CN111855648A - Dry type homocysteine test card and application thereof - Google Patents
Dry type homocysteine test card and application thereof Download PDFInfo
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- CN111855648A CN111855648A CN202010147017.5A CN202010147017A CN111855648A CN 111855648 A CN111855648 A CN 111855648A CN 202010147017 A CN202010147017 A CN 202010147017A CN 111855648 A CN111855648 A CN 111855648A
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- 238000012360 testing method Methods 0.000 title claims abstract description 40
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- 238000001914 filtration Methods 0.000 claims abstract description 21
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- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 13
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- 239000010410 layer Substances 0.000 claims description 187
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A dry type homocysteine test card and application thereof, comprising dissociation liquid R1 and dry chemical test paper, wherein the dissociation liquid R1 comprises reducing agent, serine and buffer solution; the dry chemical test paper comprises a support layer, a chromatographic membrane, an enzyme pad, a blood filtering layer, an enzyme circulation reaction layer, a diffusion layer and a plastic sheet; the chromatographic membrane, the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer are sequentially overlapped on the supporting layer from bottom to top, the length of the chromatographic membrane is greater than that of the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer, and the chromatographic membrane is fixed at one end close to the supporting layer and extends towards the other end of the supporting layer; the support layer is provided with a plastic sheet near one end far away from the enzyme pad, the plastic sheet is provided with a color development area, the plastic sheet is positioned above the chromatographic membrane and is arranged parallel to the chromatographic membrane, and the color development area covers partial area of the chromatographic membrane. Has the advantages of detecting all homocysteine in human blood and strong sensitivity.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a dry type homocysteine test card and application thereof.
Background
Homocysteine (HCY) is an amino acid in blood that contains sulfur molecules and is converted from methionine. Produced in vivo via methionine demethylation, and metabolized mainly via the re-methylation and transsulfuration pathways. Methionine synthetase, Cystathionine Beta Synthetase (CBS), vitamin B12, folic acid, and vitamin B6. Elevated homocysteine may be caused by enzyme dysfunction or vitamin deficiency, etc. HCY is normally rapidly converted to glutathione and S-adenosylmethionine. The two substances are very important in organisms, and have close relation with liver detoxification, in vivo garbage-free radical elimination and DNA repair. An increase in HCY, which means the absence of both substances, would be a significant health hazard. Therefore, homocysteine is a healthy wind vane for us.
At present, there are many methods for detecting homocysteine, including high performance liquid chromatography, enzyme-linked immunosorbent assay, chemiluminescence assay, and cycling enzyme method. In the methods, expensive large instruments such as a high performance liquid chromatography, a chemiluminescence method and a cyclic enzyme method are required for detection, or the methods are complex to operate such as an enzyme-linked immunosorbent assay, and the methods cannot meet the requirements of the primary hospital inspection site on rapidness and convenience of the detection method.
Some homocysteine detection methods suitable for on-site rapid detection are also disclosed on the intellectual property office website of China. Wherein, the website of the intellectual property office of China discloses an electrochemical test paper for quantitatively determining homocysteine and a preparation method thereof (publication number CN 104931549A); the Chinese intellectual property office website also discloses a homocysteine dry chemical detection test strip and a preparation method thereof (publication No. CN 101592656A). The two methods are convenient to operate and simple in structure, but have a major defect that most of homocysteine in blood is combined with protein through disulfide bonds to form oxidized homocysteine, and reducing agents are not used in the application documents to reduce the oxidized homocysteine, so that the methods can only detect the content of free reduced homocysteine in blood samples, and the clinical sensitivity is poor.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a dry type homocysteine test card which can detect all homocysteine in human blood and has strong sensitivity.
In order to solve the technical problems, the invention adopts the technical scheme that: a dry type homocysteine test card comprises dissociation liquid R1 and dry chemical test paper, wherein the dissociation liquid R1 comprises a reducing agent, serine and a buffer solution; the dry chemical test paper comprises a support layer, a chromatographic membrane, an enzyme pad, a blood filtering layer, an enzyme circulation reaction layer, a diffusion layer and a plastic sheet; the chromatographic membrane, the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer are sequentially overlapped on the supporting layer from bottom to top, the length of the chromatographic membrane is greater than that of the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer, and the chromatographic membrane is fixed at one end close to the supporting layer and extends towards the other end of the supporting layer; the support layer is provided with a plastic sheet near one end far away from the enzyme pad, the plastic sheet is provided with a color development area, the plastic sheet is positioned above the chromatographic membrane and is arranged parallel to the chromatographic membrane, and the color development area covers partial area of the chromatographic membrane.
By adopting the structure, the reaction principle of the invention is as follows: oxidized Homocysteine (HCY) in the sample is reduced into free HCY by a reducing agent in R1, when the sample is added into an enzyme circulation reaction layer after reduction, the free HCY reacts with serine under the action of cystathionine-beta-synthetase in the enzyme circulation layer to generate L-cystathionine, the L-cystathionine generates HCY, pyruvic acid and ammonia under the action of cystathionine-beta-lyase, and the HCY promotes the reaction to circulate back and forth; the pyruvic acid generated by the enzyme circulation reaction layer flows to the enzyme pad along with the downward seepage of the liquid, and the pyruvic acid generates acetyl phosphate, hydrogen peroxide and carbon dioxide under the action of pyruvate oxidase in the enzyme pad; the pyruvic acid generated in the enzyme pad flows onto the chromatographic membrane, the color development area of the plastic sheet is pressed at the moment, so that the color development area is contacted with the chromatographic membrane, the color development substrate carried on the color development area is dissolved and released onto the chromatographic membrane by liquid, the color development substrate reacts with hydrogen peroxide under the catalysis of peroxidase to generate color change, and the HCY is quantitatively detected by detecting the color change amount of the color development area.
Preferably, the plastic sheet is adhered to the support layer by an adhesive layer; enzyme pad, filter layer and enzyme circulation reaction layer the upper and lower surface equal and overlap the setting from top to bottom each other, the upper and lower surface of diffusion layer be greater than the upper and lower surface of enzyme pad, the one end of diffusion layer flushes with the one end of enzyme circulation reaction layer, the other end extends enzyme circulation reaction layer, enzyme pad, filter layer, enzyme circulation reaction layer the side and the diffusion layer extend enzyme circulation reaction layer part and pass through the viscose layer and support layer and bond each other.
Preferably, the dissociation solution R1 comprises a reducing agent, serine and a buffer solution; the reducing agent is one or more of tri (2-carboxyethyl) phosphine, beta-mercaptoethanol and dithiothreitol, and the preferred reducing agent is tri (2-carboxyethyl) phosphine; the buffer solution is pH 5.0-9.0, such as tris, 4-hydroxyethylpiperazine ethanesulfonic acid, 2-morpholinoethanesulfonic acid, 3- (N-morpholino) propanesulfonic acid, etc. The concentration of the reducing agent is preferably 0.05 to 0.5mmol/L, the concentration of the buffer is preferably 10 to 200mmol/L, and the concentration of serine is preferably 0.05 to 0.5 g/L.
Preferably, the support layer is made of opaque high polymer material, and the thickness is 0.10-0.30 mm; the assembly is more convenient.
Preferably, the chromatographic membrane is a nitrocellulose membrane.
Preferably, the material of the enzyme pad adopts glass fiber, polyester fiber or non-woven fabric; the specific preparation method of the enzyme pad comprises the following steps: cutting the material into a certain size, soaking in an enzyme pad treatment solution, and drying; the enzyme pad treating solution contains buffer solution, pyruvate oxidase, peroxidase, coenzyme, protecting agent, surfactant, etc.
Preferably, the buffer solution in the enzyme pad treatment solution is a buffer solution having a pH of 5.0 to 9.0, such as tris, 4-hydroxyethylpiperazine ethanesulfonic acid, 2 morpholinoethanesulfonic acid, 3- (N-morpholino) propanesulfonic acid, etc., and has a buffer solution concentration of 10mmol/L to 200 mmol/L; the protective agent is preferably one or more of sucrose, trehalose or polyvinyl alcohol, and the concentration of the protective agent is preferably 10 g/L-100 g/L; the surfactant is preferably Tween 20, and the concentration of the surfactant is preferably 0.5 g/L-10 g/L; the concentration of the pyruvate oxidase is 100-1000 KU/L; the concentration of the peroxidase is 200-2000 KU/L; the coenzyme can be one or more of magnesium chloride, magnesium sulfate and magnesium acetate, and the concentration of magnesium ions is preferably 0.5-10 mmol/L. The content or concentration ranges of the respective components in the above-mentioned treatment liquid are all ranges in the final enzyme pad treatment liquid.
Preferably, the blood filtering layer is made of porous polyester fiber, cotton fiber or non-woven fabric.
Preferably, the material of the enzyme circulation reaction layer adopts glass fiber, polyester fiber or non-woven fabric; the preparation method of the enzyme circulation reaction layer comprises the following steps: cutting the material into a certain size, soaking in an enzyme circulation reaction layer treatment solution, and drying; the treating liquid of the enzyme circulating reaction layer contains buffer solution, cystathionine-beta-synthetase, cystathionine-beta-lyase, protective agent, surfactant and the like.
Preferably, the buffer solution in the treatment solution of the enzyme cycling reaction layer is a buffer solution with a pH value of 5.0-9.0, such as tris, 4-hydroxyethyl piperazine ethanesulfonic acid, 2-morpholinoethanesulfonic acid, 3- (N-morpholino) propanesulfonic acid, etc., and the concentration of the buffer solution is 10 mmol/L-200 mmol/L; the protective agent is one or more of sucrose, trehalose or polyvinyl alcohol, and the concentration of the protective agent is 10 g/L-100 g/L; the surfactant is preferably Tween 20, and the concentration of the surfactant is 0.5 g/L-10 g/L; the concentration of the cystathionine-beta-synthetase is 50-1000 KU/L; the concentration of cystathionine-beta-lyase is 50-1000 KU/L. The contents or concentration ranges of the respective components in the above-mentioned treatment liquid are all ranges in the treatment liquid of the final enzyme circulation reaction layer.
Preferably, the diffusion layer is a 100-1000 mesh net, and the dispersion layer is used for dispersing the sample, so that the sample liquid is uniformly distributed in each region of the lower layer.
Preferably, the plastic sheet is made of transparent high polymer material, and the thickness of the plastic sheet is 0.10-0.30 mm; the plastic sheet has certain hardness and is convenient to assemble, and the transparency is convenient for the detector to detect the reflectivity.
Preferably, the color development area is fixed with 4-aminoantipyrine and a color development substrate in a dot spraying mode by a dot spraying instrument; the 4-aminoantipyrine and the chromogenic substrate are firstly prepared into solution (water), and the concentration of the 4-aminoantipyrine in the solution is 2 g/L-50 g/L; the chromogenic substrate is one of Trinder's chromogenic substrates, preferably N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline sodium salt, and the concentration of the Trinder's chromogenic substrate in the solution is 2 g/L-50 g/L.
The invention also provides the application of the dry type homocysteine test card in preparing a test substance for measuring the homocysteine content in human blood.
The invention has the advantages and beneficial effects that:
1. the reaction principle of the reagent is that the blood sample is added into the dissociation liquid R1, and the oxidation type HCY is reduced into the free type HCY under the action of the reducing agent in the dissociation liquid R1. Adding the dissociated sample into a diffusion layer, then infiltrating into an enzyme circulation reaction layer, carrying out enzyme circulation reaction on free HCY and serine under the catalytic action of cystathionine-beta-synthetase and cystathionine-beta-lyase in the enzyme circulation reaction layer to generate alanine and HCY, continuously infiltrating the sample into an oxidation reaction layer, carrying out reaction on pyruvic acid and phosphate groups under the catalysis of pyruvate oxidase to generate hydrogen peroxide and acetyl phosphoric acid, continuously infiltrating the sample into a chromatographic membrane, dissolving peroxidase in the oxidation reaction layer into the sample, carrying out chromatographic movement on the sample towards the other end under the capillary action of the chromatographic membrane 2, and mechanically pressing a color developing region to enable a novel Trinder's reagent on the color developing region and the hydrogen peroxide on the chromatographic membrane to generate a benzoquinonimine colored substance under the catalysis of peroxidase. The color change of the color development area reflects the content of HCY in the sample, and the HCY is quantitatively detected by detecting the color change of the color development area.
2. The homocysteine dry chemical detection reagent is suitable for determining the content of homocysteine in human blood.
Drawings
FIG. 1 is a schematic structural diagram of a dry homocysteine test card according to the present invention.
FIG. 2 is a graph showing the correlation between the kit of the present invention and tandem mass spectrometry.
As shown in the figure: 1. a supporting layer, 2, a chromatographic membrane, 3, an oxidation reaction layer, 4, a hemofiltration layer enzyme, 5, a circulation reaction layer, 6, a diffusion layer, 7, a plastic sheet, 7-1, a color development area, 8 and an adhesive layer.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
As shown in the attached FIG. 1, the dry homocysteine test card of the present application has a specific structure comprising: the reagent comprises a dissociation liquid R1 and dry chemical test paper, wherein the dissociation liquid R1 comprises a reducing agent and a buffer solution; the dry chemical test paper comprises a support layer 1, a chromatographic membrane 2, an enzyme pad 3, a blood filtration layer 4, an enzyme circulation reaction layer 5, a diffusion layer 6 and a plastic sheet 7; the chromatographic membrane, the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer are sequentially overlapped on the support layer 1 from bottom to top, and the length of the chromatographic membrane 2 is greater than that of the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer; the chromatographic membrane is fixed at one end close to the supporting layer and extends towards the other end of the supporting layer; the other end of the support layer is provided with a plastic sheet, the plastic sheet is provided with a color development area, the plastic sheet is positioned above the chromatographic membrane and is arranged in parallel with the chromatographic membrane, and the color development area covers partial area of the chromatographic membrane.
Specifically, a plastic sheet is arranged on the support layer near one end far away from the enzyme pad, a color development area is arranged on the plastic sheet, the height of the plastic sheet is positioned above the chromatographic membrane and is parallel to the chromatographic membrane, and the color development area of the plastic sheet in the height direction is a partial area covering the chromatographic membrane; when the color development region is pressed downward, the color development region chromatography may be attached.
Specifically, the plastic sheet 7 is adhered to the support layer 1 through an adhesive layer 8 (one end of the plastic sheet is adhered to the adhesive layer, the other end is provided with a color development area, and the free end is positioned right above part of the chromatographic membrane); the enzyme pad 3, the blood filter layer 4 and the enzyme circulation reaction layer 5 have the same upper and lower surfaces and are arranged in an overlapped manner from top to bottom (namely the enzyme pad 3, the blood filter layer 4 and the enzyme circulation reaction layer 5 have the same length and width and different thicknesses, the layers are overlapped along the thickness direction, and the length and the width directions are completely overlapped), the upper and lower surfaces of the diffusion layer 6 are larger than the upper and lower surfaces of the enzyme pad, one end of the diffusion layer is flush with the enzyme pad, and the other end of the diffusion layer extends out of the enzyme circulation reaction layer (namely the width of the diffusion layer 6 is equal to the width of the enzyme pad 3, the blood filter layer 4 and the enzyme circulation reaction layer 5, and the length of the diffusion layer is larger than the lengths of the enzyme pad 3, the blood filter layer 4 and the enzyme circulation reaction layer 5); the enzyme pad 3, the blood filtering layer 4, the enzyme circulation reaction layer 5 and the diffusion layer 6 are mutually flush at one end close to the plastic sheet 7, the diffusion layer at the other end extends out of the side surfaces of the enzyme pad 3, the blood filtering layer 4 and the enzyme circulation reaction layer 5, and then the side surfaces of the enzyme pad 3, the blood filtering layer 4 and the enzyme circulation reaction layer 5, the extended part of the diffusion layer and the supporting layer are mutually adhered together by adopting an adhesive layer 8.
Example 1:
this example provides a method for preparing a cleavage agent R1 for a dry homocysteine assay reagent, which was prepared as follows.
Dissociating agent R1-1: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 6g of tris (hydroxymethyl) aminomethane, 0.015g of beta-mercaptoethanol and 0.15g of serine, the pH value of which is adjusted to 8.0 with hydrochloric acid.
Dissociating agent R1-2: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 6g of tris (hydroxymethyl) aminomethane, 0.03g of dithiothreitol and 0.15g of serine, the pH value of which is adjusted to 8.0 with hydrochloric acid.
Dissociating agent R1-3: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 6g of tris (hydroxymethyl) aminomethane, 0.057g of tris (2-carboxyethyl) phosphine, 0.15g of serine, the pH value being adjusted to 8.0 with hydrochloric acid.
Dissociating agent R1-4: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 6g of tris (hydroxymethyl) aminomethane, 0.15g of tris (2-carboxyethyl) phosphine, 0.15g of serine, the pH value being adjusted to 8.0 with hydrochloric acid.
Example 2:
this example provides a method for preparing dry homocysteine assay paper, which is prepared as follows.
(1) Preparation of enzyme pad 3:
preparing an enzyme pad treating solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 2.4g of tris (hydroxymethyl) aminomethane, 1g of tween 20, 0.4g of magnesium chloride, 30g of trehalose, 10g of polyvinyl alcohol 10, 300KU of peroxidase, 500U of pyruvate oxidase, and adjusting the pH to 8.0 with hydrochloric acid.
Enzyme pad 3 treatment: cutting tea filter paper (Shanghai Kanning paper industry: RF17) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in oven, drying at 45 deg.C for 1 hr, and taking out for use.
(2) Preparation of the enzyme circulation reaction layer 5:
preparing an enzyme circulation reaction layer treatment solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 2.4g of tris (hydroxymethyl) aminomethane, 1g of tween 20, 0.4g of magnesium chloride, 30g of trehalose, 10g of polyvinyl alcohol 10, 200KU cystathionine-beta-synthetase, 200KU cystathionine-beta-lyase, the pH value of which is adjusted to 8.0 with hydrochloric acid.
And (3) treating an enzyme cycling reaction layer 5: cutting glass fiber (millipore G028) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(3) Preparation of plastic sheet 7:
preparing a plastic sheet treating fluid: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g sodium chloride, 2.4g tris, 1g tween 20, 10g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylanilinium sodium salt, 10g 4-aminoantipyrine, adjusted to pH 8.0 with hydrochloric acid.
Plastic sheet 7 treatment: cutting the dull polish plastic sheet (Dongguan alaqian optical material: natural dull polish PC-coarse sand) into 25mm × 300mm, spraying the above treatment liquid along one side of the plastic with a spraying device to form a color development area with a width of 15mm, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(4) Assembling:
cutting support layer 1 (Dongguan Shenghe electronic: 0.25mm PET plastic sheet) into 31.5mm × 300mm, cutting chromatographic membrane (Sidolisi CN140 nitrocellulose membrane) into 16mm × 300mm, and cutting diffusion layer 6 (upper sea screen: high tension silk screen gauze SS-PET165/25) into 5mm × 300mm
According to the structure shown in figure 1: fixing a chromatographic membrane 2, an enzyme pad 3, a blood filtration layer 4 and an enzyme circulation reaction layer 5 above a support layer 1 in sequence, dotting hot melt adhesive on the side surface of each layer, then placing a diffusion layer 6 on the enzyme circulation reaction layer 5 and the hot melt adhesive 8, and hot-pressing the diffusion layer 6 by a hot press to stick the diffusion layer 6 and the hot melt adhesive 8 together; and (3) dispensing a hot melt adhesive on the upper surface of the support layer 1 close to the other end, then placing a plastic sheet 7 on the hot melt adhesive (one end without the color developing agent coating is in contact with the hot melt adhesive), and pressing the plastic sheet 7 by a hot press to fix the plastic sheet 7 with the support layer 1 through the hot melt adhesive.
The test card (test paper) was cut into a width of 31.5mm × 5 mm.
Example 3:
this example provides a method for preparing dry homocysteine assay paper, which is prepared as follows.
(1) Preparation of enzyme pad 3:
preparing an enzyme pad treating solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 2.4g of tris (hydroxymethyl) aminomethane, 1g of tween 20, 0.4g of magnesium chloride, 30g of sucrose, 10g of polyvinyl alcohol 10, 300KU of peroxidase, 500U of pyruvate oxidase, and adjusting the pH to 8.0 with hydrochloric acid.
Enzyme pad 3 treatment: cutting tea filter paper (Shanghai Kanning paper industry: RF17) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in oven, drying at 45 deg.C for 1 hr, and taking out for use.
(2) Preparation of the enzyme circulation reaction layer 5:
preparing an enzyme circulation reaction layer treatment solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 2.4g of tris (hydroxymethyl) aminomethane, 1g of tween 20, 0.4g of magnesium chloride, 30g of sucrose, 10g of polyvinyl alcohol 10, 200KU cystathionine-beta-synthetase, 200KU cystathionine-beta-lyase, the pH value of which is adjusted to 8.0 with hydrochloric acid.
And (3) treating an enzyme cycling reaction layer 5: cutting glass fiber (millipore G028) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(3) Preparation of plastic sheet 7:
preparing a plastic sheet treating fluid: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g sodium chloride, 2.4g tris, 1g tween 20, 10g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylanilinium sodium salt, 10g 4-aminoantipyrine, adjusted to pH 8.0 with hydrochloric acid.
Plastic sheet 7 treatment: cutting the dull polish plastic sheet (Dongguan alaqian optical material: natural dull polish PC-coarse sand) into 25mm × 300mm, spraying the above treatment liquid along one side of the plastic with a spraying device to form a color development area with a width of 15mm, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(4) Assembling:
cutting support layer 1 (Dongguan Shenghe electronic: 0.25mm PET plastic sheet) into 31.5mm × 300mm, cutting chromatographic membrane (Sidolisi CN140 nitrocellulose membrane) into 16mm × 300mm, and cutting diffusion layer 6 (upper sea screen: high tension silk screen gauze SS-PET165/25) into 5mm × 300mm
Fixing a chromatographic membrane 2, an enzyme pad 3, a blood filtration layer 4 and an enzyme circulation reaction layer 5 above a support layer 1 in sequence according to the structure, dotting hot melt adhesive on the side surface of each layer, then placing a diffusion layer 6 on the enzyme circulation reaction layer 5 and the hot melt adhesive 8, and hot-pressing the diffusion layer 6 by a hot press to stick the diffusion layer 6 and the hot melt adhesive 8 together; and (3) hot-melting the other end point of the support layer 1, then placing a plastic sheet 7 on the hot-melting glue (one end without the color developing agent coating is contacted with the hot-melting glue), and pressing the plastic sheet 7 by a hot press so that the plastic sheet 7 is fixed with the support layer 1 through the hot-melting glue.
The test paper was cut to a width of 31.5mm × 5 mm.
Example 4:
this example provides a method for preparing dry homocysteine assay paper, which is prepared as follows.
(1) Preparation of enzyme pad 3:
preparing an enzyme pad treating solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 2.4g of tris (hydroxymethyl) aminomethane, 1g of tween 80, 0.4g of magnesium chloride, 30g of trehalose, 300KU of peroxidase, 500U of pyruvate oxidase, and the pH was adjusted to 8.0 with hydrochloric acid.
Enzyme pad 3 treatment: cutting tea filter paper (Shanghai Kanning paper industry: RF17) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in oven, drying at 45 deg.C for 1 hr, and taking out for use.
(2) Preparation of the enzyme circulation reaction layer 5:
preparing an enzyme circulation reaction layer treatment solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g sodium chloride, 2.4g tris (hydroxymethyl) aminomethane, 1g tween 80, 0.4g magnesium chloride, 30g trehalose, 200KU cystathionine-beta-synthetase, 200KU cystathionine-beta-lyase, adjusted to pH 8.0 with hydrochloric acid.
And (3) treating an enzyme cycling reaction layer 5: cutting glass fiber (millipore G028) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(3) Preparation of plastic sheet 7:
preparing a plastic sheet treating fluid: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g sodium chloride, 2.4g tris, 1g tween 20, 10g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylanilinium sodium salt, 10g 4-aminoantipyrine, adjusted to pH 8.0 with hydrochloric acid.
Plastic sheet 7 treatment: cutting the dull polish plastic sheet (Dongguan alaqian optical material: natural dull polish PC-coarse sand) into 25mm × 300mm, spraying the above treatment liquid along one side of the plastic with a spraying device to form a color development area with a width of 15mm, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(4) Assembling:
cutting support layer 1 (Dongguan Shenghe electronic: 0.25mm PET plastic sheet) into 31.5mm × 300mm, cutting chromatographic membrane (Sidolisi CN140 nitrocellulose membrane) into 16mm × 300mm, and cutting diffusion layer 6 (upper sea screen: high tension silk screen gauze SS-PET165/25) into 5mm × 300mm
Fixing a chromatographic membrane 2, an enzyme pad 3, a blood filtration layer 4 and an enzyme circulation reaction layer 5 above a support layer 1 in sequence according to the structure, dotting hot melt adhesive on the side surface of each layer, then placing a diffusion layer 6 on the enzyme circulation reaction layer 5 and the hot melt adhesive 8, and hot-pressing the diffusion layer 6 by a hot press to stick the diffusion layer 6 and the hot melt adhesive 8 together; and (3) hot-melting the other end point of the support layer 1, then placing a plastic sheet 7 on the hot-melting glue (one end without the color developing agent coating is contacted with the hot-melting glue), and pressing the plastic sheet 7 by a hot press so that the plastic sheet 7 is fixed with the support layer 1 through the hot-melting glue.
The test paper was cut to a width of 31.5mm × 5 mm.
Example 5:
this example provides a method for preparing dry homocysteine assay paper, which is prepared as follows.
(1) Preparation of enzyme pad 3:
preparing an enzyme pad treating solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 2.4g of tris (hydroxymethyl) aminomethane, 1g of tween 20, 0.4g of magnesium chloride, 30g of trehalose, 10g of polyvinyl alcohol 10, 300KU of peroxidase, 500U of pyruvate oxidase, and adjusting the pH to 8.0 with hydrochloric acid.
Enzyme pad 3 treatment: cutting tea filter paper (Shanghai Kanning paper industry: RF17) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in oven, drying at 45 deg.C for 1 hr, and taking out for use.
(2) Preparation of the enzyme circulation reaction layer 5:
preparing an enzyme circulation reaction layer treatment solution: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g of sodium chloride, 2.4g of tris (hydroxymethyl) aminomethane, 1g of tween 20, 0.4g of magnesium chloride, 30g of trehalose, 10g of polyvinyl alcohol 10, 200KU cystathionine-beta-synthetase, 200KU cystathionine-beta-lyase, the pH value of which is adjusted to 8.0 with hydrochloric acid.
And (3) treating an enzyme cycling reaction layer 5: cutting glass fiber (millipore G028) into 5mm × 300mm, soaking in the above treatment solution for 5 min, taking out, draining residual liquid, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(3) Preparation of plastic sheet 7:
preparing a plastic sheet treating fluid: 1L of purified water is measured, and the following substances are respectively added and stirred and mixed uniformly: 0.5g sodium chloride, 2.4g tris, 1g tween 20, 10g N-ethyl-N- (2-hydroxy-3-propanesulfo) m-toluidine, 10g 4-aminoantipyrine, adjusted to pH 8.0 with hydrochloric acid.
Plastic sheet 7 treatment: cutting the dull polish plastic sheet (Dongguan alaqian optical material: natural dull polish PC-coarse sand) into 25mm × 300mm, spraying the above treatment liquid along one side of the plastic with a spraying device to form a color development area with a width of 15mm, horizontally placing in an oven, drying at 45 deg.C for 1 hr, and taking out for use.
(4) Assembling:
cutting support layer 1 (Dongguan Shenghe electronic: 0.25mm PET plastic sheet) into 31.5mm × 300mm, cutting chromatographic membrane (Sidolisi CN140 nitrocellulose membrane) into 16mm × 300mm, and cutting diffusion layer 6 (upper sea screen: high tension silk screen gauze SS-PET165/25) into 5mm × 300mm
Fixing a chromatographic membrane 2, an enzyme pad 3, a blood filtration layer 4 and an enzyme circulation reaction layer 5 above a support layer 1 in sequence according to the structure, dotting hot melt adhesive on the side surface of each layer, then placing a diffusion layer 6 on the enzyme circulation reaction layer 5 and the hot melt adhesive 8, and hot-pressing the diffusion layer 6 by a hot press to stick the diffusion layer 6 and the hot melt adhesive 8 together; and (3) hot-melting the other end point of the support layer 1, then placing a plastic sheet 7 on the hot-melting glue (one end without the color developing agent coating is contacted with the hot-melting glue), and pressing the plastic sheet 7 by a hot press so that the plastic sheet 7 is fixed with the support layer 1 through the hot-melting glue.
The test paper was cut to a width of 31.5mm × 5 mm.
Example 6:
the linear range and the analytical sensitivity of the dry homocysteine detection reagent are detected.
The test method comprises the following steps: a25. mu.L sample was taken into an R1 tube, mixed well and reacted for 2 minutes. After the sample has reacted with R1, the tube is shaken again to mix the components thoroughly. The reagent was opened and 25. mu.L of the mixture was pipetted into the test card. And inserting the test paper into an analyzer to detect the reflectivity of 630nm wavelength, and calculating according to the calibration curve to obtain a test result.
Preparation of linear range detection samples: the samples with low value and high value of concentration of 2 mu mol/L and 100 mu mol/L are mixed into 10 concentration samples according to the proportion of 10/0, 9/1, 8/2, 7/3, 6/4, 5/5, 4/6, 3/7, 2/8, 1/9 and 0/10, and the test is carried out according to the test method.
Table 1: linear range detection results
Preparation of assay sensitivity detection samples: samples at a concentration of 20. mu. mol/L were diluted with 5% BSA to a concentration of 1. mu. mol/L, 2. mu. mol/L, 3. mu. mol/L, 4. mu. mol/L, 5. mu. mol/L, 10. mu. mol/L, 20. mu. mol/L, respectively, and tested according to the above-described test methods.
Table 2: analysis of sensitivity detection results
The linear range and the analysis sensitivity of the dissociating agent R1-3 and the test paper of the example 2 are better, which shows that the linear range and the analysis sensitivity of the combination of the dissociating agent tri (2-carboxyethyl) phosphine and the developer N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline sodium salt are better.
Example 7:
and (3) detecting the stability of the dry homocysteine detection reagent.
The test method comprises the following steps: a25. mu.L sample was taken into an R1 tube, mixed well and reacted for 2 minutes. After the sample has reacted with R1, the tube is shaken again to mix the components thoroughly. The reagent was opened and 25. mu.L of the mixture was pipetted into the test card. The test paper was inserted into the analyzer to measure the reflectance at a wavelength of 630nm, and the test results calculated from the calibration curve are shown in table 3 below.
TABLE 3 stability test results
The above results indicate that the stability of the three formulations of the strip is not substantially different, and the stability of the strip of example 4 is slightly worse.
Example 8:
the correlation test of the clinical samples with the dissociation agent R1-3 and the test paper of example 2.
124 clinical samples are taken and detected by a tandem mass spectrometry method, a dissociating agent R1-3 and test paper of example 2 respectively, the results are shown in figure 2, and correlation coefficients R2 of the two results are 0.998.
According to the embodiment, the test card can be used for detecting all homocysteine in human blood, and is high in detection sensitivity, good in stability and small in error.
Claims (11)
1. A dry homocysteine test card which is characterized in that: the reagent kit comprises a dissociation solution R1 and dry chemical test paper, wherein the dissociation solution R1 comprises a reducing agent, serine and a buffer solution; the dry chemical test paper comprises a support layer, a chromatographic membrane, an enzyme pad, a blood filtering layer, an enzyme circulation reaction layer, a diffusion layer and a plastic sheet; the chromatographic membrane, the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer are sequentially overlapped on the supporting layer from bottom to top, the length of the chromatographic membrane is greater than that of the enzyme pad, the blood filtering layer, the enzyme circulation reaction layer and the diffusion layer, and the chromatographic membrane is fixed at one end close to the supporting layer and extends towards the other end of the supporting layer; the support layer is provided with a plastic sheet near one end far away from the enzyme pad, the plastic sheet is provided with a color development area, the plastic sheet is positioned above the chromatographic membrane and is arranged parallel to the chromatographic membrane, and the color development area covers partial area of the chromatographic membrane.
2. The dry homocysteine test card according to claim 1 wherein: the plastic sheet is adhered to the supporting layer through an adhesive layer; enzyme pad, filter layer and enzyme circulation reaction layer the upper and lower surface equal and overlap the setting from top to bottom each other, the upper and lower surface of diffusion layer be greater than the upper and lower surface of enzyme pad, the one end of diffusion layer flushes with the one end of enzyme circulation reaction layer, the other end extends enzyme circulation reaction layer, enzyme pad, filter layer, enzyme circulation reaction layer the side and the diffusion layer extend enzyme circulation reaction layer part and pass through the viscose layer and support layer and bond each other.
3. The dry homocysteine test card according to claim 1 wherein: the dissociation solution R1 comprises a reducing agent, serine and a buffer solution; the reducing agent is one or more of tri (2-carboxyethyl) phosphine, beta-mercaptoethanol and dithiothreitol with the concentration of 0.05-0.5 mmol/L; the buffer solution is one or more of trihydroxymethyl aminomethane, 4-hydroxyethyl piperazine ethanesulfonic acid, 2 morpholinoethanesulfonic acid and 3- (N-morpholinyl) propanesulfonic acid with the pH value of 5.0-9.0 and the concentration of 10 mmol/L-200 mmol/L; the concentration of the serine is 0.05 g/L-0.5 g/L.
4. The dry homocysteine test card according to claim 1 wherein: the support layer is made of opaque high polymer material and has a thickness of 0.10-0.30 mm; the chromatographic membrane is a nitrocellulose membrane.
5. The dry homocysteine test card according to claim 1 wherein: the enzyme pad is made of glass fiber, polyester fiber or non-woven fabric; the specific preparation method of the enzyme pad comprises the following steps: cutting the material into a specified size, soaking in an enzyme pad treatment solution, and drying; the enzyme pad treating solution contains buffer solution, pyruvate oxidase, peroxidase, coenzyme, protecting agent and surfactant.
6. The dry homocysteine test card according to claim 5 wherein: the buffer solution in the enzyme pad treatment solution is one or more of trihydroxymethyl aminomethane, 4-hydroxyethyl piperazine ethanesulfonic acid, 2-morpholinoethanesulfonic acid and 3- (N-morpholinyl) propanesulfonic acid with the pH value of 5.0-9.0 and the concentration of 10 mmol/L-200 mmol/L; the protective agent is one or more of sucrose, trehalose or polyvinyl alcohol with the concentration of 10 g/L-100 g/L; the surfactant is Tween 20 with the concentration of 0.5 g/L-10 g/L; the concentration of the pyruvate oxidase is 100-1000 KU/L; the concentration of the peroxidase is 200-2000 KU/L; the coenzyme is one or more of magnesium chloride, magnesium sulfate and magnesium acetate with concentration of 0.5-10 mmol/L.
7. The dry homocysteine test card according to claim 1 wherein: the blood filtering layer adopts porous polyester fiber, cotton fiber or non-woven fabric; the material of the enzyme circulation reaction layer adopts glass fiber, polyester fiber or non-woven fabric; the preparation method of the enzyme circulation reaction layer comprises the following steps: cutting the material into a specified size, soaking in an enzyme circulation reaction layer treatment solution, and drying; the treating liquid of the enzyme circulating reaction layer contains buffer solution, cystathionine-beta-synthetase, cystathionine-beta-lyase, protective agent, surfactant and the like.
8. The dry homocysteine test card according to claim 7 wherein: the buffer solution in the enzyme circulation reaction layer treatment solution is one or more of trihydroxymethyl aminomethane, 4-hydroxyethyl piperazine ethanesulfonic acid, 2-morpholinoethanesulfonic acid and 3- (N-morpholinyl) propanesulfonic acid with the pH value of 5.0-9.0 and the concentration of 10 mmol/L-200 mmol/L; the protective agent is one or more of sucrose, trehalose or polyvinyl alcohol with the concentration of 10 g/L-100 g/L; the surfactant is Tween 20 with the concentration of 0.5 g/L-10 g/L; the concentration of the cystathionine-beta-synthetase is 50-1000 KU/L; the concentration of the cystathionine-beta-lyase is 50-1000 KU/L.
9. The dry homocysteine test card according to claim 1 wherein: the diffusion layer is 100-mesh and 1000-mesh net cloth; the plastic sheet is made of transparent high polymer materials, and the thickness of the plastic sheet is 0.10-0.30 mm.
10. The dry homocysteine test card according to claim 1 wherein: 4-aminoantipyrine and a color development substrate are fixed in the color development area in a dot spraying mode by a dot spraying instrument; the 4-aminoantipyrine and the chromogenic substrate are firstly prepared into a solution, and the concentration of the 4-aminoantipyrine in the solution is 2 g/L-50 g/L; the chromogenic substrate is one of Trinder's chromogenic substrates, and the concentration of the Trinder's chromogenic substrate in the solution is 2 g/L-50 g/L.
11. An application of dry-type homocysteine test card in preparing the test substance for measuring the homocysteine content in human blood.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010147017.5A CN111855648A (en) | 2020-03-05 | 2020-03-05 | Dry type homocysteine test card and application thereof |
| PCT/CN2020/123510 WO2021174873A1 (en) | 2020-03-05 | 2020-10-26 | Dry homocysteine test card and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202010147017.5A CN111855648A (en) | 2020-03-05 | 2020-03-05 | Dry type homocysteine test card and application thereof |
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| CN111855648A true CN111855648A (en) | 2020-10-30 |
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| CN202010147017.5A Pending CN111855648A (en) | 2020-03-05 | 2020-03-05 | Dry type homocysteine test card and application thereof |
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| WO (1) | WO2021174873A1 (en) |
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| CN112812963A (en) * | 2021-01-28 | 2021-05-18 | 美康生物科技股份有限公司 | Total homocysteine test card |
| CN113418916A (en) * | 2021-07-02 | 2021-09-21 | 安徽惠邦生物工程有限公司 | Creatinine quantitative rapid detection test strip and preparation method thereof |
| CN113884686A (en) * | 2021-10-18 | 2022-01-04 | 四川成电医联科技咨询有限公司 | Siphon type total homocysteine test paper and manufacturing method thereof |
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| CN114774511B (en) * | 2022-04-12 | 2024-05-10 | 广州万德康科技有限公司 | Reaction liquid for detecting magnesium ions in blood sample, dry test paper and preparation method thereof |
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| Publication number | Publication date |
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| WO2021174873A1 (en) | 2021-09-10 |
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