CA1225912A - Colorimetric ethanol analysis method and test device - Google Patents
Colorimetric ethanol analysis method and test deviceInfo
- Publication number
- CA1225912A CA1225912A CA000458581A CA458581A CA1225912A CA 1225912 A CA1225912 A CA 1225912A CA 000458581 A CA000458581 A CA 000458581A CA 458581 A CA458581 A CA 458581A CA 1225912 A CA1225912 A CA 1225912A
- Authority
- CA
- Canada
- Prior art keywords
- test device
- alcohol oxidase
- ethanol
- stabilized
- support pad
- 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
A disposable test strip device for detecting and measuring ethanol in aqueous solutions is dis-closed. The test strip includes an inert support pad that contains a stabilized dry form of the enzyme alcohol oxidase, a material having peroxidative acti-vity and an oxygen acceptor that reacts with hydrogen peroxide to give a compound of changed color. The use of this strip to determine ethanol levels colorimetri-cally is also disclosed.
A disposable test strip device for detecting and measuring ethanol in aqueous solutions is dis-closed. The test strip includes an inert support pad that contains a stabilized dry form of the enzyme alcohol oxidase, a material having peroxidative acti-vity and an oxygen acceptor that reacts with hydrogen peroxide to give a compound of changed color. The use of this strip to determine ethanol levels colorimetri-cally is also disclosed.
Description
COLORIMETRIC ETHANOL ~N~LYSIS
METHOD AND TEST DEVICE
Field of the Invention mis invention is in the field of enzyme-mediated colorimetric analysis. It concerns a colorimetric analysis method and tes~ device for determining low concentrations of ethanol in aqueous media, particularly in human body fluids.
Background of the Invention There is an increasing demand for a simple but accurate and reproducible method for determining relatively low concentrations e.g., 0.025, 0.05, 0.10 or 0.20% of ethanol in aqueous fluids. One common application of such a method could be to measure ethanol levels in human body fluids as a test for high-way intoxication or sobriety. Another would be to assure that operators of dangerous equipment, such as heavy construction equipment or military equipment are not intoxicated by alcohol. The chemical methods of choice for such tests include complex laboratory procedures such as gas chromatography for analyzing blood or urine, and a range of laboratory or field colorimetric tests. Since the late 1930's the "Drunkometer" of Stephenson Corporation has been in use. This colorimetric machine relies on the ability of ethanol in the breath to react with and discolor aqueous acidic potassium permanganate. In 1954 ~he Stephenson Corporation introduced the widely used "Breathalyzer" which relies on the decolorizing reac-tion of ethanol in the breath with acidic potassium dichromate to determine intoxication. These tests have a long history of use which has not been without difficulty and controversy. They require difficult to carry wet reagents, calibration and a reasonable level of operator competence to give good readings. They are not readily suited for rapid routine processing of large groups of samples.
Another application for the rapid determina-tion of blood alcohol levels is in the hospital emergency rooms. Approximately 1/3 of all patients currently admitted to hospital emergency rooms are tested for blood alcohol level for the purpose of making a correct judgment as to the nature of their clinical condition. Since no simple rapid method has previously existed for such a measurement current prac-tice is to take a blood sample by venipuncture and to hand carry it to the laboratory for a stat blood alcohol determination. This procedure takes from 30 minutes to a few hours.
The present invention employs the enzyme alcohol oxidase. This material was reported by Janssen et al in Biochem BioPhys Res Commun (US) 8 Sept 1965, 20, (5) p 630-4 and is presently sold by Boehringer Mannheim Biochemicals, Indianapolis, IN and Phillips Chemical, a subsididary of Phillips Petroleum.
Alcohol oxidase is a particularly unstable enzyme undergoing rapid deterioration and loss of activity. It is sold by Phillips as a solution in 70%
by weight sucrose. It is sold by Boehringer as a solid, stabilized with large amounts of the peroxide antagonist, reduced glutathione. This is not an accep~able form for this analysis which requires that hydrogen peroxide be generated and quantitated. The large amounts of peroxide antagonist would interfere with this. In addition, it has also been found that this enzyme has the interesting but difficult to deal with property of undergoing autoxidation, generating peroxide by reaction with itself.
This enzyme's use in electrode ethanol analysis systems has been reported in Chem Abstracts, 135580a, and is mentioned in USP 4,250,261 of Eggeling et al. Majkic-Singh and Berkes reported at Analy~ica Chemica Acta, 401-5 (1980) a method for determining ethanol in which the chromogen 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfona~e) (ABTS) was reacted with hydrogen peroxide generated by alcohol oxidase.
This prior method is carried out mea~uring the absorbance of solutions in a spectrophotometer in a laboratory setting. It is also characterized by careful handling of the sensitive alcohol oxidase in an effort to prevent deterioration or variation of results.
USP 4,430,427 of Hopkins also employs ethanol oxidase in a solution analysis setting.
What is now sought and provided by the present invention is a long-lived accurate and precise simple colorimetric method for determining ethanol concentrations in aqueous fluids such as human body fluids, in particular saliva and whole blood.
Statement of the Invention A device and method for detecting and measur-ing the ethanol in aqueous solution has now been found. The device is a disposable test strip which includes an inert support pad that contains a stabilized dry form of the enzyme alcohol oxidase, a material having peroxidative activity and an oxygen acceptor that reacts with hydrogen peroxide to give a compound of changed color.
When this device is contacted with an aqueous solution containing a measurable concentration of ethanol up to about 0.3% ethanol, at essentially ambient conditions, the ethanol reacts with oxygen and the alcohol oxidase to generate hydrogen peroxide (H202). The H202 reacts with the peroxidase and the oxygen acceptor to generate or consume colored compound in an amount proportional to the ethanol concentra-tion. The amount of colored compound is determined by measuring the color intensity. Color intensity can be determined using reflectance measurement such as a reflectance spectrophotometer or by comprison with standards. Thus, the device and method find ready application in at least two general areas of use.
In another aspect this invention provides a stabilized form of alcohol oxidase "stabilized alcohol oxidase" which is employed in the above-noted test device and method. "Stabilized alcohol oxidase" can be achieved by intimately combining alcohol oxidase with a stabilizing concentration of a solid aliphatic polyhy-droxyl compound having 5 to 8 carbons, and 5 to 7 hydroxyl groups, optionally with a saccharide and a chelating agent.
An additional and necessary aspect of this invention provides a neutralized form of alcohol oxidase in which the enzyme's tendency to autoxidize is eliminated or suppressed by admixture with a controlled amount of a peroxide scavenger such as ascorbic acid or cysteine.
In a further aspect, when this invention is employed to quantitate rather than qualitate ethanol, an additional controlled amount of a peroxide scavenger is admixed with the alcohol oxidase to give a more gradual color change.
~L2'~
Detailed Description of thQ Invention Brief Description of the Drawinqs The drawing contains four figures.
Fi~. 1 is an enlarged, not to scale perspec-tive view of a very simple embodiment of the device of this invention.
Figs. 2, 3 and ~ likewise are enlarged not to scale views of somewhat more complex embodiments of the device of this inventionO
The test device of this invention, in a very simple form is shown in Fig. 1 as a pad 10 made up of a hydrophilic absorbent material which can absorb the sample of aqueous material to be tested for ethanol.
The pad carries the enzyme systems and color-changing oxygen acceptor which react to generate the color change in response to ethanol. Pad 10 can be made of natural or synthetic absorptive materials. Pad 10 may be of any water-insoluble hydrophilic material includ-ing natural materials like cellulose, paper, cotton, silk, and cross-linked gelatin and synthetic materials such as cross-linked hydroxymethyacrylates and acrylates, cellulose, cellulose nitrate, cross-linked poly(vinyl alcohol), poly(vinylamine), poly(vinylsul-fonate) copolymers, poly(styrene sulfonate) and copolymers containing styrene sulfonate units, poly(vinyl acetate), poly(maleic anhydride), poly(vinyl
METHOD AND TEST DEVICE
Field of the Invention mis invention is in the field of enzyme-mediated colorimetric analysis. It concerns a colorimetric analysis method and tes~ device for determining low concentrations of ethanol in aqueous media, particularly in human body fluids.
Background of the Invention There is an increasing demand for a simple but accurate and reproducible method for determining relatively low concentrations e.g., 0.025, 0.05, 0.10 or 0.20% of ethanol in aqueous fluids. One common application of such a method could be to measure ethanol levels in human body fluids as a test for high-way intoxication or sobriety. Another would be to assure that operators of dangerous equipment, such as heavy construction equipment or military equipment are not intoxicated by alcohol. The chemical methods of choice for such tests include complex laboratory procedures such as gas chromatography for analyzing blood or urine, and a range of laboratory or field colorimetric tests. Since the late 1930's the "Drunkometer" of Stephenson Corporation has been in use. This colorimetric machine relies on the ability of ethanol in the breath to react with and discolor aqueous acidic potassium permanganate. In 1954 ~he Stephenson Corporation introduced the widely used "Breathalyzer" which relies on the decolorizing reac-tion of ethanol in the breath with acidic potassium dichromate to determine intoxication. These tests have a long history of use which has not been without difficulty and controversy. They require difficult to carry wet reagents, calibration and a reasonable level of operator competence to give good readings. They are not readily suited for rapid routine processing of large groups of samples.
Another application for the rapid determina-tion of blood alcohol levels is in the hospital emergency rooms. Approximately 1/3 of all patients currently admitted to hospital emergency rooms are tested for blood alcohol level for the purpose of making a correct judgment as to the nature of their clinical condition. Since no simple rapid method has previously existed for such a measurement current prac-tice is to take a blood sample by venipuncture and to hand carry it to the laboratory for a stat blood alcohol determination. This procedure takes from 30 minutes to a few hours.
The present invention employs the enzyme alcohol oxidase. This material was reported by Janssen et al in Biochem BioPhys Res Commun (US) 8 Sept 1965, 20, (5) p 630-4 and is presently sold by Boehringer Mannheim Biochemicals, Indianapolis, IN and Phillips Chemical, a subsididary of Phillips Petroleum.
Alcohol oxidase is a particularly unstable enzyme undergoing rapid deterioration and loss of activity. It is sold by Phillips as a solution in 70%
by weight sucrose. It is sold by Boehringer as a solid, stabilized with large amounts of the peroxide antagonist, reduced glutathione. This is not an accep~able form for this analysis which requires that hydrogen peroxide be generated and quantitated. The large amounts of peroxide antagonist would interfere with this. In addition, it has also been found that this enzyme has the interesting but difficult to deal with property of undergoing autoxidation, generating peroxide by reaction with itself.
This enzyme's use in electrode ethanol analysis systems has been reported in Chem Abstracts, 135580a, and is mentioned in USP 4,250,261 of Eggeling et al. Majkic-Singh and Berkes reported at Analy~ica Chemica Acta, 401-5 (1980) a method for determining ethanol in which the chromogen 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfona~e) (ABTS) was reacted with hydrogen peroxide generated by alcohol oxidase.
This prior method is carried out mea~uring the absorbance of solutions in a spectrophotometer in a laboratory setting. It is also characterized by careful handling of the sensitive alcohol oxidase in an effort to prevent deterioration or variation of results.
USP 4,430,427 of Hopkins also employs ethanol oxidase in a solution analysis setting.
What is now sought and provided by the present invention is a long-lived accurate and precise simple colorimetric method for determining ethanol concentrations in aqueous fluids such as human body fluids, in particular saliva and whole blood.
Statement of the Invention A device and method for detecting and measur-ing the ethanol in aqueous solution has now been found. The device is a disposable test strip which includes an inert support pad that contains a stabilized dry form of the enzyme alcohol oxidase, a material having peroxidative activity and an oxygen acceptor that reacts with hydrogen peroxide to give a compound of changed color.
When this device is contacted with an aqueous solution containing a measurable concentration of ethanol up to about 0.3% ethanol, at essentially ambient conditions, the ethanol reacts with oxygen and the alcohol oxidase to generate hydrogen peroxide (H202). The H202 reacts with the peroxidase and the oxygen acceptor to generate or consume colored compound in an amount proportional to the ethanol concentra-tion. The amount of colored compound is determined by measuring the color intensity. Color intensity can be determined using reflectance measurement such as a reflectance spectrophotometer or by comprison with standards. Thus, the device and method find ready application in at least two general areas of use.
In another aspect this invention provides a stabilized form of alcohol oxidase "stabilized alcohol oxidase" which is employed in the above-noted test device and method. "Stabilized alcohol oxidase" can be achieved by intimately combining alcohol oxidase with a stabilizing concentration of a solid aliphatic polyhy-droxyl compound having 5 to 8 carbons, and 5 to 7 hydroxyl groups, optionally with a saccharide and a chelating agent.
An additional and necessary aspect of this invention provides a neutralized form of alcohol oxidase in which the enzyme's tendency to autoxidize is eliminated or suppressed by admixture with a controlled amount of a peroxide scavenger such as ascorbic acid or cysteine.
In a further aspect, when this invention is employed to quantitate rather than qualitate ethanol, an additional controlled amount of a peroxide scavenger is admixed with the alcohol oxidase to give a more gradual color change.
~L2'~
Detailed Description of thQ Invention Brief Description of the Drawinqs The drawing contains four figures.
Fi~. 1 is an enlarged, not to scale perspec-tive view of a very simple embodiment of the device of this invention.
Figs. 2, 3 and ~ likewise are enlarged not to scale views of somewhat more complex embodiments of the device of this inventionO
The test device of this invention, in a very simple form is shown in Fig. 1 as a pad 10 made up of a hydrophilic absorbent material which can absorb the sample of aqueous material to be tested for ethanol.
The pad carries the enzyme systems and color-changing oxygen acceptor which react to generate the color change in response to ethanol. Pad 10 can be made of natural or synthetic absorptive materials. Pad 10 may be of any water-insoluble hydrophilic material includ-ing natural materials like cellulose, paper, cotton, silk, and cross-linked gelatin and synthetic materials such as cross-linked hydroxymethyacrylates and acrylates, cellulose, cellulose nitrate, cross-linked poly(vinyl alcohol), poly(vinylamine), poly(vinylsul-fonate) copolymers, poly(styrene sulfonate) and copolymers containing styrene sulfonate units, poly(vinyl acetate), poly(maleic anhydride), poly(vinyl
2-methoxyethyl ether), poly(4-vinyl phthalic acid), poly(N-vinylmorpholinone), poly(N-vinylpyrrolidone), poly(methacrylic acid), poly(acrylamide), poly(metha-crylamide), poly(ethylene oxide) and the like. The hyrophilic material may be a gel layer, porous or fibrous, or the like. Cellulose and cellulose deriva-tives are readily available, work fine and thus arepreferred hydrophilic substrates. An aqueous li~uid sample is applied to pad 10. Substrate pad 10 is sized to be smaller in volume than the sample which is applied to it. If it is larger or similar in volume to the sample, there are problems with the sample pad "chromatographing" the sample or "spreading" the sample and giving an uneven result. As depicted in Fig. 1, pad 10 is not necessarily drawn to scale. Usually it is from about 0.01 mm to 0.5 mm thick, most commonly 0.05 to 0.30 mm thick.
In the application of this device to screen humans for intoxication, the aqueous sample is whole blood, saliva or urine. In the case of blood, a 5~20 microliter volume from a minimally traumatic "finger-prick" is very preferably used. Generally, when fresh whole blood is the sample being tes~ed the test pad should be on the order of 10 mm2 to 100 mm2 in area, especially 10 mm2 to 50 mm2 in area. The sample pad contains the enzymes and chemical reagents which will react with any ethanol to produce a compound of changed color especially of an intense blue color. These will be described separately, below.
Turning to Fig. 2 a second embodiment of the device of this invention is shown. In this embodiment, pad 10 as described, is affixed to handle lOa. ~andle lOa can be the same material as pad 10, without the reagents or it can be a different material such as will be described as a backing with reference to Fig. 3.
In Fig. 3, another embodiment of the device of this invention is shown to comprise absorbent pad 10 affixed to backing 11. Backing 11 is preferably of a material that does not absorb the aqueous sample. It can be made of plastic, wood, hydrophobic paper 5~
products such as water-repellant-treated board stock, heavy paper, or the like. In one special embodiment, backing 11 and pad 10 are chosen so that pad 10 is at least partially transparent or trans-lucent and backing 11 presents a diffuse reflective surface.
In Fig. 4, yet another embodiment of -the present invention is shown in which pad 10 is overcoated with a semipermeable transparent "membrane" 12. This membrane is permeable to small molecules like water and ethanol but is impermeable to whole blood cells and similar large molecules. Such membranes are disclosed in the art of blood glucose measuring devices (See USP 4,211,845 of Genshaw and USP 3,298,789 of Mast). This is the preferred method for quantitation of ethanol in whole blood. Ethyl cellulose has been used in that application and works in the present case.
Other equivalent materials such as cellulose acetate, cellulose proprionate, polyvinylacetate and polymethylmethacrylate can be employed, as well. This membrane is of advantage when alcohol levels are being detected in a fluid like blood which is colored by large bodies (i.e., whole blood cells). The membrane keeps the colored cells out of the reagent pad. Prior to determining the color change they can be washed or wiped away and thus do not interfere with the color change in the reagent pad.
Fig. 4's depiction of membrane 12 illustrates one possible form of such a membrane and makes it easy to see how the membrane works. However, in the most common embodiments this over-coating layer is applied to the pad as a solution or "varnish" in a water impermeable organic solvent like toluene, benzene or the like which is dried. Electron micrographs of materials formed in this manner reveal that the over-coating layer is not exactly the discrete unit of Fig.
4 but rather coats the individual fibers making up pad 10. Both embodiments work the see and are included within the definition of membrane 12.
Optionally, pad 10 including coating 12 is covered by a protective cover that is not shown. This cover is removed in use but is removabl~ sealed around pad 11 prior to use. This cover can be made of foil, waterproofed paper, or water-oxygen and light-impermeable protective plastic such as poly(ethylene) or poly(vinylchloride) or the like and is provide to protect the enzymes and chemicals in pad 10 from degradation prior to the device's use in testing.
Pad 10 contains a reagent system. The reagent system is made up of stabilized alcohol oxidase, a peroxidase and a color-changing oxygen acceptor. It may contain other materials as well.
"Stabilized alcohol oxidase" is defined to mean alcohol oxidase which retains at least 50% of its activity and preferably at least 70% of its activity when stored in dry form at 56~C for 15 days. With preferred embodi-ments of this invention a "Stabilized alcohol oxidase"
can be attained which retains at least 90% of its activity under these test conditions. The stabilized alcohol oxidase is an important aspect of this inven-tion and in its simplest form comprises alcohol oxidase in intimate admixture with an effective stabilizing concentration of a solid aliphatic compound with 5 to 8 carbons and 5 to 7 hydroxyl groups. Preferably the stabilized alcohol oxidase is buffered to an essen-tially neutral pH and also includes a chelating agent, especially an amine polyacid. In addition, the stabilized alcohol oxidase optionally include~ a saccharide.
The alcohol oxidase is commercially available from two sources, Boehringer Mannheim Biochemicals (BMB) and Phillips Chemical- Neither supplier, how-ever, has been able to develop a form of the en7yme suitable for the applications described herein. BMB
offers the enzyme in a dry form which is stabilized with high levels of reduced glutathione, a peroxide scavenger. This form of the enzyme cannot be used for analytical applications based on peroxide chemistry because the high levels of glutathione interfere.
Phillips Chemical has not been able ~o develop a stable dry form of the enzyme and thus offers only solution~
of the enzyme which are shipped and stored in a frozen state. These solutions, however, can be converted to a stable, dry form using the methods described herein.
The amount of enzyme used is conventionally expressed in units of enzyme activity, i.e., 500 units etc.
Commerical solutions, when fresh generally contain about 1000 units/ml.
The solid aliphatic polyhydroxyl compound employed in the stabilized alcohol oxidase has 5 to 8 carbons and 5 to 7 hydroxyls. Preferably it has 5 or 6 carbons. Also preferably it is nonhygroscopic.
Preferred of these materials are the solid polyhydric alcohols (polyols) having from 5 to ~ carbons and 5 to 7 hydroxyl groups, preferably 5 or 6 carbons. Examples of such materials are mannitol, sorbitol, blucitol and inositol. Mannitol is the preferred polyhydric alcohol.
~L~
The chelating agent is a material that can form a chelate complex with metal ions. Amine polyacids such as, ethylenediamine tetraacetic acid (EDTA) or ethylenediamine pentacetic acid, propylene 1,3-diamine tetraacetic acid and the like and especially 2 to 4 carbon alkyl-polyamine polyacids containing from 2 to 3 amine groups and from 4 to 7 aliphatic acid groups, especially acetic acid groups, are preferred. EDTA is the most preferred chelating agent because of ready availability.
Preferably, however, the buffer employed is a zwitterion buffer, preferably a Good's buffer, especially a morpholino sulfonic acid or piperazine sulfonic acid. Examples of these materials are 3-(N-morpholino)propanesulfonic acid (acronym "MOPS"); N-2-hydroxy ethylpiperzine-N'-2-ethanesulfonic acid ("HEPES"); 2-(N-morpholino)ethanesulfonic acid ("MES"); and piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"). These materials are sold as free acids and as salts. It is possible to use either form or in a combination of forms adjusting the pH as needed with acid or base. These particular buffers offer two advantages (1) the alcohol oxidase is more stable in their pres-ence and (2) the alcohol oxidase is more easily soluble in thesebuffers than in phosphate buffers or the like.
During development of this invention it was separately determined that 3-(N-morpholino) propane sulfonic acid "MOPS"
marketed in the USA by Research Organics of Cleveland, Ohio is a particularly useful buffer component.
Optionally, the stabilized enzyme system additionally contains a saccharide. One commercial preparation of alcohol oxi-dase contains a ~r~
-IOa-substantial amount of sucrose. Other similar mono or di or tri saccharides may be added as well. These include galactose, :Eructose, maltose, çellobiose and raffinose, for example.
A stabilizing amount of polyhydroxyl compound is present with the alcohol oxidase. A "stabilizing amount" or "stabilizing concentration"
1'~ an amount sufficient to achieve a "stabilized alcohol oxidase~ as that term is herein defined. In general, such amounts involve a weight excess of the polyhydroxyl compound over the alcohol oxidase. As a guide, usually from about 0.3 to 2.5 grams of polyhydroxyl compound per 1000 units of enzyme are employed, preferably 0.4 to 1.2 grams of poly hydroxyl compound/1000 units alcohol oxidase.
The amount of chelating agent generally ranges from about 2 to about 50 mg/1000 units with use levels of about 4 to about 25 mg/1000 units being preferred.
When the optional saccharide is present it is usually present in up to about a 2 fold weight excess, based on the poly (hydroxyl) compound. Mixtures of two or more of any of the families of materials can be used in the stabilized enzyme systems, if desired.
By way of example, Table I lists a group of representative stabilized enzyme systems useful in the present invention. These systems are expressed in parts by weight based on the weight of dry alcohol oxidase present in the dry chemical mixture in the finished device.
~LX25912 TABLE I
Alcohol Oxidase 1000 units Poly hyroxyl compound 300-2500 mg Chelating agent 2-50 mg Buffer to pH 6-9 Alcohol Oxidase 1000 units Mannitol and/or Sorbitol 400-2000 mg Alkylpolyamine polyacid 2-50 mg Phosphate Buffer to pH 7-8 Alcohol Oxidase 1000 units Mannitol 400-2000 mg EDTA 2-5 mg Phosphate Buffer to pH 7-8 Alcohol Oxidase 1000 units Mannitol 400-2000 mg EDTA 2~50 mg Saccharide 400-2000 mg Buffer to pH 7-8 It will be readily understood by those skilled in the art that one does not depart from the teachings of this invention by incorporating inert materials and the like into the stabilized enzyme system. Gen-erally, however, tlle addition of other stabilizers is not required and In fact many other materials considered to stabilize enzymes are deleteriou5 when added. For example, alkali metal thiosulfates, poly-vinyl alcohol, glycerine, casein and bovine serum albumin, all possible stabilizers, in fact, are destabilizers with alcohol oxidase. While small amounts of these substances may be present in the compositions without departing from the spirit of this invention, it is preferred to exclude such amounts which would have a substantial deleterious effect on the function and goals of the present invention.
With the preferred Ngo and Lenhoff system or adaptions thereof, dimethylaminobenzoic acid ("DMAB") or a similar di(lower alkyl)amino-benzoic acid is reacted with 3-methyl-2-benzothiazoline ("MBTH") or a similar 3-lower allcyl-2-benzothiazoline to give an indomine dye. This reaction can give better analytical results, particularly a much expanded dynamic range, when the molar ratio of DMAB: MBTH (or of their analogues) is controlled to below 1.0, especially below 0.8. This is contrary to the teachings of the Ngo et al paper which uses 1 or greater. Preferred ratios are from 0.1 to 0.6.
The reagent system includes a material having peroxidative activity. This material promotes the reaction of the H202, generated by the reaction of ethanol with 2~ with the color-changing oxygen acceptor. ~hile, mo$t commonly? enzymatic plant peroxidases such as horseradi$h peroxidas~e or potato peroxidase are preferred and employed, r~
-13a-various other organic or inorganic materials having peroxidative activity can be employed. These are known to include organics such as some of the porphyrins, as well as inorganics such as ammoniur,l or alkali metal iodides, alkali metal chromic sulfates, iron II ferrocyanide, ferrous chloride and iron sulfocyanate, and the like. rhe material having peroxidative activity promotes reaction of H202 with the color-changing oxygen acceptor. A
suitable oxygen acceptor is one which undergoes a visually-detectable color change when converted from a reduced state to an oxidized state. A
large number o~ such materials have been disclosed heretofore, primarily in ~he context of glucose analyses.
Benzidine has been used as an oxygen acceptor in glucose analyses, but has carcinogenicity pro-blems. 3,3,5,5-tetraalkylbenzidine is suggested as an oxygen acceptor in glucose analysis by USP ~,211,845.
USP 3,630,847 discloses a family of parahydroxyl or paraamino pyridines as color forming oxygen aceptors.
Ngo and Lenhoff Anal Biochem, 105, 389-97 (1980) disclose using dimethylaminobenzoic acid and
In the application of this device to screen humans for intoxication, the aqueous sample is whole blood, saliva or urine. In the case of blood, a 5~20 microliter volume from a minimally traumatic "finger-prick" is very preferably used. Generally, when fresh whole blood is the sample being tes~ed the test pad should be on the order of 10 mm2 to 100 mm2 in area, especially 10 mm2 to 50 mm2 in area. The sample pad contains the enzymes and chemical reagents which will react with any ethanol to produce a compound of changed color especially of an intense blue color. These will be described separately, below.
Turning to Fig. 2 a second embodiment of the device of this invention is shown. In this embodiment, pad 10 as described, is affixed to handle lOa. ~andle lOa can be the same material as pad 10, without the reagents or it can be a different material such as will be described as a backing with reference to Fig. 3.
In Fig. 3, another embodiment of the device of this invention is shown to comprise absorbent pad 10 affixed to backing 11. Backing 11 is preferably of a material that does not absorb the aqueous sample. It can be made of plastic, wood, hydrophobic paper 5~
products such as water-repellant-treated board stock, heavy paper, or the like. In one special embodiment, backing 11 and pad 10 are chosen so that pad 10 is at least partially transparent or trans-lucent and backing 11 presents a diffuse reflective surface.
In Fig. 4, yet another embodiment of -the present invention is shown in which pad 10 is overcoated with a semipermeable transparent "membrane" 12. This membrane is permeable to small molecules like water and ethanol but is impermeable to whole blood cells and similar large molecules. Such membranes are disclosed in the art of blood glucose measuring devices (See USP 4,211,845 of Genshaw and USP 3,298,789 of Mast). This is the preferred method for quantitation of ethanol in whole blood. Ethyl cellulose has been used in that application and works in the present case.
Other equivalent materials such as cellulose acetate, cellulose proprionate, polyvinylacetate and polymethylmethacrylate can be employed, as well. This membrane is of advantage when alcohol levels are being detected in a fluid like blood which is colored by large bodies (i.e., whole blood cells). The membrane keeps the colored cells out of the reagent pad. Prior to determining the color change they can be washed or wiped away and thus do not interfere with the color change in the reagent pad.
Fig. 4's depiction of membrane 12 illustrates one possible form of such a membrane and makes it easy to see how the membrane works. However, in the most common embodiments this over-coating layer is applied to the pad as a solution or "varnish" in a water impermeable organic solvent like toluene, benzene or the like which is dried. Electron micrographs of materials formed in this manner reveal that the over-coating layer is not exactly the discrete unit of Fig.
4 but rather coats the individual fibers making up pad 10. Both embodiments work the see and are included within the definition of membrane 12.
Optionally, pad 10 including coating 12 is covered by a protective cover that is not shown. This cover is removed in use but is removabl~ sealed around pad 11 prior to use. This cover can be made of foil, waterproofed paper, or water-oxygen and light-impermeable protective plastic such as poly(ethylene) or poly(vinylchloride) or the like and is provide to protect the enzymes and chemicals in pad 10 from degradation prior to the device's use in testing.
Pad 10 contains a reagent system. The reagent system is made up of stabilized alcohol oxidase, a peroxidase and a color-changing oxygen acceptor. It may contain other materials as well.
"Stabilized alcohol oxidase" is defined to mean alcohol oxidase which retains at least 50% of its activity and preferably at least 70% of its activity when stored in dry form at 56~C for 15 days. With preferred embodi-ments of this invention a "Stabilized alcohol oxidase"
can be attained which retains at least 90% of its activity under these test conditions. The stabilized alcohol oxidase is an important aspect of this inven-tion and in its simplest form comprises alcohol oxidase in intimate admixture with an effective stabilizing concentration of a solid aliphatic compound with 5 to 8 carbons and 5 to 7 hydroxyl groups. Preferably the stabilized alcohol oxidase is buffered to an essen-tially neutral pH and also includes a chelating agent, especially an amine polyacid. In addition, the stabilized alcohol oxidase optionally include~ a saccharide.
The alcohol oxidase is commercially available from two sources, Boehringer Mannheim Biochemicals (BMB) and Phillips Chemical- Neither supplier, how-ever, has been able to develop a form of the en7yme suitable for the applications described herein. BMB
offers the enzyme in a dry form which is stabilized with high levels of reduced glutathione, a peroxide scavenger. This form of the enzyme cannot be used for analytical applications based on peroxide chemistry because the high levels of glutathione interfere.
Phillips Chemical has not been able ~o develop a stable dry form of the enzyme and thus offers only solution~
of the enzyme which are shipped and stored in a frozen state. These solutions, however, can be converted to a stable, dry form using the methods described herein.
The amount of enzyme used is conventionally expressed in units of enzyme activity, i.e., 500 units etc.
Commerical solutions, when fresh generally contain about 1000 units/ml.
The solid aliphatic polyhydroxyl compound employed in the stabilized alcohol oxidase has 5 to 8 carbons and 5 to 7 hydroxyls. Preferably it has 5 or 6 carbons. Also preferably it is nonhygroscopic.
Preferred of these materials are the solid polyhydric alcohols (polyols) having from 5 to ~ carbons and 5 to 7 hydroxyl groups, preferably 5 or 6 carbons. Examples of such materials are mannitol, sorbitol, blucitol and inositol. Mannitol is the preferred polyhydric alcohol.
~L~
The chelating agent is a material that can form a chelate complex with metal ions. Amine polyacids such as, ethylenediamine tetraacetic acid (EDTA) or ethylenediamine pentacetic acid, propylene 1,3-diamine tetraacetic acid and the like and especially 2 to 4 carbon alkyl-polyamine polyacids containing from 2 to 3 amine groups and from 4 to 7 aliphatic acid groups, especially acetic acid groups, are preferred. EDTA is the most preferred chelating agent because of ready availability.
Preferably, however, the buffer employed is a zwitterion buffer, preferably a Good's buffer, especially a morpholino sulfonic acid or piperazine sulfonic acid. Examples of these materials are 3-(N-morpholino)propanesulfonic acid (acronym "MOPS"); N-2-hydroxy ethylpiperzine-N'-2-ethanesulfonic acid ("HEPES"); 2-(N-morpholino)ethanesulfonic acid ("MES"); and piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"). These materials are sold as free acids and as salts. It is possible to use either form or in a combination of forms adjusting the pH as needed with acid or base. These particular buffers offer two advantages (1) the alcohol oxidase is more stable in their pres-ence and (2) the alcohol oxidase is more easily soluble in thesebuffers than in phosphate buffers or the like.
During development of this invention it was separately determined that 3-(N-morpholino) propane sulfonic acid "MOPS"
marketed in the USA by Research Organics of Cleveland, Ohio is a particularly useful buffer component.
Optionally, the stabilized enzyme system additionally contains a saccharide. One commercial preparation of alcohol oxi-dase contains a ~r~
-IOa-substantial amount of sucrose. Other similar mono or di or tri saccharides may be added as well. These include galactose, :Eructose, maltose, çellobiose and raffinose, for example.
A stabilizing amount of polyhydroxyl compound is present with the alcohol oxidase. A "stabilizing amount" or "stabilizing concentration"
1'~ an amount sufficient to achieve a "stabilized alcohol oxidase~ as that term is herein defined. In general, such amounts involve a weight excess of the polyhydroxyl compound over the alcohol oxidase. As a guide, usually from about 0.3 to 2.5 grams of polyhydroxyl compound per 1000 units of enzyme are employed, preferably 0.4 to 1.2 grams of poly hydroxyl compound/1000 units alcohol oxidase.
The amount of chelating agent generally ranges from about 2 to about 50 mg/1000 units with use levels of about 4 to about 25 mg/1000 units being preferred.
When the optional saccharide is present it is usually present in up to about a 2 fold weight excess, based on the poly (hydroxyl) compound. Mixtures of two or more of any of the families of materials can be used in the stabilized enzyme systems, if desired.
By way of example, Table I lists a group of representative stabilized enzyme systems useful in the present invention. These systems are expressed in parts by weight based on the weight of dry alcohol oxidase present in the dry chemical mixture in the finished device.
~LX25912 TABLE I
Alcohol Oxidase 1000 units Poly hyroxyl compound 300-2500 mg Chelating agent 2-50 mg Buffer to pH 6-9 Alcohol Oxidase 1000 units Mannitol and/or Sorbitol 400-2000 mg Alkylpolyamine polyacid 2-50 mg Phosphate Buffer to pH 7-8 Alcohol Oxidase 1000 units Mannitol 400-2000 mg EDTA 2-5 mg Phosphate Buffer to pH 7-8 Alcohol Oxidase 1000 units Mannitol 400-2000 mg EDTA 2~50 mg Saccharide 400-2000 mg Buffer to pH 7-8 It will be readily understood by those skilled in the art that one does not depart from the teachings of this invention by incorporating inert materials and the like into the stabilized enzyme system. Gen-erally, however, tlle addition of other stabilizers is not required and In fact many other materials considered to stabilize enzymes are deleteriou5 when added. For example, alkali metal thiosulfates, poly-vinyl alcohol, glycerine, casein and bovine serum albumin, all possible stabilizers, in fact, are destabilizers with alcohol oxidase. While small amounts of these substances may be present in the compositions without departing from the spirit of this invention, it is preferred to exclude such amounts which would have a substantial deleterious effect on the function and goals of the present invention.
With the preferred Ngo and Lenhoff system or adaptions thereof, dimethylaminobenzoic acid ("DMAB") or a similar di(lower alkyl)amino-benzoic acid is reacted with 3-methyl-2-benzothiazoline ("MBTH") or a similar 3-lower allcyl-2-benzothiazoline to give an indomine dye. This reaction can give better analytical results, particularly a much expanded dynamic range, when the molar ratio of DMAB: MBTH (or of their analogues) is controlled to below 1.0, especially below 0.8. This is contrary to the teachings of the Ngo et al paper which uses 1 or greater. Preferred ratios are from 0.1 to 0.6.
The reagent system includes a material having peroxidative activity. This material promotes the reaction of the H202, generated by the reaction of ethanol with 2~ with the color-changing oxygen acceptor. ~hile, mo$t commonly? enzymatic plant peroxidases such as horseradi$h peroxidas~e or potato peroxidase are preferred and employed, r~
-13a-various other organic or inorganic materials having peroxidative activity can be employed. These are known to include organics such as some of the porphyrins, as well as inorganics such as ammoniur,l or alkali metal iodides, alkali metal chromic sulfates, iron II ferrocyanide, ferrous chloride and iron sulfocyanate, and the like. rhe material having peroxidative activity promotes reaction of H202 with the color-changing oxygen acceptor. A
suitable oxygen acceptor is one which undergoes a visually-detectable color change when converted from a reduced state to an oxidized state. A
large number o~ such materials have been disclosed heretofore, primarily in ~he context of glucose analyses.
Benzidine has been used as an oxygen acceptor in glucose analyses, but has carcinogenicity pro-blems. 3,3,5,5-tetraalkylbenzidine is suggested as an oxygen acceptor in glucose analysis by USP ~,211,845.
USP 3,630,847 discloses a family of parahydroxyl or paraamino pyridines as color forming oxygen aceptors.
Ngo and Lenhoff Anal Biochem, 105, 389-97 (1980) disclose using dimethylaminobenzoic acid and
3-methylbenzothiazolinone, again in a glucose analysis setting. The text, Clinical Chemistry by Richterich, et al, John Wiley & Sons, at pages 366-367 discusses 0-dianisidine, 0-tolidine, the ammonium salt of 2,2'-azindi-[3-ethylbenzthiazoline sulfonic acid-(6)], 3-methyl-~-benzothiazolinone hydrazone/N,N-dimetyl-aniline, and phenol/4-aminophenazone as oxygen acceptors in glucose analysis. It should be emphasized that such materials are merely representative. This invention is not limited to any one specific color-changing (i.e., color forming or color decreasing) oxygen acceptor system. In general, any system which will react with H202 and give a color change proportional to the quantity of H202 can be employed.
The Ngo and Lenhoff color forming system is excellent, however. It has a high extinction coefficient - i.e., it is very intense - its blue color is very distinc-tive.
The amount of oxygen acceptor can vary and wiil in part depend upon the materials employed. As a rough rule of thumb, the amount of oxygen acceptor ranges from about 5 to about 1000 mg per 1000 units of alcohol oxidase, with amounts from 10 to about 400 mg/per 1000 units of alcohol oxidase being preferred.
A colorimetric analysis of the present general structure can be based on color endpoint or on the rate of change of color.
In the present invention, it is preferred to use endpoint measure-ments. This is because the rate can depend not only on the amount of ethanol present (i.e., the desired parameter) but also on the activity of the alcohol oxidase (a nondesired parameter).
If the test device is employed to merely give a yes-no indication of ethanol presence, the identity and amount of color-changing materials and the enzyme stabilization system can be selected to give a very large and very rapid color change with the only issue being the development of a distinctly measurable ethanol-dependent color change endpoint.
As noted in the background, alcohol oxidase undergoes an autoxidation reaction even when stabilized. This means that the system can sometimes give false positive endpoints. This can be prevented by neutralizing the peroxide functionality arising from autoxidation by adding a controlled neutralizing amount of peroxide scavenger to the reagent mixture. Such materials include ascorbic acid, cysteine, reduced glutathione, and uric acid. A
neutralizing amount is specifically defined to be from 0.2 to 12~
moles and especially 4-8~ moles per 1000 units of alcohol oxidase.
Such additions will prevent false positives and will give rise to a distinctly measurable endpoint.
2"~r3r ~
iJ ~.;3 .~Llf~, In the more common application, it is desired to not only identify the presence of ethanol but also to quantify the amount of ethanol present. In this setting, the degree of color change should be selected, based on reagents employed, concentrations, and the like to give a detectable varianc~ of color change depending on test sample ethanol levels. In such a test after a suitable period for color change develop-ment the color is read and the concentration of ethanol determined based on the color change.
The degree of color change can be moderated by adding additional peroxide scavenger to the test device. This addition has the effect of giving more separation between color changes seen with different ethanol levels. In general, additions of 4 to 80 mg of scavenger per 1000 units of alcohol oxidase will work,
The Ngo and Lenhoff color forming system is excellent, however. It has a high extinction coefficient - i.e., it is very intense - its blue color is very distinc-tive.
The amount of oxygen acceptor can vary and wiil in part depend upon the materials employed. As a rough rule of thumb, the amount of oxygen acceptor ranges from about 5 to about 1000 mg per 1000 units of alcohol oxidase, with amounts from 10 to about 400 mg/per 1000 units of alcohol oxidase being preferred.
A colorimetric analysis of the present general structure can be based on color endpoint or on the rate of change of color.
In the present invention, it is preferred to use endpoint measure-ments. This is because the rate can depend not only on the amount of ethanol present (i.e., the desired parameter) but also on the activity of the alcohol oxidase (a nondesired parameter).
If the test device is employed to merely give a yes-no indication of ethanol presence, the identity and amount of color-changing materials and the enzyme stabilization system can be selected to give a very large and very rapid color change with the only issue being the development of a distinctly measurable ethanol-dependent color change endpoint.
As noted in the background, alcohol oxidase undergoes an autoxidation reaction even when stabilized. This means that the system can sometimes give false positive endpoints. This can be prevented by neutralizing the peroxide functionality arising from autoxidation by adding a controlled neutralizing amount of peroxide scavenger to the reagent mixture. Such materials include ascorbic acid, cysteine, reduced glutathione, and uric acid. A
neutralizing amount is specifically defined to be from 0.2 to 12~
moles and especially 4-8~ moles per 1000 units of alcohol oxidase.
Such additions will prevent false positives and will give rise to a distinctly measurable endpoint.
2"~r3r ~
iJ ~.;3 .~Llf~, In the more common application, it is desired to not only identify the presence of ethanol but also to quantify the amount of ethanol present. In this setting, the degree of color change should be selected, based on reagents employed, concentrations, and the like to give a detectable varianc~ of color change depending on test sample ethanol levels. In such a test after a suitable period for color change develop-ment the color is read and the concentration of ethanol determined based on the color change.
The degree of color change can be moderated by adding additional peroxide scavenger to the test device. This addition has the effect of giving more separation between color changes seen with different ethanol levels. In general, additions of 4 to 80 mg of scavenger per 1000 units of alcohol oxidase will work,
4 to 20 ~ moles, and expressly 4 to B ~ moles/1000 units giving less separation in color change between varied ethanol levels, but concurrently greater abso-lute sensitivity to low ethanol levels while additions at the higher end of this range, i.e., 20 to B0~
moles, especially 40 to 80~ moles/1000 units giving greater separation but lower absolute sensitivity.
The time for color change development when quantitating ethanol is usually from about 10 seconds to several minutes. Generally a value equal to at least 95% of the endpoint is achieved in that time range. Longer times may be used subject to the caution that ethanol is generally more volatile than the aqueous sample in which it is contained so that it may be necessary to cover or enclose the test device in use to prevent preferential evaporation of the ethanol analyte.
25~
The degree of color change brought about by reaction of ethanol can be determined in several ways. Two manual methods include comparing the test sample with a blank and comparing a test sample with a series of standards, each depicting a given ethanol level until a match is made.
The color change can also be measured instrumentally using a spectrophotometer or the like, either by comparison of the test sample with a blank preferably after a time interval adequate to give substantial, i.e., 90+% of the total color change, or less preferably, measurement by measuring the change in the absorbance of the test sample between two or more points in time.
The instrumental measurement of color change can be carried out by reflectance or absorption measurements at a wave-length at which the color change can be detected.
The samples tested with the invention are aqueous samples, commonly human aqueous body fluids such as whole blood, serum, plasma, saliva and the like. Whole blood and saliva are usual test samples. It is well documented that the alcohol levels of these fluids are related and that these levels are in turn directly related to the subject's degree of intoxication. See, for example, Clin. Sci. (England), 56, No. 3, 283-286, 1979 and Clin.
Exp. Pharmacol. Physiol. (England), 6, No. 1, 53-59, 1979. Urine can also be the test sample, although its alcohol level is depend-ent upon a variety of factors other than only the amount of alcohol consumed and thus is not as good a measure of intoxication.
~2~ 2 When the sample being tested is saliva, urine or a like lightly colored material, the devices of any of Figs. 1 through 4 can be used. When the sample is deeply colored whole blood or the like, it is preferred, but not required, to use a device as set forth in Fig. 4 in which there is a membrane that can exclude whole blood cells and prevent their interfer-ence with the measurement of the color change.
The test method of this invention, in its broadest sense, involves applying a sample to the test strip, allowing the color change to take place, measuring the degree or rate of color change and relat-ing that measured quantity to the level of alcohol.
The protocol for such a test can be as follows for a "membrane" strip with whole blood, for example:
l. Apply sample of fresh whole blood (10-30 microliters) to pad of test strip.
2. After 20-40 seconds, blot strip, wiping of red cells and removing excess sample. (Period should be constant.) 3. Wait 20-90 seconds for color change to develop. (Again, period should be rela-tively constant.) 4. Read color change manually or instrumen-tally.
moles, especially 40 to 80~ moles/1000 units giving greater separation but lower absolute sensitivity.
The time for color change development when quantitating ethanol is usually from about 10 seconds to several minutes. Generally a value equal to at least 95% of the endpoint is achieved in that time range. Longer times may be used subject to the caution that ethanol is generally more volatile than the aqueous sample in which it is contained so that it may be necessary to cover or enclose the test device in use to prevent preferential evaporation of the ethanol analyte.
25~
The degree of color change brought about by reaction of ethanol can be determined in several ways. Two manual methods include comparing the test sample with a blank and comparing a test sample with a series of standards, each depicting a given ethanol level until a match is made.
The color change can also be measured instrumentally using a spectrophotometer or the like, either by comparison of the test sample with a blank preferably after a time interval adequate to give substantial, i.e., 90+% of the total color change, or less preferably, measurement by measuring the change in the absorbance of the test sample between two or more points in time.
The instrumental measurement of color change can be carried out by reflectance or absorption measurements at a wave-length at which the color change can be detected.
The samples tested with the invention are aqueous samples, commonly human aqueous body fluids such as whole blood, serum, plasma, saliva and the like. Whole blood and saliva are usual test samples. It is well documented that the alcohol levels of these fluids are related and that these levels are in turn directly related to the subject's degree of intoxication. See, for example, Clin. Sci. (England), 56, No. 3, 283-286, 1979 and Clin.
Exp. Pharmacol. Physiol. (England), 6, No. 1, 53-59, 1979. Urine can also be the test sample, although its alcohol level is depend-ent upon a variety of factors other than only the amount of alcohol consumed and thus is not as good a measure of intoxication.
~2~ 2 When the sample being tested is saliva, urine or a like lightly colored material, the devices of any of Figs. 1 through 4 can be used. When the sample is deeply colored whole blood or the like, it is preferred, but not required, to use a device as set forth in Fig. 4 in which there is a membrane that can exclude whole blood cells and prevent their interfer-ence with the measurement of the color change.
The test method of this invention, in its broadest sense, involves applying a sample to the test strip, allowing the color change to take place, measuring the degree or rate of color change and relat-ing that measured quantity to the level of alcohol.
The protocol for such a test can be as follows for a "membrane" strip with whole blood, for example:
l. Apply sample of fresh whole blood (10-30 microliters) to pad of test strip.
2. After 20-40 seconds, blot strip, wiping of red cells and removing excess sample. (Period should be constant.) 3. Wait 20-90 seconds for color change to develop. (Again, period should be rela-tively constant.) 4. Read color change manually or instrumen-tally.
5. Relate color reading to ethanol level via standard calibration factors or the like.
One could use this type of protocol with other samples when a membrane device is employed.
With a no membrane strip the magnitude of color change can often be greater or more pronounced because the sample fully saturates the pad and a larger amount of reaction may occur. With such devices, a typical test protocol can be as follows:
~L~
1. Apply sample of fresh test liquid to the pad of the test device in an amount that more than saturates the pad. (10 microliters or more.) 2. Blot off excess test liquid.
3. Allow color change to develop. (Usually about 95%
of total change has occured in 2 minutes with 99+~
within 4 minutes. Best results will be obtained if evaporation is minimized.
4. Read color change.
5. Correlate color change to ethanol levels as above.
When a rate measurement method is employed, this is generally best carried out in a spectrophotometer which can make readings at two or more points in time, measured from the time the sample is contacted with the pad, and from these readings calcu-late a rate of change and from that rate automatically provide a ethanol level.
The method of producing the test cevices in its general sense involves forming a solution of the enzyme, peroxidatively-active material, stabilizers and color-changing oxygen acceptors;
applying it to the pad substrate in a reproducible amount and manner and evaporating the solvent to deposit the materials on the substrate. The usual solvent is selected from water and water in combination with water-miscible (i.e. polar) inert organic solvents such as DMF, THF, DMSO, acetone or other ketones, acetonitrile or the like. Obviously, alcohols should not be used. these organics are generally employed to help solubilize the color-changing oxygen acceptor compounds which often are -2~-marginally soluble in water alone. The concentration of the components in the make-up solution can vary from about 20 to about 500 and especially 50 to 300 units of alcohol oxidase per ml, with the other components being present in the proportions set forth. The solution is usually made up by mixing all the materials except the ethanol oxidase and adding that material last. Once mixed, it is usually best to use the solution promptly. The drying of the test devices is usually carried out at mild conditions such as 15-30C and a vacuum. Similarly, it is referred to store the test devices in a cool dry environment, ideally without substantial contact with oxygen. Once used, however, the test strips are very stable, holding a constant color for two weeks or more. This is true whether or not the strips are in contact with air, moisture or moderately elevated temperatures.
The invention will be further depicted by the following examples and comparative experiments which illustrate the preparation and use of representative devices in the measurement of ethanol in aqueous fluids and compare these results with other devices not of the invention. These examples are provided only to illustrate the invention and are not to be contrued as limiting its scope.
EXAMPLE I
A test device for determining ethanol concen-trations in aqueous fluids was produced. The device used as its color-forming oxygen acceptor, a system similar to the system of Ngo and Lenhoff, Anal Biochem, 105, 389-97 (1980) using dimethylaminobenzoic acid (DMAB) and 3-methylbenzothiazolinone hydrazone (MBT~).
A first solution was prepared DMAB (72 mg) was mixed with 0.5 ml o acetonitrile and 1.0 ml of 0.4 molar disodium phosphate buffer. The buffer held the pH at a level that causes most of the DMAB to depro-tonate and ~hus go into solution. Disodium EDTA (2.4 ml of a 10 mg/ml solution) was added followed by 4.1 ml of deionized H2O and 1500 mg of mannitol (essentially to saturation). The solution was warmed to dissolve the mannitol, cooled to room temperature, and 6 mg of ascorbic acid was added. The amount of ascorbic acid added to this system is important as it serves as a means for "calibrating" of the system. Larger amounts of scorbic acid slow the system's color development while smaller amounts permit color to develop more rapidly. The pH of the solution was adjusted to 7.2 with NaOH. Horseradish peroxidase (10 mg) having an enzyme activity of 108 units/mg was then added with vigorous mixing. This solution was held temporarily.
For long ~erm storage, it is advantageous to minimize oxygen contact by degassing and/or N2 blanketing.
A second solution was made up to contain 144 mg of MBTH in ~ ml of 50/50 water/acetonitrile. Its pH
was adjusted to 7.2 with NaOH.
A solution of alcohol oxidase (1000 enzyme units/ml) in 70~ sucrose water was obtained (Phillips Petroleum). The first two solutions were mixed in a ratio of 4 parts to 1 part by volume and cooled to about 4C. 1 part by volume of the alcohol oxidase solution ( 4C) was added.
The mixed solution contained: 6 mg/ml DMAB
3 mg/ml MBTH
~ mg/ml Na2EDTA
0.03 M disodium phosphate 12.5% mannitol 14% sucrose 12% v acetonitrile 0.5 mg/ml ascorbic acid 0.83 mg/ml peroxidase 1700 units/ml of alcohol o~idase The mixture (1.0 ml) was spread on a glass plate and a 3 cm x 3 cm piece of Whatman #541 paper was gently laid on the mixture so as to minimize "wicking" of the solution and maximize the evenness of saturation. The saturated paper was turned over to saturate the oppo-site side, taken up from the plate, placed on another plate, and dried in a 100 mm Hg absolute pressure 20-22C vac oven for 30 minutes. It was then held for 16 hours at 760 mm Hg and 20-22C in a sealed container with silica gel dessicant to remove last traces of liquid.
The treated paper was cut into 0.5 cm x 0.6 cm pieces which were affixed with 3M #465 double-sided clear adhesive to white polystyrene supports to give test devices. Alternatively, and preferably the paper could be affixed by softening the polysytrene surface with solvent (toluene) and adhering the paper to ~he softened plastic.
After stressing 12 days at 56C, the paper thus prepared retains virtually all of its original color when developed with 100 mg/dl ethanol.
EXAMPLE II
The preparation of Example I was substan-tially repeated with one change. After drying the impregnated paper, the paper was over-coated with a 1.0% by weight solution of ethyl cellulose in toluene and then dried at 20-22C in vacuuo to give a micromolecule impermeable membrane coat.
After stressing 14 days at 56C, the paper thus prepared retains most of its original color when developed with 100 mg/dl ethanol. When used with whole blood to determine said blood's ethanol level, this strip has the advantage of not permitting whole blood cells to enter the strip so that they may be wiped off the strip easily.
EXAMPLE III
The preparation of Example I was repeated twice wi~h the following change. In the first repeat, instead of 0.5 mg/ml of ascorbic acid, 1.0 mg/ml was used. In the second repeat no ascorbic acid was present. The first strip was more moderated than the strip of Example I, being less sensitive to low levels of ethanol in test samples but giving better color separation between samples of differing ethanol level. The second sample containing no ascorbic acid was extremely sensitive but gave poorer separation between samples.
EXAMPLE IV
R test device for ethanol similar to Example I is preared using a single oxygen accepting dye component, 6-Di-methylamino-4-hydroxy-2-napthalenesul-fonic acid (DMAN) in place of DMAB and MBTH. The first solution is prepared as in Example I, with the omission of DMAB. The second solution is prepared as in Example I, with the substitution of DMAN for MBTH. When developed with aqueous ethanol, the prepared paper gives a color varying from tan at 0 mg/ml EtOH to golden orange-yellow at 200 mg/dl EtOH.
~L~5~
COMPARATIVE EXPERIMENTS
As a base line, Comparative Experiment H was run. This was a repeat of Example I without stabil-izers being present. That is, the mannitol and EDTA
were omitted. The sucrose in the commercial alcohol oxidase was present. After storage for 18 days at 37C
the paper thus prepared lost 68% of its color when developed with 100 mg/dl of EtOH. This result is tabulated in the Table following Example V along with the results of other comparative experiments conducted with a number of art-taught enzyme stabilizer components.
COMPARATIVE EXPERIMENT A
The preparation of Example I was substan-tially repeated, except that EDTA was omitted, 2 mg/ml casien was incorporated, and 0.8 mg/ml ascorbic acid was used. Casien is known as an enzyme stabilizer.
After storage for 18 days at 37C, the paper thus prepared lost a substantial amount of color when developed with 100 mg/dl EtOH indicating that casien was ineffective an probably destructive by comparison with Experiment H.
COMPARATIVE EXPERIMENT B
The preparation of Example I was substan-tially repeated, except that mannitol was excluded and 0.3`M tri(hydroxymethyl)aminomethane (TRIS) buffer was used in place of phosphate buffer. (TRIS) is suggested as an enzyme stabilizer but with alcohol oxidase was ineffective or even destructive.
COMPARATIVE EXPERIMENT C
The preparation of Example I was substan-tially repeated, excep~ that mannitol was excluded and 20 mg/ml bovine serum albumin (BSA), an art taught enzyme stabilizer, was incorporated. No positive effect was observed and the product was less stable than unstabilized enzyme.
COMPARATIVE EXPERIMENT D
The preparation of Example I was substan-tially repeated, except that mannitol was excluded, and 30 mg/ml polyvinyl alcohol (PVA) was incorporated.
Polyvinyl alcohol, a large molecule polyol not in accord with this invention destroyed the alcohol oxidase's activity. Another enzyme stabilizer is the three carbon polyol glycerol. This material is a liquid so not useful in a dry test strip and also has the property of reacting with alcohol oxidase and forming gels. Thus it is not useful.
COMPARATIVE EXPERIMENT E
The preparation of Example I was substan-tially repeated, except that mannitol was excluded, 0.4 M imidazole was substituted for phosphate buffer, and 25 mg/ml acacia, an art taught stabilizer was incorporated. Some stabilization was noted by compari-son to Experiment H but its loss was many times that observed in Example I.
COMPARATIVE EXPERIMENT F
The preparation of Example I was repeated with the change that the pH was buffered to p~ 5.5, a pH outside the range preferred herein. The enzyme was unstable at pH 5.5 and essentially all color forming ability was lost.
r3~,~
COMPARATIVE EXPERIMENT G
The preparation of Example I was repeated except that the EDTA
l~as omitted and Na2S2O4, an art-taught enzyme stabilizer, was added. The Na2S2O4 did not work with alcohol oxidase instead destroying the enzyme's activity.
EXAMPLE V
A series of test devices were prepared following the procedure of Example I. The ETDA, mannitol, buffer stabilizer system of Example I was replaced with a range of other stabilizers, and the pH was varied.
The test devices were then stored for 14 to 20 days and their activity was. detçrmined by measuring their response to a standard 0.1% ethanol in water mixture. The formulations and results of the tests are given in the following table.
In the solvent fusion technique an outer layer of the plastic backing is softened with a layer of solvent. With ABS plastic, ketones like methyl ethyl ketone, THF, and halohydrocarbons such as methylene chloride can be used. With acetates, ketones and halohydrocarbons work.
With acrylics, halohydrocarbons are materials of choice. With cellulosics, ketones work well, while with polyvinylchlorides, cyclohexane, THF and dichlorobenzene are materials of choice. With polystyrene and polycarbo-nates, halohydrocarbons work while with polystyrene ketones and alkyl-aromatics, toluene or xylene work well. This solvent fusion technique, while on its face merely equivalent to other adhesion methods such as adhesives and ultrasonic bonding, in fact gives much better activity for the alcohol oxidase-containing test strips.
r9~L, q~LE
Days of 37C % Color Sample Stabilizer Stress Retained - Example I 0.2~ EDT~, 10% mannitol, 17 100 pH 7.2 EXample Va 0.05 ~ Cysteine, 10% mannitol, 19 100 pH 7-8 Example Vb 0.02~ Scdium Azide, 10% 17 86 mannitol Example Vc pH 7.2 phosphate buffer, 16 90 10% mannitol Example Vd pH 9.0 phosphate buffer, 16 90 10% mannitol Comparative Experiment A casien added, EDI~ omitted 18 30 B Tris added, phcsphate omitted 18 7 mannitol omitted C BSA added, mannitol omitted 18 24 D PVA added, mannitol omitted 17 E Imidazole added, acacia added, 17 51 mannitol omitted F 0.2% EDI~, 10% mannitol, 16 3 pH 5.5 G Na2S204 added, EDI~ omitted 14 0 H No stabilizer 18 32 EXA~PLE VI
An additional test device employing this invention was prepared. A first solution was prepared by dissolving 9.64 g of anhydrous MBTH H~l, 5.03 g an anhydrous MOPS and 30.6 g of sucrose in 235 ml of acetonitrile and 231 ml of purified water. Solution pH
was adjusted to 7.2 with NaOH. A test paper was impregnated with this solution and dried. A second solution containing 3.50 g NaDMAB, 9.63 g NaMOPS, 6.53 g MOPS, 74 ml of 1% Na2EDTA. 2H2O, and 296 ml of puri-fied H2O was prepared. To this was added ~1,700 units of horseradish peroxidase and 247 mg of ascorbic acid. pH was adjusted to 7.20 with concentrated HCl and NaOH. To this solution was then added 13~,000 units of alcohol oxidase (as solution Phillips). The solution was stirred until uniform and applied to the test paper to saturation and then dried to yield a final ethanol test paper which gave superior results determining ethanol in aqueous media. This material was affixed to a polystyrene backing by a solvent fusion technique wherein the polystyrene was softened with toluene and the paper adhered to it to give a test device.
One could use this type of protocol with other samples when a membrane device is employed.
With a no membrane strip the magnitude of color change can often be greater or more pronounced because the sample fully saturates the pad and a larger amount of reaction may occur. With such devices, a typical test protocol can be as follows:
~L~
1. Apply sample of fresh test liquid to the pad of the test device in an amount that more than saturates the pad. (10 microliters or more.) 2. Blot off excess test liquid.
3. Allow color change to develop. (Usually about 95%
of total change has occured in 2 minutes with 99+~
within 4 minutes. Best results will be obtained if evaporation is minimized.
4. Read color change.
5. Correlate color change to ethanol levels as above.
When a rate measurement method is employed, this is generally best carried out in a spectrophotometer which can make readings at two or more points in time, measured from the time the sample is contacted with the pad, and from these readings calcu-late a rate of change and from that rate automatically provide a ethanol level.
The method of producing the test cevices in its general sense involves forming a solution of the enzyme, peroxidatively-active material, stabilizers and color-changing oxygen acceptors;
applying it to the pad substrate in a reproducible amount and manner and evaporating the solvent to deposit the materials on the substrate. The usual solvent is selected from water and water in combination with water-miscible (i.e. polar) inert organic solvents such as DMF, THF, DMSO, acetone or other ketones, acetonitrile or the like. Obviously, alcohols should not be used. these organics are generally employed to help solubilize the color-changing oxygen acceptor compounds which often are -2~-marginally soluble in water alone. The concentration of the components in the make-up solution can vary from about 20 to about 500 and especially 50 to 300 units of alcohol oxidase per ml, with the other components being present in the proportions set forth. The solution is usually made up by mixing all the materials except the ethanol oxidase and adding that material last. Once mixed, it is usually best to use the solution promptly. The drying of the test devices is usually carried out at mild conditions such as 15-30C and a vacuum. Similarly, it is referred to store the test devices in a cool dry environment, ideally without substantial contact with oxygen. Once used, however, the test strips are very stable, holding a constant color for two weeks or more. This is true whether or not the strips are in contact with air, moisture or moderately elevated temperatures.
The invention will be further depicted by the following examples and comparative experiments which illustrate the preparation and use of representative devices in the measurement of ethanol in aqueous fluids and compare these results with other devices not of the invention. These examples are provided only to illustrate the invention and are not to be contrued as limiting its scope.
EXAMPLE I
A test device for determining ethanol concen-trations in aqueous fluids was produced. The device used as its color-forming oxygen acceptor, a system similar to the system of Ngo and Lenhoff, Anal Biochem, 105, 389-97 (1980) using dimethylaminobenzoic acid (DMAB) and 3-methylbenzothiazolinone hydrazone (MBT~).
A first solution was prepared DMAB (72 mg) was mixed with 0.5 ml o acetonitrile and 1.0 ml of 0.4 molar disodium phosphate buffer. The buffer held the pH at a level that causes most of the DMAB to depro-tonate and ~hus go into solution. Disodium EDTA (2.4 ml of a 10 mg/ml solution) was added followed by 4.1 ml of deionized H2O and 1500 mg of mannitol (essentially to saturation). The solution was warmed to dissolve the mannitol, cooled to room temperature, and 6 mg of ascorbic acid was added. The amount of ascorbic acid added to this system is important as it serves as a means for "calibrating" of the system. Larger amounts of scorbic acid slow the system's color development while smaller amounts permit color to develop more rapidly. The pH of the solution was adjusted to 7.2 with NaOH. Horseradish peroxidase (10 mg) having an enzyme activity of 108 units/mg was then added with vigorous mixing. This solution was held temporarily.
For long ~erm storage, it is advantageous to minimize oxygen contact by degassing and/or N2 blanketing.
A second solution was made up to contain 144 mg of MBTH in ~ ml of 50/50 water/acetonitrile. Its pH
was adjusted to 7.2 with NaOH.
A solution of alcohol oxidase (1000 enzyme units/ml) in 70~ sucrose water was obtained (Phillips Petroleum). The first two solutions were mixed in a ratio of 4 parts to 1 part by volume and cooled to about 4C. 1 part by volume of the alcohol oxidase solution ( 4C) was added.
The mixed solution contained: 6 mg/ml DMAB
3 mg/ml MBTH
~ mg/ml Na2EDTA
0.03 M disodium phosphate 12.5% mannitol 14% sucrose 12% v acetonitrile 0.5 mg/ml ascorbic acid 0.83 mg/ml peroxidase 1700 units/ml of alcohol o~idase The mixture (1.0 ml) was spread on a glass plate and a 3 cm x 3 cm piece of Whatman #541 paper was gently laid on the mixture so as to minimize "wicking" of the solution and maximize the evenness of saturation. The saturated paper was turned over to saturate the oppo-site side, taken up from the plate, placed on another plate, and dried in a 100 mm Hg absolute pressure 20-22C vac oven for 30 minutes. It was then held for 16 hours at 760 mm Hg and 20-22C in a sealed container with silica gel dessicant to remove last traces of liquid.
The treated paper was cut into 0.5 cm x 0.6 cm pieces which were affixed with 3M #465 double-sided clear adhesive to white polystyrene supports to give test devices. Alternatively, and preferably the paper could be affixed by softening the polysytrene surface with solvent (toluene) and adhering the paper to ~he softened plastic.
After stressing 12 days at 56C, the paper thus prepared retains virtually all of its original color when developed with 100 mg/dl ethanol.
EXAMPLE II
The preparation of Example I was substan-tially repeated with one change. After drying the impregnated paper, the paper was over-coated with a 1.0% by weight solution of ethyl cellulose in toluene and then dried at 20-22C in vacuuo to give a micromolecule impermeable membrane coat.
After stressing 14 days at 56C, the paper thus prepared retains most of its original color when developed with 100 mg/dl ethanol. When used with whole blood to determine said blood's ethanol level, this strip has the advantage of not permitting whole blood cells to enter the strip so that they may be wiped off the strip easily.
EXAMPLE III
The preparation of Example I was repeated twice wi~h the following change. In the first repeat, instead of 0.5 mg/ml of ascorbic acid, 1.0 mg/ml was used. In the second repeat no ascorbic acid was present. The first strip was more moderated than the strip of Example I, being less sensitive to low levels of ethanol in test samples but giving better color separation between samples of differing ethanol level. The second sample containing no ascorbic acid was extremely sensitive but gave poorer separation between samples.
EXAMPLE IV
R test device for ethanol similar to Example I is preared using a single oxygen accepting dye component, 6-Di-methylamino-4-hydroxy-2-napthalenesul-fonic acid (DMAN) in place of DMAB and MBTH. The first solution is prepared as in Example I, with the omission of DMAB. The second solution is prepared as in Example I, with the substitution of DMAN for MBTH. When developed with aqueous ethanol, the prepared paper gives a color varying from tan at 0 mg/ml EtOH to golden orange-yellow at 200 mg/dl EtOH.
~L~5~
COMPARATIVE EXPERIMENTS
As a base line, Comparative Experiment H was run. This was a repeat of Example I without stabil-izers being present. That is, the mannitol and EDTA
were omitted. The sucrose in the commercial alcohol oxidase was present. After storage for 18 days at 37C
the paper thus prepared lost 68% of its color when developed with 100 mg/dl of EtOH. This result is tabulated in the Table following Example V along with the results of other comparative experiments conducted with a number of art-taught enzyme stabilizer components.
COMPARATIVE EXPERIMENT A
The preparation of Example I was substan-tially repeated, except that EDTA was omitted, 2 mg/ml casien was incorporated, and 0.8 mg/ml ascorbic acid was used. Casien is known as an enzyme stabilizer.
After storage for 18 days at 37C, the paper thus prepared lost a substantial amount of color when developed with 100 mg/dl EtOH indicating that casien was ineffective an probably destructive by comparison with Experiment H.
COMPARATIVE EXPERIMENT B
The preparation of Example I was substan-tially repeated, except that mannitol was excluded and 0.3`M tri(hydroxymethyl)aminomethane (TRIS) buffer was used in place of phosphate buffer. (TRIS) is suggested as an enzyme stabilizer but with alcohol oxidase was ineffective or even destructive.
COMPARATIVE EXPERIMENT C
The preparation of Example I was substan-tially repeated, excep~ that mannitol was excluded and 20 mg/ml bovine serum albumin (BSA), an art taught enzyme stabilizer, was incorporated. No positive effect was observed and the product was less stable than unstabilized enzyme.
COMPARATIVE EXPERIMENT D
The preparation of Example I was substan-tially repeated, except that mannitol was excluded, and 30 mg/ml polyvinyl alcohol (PVA) was incorporated.
Polyvinyl alcohol, a large molecule polyol not in accord with this invention destroyed the alcohol oxidase's activity. Another enzyme stabilizer is the three carbon polyol glycerol. This material is a liquid so not useful in a dry test strip and also has the property of reacting with alcohol oxidase and forming gels. Thus it is not useful.
COMPARATIVE EXPERIMENT E
The preparation of Example I was substan-tially repeated, except that mannitol was excluded, 0.4 M imidazole was substituted for phosphate buffer, and 25 mg/ml acacia, an art taught stabilizer was incorporated. Some stabilization was noted by compari-son to Experiment H but its loss was many times that observed in Example I.
COMPARATIVE EXPERIMENT F
The preparation of Example I was repeated with the change that the pH was buffered to p~ 5.5, a pH outside the range preferred herein. The enzyme was unstable at pH 5.5 and essentially all color forming ability was lost.
r3~,~
COMPARATIVE EXPERIMENT G
The preparation of Example I was repeated except that the EDTA
l~as omitted and Na2S2O4, an art-taught enzyme stabilizer, was added. The Na2S2O4 did not work with alcohol oxidase instead destroying the enzyme's activity.
EXAMPLE V
A series of test devices were prepared following the procedure of Example I. The ETDA, mannitol, buffer stabilizer system of Example I was replaced with a range of other stabilizers, and the pH was varied.
The test devices were then stored for 14 to 20 days and their activity was. detçrmined by measuring their response to a standard 0.1% ethanol in water mixture. The formulations and results of the tests are given in the following table.
In the solvent fusion technique an outer layer of the plastic backing is softened with a layer of solvent. With ABS plastic, ketones like methyl ethyl ketone, THF, and halohydrocarbons such as methylene chloride can be used. With acetates, ketones and halohydrocarbons work.
With acrylics, halohydrocarbons are materials of choice. With cellulosics, ketones work well, while with polyvinylchlorides, cyclohexane, THF and dichlorobenzene are materials of choice. With polystyrene and polycarbo-nates, halohydrocarbons work while with polystyrene ketones and alkyl-aromatics, toluene or xylene work well. This solvent fusion technique, while on its face merely equivalent to other adhesion methods such as adhesives and ultrasonic bonding, in fact gives much better activity for the alcohol oxidase-containing test strips.
r9~L, q~LE
Days of 37C % Color Sample Stabilizer Stress Retained - Example I 0.2~ EDT~, 10% mannitol, 17 100 pH 7.2 EXample Va 0.05 ~ Cysteine, 10% mannitol, 19 100 pH 7-8 Example Vb 0.02~ Scdium Azide, 10% 17 86 mannitol Example Vc pH 7.2 phosphate buffer, 16 90 10% mannitol Example Vd pH 9.0 phosphate buffer, 16 90 10% mannitol Comparative Experiment A casien added, EDI~ omitted 18 30 B Tris added, phcsphate omitted 18 7 mannitol omitted C BSA added, mannitol omitted 18 24 D PVA added, mannitol omitted 17 E Imidazole added, acacia added, 17 51 mannitol omitted F 0.2% EDI~, 10% mannitol, 16 3 pH 5.5 G Na2S204 added, EDI~ omitted 14 0 H No stabilizer 18 32 EXA~PLE VI
An additional test device employing this invention was prepared. A first solution was prepared by dissolving 9.64 g of anhydrous MBTH H~l, 5.03 g an anhydrous MOPS and 30.6 g of sucrose in 235 ml of acetonitrile and 231 ml of purified water. Solution pH
was adjusted to 7.2 with NaOH. A test paper was impregnated with this solution and dried. A second solution containing 3.50 g NaDMAB, 9.63 g NaMOPS, 6.53 g MOPS, 74 ml of 1% Na2EDTA. 2H2O, and 296 ml of puri-fied H2O was prepared. To this was added ~1,700 units of horseradish peroxidase and 247 mg of ascorbic acid. pH was adjusted to 7.20 with concentrated HCl and NaOH. To this solution was then added 13~,000 units of alcohol oxidase (as solution Phillips). The solution was stirred until uniform and applied to the test paper to saturation and then dried to yield a final ethanol test paper which gave superior results determining ethanol in aqueous media. This material was affixed to a polystyrene backing by a solvent fusion technique wherein the polystyrene was softened with toluene and the paper adhered to it to give a test device.
Claims (27)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A test device for detecting ethanol in an aqueous fluid which comprises an inert support pad containing stabilized alcohol oxidase, a substance having peroxidative activity and a substance which will react with hydrogen peroxide to give a compound of changed color.
2. The test device of claim 1 wherein said stabilized alcohol oxidase comprises alcohol oxidase in intimate admixture with an effective stabilizing concentration of a solid aliphatic polyhydroxyl compound having from 5 to 8 carbons and 5 to 7 hydroxyl groups.
3. The test device of claim 2 wherein said solid aliphatic poly-hydroxyl compound is a polyhydric alcohol.
4. The test device of claim 3 wherein said polyhydric alcohol comprises a member of the group consisting of mannitol, sorbitol, glucitol and inositol.
5. The test device of claim 4 wherein said polyhydric alcohol is mannitol.
6. The test device of claim 5 wherein said stabilized alcohol oxidase additionally comprises a chelating agent.
7. The test device of claim 6 wherein said chelating agent comprises a polyaminepolyacid.
8. The test device of claim 7 wherein the polyaminepolyacid is EDTA.
9. The test device of claim 8 wherein said stabilized alcohol oxidase additionally comprises a buffer that gives a pH in water of 6 to 8.
10. The test device of claim 9 wherein said buffer is a zwitterion buffer.
11. The test device of claim 10 wherein said buffer is MOPS.
12. The test device of claim 11 wherein the inert support pad additionally comprises a peroxide scavenger.
13. The test device of claim 12 wherein the peroxide scavenger is selected from the group consisting of ascorbic acid, cysteine, reduced glutathione and uric acid.
14. The test device of claim 13 wherein said support pad is over-coated with a whole cell-impermeable water-permeable material.
15. The test device of claim 14 wherein the substance which will react with hydrogen peroxide is DMAB:MBTH in a molar ratio of less than 1Ø
16. The test device of claim 15 additionally comprising an inert backing upon which said support pad is mounted.
17. The test device of claim 16 wherein the backing is plastic and the support pad is solvent bonded thereto.
18. The test device of claim 1, 2 or 3 wherein said stabilized alcohol oxidase additionally comprises a chelating agent.
19. The test device of claim 1, 2 or 3 wherein said stabilized alcohol oxidase additionally comprises a buffer that gives a pH in water of 6 to 8.
20. The test device of claim 1, 2 or 3 wherein the inert support pad additionally comprises a peroxide scavenger.
21. The test device of claim 1, 2 or 3 wherein said support pad is overcoated with a whole cell-impermeable water-permeable material.
22. The test device of claim 1, 2 or 3 wherein the substance which will react with hydrogen peroxide is DMAB:MBTH in a molar ratio of less than 1Ø
23. The test device of claim 1, 2 or 3 additionally comprising an inert backing upon which said support pad is mounted.
24. A dry stabilized alcohol oxidase formulation comprising alcohol oxidase in intimate admixture with an effective stabilizing concentration of solid aliphatic polyhydroxyl compound having from 5 to 8 carbons and 5 to 7 hydroxyl groups, chelating agent and buffer than gives a pH in water of 6 to 8.
25. A neutralized dry alcohol oxidase formulation comprising alcohol oxidase and from 0.2 to 12 µ moles per 1000 enzyme units of peroxide scavenger.
26. A neutralized, color-moderated alcohol oxidase formulation comprising alcohol oxidase and from 4 to 80 µ moles per 1000 enzyme units. of peroxide scavenger.
27. A method for determining ethanol in an aqueous sample comprising applying said sample to the device of claim 1, permitting a color change dependent upon ethanol content to develop, measure said color change and relating said color change to the ethanol content.
Applications Claiming Priority (4)
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| US51350583A | 1983-07-12 | 1983-07-12 | |
| US513,505 | 1983-07-12 | ||
| US624,774 | 1984-06-26 | ||
| US06/624,774 US4734360A (en) | 1983-07-12 | 1984-06-26 | Colorimetric ethanol analysis method and test device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1225912A true CA1225912A (en) | 1987-08-25 |
Family
ID=27057897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000458581A Expired CA1225912A (en) | 1983-07-12 | 1984-07-11 | Colorimetric ethanol analysis method and test device |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US4734360A (en) |
| EP (1) | EP0133481B1 (en) |
| JP (1) | JPS6075299A (en) |
| AT (1) | ATE30174T1 (en) |
| AU (1) | AU586150B2 (en) |
| CA (1) | CA1225912A (en) |
| DE (1) | DE3466709D1 (en) |
| DK (1) | DK340184A (en) |
| FI (1) | FI81119C (en) |
| IL (1) | IL72375A (en) |
| NO (1) | NO165316C (en) |
Families Citing this family (106)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4810633A (en) * | 1984-06-04 | 1989-03-07 | Miles Inc. | Enzymatic ethanol test |
| IT1204297B (en) * | 1986-04-09 | 1989-03-01 | Boehringer Biochemia Srl | COMPLEX OF REAGENTS FOR THE ENZYMATIC DETERMINATION OF PRIMARY ALCOHOLS C1-C4 AND RELATED METHOD |
| US4729956A (en) * | 1986-05-01 | 1988-03-08 | Phillips Petroleum Company | Stabilized alcohol oxidase compositions and method for producing same |
| US4935346A (en) * | 1986-08-13 | 1990-06-19 | Lifescan, Inc. | Minimum procedure system for the determination of analytes |
| US4874692A (en) * | 1987-07-20 | 1989-10-17 | Eastman Kodak Company | Binder composition and analytical element having stabilized peroxidase in layer containing the composition |
| US5081115A (en) * | 1987-10-15 | 1992-01-14 | The Board Of Trustees Of The Leland Stanford Junior University | Method to prevent neonatal jaundice with metalloporphyrin compositions |
| JP2660843B2 (en) * | 1988-03-07 | 1997-10-08 | 大日本印刷株式会社 | Body fluid test body |
| EP0357027B1 (en) * | 1988-08-30 | 1995-05-10 | New Oji Paper Co., Ltd. | Alcohol oxidase enzyme electrode and its use for quantitative alcohol determination. |
| US4981653A (en) * | 1988-10-06 | 1991-01-01 | Miles Inc. | Self-indicating strip |
| AU640162B2 (en) * | 1989-08-28 | 1993-08-19 | Lifescan, Inc. | Blood separation and analyte detection techniques |
| US5306623A (en) * | 1989-08-28 | 1994-04-26 | Lifescan, Inc. | Visual blood glucose concentration test strip |
| US6395227B1 (en) * | 1989-08-28 | 2002-05-28 | Lifescan, Inc. | Test strip for measuring analyte concentration over a broad range of sample volume |
| GB9002274D0 (en) * | 1990-02-01 | 1990-03-28 | Cranfield Biotech Ltd | Colorimetric analysis |
| US5139957A (en) * | 1990-05-24 | 1992-08-18 | American Sterilizer Company | Chemical indicator that includes potassium dichromate and urea and method of using the same to detect hydrogen peroxide |
| US5196302A (en) * | 1990-08-29 | 1993-03-23 | The United States Of America As Represented By The Sectetary Of The Navy | Enzymatic assays using superabsorbent materials |
| US5837546A (en) * | 1993-08-24 | 1998-11-17 | Metrika, Inc. | Electronic assay device and method |
| US7635597B2 (en) | 1995-08-09 | 2009-12-22 | Bayer Healthcare Llc | Dry reagent particle assay and device having multiple test zones and method therefor |
| US5776719A (en) * | 1997-07-07 | 1998-07-07 | Mercury Diagnostics, Inc. | Diagnostic compositions and devices utilizing same |
| US5989845A (en) | 1996-04-05 | 1999-11-23 | Mercury Diagnostics, Inc. | Diagnostic compositions and devices utilizing same |
| US5962215A (en) | 1996-04-05 | 1999-10-05 | Mercury Diagnostics, Inc. | Methods for testing the concentration of an analyte in a body fluid |
| US6040151A (en) | 1998-03-10 | 2000-03-21 | Mercury Diagnostics, Inc. | Diagnostic compositions and devices utilizing same |
| US5968839A (en) * | 1996-05-13 | 1999-10-19 | Metrika, Inc. | Method and device producing a predetermined distribution of detectable change in assays |
| US20020010406A1 (en) | 1996-05-17 | 2002-01-24 | Douglas Joel S. | Methods and apparatus for expressing body fluid from an incision |
| EP1579814A3 (en) | 1996-05-17 | 2006-06-14 | Roche Diagnostics Operations, Inc. | Methods and apparatus for sampling and analyzing body fluid |
| ATE286252T1 (en) | 1996-10-25 | 2005-01-15 | Idexx Lab Inc | IMMUNOASSAY DEVICE WITH A DIVISABLE HOUSING |
| ATE230115T1 (en) * | 1996-10-30 | 2003-01-15 | Amira Medical | SYCRONIZED ANALYTE TEST SYSTEM |
| US20050101032A1 (en) * | 1997-02-10 | 2005-05-12 | Metrika, Inc. | Assay device, composition, and method of optimizing assay sensitivity |
| US5948695A (en) | 1997-06-17 | 1999-09-07 | Mercury Diagnostics, Inc. | Device for determination of an analyte in a body fluid |
| US5968746A (en) | 1997-11-26 | 1999-10-19 | Schneider; David R. | Method and apparatus for preserving human saliva for testing |
| US6391005B1 (en) | 1998-03-30 | 2002-05-21 | Agilent Technologies, Inc. | Apparatus and method for penetration with shaft having a sensor for sensing penetration depth |
| US20060019404A1 (en) * | 1998-05-06 | 2006-01-26 | Blatt Joel M | Quantitative assay with extended dynamic range |
| US6554798B1 (en) | 1998-08-18 | 2003-04-29 | Medtronic Minimed, Inc. | External infusion device with remote programming, bolus estimator and/or vibration alarm capabilities |
| US7700305B2 (en) | 1999-09-17 | 2010-04-20 | N2Itive1 Innovations | Analyte detection |
| US6551842B1 (en) | 1999-03-26 | 2003-04-22 | Idexx Laboratories, Inc. | Method and device for detecting analytes in fluids |
| US6511814B1 (en) * | 1999-03-26 | 2003-01-28 | Idexx Laboratories, Inc. | Method and device for detecting analytes in fluids |
| US6458326B1 (en) | 1999-11-24 | 2002-10-01 | Home Diagnostics, Inc. | Protective test strip platform |
| GB0023057D0 (en) * | 2000-09-20 | 2000-11-01 | Randox Lab Ltd | Liquid reagent |
| US8641644B2 (en) | 2000-11-21 | 2014-02-04 | Sanofi-Aventis Deutschland Gmbh | Blood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means |
| US6525330B2 (en) | 2001-02-28 | 2003-02-25 | Home Diagnostics, Inc. | Method of strip insertion detection |
| US6562625B2 (en) | 2001-02-28 | 2003-05-13 | Home Diagnostics, Inc. | Distinguishing test types through spectral analysis |
| US6541266B2 (en) | 2001-02-28 | 2003-04-01 | Home Diagnostics, Inc. | Method for determining concentration of an analyte in a test strip |
| US9427532B2 (en) | 2001-06-12 | 2016-08-30 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| US8337419B2 (en) | 2002-04-19 | 2012-12-25 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| US7981056B2 (en) | 2002-04-19 | 2011-07-19 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
| US9226699B2 (en) | 2002-04-19 | 2016-01-05 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling module with a continuous compression tissue interface surface |
| US7025774B2 (en) | 2001-06-12 | 2006-04-11 | Pelikan Technologies, Inc. | Tissue penetration device |
| ES2336081T3 (en) | 2001-06-12 | 2010-04-08 | Pelikan Technologies Inc. | SELF-OPTIMIZATION PUNCTURE DEVICE WITH MEANS OF ADAPTATION TO TEMPORARY VARIATIONS IN CUTANEOUS PROPERTIES. |
| US9795747B2 (en) | 2010-06-02 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
| WO2002100254A2 (en) | 2001-06-12 | 2002-12-19 | Pelikan Technologies, Inc. | Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge |
| DE60238119D1 (en) | 2001-06-12 | 2010-12-09 | Pelikan Technologies Inc | ELECTRIC ACTUATOR ELEMENT FOR A LANZETTE |
| US6723500B2 (en) * | 2001-12-05 | 2004-04-20 | Lifescan, Inc. | Test strips having reaction zones and channels defined by a thermally transferred hydrophobic barrier |
| US6872358B2 (en) | 2002-01-16 | 2005-03-29 | Lifescan, Inc. | Test strip dispenser |
| US20030212379A1 (en) * | 2002-02-26 | 2003-11-13 | Bylund Adam David | Systems and methods for remotely controlling medication infusion and analyte monitoring |
| US7909778B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7331931B2 (en) | 2002-04-19 | 2008-02-19 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7976476B2 (en) | 2002-04-19 | 2011-07-12 | Pelikan Technologies, Inc. | Device and method for variable speed lancet |
| US8360992B2 (en) | 2002-04-19 | 2013-01-29 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US7674232B2 (en) | 2002-04-19 | 2010-03-09 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7892183B2 (en) | 2002-04-19 | 2011-02-22 | Pelikan Technologies, Inc. | Method and apparatus for body fluid sampling and analyte sensing |
| US8784335B2 (en) | 2002-04-19 | 2014-07-22 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling device with a capacitive sensor |
| US7232451B2 (en) | 2002-04-19 | 2007-06-19 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US7547287B2 (en) | 2002-04-19 | 2009-06-16 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US8221334B2 (en) | 2002-04-19 | 2012-07-17 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US9795334B2 (en) | 2002-04-19 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US7175642B2 (en) | 2002-04-19 | 2007-02-13 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
| US8267870B2 (en) | 2002-04-19 | 2012-09-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling with hybrid actuation |
| US9248267B2 (en) | 2002-04-19 | 2016-02-02 | Sanofi-Aventis Deustchland Gmbh | Tissue penetration device |
| US7491178B2 (en) | 2002-04-19 | 2009-02-17 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US9314194B2 (en) | 2002-04-19 | 2016-04-19 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| US7901362B2 (en) | 2002-04-19 | 2011-03-08 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US8702624B2 (en) | 2006-09-29 | 2014-04-22 | Sanofi-Aventis Deutschland Gmbh | Analyte measurement device with a single shot actuator |
| US8579831B2 (en) | 2002-04-19 | 2013-11-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
| US7708701B2 (en) | 2002-04-19 | 2010-05-04 | Pelikan Technologies, Inc. | Method and apparatus for a multi-use body fluid sampling device |
| US7229458B2 (en) | 2002-04-19 | 2007-06-12 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US8372016B2 (en) | 2002-04-19 | 2013-02-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling and analyte sensing |
| US7297122B2 (en) | 2002-04-19 | 2007-11-20 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
| US20030212344A1 (en) * | 2002-05-09 | 2003-11-13 | Vadim Yuzhakov | Physiological sample collection devices and methods of using the same |
| US7343188B2 (en) * | 2002-05-09 | 2008-03-11 | Lifescan, Inc. | Devices and methods for accessing and analyzing physiological fluid |
| US20030223906A1 (en) * | 2002-06-03 | 2003-12-04 | Mcallister Devin | Test strip container system |
| US20040087874A1 (en) * | 2002-10-28 | 2004-05-06 | David Schneider | Saliva collection system |
| US6927062B2 (en) * | 2002-11-25 | 2005-08-09 | Agdia, Inc. | Controls and standards for assays and method for manufacture thereof |
| US8574895B2 (en) | 2002-12-30 | 2013-11-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus using optical techniques to measure analyte levels |
| ATE476137T1 (en) | 2003-05-30 | 2010-08-15 | Pelikan Technologies Inc | METHOD AND DEVICE FOR INJECTING LIQUID |
| DK1633235T3 (en) | 2003-06-06 | 2014-08-18 | Sanofi Aventis Deutschland | Apparatus for sampling body fluid and detecting analyte |
| WO2006001797A1 (en) | 2004-06-14 | 2006-01-05 | Pelikan Technologies, Inc. | Low pain penetrating |
| EP1671096A4 (en) | 2003-09-29 | 2009-09-16 | Pelikan Technologies Inc | Method and apparatus for an improved sample capture device |
| EP1680014A4 (en) | 2003-10-14 | 2009-01-21 | Pelikan Technologies Inc | Method and apparatus for a variable user interface |
| EP1680175B1 (en) * | 2003-11-06 | 2019-06-05 | LifeScan, Inc. | Drug delivery pen with event notification means |
| EP1706026B1 (en) | 2003-12-31 | 2017-03-01 | Sanofi-Aventis Deutschland GmbH | Method and apparatus for improving fluidic flow and sample capture |
| US7822454B1 (en) | 2005-01-03 | 2010-10-26 | Pelikan Technologies, Inc. | Fluid sampling device with improved analyte detecting member configuration |
| US7150995B2 (en) * | 2004-01-16 | 2006-12-19 | Metrika, Inc. | Methods and systems for point of care bodily fluid analysis |
| US20050227370A1 (en) * | 2004-03-08 | 2005-10-13 | Ramel Urs A | Body fluid analyte meter & cartridge system for performing combined general chemical and specific binding assays |
| US8828203B2 (en) | 2004-05-20 | 2014-09-09 | Sanofi-Aventis Deutschland Gmbh | Printable hydrogels for biosensors |
| WO2005120365A1 (en) | 2004-06-03 | 2005-12-22 | Pelikan Technologies, Inc. | Method and apparatus for a fluid sampling device |
| US9775553B2 (en) | 2004-06-03 | 2017-10-03 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a fluid sampling device |
| US8652831B2 (en) | 2004-12-30 | 2014-02-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for analyte measurement test time |
| US20070031914A1 (en) * | 2005-08-05 | 2007-02-08 | Wei Zhu | Devices for analyte assays and methods of use |
| WO2009126900A1 (en) | 2008-04-11 | 2009-10-15 | Pelikan Technologies, Inc. | Method and apparatus for analyte detecting device |
| US9375169B2 (en) | 2009-01-30 | 2016-06-28 | Sanofi-Aventis Deutschland Gmbh | Cam drive for managing disposable penetrating member actions with a single motor and motor and control system |
| US8965476B2 (en) | 2010-04-16 | 2015-02-24 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
| EP2629663B1 (en) * | 2010-10-23 | 2018-01-10 | Pop Test LLC | Devices and formulations for detecting, screening and monitoring levels of certain constituents in bodily fluids and method |
| WO2015038555A2 (en) * | 2013-09-11 | 2015-03-19 | University Of Massachustetts | Rapid colorimetric detection of bacteria in water |
| FI126484B (en) | 2013-12-03 | 2017-01-13 | Goodwiller Oy | Disposable test strip device for detecting analyte in a body fluid sample |
| EP3172570A4 (en) | 2014-07-25 | 2017-12-27 | Becton, Dickinson and Company | Analyte test strip assays, and test strips and kits for use in practicing the same |
| US20160313307A1 (en) * | 2015-04-23 | 2016-10-27 | Ted Titmus | Diagnostic test strip for oral samples and method of use therefore |
| CN109477837B (en) | 2016-09-28 | 2022-09-16 | 泰尔茂株式会社 | Method, composition and chip for detecting analyte in blood sample |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3413198A (en) * | 1966-06-30 | 1968-11-26 | Calbiochem | Reagents and method for assaying biological samples |
| US3992158A (en) * | 1973-08-16 | 1976-11-16 | Eastman Kodak Company | Integral analytical element |
| US3926736A (en) * | 1974-08-30 | 1975-12-16 | Calbiochem | Enzymatic ethanol assay |
| CA1048390A (en) * | 1975-02-14 | 1979-02-13 | James B. Dugle | Method, composition, and device for determining the specific gravity of a liquid |
| ZA76233B (en) * | 1976-01-15 | 1977-08-31 | Chembro Holdings Pty Ltd | Measurement of alcohol levels in body fluids |
| US4430427A (en) * | 1980-11-04 | 1984-02-07 | Phillips Petroleum Company | Red absorbing combination of alcohol oxidase and an azide compound |
| JPS57197466A (en) * | 1981-04-29 | 1982-12-03 | Konishiroku Photo Ind Co Ltd | Analysis element |
| JPS589688A (en) * | 1981-07-06 | 1983-01-20 | Toyobo Co Ltd | Stable enzymic composition |
| US4414334A (en) * | 1981-08-07 | 1983-11-08 | Phillips Petroleum Company | Oxygen scavenging with enzymes |
| JPS5886083A (en) * | 1981-11-12 | 1983-05-23 | Wako Pure Chem Ind Ltd | Stabilizing agent for glycerol-3-phosphoric acid oxidase |
| US4450153A (en) * | 1982-09-30 | 1984-05-22 | Phillips Petroleum Company | Alcohol removal from blood with alcohol oxidase |
| CA1205731A (en) * | 1982-11-01 | 1986-06-10 | Roger C. Phillips | Test device and method for measurement of analyte levels in colored aqueous fluids |
| US4642286A (en) * | 1984-05-07 | 1987-02-10 | Moldowan Mervin J | Composition and method for ethanol determination |
-
1984
- 1984-06-26 US US06/624,774 patent/US4734360A/en not_active Expired - Fee Related
- 1984-07-11 FI FI842790A patent/FI81119C/en not_active IP Right Cessation
- 1984-07-11 NO NO842827A patent/NO165316C/en unknown
- 1984-07-11 AT AT84108115T patent/ATE30174T1/en active
- 1984-07-11 EP EP84108115A patent/EP0133481B1/en not_active Expired
- 1984-07-11 CA CA000458581A patent/CA1225912A/en not_active Expired
- 1984-07-11 IL IL72375A patent/IL72375A/en unknown
- 1984-07-11 DE DE8484108115T patent/DE3466709D1/en not_active Expired
- 1984-07-11 DK DK340184A patent/DK340184A/en not_active Application Discontinuation
- 1984-07-12 JP JP59143383A patent/JPS6075299A/en active Granted
- 1984-07-12 AU AU30539/84A patent/AU586150B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| NO165316B (en) | 1990-10-15 |
| AU3053984A (en) | 1985-01-17 |
| NO165316C (en) | 1991-01-23 |
| JPH0549275B2 (en) | 1993-07-23 |
| ATE30174T1 (en) | 1987-10-15 |
| NO842827L (en) | 1985-01-14 |
| FI842790A0 (en) | 1984-07-11 |
| FI81119B (en) | 1990-05-31 |
| EP0133481A1 (en) | 1985-02-27 |
| DK340184D0 (en) | 1984-07-11 |
| IL72375A (en) | 1988-04-29 |
| FI81119C (en) | 1990-09-10 |
| JPS6075299A (en) | 1985-04-27 |
| EP0133481B1 (en) | 1987-10-07 |
| DE3466709D1 (en) | 1987-11-12 |
| FI842790A (en) | 1985-01-13 |
| US4734360A (en) | 1988-03-29 |
| AU586150B2 (en) | 1989-07-06 |
| DK340184A (en) | 1985-01-13 |
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