, used in the high concentrations required to produce a sensitive dry solid state test device, do not exhibit this high degree of false positive response.
The present invention solves the false positive problem seen in the reagent strip format, either by the use of a srecialized incorporation procedure or by the addition of an azide to the test composition.
The present inventors speculate, but do not base their invention on the premise that the ubiquity of low molecular weight primary alcohols in reagents used may contribute to ~he false positive problem. In addition, there is some evidence that alcohol oxidase may react with the primary hydroxy groups of serine 1;~5~ 7 residues on the enzyme itself, an autooxidation process unknown in other oxidase enzymes.
Two approaches have been used to obyiate the false positive problem seen when the solution assay composition, in increased concentration levels, is incorporated into a carrier matrix to produce a solid state test device. One approach is used when the chromogenic indicator system is composed of a coupled pair of indicator components. In that case, one component is incorporated into the carrier matrix with the peroxidatively active substance and alcohol oxi-dase, and the impregnated carrier is dried prior to the incorporation of the second chromogenic component.
A second approach to eliminating the false posi-tive problem is the addition of an azide, such as sodium azide. While the azide performs as a competi-tive inhibitor, allowing the quantitative assay of ethanol at high concentrations, it is speculated that it also protects the enzyme from its self-destructive tendencies, which tendencies may contribute to the false positive problem. The addition of azide is the best mode known to the inventors for producing a solid state test device which is sensitive to at least 100 mg/dL ethanol and yet the chromogenic component of an indicator system is substantially in the reduced form (i.e., no false positive reaction). The addition of azide is preferable even when specialized incor-poration techniques are utilize'.
A. Test Components Preparations exhibiting alcohol oxidase activity are available from a variety of scurces including species of fungae and yeasts. Preparations from Pichia-Type yeasts are a preferred source of alcohol g oxidase. Such yeast sources are listed in U.S. Patent No. 4r430~427~ The alcohol oxidase preparations used react with ethanol and other lower primary alcohols, such as methanol and l-propanol.
Peroxidatively active substances, useful in the present invention, can be chosen from various organic and inorganic sources. Plant peroxidases, such as horseradish peroxidase or potato peroxidase, can be used. In addition, even though less satisfactory, hemin and hemin derivatives, hemoglobins and hematin can be used.
The chromogenic indicator system can be either a single component or two components forming a coupled chromogenic indicator system. When a single chromo-genic component, such as 3,3',5,5'-tetramethylbenzi-dine, o-tolidine, 2-amino-8-naphthol-3,6-disulfonic acid, the natural product gum guaiac or p-anisidine, is used, an azide is preferably included in the test composition to obviate the false positive problem.
~he patent suggests that such a system can be in-corporated into a solid state test device. However, there is no published information of which the in-ventors are aware on how to incorporate an alcohol oxidase solution assay into a test device.
'7 SUMMAR~ OF THE INVENTION
The present invention provides a test device for the determination of ethanol in an aqueous test sam-ple, a method for its preparation and a method for its use. The test device is composed of a carrier matrix incorporated with alcohol oxidase, a peroxidatively active substance and a chromogenic indicator system capable of providing a detectable colorimetric res-ponse, wherein the chromogenic indicator system is substantially in the reduced (uncolored) form and the alcohol oxidase is present in a quantity sufficient to provide a colorimetric response to at least 100 mg/dL
ethanol in less than 5 minutes. When azide is ad-ditionally incorporated, the device can be used to quantitatively determine ethanol content. The use of alcohol oxidase, a peroxidatively active substance and a suitable chromogenic indicator incorporated into a carrier matrix for determining ethanol in an aqueous test sample is also considered to be part of the invention.
In use, the aqueous test sample is contacted with the test device. The presence of 100 mg/dL ethanol and/or concentration of ethanol in the test sample is then determined by observing any detectable colori-metric response produced in less than about 5 minutes.
The test device of the present invention over-comes the false positive problem seen when increased concentrations of solution assay components, used to provide sufficient sensltivity to detect at least 100 mg/dL ethanol in an aqueous test sample, are simply incorporated into a carrier matrix. The test device provides rapid results, sufficient detectable response forming in less than 5 minutes. The device ~5~ 7 can be prepared with ordinary drying techniques and does not require lyophilization.
DETAILED DESCRIPTIO~ 0~ THE I~rVEZl1TIOlV
Solution assay reagent concentrations are usually too low to provide a sensitive assay when incorporated into a solid state test device. Therefore, the rea-gent concentrations incorporated into a carrier matrix must be increased and of course the concentra-tions in the final dry solid state test device are even higher. This concentration change itself often leads to peculiar problems related to differences in interactions and stabilities of the components in the solid state test device. These problems are particu-larly egregious when enzymes are involved.
In the case of an alcohol oxidase based ethanol test, direct incorporation of the solution assay rea-gent concentrations into a carrier matrix does not produce a test device with satisfactory sensitivity.
Required sensitivity can vary with the intended use of the test device. For compliance programs, very low levels of blood ethanol - 25 to 50 mg/dL (milligrams per deciliter) - would indicate noncompliance. For law enforcement, tests must be sensitive to the legally set limit, which in the United States is usually about lO0 mg/dL blood ethanol. The experimental protocols used herein required sensitivity to at least 100 mg/dL
ethanol in an aqueous fluid sample with a colorimetric response in less than about 5 minutes. A preferred test device can indicate the presence of lO0 mg/dL
ethanol in about l to 2 minutes, most preferably 1 minute o. less.
~Z~ JL'7 When solution assay reagent concentrations are increased to provide the requisite sensitivity as defined above, incorporated into a carrier matrix and dried, the indicator is substantially in the oxidized form (i.e., colored) even prior to contact with an aqueous test sample containing ethanol. In other words, a test device so prepared gives an instantaneous and ubiquitous false positive test for ethanol. While false positives are always undesirable, they are particularly detrimental in ethanol testing. False indications of noncompliance could seriously damage a program's ability to rehabilitate problem drinkers.
of course, a false positive test indicating a blood alcohol level over the legal limit would have par-ticularly serious consequences for a driver.
The false positive problem is either not seen inthe dilute solution assay or is perhaps negated by the use of a blank (i.e., comparison with an unreacted portion of the test composition). The problem also appears to be unique to alcohol oxidase since other oxidase er.zymes such as glucose oxidase, cholesterol oxidase, uricase and galactose oxidase, used in the high concentrations required to produce a sensitive dry solid state test device, do not exhibit this high degree of false positive response.
The present invention solves the false positive problem seen in the reagent strip format, either by the use of a srecialized incorporation procedure or by the addition of an azide to the test composition.
The present inventors speculate, but do not base their invention on the premise that the ubiquity of low molecular weight primary alcohols in reagents used may contribute to ~he false positive problem. In addition, there is some evidence that alcohol oxidase may react with the primary hydroxy groups of serine 1;~5~ 7 residues on the enzyme itself, an autooxidation process unknown in other oxidase enzymes.
Two approaches have been used to obyiate the false positive problem seen when the solution assay composition, in increased concentration levels, is incorporated into a carrier matrix to produce a solid state test device. One approach is used when the chromogenic indicator system is composed of a coupled pair of indicator components. In that case, one component is incorporated into the carrier matrix with the peroxidatively active substance and alcohol oxi-dase, and the impregnated carrier is dried prior to the incorporation of the second chromogenic component.
A second approach to eliminating the false posi-tive problem is the addition of an azide, such as sodium azide. While the azide performs as a competi-tive inhibitor, allowing the quantitative assay of ethanol at high concentrations, it is speculated that it also protects the enzyme from its self-destructive tendencies, which tendencies may contribute to the false positive problem. The addition of azide is the best mode known to the inventors for producing a solid state test device which is sensitive to at least 100 mg/dL ethanol and yet the chromogenic component of an indicator system is substantially in the reduced form (i.e., no false positive reaction). The addition of azide is preferable even when specialized incor-poration techniques are utilize'.
A. Test Components Preparations exhibiting alcohol oxidase activity are available from a variety of scurces including species of fungae and yeasts. Preparations from Pichia-Type yeasts are a preferred source of alcohol g oxidase. Such yeast sources are listed in U.S. Patent No. 4r430~427~ The alcohol oxidase preparations used react with ethanol and other lower primary alcohols, such as methanol and l-propanol.
Peroxidatively active substances, useful in the present invention, can be chosen from various organic and inorganic sources. Plant peroxidases, such as horseradish peroxidase or potato peroxidase, can be used. In addition, even though less satisfactory, hemin and hemin derivatives, hemoglobins and hematin can be used.
The chromogenic indicator system can be either a single component or two components forming a coupled chromogenic indicator system. When a single chromo-genic component, such as 3,3',5,5'-tetramethylbenzi-dine, o-tolidine, 2-amino-8-naphthol-3,6-disulfonic acid, the natural product gum guaiac or p-anisidine, is used, an azide is preferably included in the test composition to obviate the false positive problem.
3,3',5,5'-Tetramethylbenzidine, o-tolidine and gum guaiac are preferred single chromogenic indicator components. Coupled chromogenic components, such as 3,5-dichloro-2-hydroxybenzene sulfonic acid (DHSA)/4-aminoantipyrine, 3,5-dichloro-2-hydroxybenzene sul-fonic acid/3-methyl-2-benzothiazoline hydrazone or m-anisidine/4-aminoantipyrine, among others, can be used to prepare the test device. The chromogenic pair, DHSA/4-aminoantipyrine is a preferred coupled indi-cator system.
Choice of the chromogenic indicator system can affect the ability of the test device to fulfill the stated criteria of providing a colorimetric response to the presence of 100 mg/dL ethanol in an aqueous test sample in less than about 5 minutes. Given the present disclosure, choice of a sufficiently sensitive indicator system can be made by one of ordinary skill in the art.
Preferred azides are the metal salt azides, par-ticuarly the electropositive metal azides which are not explosive. Particularly preferred are the metal azides selected from the Group lA of the Periodic Table according to Mendeleeff, such as lithium azide, sodium azide, potassium azide and the like. Of these sodium azide was found to be the most readily avail-able commercially.
The pH optimum of alcohol oxidase is around pH
7.5. Therefore, although not required for all aqueous test samples, a buffer is preferably incorporated into the test device. This is especially true if the test device is formulated for use with a body fluid such as urine where the pH of the sample may be as low as 4 to 5. Any buffer capable of providing a pH in the range of 5.0 to 9.0 can be used in the invention. Useful buffers include sodium phosphate, sodium citrate and tristhydroxymethyl)aminomethane and tris(hydroxy-methyl)-aminomethane glutamate. Other buffers can readily be chosen by those of ordinary skill in the art, given the present disclosure.
Additional components, such as wetting substances and color or shelf-life stabilizers, can be included as long as they do not interfere with the enzymatic reaction of alcohol oxidase with ethanol or that of the peroxidatively active substance with the generated peroxide. Suitable wetting substances include polymers, such as polyvinyl pyrrolidone, and surfactants. A
polyethoxylated fatty alcohol ~obtained under the -trademark Emulphor~ ON 870 from GAF, New York, NY) can also be used.
1 ~ ~4 1 i 7 Dioctyl sodium sulfosuccinate ~obtained under the trademark Aerosol OT from Aldrich Chemical Co., Milwaukee, Wisconsin) acts both as a surfactant and as a color stabilizer. Useful shelf-life stabilizers include sorbitol and tris~hydroxymethyl~aminomethane glutamate; sorbitol is particularly preferred.
The carrier matrix can be any substance capable of being incorporated with the components of the test composition. Thus, the matrix can take on many known forms, such as those utilized for reagent strips for solution analysis. For example, U.S. Patent No.
3,846,247 teaches the use of felt, porous ceramic strips and woven or matted glass fibers. As sub-stitutes for paper, U.S. Patent No. 3,522,928 teaches the use of wood sticks, cloth, sponge material and argillaceous substances. French Patent No. 2,170,397 teaches the use of carrier matrices having greater than 50% polyamide fibers therein. Another approach to carrier matrices is disclosed in U.S. Patent No.
Choice of the chromogenic indicator system can affect the ability of the test device to fulfill the stated criteria of providing a colorimetric response to the presence of 100 mg/dL ethanol in an aqueous test sample in less than about 5 minutes. Given the present disclosure, choice of a sufficiently sensitive indicator system can be made by one of ordinary skill in the art.
Preferred azides are the metal salt azides, par-ticuarly the electropositive metal azides which are not explosive. Particularly preferred are the metal azides selected from the Group lA of the Periodic Table according to Mendeleeff, such as lithium azide, sodium azide, potassium azide and the like. Of these sodium azide was found to be the most readily avail-able commercially.
The pH optimum of alcohol oxidase is around pH
7.5. Therefore, although not required for all aqueous test samples, a buffer is preferably incorporated into the test device. This is especially true if the test device is formulated for use with a body fluid such as urine where the pH of the sample may be as low as 4 to 5. Any buffer capable of providing a pH in the range of 5.0 to 9.0 can be used in the invention. Useful buffers include sodium phosphate, sodium citrate and tristhydroxymethyl)aminomethane and tris(hydroxy-methyl)-aminomethane glutamate. Other buffers can readily be chosen by those of ordinary skill in the art, given the present disclosure.
Additional components, such as wetting substances and color or shelf-life stabilizers, can be included as long as they do not interfere with the enzymatic reaction of alcohol oxidase with ethanol or that of the peroxidatively active substance with the generated peroxide. Suitable wetting substances include polymers, such as polyvinyl pyrrolidone, and surfactants. A
polyethoxylated fatty alcohol ~obtained under the -trademark Emulphor~ ON 870 from GAF, New York, NY) can also be used.
1 ~ ~4 1 i 7 Dioctyl sodium sulfosuccinate ~obtained under the trademark Aerosol OT from Aldrich Chemical Co., Milwaukee, Wisconsin) acts both as a surfactant and as a color stabilizer. Useful shelf-life stabilizers include sorbitol and tris~hydroxymethyl~aminomethane glutamate; sorbitol is particularly preferred.
The carrier matrix can be any substance capable of being incorporated with the components of the test composition. Thus, the matrix can take on many known forms, such as those utilized for reagent strips for solution analysis. For example, U.S. Patent No.
3,846,247 teaches the use of felt, porous ceramic strips and woven or matted glass fibers. As sub-stitutes for paper, U.S. Patent No. 3,522,928 teaches the use of wood sticks, cloth, sponge material and argillaceous substances. French Patent No. 2,170,397 teaches the use of carrier matrices having greater than 50% polyamide fibers therein. Another approach to carrier matrices is disclosed in U.S. Patent No.
4,046,513, wherein the concept of printing reagents onto a sllitable carrier matrix is employed. U.S.
Patent No. 4,046,514 discloses the interweaving or knitting of filaments bearing reagents in a reactant system.
~ It is, therefore, to be appreciated that in producing a test device of the invention all such carrier matrix concepts can be employed, as can others. The matrix can include a system which physi-cally entraps any or all of these ingredients, such as polymeric microcapsules which rupture upon contact with an aqueous solution. For example, alcohol oxidase and the chromogenic indicator system or one component of a coupled chromogenic indicator, can be maintained separately within the same carrier matrix without interaction until contacted with a solution.
The matrix can also comprise a system wherein the composition ingredients are homogeneously combined in a fluid or semifluid state, which later hardens or sets, thereby incorporating the ingredients.
Other matrix formats are contemplated, including the use of a microporous membrane or polymer film mat-rices. Microporous membranes are available as pre-formed membranes or can be prepared by such tech-niques as phase inversion. Suitable polymer films can be produced with commercially available latex formulations based on latex polymer suspensions such as those formed from a 60:40 copolymer of styrene and butadiene. Other natural or synthetic polymers or mixtures thereof can also be used. Examples of such film formulations can be found in U.S. Patents Nos.
3,630,957 and 4,312,834.
The presently preferred method is to impregnate a bibulous carrier matrix, for example filter paper, with the composition followed by affixing the impreg-nated matrix to a support member. When a whole bloodsample is tested, the impregnated carrier matrix can be coated to allow excess sample to be washed or wiped off.
B. Method of Preparation Preferably, the reagent test strip or test device is prepared by sequential incorporation of the carrier matrix with drying between incorporation steps.
Drying can be accomplished by any means which will not deleteriously affect the incorporated composition, usually by means of an air oven. The dried paper can thereafter be cut and mounted on one end of a support member, for example, a rigid or semi-rigid polystyrene film strip. Mounting of the paper on the strip can be accomplished through use of a double-faced adhesive ~ 7 tape, such as that commercially available from the 3M
Co. as DOUBLE STICK .
The following examples illustrate the preferred methods of incorporation by a~ incorporating any organic soluble single chromogenic component prior to incorporation of alcohol oxidase and any remaining components, and drying thoroughly, ~) incorporating one component of a coupled chromogenic indicator into the carrier matrix with the alcohol oxidase with drying prior to the incorporation of the second chromogenic component or c) incorporating a dried microporous polymer film containing alcohol oxidase and peroxidase with a chromogenic indicator. When split incorporation is used, the test strip can ex-hibit a slight coloration visibly different from thewhite of a paper carrier matrix. This coloration may intensify slightly when contacted with water. How-ever, the chromogenic components remain substantially in the reduced (uncolored) form and there is a sign-ificant change in color in less than about 5 minuteswhen the device is contacted with an aqueous sample containing 100 mg/dL ethanol. Due to this slight coloration, however, it is preferred to incorporate an azide with the alcohol oxidase even when the coupled chromogenic components are split for incorporation.
The test device could also be prepared by com-bining the assay ingredients in a polymer matrix in a fluid or semi-fluid state and applying to a support.
Examples of such procedures are also provided.
Incorporation can be accomplished by any method such as dipping spreading or spraying which allows the carrier matrix to be substantially uniformly incorporated with the assay composition.
41:~7 C. Concentration Ranges of Test Components The concentration ranges of the test components are substantially higher than the concentrations re-quired for solution assays of ethanol. The ~hoice of appropriate concentrations is further complicated by the apparent interaction of azide with peroxidase ["Deleterious Effect of Sodium Azide on Activity of Peroxidase", 36 J. CZin. PathoZogy 10, 1983], as well as with alcohol oxidase (U.S. Patent No. 4,430,427).
In addition, appropriate concentrations vary slightly depending on whether the test sample is urine, saliva, or blood. Preferably the required concentration of alcohol oxidase (AOD), which with the indicator system of choice can provide a sufficiently sensitive ethanol test, is determined first. The concentration of al-cohol oxidase required is inversely related to the sensitivity of the indicator. An excess of peroxidase is used and preferably the ratio of azide to per-oxidase ~POD) is no more than 0.4:1 by weight, an amount which is sufficient to prevent a false positive indication without inhibiting the necessary activity of peroxidase.
The following table provides a guide to the working and preferred concentration ranges for com-ponents in the reagent soluti~n used to prepare thetest device of the present .nvention with the sensi-tivity and reaction time stated previously. (Defini-tion of units can be found under the heading "Ex-amples").
11'7 saliva or serum working ~referred -AOD 10-250 IU/mL 2Q-100 IU/mL
POD 24-3600 IU/mL 50-200 IU/mL
azide 0 mM to 15 mM 2.5 mM to 5 mM
Patent No. 4,046,514 discloses the interweaving or knitting of filaments bearing reagents in a reactant system.
~ It is, therefore, to be appreciated that in producing a test device of the invention all such carrier matrix concepts can be employed, as can others. The matrix can include a system which physi-cally entraps any or all of these ingredients, such as polymeric microcapsules which rupture upon contact with an aqueous solution. For example, alcohol oxidase and the chromogenic indicator system or one component of a coupled chromogenic indicator, can be maintained separately within the same carrier matrix without interaction until contacted with a solution.
The matrix can also comprise a system wherein the composition ingredients are homogeneously combined in a fluid or semifluid state, which later hardens or sets, thereby incorporating the ingredients.
Other matrix formats are contemplated, including the use of a microporous membrane or polymer film mat-rices. Microporous membranes are available as pre-formed membranes or can be prepared by such tech-niques as phase inversion. Suitable polymer films can be produced with commercially available latex formulations based on latex polymer suspensions such as those formed from a 60:40 copolymer of styrene and butadiene. Other natural or synthetic polymers or mixtures thereof can also be used. Examples of such film formulations can be found in U.S. Patents Nos.
3,630,957 and 4,312,834.
The presently preferred method is to impregnate a bibulous carrier matrix, for example filter paper, with the composition followed by affixing the impreg-nated matrix to a support member. When a whole bloodsample is tested, the impregnated carrier matrix can be coated to allow excess sample to be washed or wiped off.
B. Method of Preparation Preferably, the reagent test strip or test device is prepared by sequential incorporation of the carrier matrix with drying between incorporation steps.
Drying can be accomplished by any means which will not deleteriously affect the incorporated composition, usually by means of an air oven. The dried paper can thereafter be cut and mounted on one end of a support member, for example, a rigid or semi-rigid polystyrene film strip. Mounting of the paper on the strip can be accomplished through use of a double-faced adhesive ~ 7 tape, such as that commercially available from the 3M
Co. as DOUBLE STICK .
The following examples illustrate the preferred methods of incorporation by a~ incorporating any organic soluble single chromogenic component prior to incorporation of alcohol oxidase and any remaining components, and drying thoroughly, ~) incorporating one component of a coupled chromogenic indicator into the carrier matrix with the alcohol oxidase with drying prior to the incorporation of the second chromogenic component or c) incorporating a dried microporous polymer film containing alcohol oxidase and peroxidase with a chromogenic indicator. When split incorporation is used, the test strip can ex-hibit a slight coloration visibly different from thewhite of a paper carrier matrix. This coloration may intensify slightly when contacted with water. How-ever, the chromogenic components remain substantially in the reduced (uncolored) form and there is a sign-ificant change in color in less than about 5 minuteswhen the device is contacted with an aqueous sample containing 100 mg/dL ethanol. Due to this slight coloration, however, it is preferred to incorporate an azide with the alcohol oxidase even when the coupled chromogenic components are split for incorporation.
The test device could also be prepared by com-bining the assay ingredients in a polymer matrix in a fluid or semi-fluid state and applying to a support.
Examples of such procedures are also provided.
Incorporation can be accomplished by any method such as dipping spreading or spraying which allows the carrier matrix to be substantially uniformly incorporated with the assay composition.
41:~7 C. Concentration Ranges of Test Components The concentration ranges of the test components are substantially higher than the concentrations re-quired for solution assays of ethanol. The ~hoice of appropriate concentrations is further complicated by the apparent interaction of azide with peroxidase ["Deleterious Effect of Sodium Azide on Activity of Peroxidase", 36 J. CZin. PathoZogy 10, 1983], as well as with alcohol oxidase (U.S. Patent No. 4,430,427).
In addition, appropriate concentrations vary slightly depending on whether the test sample is urine, saliva, or blood. Preferably the required concentration of alcohol oxidase (AOD), which with the indicator system of choice can provide a sufficiently sensitive ethanol test, is determined first. The concentration of al-cohol oxidase required is inversely related to the sensitivity of the indicator. An excess of peroxidase is used and preferably the ratio of azide to per-oxidase ~POD) is no more than 0.4:1 by weight, an amount which is sufficient to prevent a false positive indication without inhibiting the necessary activity of peroxidase.
The following table provides a guide to the working and preferred concentration ranges for com-ponents in the reagent soluti~n used to prepare thetest device of the present .nvention with the sensi-tivity and reaction time stated previously. (Defini-tion of units can be found under the heading "Ex-amples").
11'7 saliva or serum working ~referred -AOD 10-250 IU/mL 2Q-100 IU/mL
POD 24-3600 IU/mL 50-200 IU/mL
azide 0 mM to 15 mM 2.5 mM to 5 mM
5 urine working preferred AOD 10-500 IU/mL 20-200 IU/mL
POD 24-3600 IU/mL 50-200 IU/mL
azide 0 mM to 5.0 mM 0.5 mM to 3.0 mM
Most preferably the concentration of alcohol oxidase for a saliva/serum test device is 40 to 70 IU/mL and for urine is 50-150 IU/mL. In any case, the concen-tration of the single chromogenic component or each of the couple chromogenic components will be about 0.01 to 0.2 M ~molar~, preferably about 0.025 to 0.1 M.
The buffer should provide a pH of from about 5 to 9, preferably from about 7 to 8. These concentrations ranges and relative concentrations of components are viable whether the solution is an aqueous impregnating solution or a polymer suspension.
D. Method of Use The test device is advantageously used by momen-tarily dipping it in a test sample or by otherwise introducing a test sample onto the carrier iilatrix, whereby a de~ectable colorimetric change results when 100 mg/dL ethanol is present. Contact with the test sample can also be made by pipette, swab or spatula.
Although dipping is a highly satisfactory method of contact when urine is used, a blood sample will nor-mally require pipetting and the latter methods can be useful in testing saliva.
The following examples describe experiments which were performed in developing the present invention.
While the examples serve to illustrate the invention, they are not to be interpreted as limiting its scope which is defined solely by the claims appended hereto.
One skilled in the art will be able to make such variations, substitutions and changes in the com-ponents of the composition and ingredients and reac-tion parameters as may seem desirable.
EXA~PLES
Alcohol oxidase (AOD, EC 1.1.3.13) was obtained from Phillips Chemical Co., Bartlesville, OK. The activity of the alcohol oxidase used is given in International Units per milliliter (IU/mL) of stock solution. One International Unit (IU) of activity catalyzes the formation of 1 micromole aldehyde and hydrogen peroxide in an air-saturated solution at pH
7.5, 25C. The peroxidase used was horseradish per-oxidase obtained from Miles Laboratories, Inc., Kankakee, Ill.. with an activity of 130 IU/mg.
Aerosol~ OT is the trademark used by Aldrich Chemical Co., Milwaukee, WI, for dioctyl sodium sul-fosuccinate. Emulphor~ ON 870 is the trademark used by GAF, New York, NY. The other reagents used are com-mercially available.
~IS-1337 t,~
The following ab~re~iations are used in the examples:
dL deciliter mL milliliters M molar mM millimolar C degrees centrigrade mg milligrams IU International Unit TMB 3,3',5,5'-tetramethylbenzidine DHSA 3,5-dichloro-2-hydroxybenzene sulfonic acid MBTH 3-methyl-2-benzothiazoline hydrazone POD peroxidase AOD alcohol oxidase Percentages used indicate weight in milligrams per 100 militers of aqueous solution (%w/v) unless otherwise indicated.
A. Direct Incorporation Solution Chemistry Attempts were made to incorporate the solution assay for ethanol, described by Phillips Chemical Co.
in a Biochemicals Technical Bulletin on Alcohol Oxi-dase, into a solid state test device. The assay follows the production of peroxide with an indicator system comprising a chromogenic indicator and per-oxidase.
1~41~7 t~hatman 3~ filter paper was dipped into a solu-tion using enzyme concentrations compara~le to those used in the Phillips assav ci.e., 5 IU/mL alcohol oxidase in the impregnating solution? and dried 10 minutes at 60~C in an air oven.
solution o-dianisidine 2HCl ~0.008%
in 0.1 M phosphate buffer, pH 7.5) 10 mL
P9D, 12 mg/mL 0.004 mL
AOD ~500 IU/mL) 0.1 mL
The carrier so incorporated was white in color. How-ever, when contacted with an aqueous test sample containing 100 mg/dL ethanol, there was na apparent color development, i.e., the test device produced by direct incorporation of the liquid assay reagents was not sufficiently sensitive to ethanol to produce a useful test device.
A second attempt was made to produce a test device based on the liquid assay of Phillips by in-creasing the concentrations of the en~ymes to provide a more sensitive test. Whatman 3~M filter paper was dipped in a solution containing about 10 times the concentration of alcohol oxidase and 100 times the concentration peroxidase used previously and dried 10 minutes at 60~C in an air oven.
solution:
o-dianisidine-2HCl (0.008o in 0.1l~
phosphate, pH 7.5) 8.5 ml POD, 12 mg/mL 0.5 ml AOD (500 IU/mL) l.O ml MS-13~7 * Trade Mark lI7 The dried carrier matrix developed a pale pink when contacted with either a blank ~water only~ or an aqueous test sample containing 100 mg/dL ethanol.
There was very little differentiation between the 0 and 100 mg/dL level of ethanol. Therefore the test device so prepared is still not sufficiently sensitive to ethanol. o-dianisidine is believed to be an in-sufficiently sensitive indicator to provide a test device for 100 mg/dL ethanol which develops a colori-metric response in less than 5 minutes.
A similar lack of sensitivity was seen when aPhillips assay which follows the aldehyde produced by the action of alcohol oxidase on ethanol. Test devices were prepared by dipping Whatman 3MM filter paper into Solution l or Solution 2, below. Each type was dried for 10 minutes at 60C in an air oven.
Solution 1 Solution 2 MBTH-HCl~0.04%) 2.0 ml 2.0 ml AOD (500 IU/mL) 0.05 ml 1.0 ml Ferric chloride (0,2% in 0.lN HCl) 8.0 ml 7.0 ml In Solution 2, the concentration of alcohol oxidase was increased 20 times over that in Solution l. Test devices prepared in either manner showed no reaction when contacted by an aqueous test sample containing 100 mg/dL ethanol. This result was not unexpected since the Phillips aldehyde based assay required one hour incubation of the ethanol with the assay solu-tion, making it unsuitable for a rapid dip-and-read test.
L'7 . - 20 -B: False Positive Produced if Solution Assay Reagent Concentration Increased to Produce Sensitive Test Since it was believed that the insensitivity of the test device, produced in portion A, to 1-00 mg/dL
of ethanol might be due to the indicator, o-dianisi-dine, used in solution assays, two further experiments were performed. In the first, (I), the concentrations of o-dianisidine and alcohol oxidase were increased further; in the second, ~II), a more sensitive indica-tor, 3,3',5,5'-tetramethylbenzidine (TMB) was substi-tuted.
o-dianisidine-2HC1 45 ml (0.1% in 0.lM phosphate, pH 7.5) POD, (12 mg/ml) 0.5 ml AOD (250 IU/ml) 5.0 ml first dip 0.05 M TMB
Aerosol~ O.T. in toluene second dip 0.1 phosphate, pH 7.5 4.5 ml POD (12 mg/ml) 0.5 ml AOD (500 IU/ml) 1.0 ml water 4.0 ml Composition I was incorporated onto Whatman 3~
paper by dipping. The paper immediately turned light brown, a color which should have been an indication of the presence of ethanol. The impregnated paper was dried 15 minutes at 60 in an air oven. The brown color continued to develop and became more intense with time. When the dried paper was contacted with an aqueous sample containing lO0 mg/dL of ethanol, in less than 5 minutes the color was discernably different from the false positive color. Therefore, the strips exhibited sufficient sensitivity, but their false positive reaction made them unsuitable for use.
Composition II was incorporated into Whatman 3~
paper by a two-step impregnation with drying in the air oven between impregnations. The paper was first dipped in the TMB, Aerosol~ O.T., toluene solution and dried 3 minutes at 60C. After dipping in the second solution containing alcohol oxidase, the strips developed an intense blue color. The intensity of the color diminished somewhat on drying, but the dried doubly impregnated paper was still a pronounced blue-green. Although the strip exhibited a discernable color change in less than 5 minutes when contacted with an aqueous sample containing lO0 mg/dL ethanol, the false positive color seen in uncontacted strips ~made the formulation unsuitable for use.
Both strip formulations also exhibit~d a small increase in color when contacted with water.
'7 C: Single Chromogenic Component Whatman 3MM paper was incorporated with the test composition including an azide, peroxidase and 3,3',5,5'-tetramethyl~enzidene ~TMB) as the chromogenic indi-cator system. The chromogenic indicator, TMs, wasincorporated first by dipping the paper into a solu-tion of TMB in toluene containing 0.5% Aerosol OT.
The paper was then dried in an air oven at ~0C for 10 minutes.
The dried paper was then dipped into a solution containing:
polyvinylpyrolidone (IC-30) 2.0 mL
15% in water Emulphor~ ON 870 5% in water 2.0 mL
phosphate buffer 0.5M, pH 8.5 2.0 mL
water 2.25 mL
sodium azide, 0.03M 0.25 mL
POD, 12 mg/mL 0.5 mL
AOD, 400 IU/mL 1.0 mL
The doubly incorporated paper was dried 15 min-utes at 60C. This paper exhibited no cclor (false positive) prior to contact with an ethanol containing sample.
A 0.5 x 0.5 cm piece of the doubly dried and incorporated paper was affixed to support member formed by an elongated piece of rigid nonreactive material such as polystyrene which will then act as a handle.
This formulation is the best mode known to the inventors for preparing a test device sensitive to at least 100 mg/dL ethanol in an aqueous test sample.
1~'5~
The test device reacts with this sensitivity to provide a detectable colorimetric response in one to two minu-tes.
D: Test Device Without Azide Whatman 31 ET paper was first dipped into a solution containing peroxidase, alcohol oxidase and one component of a coupled chromogenic indicator and dried 15 minutes at 60C in an air oven. The dried paper was then dipped in solution 2 and dried 5 minu-tes at 60C.
Solution 1 POD
~3 mg/mL in 1.0 M sodium phosphate, pH 7.5) 4.0 mL
Emulphor~ ON 870, (5~ in water) 4.0 mL
AOD
(850 IU/mL 3.5 mL
DHSA ~0.2 M in water) 2.0 mL
water 6.5 mL
Solution 2 4-aminoantipyrine, 0.02 M, in toluene.
When a device is prepared without the addition of an azide, the paper exhibits a slight coloration However, the components remain substantially in the reduced (uncolored) form in the final device, the device is sensitive to at least 100 mg/dL ethanol in an aqueous te~t sample and when contacted with such a sample, the device exhibits a detectable . 30 colorimetric response in one to two minutes. Such a device is particularly suitable for a presumptive test for ethanol wherein the presence of ethanol would be indicated by a strong "positive" color.
E: Separation of Chromogenic Components Plus Azide Although a solid phase ethanol device can be prepared as shown in portion D, even when using coupled chromogenic indicators which can be separated for incorporation, it is preferred to include an azide.
Whatman 31 ET paper was dipped in Solution 1 which includes sodium azide and dried in an air oven 15 minutes at 50C. The dried paper was then dipped in Solution 2 and dried. The doubly dried and in-corporated paper can be cut and affixed to one end of a support member formed by an elongated plastic handle for convenient handling.
Solution 1 polyvinyl pyrolidone (15% in water) 2.0 mL
POD
~12 mg/mL in l.OM
sodium phosphate, pH 7.5) 0.5 mL
Emulphor~ ON 870 (5% in water) 2.0 mL
AOD
~800 IU/mL) 3.0 mL
25 ` DHSA (0.lM) 2.0 mL
sodium azide (0.02M in water) 0.5 mL
Solution 2 0.02M MBTH base in toluene.
lZ`~ '7 Devices prepared in thls manner e~ibit essen-tially no coloration prior to contact with an ethanol containing sample and can be used for either a pre-sumptive test or for quantitation of the ethanol present in the sample.
F: Polymer Film Carrier Matrix A test device having the specified characteristics can also be prepared using a polymer film as the carrier matri~ using the film formulation presented below:
First layer Late~ 2.0 mL
TMB 2HCl lS7 mg Avicel RC 591-F 3 gm Water 8.0 mL
Second la~er Phosphate, 0.SM, pH 7.5 2.0 mL
POD, 10 mg/mL 0.5 mL
Sodium a ide, (8mM)0.5 mL
Emulphor~ ON 870, (30%) 1.0 mL
Late~ 2,0 mL
AOD, 500 IU/mL1.0 mL
Water 3.0 mL
Avicel RC 591-F3.0 gm The late~ is Polysar ~E 46S, a 60:40 styrene-butadiene copolymer containing 50O solids, obtained from Polysar Incorporated, Monaca, PA.
MS-13;7 * Trade Mark Avicel RC 5~1-F is a microcrystalline cellulose ob-tained from FMC Corp., Food and Pharmaceutical pro-ducts Div., Philadelphia, PA.
The film is applied in two layers using a doctor blade. A preferred film thickness is about 30 to 40 microns giving a dry film thickness of about 25-35 microns. However, the thickness of the film is not critical to the performance of the device. The film can be air dried or dried in an air oven at 60C for lO minutes.
G: Phase Inversion Membranous Film as the Carrier Matrix A solid state test device which is sensitive to at least 100 mg/dL ethanol and provides a colorimetric response in less than 5 minutes can also be prepared using phase inversion membraneous films.
A white hydrophilic membrane is prepared as follows:
With high speed stirring Solution 2 is slowly added (dropwise) to Solutionl. The "cloud point"
will be reached upon completion of addition. To this add slowly with good stirring 2.0 grams Cab-o-sil.
Using a doctor blade case the material at 505 ' mL thickness on clear Trycite support member.
Dry the film at 75C for 8-10 minutes.
.
Solution l Cellulose acetate (viscosity 45) 15% plus 1.5% KP-140 in acetone 36.0 gm 15% SMA 2625A plus 1.5%
KP-140 in acetone 4.0 gm Toluene 2.0 mL
Solution 2 Sorbitol ~60%~ 1.8 mL
Sodium azide (0.03 M) 1.8 mL
Emulphor~ ON 870 ~10%) l.Q mL
S POD (20 mg/dL) O.S mL
AOD (500 IU/mL) 1.8 mL
2-propanol 6.0 mL
Phosphate buffer, pH 7.5, 0.05M 11.2 mL
Cellulose acetate ~viscosity 45) can be obtained from Eastman Chemical Products, Inc., Kingspoint, TN.
KP-140 is tributoxyethylphosphate, a plasticizer, obtained from FMC Corp., Industrial Chemical Group, Philadelphia, PA. SMA 2625A is a styrene-maleic an-hydride copolymer from Atlantic Richfield Co., Phila-delphia, PA. Cab-o-sil is a fused silica polymer ob-tained from Cabot Corp., Boston, MA.
The completed white, hydrophilic membrane is then impregnated with a chromogenic indicator system. One impregnating solution would be 0.05 M 3,3',5,5'-tetramethylbenzidine with 0.5% Aerosol~ OT in toluene.
Another impregnating solution would be 0.5% gum guaiac in chloroform ~CHC13). Either solution can be im-pregnated into the phase inverted film by roller application to the surface of the film at speeds of 10 ~o SO feet per minute. The wetted film is then dried at the flash point of the solvent.
Although the invention has been described with a certain degree of particularity, it is understood that the present disclosure has been made only by way of example and that numerous changes in the details can be resorted to without departing from the scope of the lnvent lon .
POD 24-3600 IU/mL 50-200 IU/mL
azide 0 mM to 5.0 mM 0.5 mM to 3.0 mM
Most preferably the concentration of alcohol oxidase for a saliva/serum test device is 40 to 70 IU/mL and for urine is 50-150 IU/mL. In any case, the concen-tration of the single chromogenic component or each of the couple chromogenic components will be about 0.01 to 0.2 M ~molar~, preferably about 0.025 to 0.1 M.
The buffer should provide a pH of from about 5 to 9, preferably from about 7 to 8. These concentrations ranges and relative concentrations of components are viable whether the solution is an aqueous impregnating solution or a polymer suspension.
D. Method of Use The test device is advantageously used by momen-tarily dipping it in a test sample or by otherwise introducing a test sample onto the carrier iilatrix, whereby a de~ectable colorimetric change results when 100 mg/dL ethanol is present. Contact with the test sample can also be made by pipette, swab or spatula.
Although dipping is a highly satisfactory method of contact when urine is used, a blood sample will nor-mally require pipetting and the latter methods can be useful in testing saliva.
The following examples describe experiments which were performed in developing the present invention.
While the examples serve to illustrate the invention, they are not to be interpreted as limiting its scope which is defined solely by the claims appended hereto.
One skilled in the art will be able to make such variations, substitutions and changes in the com-ponents of the composition and ingredients and reac-tion parameters as may seem desirable.
EXA~PLES
Alcohol oxidase (AOD, EC 1.1.3.13) was obtained from Phillips Chemical Co., Bartlesville, OK. The activity of the alcohol oxidase used is given in International Units per milliliter (IU/mL) of stock solution. One International Unit (IU) of activity catalyzes the formation of 1 micromole aldehyde and hydrogen peroxide in an air-saturated solution at pH
7.5, 25C. The peroxidase used was horseradish per-oxidase obtained from Miles Laboratories, Inc., Kankakee, Ill.. with an activity of 130 IU/mg.
Aerosol~ OT is the trademark used by Aldrich Chemical Co., Milwaukee, WI, for dioctyl sodium sul-fosuccinate. Emulphor~ ON 870 is the trademark used by GAF, New York, NY. The other reagents used are com-mercially available.
~IS-1337 t,~
The following ab~re~iations are used in the examples:
dL deciliter mL milliliters M molar mM millimolar C degrees centrigrade mg milligrams IU International Unit TMB 3,3',5,5'-tetramethylbenzidine DHSA 3,5-dichloro-2-hydroxybenzene sulfonic acid MBTH 3-methyl-2-benzothiazoline hydrazone POD peroxidase AOD alcohol oxidase Percentages used indicate weight in milligrams per 100 militers of aqueous solution (%w/v) unless otherwise indicated.
A. Direct Incorporation Solution Chemistry Attempts were made to incorporate the solution assay for ethanol, described by Phillips Chemical Co.
in a Biochemicals Technical Bulletin on Alcohol Oxi-dase, into a solid state test device. The assay follows the production of peroxide with an indicator system comprising a chromogenic indicator and per-oxidase.
1~41~7 t~hatman 3~ filter paper was dipped into a solu-tion using enzyme concentrations compara~le to those used in the Phillips assav ci.e., 5 IU/mL alcohol oxidase in the impregnating solution? and dried 10 minutes at 60~C in an air oven.
solution o-dianisidine 2HCl ~0.008%
in 0.1 M phosphate buffer, pH 7.5) 10 mL
P9D, 12 mg/mL 0.004 mL
AOD ~500 IU/mL) 0.1 mL
The carrier so incorporated was white in color. How-ever, when contacted with an aqueous test sample containing 100 mg/dL ethanol, there was na apparent color development, i.e., the test device produced by direct incorporation of the liquid assay reagents was not sufficiently sensitive to ethanol to produce a useful test device.
A second attempt was made to produce a test device based on the liquid assay of Phillips by in-creasing the concentrations of the en~ymes to provide a more sensitive test. Whatman 3~M filter paper was dipped in a solution containing about 10 times the concentration of alcohol oxidase and 100 times the concentration peroxidase used previously and dried 10 minutes at 60~C in an air oven.
solution:
o-dianisidine-2HCl (0.008o in 0.1l~
phosphate, pH 7.5) 8.5 ml POD, 12 mg/mL 0.5 ml AOD (500 IU/mL) l.O ml MS-13~7 * Trade Mark lI7 The dried carrier matrix developed a pale pink when contacted with either a blank ~water only~ or an aqueous test sample containing 100 mg/dL ethanol.
There was very little differentiation between the 0 and 100 mg/dL level of ethanol. Therefore the test device so prepared is still not sufficiently sensitive to ethanol. o-dianisidine is believed to be an in-sufficiently sensitive indicator to provide a test device for 100 mg/dL ethanol which develops a colori-metric response in less than 5 minutes.
A similar lack of sensitivity was seen when aPhillips assay which follows the aldehyde produced by the action of alcohol oxidase on ethanol. Test devices were prepared by dipping Whatman 3MM filter paper into Solution l or Solution 2, below. Each type was dried for 10 minutes at 60C in an air oven.
Solution 1 Solution 2 MBTH-HCl~0.04%) 2.0 ml 2.0 ml AOD (500 IU/mL) 0.05 ml 1.0 ml Ferric chloride (0,2% in 0.lN HCl) 8.0 ml 7.0 ml In Solution 2, the concentration of alcohol oxidase was increased 20 times over that in Solution l. Test devices prepared in either manner showed no reaction when contacted by an aqueous test sample containing 100 mg/dL ethanol. This result was not unexpected since the Phillips aldehyde based assay required one hour incubation of the ethanol with the assay solu-tion, making it unsuitable for a rapid dip-and-read test.
L'7 . - 20 -B: False Positive Produced if Solution Assay Reagent Concentration Increased to Produce Sensitive Test Since it was believed that the insensitivity of the test device, produced in portion A, to 1-00 mg/dL
of ethanol might be due to the indicator, o-dianisi-dine, used in solution assays, two further experiments were performed. In the first, (I), the concentrations of o-dianisidine and alcohol oxidase were increased further; in the second, ~II), a more sensitive indica-tor, 3,3',5,5'-tetramethylbenzidine (TMB) was substi-tuted.
o-dianisidine-2HC1 45 ml (0.1% in 0.lM phosphate, pH 7.5) POD, (12 mg/ml) 0.5 ml AOD (250 IU/ml) 5.0 ml first dip 0.05 M TMB
Aerosol~ O.T. in toluene second dip 0.1 phosphate, pH 7.5 4.5 ml POD (12 mg/ml) 0.5 ml AOD (500 IU/ml) 1.0 ml water 4.0 ml Composition I was incorporated onto Whatman 3~
paper by dipping. The paper immediately turned light brown, a color which should have been an indication of the presence of ethanol. The impregnated paper was dried 15 minutes at 60 in an air oven. The brown color continued to develop and became more intense with time. When the dried paper was contacted with an aqueous sample containing lO0 mg/dL of ethanol, in less than 5 minutes the color was discernably different from the false positive color. Therefore, the strips exhibited sufficient sensitivity, but their false positive reaction made them unsuitable for use.
Composition II was incorporated into Whatman 3~
paper by a two-step impregnation with drying in the air oven between impregnations. The paper was first dipped in the TMB, Aerosol~ O.T., toluene solution and dried 3 minutes at 60C. After dipping in the second solution containing alcohol oxidase, the strips developed an intense blue color. The intensity of the color diminished somewhat on drying, but the dried doubly impregnated paper was still a pronounced blue-green. Although the strip exhibited a discernable color change in less than 5 minutes when contacted with an aqueous sample containing lO0 mg/dL ethanol, the false positive color seen in uncontacted strips ~made the formulation unsuitable for use.
Both strip formulations also exhibit~d a small increase in color when contacted with water.
'7 C: Single Chromogenic Component Whatman 3MM paper was incorporated with the test composition including an azide, peroxidase and 3,3',5,5'-tetramethyl~enzidene ~TMB) as the chromogenic indi-cator system. The chromogenic indicator, TMs, wasincorporated first by dipping the paper into a solu-tion of TMB in toluene containing 0.5% Aerosol OT.
The paper was then dried in an air oven at ~0C for 10 minutes.
The dried paper was then dipped into a solution containing:
polyvinylpyrolidone (IC-30) 2.0 mL
15% in water Emulphor~ ON 870 5% in water 2.0 mL
phosphate buffer 0.5M, pH 8.5 2.0 mL
water 2.25 mL
sodium azide, 0.03M 0.25 mL
POD, 12 mg/mL 0.5 mL
AOD, 400 IU/mL 1.0 mL
The doubly incorporated paper was dried 15 min-utes at 60C. This paper exhibited no cclor (false positive) prior to contact with an ethanol containing sample.
A 0.5 x 0.5 cm piece of the doubly dried and incorporated paper was affixed to support member formed by an elongated piece of rigid nonreactive material such as polystyrene which will then act as a handle.
This formulation is the best mode known to the inventors for preparing a test device sensitive to at least 100 mg/dL ethanol in an aqueous test sample.
1~'5~
The test device reacts with this sensitivity to provide a detectable colorimetric response in one to two minu-tes.
D: Test Device Without Azide Whatman 31 ET paper was first dipped into a solution containing peroxidase, alcohol oxidase and one component of a coupled chromogenic indicator and dried 15 minutes at 60C in an air oven. The dried paper was then dipped in solution 2 and dried 5 minu-tes at 60C.
Solution 1 POD
~3 mg/mL in 1.0 M sodium phosphate, pH 7.5) 4.0 mL
Emulphor~ ON 870, (5~ in water) 4.0 mL
AOD
(850 IU/mL 3.5 mL
DHSA ~0.2 M in water) 2.0 mL
water 6.5 mL
Solution 2 4-aminoantipyrine, 0.02 M, in toluene.
When a device is prepared without the addition of an azide, the paper exhibits a slight coloration However, the components remain substantially in the reduced (uncolored) form in the final device, the device is sensitive to at least 100 mg/dL ethanol in an aqueous te~t sample and when contacted with such a sample, the device exhibits a detectable . 30 colorimetric response in one to two minutes. Such a device is particularly suitable for a presumptive test for ethanol wherein the presence of ethanol would be indicated by a strong "positive" color.
E: Separation of Chromogenic Components Plus Azide Although a solid phase ethanol device can be prepared as shown in portion D, even when using coupled chromogenic indicators which can be separated for incorporation, it is preferred to include an azide.
Whatman 31 ET paper was dipped in Solution 1 which includes sodium azide and dried in an air oven 15 minutes at 50C. The dried paper was then dipped in Solution 2 and dried. The doubly dried and in-corporated paper can be cut and affixed to one end of a support member formed by an elongated plastic handle for convenient handling.
Solution 1 polyvinyl pyrolidone (15% in water) 2.0 mL
POD
~12 mg/mL in l.OM
sodium phosphate, pH 7.5) 0.5 mL
Emulphor~ ON 870 (5% in water) 2.0 mL
AOD
~800 IU/mL) 3.0 mL
25 ` DHSA (0.lM) 2.0 mL
sodium azide (0.02M in water) 0.5 mL
Solution 2 0.02M MBTH base in toluene.
lZ`~ '7 Devices prepared in thls manner e~ibit essen-tially no coloration prior to contact with an ethanol containing sample and can be used for either a pre-sumptive test or for quantitation of the ethanol present in the sample.
F: Polymer Film Carrier Matrix A test device having the specified characteristics can also be prepared using a polymer film as the carrier matri~ using the film formulation presented below:
First layer Late~ 2.0 mL
TMB 2HCl lS7 mg Avicel RC 591-F 3 gm Water 8.0 mL
Second la~er Phosphate, 0.SM, pH 7.5 2.0 mL
POD, 10 mg/mL 0.5 mL
Sodium a ide, (8mM)0.5 mL
Emulphor~ ON 870, (30%) 1.0 mL
Late~ 2,0 mL
AOD, 500 IU/mL1.0 mL
Water 3.0 mL
Avicel RC 591-F3.0 gm The late~ is Polysar ~E 46S, a 60:40 styrene-butadiene copolymer containing 50O solids, obtained from Polysar Incorporated, Monaca, PA.
MS-13;7 * Trade Mark Avicel RC 5~1-F is a microcrystalline cellulose ob-tained from FMC Corp., Food and Pharmaceutical pro-ducts Div., Philadelphia, PA.
The film is applied in two layers using a doctor blade. A preferred film thickness is about 30 to 40 microns giving a dry film thickness of about 25-35 microns. However, the thickness of the film is not critical to the performance of the device. The film can be air dried or dried in an air oven at 60C for lO minutes.
G: Phase Inversion Membranous Film as the Carrier Matrix A solid state test device which is sensitive to at least 100 mg/dL ethanol and provides a colorimetric response in less than 5 minutes can also be prepared using phase inversion membraneous films.
A white hydrophilic membrane is prepared as follows:
With high speed stirring Solution 2 is slowly added (dropwise) to Solutionl. The "cloud point"
will be reached upon completion of addition. To this add slowly with good stirring 2.0 grams Cab-o-sil.
Using a doctor blade case the material at 505 ' mL thickness on clear Trycite support member.
Dry the film at 75C for 8-10 minutes.
.
Solution l Cellulose acetate (viscosity 45) 15% plus 1.5% KP-140 in acetone 36.0 gm 15% SMA 2625A plus 1.5%
KP-140 in acetone 4.0 gm Toluene 2.0 mL
Solution 2 Sorbitol ~60%~ 1.8 mL
Sodium azide (0.03 M) 1.8 mL
Emulphor~ ON 870 ~10%) l.Q mL
S POD (20 mg/dL) O.S mL
AOD (500 IU/mL) 1.8 mL
2-propanol 6.0 mL
Phosphate buffer, pH 7.5, 0.05M 11.2 mL
Cellulose acetate ~viscosity 45) can be obtained from Eastman Chemical Products, Inc., Kingspoint, TN.
KP-140 is tributoxyethylphosphate, a plasticizer, obtained from FMC Corp., Industrial Chemical Group, Philadelphia, PA. SMA 2625A is a styrene-maleic an-hydride copolymer from Atlantic Richfield Co., Phila-delphia, PA. Cab-o-sil is a fused silica polymer ob-tained from Cabot Corp., Boston, MA.
The completed white, hydrophilic membrane is then impregnated with a chromogenic indicator system. One impregnating solution would be 0.05 M 3,3',5,5'-tetramethylbenzidine with 0.5% Aerosol~ OT in toluene.
Another impregnating solution would be 0.5% gum guaiac in chloroform ~CHC13). Either solution can be im-pregnated into the phase inverted film by roller application to the surface of the film at speeds of 10 ~o SO feet per minute. The wetted film is then dried at the flash point of the solvent.
Although the invention has been described with a certain degree of particularity, it is understood that the present disclosure has been made only by way of example and that numerous changes in the details can be resorted to without departing from the scope of the lnvent lon .
Claims (28)
1. A test device for determining the presence of ethanol in an aqueous test sample, the test device comprising a carrier matrix incorporated with alcohol oxidase, a peroxidatively active substance and a chromogenic indicator system capable of providing a detectable response wherein a chromogenic component of the indicator system is substantially in the reduced form, the alcohol oxidase being present in a quanity sufficient to provide a detectable colorimetric response to the presence of 100 mg/dL ethanol in the test sample in less than about 5 minutes.
2. The test device of claim 1 in which the amount of alcohol oxidase present is that amount re-sulting from the incorporation of the carrier with a solution containing at least 10 IU/mL alcohol oxidase and drying.
3. The test device of claim 1 in which the car-rier matrix is paper.
4. The test device of claim 1 in which the carrier matrix is additionally incorporated with an azide.
5. The test device of claim 1 in which the carrier matrix is additionally incorporated with a buffering substance capable of providing a pH in the range of from about 5 to 9.
6. The test device of claim 1 in which the carrier matrix is additionally incorporated with a stabilizing agent.
7. The test device of claim 6 in which the stabilizing agent is sorbitol.
8. The test device of claim 1 in which the peroxidatively active substance is peroxidase.
9. The test device of claim 4 in which the chromogenic indicator system is a single chromogenic component chosen from gum guaiac, o-tolidine, 3,3',5,5'-tetramethylbenzidine or mixtures thereof.
10. The test device of claim 1 in which the chromogenic indicator system comprises the coupled chromogenic components: 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminoantipyrine; 3,5-dichloro-2-hydroxybenzene sulfonic acid/3-methyl-2-benzothia-zoline hydrazone; m-anisidine/4-aminoantipyrine; or mixtures thereof.
11. The test device of claim 10 in which the carrier matrix is additionally incorporated with an azide.
12. A method for preparing the test device of claim 1 which comprises the steps of:
a) preparing a first mixture of one component of a coupled chromogenic indicator system and an organic solvent;
b) preparing an aqueous second mixture of a peroxidatively active substance, and any re-maining components of the chromogenic indica-tor system, and alcohol oxidase;
c) incorporating the carrier matrix with one of the first or second mixtures and drying;
and d) incorporating the carrier with the other of the first or second mixtures and drying.
a) preparing a first mixture of one component of a coupled chromogenic indicator system and an organic solvent;
b) preparing an aqueous second mixture of a peroxidatively active substance, and any re-maining components of the chromogenic indica-tor system, and alcohol oxidase;
c) incorporating the carrier matrix with one of the first or second mixtures and drying;
and d) incorporating the carrier with the other of the first or second mixtures and drying.
13. The method of claim 12 in which the aqueous second mixture contains at least 10 IU/mL
alcohol oxidase.
alcohol oxidase.
14. The method of claim 12 in which an azide is additionally included in the aqueous second mixture.
15. The method of claim 12 in which a buffering substance capable of providing a pH in the range of from about 5 to 9 is additionally included in the aqueous second mixture.
16. The method of claim 12 in which a stabiliz-ing agent is additionally included in the aqueous second mixture.
17. The method of claim 16 in which the sta-bilizing agent is sorbitol.
18. A method for preparing a test device of claim 1 which comprises the steps of:
a) preparing a first mixture of a chromogenic component and a latex polymer, b) preparing a second mixture of a peroxida-tively active substance, an azide, alcohol oxi-dase, any remaining components of the chromo-genic indicator system and a latex polymer, c) applying the first or second mixture on a support member;
d) drying e) applying the other of the first or second mixture on the support member; and f) drying.
a) preparing a first mixture of a chromogenic component and a latex polymer, b) preparing a second mixture of a peroxida-tively active substance, an azide, alcohol oxi-dase, any remaining components of the chromo-genic indicator system and a latex polymer, c) applying the first or second mixture on a support member;
d) drying e) applying the other of the first or second mixture on the support member; and f) drying.
19. The method of claim 18 in which the second mixture contains at least 10 IU/mL alcohol oxidase.
20. A method of preparing a test device of claim 1 which comprises the steps of:
a) preparing a phase inversion polymer film from solutions containing alcohol oxidase and a peroxidatively active substance, b) impregnating the phase inversion polymer film with a solution containing a chromogenic indicator and an organic solvent.
a) preparing a phase inversion polymer film from solutions containing alcohol oxidase and a peroxidatively active substance, b) impregnating the phase inversion polymer film with a solution containing a chromogenic indicator and an organic solvent.
21. The method of claim 20 in which a solution used to prepare the phase inversion polymer film contains at least 10 IU/mL alcohol oxidase.
22. The method of claim 21 in which a solution used to prepare the phase inversion polymer film additionally contains an azide.
23. A method for preparing the test device of claim 10 which comprises the steps of:
a) preparing a first mixture of the single chromogenic component in an organic solvent;
b) preparing an aqueous second mixture of a peroxidatively active substance, alcohol oxi-dase and an azide;
c) incorporating the carrier matrix with the first mixture and drying; and d) incorporating the carrier matrix with the second mixture and drying.
a) preparing a first mixture of the single chromogenic component in an organic solvent;
b) preparing an aqueous second mixture of a peroxidatively active substance, alcohol oxi-dase and an azide;
c) incorporating the carrier matrix with the first mixture and drying; and d) incorporating the carrier matrix with the second mixture and drying.
24. The method of claim 23 in which the aqueous second mixture contains at least 10 IU/mL of alcohol oxidase.
25. The method of claim 23 in which a buffering substance capable of providing a pH in the range of from about 5 to 9 is additionally included in the aqueous second mixture.
26. The method of claim 23 in which a stabiliz-ing agent is additionally included in the aqueous second mixture.
27. The method of claim 26 in which the stabi-lizing agent is sorbitol.
28. A process for determining the presence of ethanol in an aqueous test sample, the process com-prising contacting the test sample with the test device of claim 1 and observing any detectable colori-metric response in less than 5 minutes.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US616,732 | 1975-09-25 | ||
| US06/616,732 US4810633A (en) | 1984-06-04 | 1984-06-04 | Enzymatic ethanol test |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1254117A true CA1254117A (en) | 1989-05-16 |
Family
ID=24470741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000480608A Expired CA1254117A (en) | 1984-06-04 | 1985-05-02 | Enzymatic ethanol test |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4810633A (en) |
| EP (1) | EP0164008B1 (en) |
| JP (1) | JPS60262598A (en) |
| AT (1) | ATE51028T1 (en) |
| AU (1) | AU560448B2 (en) |
| CA (1) | CA1254117A (en) |
| DE (1) | DE3576528D1 (en) |
| DK (1) | DK249085A (en) |
| FI (1) | FI852225L (en) |
| IL (1) | IL75065A (en) |
| NO (1) | NO852070L (en) |
| ZA (1) | ZA853430B (en) |
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| JPS6348457A (en) * | 1986-08-19 | 1988-03-01 | Fuji Photo Film Co Ltd | Dry-type multi-layered analytical element |
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| EP0357027B1 (en) * | 1988-08-30 | 1995-05-10 | New Oji Paper Co., Ltd. | Alcohol oxidase enzyme electrode and its use for quantitative alcohol determination. |
| US5508171A (en) * | 1989-12-15 | 1996-04-16 | Boehringer Mannheim Corporation | Assay method with enzyme electrode system |
| DE69020908T2 (en) * | 1989-12-15 | 1996-02-15 | Boehringer Mannheim Corp | REDOX MEDIATION REAGENT AND BIOSENSOR. |
| US5532138A (en) * | 1990-04-26 | 1996-07-02 | Behringwerke Ag | Method and kits for determining peroxidatively active catalysts |
| US5141854A (en) * | 1990-12-13 | 1992-08-25 | Hoffman-La Roche, Inc. | Enzymatic composition for ethanol assay |
| CA2081870A1 (en) * | 1991-11-15 | 1993-05-16 | Robert Bauer | Detection of analytes in saliva using peroxide-peroxidase test systems |
| US5332662A (en) * | 1992-07-31 | 1994-07-26 | Syntex (U.S.A.) Inc. | Methods for determining peroxidatively active substances |
| US5416004A (en) * | 1993-01-19 | 1995-05-16 | Detwiler; Richard L. | Multilayer analytical element containing primary amine buffer and method for the determination of ethanol |
| US5429931A (en) * | 1993-05-24 | 1995-07-04 | Eastman Kodak Company | Multilayer analytical element containing crosslinked binder and method for the determination of ethanol |
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-
1984
- 1984-06-04 US US06/616,732 patent/US4810633A/en not_active Expired - Fee Related
-
1985
- 1985-05-01 IL IL75065A patent/IL75065A/en unknown
- 1985-05-02 CA CA000480608A patent/CA1254117A/en not_active Expired
- 1985-05-07 ZA ZA853430A patent/ZA853430B/en unknown
- 1985-05-18 DE DE8585106126T patent/DE3576528D1/en not_active Expired - Fee Related
- 1985-05-18 AT AT85106126T patent/ATE51028T1/en not_active IP Right Cessation
- 1985-05-18 EP EP85106126A patent/EP0164008B1/en not_active Expired - Lifetime
- 1985-05-23 NO NO852070A patent/NO852070L/en unknown
- 1985-05-24 AU AU42852/85A patent/AU560448B2/en not_active Ceased
- 1985-06-03 JP JP60118980A patent/JPS60262598A/en active Granted
- 1985-06-03 DK DK249085A patent/DK249085A/en not_active Application Discontinuation
- 1985-06-03 FI FI852225A patent/FI852225L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0164008A3 (en) | 1988-10-26 |
| ATE51028T1 (en) | 1990-03-15 |
| US4810633A (en) | 1989-03-07 |
| FI852225L (en) | 1985-12-05 |
| EP0164008A2 (en) | 1985-12-11 |
| AU560448B2 (en) | 1987-04-09 |
| DK249085A (en) | 1985-12-05 |
| JPS60262598A (en) | 1985-12-25 |
| IL75065A0 (en) | 1985-09-29 |
| DK249085D0 (en) | 1985-06-03 |
| AU4285285A (en) | 1985-12-12 |
| NO852070L (en) | 1985-12-05 |
| ZA853430B (en) | 1985-12-24 |
| DE3576528D1 (en) | 1990-04-19 |
| IL75065A (en) | 1989-09-10 |
| EP0164008B1 (en) | 1990-03-14 |
| FI852225A0 (en) | 1985-06-03 |
| JPH0586197B2 (en) | 1993-12-10 |
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| Date | Code | Title | Description |
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| MKEX | Expiry | ||
| MKEX | Expiry |
Effective date: 20060516 |