Dry chemical reagent tablet for quantitative detection of creatine kinase and preparation method thereof

Abstract

The present invention provides a dry chemical reagent sheet for quantitatively detecting creatine kinase and a preparation method thereof, and relates to the field of dry chemical reagent sheets. The dry chemical reagent sheet includes a light-transmitting layer, a reagent layer and a diffusion layer, wherein the reagent layer is arranged above the light-transmitting layer, the diffusion layer is above the reagent layer, the diffusion layer includes a diffusion layer reagent or is formed by a diffusion layer reagent, the diffusion layer reagent includes a hydrophilic polymer, a surfactant and particulate matter, wherein the particulate matter includes a first particulate matter and a second particulate matter; the number ratio of the first particulate matter to the second particulate matter is 1:(0.25-4), and the difference in particle size between the first particulate matter and the second particulate matter is 1-4 μm. The dry chemical reagent sheet provided by the present invention can quickly and accurately quantitatively detect the content of creatine kinase in a liquid sample, has good repeatability, and has a simple preparation process, a small sample size, and is easy to store and transport.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims the priority of Chinese patent application CN 202411438738.6, entitled “Dry chemical reagent sheet for quantitative detection of creatine kinase and preparation method thereof”, filed on October 15, 2024. The entire contents of the above application are incorporated herein by reference. Technical Field

[0003] The invention relates to the field of dry chemical reagent tablets, and in particular to a dry chemical reagent tablet for quantitatively detecting creatine kinase and a preparation method thereof. Background Art

[0004] Creatine kinase (CK), also known as creatine phosphokinase, is mainly found in the cytoplasm and mitochondria. CK is a dimeric enzyme composed of two monomeric subunits, M (muscle) type and B (brain) type, which is directly related to intracellular energy circulation, muscle contraction and ATP regeneration. CK is mainly present in skeletal muscle and myocardium, and is also distributed in brain tissue. Increased CK is common in patients with myocardial infarction, viral myocarditis, cerebrovascular accident, dermatomyositis and muscular dystrophy; decreased CK is common in hyperthyroidism. As one of the important enzymatic indicators for diagnosing myocardial infarction, the activity determination of CK can also be used for the diagnosis of skeletal muscle diseases and other myocardial diseases.

[0005] However, the wet method commonly used in clinical practice usually requires a large number of manual operation steps such as sample preparation, reagent addition, and reaction monitoring. The operation is complicated and has a high risk of error. At the same time, the detection process usually takes a long time to complete and the cost is relatively high, so its application has always been relatively limited.

[0006] Quantitative dry film testing belongs to dry chemical analysis, which is a technical method widely used in clinical testing and biochemical research. Its core feature is to pre-dry a variety of reaction reagents and fix them on paper or other carriers. Then, during the actual test, the liquid in the sample (such as whole blood, serum, plasma, urine, etc.) is used as the reaction medium to make the components to be tested in the sample react directly with the dry reagent. This reaction usually produces a color change, and qualitative or quantitative analysis is performed through naked eye observation or instrument detection. Compared with traditional wet chemistry, quantitative dry film testing has many advantages such as simple and convenient operation, fast results, low cost, small dosage, easy storage, easy to combine with automated equipment, strong adaptability, and environmental friendliness.

[0007] To this end, we hope to develop a dry film for quantitative detection of creatine kinase, which can not only effectively simplify the detection process of creatine kinase and reduce the cost of use, but also optimize the application environment and realize convenient and fast POCT (point-of-care testing). Summary of the invention

[0008] In view of the above analysis, the present invention aims to provide a dry chemical reagent sheet for quantitative detection of creatine kinase and a preparation method thereof, so as to solve at least one of the following existing problems: large amount of blood drawn, long analysis cycle, complicated process, inability to measure in real time, etc.

[0009] The purpose of the present invention is mainly achieved through the following technical solutions:

[0010] In the first aspect, the present invention provides a dry chemical reagent sheet for quantitatively detecting creatine kinase, comprising a light-transmitting layer, a reagent layer and a diffusion layer, wherein the reagent layer is arranged above the light-transmitting layer, and the diffusion layer is above the reagent layer. The diffusion layer comprises a diffusion layer reagent or is formed by a diffusion layer reagent, and the diffusion layer reagent comprises a hydrophilic polymer, a surfactant and particulate matter, wherein the particulate matter comprises a first particulate matter and a second particulate matter; the number ratio of the first particulate matter to the second particulate matter is 1:(0.25-4), preferably 1:(0.5-1.5), and the difference in particle size between the first particulate matter and the second particulate matter is 1-4 μm, preferably 1.5-2.5 μm.

[0011] Preferably, the particle size of the first particles is 4 to 8 μm, preferably 4.5 to 5.5 μm.

[0012] Preferably, the particle size of the second particles is 1 to 4 μm, preferably 2.5 to 3.5 μm.

[0013] Preferably, the amount of the hydrophilic polymer in the diffusion layer reagent is 1 to 25 g / m 2 , preferably 10 to 20 g / m 2 ; The dosage of surfactant is 5~50g / m 2 , preferably 5 to 15 g / m 2 ; The amount of particles is 10~500g / m 2 , preferably 100 to 200 g / m 2 .

[0014] Preferably, in the diffusion layer reagent, the hydrophilic high molecular polymer includes at least one of cellulose acetate and cellulose nitrate. Preferably, the viscosity of the hydrophilic high molecular polymer is 30 to 45 poise, preferably 35 to 40 poise.

[0015] Preferably, in the diffusion layer reagent, the surfactant includes at least one of Tween, Triton and Surfynol; preferably, in the diffusion layer reagent, the surfactant includes Tween, Triton and Surfynol, and the mass ratio of Tween, Triton and Surfynol is (1-3):1:(1-3).

[0016] Preferably, the first particulate matter and the second particulate matter are the same or different, and each independently comprises at least one of titanium dioxide, barium sulfate, diatomaceous earth, kaolin, barium titanate, and polystyrene microspheres.

[0017] Preferably, the reagent layer includes a reagent layer reagent or is formed by a reagent layer reagent, and the reagent layer reagent includes a buffer system, a biological enzyme, a color developer, a surfactant and a colloid, wherein the biological enzyme includes peroxidase, glycerol phosphate oxidase, and glycerol kinase, wherein the activity of peroxidase is 250-300 U / mg, preferably 270-290 U / mg, and more preferably 275-285 U / mg; the activity of glycerol kinase is 50-100 U / mg, preferably 60-90 U / mg, and more preferably 70-80 U / mg; the activity of L-α-glycerol phosphate oxidase is 8-45 U / mg, preferably 30-45 U / mg, and more preferably 35-40 U / mg.

[0018] Preferably, the amount of the buffer system in the reagent layer reagent is 5 to 20 mol / m 2 ; preferably 9 to 15 mol / m 2 ; The dosage of glycerol kinase is 3000~10000U / m 2 , preferably 4000~6000U / m 2 ; The dosage of peroxidase is 30000~60000U / m 2 , preferably 40000~50000U / m 2 ; The dosage of L-α-glycerophosphate oxidase is 3000~10000U / m 2 , preferably 3000~5000U / m 2 ; The dosage of the developer is 200~300mmol / m 2 ; preferably 240 to 280 mmol / m 2 ; The dosage of surfactant is 5~20g / m 2 ; preferably 5 to 10 g / m 2 ; The amount of colloid used is 1 to 50 g / m 2 ; preferably 1 to 15 g / m 2 .

[0019] Preferably, in the reagent layer reagent, the buffer system includes at least one of a phosphate buffer system, a Tris buffer system, and a boric acid buffer system; and the pH of the buffer system is 6.5 to 9.5.

[0020] Preferably, in the reagent layer reagent, the color developing agent includes 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-p-(4-dimethylaminophenyl)imidazole.

[0021] Preferably, in the reagent layer reagent, the surfactant includes at least one of Tween, Triton, Surfynol, and S24; preferably, in the reagent layer reagent, the surfactant includes S24 and Triton, wherein: the amount of S24 is 5 to 15 g / m 2 , preferably 5 to 9 g / m 2 ; The dosage of Triton is 0.1~5g / m 2 , preferably 0.1 to 1 g / m 2 .

[0022] Preferably, the colloid comprises at least one of gelatin, pullulan, polyvinyl alcohol, polyacrylamide, and hydrophilic cellulose derivatives. Preferably, the gelatin is selected from at least one of photographic gelatin and edible gelatin.

[0023] Preferably, the reagent layer reagent also includes N-acetyl-L-cysteine, creatine phosphate, ADP, magnesium salt and glycerol, wherein: the amount of N-acetyl-L-cysteine ​​is 10-100 mg / m 2 ; The dosage of creatine phosphate is 7-9 g / m 2 ; The dosage of ADP is 100~300mg / m 2 ; The dosage of magnesium salt is 200~300mg / m 2 The dosage of glycerol is 0.1~5g / m 2 .

[0024] Preferably, the thickness of the reagent layer is 10-100 μm, the thickness of the diffusion layer is 200-600 μm, and the thickness of the light-transmitting layer is 30-300 μm.

[0025] In a second aspect, the present invention provides a method for preparing the dry chemical reagent sheet, comprising the following steps:

[0026] Step 1: Apply the reagent layer reagent on the light-transmitting layer and dry it;

[0027] Step 2: Apply the diffusion layer reagent on the reagent layer and dry.

[0028] Beneficial effects:

[0029] The dry chemical reagent sheet provided by the present invention is easy to operate and can quickly and accurately quantitatively detect the content of creatine kinase in a liquid sample. The test result has high accuracy and good repeatability. The dry chemical reagent sheet has a simple preparation process, a small sample size, is easy to store and transport, has low cost, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic structural diagram of the dry chemical reagent tablet provided by the present invention.

[0031] Figure 2This is a color comparison diagram of the dry chemical reagent sheet of Example 1 provided by the present invention.

[0032] In the figure, 1-upper outer frame, 2-sample loading hole of upper outer frame, 3-diffusion layer, 4-reagent layer, 5-light-transmitting layer, 6-light-transmitting hole of lower outer frame, 7-lower outer frame. DETAILED DESCRIPTION

[0033] Preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of the present invention and are used to explain the principles of the present invention together with the embodiments of the present invention.

[0034] The present invention provides a dry chemical reagent sheet for rapidly measuring the content of creatine kinase. The dry chemical reagent sheet is composed of an upper outer frame, a diffusion layer, a reagent layer, a light-transmitting layer and a lower outer frame from top to bottom. The diffusion layer is a porous cellulose membrane, including a cellulose solution and one or more of titanium dioxide, barium sulfate, barium titanate, polystyrene microspheres and the like. The reagent layer includes a buffer system, a biological enzyme, a color developer, a surfactant, a colloid and the like. The diffusion layer, the reagent layer and the light-transmitting layer are sequentially bonded together through a coating process. A sample addition hole is provided at the center of the upper outer frame, and a light-transmitting hole is provided at the center of the lower outer frame. A liquid sample is dropped into the sample addition hole, and after being evenly dispersed through the diffusion layer, it reacts with a preset reagent in the reagent layer to produce a color-developing circular spot. The concentration of creatine kinase is quantitatively determined according to the depth of the color of the circular spot. Compared with the wet chemical detection method, the dry chemical reagent sheet has the advantages of fast speed, high accuracy, simple and convenient operation, and reliable results, and is an ideal method for creatine kinase detection. It can be widely used in many fields such as in vitro diagnosis, drug synthesis and screening, environmental monitoring and protection, health quarantine, and forensic identification.

[0035] Hereinafter, the present invention will be described in detail.

[0036] In a first aspect, the present invention provides a dry chemical reagent sheet for quantitatively detecting creatine kinase, comprising a light-transmitting layer and a reagent layer, wherein the reagent layer is arranged above the light-transmitting layer, the reagent layer comprises a reagent layer reagent or is formed by a reagent layer reagent, and the reagent layer reagent comprises a buffer system, a biological enzyme, a color developer, a surfactant and a colloid.

[0037] As an embodiment of the present invention, the buffer system is at least one of a phosphate buffer system, a Tris buffer system, and a boric acid buffer system, and the pH is 6.5 to 9.5. The amount of the buffer system is 5 to 20 mol / m 2 , preferably 9 to 15 mol / m 2 .

[0038] As a specific embodiment of the present invention, the phosphate buffer system includes disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0039] As a specific embodiment of the present invention, the boric acid buffer system includes boric acid and sodium borate.

[0040] As a specific embodiment of the present invention, the Tris buffer system includes Tris and hydrochloric acid.

[0041] As a specific embodiment of the present invention, the biological enzyme includes peroxidase, L-α-phosphoglycerate oxidase, and glycerol kinase.

[0042] The dosage of glycerol kinase is 3000~10000U / m 2 , preferably 4000~6000U / m 2 ;

[0043] The dosage of peroxidase is 30000~60000U / m 2 , preferably 40000~50000U / m 2 ;

[0044] The dosage of L-α-glycerophosphate oxidase is 3000~10000U / m 2 , preferably 3000~5000U / m 2 .

[0045] As a specific embodiment of the present invention, the activity of peroxidase is 250-300 U / mg, preferably 270-290 U / mg, more preferably 275-285 U / mg; the activity of glycerol kinase is 50-100 U / mg, preferably 60-90 U / mg, more preferably 70-80 U / mg; the activity of L-α-glycerophosphate oxidase is 8-45 U / mg, preferably 30-45 U / mg, more preferably 35-40 U / mg.

[0046] As a specific embodiment of the present invention, in the reagent layer reagent, the color developer is selected from 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-p-(4-dimethylaminophenyl)imidazole, and the amount of the color developer is 200 to 300 mmol / m 2 , preferably 240 to 280 mmol / m 2 .

[0047] As a specific embodiment of the present invention, in the reagent layer reagent, the surfactant is one or more of Tween (model may be Teween-80), Triton (model may be TritonX-100), Surfynol (model may be Surfynol 485), S24 (model may be SURFACTANT 10G). The amount of the surfactant used is 5 to 20 g / m 2 , preferably 5 to 10 g / m 2 .

[0048] As a specific embodiment of the present invention, in the reagent layer reagent, the surfactant includes S24 and Triton, wherein:

[0049] The dosage of S24 is 5-15g / m 2 , preferably 5 to 9 g / m 2 ;

[0050] The dosage of Triton is 0.1~5g / m 2 , preferably 0.1 to 1 g / m 2 .

[0051] As a specific embodiment of the present invention, in the reagent layer reagent, the colloid is selected from at least one of gelatin, pullulan, polyvinyl alcohol, polyacrylamide, and hydrophilic cellulose derivatives; the amount of the colloid is 1 to 50 g / m 2 , preferably 1 to 15 g / m 2 .

[0052] As a specific embodiment of the present invention, in the reagent layer reagent, the gelatin is selected from at least one of photographic gelatin and edible gelatin.

[0053] As a specific embodiment of the present invention, the reagent layer reagent also includes: N-acetyl-L-cysteine, creatine phosphate, adenosine diphosphate (ADP), MgCl2·6H2O and glycerol, wherein:

[0054] The dosage of N-acetyl-L-cysteine ​​is 10-100 mg / m 2 , preferably 64.8 mg / m 2 ;

[0055] The dosage of creatine phosphate is 7-9 g / m 2 , preferably 7.9 g / m 2 ;

[0056] The dosage of ADP is 100-300 mg / m 2 , preferably 200 mg / m 2 ;

[0057] The dosage of MgCl2·6H2O is 200~300mg / m 2 , preferably 266.7 mg / m 2 ;

[0058] The dosage of glycerol is 0.1~5g / m 2 , preferably 0.2g / m 2 .

[0059] As a specific embodiment of the present invention, the reagent layer has a wet film thickness of 200 to 500 μm, a dry film thickness of 10 to 100 μm, and preferably a dry film thickness of 15 to 30 μm.

[0060] As a specific embodiment of the present invention, the reagent sheet further includes a diffusion layer disposed above the reagent layer, the diffusion layer includes a diffusion layer reagent or is formed by a diffusion layer reagent, and the diffusion layer reagent includes a hydrophilic polymer solution, a surfactant, and particulate matter.

[0061] As a specific embodiment of the present invention, in the diffusion layer reagent, the hydrophilic polymer is selected from at least one of cellulose acetate and cellulose nitrate, wherein the amount of the hydrophilic polymer is 1 to 25 g / m 2 , preferably 10 to 20 g / m 2 .

[0062] As a specific embodiment of the present invention, the viscosity of the hydrophilic high molecular polymer is 30 to 45 poise, for example, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 poise, etc., preferably 35 to 40 poise. It should be noted that the viscosity determination method of the hydrophilic high molecular polymer such as cellulose acetate is to determine the intrinsic viscosity of cellulose acetate by the viscosity method, and the intrinsic viscosity of cellulose acetate in tetrahydrofuran solvent is determined by using the viscosity method, that is, the constant in the molecular weight equation.

[0063] As a specific embodiment of the present invention, in the diffusion layer reagent, the surfactant is one or more of Tween (model may be Teween-80), Triton (model may be TritonX-100), Surfynol (model may be Surfynol 485). The amount of the surfactant used is 5 to 50 g / m 2 , preferably 5 to 15 g / m 2 .

[0064] As a specific embodiment of the present invention, in the diffusion layer reagent, the surfactant includes Tween, Triton and Surfynol, wherein the mass ratio of Tween, Triton and Surfynol is (1-3):1:(1-3).

[0065] As a specific embodiment of the present invention, in the diffusion layer reagent, the particles include first particles and second particles, the first particles and the second particles are the same or different, and are independently selected from at least one of titanium dioxide, barium sulfate, diatomaceous earth, kaolin, barium titanate, and polystyrene microspheres; the amount of the particles is 10 to 500 g / m 2 , preferably 100 to 200 g / m 2 .

[0066] As a specific embodiment of the present invention, the average particle size of the first particles is 4 to 8 μm, for example, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 μm, etc., preferably 4.5 to 5.5 μm, and more preferably 5 μm.

[0067] As a specific embodiment of the present invention, the average particle size of the second particulate matter is 1 to 4 μm, for example, 1.5, 2.0, 2.5, 3.0, 3.5 μm, etc., preferably 2.5 to 3.5 μm, and more preferably 3 μm.

[0068] As a specific embodiment of the present invention, the number ratio of the first particles to the second particles is 1:(0.25-4), preferably 1:(0.5-1.5), and more preferably 1:1.

[0069] As a specific embodiment of the present invention, the difference in particle size between the first particle and the second particle is 1-4 μm, such as 1.5, 2.0, 2.5, 3.0, 3.5 μm, etc., preferably 1.5-2.5 μm. It should be noted that the difference in the present invention refers to the absolute value.

[0070] In the present invention, the combination of the first particulate matter and the second particulate matter will change the pore size of the porous cellulose membrane of the diffusion layer, thereby further effectively improving the porosity of the diffusion layer, and further improving the diffusion speed of the droplets on the diffusion layer and the uniformity of the diffusion circular spots. It should be noted that in the present invention, unless otherwise specified, the particle size of the particulate matter is the average particle size.

[0071] As a specific embodiment of the present invention, the diffusion layer has a wet film thickness of 500 to 1200 μm, a dry film thickness of 200 to 600 μm, and preferably a dry film thickness of 300 to 500 μm.

[0072] As a specific embodiment of the present invention, the light-transmitting layer comprises a transparent plastic material, and the material is selected from at least one of polyethylene PE, polypropylene PP, polyester methyl methacrylate PMMA, polystyrene PS, polycarbonate PC, and polyethylene terephthalate PET, with PET being the best.

[0073] As a specific embodiment of the present invention, the thickness of the light-transmitting layer is 30 to 300 μm, preferably 100 to 200 μm.

[0074] It should be noted that the light transmittance of the light-transmitting layer to visible light with a wavelength of 670 nm is above 80%, preferably above 95%. Both the incident light source and the reflected light can pass through the light-transmitting layer.

[0075] As a specific embodiment of the present invention, the surface of the light-transmitting layer is pre-treated by corona, plasma irradiation, ultraviolet irradiation, etc.

[0076] As a specific embodiment of the present invention, the reagent sheet further comprises an upper outer frame, which is arranged above the diffusion layer. A sample addition hole is arranged at the center of the upper outer frame, and the diameter of the hole is 5 to 10 mm.

[0077] As a specific embodiment of the present invention, the reagent sheet further comprises a lower outer frame, which is arranged below the light-transmitting layer. A light-transmitting hole is arranged at the center of the lower outer frame, and the diameter of the hole is 5 to 10 mm.

[0078] As a specific embodiment of the present invention, a glue layer is attached to the lower outer frame for bonding the upper outer frame. The upper outer frame and the lower outer frame can fix the multi-layer film structure and prevent the oxidation of the pre-set reagent in the reagent layer, thereby extending the storage time of the reagent.

[0079] The dry chemical reagent sheet provided by the present invention has the advantages of being easy to operate and capable of quickly and accurately quantitatively detecting the content of creatine kinase in a liquid sample. The detection result has high accuracy and good repeatability, and the dry chemical reagent sheet has a simple preparation process, a small sample size, is easy to store and transport, has low cost, and has a wide range of applications.

[0080] In the present invention, when the sample is dropped onto the dry sheet, it diffuses into the reagent layer through the diffusion layer, and N-acetyl-L-cysteine ​​(NAC) and Mg 2+ Activate creatine kinase. Under the action of creatine kinase, creatine phosphate and ADP are converted into creatine and ATP. In the presence of glycerol kinase (GK), glycerol undergoes phosphorylation under the action of ATP and is converted into L-α-glycerophosphate. Under the action of L-α-glycerophosphate oxidase (α-GPO), L-α-glycerophosphate is oxidized into dihydroxyacetone phosphate and hydrogen peroxide. Finally, hydrogen peroxide oxidizes the colorless dye under the action of peroxidase to generate dye for color development.

[0081] In a second aspect, the present invention provides a method for preparing a dry chemical reagent sheet for quantitatively detecting creatine kinase, comprising the following steps:

[0082] Step 1: Apply the reagent layer reagent on the light-transmitting layer and dry it;

[0083] Step 2: Apply the diffusion layer reagent on the reagent layer and dry;

[0084] Step 3: Assemble the upper outer frame and the lower outer frame.

[0085] As a specific embodiment of the present invention, the structure of the dry chemical reagent sheet provided by the present invention can be as follows: Figure 1 shown. Figure 1 In the figure, 1 is an upper outer frame, 2 is a sample loading hole of the upper outer frame, 3 is a diffusion layer, 4 is a reagent layer, 5 is a light-transmitting layer, 6 is a light-transmitting hole of the lower outer frame, and 7 is a lower outer frame.

[0086] The preferred embodiments of the present invention are described in detail below to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0087] It should be noted that, in the present invention, the amount of each component in the reagent layer reagent and the diffusion layer reagent is the amount of the listed substances, and does not include the amount of the solvent for dissolving the component.

[0088] Unless otherwise specified, the reagents used in the embodiments of the present invention are all commercially available products.

[0089] Example 1

[0090] This embodiment provides a dry chemical reagent sheet for quantitative determination of creatine kinase and a preparation method thereof. The dry chemical reagent sheet is composed of an upper outer frame 1, a diffusion layer 3, a reagent layer 4, a light-transmitting layer 5 and a lower outer frame 7 from top to bottom (eg, Figure 1 A sample adding hole 2 is provided at the center of the upper outer frame, and a light transmission hole 6 is provided at the center of the lower outer frame.

[0091] Use Figure 1 The dry chemical reagent sheet structure shown in the figure has the following specific operation steps:

[0092] The light-transmitting layer 5 is made of PET film with a thickness of 125 μm and a light transmittance of more than 95% at a wavelength of 670 nm. The formulation of the reagent layer 4 is shown in Table 1. The prepared reagent layer coating liquid is evenly coated on the light-transmitting layer PET film, and the wet film thickness is 300 μm. Place it in a blast drying oven at 37°C, and continue to coat the diffusion layer 3 after it is completely dried, with a wet film thickness of 1000 μm. The diffusion layer formulation is shown in Table 2. After drying in a fume hood, cut into 1 cm 2 The reagent sheet is assembled by using a plastic sheet as the upper outer frame 1 and the lower outer frame 7 of the reagent sheet. The central opening diameter of the upper and lower outer frames is 10 mm.

[0093] Table 1 Reagent layer formula

[0094]

[0095]

[0096] Table 2 Diffusion layer formula

[0097]

[0098] Example 2

[0099] This embodiment is basically the same as embodiment 1, except that in the reagent layer,

[0100] Peroxidase activity 281U / mg,

[0101] The activity of glycerol kinase is 74.6U / mg,

[0102] The activity of L-α-glycerophosphate oxidase was 9.33 U / mg.

[0103] Example 3

[0104] This embodiment is basically the same as Embodiment 1, except that the colloid in the reagent layer is edible gelatin.

[0105] Example 4

[0106] This embodiment is basically the same as embodiment 1, except that the amount of buffer solution in the reagent layer is 18 mol / m 2 .

[0107] Example 5

[0108] This embodiment is substantially the same as the embodiment 1, except that the viscosity of the cellulose acetate in the diffusion layer is 30 poise.

[0109] Example 6

[0110] This embodiment is basically the same as the embodiment 1, except that the particles in the diffusion layer are barium sulfate (the ratio of the number of barium sulfate with particle sizes of 5 μm and 3 μm is 1:1).

[0111] Example 7

[0112] This embodiment is basically the same as embodiment 1, except that in the reagent layer,

[0113] The activity of peroxidase was 273U / mg.

[0114] The activity of glycerol kinase was 74.6 U / mg.

[0115] The activity of L-α-glycerophosphate oxidase was 37.7 U / mg.

[0116] Example 8

[0117] This embodiment is substantially the same as the embodiment 1, except that the viscosity of the cellulose acetate in the diffusion layer is 45 poise.

[0118] Example 9

[0119] This embodiment is basically the same as the embodiment 1, except that titanium dioxide particles with particle sizes of 5 μm and 3 μm are added in a quantity ratio of 1:2 into the diffusion layer.

[0120] Example 10

[0121] This embodiment is basically the same as the embodiment 1, except that titanium dioxide particles with particle sizes of 6 μm and 2 μm are added in a quantity ratio of 1:1 into the diffusion layer.

[0122] Embodiment 11

[0123] This embodiment is basically the same as the embodiment 1, except that, during the preparation of the diffusion layer, titanium dioxide particles with particle sizes of 8 μm and 4 μm are added in a quantity ratio of 1:1.

[0124] Example 12

[0125] This embodiment is basically the same as the embodiment 1, except that titanium dioxide particles with particle sizes of 5 μm and 3 μm are added in a quantity ratio of 1:5 in the diffusion layer.

[0126] Comparative Example 1

[0127] This comparative example is substantially the same as Example 1, except that in the diffusion layer, the particles only include titanium dioxide with a particle size of 3 μm.

[0128] Comparative Example 2

[0129] This comparative example is substantially the same as Example 1, except that in the diffusion layer, the particles only include titanium dioxide with a particle size of 5 μm.

[0130] Experimental example

[0131] Prepare 5 serum samples with creatine kinase concentrations of 0.04U / mL, 0.37U / mL, 0.70U / mL, 1.175U / mL, and 1.65U / mL (among which, serum samples with concentrations of 0.04U / mL, 0.70U / mL, and 1.65U / mL are commercial composite calibrators, and the remaining concentrations are dilution concentrations of commercial composite calibrators). 11μL of the sample to be tested is added dropwise to the upper outer frame sample well 2. The sample penetrates the reagent layer evenly and quickly through the diffusion layer, reacts at 37°C for 5min, and uses an optical density meter to measure the optical density value of the colored circular spot at a wavelength of 670nm. The test results are shown in Table 3.

[0132] Commercially available composite calibrants with concentrations of 0.04 U / mL, 0.70 U / mL, and 1.65 U / mL were added dropwise to the Example and the Comparative Example for reaction. The coefficient of variation CV was calculated by repeating 10 times. The results are shown in Table 4.

[0133] The color contrast results of Example 1 are as follows Figure 2 As shown, the three circular spots with creatine kinase concentrations from low to high are uniform in size and color, and the colors change from light to dark ( Figure 2 In the figure, from top to bottom, the corresponding concentrations are 0.04 U / mL, 0.70 U / mL, and 1.65 U / mL).

[0134] Table 3 Performance parameters of embodiments and comparative examples 1

[0135]

[0136]

[0137] It can be seen from Table 3 that the slope of Example 1 is 0.9313, which is higher than that of Comparative Examples 1-2, indicating that the difference in optical density between high concentration and low concentration is large and the color difference is obvious; the correlation coefficient r of Example 1 is 2 The value was 0.9995, which was higher than that of Comparative Example 1-2, indicating that the concentration of creatine kinase and the optical density showed a good linear relationship.

[0138] Table 4 Coefficient of variation CV of the embodiments and comparative examples

[0139]

[0140]

[0141] It can be seen from Table 4 that the coefficient of variation CV of Example 1 is lower than that of Comparative Examples 1-2, indicating that Example 1 has better precision.

[0142] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A dry chemical reagent sheet for quantitatively detecting creatine kinase, comprising a light-transmitting layer, a reagent layer and a diffusion layer, wherein the reagent layer is arranged above the light-transmitting layer, the diffusion layer is above the reagent layer, the diffusion layer comprises a diffusion layer reagent or is formed by a diffusion layer reagent, the diffusion layer reagent comprises a hydrophilic polymer, a surfactant and particulate matter, wherein: The particles include first particles and second particles; The number ratio of the first particles to the second particles is 1:(0.25-4), preferably 1:(0.5-1.5), The difference in particle size between the first particulate matter and the second particulate matter is 1 to 4 μm, preferably 1.5 to 2.5 μm.

2. The dry chemical reagent sheet according to claim 1, characterized in that: The particle size of the first particles is 4 to 8 μm, preferably 4.5 to 5.5 μm; And / or, the particle size of the second particulate matter is 1 to 4 μm, preferably 2.5 to 3.5 μm.

3. The dry chemical reagent sheet according to any one of claims 1-2, characterized in that: In the diffusion layer reagent, The amount of hydrophilic polymer is 1 to 25 g / m 2 , preferably 10 to 20 g / m 2 ; And / or, the amount of surfactant is 5 to 50 g / m 2 , preferably 5 to 15 g / m 2 ; And / or, the amount of particles is 10 to 500 g / m 2 , preferably 100 to 200 g / m 2 .

4. The dry chemical reagent sheet according to any one of claims 1 to 3, characterized in that: In the diffusion layer reagent, the hydrophilic polymer includes at least one of cellulose acetate and cellulose nitrate. Preferably, the viscosity of the hydrophilic polymer is 30 to 45 poise, preferably 35 to 40 poise. And / or, in the diffusion layer reagent, the surfactant includes at least one of Tween, Triton and Surfynol; preferably, in the diffusion layer reagent, the surfactant includes Tween, Triton and Surfynol, and preferably, the mass ratio of Tween, Triton and Surfynol is (1-3):1:(1-3); And / or, the first particulate matter and the second particulate matter are the same or different, and each independently comprises at least one of titanium dioxide, barium sulfate, diatomaceous earth, kaolin, barium titanate, and polystyrene microspheres.

5. The dry chemical reagent sheet according to any one of claims 1 to 4, characterized in that: The reagent layer includes or is formed by a reagent layer reagent, wherein the reagent layer reagent includes a buffer system, a biological enzyme, a color developer, a surfactant and a colloid, wherein the biological enzyme includes peroxidase, L-α-phosphoglycerate oxidase, and glycerol kinase. Preferably, The activity of peroxidase is 250-300 U / mg, preferably 270-290 U / mg, more preferably 275-285 U / mg; and / or, the activity of glycerol kinase is 50 to 100 U / mg, preferably 60 to 90 U / mg, more preferably 70 to 80 U / mg; And / or, the activity of L-α-glycerophosphate oxidase is 8 to 45 U / mg, preferably 30 to 45 U / mg, more preferably 35 to 40 U / mg.

6. The dry chemical reagent sheet according to any one of claims 1 to 5, characterized in that: In the reagent layer reagent, The amount of buffer system is 5~20mol / m 2 ; preferably 9 to 15 mol / m 2 ; And / or, the dosage of glycerol kinase is 3000~10000U / m 2 , preferably 4000~6000U / m 2 ; And / or, the dosage of peroxidase is 30000~60000U / m 2 , preferably 40000~50000U / m 2 ; and / or, the dosage of L-α-glycerophosphate oxidase is 3000~10000U / m 2 , preferably 3000~5000U / m 2 ; And / or, the amount of the developer is 200 to 300 mmol / m 2 ; preferably 240 to 280 mmol / m 2 ; And / or, the amount of surfactant is 5 to 20 g / m 2 ; preferably 5 to 10 g / m 2 ; And / or, the amount of colloid is 1 to 50 g / m 2 ; preferably 1 to 15 g / m 2 .

7. The dry chemical reagent sheet according to any one of claims 1 to 6, characterized in that: In the reagent layer reagent, the buffer system includes at least one of a phosphate buffer system, a Tris buffer system, and a boric acid buffer system; the pH of the buffer system is 6.5 to 9.5; and / or, in the reagent layer reagent, the color developing agent includes 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-p-(4-dimethylaminophenyl)imidazole; And / or, in the reagent layer reagent, the surfactant includes at least one of Tween, Triton, Surfynol, and S24; preferably, in the reagent layer reagent, the surfactant includes S24 and Triton, wherein: the amount of S24 is 5 to 15 g / m 2 , preferably 5 to 9 g / m 2 ; The dosage of Triton is 0.1~5g / m 2 , preferably 0.1 to 1 g / m 2 ; And / or, the colloid comprises at least one of gelatin, pullulan, polyvinyl alcohol, polyacrylamide, and hydrophilic cellulose derivatives. Preferably, the gelatin is selected from at least one of photographic gelatin and edible gelatin.

8. The dry chemical reagent sheet according to any one of claims 1 to 7, characterized in that: The reagent layer reagents also include N-acetyl-L-cysteine, creatine phosphate, ADP, magnesium salt and glycerol, wherein: The dosage of N-acetyl-L-cysteine ​​is 10-100 mg / m 2 ; and / or, creatine phosphate in an amount of 7-9 g / m 2 ; and / or, ADP in an amount of 100 to 300 mg / m 2 ; And / or, the dosage of magnesium salt is 200-300 mg / m 2 ; And / or, the amount of glycerol is 0.1 to 5 g / m 2 .

9. The dry chemical reagent sheet according to any one of claims 1 to 8, characterized in that: The thickness of the reagent layer is 10 to 100 μm. and / or the diffusion layer has a thickness of 200 to 600 μm, And / or, the thickness of the light-transmitting layer is 30 to 300 μm.

10. A method for preparing a dry chemical reagent sheet according to any one of claims 1 to 9, characterized in that: The steps include: Step 1: Apply the reagent layer reagent on the light-transmitting layer and dry it; Step 2: coating the diffusion layer reagent on the reagent layer and drying to obtain the dry chemical reagent sheet.

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