Reagent and kit for detecting creatine kinase and application

Abstract

The invention belongs to the technical field of creatine kinase detection kits, and particularly relates to a creatine kinase detection reagent, a creatine kinase detection kit and application, the creatine kinase detection reagent comprises a buffer solution, creatine phosphate, glycerol, glycerol kinase, glycerophosphate oxidase, peroxidase and a color developing agent, and further comprises a CK activation reducing agent and perfluoro and polyfluoroalkyl substances, the CK activation reducing agent is a sulfur-containing reducing agent. According to the method, the detection accuracy can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of creatine kinase detection kits, and specifically relates to a reagent, a kit and a use for detecting creatine kinase. Background Art

[0002] Creatine kinase (CK) is mainly present in skeletal muscle and myocardium, and also exists in brain tissue. The activity of CK begins to increase 2 - 4 hours after acute myocardial infarction, and can be as high as 10 - 12 times the normal upper limit. CK is more specific for the diagnosis of myocardial infarction than AST and LDH.

[0003] For the detection of CK by conventional biochemical analyzers, venous blood needs to be drawn. For the diagnosis of acute myocardial infarction and the like, the waiting time is too long, so there is a need for rapid detection of CK. The rapid detection of CK can be roughly divided into the dehydrogenase route and the oxidase route.

[0004] Dehydrogenase Route:

[0005]

[0006] The absorbance of NADPH at 340 nm is measured, and the CK activity is calculated. It is also possible to couple with a tetrazolium compound such as NBT and use diaphorase for color development.

[0007] Oxidase Route:

[0008]

[0009] The absorbance generated by a chromogenic reagent (such as TMB) at its wavelength (650 nm for TMB) is measured, and the CK activity is calculated.

[0010] In conventional biochemical analyzers, the dehydrogenase route is generally adopted. However, in rapid detection methods based on dry chemistry technology, the dehydrogenase route is difficult to apply due to the low molar extinction coefficient of its color development system.

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

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

[0013] An embodiment of the present invention provides a reagent for detecting creatine kinase, which includes a buffer, creatine phosphate, glycerol, glycerol kinase, glycerol phosphate oxidase, peroxidase, and a chromogenic agent. It also contains a CK activation reducing agent and perfluoro and polyfluoroalkyl substances, and the CK activation reducing agent is a sulfur-containing reducing agent.

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

[0015] Preferably, the perfluoro and polyfluoroalkyl substances are one or more of perfluorooctane sulfonic acid, perfluorooctanoic 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-methylglucamine, N-nonanoyl-N-methylglucosamine, and N-decanoyl-N-methylglucamine.

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

[0019] Preferably, it also includes a surfactant, ATP, magnesium chloride, BSA, trehalose, and polyvinylpyrrolidone. The buffer is a HEPES buffer, the surfactant is Tween-20, and the chromogenic agent is TMB.

[0020] An embodiment of the present invention provides a kit for detecting creatine kinase, which includes a bottom layer, a chromogenic layer, a blood filtering layer, a diffusion layer, and an upper layer arranged in sequence from bottom to top. The chromogenic layer is impregnated with the reagent for detecting creatine kinase.

[0021] Preferably, the material of the chromogenic layer is glass fiber, filter paper, non-woven fabric, or synthetic membrane; the material of the blood filtering layer is glass fiber or polysulfone membrane; the material of the diffusion layer is filter paper, glass fiber, non-woven fabric, mesh cloth, or synthetic fiber; the materials of the bottom layer and the upper layer are polyethylene, polyvinyl chloride, polystyrene, or polyester fiber.

[0022] An embodiment of the present invention provides the use of perfluoro and polyfluoroalkyl substances in the preparation of a reagent for detecting creatine kinase.

[0023] The beneficial effect of the present invention is that in the detection reagent, the CK activation reducing agent is replaced from cysteine-based reducing agents with sulfur-containing inorganic reducing agents, and at the same time, perfluoro and polyfluoroalkyl substances are added, which can avoid the interference of cysteine-based reducing agents on the chromogenic agent and improve the accuracy of the detection results.

[0024] The inventors found that a combination of multiple perfluoro and polyfluoroalkyl substances, with the addition of glucosamine surfactants, can better reduce interference and improve the accuracy of detection results compared to a combination of individual raw materials.

[0025] The reagent for detecting creatine kinase of the present invention can be used in conjunction with a small detection instrument, such as a spectrophotometer, preferably a reflective spectrophotometer. The spectrophotometer monitors the change in the reflected light density at a specific wavelength after the reaction of the dry chemical test strip. Since the reagent for detecting creatine kinase of the present invention can eliminate the influence of CK activator on the color reaction, more accurate detection can be achieved. In addition, the test strip of this method is easy to operate and does not require professional personnel to operate, which can meet the market demand under special conditions, especially for emergency applications. Compared with the traditional method of detecting by liquid biochemical reagents, the dry chemical detection reagent of the present invention can provide convenient and fast detection for emergency departments, primary hospitals, families, small clinics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic structural diagram of the kit of the present invention.

[0027] In the figure, detection hole 107, bottom layer 106, color development layer 105, blood filtering layer 104, diffusion layer 103, upper layer 102, sample adding hole 101, detection kit 10. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] It should be noted that when an element is referred to as being "provided on" another element, it can be directly on the other element or there may also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0031] EXAMPLE

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

[0033] The material of the blood filtering layer 104 is glass fiber or polysulfone membrane.

[0034] The materials of the bottom layer 106 and the upper layer 102 can be selected from 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 cloth, or synthetic fiber. Preferably, the material of the diffusion layer 103 is a hydrophilic material. More preferably, the diffusion layer is surface pretreated with a surfactant; the function of the diffusion layer is to uniformly penetrate the tested sample into the next layer.

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

[0037] Comparative Example 1

[0038] Soak the mesh cloth with a 0.05% (v / v) Triton X100 solution for 10 min, and then dry it at 37°C to obtain the diffusion layer 103.

[0039] Use Pall Corporation 1660 as the blood filtering layer without treatment.

[0040] Soak the Pall Corporation Biodye C 0.45μm membrane material with the following reagent for detecting creatine kinase for 10 min.

[0041]

[0042]

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

[0044] Comparative Example 2

[0045] Change N-acetylcysteine in Comparative Example 1 to sodium bisulfite (the concentration is still 5 mmol / L), and the others are exactly the same.

[0046] Comparative Example 3

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

[0048] Add perfluorooctanesulfonic acid (CAS No.: 27619-97-2) to the treatment solution of the color developing layer 105 in Comparative Example 1 to make its concentration 0.005% (w / v). Then, treat the color developing layer 105 using the same method as in Comparative Example 1, and the material of the color developing layer 105 is also the same as that 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] Add perfluorooctanoic acid (CAS No.: 335-67-1) to the treatment solution of the color developing layer 105 in Comparative Example 1 to make its concentration 0.01% (w / v). Then, treat the color developing layer 105 using the same method as in Comparative Example 1, and the material of the color developing layer 105 is also the same as that 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] Add perfluorooctanesulfonic acid (CAS No.: 27619-97-2) to the treatment solution of the color developing layer 105 in Comparative Example 1 to make its concentration 0.005% (w / v), and change N-acetylcysteine to sodium bisulfite (the concentration remains 5 mmol / L). Then, treat the color developing layer 105 using the same method as in Comparative Example 1, and the material of the color developing layer 105 is also the same as that 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] Add perfluorooctanoic acid (CAS No.: 335-67-1) to the treatment solution of the color developing layer 105 in Comparative Example 1 to make its concentration 0.01% (w / v), and change N-acetylcysteine to sodium sulfite (the concentration remains 5 mmol / L). Then, treat the color developing layer 105 using the same method as in Comparative Example 1, and the material of the color developing layer 105 is also the same as that 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] Add perfluorononanoic acid (CAS No.: 375-95-1) to the treatment solution of the color developing layer 105 in Comparative Example 1 to make its concentration 0.01% (w / v), and change N-acetylcysteine to sodium thiosulfate (the concentration remains 5 mmol / L). Then, treat the color developing layer 105 using the same method as in Comparative Example 1, and the material of the color developing layer 105 is also the same as that 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] Perfluorododecanoic acid (CAS No.: 307-55-1) was added to the treatment solution of the color development layer 105 in Comparative Example 1 to make its concentration 0.05% (w / v), and N-acetylcysteine was changed to sodium thiosulfate (the concentration is still 5 mmol / L). Then, the color development layer 105 was treated in the same manner as in Comparative Example 1, and the material of the color development layer 105 was also the same as that 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] Perfluorohexanesulfonic acid (CAS No.: 355-46-4) was added to the treatment solution of the color development layer 105 in Comparative Example 1 to make its concentration 0.1% (w / v), and N-acetylcysteine was changed to sodium sulfide (the concentration is still 5 mmol / L). Then, the color development layer 105 was treated in the same manner as in Comparative Example 1, and the material of the color development layer 105 was also the same as that 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] Perfluorooctanesulfonic acid (CAS No.: 27619-97-2) and perfluorododecanoic acid (CAS No.: 307-55-1) were added to the treatment solution of the color development layer 105 in Comparative Example 1 to make their concentrations 0.005% (w / v) and 0.015% (w / v) respectively, and N-acetylcysteine was changed to sodium bisulfite (the concentration is still 5 mmol / L). Then, the color development layer 105 was treated in the same manner as in Comparative Example 1, and the material of the color development layer 105 was also the same as that 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 the treatment solution of the color display layer 105 in Comparative Example 1, add pentadecafluorooctanoic acid (CAS No.: 335-67-1), perfluorohexanesulfonic acid (CAS No.: 355-46-4), n-perfluorododecanoic acid (CAS No.: 307-55-1), and N-octanoyl-N-methylglucamine (CAS No.: 85316-98-9) so that their concentrations are 0.005% (w / v), 0.015% (w / v), 0.01% (w / v), and 0.1% (w / v) respectively, and change N-acetylcysteine to sodium sulfite (the concentration remains 5 mmol / L). Then, treat the color display layer 105 using the same method as in Comparative Example 1, and the material of the color display layer 105 is also the same as that 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 the treatment solution of the color display layer 105 in Comparative Example 1, add tridecafluorooctanesulfonic acid (CAS No.: 27619-97-2), n-perfluorododecanoic acid (CAS No.: 307-55-1), and N-octanoyl-N-methylglucamine (CAS No.: 85316-98-9) so that their concentrations are 0.005% (w / v), 0.015% (w / v), and 0.1% (w / v) respectively, and change N-acetylcysteine to sodium bisulfite (the concentration remains 5 mmol / L). Then, treat the color display layer 105 using the same method as in Comparative Example 1, and the material of the color display layer 105 is also the same as that 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 the treatment solution of the color display layer 105 in Comparative Example 1, add pentadecafluorooctanoic acid (CAS No.: 335-67-1), perfluorohexanesulfonic acid (CAS No.: 355-46-4), n-perfluorododecanoic acid (CAS No.: 307-55-1), N-octanoyl-N-methylglucamine (CAS No.: 85316-98-9), and N-decanoyl-N-methylglucamine (CAS No.: 85261-20-7) so that their concentrations are 0.005% (w / v), 0.015% (w / v), 0.01% (w / v), 0.1% (w / v), and 0.2% (w / v) respectively, and change N-acetylcysteine to sodium sulfite (the concentration remains 5 mmol / L). Then, treat the color display layer 105 using the same method as in Comparative Example 1, and the material of the color display layer 105 is also the same as that 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 the treatment liquid of the color developing layer 105 in Comparative Example 1, add perfluorooctanesulfonic acid (CAS No.: 27619-97-2), perfluorohexanesulfonic acid (CAS No.: 355-46-4), perfluoroheptanoic 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) so that their concentrations are 0.005% (w / v), 0.005% (w / v), 0.015% (w / v), 0.15% (w / v), 0.2% (w / v) respectively, and change N-acetylcysteine to sodium sulfide (the concentration is still 5 mmol / L). Then, treat the color developing layer 105 by the same method as in Comparative Example 1, and the material of the color developing layer 105 is also the same as that in Comparative Example 1.

[0082] Example 9

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

[0084] In the treatment liquid of the color developing layer 105 in Comparative Example 1, add perfluorooctanoic acid (CAS No.: 335-67-1) so that its concentration is 0.01% (w / v), and change N-acetylcysteine to sodium sulfite (the concentration is still 5 mmol / L).

[0085] In addition, add N-nonanoyl-N-methylglucosamine (CAS No.: 85261-19-4) so that its concentration is 0.1% (w / v), and at the same time, control the concentration of PVP K30 to be 2.4% (w / v). Then, treat the color developing layer 105 by the same method as in Comparative Example 1, and the material of the color developing layer 105 is also the same as that in Comparative Example 1.

[0086] Example 10

[0087] The upper layer 102 and the bottom layer 106 adopt polyvinyl chloride (PVC) sheets with one-sided adhesive, and the sample adding holes 101 and the detection holes 107 are respectively punched with a punching machine, and the hole diameter is 5 mm. Cut the diffusion layer 103, the blood filtering layer 104, and the color developing layer 105 into a width of 7 mm. Paste the diffusion layer 103 and the color developing layer 105 on the adhesives of the upper layer 102 and the bottom layer 106 respectively, covering the sample adding hole 101 and the detection hole 107. Then sandwich the blood filtering layer 104 between the upper layer with the diffusion layer 103 pasted and the lower layer with the color developing layer 105 pasted, align the positions of the sample adding hole 101 and the detection hole 107, and paste the upper layer 102 and the bottom layer 106 together with the adhesives in alignment.

[0088] Example 11

[0089] Add 20 μL of the sample onto the sample loading well 101, place it on a reflectometer that can maintain a constant temperature of 37 °C, delay for 60 s, and read the change rate ΔA of the absorbance of the test strip at 650 nm from 61 - 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 can be clearly seen that in Comparative Example 1, the color development interference of the reducing activator on hydrogen peroxide is very obvious, and the color development intensity is very low.

[0095] It can be seen from Comparative Example 2 that simply changing N - acetylcysteine to an inorganic reducing agent containing sulfur basically does not improve the results.

[0096] It can be seen from Comparative Example 3 and Comparative Example 4 that the detection results have been significantly improved by simply adding perfluoro and polyfluoroalkyl substances.

[0097] It can be seen from Examples 1 - 5 that by adding perfluoro and polyfluoroalkyl substances, the interference of the reducing activator on hydrogen peroxide is greatly reduced, the color development signal is significantly enhanced, and the detection accuracy is improved.

[0098] It can be seen from Examples 5.1 and 5.2 that the effect is further improved by adding multiple perfluoro and polyfluoroalkyl substances.

[0099] It can be seen from Examples 6 and 8 that the effect reaches the optimum by adding multiple perfluoro and polyfluoroalkyl substances and glucosamine - type surfactants.

[0100] The technical features of the above - described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above - described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0101] The above - described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

[0102] Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; under the concept of this application, the technical features in 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 in this application as described above, and for the sake of brevity, they are not provided in detail.

[0103] One or more embodiments of 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 of this application shall be included within the scope of protection of this application.

Claims

1. A reagent for detecting creatine kinase, characterized in that: The invention comprises a buffer, creatine phosphate, glycerol, glycerol kinase, glycerol phosphate oxidase, peroxidase and a color developer, and also comprises a CK activating reducing agent and perfluoro and polyfluoroalkyl substances. The CK activating reducing agent is a reducing agent containing sulfur.

2. The reagent for detecting creatine kinase as claimed in claim 1, characterized in that: The mass concentration of the perfluoroalkyl and polyfluoroalkyl substances is 0.001-0.1%.

3. The reagent for detecting creatine kinase as claimed in claim 1, characterized in that: The perfluoro and polyfluoroalkyl substances are one or more of perfluorooctane sulfonic acid, perfluorooctane carboxylic acid, perfluorononanoic acid, perfluorododecanoic acid, and perfluorohexane sulfonic acid.

4. The reagent for detecting creatine kinase as claimed in claim 3, characterized in that: It also contains glucosamine surfactant.

5. The reagent for detecting creatine kinase as claimed in claim 4, characterized in that: The glucosamine surfactant is one or more of N-octanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, and N-decanoyl-N-methylglucamine.

6. The reagent for detecting creatine kinase according to any one of claims 1 to 5, characterized in that: The CK activation reducing agent is one or more of sodium sulfide, sodium bisulfite, sodium sulfite, and sodium thiosulfate.

7. The reagent for detecting creatine kinase according to any one of claims 1 to 5, characterized in that: The method further comprises a surfactant, ATP, magnesium chloride, BSA, trehalose and polyvinyl pyrrolidone. The buffer is a HEPES buffer, the surfactant is Tween-20, and the color developer is TMB.

8. A kit for detecting creatine kinase, characterized in that: The invention comprises a bottom layer (106), a color developing layer (105), a blood filtering layer (104), a diffusion layer (103) and an upper layer (102) which are arranged in sequence from bottom to top, wherein the color developing layer (105) is impregnated with a reagent for detecting creatine kinase according to any one of claims 1 to 7.

9. A kit for detecting creatine kinase, characterized in that: The material of the color development layer (105) is glass fiber, filter paper, non-woven fabric or synthetic membrane; the material of the blood filtering layer (104) is glass fiber or polysulfone membrane; the material of the diffusion layer (103) is filter paper, glass fiber, non-woven fabric, mesh or synthetic fiber; the material of the bottom layer (106) and the upper layer (102) is polyethylene, polyvinyl chloride, polystyrene or polyester fiber.

10. Use of perfluoroalkyl and polyfluoroalkyl substances in the preparation of a reagent for detecting creatine kinase.

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