A reagent, kit and use for detecting creatine kinase

Abstract

This invention belongs to the technical field of creatine kinase detection kits, specifically relating to a reagent, kit, and application for detecting creatine kinase, including a buffer, creatine phosphate, glycerol, glycerol kinase, glycerol phosphate oxidase, peroxidase, and a chromogenic agent, and also containing a CK activating reducing agent, as well as perfluorinated and polyfluoroalkyl substances. The CK activating reducing agent is a sulfur-containing reducing agent. This invention can improve the accuracy of detection.

Description

Technical Field

[0001] This invention belongs to the technical field of creatine kinase detection kits, specifically relating to a reagent, kit, and application for detecting creatine kinase. Background Technology

[0002] Creatine kinase (CK) is mainly found in skeletal muscle and cardiac muscle, and also in brain tissue. CK activity begins to increase 2-4 hours after an acute myocardial infarction, reaching levels up to 10-12 times the upper limit of normal. CK is more specific than AST and LDH for the diagnosis of myocardial infarction.

[0003] Conventional CK testing using biochemical analyzers requires drawing venous blood, which takes too long for diagnosing conditions such as acute myocardial infarction. Therefore, there is a need for rapid CK testing. Rapid CK testing can be broadly categorized into dehydrogenase and oxidase pathways.

[0004] Dehydrogenase pathway:

[0005]

[0006] The absorbance of NADPH at 340 nm was measured, and the CK activity was calculated. Alternatively, a tetrazolium compound such as NBT could be coupled in, and colorimetric analysis using a yellow transfectant enzyme could be employed.

[0007] Oxidase pathway:

[0008]

[0009] The CK activity is calculated by testing the absorbance of the colorimetric reagent (such as TMB) at its wavelength (650 nm for TMB).

[0010] Conventional biochemical analyzers typically use the dehydrogenase route. However, in rapid detection methods based on dry chemistry technology, the dehydrogenase route is difficult to apply due to the low molar absorptivity of its colorimetric system.

[0011] The oxidase route faces a challenge: the detection of CK requires activation with reducing reagents such as cysteine ​​and N-acetylcysteine, but these reducing reagents react with hydrogen peroxide, affecting the color development of the chromogenic agent (such as TMB) and causing deviations in the results. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a reagent, kit and application for detecting creatine kinase, thereby improving the accuracy of detection.

[0013] This invention provides a reagent for detecting creatine kinase, comprising a buffer solution, creatine phosphate, glycerol, glycerol kinase, glycerol phosphate oxidase, peroxidase, and a chromogenic agent, and further comprising a CK activating reducing agent, as well as perfluorinated and polyfluoroalkyl substances, wherein the CK activating reducing agent is a sulfur-containing reducing agent.

[0014] Preferably, the mass concentration of the perfluorinated and polyfluoroalkyl substances is 0.001-0.1%.

[0015] Preferably, the perfluorinated and polyfluoroalkyl substances are one or more of perfluorooctane sulfonic acid, perfluorooctane carboxylic acid, perfluorononanoic acid, perfluorododecanoic acid, and perfluorohexane sulfonic acid.

[0016] Preferably, it also contains a glucosamine surfactant. Its mass concentration is preferably 0.05-0.5%.

[0017] Preferably, the glucosamine surfactant is one or more of N-octanoyl-N-methylglucosamine, N-nonanoyl-N-methylglucosamine, and N-decanoyl-N-methylglucosamine.

[0018] Preferably, the CK activating reducing agent is one or more of sodium sulfide, sodium bisulfite, sodium sulfite, and sodium thiosulfate.

[0019] Preferably, the buffer also includes a surfactant, ATP, magnesium chloride, BSA, trehalose, and polyvinylpyrrolidone, wherein the buffer is HEPES buffer, the surfactant is Tween-20, and the colorimetric reagent is TMB.

[0020] This invention provides a kit for detecting creatine kinase, comprising, from bottom to top, a bottom layer, a chromogenic layer, a blood filtration layer, a diffusion layer, and a top layer, wherein the chromogenic layer is impregnated with the reagent for detecting creatine kinase.

[0021] Preferably, the color developing layer is made of glass fiber, filter paper, non-woven fabric, or synthetic membrane; the blood filtration layer is made of glass fiber or polysulfone membrane; the diffusion layer is made of filter paper, glass fiber, non-woven fabric, mesh fabric, or synthetic fiber; and the bottom and top layers are made of polyethylene, polyvinyl chloride, polystyrene, or polyester fiber.

[0022] This invention provides the use of perfluorinated and polyfluoroalkyl substances in the preparation of reagents for detecting creatine kinase.

[0023] The beneficial effect of this invention is that by replacing the CK activating reducing agent with a sulfur-containing inorganic reducing agent in the detection reagent, and by adding perfluorinated and polyfluoroalkyl substances, the interference of cysteine-based reducing agents on the colorimetric reagent can be avoided, thereby improving the accuracy of the detection results.

[0024] The inventors discovered that combinations of multiple perfluorinated and polyfluoroalkyl substances, along with the addition of glucosamine surfactants, can better reduce interference and improve the accuracy of detection results compared to combinations of single raw materials.

[0025] The reagent for detecting creatine kinase of the present invention can be used with small detection instruments, such as a spectrophotometer, preferably a reflectance spectrophotometer. This spectrophotometer monitors the change in reflected light density at a specific wavelength after the dry chemical test strip reaction. Because the reagent for detecting creatine kinase of the present invention eliminates the influence of CK activator on the colorimetric reaction, it can achieve more accurate detection. Furthermore, the test strip of this method is easy to operate, requiring no professional personnel, and can meet market demands under special conditions, especially in emergency applications. Compared with traditional methods using liquid biochemical reagents, the dry chemical test reagent of the present invention can provide convenient and rapid testing for emergency departments, primary hospitals, homes, and small clinics. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the kit of the present invention.

[0027] In the figure, the detection well is 107, the bottom layer is 106, the color development layer is 105, the blood filtration layer is 104, the diffusion layer is 103, the top layer is 102, the sample application well is 101, and the test kit is 10. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0029] It should be noted that when a component is referred to as being "located on" another component, it can be directly on the other component or there may be an intervening component. When a component is referred to as being "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0031] Example

[0032] like Figure 1As shown, the test kit 10 of one embodiment of the present invention includes a bottom layer 106, a color development layer 105, a blood filtration layer 104, a diffusion layer 103, and an upper layer 102 arranged sequentially from bottom to top. The upper layer 102 is provided with a sample addition well 101 for adding samples; the bottom layer 106 is provided with a detection well 107.

[0033] The blood filtration layer 104 is made of glass fiber or polysulfone membrane.

[0034] The bottom layer 106 and the top layer 102 can be made of polyethylene, polyvinyl chloride, polystyrene or polyester fiber.

[0035] The material of the diffusion layer 103 can be selected from filter paper, glass fiber, non-woven fabric, mesh fabric or synthetic fiber. Preferably, the material of the diffusion layer 103 is a hydrophilic material. More preferably, the diffusion layer is pretreated with a surfactant. The function of the diffusion layer is to uniformly penetrate the test sample into the next layer.

[0036] The carrier material for the color development layer 105 can be selected from glass fiber, filter paper, non-woven fabric or synthetic membrane.

[0037] Comparative Example 1

[0038] The mesh was soaked in 0.05% (v / v) Triton X100 solution for 10 min and then dried at 37°C to obtain diffusion layer 103.

[0039] Pall 1660 was used as the blood filtration layer, requiring no further processing.

[0040] The Pall Biodye C 0.45μm membrane was soaked for 10 min using the reagents for detecting creatine kinase.

[0041]

[0042]

[0043] Then it is dried at 37°C to obtain the color development layer 105.

[0044] Comparative Example 2

[0045] In Comparative Example 1, N-acetylcysteine ​​was replaced with sodium bisulfite (concentration remained at 5 mmol / L), while everything else remained exactly the same.

[0046] Comparative Example 3

[0047] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0048] Tridecylfluorooctanoic acid (CAS No.: 27619-97-2) was added to the treatment solution of the chromogenic layer 105 in Comparative Example 1 to a concentration of 0.005% (w / v). Then, the chromogenic layer 105 was treated in the same way as in Comparative Example 1, and the material of the chromogenic layer 105 was also the same as in Comparative Example 1.

[0049] Comparative Example 4

[0050] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0051] In Comparative Example 1, pentafluorooctanoic acid (CAS No.: 335-67-1) was added to the treatment solution of the chromogenic layer 105 to a concentration of 0.01% (w / v). Then, the chromogenic layer 105 was treated in the same way as in Comparative Example 1, and the material of the chromogenic layer 105 was also the same as in Comparative Example 1.

[0052] Example 1

[0053] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0054] In Comparative Example 1, tridecanoic acid (CAS No.: 27619-97-2) was added to the treatment solution of the chromogenic layer 105 to a concentration of 0.005% (w / v), and N-acetylcysteine ​​was replaced with sodium bisulfite (concentration remained at 5 mmol / L). Then, the chromogenic layer 105 was treated using the same method as in Comparative Example 1, and the material of the chromogenic layer 105 was also the same as in Comparative Example 1.

[0055] Example 2

[0056] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0057] In Comparative Example 1, pentafluorooctanoic acid (CAS No.: 335-67-1) was added to the treatment solution of chromogenic layer 105 to a concentration of 0.01% (w / v), and N-acetylcysteine ​​was replaced with sodium sulfite (concentration remained at 5 mmol / L). Then, chromogenic layer 105 was treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0058] Example 3

[0059] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0060] In Comparative Example 1, heptadecanoic acid (CAS No.: 375-95-1) was added to the treatment solution of chromogenic layer 105 to a concentration of 0.01% (w / v), and N-acetylcysteine ​​was replaced with sodium thiosulfate (concentration remained at 5 mmol / L). Then, chromogenic layer 105 was treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0061] Example 4

[0062] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0063] In Comparative Example 1, perfluorododecanoic acid (CAS No.: 307-55-1) was added to the treatment solution of chromogenic layer 105 to a concentration of 0.05% (w / v), and N-acetylcysteine ​​was replaced with sodium thiosulfate (concentration remained at 5 mmol / L). Then, chromogenic layer 105 was treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0064] Example 5

[0065] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0066] Perfluorohexyl sulfonic acid (CAS No.: 355-46-4) was added to the treatment solution of chromogenic layer 105 in Comparative Example 1 to a concentration of 0.1% (w / v), and N-acetylcysteine ​​was replaced with sodium sulfide (concentration remained at 5 mmol / L). Then, chromogenic layer 105 was treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0067] Example 5.1

[0068] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0069] In Comparative Example 1, tridecanoic acid (CAS No.: 27619-97-2) and perfluorododecanoic acid (CAS No.: 307-55-1) were added to the treatment solution of chromogenic layer 105, with concentrations of 0.005% (w / v) and 0.015% (w / v), respectively. N-acetylcysteine ​​was replaced with sodium bisulfite (concentration remained at 5 mmol / L). Chromogenic layer 105 was then treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0070] Example 5.2

[0071] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0072] In Comparative Example 1, pentafluorooctanoic acid (CAS No.: 335-67-1), perfluorohexylsulfonic acid (CAS No.: 355-46-4), perfluorododecanoic acid (CAS No.: 307-55-1), and N-octanoyl-N-methylglucosamine (CAS No.: 85316-98-9) were added to the treatment solution of chromogenic layer 105 to achieve concentrations of 0.005% (w / v), 0.015% (w / v), 0.01% (w / v), and 0.1% (w / v), respectively, and N-acetylcysteine ​​was replaced with sodium sulfite (concentration remained at 5 mmol / L). Chromogenic layer 105 was then treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0073] Example 6

[0074] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0075] In Comparative Example 1, tridecanoic acid (CAS No.: 27619-97-2), perfluorododecanoic acid (CAS No.: 307-55-1), and N-octanoyl-N-methylglucosamine (CAS No.: 85316-98-9) were added to the treatment solution of chromogenic layer 105 to achieve concentrations of 0.005% (w / v), 0.015% (w / v), and 0.1% (w / v), respectively, and N-acetylcysteine ​​was replaced with sodium bisulfite (concentration remained at 5 mmol / L). Chromogenic layer 105 was then treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0076] Example 7

[0077] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0078] In Comparative Example 1, pentafluorooctanoic acid (CAS No.: 335-67-1), perfluorohexylsulfonic acid (CAS No.: 355-46-4), perfluorododecanoic acid (CAS No.: 307-55-1), N-octanoyl-N-methylglucosamine (CAS No.: 85316-98-9), and N-decanoyl-N-methylglucosamine (CAS No.: 85261-20-7) were added to the treatment solution of chromogenic layer 105 to achieve concentrations of 0.005% (w / v), 0.015% (w / v), 0.01% (w / v), 0.1% (w / v), and 0.2% (w / v), respectively. N-acetylcysteine ​​was replaced with sodium sulfite (concentration remained at 5 mmol / L). Chromogenic layer 105 was then treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0079] Example 8

[0080] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0081] In Comparative Example 1, tridecanoic acid (CAS No.: 27619-97-2), perfluorohexylsulfonic acid (CAS No.: 355-46-4), heptadecafluorononanoic acid (CAS No.: 375-95-1), N-nonanoyl-N-methylglucosamine (CAS No.: 85261-19-4), and N-decanoyl-N-methylglucosamine (CAS No.: 85261-20-7) were added to the treatment solution of chromogenic layer 105 to achieve concentrations of 0.005% (w / v), 0.005% (w / v), 0.015% (w / v), 0.15% (w / v), and 0.2% (w / v), respectively. N-acetylcysteine ​​was replaced with sodium sulfide (concentration remained at 5 mmol / L). Chromogenic layer 105 was then treated using the same method as in Comparative Example 1, and the material of chromogenic layer 105 was also the same as in Comparative Example 1.

[0082] Example 9

[0083] The diffusion layer 103 and the blood filtration layer 104 are the same as those in Comparative Example 1.

[0084] In Comparative Example 1, pentafluorooctanoic acid (CAS No.: 335-67-1) was added to the chromogenic layer 105 treatment solution to a concentration of 0.01% (w / v), and N-acetylcysteine ​​was replaced with sodium sulfite (concentration remained at 5 mmol / L).

[0085] In addition, N-nonanoyl-N-methylglucosamine (CAS No.: 85261-19-4) was added to a concentration of 0.1% (w / v), while the concentration of PVP K30 was controlled at 2.4% (w / v). Then, the colorimetric layer 105 was treated in the same way as in Comparative Example 1, and the material of the colorimetric layer 105 was also the same as in Comparative Example 1.

[0086] Example 10

[0087] The upper layer 102 and the lower layer 106 are made of polyvinyl chloride (PVC) sheets with adhesive on one side. Sample application holes 101 and detection holes 107 are punched out using a punching machine, with a hole diameter of 5 mm. The diffusion layer 103, blood filtration layer 104, and color development layer 105 are all cut to a width of 7 mm. The diffusion layer 103 and color development layer 105 are respectively adhered to the adhesive on the upper layer 102 and the lower layer 106, covering the sample application hole 101 and the detection hole 107. Then, the blood filtration layer 104 is sandwiched between the upper layer with the diffusion layer 103 and the lower layer with the color development layer 105, aligning the positions of the sample application hole 101 and the detection hole 107. The upper layer 102 and the lower layer 106 are then adhered together with the adhesive on.

[0088] Example 11

[0089] Add 20 μL of sample to sample well 101, place it on a reflectometer that can maintain a constant temperature of 37℃, delay for 60 s, and read the rate of change ΔA of absorbance at 650 nm of the test paper from 61 to 120 s. The results are shown in Table 1-2 below:

[0090] Table 1

[0091] CK U / L Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Example 1 Example 2 Example 3 Example 4 50 0.0002 0.0004 0.0003 0.0004 0.0008 0.0009 0.0007 0.0007 400 0.0008 0.0011 0.0011 0.0011 0.0018 0.0021 0.0019 0.0022 800 0.0011 0.0012 0.0018 0.0022 0.0035 0.0034 0.0030 0.0031 1200 0.0019 0.0022 0.0025 0.0033 0.0051 0.0052 0.0044 0.0048 1600 0.0019 0.0023 0.0037 0.0044 0.0061 0.0063 0.0058 0.0066

[0092] Table 2

[0093] CK U / L Example 5 Example 5.1 Example 5.2 Example 6 Example 7 Example 8 50 0.0005 0.0008 0.0009 0.0013 0.0012 0.0020 400 0.0024 0.0033 0.0038 0.0035 0.0035 0.0044 800 0.0035 0.0055 0.0051 0.0075 0.0072 0.0090 1200 0.0052 0.0077 0.0083 0.0112 0.0102 0.0133 1600 0.0077 0.0110 0.0121 0.0158 0.0154 0.0175

[0094] It is evident that in Comparative Example 1, the reducing activator significantly interferes with the color development of hydrogen peroxide, resulting in very low color intensity.

[0095] As can be seen from Comparative Example 2, simply replacing N-acetylcysteine ​​with a sulfur-containing inorganic reducing agent does not significantly improve the results.

[0096] Comparative Examples 3 and 4 show that the addition of perfluorinated and polyfluoroalkyl substances alone has significantly improved the test results.

[0097] As can be seen from Examples 1-5, the addition of perfluorinated and polyfluoroalkyl substances greatly reduces the interference of reducing activators on hydrogen peroxide, significantly enhances the colorimetric signal, and improves the accuracy of detection.

[0098] As can be seen from Examples 5.1 and 5.2, the effect is further improved by adding a variety of perfluorinated and polyfluoroalkyl substances.

[0099] As can be seen from Examples 6 and 8, the addition of various perfluorinated and polyfluoroalkyl substances, as well as glucosamine surfactants, achieves the best results.

[0100] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0101] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

[0102] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0103] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A reagent for detecting creatine kinase, characterized in that, It includes a buffer solution, creatine phosphate, glycerol, glycerol kinase, glycerol phosphate oxidase, peroxidase, and a colorimetric reagent, and also contains a CK activator / reducer, as well as perfluorinated and polyfluoroalkyl substances, wherein the CK activator / reducer is one or more of sodium sulfide, sodium bisulfite, sodium sulfite, and sodium thiosulfate; The mass concentration of the perfluorinated and polyfluoroalkyl substances is 0.005-0.1%; the perfluorinated and polyfluoroalkyl substances are one or more of tridecylfluorooctanoic acid, pentadecylfluorooctanoic acid, heptadecanoic acid, perfluorododecanoic acid, and perfluorohexylsulfonic acid.

2. The reagent for detecting creatine kinase according to claim 1, wherein It also contains glucosamine surfactants.

3. The reagent for detecting creatine kinase as described in claim 2, characterized in that, The glucosamine surfactant is one or more of N-octanoyl-N-methylglucosamine, N-nonanoyl-N-methylglucosamine, and N-decanoyl-N-methylglucosamine.

4. The reagent for detecting creatine kinase as described in any one of claims 1-3, characterized in that, It also includes surfactants, ATP, magnesium chloride, BSA, trehalose, and polyvinylpyrrolidone, wherein the buffer is HEPES buffer, the surfactant is Tween-20, and the colorimetric agent is TMB.

5. A kit for detecting creatine kinase, characterized in that, The mixture comprises, from bottom to top, a bottom layer (106), a color development layer (105), a blood filtration layer (104), a diffusion layer (103), and a top layer (102), wherein the color development layer (105) is impregnated with a reagent for detecting creatine kinase as described in any one of claims 1-4.

6. The kit for detecting creatine kinase as described in claim 5, characterized in that, The color development layer (105) is made of glass fiber, filter paper, non-woven fabric or synthetic membrane; the blood filtration layer (104) is made of glass fiber or polysulfone membrane; the diffusion layer (103) is made of filter paper, glass fiber, non-woven fabric, mesh or synthetic fiber; the bottom layer (106) and the top layer (102) are made of polyethylene, polyvinyl chloride, polystyrene or polyester fiber.

7. The use of a perfluorinated and polyfluoroalkyl substance in the preparation of a reagent for detecting creatine kinase, characterized in that, The mass concentration of the perfluorinated and polyfluoroalkyl substances is 0.005-0.1%; the perfluorinated and polyfluoroalkyl substances are one or more of tridecylfluorooctanoic acid, pentadecylfluorooctanoic acid, heptadecanoic acid, perfluorododecanoic acid, and perfluorohexylsulfonic acid.

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