A creatinine detection device

Abstract

The application 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 component and a reagent 2 component. The reagent 1 component comprises a flow pad, an enzyme pad and a blood filtration membrane. The reagent 2 component comprises a start inhibition layer, a separation layer and a chromogenic layer. The reagent 1 component and the reagent 2 component are stored in a non-contact state in a normal state, and are contacted to start a reaction in the test. The separation layer contains perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid. The creatinine detection device provided by the application can eliminate the interference of endogenous ammonia, improve the diffusion capacity of ammonia and improve the sensitivity of detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in-vitro diagnosis equipment, in particular to a creatinine detection device. BACKGROUND

[0002] Creatinine can reflect the filtration function of glomerulus to a certain extent. When the filtration function of glomerulus is reduced to less than 30% of normal, the creatinine level begins to rise, and the higher the creatinine, the worse the filtration function of glomerulus.

[0003] The determination of creatinine mainly includes chemical determination method (Jaffe method), enzyme method, high performance liquid chromatography and capillary electrophoresis method, etc.

[0004] The chemical determination method is low in cost and simple in operation, and is one of the most commonly used methods for determining creatinine in China at present. The principle is that creatinine in the sample reacts with picrate to generate yellow-red picric acid creatinine complex. The disadvantage of this method is that the specificity is not high, and vitamin C, acetone, acetylacetate, methyldopa, as well as high concentration of glucose, protein and some antibiotics such as penicillin G, cefoxitin and cefazolin can also react with alkaline picric acid to generate red color.

[0005] High performance liquid chromatography (HPLC), creatinine is positively charged in weak acid environment, and can be well separated from other components through cation exchange chromatography column, and its light absorption is measured at 234 nm. This method has high precision and good specificity, but it is not suitable for large-scale clinical sample analysis, and is usually used as a reference method for evaluating commercially available creatinine determination kits and some scientific research purposes.

[0006] Capillary electrophoresis method, serum samples are pretreated by high-speed centrifugation, and urine samples can be pretreated by low-speed centrifugation to remove formed elements, and the supernatant is separated by micellar electrokinetic capillary electrophoresis and then the absorbance at 235 nm is measured. This method has a wide linear range and is relatively simple to operate, but special equipment is needed and serum samples need to be pretreated, which makes it difficult to use in clinical routine.

[0007] Enzymatic determination method is a creatinine detection method that takes into account simplicity and accuracy, and is suitable for clinical use, mainly including creatinine deiminase (Creatinine deiminase; EC 3.5.4.21) and creatininase (Creatininase; EC 3.5.2.10).

[0008] The principle of creatininase method is as follows:

[0009]

[0010] The target analyte creatinine is hydrolyzed into creatine by creatininase, and the creatine is hydrolyzed into sarcosine by creatine hydrolase, and the generated sarcosine is oxidized into hydrogen peroxide by sarcosine oxidase, and the hydrogen peroxide reacts with peroxidase and chromogenic agent to produce a color change that can be detected. This method requires the participation of four enzymes, making the detection cost higher.

[0011] The principle of creatinine deiminase is as follows:

[0012]

[0013] Creatinine is converted into ammonia by creatinine deiminase, and then there are two detection routes. One is to use glutamate dehydrogenase, and ammonia and alpha-ketoglutarate are catalyzed by glutamate dehydrogenase to oxidize reduced coenzyme NADH or NADPH (340 nm has an absorption peak) to generate oxidized coenzyme NAD or NADP (340 nm has no absorption peak). After the reaction is completed, the content of creatinine can be detected by testing the decrease of absorbance at 340 nm.

[0014]

[0015] The other route uses an indicator to detect the generation of ammonia.

[0016]

[0017] Obviously, creatinine deiminase method requires fewer enzymes than creatininase method, so the cost of detection reagents will be lower.

[0018] Based on the two detection routes of creatinine deiminase, the endogenous ammonia concentration in blood will directly affect the creatinine content test.

[0019] In order to remove the interference of endogenous ammonia, US5008078 discloses a plasma creatinine test strip, which is divided into a transparent base sheet, an ammonia color developing layer, a gas permeable layer, an ammonia producing layer, an ammonia blocking layer, and an ammonia removing layer from bottom to top. The sample is added to the test strip from top to bottom, first passes through the ammonia removing layer to remove the endogenous ammonia, then flows through the ammonia blocking layer, and under the action of creatinine deiminase in the ammonia producing layer, ammonia is generated, the ammonia gas permeates through the gas permeable layer, and the ammonia color developing layer develops color. However, the sample passing time through the ammonia removing layer is determined by the sample viscosity and other factors, and it is difficult to ensure sufficient removal time, and in addition, the ammonia removal reagent of the removal layer will be redissolved in the sample and brought to the ammonia producing layer, decomposing the generated ammonia, causing the deviation of the result.

[0020] Chinese patent CN102288747 discloses a dry chemical test strip for removing interference, which can realize the reaction of sample with reagent 1 to remove interference, and the detection reaction of target analyte is started after adding reagent 2 at a set time. The problem of insufficient removal time in US5008078 is solved, but the test strip is only suitable for detecting glutathione transaminase, glutathione transaminase and creatinine enzyme method detection of creatinine, and cannot meet the requirements of creatinine deiminase method detection of creatinine. SUMMARY

[0021] The present application provides a creatinine deiminase method creatinine detection device which can remove endogenous ammonia interference and has high ammonia gas absorption efficiency.

[0022] The creatinine detection device provided by the present application comprises 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 they are kept in non-contact in a normal state and can be in contact with each other in a test state, the reagent 1 component is directly fixed on the substrate, and the reagent 2 component is connected to the substrate through an adhesive member, the adhesive member has a sufficient height to suspend the reagent 2 component above the reagent 1 component, and the reagent 2 component can be kept in contact with the reagent 1 component by being pressed down, the reagent 1 component comprises a flow pad, an enzyme pad and a blood filtration membrane, and the reagent 2 component comprises a start inhibition layer, a separation layer and a color development layer, the separation layer contains perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid.

[0023] The preferred perfluorooctane sulfonic acid is tridecafluorooctane sulfonic acid, and the preferred perfluorooctane carboxylic acid is pentadecafluorooctanoic acid.

[0024] Preferably, the mass concentration of the perfluorooctane sulfonic acid and the perfluorooctane carboxylic acid is 0.001-0.005%.

[0025] Preferably, the separation layer further contains a long-chain fatty acid ester.

[0026] Preferably, the long-chain fatty acid ester is methyl nonadecanoate, methyl heptadecanoate or methyl pentadecanoate.

[0027] Preferably, the mass concentration of the long-chain fatty acid ester is 0.002-0.05%.

[0028] Preferably, the separation layer is a gas-permeable hydrophobic polytetrafluoroethylene membrane.

[0029] The enzyme pad is fixed with a reagent for removing endogenous ammonia, which is preferably glutamate dehydrogenase, NADP and α-ketoglutaric acid.

[0030] The start inhibition layer is fixed with a reagent creatinine deiminase which reacts with creatinine.

[0031] Preferably, the start inhibition layer contains glutamate dehydrogenase inhibitor, preferably mouse anti-glutamate dehydrogenase antibody.

[0032] The color developing layer can use acid-base indicator, preferably bromophenol blue.

[0033] Preferably, the flow pad is nitrocellulose membrane.

[0034] Preferably, the reagent 1 component further comprises mesh cloth.

[0035] After the whole blood sample is added to the reagent 1 component, the filter membrane in the reagent 1 component traps the blood cells in the sample, and the plasma re-dissolves the reagents such as glutamate dehydrogenase, NADP and α-ketoglutaric acid fixed on the enzyme pad, and flows to the flow pad, and consumes the endogenous ammonia in the sample according to the following reaction.

[0036]

[0037] After the set time, the endogenous ammonia has been fully removed. The reagent 2 component is pressed by the instrument or manually, the start inhibition layer in the reagent 2 component contacts with the flow pad in the reagent 1 component, and the creatinine desiminase in the start inhibition layer decomposes creatinine according to the following reaction formula to produce ammonia.

[0038]

[0039] At the same time, the glutamate inhibitor in the start inhibition layer inhibits the activity of glutamate dehydrogenase, preventing the ammonia produced by creatinine from being consumed. The ammonia produced by the start inhibition layer can diffuse into the color developing layer, and the indicator in the color developing layer reacts with the ammonia to produce a detectable signal change.

[0040] The separation layer separates the liquid contact between the start inhibition layer and the color developing layer, but needs to allow the ammonia produced in the start inhibition layer to diffuse into the color developing layer. It is found in experiments that the detection signal is obviously improved after adding perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid in the separation layer, and the detection signal is improved more obviously if long-chain fatty acid ester is also added, and the liquid in the start inhibition layer does not come to the color developing layer, causing abnormal coloration.

[0041] The creatinine detection device provided by the application can control the reaction time of removing endogenous ammonia, fully remove the interference of endogenous ammonia, and the ammonia produced by creatinine can better diffuse into the color developing layer in the form of gas, greatly improving the detection sensitivity.

[0042] The detection device of the present application can be used with a small detection instrument, such as a spectrophotometer, preferably a reflective spectrophotometer. The spectrophotometer can be used to detect the change in reflectance density of the creatinine detection device after the reaction at a specific wavelength, and the creatinine concentration is given by an algorithm. The creatinine detection device of the present application can eliminate the influence of endogenous ammonia on the color reaction, and improve the diffusion of ammonia, thus improving the accuracy and sensitivity, so that the detection is more accurate. In addition, the detection device of the present application is easy to operate, and does not require professional operation, which can meet the market demand under special conditions, especially for emergency, home application. Compared with the traditional detection method by liquid biochemical reagent, the detection device of the present application can provide convenient and fast detection for emergency department, primary hospital, family and small clinic, etc. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Fig. 1 is a structural schematic diagram of the detection device of the present application.

[0044] Figure 2 Fig. 4 is a schematic diagram of the placement position of the substrate and the flow pad in the manufacture of the large plate of the detection device of the present application.

[0045] Figure 3 Fig. 6 is a schematic diagram of the overlapping area of the transparent carrier and the flow pad of the detection device of the present application.

[0046] In the figure, the substrate 101, the enzyme pad 102, the blood filter film 103, the glue A 104, the mesh cloth 105, the flow pad 106, the start inhibition layer 107, the separation layer 108, the color development layer 109, the transparent carrier 110, the bonding piece 111, the glue B 112, the glue C 113, the overlapping area 201, the detection device 10. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0048] It should be noted that when an element is referred to as "provided on" another element, it can be directly on the other element or there can be a middle element. When an element is referred to as "connected" to another element, it can be directly connected to the other element or there can be a middle element.

[0049] 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.

[0050] Example

[0051] 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.

[0052] 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.

[0053]

[0054] 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.

[0055] Example 1

[0056] A 0.35mm thick white PET board measuring 20cm x 8cm was used as the substrate.

[0057] Preparation of component 1 of reagent:

[0058] Cut a 20cm length from a 25mm wide Pall Vivid 90 nitrocellulose membrane to serve as a flow pad.

[0059] Cut the Jelena bioglass cellulose membrane GL048 into 20 cm x 0.5 cm, and soak it in the following solution for 10 min,

[0060] HEPES 0.1 mol / L

[0061] Tween 20 0.5% (v / v)

[0062] Glutamate dehydrogenase 100 U / mL

[0063] NADP 5 mmol / L

[0064] Alpha-ketoglutarate 20 mmol / L

[0065] BSA 2% (w / v)

[0066] pH = 7.5

[0067] Then take it out and put it in 37°C drying, to get the enzyme pad.

[0068] Cut the Sartorius membrane material 1660 into 20 cm x 0.5 cm, as a blood filter membrane.

[0069] Soak the 20 cm x 0.5 cm gauze in 0.05% (v / v) Triton X100 solution for 10 min, and then dry it at 37°C, to get the mesh cloth.

[0070] Align one end of the flow pad, enzyme pad, blood filter membrane, and mesh cloth, and place them on the substrate in order according to Figure 1 , Figure 2 The edge distance from the substrate edge is 4 cm, and the end is left empty as the hand-held end (the right end of Figure 1 , Figure 2 The lower end of). Glue A at the aligned end point, and fix the substrate, flow pad, enzyme pad, blood filter membrane, and mesh cloth.

[0071] The above completes the preparation of reagent 1 component, and it is fixed on the substrate.

[0072] Preparation of reagent 2 component:

[0073] Cut the 0.25 mm thick transparent PET sheet into 20 cm x 2.5 cm, as a transparent carrier.

[0074] Spray the following solution on the transparent carrier to form a 2 cm wide zone as a color developing layer. The edge of the zone is 3 mm away from one end of the transparent carrier, to facilitate glue dispensing.

[0075] Bromophenol blue 4 mg / mL

[0076] Tween 20 0.5% (v / v)

[0077] Gelatin 2% (w / v)

[0078] A Corning hydrophobic polytetrafluoroethylene membrane with a pore size of 0.45 μm was cut into 20 cm x 2 cm and used as the spacer layer.

[0079] A Jierui bioglass cellulose membrane GL048 was cut into 20 cm x 2 cm and soaked in the following solution for 10 min,

[0080] HEPES 0.1 mol / L

[0081] Tween 20 0.5% (v / v)

[0082] Creatinine deiminase 100 U / mL

[0083] Mouse anti-glutamate dehydrogenase antibody 1 mg / mL

[0084] BSA 2% (w / v)

[0085] NaCl 0.9% (w / v)

[0086] pH = 7.5 Then take out and place in a 37°C oven for 2 h to obtain the start inhibition layer.

[0087] The spacer layer and the start inhibition layer were placed in order on the chromogenic layer, and a silicon-based elastic glue was fixed on both end points to obtain reagent 2 component.

[0088] The transparent carrier in the reagent 2 component was fixed on the substrate through an adhesive and overlapped with the flow pad in a length of 1.5 cm in the overlapping area 201, as shown, to obtain a detection device large plate. Figure 3

[0089] The large plate was cut into a strip with a width of 5 mm and a length of 8 cm along the length direction, and the detection device 10 was obtained.

[0090] Example 2

[0091] A Corning hydrophobic polytetrafluoroethylene membrane with a pore size of 0.45 μm was cut into 20 cm x 2 cm and soaked in a 0.002% (w / v) tridecafluorooctanesulfonic acid (CAS No.: 27619-97-2) acetone solution for 1 h and dried at 37°C to obtain the spacer layer.

[0092] The others were the same as in Example 1.

[0093] Example 3

[0094] A Corning hydrophobic polytetrafluoroethylene membrane with a pore size of 0.45 μm was cut into 20 cm x 2 cm and soaked in a 0.004% (w / v) pentadecafluorooctanoic acid (CAS No.: 335-67-1) acetone solution for 1 h and dried at 37°C to obtain the spacer layer. ​

[0095] Otherwise as in Example 1.

[0096] Example 4

[0097] A Millipore hydrophobic polytetrafluoroethylene membrane with a pore size of 0.45 pm was cut into 20 cm x 2 cm, soaked in a 0.001% (w / v) pentadecafluorooctanoic acid (CAS No.: 335-67-1) and 0.005% (w / v) pentadecafluorooctanoic acid (CAS No.: 335-67-1) acetone solution for 1 h, and dried at 37 °C as a spacer layer.

[0098] Otherwise as in Example 1.

[0099] Example 5

[0100] A Millipore hydrophobic polytetrafluoroethylene membrane with a pore size of 0.45 pm was cut into 20 cm x 2 cm, soaked in a 0.004% (w / v) pentadecafluorooctanoic acid (CAS No.: 335-67-1), 0.002% (w / v) pentadecafluorooctanoic acid (CAS No.: 335-67-1), 0.002% (w / v) methyl nonadecanoate (CAS No.: 1731-94-8) acetone:chloroform = 1 : 1 solution for 1 h, and dried at 37 °C as a spacer layer.

[0101] Otherwise as in Example 1.

[0102] Example 6

[0103] A Millipore hydrophobic polytetrafluoroethylene membrane with a pore size of 0.45 pm was cut into 20 cm x 2 cm, soaked in a 0.003% (w / v) pentadecafluorooctanoic acid (CAS No.: 335-67-1), 0.003% (w / v) pentadecafluorooctanoic acid (CAS No.: 335-67-1), 0.05% (w / v) methyl heptadecanoate (CAS No.: 1731-92-6), 0.01% (w / v) methyl pentadecanoate (CAS No.: 7132-64-1) acetone:chloroform = 1 : 1 solution for 1 h, and dried at 37 °C as a spacer layer.

[0104] Otherwise as in Example 1.

[0105] Example 7

[0106] The ability of each example to remove endogenous interference was investigated.

[0107] Solution A was prepared:

[0108] Creatinine 50 pmol / L

[0109] BSA 5% (w / v)

[0110] NaCl 0.9% (w / v) Solution B was prepared:

[0111] Creatinine 50 μmol / L

[0112] NH3H20 1 mmol / L

[0113] BSA 5% (w / v)

[0114] NaCl 0.9% (w / v)

[0115] Add 30 μL solution to the mesh cloth of the detection device, press the transparent carrier after 1 min, and keep the starting inhibition layer in contact with the flow pad for 1 min. Read the reflectance of the chromogenic layer at 600 nm from the transparent carrier method, and the results are shown in the following table:

[0116] Table 1:

[0117]

[0118] As can be seen from the above table, in the case of creatinine concentration of only 50 μmol / L, the addition of 1 mmol / L of ammonia, and the concentration of the interferent is 20 times that of the target analyte creatinine, the detection reflectance is basically the same, that is, ammonia does not interfere with the detection of the target creatinine.

[0119] Example 8

[0120] Investigate the sensitivity of the detection of each example.

[0121] Prepare the mother liquor:

[0122] BSA 5% (w / v)

[0123] NaCl 0.9% (w / v) Then prepare creatinine solutions with concentrations of 0, 10, 20, 50, and 100 μmol / L, respectively, using the mother liquor. Use the detection device of each example to detect the above solutions, and the detection method and wavelength for reading reflectance are the same as in Example 7. The results are shown in the following table:

[0124] Table 2:

[0125]

[0126] From the above table, it can be seen that there is no substantial difference between the reflectance of the creatinine solution of 0, 10, 20 μmol / L in Example 1, and it is difficult to detect creatinine with a concentration below 50 μmol / L. Examples 2, 3, and 4 can barely distinguish the creatinine solution of 0, 10, 20 μmol / L, but the reflectance of the creatinine solution of 0, 10 μmol / L is less different, and Examples 5 and 6 have good distinction for each concentration. It can be seen that the addition of perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid in the separation layer improves the diffusion of ammonia and improves the detection sensitivity. Further addition of long-chain fatty acid ester in the presence of perfluorooctane sulfonic acid and / or perfluorooctane carboxylic acid further improves the sensitivity.

[0127] The above only describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application. The technical features of the above-described embodiments can be combined arbitrarily, and for the sake of brevity, not all possible combinations of the technical features in the above-described embodiments are described, however, as long as the combinations of the technical features do not contradict, they shall be considered as the scope described in the present application.

[0128] Those skilled in the art should understand that the discussion of any embodiment above is only exemplary and is not intended to suggest that the protection scope of the present application is limited to these examples; under the idea of the present application, the above embodiments or technical features in different embodiments can also be combined, the steps can be implemented in any order, and there are many other changes of different aspects of one or more embodiments of the present application as described above, which are not provided in details for the sake of brevity.

[0129] One or more embodiments of the present application are intended to cover all such alternatives, modifications, and variations falling within the broad scope of the present application. Therefore, any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principle of one or more embodiments of the present application shall be included in the protection scope of the present 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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