A dry chemical enzymatic uric acid detection reagent card

Abstract

The present invention discloses a dry chemical enzyme method uric acid detection reagent card, which effectively solves the technical problems of complicated, low-efficiency and inaccurate uric acid detection in the existing technology. The reagent card comprises a PVC back plate, on which a sample processing section, an enzymatic reaction section, a substrate mixing section and an enzymatic detection section are sequentially adhered from left to right. A card shell is arranged above the reagent card, and a sample addition hole arranged above the sample processing section and a detection hole arranged above the enzymatic detection section are arranged on the card shell. The present invention is exquisitely designed, and utilizes chromatography power. The blood sample sequentially passes through the sample processing section to remove red blood cells, is catalytically decomposed in the enzymatic reaction section, and is mixed with a detection substrate in the substrate mixing section, so as to achieve final color development or luminescence in the detection section. The chromatography filter paper used in the detection section is both the power of sample chromatography and the carrier for collecting reaction products for final detection.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro medical diagnosis, and particularly to a dry chemical enzymatic uric acid detection reagent card. Background Art

[0002] Uric acid (2,4,6-trihydroxypurine, abbreviated as UA) is a metabolite of the human body. Under normal circumstances, 70% of uric acid comes from the body's metabolism, and 30% comes from diet. Most of it is finally excreted by the kidneys through the circulatory system. Any problem in this pathway may lead to hyperuricemia. Gout is clearly related to the increase in blood uric acid level. Among the population with hyperuricemia (HUA) without gout symptoms, the annual incidence of gout in the group with blood uric acid greater than 9 mg / dl is 4.9%, the annual incidence in the group of 7-9 mg / dl is 0.5%, and the group below 7 mg / dl is only 0.1%. Lowering the blood uric acid of gout patients below 6 mg / dl can reduce both gout attacks and crystal deposition in joints. Recent studies have found that gout is closely related to metabolic syndrome. The ratio of gout patients with metabolic syndrome is 3.05 times that of non-gout patients. HUA related to gout is also a risk factor for various metabolic-related diseases such as diabetes, chronic kidney disease, obesity, hypertension, kidney stones, myocardial infarction, heart failure, and stroke.

[0003] Blood uric acid monitoring is currently a necessary means for the treatment of diseases such as gout. The importance of the long-term management of gout and HUA has attracted increasing attention. However, at present, most of the uric acid detections of gout patients are still carried out in the hospital during the acute attack period, which is too late and will bring great pain to the patients. Therefore, it is of great significance for uric acid detection to enter primary medical care and even families. At present, hospitals still use biochemical analyzers to detect uric acid. Large instruments are expensive and difficult to calibrate, so they cannot enter primary clinics and families, which is not conducive to the timely prevention and treatment of uric acid-related diseases.

[0004] There is currently research on detecting uric acid using the immunological antibody-antigen method. However, uric acid is a small molecule compound (molecular weight 168), and there are many interfering molecules with similar structures in the blood. Neither monoclonal antibodies nor polyclonal antibodies can eliminate interference at present, resulting in inaccurate medical detection. Therefore, there are no ELISA or colloidal gold kits registered as medical devices using immunological principles at present.

[0005] On this basis, the present invention provides a dry chemical enzymatic uric acid detection reagent card to solve this problem. Summary of the Invention

[0006] In view of the above situation, to overcome the deficiencies of the prior art, the present invention provides a dry chemical enzymatic uric acid detection reagent card, effectively solving the technical problems of complex uric acid detection, low efficiency, and inaccurate detection in the prior art.

[0007] A dry chemical enzymatic uric acid detection reagent card, characterized in that it includes a reagent card, and the reagent card includes a PVC backplane. On the PVC backplane, a sample treatment section, an enzymatic reaction section, a substrate mixing section, and an enzymatic detection section are adhered in sequence from left to right. A card shell is provided above the reagent card, and a sample addition hole placed above the sample treatment section and a detection hole placed above the enzymatic detection section are opened on the card shell.

[0008] Preferably, the sample treatment section is made by soaking glass fiber paper in the sample treatment solution and then drying it in a constant temperature air blast drying oven, with the temperature adjusted to 30 - 35°C. The formula of the sample treatment solution is as follows:

[0009] PB buffer solution with a pH of 7.4, 20 - 50 mmol / L

[0010] Sodium chloride, 8.0 - 8.2 g / L

[0011] Bovine serum albumin, 10 - 25 g / L

[0012] Sucrose, 10 - 25 g / L

[0013] Disodium ethylenediaminetetraacetate, 0.1 - 0.2 g / L

[0014] Phytohemagglutinin (such as concanavalin A), 5 - 20 ng / L

[0015] Proclin 300, 1 - 5 mL / L.

[0016] Preferably, the enzymatic reaction section is made by spraying the enzymatic reaction solution on an acetate fiber membrane or a nitrocellulose membrane and then drying it in a constant temperature air blast drying oven, with the temperature adjusted to 30 - 35°C. The formula of the enzymatic reaction solution is as follows:

[0017] PB buffer solution with a pH of 7.4, 20 - 50 mmol / L

[0018] Sodium chloride, 8.0 - 8.2 g / L

[0019] Bovine serum albumin, 10 - 25 g / L

[0020] Sucrose, 10 - 25 g / L

[0021] Uricase, 5 - 10 KU / L

[0022] Proclin 300, 1 - 5 mL / L.

[0023] Preferably, the substrate mixing section is prepared by spraying the substrate solution on glass fiber paper and then drying it in a constant temperature air blast drying oven with the temperature adjusted to below 50°C. The formula of the substrate solution is as follows:

[0024] Acetic acid buffer solution with a pH of 4.0, 20 - 50 mmol / L

[0025] Glycerol, 10 - 20 ml / L

[0026] TMB, 0.1 - 0.5 g / L

[0027] Preferably, the enzymatic detection section is prepared by spraying the detection solution on chromatography filter paper and then drying it in a constant temperature air blast drying oven with the temperature adjusted to 30 - 35°C. The formula of the detection solution is as follows:

[0028] PB buffer solution with a pH of 7.4, 5 - 10 mmol / L

[0029] Sodium chloride, 8.0 - 8.2 g / L

[0030] Bovine serum albumin, 10 - 25 g / L

[0031] Sucrose, 10 - 25 g / L

[0032] Peroxidase, 0.05 - 0.2 KU / L

[0033] Proclin 300, 1 - 5 mL / L.

[0034] Preferably, the PVC backplane is pasted to the sample treatment section, enzymatic reaction section, substrate mixing section, and enzymatic detection section respectively with adhesive.

[0035] Preferably, the reagent card is also equipped with a termination solution, and the termination solution is sulfuric acid or hydrochloric acid with a concentration of 2 mol / mL.

[0036] Compared with the prior art, the present invention has the following technical effects:

[0037] 1. The present invention can provide a simple, fast, and accurate reagent card.

[0038] 2. The present invention is exquisitely designed, all raw materials are commercialized, inexpensive, has strong detection specificity and high sensitivity.

[0039] 3. The detection operation of the present invention is simple and convenient, fingertip whole blood can be used for detection, and a colorimetric card or a card reader is equipped when using a chromogenic substrate, and the uric acid content can be simply determined at home.

[0040] 4. The present invention uses a luminescent substrate in primary hospitals, and an optoelectronic reader can be equipped to accurately measure the uric acid content. Description of the Drawings

[0041] Figure 1 This is a three-dimensional schematic diagram of the overall structure of the reagent card of the present invention. Detailed implementation manners

[0042] Regarding the foregoing and other technical contents, features and effects of the present invention, they will be clearly presented in the following detailed description of the embodiments in conjunction with the attached Figure 1 drawings. The structural contents mentioned in the following embodiments are all with reference to the drawings of the specification.

[0043] The exemplary embodiments of the present invention will be described below with reference to the drawings.

[0044] The present invention relates to a uric acid detection reagent card using a dry chemical enzymatic method, which includes a reagent card 1. The reagent card 1 is in a long strip shape and includes a PVC backplane 2. Above the PVC backplane 2, a sample treatment section 3, an enzymatic reaction section 4, a substrate mixing section 5, and an enzymatic detection section 6 are successively adhered from left to right by an adhesive. The thickness of the sample treatment section 3 is higher than that of the enzymatic reaction section 4, the thickness of the enzymatic reaction section 4 is lower than that of the substrate mixing section 5, and the thickness of the substrate mixing section 5 is higher than that of the enzymatic detection section 6.

[0045] Above the reagent card 1, there is a card shell 7. The card shell 7 is in a long strip shape and matches the reagent card 1. It is buckled above the reagent card 1. Then, a sample addition hole 8 and a detection hole 9 are opened on the card shell 7. The sample addition hole 8 is placed above the sample treatment section 3, and the detection hole 9 is placed above the enzymatic detection section 6. The sample addition hole 8 is used to add a blood sample, and the detection hole 9 is used to observe luminescence or color development. The flow direction of the blood sample is from left to right.

[0046] The manufacturing method of the reagent card is as follows:

[0047] 1. Manufacture the sample treatment section;

[0048] Prepare a sample treatment solution, and the formula of the sample treatment solution is:

[0049] PB buffer solution with a pH of 7.4, 20 - 50 mmol / L

[0050] Sodium chloride, 8.0 - 8.2 g / L

[0051] Bovine serum albumin, 10 - 25 g / L

[0052] Sucrose, 10 - 25 g / L

[0053] Disodium ethylenediaminetetraacetate, 0.1 - 0.2 g / L

[0054] Phytohemagglutinin (such as concanavalin A), 5 - 20 ng / L

[0055] Proclin 300, 1 - 5 mL / L

[0056] Then, the sample treatment solution is evenly sprayed on the glass fiber paper and then dried in a constant temperature forced air drying oven. The drying temperature of the constant temperature forced air drying oven is adjusted to 30 - 35 °C, and the drying time is 4 - 8 hours.

[0057] 2. Preparation of the enzymatic reaction section:

[0058] Prepare the enzymatic reaction solution. The formula of the enzymatic reaction solution is:

[0059] PB buffer solution with a pH of 7.4, 20 - 50 mmol / L

[0060] Sodium chloride, 8.0 - 8.2 g / L

[0061] Bovine serum albumin, 10 - 25 g / L

[0062] Sucrose, 10 - 25 g / L

[0063] Uricase, 5 - 10 KU / L

[0064] Proclin 300, 1 - 5 mL / L

[0065] Then, the enzymatic reaction solution is evenly sprayed on the cellulose acetate membrane or nitrocellulose membrane and then dried in a constant temperature forced air drying oven. The drying temperature of the constant temperature forced air drying oven is adjusted to 30 - 35 °C, and the drying time is 4 - 8 hours.

[0066] 3. Preparation of the substrate mixing section:

[0067] Prepare the substrate solution. The formula of the substrate solution is:

[0068] Chromogenic substrate:

[0069] Acetic acid buffer solution with a pH of 4.0, 20 - 50 mmol / L

[0070] Glycerol, 10 - 20 ml / L

[0071] TMB, 0.1 - 0.5 g / L

[0072] Another formula is:

[0073] Luminescent substrate:

[0074] CB buffer solution with a pH of 9.0, 20 - 50 mmol / L

[0075] p-Iodophenol, 10 - 20 ml / L

[0076] Isoluminol, 0.1 - 0.5 g / L

[0077] Then, the substrate solution is evenly sprayed on the glass fiber paper and then dried in a constant temperature air blast drying oven. The drying temperature of the constant temperature air blast drying oven is adjusted to below 50°C, preferably 30 - 35°C, and the drying time is 4 - 8 hours.

[0078] 4. Preparation of the enzyme-catalyzed detection section:

[0079] Prepare the detection solution. The formula of the detection solution is as follows:

[0080] PB buffer solution with a pH of 7.4, 5 - 10 mmol / L

[0081] Sodium chloride, 8.0 - 8.2 g / L

[0082] Bovine serum albumin, 10 - 25 g / L

[0083] Sucrose, 10 - 25 g / L

[0084] Peroxidase, 0.05 - 0.2 KU / L

[0085] Proclin 300, 1 - 5 mL / L

[0086] Then, the detection solution is evenly sprayed on the chromatography filter paper and then dried in a constant temperature air blast drying oven. The drying temperature of the constant temperature air blast drying oven is adjusted to 30 - 35°C, and the drying time is 4 - 8 hours.

[0087] 5. Preparation of the reagent card:

[0088] Then, the sample processing section 3, the enzyme-catalyzed reaction section 4, the substrate mixing section 5, and the enzyme-catalyzed detection section 6 are successively adhered to the PVC backplane 2 with adhesive to form a reagent card 1 with a width of 3 - 8 mm.

[0089] 6. Install a card shell 7 above the reagent card 1. Sampling holes 8 and detection holes 9 are reserved on the card shell 7. The sampling hole 8 is above the sample processing section 3, and the detection hole 9 is above the enzyme-catalyzed detection section 6 to form a complete uric acid detection reagent card.

[0090] 7. During detection, 80 - 120 μL of blood sample is added to the sampling hole, and the result is observed through the detection hole.

[0091] 8. Set a good time. For example, if you want to see the luminescence or color development degree at 10 minutes, that is, at 10 minutes, add a termination solution to the reagent card. The termination solution is sulfuric acid or hydrochloric acid with a concentration of 2 mol / mL. After adding the termination solution, the luminescence or display degree can be fixed to facilitate observing the result and making a comparison.

[0092] Uric acid detection is mainly used for assessing the severity of gout and the efficacy of drug treatment, and for monitoring the uric acid level in patients. The traditional method is the biochemical method, which requires large instruments and is generally only available in the laboratories of hospitals above the second level. Venous blood samples need to be collected and centrifuged before testing. The reagent card of the present invention can be directly observed with the naked eye or detected and recorded using a CCD scanning imaging instrument. The instrument is simple. It can be immediately detected using fingertip blood, and the results can be obtained in 10-15 minutes. It can be widely used in department diagnosis, hospitals below the second level, community hospitals, and even clinics, etc., facilitating the treatment and monitoring of chronic diseases. It can also be installed on a physical examination vehicle for chronic disease surveys, etc., greatly facilitating the diagnosis and treatment of people with limited mobility due to gout nearby. The single test card and colorimetric card can be used directly at home, and patients can monitor the fluctuations of their own uric acid levels in their bodies and reasonably adjust their diet and medication.

[0093] The present invention is exquisitely designed. Utilizing chromatographic dynamics, the blood sample sequentially passes through the sample treatment section to remove red blood cells, is catalytically decomposed in the enzymatic reaction section, is mixed with the detection substrate in the substrate mixing section, and finally develops color or emits light at the detection section. The chromatographic filter paper used in the detection section is both the driving force for sample chromatography and the carrier for collecting reaction products for the final detection. The specific principle is that in the sample treatment section of the whole blood sample, red blood cells aggregate and coagulate under the action of lectin. The aggregated red blood cells are blocked in the sample treatment section and do not affect subsequent reactions. The blood from which red blood cells have been removed continues to chromatograph to the enzymatic reaction section, where uric acid in the liquid is catalytically decomposed by uricase to produce hydrogen peroxide. Under the action of chromatography, the liquid carries the reaction product hydrogen peroxide to dissolve the substrate in the substrate mixing section and is fully mixed during the chromatography process. Finally, the mixed liquid reaches the detection section, where peroxidase (HRP) catalytically decomposes hydrogen peroxide and oxidizes the substrate to emit light or develop color at the same time. The content of hydrogen peroxide is calculated by detecting the color development depth or light emission intensity, and thus the concentration of uric acid is indirectly detected.

[0094] Compared with the prior art, the present invention has the following technical effects:

[0095] 1. The present invention can provide a simple, fast, and accurate reagent card.

[0096] 2. The present invention is exquisitely designed, all raw materials are commercialized, the price is cheap, the detection specificity is strong, and the sensitivity is high.

[0097] 3. The detection operation of the present invention is simple and convenient. It can use fingertip whole blood for detection. When using a color-developing substrate, a colorimetric card or a card reader is equipped, and the uric acid content can be simply determined at home.

[0098] 4. The present invention uses a luminescent substrate in primary hospitals and is equipped with a photoelectric reading instrument to accurately determine the uric acid content.

Claims

1. A dry chemical enzymatic uric acid detection reagent card, characterized in that, It includes a reagent card (1), and the reagent card (1) includes a PVC backplane (2). On the PVC backplane (2), a sample treatment section (3), an enzymatic reaction section (4), a substrate mixing section (5), and an enzymatic detection section (6) are adhered in sequence from left to right. Above the reagent card (1), there is a card shell (7). On the card shell (7), a sample addition hole (8) placed above the sample treatment section (3) and a detection hole (9) placed above the enzymatic detection section (6) are provided. The sample treatment section (3) is made by soaking glass fiber paper in the sample treatment solution and then drying it in a constant temperature air blast drying oven with the temperature adjusted to 30 - 35°C. The formula of the sample treatment solution is as follows: PB buffer solution with a pH of 7.4, 20 - 50 mmol / L, sodium chloride 8.0 - 8.2 g / L, bovine serum albumin 10 - 25 g / L, sucrose 10 - 25 g / L, disodium ethylenediaminetetraacetate 0.1 - 0.2 g / L, concanavalin A 5 - 20 ng / L, and Proclin 300 1 - 5 mL / L; The enzymatic reaction section (4) is made by spraying the enzymatic reaction solution on an acetate fiber membrane or a nitrocellulose membrane and then drying it in a constant temperature air blast drying oven with the temperature adjusted to 30 - 35°C. The formula of the enzymatic reaction solution is as follows: PB buffer solution with a pH of 7.4, 20 - 50 mmol / L, sodium chloride 8.0 - 8.2 g / L, bovine serum albumin 10 - 25 g / L, sucrose 10 - 25 g / L, uricase 5 - 10 KU / L, and Proclin 300 1 - 5 mL / L; The substrate mixing section (5) is made by spraying the substrate solution on glass fiber paper and then drying it in a constant temperature air blast drying oven with the temperature adjusted to below 50°C. The formula of the substrate solution is as follows: Acetic acid buffer solution with a pH of 4.0, 20 - 50 mmol / L, glycerol 10 - 20 ml / L, and TMB 0.1 - 0.5 g / L; The enzymatic detection section (6) is made by spraying the detection solution on chromatographic filter paper and then drying it in a constant temperature air blast drying oven with the temperature adjusted to 30 - 35°C. The formula of the detection solution is as follows: PB buffer solution with a pH of 7.4, 5 - 10 mmol / L, sodium chloride 8.0 - 8.2 g / L, bovine serum albumin 10 - 25 g / L, sucrose 10 - 25 g / L, peroxidase 0.05 - 0.2 KU / L, and Proclin 300 1 - 5 mL / L.

2. The uric acid detection reagent card using a dry chemical enzymatic method according to claim 1, characterized in that, The PVC backplane (2) is adhered to the sample treatment section (3), the enzymatic reaction section (4), the substrate mixing section (5), and the enzymatic detection section (6) respectively with self - adhesive.

3. The uric acid detection reagent card of a dry chemical enzymatic method according to claim 1, wherein The reagent card (1) is also equipped with a termination solution, and the termination solution is sulfuric acid or hydrochloric acid with a concentration of 2 mol / mL.

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