MULTILAYER ANALYTICAL ELEMENT

Abstract: The invention relates to a multilayer analytical element for quantitatively measuring a component of a sample in the presence of a reagent capable of interacting with the sample. The multilayer analytical element of the invention comprises a solid support and a hydrophilic layer thereon, the hydrophilic layer being made of gelatin, polyvinyl alcohol or a mixture thereof and containing a developing agent and a color-producing agent in a total amount of 5 to 30 weight %, the developing agent and color-producing agent being present in a weight ratio of 1:0.3 to 1:3.0. The developing agent is capable of undergoing oxidation caused by the reagent in the presence of an oxidizing agent, whereby the developing agent in oxidized form reacts with the color-producing agent to form a colored product. The multilayer analytical element according to the invention can be used in medicine and pharmacology as well as in criminal law. It can be utilized for measuring cholesterol, chloride ions and glucose, thereby enabling the diagnosis of hypercholesterolemia, cysticfibrosis and diabetes.

Description: "MULTILAYER ANALYTICAL ELEMENT" BACKGROUND OF THE INVENTION The present invention relates to improvements in the field of material analysis. More particularly, the invention relates to a multilayer analytical element for measuring the level of cholesterol in skin or other solid material. USSR Author Certificate No. 1,054,784 describes an analytical element which is used in thin- layer chromatography of lipids and which consists of a support (such as a glass plate) , having thereon a layer of an adsorbing material such as a mixture of silica gel, gypsum, ammonium sulfate and copper acetate. Such an analytical element is not suitable for measuring cholesterol in human skin. USSR Author Certificate No. 1,495,378 describes a two-layer analytical element which comprises a support such as a polyethylene film and a layer of gelatin with immobilized oxidase as an enzyme. The element changes color when amines or aqueous solutions thereof contact the gelatin layer. Such an analytical element is also not suitable for measuring cholesterol in human skin. Canadian Patent No. 1,335,968 describes a method of measuring cholesterol by contacting the skin or another cholesterol-containing surface with an affinity-enzymatic compound of the general formula A-C- B, wherein A is a detecting agent having affinity for cholesterol, B is an enzymatic visualizing agent capable of reacting with a color-producing agent to form a colored product, and C is a binding agent linking the detecting and visualizing agents together. SHEET The presence of cholesterol is determined by a change of color of the color-producing agent. Such a method has drawbacks in that the solutions and colored reaction product are not sufficiently stable and that at low cholesterol levels the sensitivity and reproducibility are rather poor. Due to the drawbacks associated with the above known analytical elements and with the above known method of measuring cholesterol, it is necessary to develop an analytical element combining the advantages of the known liquid-phase method for choles¬ terol assay and the convenience of practical applica¬ tion of multilayer analytical elements utilizing solid supports. A multilayer analytical element has been proposed in US Patent No. 4,895,704, which comprises a water-impermeable light-transmitting solid support and a hydrophilic layer containing at least one reagent capable of reacting with a component of a sample to form a substance which can be detected by light radiation. The analytical element includes an additional layer containing light-scattering particles in an amount ensuring light transmittance in the range from 10 to 2.5%. Drawbacks to such a known multilayer analytical element are its non-suitability for measur¬ ing cholesterol levels in skin, low intensity of color reaction and high contrast of analytical effect at low levels of substance being assayed. SUMMARY OF THE INVENTION It is therefore an object of the present invention to enhance the sensitivity, reproducibility and intensity of color reaction of analytical elements, to increase time interval for which the color developed during enzymatic reaction is preserved, to intensify the procedure of measuring cholesterol levels in skin and to widen the possibilities for urgent diagnosis of cholesterol. According to a broad aspect of the invention, there is provided a multilayer analytical element for quantitatively measuring a component of a sample in the presence of a reagent capable of intereacting with the sample. The multilayer analytical element of the inven¬ tion comprises a solid support and a hydrophilic layer thereon, the hydrophilic layer being made of gelatin, polyvinyl alcohol or a mixture thereof and containing a developing agent and a color-producing agent in a total amount of 5 to 30 weight %, the developing agent and color-producing agent being present in a weight ratio of 1:0.3 to 1:3.0. The developing agent is capable of undergoing oxidation caused by the reagent in the presence of an oxidizing agent, whereby the developing agent in oxidized form reacts with the color-producing agent to form a colored product. Where the multilayer analytical element of the invention is used for cholesterol assay in skin, the reagent utilized is an affinity-enzymatic compound of the formula A-C-B as defined above, in which the enzymatic visualizing agent B is a peroxidase. In this case, the developing agent is capable of undergoing oxidation caused by the peroxidase in the presence of hydrogen peroxide, whereby the developing agent in oxidized form reacts with the color-producing agent to form a colored product indicative of cholesterol level in the skin. DESCRIPTION OF PREFERRED EMBODIMENTS The developing agent used is preferably a compound of the formula: wherein R, R' and R" are identical or different and each represent a hydrogen atom, an alkyl or alkanol group. Examples of such compounds include N,N-diethyl- paraphenylenediamine, paraethylamino-orthotoluidine, para- [N-ethyl-N- (3-hydroxyvinyl) amino] -aniline, para- [N-ethyl-N- (3-methylsulfonylaminoethyl) amino] -ortho- toluidine, para- [N-ethyl-N- (jS-hydroxyethyl) amino] - orthotoluidine and para- [N-butyl-N- (7-sulfopropyl) - amino] -aniline. Use can also be made of diethyl-p-toly- lenediamine, ethyloxyethyl-p-phenylenediamine, ethyl- methanesulfoaminoethyl-p-tolylenediamine, ethyloxy- ethyl-p-tolylenediamine, butylsulfopropyl-p-phenylene- diamine or diethyl-p-phenylenediamine as developing agent. As color-producing agents, derivatives of pyrazolone-5, benzoylacetic acid or naphthoic acid can be used. Examples of suitable color-producing agents include 1-(2,4,6-trichlorophenyl)-3-pentadecylamino- phenyl-aminopyrazolone, 3,5-dicarboxyphenyloctadecyla- mide-l-oxy-2-naphthoic acid, 2-chloro-5-[7- (2,4- ditetraminephenoxy)-butiroylamino]-anilide-α-N-ben- zoylhidantoylpivaloyl acetic acid, meta- (N-methyl-N- octadecyl)-amino-parabenzoylacetamidobenzenesulfonic acid, metacarboxy-meta-{N-[parachlorometa-(N-orthooc- tadecyl-oxybenzoylacetyl) amido]benzenesulfonylamido}- carboxylic acid, orthophenoxy-meta(5-oxo-l,2-pyrazol-3- ino)-benzenesulfonic acid, 2 l -methyloctadecylamino-5*- sulfanilidebenzoylacetic acid, 2'-chloro-5'- (3",5"- dicarboxyphenylsulfamide)-anilide-2-octadecyloxyben- zoylacetic acid, octadecylamide-l-oxy-4-sulfo-2- naphthoic acid, 2'-methyloctadecylamino-S'-sulfanilide- 1,2-oxynaphthoic acid and 1- (4'-phenoxy-3'- sulfophenyl)-3-steroyl-aminopyrazolone. The color-producing agents are evenly distributed in the hydrophilic layer as microcapsules, microparticles or separate molecules depending on the nature of color-producing agent. To achieve a micro- particular or molecular distribution of the color- producing agent in the hydrophilic layer, compounds with hydrophilic (polar) side chains are used. To achieve a microcapsular distribution of the color- producing agent, compounds with hydrophobic (nonpolar) side chains are used. As a support, use is preferably made of a hydrophylized film of polyethyleneterephthalate, poly¬ ethylene or trinitrocellulose. A noncolored polymer, paper or polyethylene-covered paper can also be used. The hydrophilic layer can also contain moistening and tanning agents. Moistening agents ensure a uniform application on the support of a hydrophilic layer with a constant thickness, preferably of 2-100 microns. Tanning agents ensure the required level of physico-mechanical properties of the multilayer analytical element. Chrome acetate and the monosodium salt of 2,4-dichloro-6-hydroxytriazin-l,3, 5 can be used as tanning agents. The multilayer analytical element according to the invention can be used in medicine and pharma¬ cology as well as in criminal law. It can be utilized for measuring not only cholesterol, but also chloride ions and glucose, thereby enabling the diagnosis of cysticfibrosis and diabetes in addition to hypercholes- terolemia. The following non-limiting examples further illustrate the invention. EXAMPLE 1 A multilayer analytical element for choles¬ terol assay in skin was produced by applying on a solid support consisting of a polyethyleneterephthalate film having a thickness of 50 microns, a layer of photo¬ graphic gelatin containing 7.5 weight % of a hydro- phobic purple color-producing agent consisting of 1- (2, 4, 6-trichlorophenyl)-3-pentadecyl-aminophenyl-amino- pyrazolone. The color-producing agent had an average particle size less than 0.1 micron. The gelatin layer contained 7.5 weight % of N,N-diethyl-paraphenylene- diamine as a developing agent distributed at molecular level. The layer also contained chrome acetate as a tanning agent. This multilayer analytical element was used for the assay of cholesterol level in skin integument from the hand of a patient suffering from athero¬ sclerosis as well as in the skin of a healthy person. One drop of an aqueous solution of an affinity- enzymatic compound of the formula A-C-B as defined above, in which the enzymatic visualizing agent B is a peroxidase, was applied on the skin of each person's palm. Incubation was performed for 1 minute, then the liquid was removed with the help of dry and moistened tampons alternately. In the same area, one drop of hydrogen peroxide was applied and immediately there¬ after the multilayer analytical element was placed firmly and pressed against the skin surface for 1 minute. The analytical element was then washed for 15-20 seconds in running water. The result of the assay was determined by comparing the color imprint obtained with a graduated scale or by measuring the intensity of color spot spectrophotometrically such as with a LCD (liquid crystal display) spectrophotometer. Table 1 shows the results obtained by using known liquid-phase methods for measurement of blood and skin cholesterol levels as well as by using the multi- layer analytical element according to the invention. TABLE 1 Group Known assay Present assay Blood Skin Skin cholesterol cholesterol Cholesterol Healthy 185 ± 25 1.9 ± 0.3 0.13 ± 0.04 persons (24) Atheros¬ clerotic 228 ± 32 2.8 ± 0.5 0.32 ± 0.07 patients (29) The reproducibility of color reaction was studied on the same patients using 10 subsequent assays. The standard deviation was not higher than 8%. Measurements performed using multilayer analytical elements which were stocked either for 1 week or for 6 months showed the same reproducibility of the results. The assay took 2-3 minutes, and preparation of the assay took about 3 minutes. Kits for self- testing patients can thus be manufactured, incorporat¬ ing the multilayer analytical element. EXAMPLE 2 A multilayer analytical element for measuring cholesterol level in skin was produced by applying on a support consisting of a white polyethylene-covered photographic paper, a layer of polyvinyl alcohol containing a blue color-producing agent consisting of 3,5-dicarboxyphenyloctadecylamide-l-oxy-2-naftoic acid. The polyvinyl alcohol layer also contained para- ethylamino-orthotoluidine as a developing agent. This multilayer analytical element was used for cholesterol assay in skin, according to the same procedure as described in Example 1. Essentially the same results were obtained. EXAMPLE 3 A flexible multilayer analytical element was produced by applying on a support consisting of a poly¬ ethylene film with a thickness of 100 microns treated with plasma of corona discharge for its hydrophyliza- tion, a layer of gelatin in admixture with polyvinyl alcohol and containing a dispersion of a hydrophobic yellow color-producing agent, i.e. 2-chloro-50[7- (2, 4- ditetraminephenoxy)-butiroylamino]-anilide-α-N-benzoyl- hidantoylpivaloyl acetic acid. The layer of gelatin and polyvinyl alcohol also contained para-[N-ethyl-N- (β- hydroxyvinyl)amino]-aniline as a developing agent. This multilayer analytical element was used for cholesterol assay in skin, according to the same procedure as described in Example 1. Essentially the same results were obtained.

Claims: CLAIMS : 1. A multilayer analytical element for quantita¬ tively measuring a component of a sample in the presence of a reagent capable of intereacting with the sample, said multilayer analytical element comprising a solid support and a hydrophilic layer thereon, said hydrophilic layer being made of gelatin, polyvinyl alcohol or a mixture thereof and containing a develop- ing agent and a color-producing agent in a total amount of 5 to 30 weight %, said developing agent and said color-producing agent being present in a weight ratio of 1:0.3 to 1:3.0, said developing agent being capable of undergoing oxidation caused by the reagent in the presence of an oxidizing agent, whereby the developing agent in oxidized form reacts with said color-producing agent to form a colored product. 2. A multilayer analytical element as claimed in claim 1, wherein said developing agent is a compound of formula: wherein R, R 1 and R" are identical or different and each represent a hydrogen atom, an alkyl or alkanol group. 3. A multilayer analytical element as claimed in claim 2, wherein said developing agent is a compound selected from the group consisting of N,N-diethyl-para- phenylenediamine, paraethyla ino-orthotoluidine, para- [N-ethyl-N-(S-hydroxyvinyl)amino]-aniline, para-[N- ethyl-N- (jβ-methylsulfonylaminoethyl)amino]-ortho- toluidine, para-[N-ethyl-N- (0-hydroxyethyl)amino]- orthotoluidine and para-[N-butyl-N-(7-sulfopropyl)- amino]-aniline. . A multilayer analytical element as claimed in claim 1, wherein said developing agent is a compound selected from the group consisting of diethyl-p-toly- lenediamine, ethyloxyethyl-p-phenylenediamine, ethyl- methanesulfoaminoethyl-p-tolylenediamine, ethyloxy- ethyl-p-tolylenediamine, butylsulfopropyl-p-phenylene- diamine and diethyl-p-phenylenediamine as developing agent. 5. A multilayer analytical element as claimed in claim 1, wherein said color-producing agent is a compound selected from the group consisting of derivatives of pyrazolone-5, benzoylacetic acid and naphthoic acid. 6. A multilayer analytical element as claimed in claim 5, wherein said color-producing agent is a compound selected from the group consisting of 1- (2,4, 6-trichlorophenyl)-3-pentadecylaminophenyl-amino- pyrazolone, 3,5-dicarboxyphenyloctadecylamide-l-oxy-2- naphthoic acid, 2-chloro-5-[7- (2,4-ditetraminephenoxy)- butiroylamino]-anilide-α-N-benzoylhidantoylpivaloyl acetic acid, meta-(N-methyl-N-octadecyl)amino-para- benzoylacetamidobenzenesulfonic acid, metacarboxy-meta- {N-[parachlorometa- (N-orthooctadecyloxybenzoylacetyl)- amido]benzenesulfonylamido}-carboxylic acid, ortho- phenoxy-meta(5-oxo-l,2-pyrazol-3-ino)-benzenesulfonic acid, 2'-methyloctadecylamino-5'-sulfanilidebenzoyl- acetic acid, 2'-chloro-5'- (3",5"-dicarboxyphenylsulfa- mide)-anilide-2-octadecyloxybenzoylacetic acid, octa- decylamide-l-oxy-4-sulfo-2-naphthoic acid, 2'-methyl- octadecylamino-5'-sulfanilide-l,2-oxynaphthoic acid and 1- (4'-phenoxy-3'-sulfophenyl)-3-steroyl-aminopyrazo- lone. 7. A multilayer analytical element as claimed in claim 5, wherein said color-producing agent is a hydro- phobic compound having a particle size less than 0.1 μ. 8. A multilayer analytical element as claimed in claim 7, wherein said hydrophobic compound is 1- (2,4,6- trichlorophenyl) -3-pentadecylaminophenyl-aminopyra- zolone or 2-chloro-5-[7- (2,4-ditetraminephenoxy)- butiroylamino]-anilide-α-N-benzoylhidantoylpivaloyl acetic acid. 9. A multilayer analytical element as claimed in claim 1, wherein said hydrophilic layer has a thickness ranging from 2 to 100 μ. 10. A multilayer analytical element as claimed in claim 1, wherein said hydrophilic layer further contains at least one moistening agent. 11. A multilayer analytical element as claimed in claim 1, wherein said hydrophilic layer further contains at least one tanning agent. 12. A multilayer analytical element for choles- terol assay in the presence of an affinity-enzymatic compound of formula A-C-B, wherein A is a detecting agent having affinity for cholesterol, B is an enzymatic visualizing agent consisting of a peroxidase and C is a binding agent linking the detecting and visualizing agents together, said multilayer analytical element comprising a solid support and a hydrophilic layer thereon, said hydrophilic layer being made of gelatin, polyvinyl alcohol or a mixture thereof and containing a developing agent and a color-producing agent in a total amount of 5 to 30 weight %, said developing agent and said color-producing agent being present in a weight ratio of 1:0.3 to 1:3.0, said developing agent being capable of undergoing oxidation caused by said peroxidase in the presence of hydrogen peroxide, whereby the developing agent in oxidized form reacts with said color-producing agent to form a colored product. 13. A multilayer analytical element as claimed in claim 12, wherein said developing agent is a compound of formula: wherein R, R' and R" are identical or different and each represent a hydrogen atom, an alkyl or alkanol group. - 14 - 14. A multilayer analytical element as claimed in claim 13, wherein said developing agent is a compound selected from the group consisting of N,N-diethyl-para- phenylenediamine, paraethylamino-orthotoluidine, para- [N-ethyl-N- (j8-hydroxyvinyl)amino]-aniline, para-[N- ethyl-N- (0-methylsulfonylaminoethyl)amino]-ortho- toluidine, para-[N-ethyl-N- (3-hydroxyethyl)amino]- orthotoluidine and para-[N-butyl-N- (7-sulfopropyl)- amino]-aniline. 15. A multilayer analytical element as claimed in claim 12, wherein said developing agent is a compound selected from the group consisting of diethyl-p-toly- lenediamine, ethyloxyethyl-p-phenylenediamine, ethyl- methanesulfoaminoethyl-p-tolylenediamine, ethyloxy- ethyl-p-tolylenediamine, butylsulfopropyl-p-phenylene- diamine and diethyl-p-phenylenediamine as developing agent. 16. A multilayer analytical element as claimed in claim 12, wherein said color-producing agent is a compound selected from the group consisting of deriva¬ tives of pyrazolone-5, benzoylacetic acid and naphthoic acid. 17. A multilayer analytical element as claimed in claim 16, wherein said color-producing agent is a compound selected from the group consisting of 1- (2,4, 6-trichlorophenyl)-3-pentadecylaminophenyl-amino- pyrazolone, 3, 5-dicarboxyphenyloctadecylamino-l-oxy-2- naphthoic acid, 2-chloro-5-[7- (2, 4-ditetraminephenoxy)- butiroylamino]-anilide-α-N-benzoylhidantoylpivaloyl acetic acid, meta- (N-methyl-N-octadecyl) amino-para- benzoylacetamidobenzenesulfonic acid, metacarboxy-meta- {N-[parachlorometa- (N-orthooctadecyloxybenzoylacetyl)- 98 - 15 - amido]benzenesulfonylamido}-carboxylic acid and ortho- phenoxy-meta(5-oxo-l,2-pyrazol-3-ino)-benzenesulfonic acid, 2'-methyloctadecylamino-5'-sulfanilidebenzoyl- acetic acid, 2'-chloro-5'- (3",5"-dicarboxyphenylsulfa- mide)-anilide-2-octadecyloxybenzoylacetic acid, octa- decylamide-l-oxy-4-sulfo-2-naphthoic acid, 2'- methyloctadecylamino-5'-sulfanilide-l,2-oxynaphthoic acid and 1-(4'-phenoxy-3'-sulfophenyl)-3-steroyl- aminopyrazolone 18. A multilayer analytical element as claimed in claim 16, wherein said color-producing agent is a hydrophobic compound having a particle size less than 0.1 μ. 19. A multilayer analytical element as claimed in claim 18, wherein said hydrophobic compound is 1- (2,4, 6-trichlorophenyl)-3-pentadecylaminophenyl-amino- pyrazolone or 2-chloro-5-[7- (2,4-ditetraminephenoxy)- butiroylamino]-anilide-α-N-benzoylhidantoylpivaloyl acetic acid. 20. A multilayer analytical element as claimed in claim 12, wherein said ' hydrophilic layer has a thickness ranging from 2 to 100 μ. 21. A multilayer analytical element as claimed in claim 12, wherein said hydrophilic layer further contains at least one moistening agent. 22. A multilayer analytical element as claimed in claim 12, wherein said hydrophilic layer further contains at least one tanning agent.

Inventors: BABLYUK YEVGENEY BORISOVICH, KOCHETOV IVAN ALEXANDROVICH, LOPUKHIN YURY MICHAELOVICH, MALEEV ALEXANDER MICHAELOVICH, PARFENOV ALEXANDR SERGEIVICH, RABOVSKI ALEXANDR B, BABLYUK, YEVGENEY BORISOVICH, KOCHETOV, IVAN ALEXANDROVICH, LOPUKHIN, YURY MICHAELOVICH, MALEEV, ALEXANDER MICHAELOVICH, PARFENOV, ALEXANDR SERGEIVICH, RABOVSKI, ALEXANDR B.
Applicants: 2860601 CANADA INC, BABLYUK YEVGENEY BORISOVICH, KOCHETOV IVAN ALEXANDROVICH, LOPUKHIN YURY MICHAELOVICH, MALEEV ALEXANDER MICHAELOVICH, PARFENOV ALEXANDR SERGEIVICH, RABOVSKI ALEXANDR B, 2860601 CANADA INC., BABLYUK, YEVGENEY BORISOVICH, KOCHETOV, IVAN ALEXANDROVICH, LOPUKHIN, YURY MICHAELOVICH, MALEEV, ALEXANDER MICHAELOVICH, PARFENOV, ALEXANDR SERGEIVICH, RABOVSKI, ALEXANDR B.
Application year: 1995
Main classification: G01N33
IPC codes: C12Q1, G01N31, G01N33