CA2026586A1 - Method and element for assay of catechol and catechol generating substances - Google Patents
Method and element for assay of catechol and catechol generating substancesInfo
- Publication number
- CA2026586A1 CA2026586A1 CA002026586A CA2026586A CA2026586A1 CA 2026586 A1 CA2026586 A1 CA 2026586A1 CA 002026586 A CA002026586 A CA 002026586A CA 2026586 A CA2026586 A CA 2026586A CA 2026586 A1 CA2026586 A1 CA 2026586A1
- Authority
- CA
- Canada
- Prior art keywords
- catechol
- salicylate
- sample
- nadh
- leuco dye
- 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.)
- Abandoned
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
- Y10S435/808—Optical sensing apparatus
-
- 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
- Y10S435/97—Test strip or test slide
Abstract
METHOD AND ELEMENT FOR AS SAY OF CATECHOL AND
CATECHOL GENERATING SUBSTANCES
Abstract of the Disclosure A method and multilayer analytical element for the determination of catechol and catechol generating substances such as salicylate is described. A series of enzymatic conversions involving tyrosinase is used to convert catechol to o-quinone and the latter to convert a leuco dye to a colored dye.
CATECHOL GENERATING SUBSTANCES
Abstract of the Disclosure A method and multilayer analytical element for the determination of catechol and catechol generating substances such as salicylate is described. A series of enzymatic conversions involving tyrosinase is used to convert catechol to o-quinone and the latter to convert a leuco dye to a colored dye.
Description
0 2 6 ~
METHOD AND ELEMENT FOR ASSAY OF CATECHOL AND
CATECHOL GENERATING SUBSTANCES
Fie~d Qf ~he_Invention This invention relates to clinical analyses 5 and, in particular, to a method and element for the assaying catechol, catechol ~enerating substances such as salicylate and enzymes that catalyze generation of catechol.
BACKGROUND!~E~DI~D~ENTION
The determination of the catechol generating substance salicylate in biological fluids such as human serum, has diagnostic significance.
Acetylsalicylic acid (aspirin) is used as an analgesic and as an anti-inflammatory drug for 15 arthritis. It rapidly hydrolyzes to salicylate which has the therapeutic effect. The therapeutic level as an analgesic is up to ?O mg/dl. For arthritis the level is up to 30 mg/dl. Problems such as headaches, tinnitus, flushing and hyperventilation occur at 20 higher salicylate levels followed by imbalances in the acid-base level. Salicylate levels above 60 mg/dl can be lethal.
One method for assaying salicylate employs the enzymatic conversion of salicylate to catechol 25 catalyzed by salicylate hydroxylase with the accompanying conversion of NADH to NAD+. There is a quantitative correlation between disappearance of NADH, as reflected in a change in optical density at 340 nm, and the concentration of salicylate (You and 30 Bittikofer, Clin. Chem., 30:1549, 1984). The problem is that this method is suitable for incorporation into a dry format to be stored over time because of the instability of NADH at low pH and the low extinction coefficient of NADH
Other problems of existing methods for the determination of salicylate suffer from poor -2- 202S5~
sensitivity NADH method, interferences from phenolic and ketoacids normally present in serum Ferric chloride, time-consuming procedures, ina~ility to measure protein bound salicylate (measure only free 5 salicylate refers to ion selective electrodes), or unsuitability for dry format.
SUMMARY OF THE INVENTION
The present invention provides a colori-metric method for a quantitative assay of catechol, 10 including catechol generating substances such as salicylate, and enzymes that catalyze generation of catechol. The method comprises the steps of:
a) providing a sample suspected of containing catechol, a catechol generating substance such as salicylate or an enzyme that catalyzes generation of catechol; and b) admixing with the sample NADH, tyrosinase, a colorless leuco dye; or alternatively i) admixing with the admixture of b) an enzyme, such as salicylate hydroxylase, selected to catalyze the conversion of the catechol generating substance to catechol; or ii) admixing with the admixture of b) an enzyme substrate that generates catechol.
c) determining colorimetrically the quantita-tive presence in the sample of catechol, the catechol generating substance such as salicylate, or the enzyme that catalyzes generation of catechol from the enzyme substrate.
The present invention also provides an analytical element for assaying catechol, including 35 any catechol generating substances such as salicylate, or an enzyme that catalyzes generation of _3_ 202~3~
catechol from the catechol generating substance (enzyme substrate). The element comprises one layer containing tyrosinase and another layer comprising NADH and a colorless form of a leuco dye in either 5 layer The NADH free layer contains the enzyme selected to catalyzed generation of catechol by reaction with NADH when the assay is directed to such catechol generating substances. Alternatively the 10 element contains a substrate for a particular enzyme when the objective of the assay is that particular enzyme. The inclusion in the element of salicylate hydroxylase for the assay of its substrate, salicylate is a preferred embodiment of this aspect 15 of the invention.
The present invention further provides a dry multilayer analytical element for quantitatively assaying salicylate comprising a support bearing at least 2 layers, wherein:
a) one layer comprises NADH, and b) the other layer comprises tyrosinase and a colorless leuco dye.
This element also includes salicylate hydroxylase when the assay is conducted by omitting a step of 25 adding the salicylate hydroxylase to the sample to be assayed.
The method and element o~ this invention obviates the problems associated with measuring the disappearance of NADH since the measurement is made 30 of a dye. Thus the instability and low extinction coefficient of NADH at 340 nm is eliminated as an assay limiting factor. Moreover many of the other mentioned problems are ameliorated or entirely eliminated.
DETAILED DESCRI?TION OF THE INVENTION
The present invention provides a method and analytical elements for assaying salicylate, catechol -4- 202~a~
and other catechol generating substances. The method and elements are based on a series of enzymatic conversions. The chemical reactions involved a~e illustrated using salicylate. However it will be 5 clear to all analytical chemists that this set of reactions can be easily adapted to the teachings herein for the analysis of catechol, any other catechol generating substance and the enzymes catalyst used in the reactions.
Salicylate hydroxylase in the presence of NADH converts salicylate to pyrocatechol (Eq. 1) which is oxidized by oxygen in the presence of tyrosinase to o-quinone (Eq. 2). The o-quinone is then quantitatively determined by the oxidation of a 15 suitable leuco dye or the oxidative coupling of a developer and a coupler, to yield a dye (Eq. 3).
Equation 1~:
20 I 0 + NADH SallCvlate ~ I `0' co NAD
Equation 2):
2 I + 2 Tylo i_a Q> 2 I I + 2 H20 Equation 3~:
I I + Leuco~ e, Dye ~0 In versions of the elements for assay of catechol the assay is carried out by simple spotting 35 of the sample on the element. In versions for assaying a catechol generating substances such as -5- 2~2~8~
salicylate, the enzyme may be included in the element or added to the sample to be analyzed. In versions for assaying an enzyme the enzyme substrate may be added to the sample or to the element. The assays 5 can be carried out both qualitatively and quantita-tively in biological fluids in animals or humans, but preferably of humans. Such fluids include, but are not limited to, whole blood, plasma, sera, lymph, bile, urine, spinal fluid, sputum, perspiration and 10 the like as well as stool secretions. It is also possible to assay fluid preparations of human or animal tissue such as skeletal muscle, heart, kidney, lungs, brains, bone marrow, skin and the like.
Examples of catechol generating substances 15 other than salicylate are known. For e2ample, o-hydroxyphenyl phosphate is hydrolyzed to catechol by alkaline or acid phosphatase. Tyrosine 0-phosphate can be hydrolyzed by prostatic acid phosphatase. In each of the these hydrolyzations 20 samples containing o--hydroxyphenyl phosphate and tyrosine can be assayed quantitatively. Indeed acid phosphatase can be assayed in a single step.
The various reagents used in the method and elements are available commercially. Tyrosinase is 25 also available commercially.
Any leuco dye which changes from colorless to a measurable color will be useful in the present invention. Such dyes include triarylimidazoles and triarylmethanes compounds. A method for identifying 30 useful dyes for detecting o-quinone and o-quinone generating materials such as catechol and salicylate is disclosed in U.S.Patent 4,089,747. Useful leuco dyes are also disclosed in the latter patent. Thus leuco dyes from which useful substances for the 35 present invention can be chosen, using an objective and easily performed test are well know in the -6- 202~8~
analytical arts. Particularly useful dyes are triaryimidazoles, triarylmethanes and reduced indophenols.
Elements of the invention can be configured 5 in a variety of forms, including elongated tapes of any desired width, sheets, slides or chips.
The elements can be used in manual or automated assay techniques. In general, in using the elements, assays are made by taking the element from 10 a supply roll, chip packet or other source and physically contacting it with a sample (for example, up to 200 yl) of the liquid to be tested so that the sample and reagents interact sequentially within the element become mixed. Such contact can be 15 accomplished in any suitable manner, for example, by dipping or immersing the element into the sample or, preferably, by spotting the element by hand or machine with a drop of the sample with a suitable dispensing means.
After sample application, the element is incubated, for a period of up to 5 minutes, to facilitate color development. By incubation, we simply mean that the reagents are maintained in contact with each other for a period of up to 5 25 minutes before color measurements are made.
The dry analytical elements of this invention are multilayered. At least one of the layers has a spreading function as that function is known in the art. The layers include the reagents 30 used in the method of the invention. The reagents are in two distinct layers or two distinct zones.
The NADH is in a zone or layer separate from the other reagents except the leuco dye which can be in either zone or layer. All of the foregoing layers 35 are coated on a support. The layers are generally in fluid contact with each other, meaning that fluids, -7- 2026~86 reagents and reaction products (for example, color dyes) can pass or be transported between superposed regions of adjacent zones. In other words, when the element is contacted with an aqueous flui~, all reagents of the analytical composition of this invention mixed sequentially as stated hereinbefore and can readily move within the element as a composition. Each layer can be separate or two or more zones can be separate areas in a single layer of the element. Besides the references noted above, suitable element components are described also, for example, in U. S. Patents 4,042,335 (issued August 16, 1977 to Clément), 4,132,528 (issued January 2, 1979 to Eikenberry et al), and 4,144,306 (issued 15 March 13, 1979 to Figueras).
Useful spreading layers can be prepared using fibrous materials, either mixed with a suitable binder material or woven into a fabric, as described in U. S. Patent 4,292,272 (issued September 29, 1981 to Kitajima et al), polymeric compositions or particulate materials, for example a blush polymer such as disclosed in U.S. Patent 3,992,158, beads bound together with or without binding adhesives, as described in U. S. Patents 4,258,001 (issued March 24, 1981 to Pierce et al) and 4,430,436 (issued February 7, 1984 to Koyama et al) and Japanese Patent Publication 57(1982)-101760. Particularly useful spreading layers comprise barium sulphate or titanium dioxide. Since the sample is generally applied directly to the spreading layer, it is desirable that the spreading layer be isotropically porous, meaning that the porosity is the same in each direction in the layer as caused by interconnected spaces or pores between particles, fibers or polymeric strands.
The layers can be coated on transparent supports such as polyethylene terephthalate. Other supports are well known in the art.
-8- 202~
The elements of this invention can also contain one or more other addenda commonly put in the elements for various manufacturing or operational advantages. Such addenda include surfactants, 5 buffers, solvents, hardeners and other materials known in the art.
The following examples clearly establish the improved aspects of the present invention.
' ~Surfactants TX-100, TX-405, lOG, Alkanol XC and Zonyl 10 FSN and the enzyme, tyrosinase and the other chemicals used in the following examples are all ~commercially available. Triton X-100 (TX-100), and Triton X-405 (TX-405) are from Rohm and Haas;
` Peroxidase and Tyrosinase are from Sigma. The leuco 15 dye used was 2-(4-hydroxy-4,5-dimethoxyphenyl)-4,5-bis(4-dimethylaminophenyl)imidazole. Unless otherwise stated, all other materials were prepared by or obtained from Eastman Kodak Company 0 Example 1: Determination of Salicvlate Concen-trat_Q~on Element of Invention The element used in this example had the following configuration and reagent content.
202~
_9_ Preferred Concentration Concentration _ _ __ Cglm2? _ ____ lelg~//
Reagent Layer:
Tyrosinase 10,000 - 60,000 U/m225,000 U/m2 K2HP04 0.3 - 2.4 0.76 KH2PO4 0.2 - 1.6 0.40 10G Surfactant Q.03 - 0.25 0.11 Reagent/Spreading Zone:
BaSO4 50 - 175 108 Cellulose Acetane 0.4 - 1,2 0.6 ~Polyurethane 1 - 5 1.08 TX-100 0.2 - 2.5 1.35 K2HPO4 0.4 - 3 5 1.15 NaH2PO4 0.25 - 2 0.70 **Dimedone 0.08 - 1.44 0.32 932480* 0.01 - 0.1 0.03 185421* 0.6 - 1.8 1.08 Sub Layer:
poly-n-isopropyl-acrvlamide 0 05 - 2.0 _ _ 0.27 _ _ Gelatin Layer (pH 7.0):
Gelatin 3 - 20 10 TX-100 0.01 - 0.1 0.03 TX-405 0.01 - 0.1 0.03 NaH2P4 0 09 - 0.81 0.27 ***MWaMt 2 - 12 5 . _ . . . _ L ~L_I I I 1_1 1 i I l I L I I I I I I I 1_1 I L l I
* Polyurethane from B.F. Goodrich ** Antioxidant 35 ~*Copoly[methacrylamide-2-(sulfo-1,1-dimethyl-acrylamide,Na salt)-~2-acetoacetoxyethyl methacrylate)]
~lo- 202~
A separate series of solutions containing 1,2,4,6 and lO mM/L of salicylic acid and NADH were combined in a volume ratio of 1:1. The result was a series of test solutions containing 0.5, 1.0, 2.0, 5 3.0 and 5.0 mM/mL of salicylic acid and NADH ~s shown in Table I Each of the listed solutions in column A
was combined with 1.0 mL of a solution containing 100 U/mL of salicylate hydroxylase. Each of the resulting solutions were spotted on separate dry 10 analytical elements having the configuration and reagent content described above. The reflectance density at 670 nm for each solution is presented in Table I.
T A B L E
Test Solution Salicylic Acid Reflectance No. Concentration mM/mL Densitv_at 670 nm 1 0.5 .145 2 1.0 .240 3 2.~ 300 4 3.0 .405 5.0 .535 The reflectance density shows that the 25 method and element of this invention are well suited for assaying salicylate in aqueous fluids. Other experiments conducted with this same element show that it is all well suited for assaying catechol, including catechol generating substances.
Example 2:
Another element embodiment embodying the method of this invention is presented below.
--11 2 02 ~
Prefer r ed Concentration Concentration (g/m2~ (g/m2) _ _ _ _ _ _ _ _ _ Tio2 Spreading Layer _ _ _ _ _ _ __ Subbing Layer:
poly-n-isopropyl-acrylamide I-100 Layer Range (g/m2) Reagent Layer Zone 1:
Gelatin 6.0 g/m2 3 - 12 Zonyl FSN
(surfactant) 0 03 g/m2 0.01 - 0.2 K2HP04 1-40 g/m2 0.7 - 2.3 KH2P4 0.13 g/m2 0.06 0.19 NaCl 0 74 g/m2 0.5 - 1.5 bisvinylsulfonyl-methyl ether 0 03 g/m2 0.15 - 1.2 NADH 0-76 g/m2 0.5 - 1.5 . _ _ . _ Reagent Layer Zone 2:
Gelatin 6.0 g/m2 3 - 12 Zonyl FSN
(surfactant) 0.03 g/m 0.01 - 0.2 K2HPO4 1-40 g/m2 0.7 - 2.3 KH2PO4 0.13 g/m 0.06 - 0.19 NaCl 0.74 g/m 0.25 - 1.0 Alkanol XC 0.21 g/m2 0.05 - 0.5 Leuco-Dye 0.53 g/m 0.2 - 0.8 di-n-pentyl phenol 5 35 g/m2 2 - 8 Dimedone 0.13 g/m2 0.05 - 0.3 Salicylate Hydroxylase 1,000 U/m2 400 - 2,500 TYROSINASE 100,000 U/m2 25,000-150,000 _ ~X 7.6 __ ESTAR _ _ -12- 202~5~6 A series of solutions were prepared containing 0.2, 1.4, 2.9, 4.3, 5.8 and 7.2 mM/L of salicylic acid (sodium salt). Each of the solutions was spotted on separate dry analytical elements 5 having the configuration and reagent content described above. The reflectance density at 670 nm for each solution is presented in table II.
T A B L E II
10 Test SolutionSodium SalicylateReflectance No. mM/mQ _ Density at 670 nm 1 0.2 0.22 2 1.4 0.36 3 2.9 0.38 4 4.3 0.43 5.8 0.45 6 7.2 0.47 The reflectance density again shows that the method 20 and element of this invention are well suited for assaying salicylate in aqueous fluids.
The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations 25 and modifications can be effected within the spirit and scope of the invention.
METHOD AND ELEMENT FOR ASSAY OF CATECHOL AND
CATECHOL GENERATING SUBSTANCES
Fie~d Qf ~he_Invention This invention relates to clinical analyses 5 and, in particular, to a method and element for the assaying catechol, catechol ~enerating substances such as salicylate and enzymes that catalyze generation of catechol.
BACKGROUND!~E~DI~D~ENTION
The determination of the catechol generating substance salicylate in biological fluids such as human serum, has diagnostic significance.
Acetylsalicylic acid (aspirin) is used as an analgesic and as an anti-inflammatory drug for 15 arthritis. It rapidly hydrolyzes to salicylate which has the therapeutic effect. The therapeutic level as an analgesic is up to ?O mg/dl. For arthritis the level is up to 30 mg/dl. Problems such as headaches, tinnitus, flushing and hyperventilation occur at 20 higher salicylate levels followed by imbalances in the acid-base level. Salicylate levels above 60 mg/dl can be lethal.
One method for assaying salicylate employs the enzymatic conversion of salicylate to catechol 25 catalyzed by salicylate hydroxylase with the accompanying conversion of NADH to NAD+. There is a quantitative correlation between disappearance of NADH, as reflected in a change in optical density at 340 nm, and the concentration of salicylate (You and 30 Bittikofer, Clin. Chem., 30:1549, 1984). The problem is that this method is suitable for incorporation into a dry format to be stored over time because of the instability of NADH at low pH and the low extinction coefficient of NADH
Other problems of existing methods for the determination of salicylate suffer from poor -2- 202S5~
sensitivity NADH method, interferences from phenolic and ketoacids normally present in serum Ferric chloride, time-consuming procedures, ina~ility to measure protein bound salicylate (measure only free 5 salicylate refers to ion selective electrodes), or unsuitability for dry format.
SUMMARY OF THE INVENTION
The present invention provides a colori-metric method for a quantitative assay of catechol, 10 including catechol generating substances such as salicylate, and enzymes that catalyze generation of catechol. The method comprises the steps of:
a) providing a sample suspected of containing catechol, a catechol generating substance such as salicylate or an enzyme that catalyzes generation of catechol; and b) admixing with the sample NADH, tyrosinase, a colorless leuco dye; or alternatively i) admixing with the admixture of b) an enzyme, such as salicylate hydroxylase, selected to catalyze the conversion of the catechol generating substance to catechol; or ii) admixing with the admixture of b) an enzyme substrate that generates catechol.
c) determining colorimetrically the quantita-tive presence in the sample of catechol, the catechol generating substance such as salicylate, or the enzyme that catalyzes generation of catechol from the enzyme substrate.
The present invention also provides an analytical element for assaying catechol, including 35 any catechol generating substances such as salicylate, or an enzyme that catalyzes generation of _3_ 202~3~
catechol from the catechol generating substance (enzyme substrate). The element comprises one layer containing tyrosinase and another layer comprising NADH and a colorless form of a leuco dye in either 5 layer The NADH free layer contains the enzyme selected to catalyzed generation of catechol by reaction with NADH when the assay is directed to such catechol generating substances. Alternatively the 10 element contains a substrate for a particular enzyme when the objective of the assay is that particular enzyme. The inclusion in the element of salicylate hydroxylase for the assay of its substrate, salicylate is a preferred embodiment of this aspect 15 of the invention.
The present invention further provides a dry multilayer analytical element for quantitatively assaying salicylate comprising a support bearing at least 2 layers, wherein:
a) one layer comprises NADH, and b) the other layer comprises tyrosinase and a colorless leuco dye.
This element also includes salicylate hydroxylase when the assay is conducted by omitting a step of 25 adding the salicylate hydroxylase to the sample to be assayed.
The method and element o~ this invention obviates the problems associated with measuring the disappearance of NADH since the measurement is made 30 of a dye. Thus the instability and low extinction coefficient of NADH at 340 nm is eliminated as an assay limiting factor. Moreover many of the other mentioned problems are ameliorated or entirely eliminated.
DETAILED DESCRI?TION OF THE INVENTION
The present invention provides a method and analytical elements for assaying salicylate, catechol -4- 202~a~
and other catechol generating substances. The method and elements are based on a series of enzymatic conversions. The chemical reactions involved a~e illustrated using salicylate. However it will be 5 clear to all analytical chemists that this set of reactions can be easily adapted to the teachings herein for the analysis of catechol, any other catechol generating substance and the enzymes catalyst used in the reactions.
Salicylate hydroxylase in the presence of NADH converts salicylate to pyrocatechol (Eq. 1) which is oxidized by oxygen in the presence of tyrosinase to o-quinone (Eq. 2). The o-quinone is then quantitatively determined by the oxidation of a 15 suitable leuco dye or the oxidative coupling of a developer and a coupler, to yield a dye (Eq. 3).
Equation 1~:
20 I 0 + NADH SallCvlate ~ I `0' co NAD
Equation 2):
2 I + 2 Tylo i_a Q> 2 I I + 2 H20 Equation 3~:
I I + Leuco~ e, Dye ~0 In versions of the elements for assay of catechol the assay is carried out by simple spotting 35 of the sample on the element. In versions for assaying a catechol generating substances such as -5- 2~2~8~
salicylate, the enzyme may be included in the element or added to the sample to be analyzed. In versions for assaying an enzyme the enzyme substrate may be added to the sample or to the element. The assays 5 can be carried out both qualitatively and quantita-tively in biological fluids in animals or humans, but preferably of humans. Such fluids include, but are not limited to, whole blood, plasma, sera, lymph, bile, urine, spinal fluid, sputum, perspiration and 10 the like as well as stool secretions. It is also possible to assay fluid preparations of human or animal tissue such as skeletal muscle, heart, kidney, lungs, brains, bone marrow, skin and the like.
Examples of catechol generating substances 15 other than salicylate are known. For e2ample, o-hydroxyphenyl phosphate is hydrolyzed to catechol by alkaline or acid phosphatase. Tyrosine 0-phosphate can be hydrolyzed by prostatic acid phosphatase. In each of the these hydrolyzations 20 samples containing o--hydroxyphenyl phosphate and tyrosine can be assayed quantitatively. Indeed acid phosphatase can be assayed in a single step.
The various reagents used in the method and elements are available commercially. Tyrosinase is 25 also available commercially.
Any leuco dye which changes from colorless to a measurable color will be useful in the present invention. Such dyes include triarylimidazoles and triarylmethanes compounds. A method for identifying 30 useful dyes for detecting o-quinone and o-quinone generating materials such as catechol and salicylate is disclosed in U.S.Patent 4,089,747. Useful leuco dyes are also disclosed in the latter patent. Thus leuco dyes from which useful substances for the 35 present invention can be chosen, using an objective and easily performed test are well know in the -6- 202~8~
analytical arts. Particularly useful dyes are triaryimidazoles, triarylmethanes and reduced indophenols.
Elements of the invention can be configured 5 in a variety of forms, including elongated tapes of any desired width, sheets, slides or chips.
The elements can be used in manual or automated assay techniques. In general, in using the elements, assays are made by taking the element from 10 a supply roll, chip packet or other source and physically contacting it with a sample (for example, up to 200 yl) of the liquid to be tested so that the sample and reagents interact sequentially within the element become mixed. Such contact can be 15 accomplished in any suitable manner, for example, by dipping or immersing the element into the sample or, preferably, by spotting the element by hand or machine with a drop of the sample with a suitable dispensing means.
After sample application, the element is incubated, for a period of up to 5 minutes, to facilitate color development. By incubation, we simply mean that the reagents are maintained in contact with each other for a period of up to 5 25 minutes before color measurements are made.
The dry analytical elements of this invention are multilayered. At least one of the layers has a spreading function as that function is known in the art. The layers include the reagents 30 used in the method of the invention. The reagents are in two distinct layers or two distinct zones.
The NADH is in a zone or layer separate from the other reagents except the leuco dye which can be in either zone or layer. All of the foregoing layers 35 are coated on a support. The layers are generally in fluid contact with each other, meaning that fluids, -7- 2026~86 reagents and reaction products (for example, color dyes) can pass or be transported between superposed regions of adjacent zones. In other words, when the element is contacted with an aqueous flui~, all reagents of the analytical composition of this invention mixed sequentially as stated hereinbefore and can readily move within the element as a composition. Each layer can be separate or two or more zones can be separate areas in a single layer of the element. Besides the references noted above, suitable element components are described also, for example, in U. S. Patents 4,042,335 (issued August 16, 1977 to Clément), 4,132,528 (issued January 2, 1979 to Eikenberry et al), and 4,144,306 (issued 15 March 13, 1979 to Figueras).
Useful spreading layers can be prepared using fibrous materials, either mixed with a suitable binder material or woven into a fabric, as described in U. S. Patent 4,292,272 (issued September 29, 1981 to Kitajima et al), polymeric compositions or particulate materials, for example a blush polymer such as disclosed in U.S. Patent 3,992,158, beads bound together with or without binding adhesives, as described in U. S. Patents 4,258,001 (issued March 24, 1981 to Pierce et al) and 4,430,436 (issued February 7, 1984 to Koyama et al) and Japanese Patent Publication 57(1982)-101760. Particularly useful spreading layers comprise barium sulphate or titanium dioxide. Since the sample is generally applied directly to the spreading layer, it is desirable that the spreading layer be isotropically porous, meaning that the porosity is the same in each direction in the layer as caused by interconnected spaces or pores between particles, fibers or polymeric strands.
The layers can be coated on transparent supports such as polyethylene terephthalate. Other supports are well known in the art.
-8- 202~
The elements of this invention can also contain one or more other addenda commonly put in the elements for various manufacturing or operational advantages. Such addenda include surfactants, 5 buffers, solvents, hardeners and other materials known in the art.
The following examples clearly establish the improved aspects of the present invention.
' ~Surfactants TX-100, TX-405, lOG, Alkanol XC and Zonyl 10 FSN and the enzyme, tyrosinase and the other chemicals used in the following examples are all ~commercially available. Triton X-100 (TX-100), and Triton X-405 (TX-405) are from Rohm and Haas;
` Peroxidase and Tyrosinase are from Sigma. The leuco 15 dye used was 2-(4-hydroxy-4,5-dimethoxyphenyl)-4,5-bis(4-dimethylaminophenyl)imidazole. Unless otherwise stated, all other materials were prepared by or obtained from Eastman Kodak Company 0 Example 1: Determination of Salicvlate Concen-trat_Q~on Element of Invention The element used in this example had the following configuration and reagent content.
202~
_9_ Preferred Concentration Concentration _ _ __ Cglm2? _ ____ lelg~//
Reagent Layer:
Tyrosinase 10,000 - 60,000 U/m225,000 U/m2 K2HP04 0.3 - 2.4 0.76 KH2PO4 0.2 - 1.6 0.40 10G Surfactant Q.03 - 0.25 0.11 Reagent/Spreading Zone:
BaSO4 50 - 175 108 Cellulose Acetane 0.4 - 1,2 0.6 ~Polyurethane 1 - 5 1.08 TX-100 0.2 - 2.5 1.35 K2HPO4 0.4 - 3 5 1.15 NaH2PO4 0.25 - 2 0.70 **Dimedone 0.08 - 1.44 0.32 932480* 0.01 - 0.1 0.03 185421* 0.6 - 1.8 1.08 Sub Layer:
poly-n-isopropyl-acrvlamide 0 05 - 2.0 _ _ 0.27 _ _ Gelatin Layer (pH 7.0):
Gelatin 3 - 20 10 TX-100 0.01 - 0.1 0.03 TX-405 0.01 - 0.1 0.03 NaH2P4 0 09 - 0.81 0.27 ***MWaMt 2 - 12 5 . _ . . . _ L ~L_I I I 1_1 1 i I l I L I I I I I I I 1_1 I L l I
* Polyurethane from B.F. Goodrich ** Antioxidant 35 ~*Copoly[methacrylamide-2-(sulfo-1,1-dimethyl-acrylamide,Na salt)-~2-acetoacetoxyethyl methacrylate)]
~lo- 202~
A separate series of solutions containing 1,2,4,6 and lO mM/L of salicylic acid and NADH were combined in a volume ratio of 1:1. The result was a series of test solutions containing 0.5, 1.0, 2.0, 5 3.0 and 5.0 mM/mL of salicylic acid and NADH ~s shown in Table I Each of the listed solutions in column A
was combined with 1.0 mL of a solution containing 100 U/mL of salicylate hydroxylase. Each of the resulting solutions were spotted on separate dry 10 analytical elements having the configuration and reagent content described above. The reflectance density at 670 nm for each solution is presented in Table I.
T A B L E
Test Solution Salicylic Acid Reflectance No. Concentration mM/mL Densitv_at 670 nm 1 0.5 .145 2 1.0 .240 3 2.~ 300 4 3.0 .405 5.0 .535 The reflectance density shows that the 25 method and element of this invention are well suited for assaying salicylate in aqueous fluids. Other experiments conducted with this same element show that it is all well suited for assaying catechol, including catechol generating substances.
Example 2:
Another element embodiment embodying the method of this invention is presented below.
--11 2 02 ~
Prefer r ed Concentration Concentration (g/m2~ (g/m2) _ _ _ _ _ _ _ _ _ Tio2 Spreading Layer _ _ _ _ _ _ __ Subbing Layer:
poly-n-isopropyl-acrylamide I-100 Layer Range (g/m2) Reagent Layer Zone 1:
Gelatin 6.0 g/m2 3 - 12 Zonyl FSN
(surfactant) 0 03 g/m2 0.01 - 0.2 K2HP04 1-40 g/m2 0.7 - 2.3 KH2P4 0.13 g/m2 0.06 0.19 NaCl 0 74 g/m2 0.5 - 1.5 bisvinylsulfonyl-methyl ether 0 03 g/m2 0.15 - 1.2 NADH 0-76 g/m2 0.5 - 1.5 . _ _ . _ Reagent Layer Zone 2:
Gelatin 6.0 g/m2 3 - 12 Zonyl FSN
(surfactant) 0.03 g/m 0.01 - 0.2 K2HPO4 1-40 g/m2 0.7 - 2.3 KH2PO4 0.13 g/m 0.06 - 0.19 NaCl 0.74 g/m 0.25 - 1.0 Alkanol XC 0.21 g/m2 0.05 - 0.5 Leuco-Dye 0.53 g/m 0.2 - 0.8 di-n-pentyl phenol 5 35 g/m2 2 - 8 Dimedone 0.13 g/m2 0.05 - 0.3 Salicylate Hydroxylase 1,000 U/m2 400 - 2,500 TYROSINASE 100,000 U/m2 25,000-150,000 _ ~X 7.6 __ ESTAR _ _ -12- 202~5~6 A series of solutions were prepared containing 0.2, 1.4, 2.9, 4.3, 5.8 and 7.2 mM/L of salicylic acid (sodium salt). Each of the solutions was spotted on separate dry analytical elements 5 having the configuration and reagent content described above. The reflectance density at 670 nm for each solution is presented in table II.
T A B L E II
10 Test SolutionSodium SalicylateReflectance No. mM/mQ _ Density at 670 nm 1 0.2 0.22 2 1.4 0.36 3 2.9 0.38 4 4.3 0.43 5.8 0.45 6 7.2 0.47 The reflectance density again shows that the method 20 and element of this invention are well suited for assaying salicylate in aqueous fluids.
The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations 25 and modifications can be effected within the spirit and scope of the invention.
Claims (14)
1. An analytical element for assaying catechol, including any catechol generating substances comprising one layer containing tyrosinase and another layer comprising NADH and a colorless form of a leuco dye in either layer.
2. A colorimetric method for a quantita-tive assay catechol, including any catechol generating substance comprising:
a) providing a sample suspected of containing catechol,including any catechol generating substance;
b) admixing with the sample NADH, tyrosinase, a colorless leuco dye; and c) determining the quantity of catechol present in the sample by colorimetric measurement.
a) providing a sample suspected of containing catechol,including any catechol generating substance;
b) admixing with the sample NADH, tyrosinase, a colorless leuco dye; and c) determining the quantity of catechol present in the sample by colorimetric measurement.
3. The method of claim 2 wherein the assay is for a catechol generating substance; step b) includes an enzyme selected to catalyze the conversion of the catechol generating substance to catechol and step c) is colorimetric determination of the catechol generating substance.
4. The method of claim 3 wherein the catechol generating substance is selected from the group consisting of o-hydroxyphenyl phosphate, or tyrosine o-phosphate.
5. A colorimetric method for a quantita-tive assay for salicylate comprising:
a) providing a sample suspected of containing salicylate;
b) admixing with the sample NADH, salicylate hydroxylase, tyrosinase, a colorless leuco dye; and c) determining the quantity of salicylate present in the sample by colorimetric measurement.
a) providing a sample suspected of containing salicylate;
b) admixing with the sample NADH, salicylate hydroxylase, tyrosinase, a colorless leuco dye; and c) determining the quantity of salicylate present in the sample by colorimetric measurement.
6. The method of claim 5 wherein each of the enumerated chemicals are added to the sample in the following order:
a) NADH and salicylate hydroxylase;
b) tyrosinase; and c) a colorless form of the leuco dye.
a) NADH and salicylate hydroxylase;
b) tyrosinase; and c) a colorless form of the leuco dye.
7. The method of claim 5 or 6 wherein the enumerated chemicals of step b) are admixed with the sample in the following concentrations;
a) 1 to 2.OmM/mL NADH;
b) 20 to 300 U/mL salicylate hydroxylase;
c) 50 to 1000 U/mL tyrosinase; and d) 0.2 to 10 ug/mL colorless leuco dye.
a) 1 to 2.OmM/mL NADH;
b) 20 to 300 U/mL salicylate hydroxylase;
c) 50 to 1000 U/mL tyrosinase; and d) 0.2 to 10 ug/mL colorless leuco dye.
8. The method of claim 5 or 6 wherein NADH
and salicylate hydroxylase are mixed with the sample in a 1:1 ratio.
and salicylate hydroxylase are mixed with the sample in a 1:1 ratio.
9. The method of claim 1 or 2 wherein the colorless leuco dye is selected form the group consisting of triarylimidazole reduced triaryl methanes and reduced indophenols.
10. The method of claim 5 or 6 wherein the colorless leuco dye is 2(4-hydroxy 3,5 dimethoxy-phenyl) 4,5 bis-(4-dimethylaminophenyl)imidazole.
11. The method of claim 5 or 6 wherein the assay is conducted in pH range of 6 to 8.5.
12. A dry multilayer analytical element for quantitatively assaying salicylate comprising a support bearing at least 2 layers, wherein:
a) one layer comprises NADH, and b) the other layer comprises tyrosinase and a colorless leuco dye.
a) one layer comprises NADH, and b) the other layer comprises tyrosinase and a colorless leuco dye.
13. A dry multilayer analytical element according to claim 12 wherein the other layer in b) also includes salicylate hydroxylase.
14. The method of claim 2 wherein the assay is directed to an enzyme that catalyzes generation of catechol from the catechol generating substances.
admixing with the admixture of step b) an enzyme substrate for the enzyme and in step c) determining the activity of the enzyme colorimetrically.
admixing with the admixture of step b) an enzyme substrate for the enzyme and in step c) determining the activity of the enzyme colorimetrically.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/548,395 US5320946A (en) | 1990-07-05 | 1990-07-05 | Method and element for assay of catechol and catechol generating substances |
US548,395 | 1990-07-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2026586A1 true CA2026586A1 (en) | 1992-01-06 |
Family
ID=24188682
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002026586A Abandoned CA2026586A1 (en) | 1990-07-05 | 1990-10-01 | Method and element for assay of catechol and catechol generating substances |
Country Status (9)
Country | Link |
---|---|
US (2) | US5320946A (en) |
EP (1) | EP0464934B1 (en) |
JP (1) | JPH07114710B2 (en) |
AT (1) | ATE130042T1 (en) |
CA (1) | CA2026586A1 (en) |
DE (1) | DE69114376T2 (en) |
DK (1) | DK0464934T3 (en) |
ES (1) | ES2081422T3 (en) |
GR (1) | GR3018763T3 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5362630A (en) * | 1981-07-28 | 1994-11-08 | Duke University | Isolation of pseudomonas salicylate hydroxlase and its use for the identification and quantitation of salicylate in body fluids |
US5474907A (en) * | 1994-03-25 | 1995-12-12 | Eastman Kodak Company | Multilayer analytical element for salicylate assay |
US5496702A (en) * | 1994-09-01 | 1996-03-05 | Johnson & Johnson Clinical Diagnostics, Inc. | Immunoassay elements having stable leuco dye coatings |
CA2928581C (en) * | 2013-11-08 | 2021-12-07 | Seeleaf, Inc. | Methods and articles for the detection of exposure to polyhydroxyaromatic skin irritants |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3992158A (en) * | 1973-08-16 | 1976-11-16 | Eastman Kodak Company | Integral analytical element |
US4089747A (en) * | 1976-08-09 | 1978-05-16 | Eastman Kodak Company | Compositions for the detection of hydrogen peroxide |
CA1095819A (en) * | 1977-01-14 | 1981-02-17 | Eastman Kodak Company | Element for analysis of liquids |
US4110079A (en) * | 1977-06-09 | 1978-08-29 | Eastman Kodak Company | Analytical element for clinical analysis |
JPS575519A (en) * | 1980-06-13 | 1982-01-12 | Toshiba Corp | Gas turbine plant using low calorific power gas |
DE3046741A1 (en) * | 1980-12-11 | 1982-07-15 | Boehringer Mannheim Gmbh, 6800 Mannheim | DETECTION OF NAD (P) H OR SALICYLATE |
DE3332144A1 (en) * | 1982-09-06 | 1984-03-08 | Konishiroku Photo Industry Co., Ltd., Tokyo | Analytical element |
GB8508677D0 (en) * | 1985-04-03 | 1985-05-09 | Genetics Int Inc | Assay for salicylate |
GB8728296D0 (en) * | 1987-12-03 | 1988-01-06 | Health Lab Service Board | Assay of sailicylates/reduced pyridine nucleotides |
US5185249A (en) * | 1990-04-04 | 1993-02-09 | Eastman Kodak Company | Dry analytical element for assaying salicylate |
-
1990
- 1990-07-05 US US07/548,395 patent/US5320946A/en not_active Expired - Lifetime
- 1990-10-01 CA CA002026586A patent/CA2026586A1/en not_active Abandoned
-
1991
- 1991-06-28 DK DK91201661.5T patent/DK0464934T3/en active
- 1991-06-28 ES ES91201661T patent/ES2081422T3/en not_active Expired - Lifetime
- 1991-06-28 DE DE69114376T patent/DE69114376T2/en not_active Expired - Lifetime
- 1991-06-28 EP EP91201661A patent/EP0464934B1/en not_active Expired - Lifetime
- 1991-06-28 AT AT91201661T patent/ATE130042T1/en not_active IP Right Cessation
- 1991-07-04 JP JP3163195A patent/JPH07114710B2/en not_active Expired - Fee Related
-
1993
- 1993-01-21 US US08/008,037 patent/US5506116A/en not_active Expired - Lifetime
-
1996
- 1996-01-24 GR GR960400161T patent/GR3018763T3/en unknown
Also Published As
Publication number | Publication date |
---|---|
US5506116A (en) | 1996-04-09 |
JPH04252197A (en) | 1992-09-08 |
ES2081422T3 (en) | 1996-03-16 |
EP0464934B1 (en) | 1995-11-08 |
US5320946A (en) | 1994-06-14 |
GR3018763T3 (en) | 1996-04-30 |
DE69114376T2 (en) | 1996-04-18 |
DK0464934T3 (en) | 1996-01-22 |
EP0464934A1 (en) | 1992-01-08 |
JPH07114710B2 (en) | 1995-12-13 |
ATE130042T1 (en) | 1995-11-15 |
DE69114376D1 (en) | 1995-12-14 |
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EEER | Examination request | ||
FZDE | Discontinued |