Creatinine detection device

Abstract

The invention discloses a creatinine detection device, and belongs to the field of in-vitro diagnosis equipment. The creatinine detection device comprises a substrate, a reagent 1 assembly and a reagent 2 assembly, the reagent 1 assembly comprises a flow pad, an enzyme pad and a blood filter membrane, the reagent 2 assembly comprises a start inhibition layer, a separation layer and a color development layer, the reagent 1 assembly and the reagent 2 assembly are stored in a non-contact mode in a normal state, and the reagent 1 assembly and the reagent 2 assembly are in contact to start a reaction during testing; the separation layer contains perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid. According to the creatinine detection device provided by the invention, interference of endogenous ammonia can be eliminated, the diffusivity of the ammonia is improved, and the detection sensitivity is improved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic equipment, and in particular relates to a creatinine detection device. Background Technology

[0002] Creatinine can reflect glomerular filtration function to some extent. When glomerular filtration function drops below 30% of normal, creatinine levels begin to rise; the higher the creatinine, the worse the glomerular filtration function.

[0003] Creatinine is mainly determined by chemical methods (Jaffe method), enzymatic methods, high performance liquid chromatography, and capillary electrophoresis.

[0004] Chemical assays are inexpensive and easy to operate, making them one of the most commonly used methods for determining creatinine in China. The principle is that creatinine in the sample reacts with picrates to form a yellow-red picrate-creatinine complex. The disadvantage of this method is its low specificity; vitamin C, acetone, acetoacetic acid, methyldopa, as well as high concentrations of glucose, proteins, and some antibiotics such as penicillin G, cefoxitin, and cefazolin can also react with alkaline picrates to produce a red color.

[0005] High-performance liquid chromatography (HPLC) is used to analyze creatinine, which carries a positive charge in a weakly acidic environment. Creatinine can be well separated from other components by a cation exchange chromatography column, and its absorbance is measured at 234 nm. This method offers high precision and specificity, but it is not suitable for large-scale clinical sample analysis. It is typically used as a reference method for creatinine determination, evaluating commercially available creatinine measurement kits, and for certain research purposes.

[0006] In capillary electrophoresis, serum samples are pretreated by high-speed centrifugation, while urine samples can be centrifuged at low speed to remove formed elements. The supernatant is then separated by micellar electrokinetic capillary electrophoresis, and the absorbance at 235 nm is measured. This method has a wide linear range and is relatively simple to operate, but it requires specialized equipment and serum sample pretreatment, making routine clinical use difficult.

[0007] Enzymatic assays are a clinically suitable method for detecting creatinine, combining simplicity and accuracy. They mainly fall into two categories: creatinine deiminase (EC 3.5.4.21) and creatininase (EC 3.5.2.10).

[0008] The principle of the creatinine enzymatic method is as follows: In the creatinine enzymatic method, the target analyte creatinine is hydrolyzed into creatine by creatinine enzymes. Creatine is then hydrolyzed into sarcosine by creatine hydrolases. The resulting sarcosine is then converted into hydrogen peroxide by sarcosine oxidases. The hydrogen peroxide reacts with peroxidase and a chromogenic agent to produce a detectable color change. This method requires the participation of four enzymes, making its detection cost relatively high.

[0009] The mechanism of creatinine deiminase is as follows: Creatinine is converted to ammonia by creatinine deiminase. There are two detection routes. One uses glutamate dehydrogenase, where ammonia reacts with α-ketoglutarate, catalyzing the oxidation of reduced coenzymes NADH or NADPH (with an absorption peak at 340 nm) to oxidized coenzymes NAD or NADP (with no absorption peak at 340 nm). The creatinine content can then be detected by measuring the decrease in absorbance at 340 nm after the reaction.

[0010] Another approach uses indicators to detect ammonia formation.

[0011] Obviously, the creatinine deiminase method requires fewer enzymes than the creatinine enzymatic method, therefore the cost of the detection reagents will be lower.

[0012] Based on the two detection routes for creatinine deiminase mentioned above, the concentration of endogenous ammonia in the blood will directly affect the creatinine content test.

[0013] To remove interference from endogenous ammonia, US Patent 5008078 discloses a plasma creatinine test strip, which consists of, from bottom to top, a transparent substrate, an ammonia development layer, a breathable layer, an ammonia-producing layer, an ammonia-blocking layer, and an ammonia-removing layer. The sample is added to the strip from top to bottom, first passing through the ammonia-removing layer to remove endogenous ammonia, then flowing through the ammonia-blocking layer. In the ammonia-producing layer, creatinine deiminase produces ammonia, which permeates through the breathable layer and develops color in the ammonia development layer. However, in this method, the time it takes for the sample to pass through the ammonia-removing layer is determined by factors such as sample viscosity, making it difficult to ensure sufficient removal time. Furthermore, the ammonia removal reagent in the removal layer can be redissolved in the sample and carried to the ammonia-producing layer, decomposing the generated ammonia and causing deviations in the results.

[0014] Chinese patent CN102288747 discloses a dry chemical strip for removing interferences. This strip allows the sample to react with reagent 1 first to remove interfering substances, and then reagent 2 is added after a set time to initiate the detection reaction of the target analyte. This solves the problem of insufficient removal time in US5008078. However, this strip is only suitable for the enzymatic detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine, and does not meet the requirements for the enzymatic detection of creatinine deiminase. Summary of the Invention

[0015] This invention provides a creatinine deiminase method creatinine detection device that removes interference from endogenous ammonia and has high ammonia absorption efficiency.

[0016] The present invention provides a creatinine detection device, comprising a substrate, a reagent 1 component, and a reagent 2 component. The reagent 1 component and the reagent 2 component are arranged on the detection device in a manner that keeps them non-contact under normal conditions but allows them to contact each other under testing conditions. The reagent 1 component is directly fixed to the substrate, and the reagent 2 component is connected to the substrate by an adhesive member. The adhesive member has sufficient height to suspend the reagent 2 component above the reagent 1 component, and the reagent 2 component can be pressed down to maintain contact with the reagent 1 component. The reagent 1 component includes a flow pad, an enzyme pad, and a blood filtration membrane. The reagent 2 component includes an activation inhibition layer, a separation layer, and a colorimetric layer. The separation layer contains perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid.

[0017] The preferred perfluorooctane sulfonic acid is tridecylfluorooctane sulfonic acid, and the preferred perfluorooctane carboxylic acid is pentadecylfluorooctane.

[0018] Preferably, the mass concentration of the perfluorooctane sulfonic acid and perfluorooctane carboxylic acid is 0.001-0.005%.

[0019] Preferably, the spacer layer further contains long-chain fatty acid esters.

[0020] Preferably, the long-chain fatty acid ester is methyl nonadecanoate, methyl heptadecanoate, or methyl pentadecanoate.

[0021] Preferably, the mass concentration of the long-chain fatty acid ester is 0.002-0.05%.

[0022] Preferably, the separating layer is a breathable hydrophobic polytetrafluoroethylene membrane.

[0023] The enzyme pad contains an immobilized reagent for removing endogenous ammonia, preferably glutamate dehydrogenase, NADP, or α-ketoglutarate.

[0024] The activation inhibition layer contains immobilized creatinine deiminase, a reagent that reacts with creatinine.

[0025] Preferably, the activation inhibition layer contains a glutamate dehydrogenase inhibitor, and the preferred inhibitor is a mouse anti-glutamate dehydrogenase antibody.

[0026] The colorimetric layer can use an acid-base indicator, preferably bromophenol blue.

[0027] Preferably, the flow pad is a nitrocellulose membrane.

[0028] Preferably, reagent component 1 also includes a mesh fabric.

[0029] After the whole blood sample is added to reagent component 1, the blood filtration membrane in reagent component 1 retains the blood cells in the sample. The plasma is reconstituted and fixed on the enzyme pad with reagents such as glutamate dehydrogenase, NADP, and α-ketoglutarate, and flows onto the flow pad to consume the endogenous ammonia in the sample according to the following reaction.

[0030] After the set time, the endogenous ammonia has been fully removed. Press down on reagent component 2, either manually or by instrument. The initiation inhibition layer in reagent component 2 comes into contact with the flow pad in reagent component 1. The creatinine deiminase in the initiation inhibition layer decomposes creatinine according to the following reaction formula, producing ammonia.

[0031] Simultaneously, the glutamate inhibitor in the inhibition layer suppresses the activity of glutamate dehydrogenase, preventing the ammonia produced from creatinine from being consumed. The generated ammonia permeates through the breathable septum and reacts with the indicator in the chromogenic layer, producing a detectable signal change.

[0032] The separator layer separates the initiation inhibition layer from the liquid in the developing layer, but it is necessary to allow the ammonia generated in the initiation inhibition layer to diffuse into the developing layer. Experiments showed that adding perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid to the separator layer significantly improved the detection signal. If long-chain fatty acid esters were added simultaneously, the improvement in detection signal was even more pronounced, and the liquid in the initiation inhibition layer would not reach the developing layer, causing abnormal color development.

[0033] The creatinine detection device provided by this invention can control the reaction time to remove endogenous ammonia, fully remove the interference of endogenous ammonia, and the ammonia generated by creatinine can better diffuse into the colorimetric layer in gaseous form, which greatly improves the detection sensitivity.

[0034] The detection device of this invention can be used in conjunction with small detection instruments, such as a spectrophotometer, preferably a reflectance spectrophotometer. This spectrophotometer can detect the change in reflected light density at a specific wavelength after the creatinine detection device reacts, and the creatinine concentration is calculated using an algorithm. Because the creatinine detection device of this invention eliminates the influence of endogenous ammonia on the colorimetric reaction and improves ammonia diffusion, it enhances accuracy and sensitivity, thus making the detection more accurate. Furthermore, the detection device of this method is easy to operate, requiring no professional personnel, and can meet market demands under special conditions, especially in emergency and home applications. Compared to traditional methods using liquid biochemical reagents, the detection device of this invention provides convenient and rapid testing for emergency departments, primary hospitals, homes, and small clinics. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the detection device of the present invention.

[0036] Figure 2 This is a schematic diagram showing the placement of the substrate and the flow pad during the fabrication of the large plate of the detection device of the present invention.

[0037] Figure 3 This is a schematic diagram of the overlapping area between the transparent carrier and the flow pad in the detection device of the present invention.

[0038] In the figure, the components are: substrate 101, enzyme pad 102, blood filtration membrane 103, adhesive A 104, mesh fabric 105, flow pad 106, initiation inhibition layer 107, separation layer 108, color development layer 109, transparent carrier 110, adhesive 111, adhesive B 112, adhesive C 113, overlapping area 201, and detection device 10. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] It should be noted that when a component is said to be "located" on another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected" to another component, it can be directly connected to the other component or there may be an intervening component.

[0041] 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.

[0042] Example like Figure 1 As shown, the creatinine detection device 10 provided by the present invention includes a substrate 101, a flow pad 106, an enzyme pad 102, a blood filtration membrane 103, and a mesh fabric 105, which are adhered to the substrate 101 by adhesive A 104. An activation inhibition layer 107, a separating layer 108, and a color developing layer 109 are adhered to a transparent carrier 110 by adhesives B 112 and C 113. The transparent carrier 110 is a transparent plastic sheet, preferably a 0.25mm polystyrene sheet, PET, or polymethyl methacrylate sheet. The transparent carrier is adhered to the substrate 101 by an adhesive 111, which has a certain height to keep the activation inhibition layer 107 separated from the flow pad 106. The adhesive can be various adhesives or double-sided tape.

[0043] During testing, a whole blood sample is placed on the mesh 105. The sample is evenly distributed onto the filtration membrane 103 by the mesh. Blood cells are retained by the filtration membrane. The reagent on the plasma rehydration pad 102, which removes endogenous ammonia interference, is transferred to the flow pad. After a period of time, the endogenous ammonia is removed. The instrument or manual pressure is applied to the transparent carrier 110, causing the activation inhibition layer 107 to come into contact with the flow pad 106. The creatinine deiminase in the activation inhibition layer decomposes creatinine according to the following reaction formula, producing ammonia.

[0044] Simultaneously, the glutamate dehydrogenase inhibitor in the inhibition layer suppresses the activity of glutamate dehydrogenase, preventing the ammonia produced from creatinine from being consumed. The generated ammonia permeates through the breathable septum and reacts with the indicator in the chromogenic layer, producing a detectable signal change.

[0045] Example 1 A 0.35mm thick white PET board measuring 20cm x 8cm was used as the substrate.

[0046] Preparation of reagent component 1: Cut a 20cm length from a 25mm wide Pall Vivid 90 nitrocellulose membrane to serve as a flow pad.

[0047] Cut the Jieyi Bioglass cellulose membrane GL048 into 20cm × 0.5cm pieces and immerse them in the following solution for 10 minutes. HEPES 0.1 mol / L Tween 20 0.5% (v / v) Glutamate dehydrogenase 100 U / mL NADP 5 mmol / L α-Ketoglutarate 20 mmol / L BSA 2% (w / v) pH=7.5 Then remove it and dry it at 37°C to obtain the enzyme pad.

[0048] The Sitopan membrane material 1660 was cut into 20cm × 0.5cm pieces to be used as blood filtration membranes.

[0049] A 20cm × 0.5cm mesh was soaked in a 0.05% (v / v) Triton X100 solution for 10 minutes and then dried at 37℃ to obtain the mesh fabric.

[0050] Align one end of the flow pad, enzyme pad, blood filtration membrane, and mesh fabric, and follow the instructions. Figure 1 , Figure 2As shown, place them sequentially on the substrate, with the edge 4cm away from the edge of the substrate, leaving this end empty as the handle. Figure 1 The right end Figure 2 (Lower end). Apply glue A to this aligned end to fix the substrate, flow pad, enzyme pad, blood filtration membrane, and mesh.

[0051] The preparation of reagent component 1 was completed and it was fixed on the substrate.

[0052] Preparation of reagent component 2: Cut a 0.25mm thick transparent PET sheet into 20cm x 2.5cm pieces to serve as a transparent carrier.

[0053] Spray the following solution onto a transparent substrate to form a 2cm wide band, which serves as the developing layer. The edge of the band should be 3mm from one end of the transparent substrate for easy dispensing.

[0054] Bromophenol blue 4 mg / mL Tween 200.5% (v / v) Gelatin 2% (w / v) The 0.45μm pore size hydrophobic polytetrafluoroethylene membrane from the COBBAT was cut into 20cm×2cm pieces to serve as a separator layer.

[0055] Cut the Jieyi Bioglass cellulose membrane GL048 into 20cm × 2cm pieces and immerse them in the following solution for 10 minutes. HEPES 0.1 mol / L Tween 200.5% (v / v) Creatinine deiminase 100 U / mL Mouse anti-glutamate dehydrogenase antibody 1 mg / mL BSA 2% (w / v) NaCl 0.9% (w / v) The pH is 7.5. Then, it is taken out and dried at 37°C for 2 hours to obtain the start-up inhibition layer.

[0056] The separating layer and the initiation inhibition layer were placed sequentially on the color development layer, and then fixed with silicone-based elastic adhesive at both ends to obtain reagent component 2.

[0057] The transparent carrier from reagent component 2 is fixed to the substrate using an adhesive, ensuring that the overlap area 201 between the transparent carrier and the flow pad is 1.5 cm in length. Figure 3 As shown, the large plate of the detection device is obtained.

[0058] The large plate is cut into strips 5mm wide and 8cm long along its length to obtain the detection device 10.

[0059] Example 2 A 0.45μm pore size hydrophobic polytetrafluoroethylene membrane from Corbett was cut into 20cm×2cm pieces, immersed in 0.002% (w / v) tridecylfluorooctane sulfonic acid (CAS No.: 27619-97-2) in acetone solution for 1 hour, and dried at 37℃ to serve as a separator layer.

[0060] Everything else is the same as in Example 1.

[0061] Example 3 A 0.45μm pore size hydrophobic polytetrafluoroethylene membrane from Corbett was cut into 20cm×2cm pieces, immersed in a 0.004% (w / v) solution of pentadecanoic acid (CAS No.: 335-67-1) in acetone for 1 hour, and dried at 37℃ to serve as a separator layer.

[0062] Everything else is the same as in Example 1.

[0063] Example 4 A 0.45μm pore size hydrophobic polytetrafluoroethylene membrane from Corbett was cut into 20cm×2cm pieces and immersed in an acetone solution containing 0.001% (w / v) pentafluorooctanoic acid (CAS No.: 335-67-1) and 0.005% (w / v) pentafluorooctanoic acid (CAS No.: 335-67-1) for 1 hour, and then dried at 37°C to serve as a separator layer.

[0064] Everything else is the same as in Example 1.

[0065] Example 5 A 0.45μm pore size hydrophobic polytetrafluoroethylene membrane from Cobot was cut into 20cm×2cm pieces and immersed in a solution of 0.004% (w / v) pentadecanoic acid (CAS No.: 335-67-1), 0.002% (w / v) pentadecanoic acid (CAS No.: 335-67-1), and 0.002% (w / v) methyl nonadecanoate (CAS No.: 1731-94-8) in acetone:chloroform = 1:1 for 1 hour, and then dried at 37℃ to serve as a separating layer.

[0066] Everything else is the same as in Example 1.

[0067] Example 6 A 0.45μm pore size hydrophobic polytetrafluoroethylene membrane from Cobot was cut into 20cm×2cm pieces and immersed in a 1:1 solution of 0.003% (w / v) pentafluorooctanoic acid (CAS No.: 335-67-1), 0.003% (w / v) pentafluorooctanoic acid (CAS No.: 335-67-1), 0.05% (w / v) methyl heptanoate (CAS No.: 1731-92-6), and 0.01% (w / v) methyl pentadecanoate (CAS No.: 7132-64-1) in acetone:chloroform for 1 hour. The membrane was then dried at 37°C to serve as a separating layer.

[0068] Everything else is the same as in Example 1.

[0069] Example 7 The ability of each embodiment to remove intrinsic interference was examined.

[0070] Preparation of solution A: Creatinine 50 μmol / L BSA 5% (w / v) NaCl 0.9% (w / v) solution B: Creatinine 50 μmol / L NH3·H2O 1mmol / L BSA 5% (w / v) NaCl 0.9% (w / v) Add 30 μL of solution to the mesh of the detection device. After 1 min, press down on the transparent carrier and keep the start-up inhibition layer in contact with the flow pad for 1 min. Read the reflectance of the colorimetric layer at 600 nm using the transparent carrier method. The results are shown in the table below: Table 1: As can be seen from the table above, when the creatinine concentration was only 50 μmol / L, the addition of 1 mmol / L ammonia water, which was 20 times the concentration of the interfering substance, resulted in virtually no difference in the detection reflectance. This means that ammonia did not interfere with the detection of the target creatinine.

[0071] Example 8 The sensitivity of the detection in each embodiment was examined.

[0072] Preparation of mother liquor: BSA 5% (w / v) NaCl 0.9% (w / v) was used to prepare creatinine solutions with concentrations of 0, 10, 20, 50, and 100 μmol / L. The solutions were then tested using the detection devices of each embodiment, with the detection method and wavelength for reading reflectance being the same as in Example 7. The results are shown in the table below: Table 2: As can be seen from the table above, in Example 1, there was essentially no difference in reflectance among the 0, 10, and 20 μmol / L creatinine solutions, making it difficult to detect creatinine concentrations below 50 μmol / L. Examples 2, 3, and 4 could barely distinguish between 0, 10, and 20 μmol / L creatinine solutions, but the reflectance difference between 0 and 10 μmol / L solutions was small. Examples 5 and 6 showed good differentiation across all concentrations. It is evident that adding perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid to the separator layer improved ammonia diffusion and increased detection sensitivity. Further addition of long-chain fatty acid esters in addition to perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid further improved sensitivity.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification.

[0074] 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.

[0075] 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 creatinine detection device, characterized in that, The device includes a substrate, reagent component 1, and reagent component 2. Reagent component 1 and reagent component 2 are arranged on the detection device in a manner that keeps them non-contact under normal conditions but allows them to contact each other under testing conditions. Reagent component 1 is directly fixed to the substrate, and reagent component 2 is connected to the substrate by an adhesive member. The adhesive member has sufficient height to suspend reagent component 2 above reagent component 1, and reagent component 2 can be pressed down to maintain contact with reagent component 1. Reagent component 1 includes a flow pad, an enzyme pad, and a blood filtration membrane. Reagent component 2 includes an activation inhibition layer, a separation layer, and a color development layer. The separation layer contains perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid.

2. The creatinine detection device according to claim 1, characterized in that, The perfluorooctane sulfonic acid is tridecylfluorooctane sulfonic acid, and the perfluorooctane carboxylic acid is pentadecylfluorooctane.

3. The creatinine detection device according to claim 2, characterized in that, The mass concentration of the perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid is 0.001-0.005%.

4. The creatinine detection device according to claim 3, characterized in that, The separating layer also contains long-chain fatty acid esters.

5. The creatinine detection device according to claim 4, characterized in that, The long-chain fatty acid ester is methyl nonadecanoate, methyl heptadecanoate, or methyl pentadecanoate.

6. The creatinine detection device according to claim 5, characterized in that, The mass concentration of the long-chain fatty acid ester is 0.002-0.05%.

7. The creatinine detection device according to claim 1, characterized in that, The separating layer is a breathable hydrophobic polytetrafluoroethylene membrane, the enzyme pad contains a reagent for removing endogenous ammonia, and the initiation inhibition layer contains inhibitors of creatinine deiminase and glutamate dehydrogenase.

8. The creatinine detection device according to claim 7, characterized in that, The reagents for removing endogenous ammonia are glutamate dehydrogenase, NADP, and α-ketoglutarate, and the glutamate dehydrogenase inhibitor is mouse anti-glutamate dehydrogenase antibody.

9. The creatinine detection device according to claim 1, characterized in that, The color development layer contains bromophenol blue, and the flow pad is a nitrocellulose membrane.

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