CN116144731A - Reactive enzyme liquid, test paper and device system for electrochemical urea nitrogen detection - Google Patents

Reactive enzyme liquid, test paper and device system for electrochemical urea nitrogen detection Download PDF

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CN116144731A
CN116144731A CN202310362599.2A CN202310362599A CN116144731A CN 116144731 A CN116144731 A CN 116144731A CN 202310362599 A CN202310362599 A CN 202310362599A CN 116144731 A CN116144731 A CN 116144731A
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urea nitrogen
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enzyme solution
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闫琦
葛艳秋
秦玉
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Nanjing Jingjie Biotechnology Co ltd
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Abstract

The invention relates to a reaction enzyme solution, test paper and a device system for electrochemical urea nitrogen detection, wherein the reaction enzyme solution comprises urease, glutamate dehydrogenase, alpha-ketoglutaric acid, reduced coenzyme I, glutamate oxidase, peroxidase and an electron mediator; the mass concentration of the urease in the reaction enzyme liquid is 4-6%, and the mass ratio of the urease, the glutamate dehydrogenase, the alpha-ketoglutaric acid and the reduced coenzyme I in the reaction enzyme liquid is 1 (0.5-1): 1-1.5): 0.5-1. The test paper for electrochemical urea nitrogen detection belongs to a current type sensor, and can be used for accurately detecting the urea nitrogen content in a whole blood sample within 2-10 seconds.

Description

Reactive enzyme liquid, test paper and device system for electrochemical urea nitrogen detection
Technical Field
The invention relates to the technical field of medical detection, in particular to urea nitrogen detection, and especially relates to a reactive enzyme solution, test paper and device system for electrochemical urea nitrogen detection.
Background
Urea nitrogen is human eggThe main end product of white matter metabolism. Deamination of amino acids to produce NH 3 And CO 2 Both synthesize urea in the liver, producing 0.3g of urea per gram of protein metabolism. The nitrogen content in urea is 28/60, almost half. Generally, the kidney is the main organ for excreting urea, and after the urea is filtered from glomeruli, the urea can be reabsorbed in each section of tubular, but the faster the flow rate of urine in the tubular, the smaller the reabsorption is, i.e. the maximum clearance is achieved. Like creatinine, blood urea nitrogen can be in the normal range in the early stages of kidney function impairment. The concentration of blood urea nitrogen increases rapidly when glomerular filtration rate falls below 50% of normal. Under normal conditions, the ratio of blood urea nitrogen to creatinine (BUN/Scr) is about 10.1, and the ratio can be increased, even can reach 20-30, for high protein diets, high catabolic states, water deficiency, renal ischemia, hypovolemia and certain acute glomerulonephritis; whereas low protein diets, liver disease often results in a decrease in the ratio, which may be referred to as hypoazotemia. Various kidney parenchymal lesions, such as glomerulonephritis, interstitial nephritis, acute and chronic renal failure, intrarenal space occupation and destructive lesions can increase blood urea nitrogen. Multiple extra-renal factors can also cause the elevation of blood urea nitrogen, and if the extra-renal factors are eliminated, BUN concentration reaches 21.4mmol/L (60 mg/dL), which is one of the uremia diagnosis indexes.
The blood urea detection is a conventional kidney function examination item, is one of indexes reflecting kidney functions, can sensitively reflect kidney function damage conditions of diabetic nephropathy patients, has important diagnostic significance for diabetic kidney damage, and is an important biochemical index for clinically observing and diagnosing various kidney diseases, so that the measurement of blood urea content has important significance in clinical diagnosis, screening and monitoring of kidney diseases. Currently, the detection method of the haematuria mainly comprises a diacetyl monoxime color development method, a urease-Bose color comparison method, an enzyme coupling rate method, a urease biosensor method and the like.
The principle of the diacetyl-oxime chromogenic method for measuring blood urea is that diacetyl is condensed with urea to form red 4, 5-dimethyl-2-oxo-imidazole compound in a strong acid solution under the condition that thiosemicarbazide and cadmium ions coexist, the color depth is in direct proportion to the urea nitrogen content, and the urea nitrogen content in a sample needs to be compared with a urea standard liquid treated in the same way. However, diacetyl is generally produced by reacting diacetyl monooxime with a strong acid in the reaction system, followed by condensation with urea to form red 4, 5-dimethyl-2-oxoimidazole (i.e., the Fearon reaction), because diacetyl is unstable. The method is only suitable for laboratory standard operation and is generally not suitable for rapid detection on site. Although the method has the advantages of available reagents and low cost, the reagents are required to be heated and boiled during operation, have corrosiveness and toxicity, are easy to pollute the environment, and have a narrow linear range.
The principle of urease-Bosch colorimetry is that urea is catalyzed by urease to hydrolyze to produce ammonium and carbon dioxide. Sodium nitrosoferricyanide catalyzed NH under alkaline conditions 4+ Reacts with phenol and hypochlorite to produce a blue compound. The method is widely applied in the prior art, and comprises a plurality of fields such as clinical medicine, environment and the like. However, after comparing the o-phthalaldehyde method and the urease-Bosch colorimetric method to determine urea in swimming pool water, it was concluded that the urease Bosch colorimetric method is good in linearity without interference, but when CuSO 4 When the content of urease reaches 0.3mg/L in swimming pool water, the urease and copper ions generate insoluble salt to be deactivated, so that the reaction cannot be performed normally, and the method has certain limitation.
The principle of the enzyme coupling rate method is that urea is hydrolyzed under the catalysis of urease to generate ammonia and carbon dioxide, ammonia is catalyzed by glutamate dehydrogenase to generate glutamic acid in the presence of alpha-ketoglutaric acid and reduced coenzyme I, and the reduced coenzyme I is oxidized to oxidized coenzyme I, the reduced coenzyme I is absorbed at 340nm, and the absorbance reduction rate is in direct proportion to the urea content in a sample to be tested. However, in clinical medicine, hemoglobin in a blood sample to be detected may cause a certain interference to measurement, and it is necessary to avoid hemolysis of the blood sample to be detected, so that there is a certain limitation in this method.
The urease biosensor method is a method which utilizes an instrument sensitive to biological substances and converting the concentration of the biological substances into an electric signal for detection, and has the advantages of high accuracy, rapid detection and the like. At present, various large-scale full-automatic biochemical analyzers are commonly adopted for detection by the urease biosensor method, but because the large-scale full-automatic biochemical analyzers are high in equipment price and complex in operation, operators need to have related professional knowledge and receive corresponding training, the requirements on using matched conditions are high, a regulated power supply, a water purifier and the like are required to be equipped, the occupied area is large, the maintenance cost is high, and the professionals are required to maintain regularly, so that basic medical institutions or family individuals do not have the condition of purchasing and using the urease biosensor. In addition, a large number of patients in a large hospital have complicated detection procedures, long flow and long waiting time, and the large time burden is brought to the patients.
Along with popularization and use of a large-scale full-automatic biochemical analyzer and updating of the biochemical analyzer, transition from a traditional large-scale biochemical analyzer to a small-scale convenient instrument is a development great direction of biomedical instruments. Therefore, developing an electrochemical urea nitrogen detection instrument which is convenient, quick and accurate and does not need large equipment has important significance in improving detection accuracy and sensitivity and shortening detection time.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides a reaction enzyme solution, test paper and a device system for electrochemical urea nitrogen detection, wherein urea generates glutamic acid under the action of urease and glutamate dehydrogenase in the test paper detection process of the electrochemical urea nitrogen detection, the glutamic acid generates hydrogen peroxide under the action of glutamate oxidase, the hydrogen peroxide changes a reduced electronic mediator from a reduced state to an oxidized state under the action of catalase, and an externally applied low negative voltage is generated under the action of excitation potential, so that the electronic mediator is changed from the oxidized state back to the reduced state and simultaneously generates a reduction current signal, and the transfer rate of electrons is proportional to the concentration of urea nitrogen based on the transfer rate of electrons, so that the concentration of urea nitrogen in a sample to be detected can be obtained, and the aim of accurately detecting the content of urea nitrogen in a whole blood sample within 2-10 seconds can be realized.
To achieve the purpose, the invention adopts the following technical scheme:
the invention aims to provide a reaction enzyme solution for electrochemical urea nitrogen detection, which comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I, glutamate oxidase, peroxidase and an electron mediator; the mass concentration of the urease in the reaction enzyme liquid is 4-6%, and the mass ratio of the urease, the glutamate dehydrogenase, the alpha-ketoglutaric acid and the reduced coenzyme I in the reaction enzyme liquid is 1 (0.5-1): 1-1.5): 0.5-1.
The mass concentration of urease in the reaction enzyme solution is 4-6%, for example, 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, 5.8% or 6%, and the mass ratio of urease, glutamate dehydrogenase, α -ketoglutarate to reduced coenzyme i in the reaction enzyme solution is 1 (0.5-1.5): 0.5-1, for example, 1:0.5:1:0.5, 1:0.55:0.5, 1:0.50:1:0.5, 1:0.6:1:0.5, 1:0.65:1:0.5, 1:0.75:1.2:0.5, 1:0.8:1.5:0.5, 1:1.0:1.0:0, etc.
As a preferable technical scheme of the invention, the mass concentration of urease in the reaction enzyme solution is 5%. With better test sensitivity at this mass concentration.
According to the preferred technical scheme of the invention, the mass concentration of the glutamate oxidase in the reaction enzyme solution is 4-10%, for example, 4%, 4.5%, 5%, 7%, 8%, 8.2%, 8.5%, 8.8%, 9.0%, 9.2%, 9.5%, 9.9% or 10%, etc., and the mass ratio of the glutamate oxidase, the peroxidase and the electron mediator in the reaction enzyme solution is 1 (0.4-1): 0.4-1, for example, 1:0.4:0.4, 1:0.5:0.4, 1:0.4:0.5, 1:0.6:0.4, 1:0.4:0.6, 1:0.6:1, 1:0.6:0.7, 1:0.6:0.5 or 1:1:1, etc.
As a preferable technical scheme of the invention, the mass ratio of the glutamate oxidase to the urease in the reaction enzyme solution is (1.5-2.5): 1, for example, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1, etc.
The invention preferably controls the proportion of each component in the range, and the detection accuracy is higher
As a preferred embodiment of the present invention, the electron mediator is a water-soluble reduced electron mediator including potassium ferrocyanide.
As a preferred embodiment of the present invention, the solvent of the reaction enzyme solution includes any one or a combination of at least two of Tris-HCl buffer, phosphate buffer, ACES buffer, sodium acetate buffer, MES buffer, good's buffer, glycine buffer, and the pH of the solvent is controlled to 7.4-9.0, for example, 7.4, 7.5, 7.8, 8.0, 8.2, 8.4, 8.5, 8.7 or 9.0, etc., but the present invention is not limited to the above-mentioned values, and other non-mentioned values within the above-mentioned range are applicable.
The solvent of the reaction enzyme solution is preferably Tris-HCl buffer solution.
As a preferable technical scheme of the invention, the reaction enzyme solution also comprises a thickening agent, a protective agent and a surfactant; the mass ratio of the urease to the thickener to the protective agent in the reaction enzyme liquid is 1 (0.1-2) (0.5-1), and can be 1:0.2:0.5, 1:2:0.5, 1:1.5:1, 1:0.1:1, and the like.
The thickener includes any one or a combination of at least two of methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, sodium starch phosphate, propylene glycol alginate, or carboxymethyl cellulose, wherein typical but non-limiting combinations are combinations of cellulose and methyl cellulose, combinations of hydroxypropyl methyl cellulose and methyl cellulose, combinations of cellulose and hydroxypropyl methyl cellulose, combinations of propylene glycol alginate and methyl cellulose, and the like.
The protective agent includes any one or a combination of at least two of sucrose, lactose, mannitol, bovine serum albumin, gelatin or trehalose, wherein typical but non-limiting combinations are combinations of sucrose and lactose, combinations of sucrose and mannitol, combinations of mannitol and lactose, combinations of bovine serum albumin and lactose, and combinations of trehalose and lactose.
The surfactant includes any one or a combination of at least two of Triton X-100, tween or sodium dodecyl sulfate, wherein typical but non-limiting combinations are Triton X-100 and Tween combinations, triton X-100 and sodium dodecyl sulfate combinations, and sodium dodecyl sulfate and Tween combinations.
It should be noted that the thickener, the protectant and the surfactant according to the present invention are collectively referred to as non-reactive materials, and all three have different effects even if they do not participate in the reaction: the thickener can enable the components in the reaction enzyme liquid to be uniformly distributed, especially in the drying process of preparing the test paper for electrochemical urea nitrogen detection, and the thickener can enable the enzyme layer to be more uniform in the covering and drying processes; the protective agent can protect the activity of the enzyme, enhance the stability of the enzyme, and especially in the drying process of preparing the test paper for electrochemical urea nitrogen detection, the protective agent can protect the activity of the enzyme for a longer time under the drying condition, so that the stability of the test paper for electrochemical urea nitrogen detection can be stored for a long time is enhanced; the surfactant has the functions of reducing the surface tension of liquid, increasing the contact area between a sample and a reaction zone and promoting the reaction.
The second object of the present invention is to provide a test strip for electrochemical urea nitrogen detection, wherein the test strip is the reactive enzyme solution of one object of the present invention.
As a preferable technical scheme of the invention, the test paper for electrochemical urea nitrogen detection comprises an insulating substrate, an electrode layer and a hydrophilic film layer, wherein the electrode layer and the hydrophilic film layer are positioned on the insulating substrate; one end of the hydrophilic film layer is provided with a reagent window; the hydrophilic film layer is adhered to one side of the electrode layer, so that one end part of the electrode layer is exposed along the direction away from the reagent window; a reaction area is arranged on the electrode layer at a position corresponding to the reagent window;
wherein, the reaction area is provided with a reaction enzyme solution and a sample to be tested.
The reaction principle of the test paper for electrochemical urea nitrogen detection provided by the invention is as follows:
（i）
Figure SMS_1
（ii）
Figure SMS_2
（iii）
Figure SMS_3
（iv）
Figure SMS_4
the test paper for electrochemical urea nitrogen detection provided by the invention is based on the test paper for electrochemical urea nitrogen detection, and can enable a sample to be tested to pass through a reaction zone under the siphon action, and specifically comprises the following components: urea in a sample to be detected firstly generates glutamic acid under the action of urease and glutamate dehydrogenase in a reaction zone, so that the glutamic acid generates hydrogen peroxide under the action of glutamate oxidase, and the hydrogen peroxide participates in reaction (iv) under the action of catalase, namely, a reduced electron mediator is changed into an oxidation state from a reduction state, and an externally applied low negative voltage is generated under the action of excitation potential, so that the electron mediator is changed back into the reduction state from the oxidation state and simultaneously generates a reduction current signal, and the urea nitrogen concentration in the sample to be detected can be obtained based on the fact that the transfer rate of electrons is proportional to the urea nitrogen concentration. The test paper for electrochemical urea nitrogen detection provided by the invention belongs to a current type sensor, has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like, can realize the aim of accurately detecting the urea nitrogen content in a whole blood sample within 2-10 s, and can provide a quick and accurate detection test paper with high sensitivity and convenience in use for household and medical people in the process of screening kidney injury hidden danger.
In a preferred embodiment of the present invention, the insulating substrate is made of a polymer material, and the polymer material includes any one of a PET sheet, a PVC sheet, and a PP sheet, and the thickness of the insulating substrate is 1 to 1.5mm, for example, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, or 1.5mm, but the present invention is not limited to the above-mentioned values, and other values not shown in the above-mentioned numerical ranges are equally applicable.
The electrode layer is a metal thin layer or printing carbon paste attached to the insulating substrate; the metal material corresponding to the metal thin layer comprises any one or an alloy formed by at least two of gold, platinum, palladium, nickel or titanium, the thickness of the metal thin layer is 5-20 nm, for example, 5nm, 8nm, 10nm, 11nm, 13nm, 15nm, 16nm, 18nm or 20nm, etc., but the metal thin layer is not limited to the listed values, and other non-listed values in the above numerical range are equally applicable.
As a preferable technical scheme of the invention, the volume ratio of the sample to be detected to the reaction enzyme solution is 1 (1-4), for example, 1:1.0, 1:1.2, 1:1.5, 1:1.8, 1:2.0, 1:2.2, 1:2.5, 1:2.8, 1:3.0, 1:3.2, 1:3.5, 1:3.8 or 1:4 can be adopted.
As a preferable technical scheme of the invention, the reaction zone comprises a first reaction zone and a second reaction zone, wherein the first reaction zone is arranged at the front end of the reagent window, and the second reaction zone is arranged at the rear end of the reagent window and is in contact with the second reaction zone;
as a preferable technical scheme of the invention, the first reaction zone is provided with urease, glutamate dehydrogenase, alpha-ketoglutarate and reduced coenzyme I in a reaction enzyme solution;
as a preferable technical scheme of the invention, the second reaction zone is provided with glutamate oxidase, peroxidase and an electron mediator in a reaction enzyme solution;
as a preferable technical scheme of the invention, the sample to be detected is dripped into the first reaction zone and is infiltrated and extended to the second reaction zone.
It is worth to say that the electrode layer is a metal thin layer or printing carbon paste attached to the insulating substrate, and the electrode with the same function is realized through screen mask sputtering/electroplating or laser etching; in addition, the electrode layer is preferably a pure gold electrode layer prepared by using a screen mask sputtering process, has strong conductivity, can well promote electron transfer between an insulating substrate and a hydrophilic film layer, further accelerates the electron conduction rate between an electrode and an electroactive substance in the electrode layer, can make a current signal stronger, shortens the measurement time and improves the sensitivity.
The electrode layer comprises a working electrode, a reference electrode, a liquid flowing in-place electrode and a connecting electrode; the invention does not make specific requirements on the positions of the electrodes, and the person skilled in the art can select according to actual conditions on the basis of meeting the reaction principle of an electrochemical urea nitrogen detection method.
The invention further aims to provide a device system for electrochemical urea nitrogen detection, which comprises the test paper for electrochemical urea nitrogen detection.
As a preferred technical solution of the present invention, the device system further includes: the mixing component is used for mixing the sample to be detected and the reaction enzyme solution to obtain a reaction solution; the test paper for detecting the electrochemical urea nitrogen receives the reaction liquid in the mixing component to generate excitation potential and generate electrochemical reaction;
the device system further comprises a measuring component which is connected with the test paper for detecting the electrochemical urea nitrogen and measures the electrochemical reaction current value;
and a calculating and displaying part for calculating and displaying the urea nitrogen content in the sample to be measured according to the urea nitrogen concentration-current value standard curve.
The device system for electrochemical urea nitrogen detection firstly sequentially generates glutamic acid from urea in a sample to be detected under the action of urease and glutamate dehydrogenase, the glutamic acid generates hydrogen peroxide under the action of glutamate oxidase, the hydrogen peroxide participates in reaction (iv) under the action of catalase, namely, a reduced electronic mediator is changed into an oxidation state, and an externally applied low negative voltage is generated under the action of excitation potential, so that the electronic mediator is changed into the reduction state from the oxidation state and simultaneously generates a reduction current signal, and the transfer rate based on electrons is proportional to the concentration of urea nitrogen, thereby obtaining the concentration of urea nitrogen in the sample to be detected. The test paper for electrochemical urea nitrogen detection provided by the invention belongs to current type test paper, has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like, and can realize the aim of accurately detecting the urea nitrogen content in a whole blood sample within 2-10 seconds.
As a preferred embodiment of the present invention, the excitation potential is-50 to-300 mV, for example, -50mV, -80mV, -100mV, -120mV, -150mV, -180mV, -200mV, -230mV, -250mV, -270mV or-300 mV, etc., but the excitation potential is not limited to the values listed, and other values not listed in the above-mentioned value ranges are equally applicable.
The preparation method of the test paper for electrochemical urea nitrogen detection comprises the following steps of:
(a) Taking an insulating substrate, and then adopting modes such as screen mask sputtering/electroplating or laser etching and the like to attach a metal thin layer or print carbon paste on the insulating substrate as an electrode layer, wherein the electrode layer comprises a working electrode, a reference electrode, a flowing liquid in-place electrode and a connecting electrode;
(b) Covering a layer of reaction enzyme liquid on the position of the reaction area on the electrode layer in a liquid-dropping mode, controlling the load to be 1-2mg, and drying for 20-120 min at 45-55 ℃;
(c) After the drying operation is finished, a hydrophilic film layer with a reagent window at one end is covered on the electrode layer by using double faced adhesive tape or glue, so that a reaction area at the reagent window and the hydrophilic film layer form a siphon pool capable of sucking a sample to be detected together, and finally, after lamination and cutting, the obtained test paper for electrochemical urea nitrogen detection exists in a sealed plastic cylinder with a molecular sieve drying agent.
The application method of the test paper for electrochemical urea nitrogen detection comprises the following steps of:
the method comprises the steps of inserting a part of exposed electrode of test paper for electrochemical urea nitrogen detection into a socket of detection equipment matched with the electrode, dripping a sample to be detected from an inlet of a siphon pool of the test paper for electrochemical urea nitrogen detection, flowing into a reaction area through siphoning, enabling urea in the sample to be detected to generate glutamic acid under the action of urease and glutamate dehydrogenase in the reaction area, enabling the glutamic acid to generate hydrogen peroxide under the action of glutamate oxidase, enabling the hydrogen peroxide to participate in reaction (iv) under the action of catalase, namely, changing a reduced electronic mediator from a reduced state to an oxidized state, generating an externally applied low negative voltage under the action of excitation potential, enabling the electronic mediator to be changed from the oxidized state back to the reduced state and simultaneously generating a reduction current signal, transmitting the reduction current signal to a measuring part through electrode conduction on a working electrode and a reference electrode, enabling the current to be proportional to the concentration of urea nitrogen based on the transfer rate of electrons, and obtaining urea nitrogen content in the sample by calculating and displaying the part matched with the urea nitrogen content; in the use process of the test paper for electrochemical urea nitrogen detection, the test can be completed by only relying on a blood taking needle and a matched tester, and a matched large-scale full-automatic biochemical analyzer is not needed.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) According to the test paper for electrochemical urea nitrogen detection, urea in a sample to be detected firstly sequentially generates glutamic acid under the action of urease and glutamate dehydrogenase, the glutamic acid generates hydrogen peroxide under the action of glutamate oxidase, the hydrogen peroxide changes a reduced electronic mediator from a reduced state to an oxidized state under the action of catalase, and externally applied low negative voltage is generated under the action of excitation potential, so that the electronic mediator is changed from the oxidized state back to the reduced state and simultaneously generates a reduction current signal, the urea nitrogen concentration in the sample to be detected can be obtained based on the fact that the transfer rate of electrons is proportional to the urea nitrogen concentration, and the aim of detecting urea nitrogen by utilizing the principle of electrochemical current type for the first time is fulfilled;
(2) The device system for detecting the urea nitrogen by electrochemistry belongs to a current type sensor, has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like, can realize the aim of accurately detecting the urea nitrogen content in a whole blood sample within 2-10 s, and can provide a detection test paper which is quick, accurate, high in sensitivity and convenient to use for household and medical people in the process of screening kidney injury hidden danger;
(3) The test paper for electrochemical urea nitrogen detection has the characteristics of good correlation, wider detection linear range, higher detection speed, simplicity in operation, no need of large-scale instruments and small required sample size, the square of the correlation coefficient is larger than 0.99, the current value CV is smaller than 5%, and the lowest concentration can be tested to 1.71mmol/L.
Drawings
FIG. 1 is a schematic diagram of a test strip for electrochemical urea nitrogen detection according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of a reagent window of a test strip for electrochemical urea nitrogen detection according to an embodiment of the present invention.
In the figure: 1-a working electrode; 2-a reference electrode; a 3-reaction zone; 4-a detection electrode; 5-switching on the electrode; 6-reagent window; 7-hydrophilic film layer.
Detailed Description
The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings.
For a better illustration of the present invention, which is convenient for understanding the technical solution of the present invention, exemplary but non-limiting examples of the present invention are as follows:
as a specific embodiment of the invention, a test paper for electrochemical urea nitrogen detection is provided, and a schematic diagram of the test paper is shown in fig. 1-2, wherein the test paper for electrochemical urea nitrogen detection comprises an insulating substrate, an electrode layer and a hydrophilic film layer 7, wherein the electrode layer and the hydrophilic film layer are positioned on the insulating substrate; one end of the hydrophilic film layer 7 is provided with a reagent window 6; the hydrophilic film layer 7 is adhered to one side of the electrode layer, so that one end part of the electrode layer is exposed along the direction away from the reagent window 6; a reaction zone 3 is arranged on the electrode layer at a position corresponding to the reagent window 6;
wherein, the reaction zone 3 is provided with a reaction enzyme solution and a sample to be tested; the reaction enzyme solution comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I, glutamate oxidase, peroxidase and an electron mediator.
The electrode layer at least comprises a working electrode 1 and a reference electrode 2; besides the working electrode 1 and the reference electrode 2, the sample in-place detection electrode 4, the switch-on electrode 5 and the like can be designed according to the requirements; the invention does not require specific positions of the electrodes.
The following is an explanation of specific examples.
Example 1 preparation of test paper for electrochemical urea Nitrogen detection
The preparation method of the test paper for electrochemical urea nitrogen detection in the embodiment comprises the following steps:
preparing a reaction enzyme solution: firstly, preparing Tris-HCl buffer solution with the concentration of 0.2mol/L and the pH value of 8.0 as a solvent; weighing 1.5% of trehalose, 2% of bovine serum albumin, 3% of hydroxyethyl cellulose, 3-1001% of Triton X and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the Tris-HCl buffer solution is completely dissolved; finally weighing 5% of urease, 5% of glutamate dehydrogenase, 6% of alpha-ketoglutarate sodium, 3% of reduced coenzyme I, 1.5% of trehalose, 2% of bovine serum albumin, 3% of hydroxyethyl cellulose and 3% of TritonX-1001% according to the mass percentage, and mixing until the components are completely dissolved; finally weighing 5% of glutamate oxidase, 5% of peroxidase and 5% of potassium ferrocyanide serving as an electron mediator according to the mass percentage, mixing and stirring for 20min until the mixture is completely dissolved, and obtaining a reaction enzyme solution.
Preparation of a metal electrode layer: and (3) taking the PET sheet as an insulating substrate of a high polymer material, then sputtering a pure gold layer with the thickness of 20nm on the insulating substrate in vacuum, and engraving electrode systems such as a working electrode, a reference electrode, a flowing liquid in-place electrode, a connecting electrode and the like on the pure gold layer through a laser engraving technology.
Preparation of test paper for electrochemical urea nitrogen detection: and covering a layer of reaction enzyme liquid on the obtained metal electrode layer in a liquid-dropping mode at the position corresponding to the reaction zone, controlling the load to be 5-6mg, and carrying out heat treatment and drying for 60min at a drying channel section of 45 ℃. After drying, a hydrophilic film layer with a reagent window at one end is covered on the electrode layer by double faced adhesive tape or glue, so that a reaction area at the reagent window and the hydrophilic film layer between the reagent window form a siphon pool capable of sucking a sample to be tested, and finally, after lamination and cutting, the obtained test paper for electrochemical urea nitrogen detection exists in a sealed plastic cylinder with a molecular sieve drying agent.
The embodiment also provides a device system for electrochemical urea nitrogen detection, the device system includes the test paper for electrochemical urea nitrogen detection, the device system further includes:
the mixing component is used for mixing the sample to be detected and the reaction enzyme solution to obtain a reaction solution; the test paper for detecting the electrochemical urea nitrogen receives the reaction liquid in the mixing component to generate excitation potential and generate electrochemical reaction;
the device system further comprises a measuring component which is connected with the test paper for detecting the electrochemical urea nitrogen and measures the electrochemical reaction current value;
and a calculating and displaying part for calculating and displaying the urea nitrogen content in the sample to be measured according to the urea nitrogen concentration-current value standard curve.
Example 2 preparation of test paper for electrochemical urea Nitrogen detection
The preparation method of the test paper for electrochemical urea nitrogen detection in this example was the same as in example 1, except that glutamate dehydrogenase was 10% and glutamate oxidase was 10%.
Example 3 preparation of test paper for electrochemical urea Nitrogen detection
The preparation method of the test paper for electrochemical urea nitrogen detection in this example is the same as in example 1 except that glutamate oxidase is 10%.
Example 4 preparation of test paper for electrochemical urea Nitrogen detection
The preparation method of the test paper for electrochemical urea nitrogen detection in this example was the same as in example 1 except that glutamate dehydrogenase was 10%.
Example 5 preparation of test paper for electrochemical urea Nitrogen detection
The preparation method of the test paper for electrochemical urea nitrogen detection in this example was the same as in example 1, except that glutamate dehydrogenase was 6% and glutamate oxidase was 8%.
Example 6 preparation of test paper for electrochemical urea Nitrogen detection
Preparing a reaction enzyme solution: firstly, preparing Tris-HCl buffer solution with the concentration of 0.2mol/L and the pH value of 8.0 as a solvent; weighing 1.5% of trehalose, 2% of bovine serum albumin, 3% of hydroxyethyl cellulose, 3-1001% of Triton X and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the Tris-HCl buffer solution is completely dissolved; finally weighing 5% of urease, 5% of glutamate dehydrogenase, 6% of alpha-ketoglutarate sodium, 4% of reduced coenzyme I, 1.5% of trehalose, 2% of mannitol, 3% of sodium starch phosphate and 0.8% of tween according to the mass percentage, and mixing until the materials are completely dissolved; finally weighing 5% of glutamate oxidase, 5% of peroxidase and 5% of potassium ferrocyanide serving as an electron mediator according to the mass percentage, mixing and stirring for 25min until the mixture is completely dissolved, and obtaining a reaction enzyme solution.
Preparation of a metal electrode layer: and (3) taking the PET sheet as an insulating substrate of a high polymer material, then sputtering a pure gold layer with the thickness of 20nm on the insulating substrate in vacuum, and engraving electrode systems such as a working electrode, a reference electrode, a flowing liquid in-place electrode, a connecting electrode and the like on the pure gold layer through a laser engraving technology.
Preparation of test paper for electrochemical urea nitrogen detection: and covering a layer of reaction enzyme liquid on the obtained metal electrode layer in a liquid-dropping mode at the position corresponding to the reaction zone, controlling the load to be 5-6mg, and carrying out heat treatment and drying for 70min at a drying channel section of 45 ℃. After drying, a hydrophilic film layer with a reagent window at one end is covered on the electrode layer by double faced adhesive tape or glue, so that a reaction area at the reagent window and the hydrophilic film layer between the reagent window form a siphon pool capable of sucking a sample to be tested, and finally, after lamination and cutting, the obtained test paper for electrochemical urea nitrogen detection exists in a sealed plastic cylinder with a molecular sieve drying agent.
Example 7 preparation of test paper for electrochemical urea Nitrogen detection
Preparing a reaction enzyme solution: firstly, preparing Tris-HCl buffer solution with the concentration of 0.2mol/L and the pH value of 8.0 as a solvent; weighing 1.5% of trehalose, 2% of bovine serum albumin, 3% of hydroxyethyl cellulose, 3-1001% of Triton X and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the Tris-HCl buffer solution is completely dissolved; finally weighing 5% of urease, 5% of glutamate dehydrogenase, 6% of alpha-ketoglutarate sodium, 4% of reduced coenzyme I, 1.5% of sucrose, 2% of gelatin, 3% of sodium starch phosphate and 1% of sodium dodecyl sulfate according to the mass percentage, and mixing until the components are completely dissolved; finally, weighing 6% of glutamate oxidase, 5% of peroxidase and 5% of potassium ferrocyanide serving as an electron mediator according to the mass percentage, mixing and stirring for 25min until the mixture is completely dissolved, and obtaining a reaction enzyme solution.
Preparation of a metal electrode layer: and (3) taking the PET sheet as an insulating substrate of a high polymer material, then sputtering a pure gold layer with the thickness of 20nm on the insulating substrate in vacuum, and engraving electrode systems such as a working electrode, a reference electrode, a flowing liquid in-place electrode, a connecting electrode and the like on the pure gold layer through a laser engraving technology.
Preparation of test paper for electrochemical urea nitrogen detection: and covering a layer of reaction enzyme liquid on the obtained metal electrode layer in a liquid-dropping mode at the position corresponding to the reaction zone, controlling the load to be 5-6mg, and carrying out heat treatment and drying for 55min at a drying channel section of 45 ℃. After drying, a hydrophilic film layer with a reagent window at one end is covered on the electrode layer by double faced adhesive tape or glue, so that a reaction area at the reagent window and the hydrophilic film layer between the reagent window form a siphon pool capable of sucking a sample to be tested, and finally, after lamination and cutting, the obtained test paper for electrochemical urea nitrogen detection exists in a sealed plastic cylinder with a molecular sieve drying agent.
Example 8 preparation of test paper for electrochemical urea Nitrogen detection
In this example, the mass ratio of the remaining substances was kept constant, except that the mass concentration of urease in the reaction enzyme solution was 6%, and the same as in example 1 was repeated.
Test methods referring to the test methods in examples 1 to 7, the results showed that the sensitivity in example 8 was decreased, the average value of the current values at different concentrations was changed from 88 to 520, and the accuracy of the test was decreased compared with example 1.
Example 9 preparation of test paper for electrochemical Urea Nitrogen detection and device System thereof
This example differs from example 1 in that two reaction zones are provided, specifically as follows:
preparing a reaction enzyme solution: preparing Tris-HCl buffer solution with the concentration of 0.2mol/L and the pH of 8.0 as a solvent; weighing 1.5% of trehalose, 2% of bovine serum albumin, 3% of hydroxyethyl cellulose, 3% of TritonX-1001% of the rest of the Tris-HCl buffer solution obtained above according to mass percentage, mixing until the Tris-HCl buffer solution is completely dissolved, and dividing the mixed solution into two parts, namely a first solution and a second solution;
preparing a reaction enzyme solution of a first reaction zone: weighing 5% of urease, 5% of glutamate dehydrogenase, 6% of alpha-ketoglutarate sodium and 3% of reduced coenzyme I according to the mass percentage, mixing the materials into a first solution, stirring the first solution for 20 minutes until the first solution is completely dissolved, and obtaining a reaction enzyme solution of a first reaction zone;
preparing a reaction enzyme solution in the second reaction zone: weighing 5% of glutamate oxidase, 5% of peroxidase and 5% of potassium ferrocyanide serving as an electron mediator according to the mass percentage, mixing the mixture into a second solution, and stirring the second solution for 20min until the second solution is completely dissolved to obtain a reaction enzyme solution of a second reaction zone.
Preparation of a metal electrode layer: and (3) taking the PET sheet as an insulating substrate of a high polymer material, then sputtering a pure gold layer with the thickness of 20nm on the insulating substrate in vacuum, and engraving electrode systems such as a working electrode, a reference electrode, a flowing liquid in-place electrode, a connecting electrode and the like on the pure gold layer through a laser engraving technology.
Preparation of test paper for electrochemical urea nitrogen detection: and covering a layer of the reactive enzyme liquid of the first reaction zone on the obtained metal electrode layer in a liquid-dropping mode corresponding to the position of the first reaction zone, covering a layer of the reactive enzyme liquid of the second reaction zone in a liquid-dropping mode corresponding to the position of the second reaction zone, controlling the total load to be 5-6mg, and carrying out heat treatment and drying for 60min at a drying channel section of 45 ℃. After drying, a hydrophilic film layer with a reagent window at one end is covered on the electrode layer by double faced adhesive tape or glue, so that a reaction area at the reagent window and the hydrophilic film layer between the reagent window form a siphon pool capable of sucking a sample to be tested, and finally, after lamination and cutting, the obtained test paper for electrochemical urea nitrogen detection exists in a sealed plastic cylinder with a molecular sieve drying agent.
The embodiment also provides a device system for electrochemical urea nitrogen detection, the device system includes the test paper for electrochemical urea nitrogen detection, the device system further includes:
the mixing component is used for mixing the sample to be detected and the reaction enzyme solution to obtain a reaction solution; the test paper for detecting the electrochemical urea nitrogen receives the reaction liquid in the mixing component to generate excitation potential and generate electrochemical reaction;
the device system further comprises a measuring component which is connected with the test paper for detecting the electrochemical urea nitrogen and measures the electrochemical reaction current value;
and a calculating and displaying part for calculating and displaying the urea nitrogen content in the sample to be measured according to the urea nitrogen concentration-current value standard curve.
In this example, the square of the correlation coefficient of the trend line is 0.999, and the current value CV is less than 2%, so that the variable number of the reaction in a single reaction is reduced, and the repeatability is further improved compared with that of example 1. It is thus shown that the invention preferably divides into two reaction zones, further improving the reproducibility of the test.
The operation method of the device system for electrochemical urea nitrogen detection according to embodiments 1 to 7 includes the following steps:
the method comprises the steps of inserting a part of exposed electrode of test paper for electrochemical urea nitrogen detection into a socket of matched detection equipment, dripping a sample to be detected from an inlet of a siphon pool of the test paper for electrochemical urea nitrogen detection, flowing into a reaction area through siphoning, enabling urea in the sample to be detected to generate glutamic acid under the action of urease and glutamate dehydrogenase in the reaction area, then generating hydrogen peroxide under the action of glutamate oxidase in the reaction area, enabling the hydrogen peroxide to change a reduced electronic mediator from a reduced state to an oxidized state under the action of catalase, generating an externally applied low negative voltage under the action of excitation potential-150 mV, enabling the electronic mediator to be changed back to the reduced state from the oxidized state and simultaneously generating a reduction current signal, transmitting current to a measuring part through electrode conduction on a working electrode and a reference electrode, and obtaining urea nitrogen content in the sample to be detected through a matched calculation display part based on the transfer rate of electrons.
The following performance tests are now carried out according to the operating method of the device system for electrochemical urea nitrogen detection described above:
experiment (one) Urea Nitrogen concentration-Current value Standard curve
The venous whole blood of a normal person is used, urea with different amounts is added to prepare 9 urea whole blood samples with different concentrations, and the concentrations measured by a large-scale full-automatic biochemical analyzer are respectively as follows: 1.71mmol/L, 3.62mmol/L, 5.93 mmol/L, 8.01mmol/L, 10.5mmol/L, 16.1mmol/L, 25.8mmol/L, 30.6mmol/L, 39.2mmol/L; and then according to the using method of the electrochemical urea nitrogen detection test paper, controlling excitation potential to be-150 mV, respectively testing current responses to urea whole blood samples with 9 different concentrations, taking current values when the reaction time is 10s, testing each urea whole blood sample in parallel for 10 times, and taking an average value, so as to judge repeatability, relative sensitivity and correlation with the biochemical analyzer test concentration, wherein the results are shown in the accompanying tables 1-5.
TABLE 1
Figure SMS_5
TABLE 2
Figure SMS_6
TABLE 3 Table 3
Figure SMS_7
TABLE 4 Table 4
Figure SMS_8
TABLE 5
Figure SMS_9
From tables 1-5 and it can be seen that each example was tested to a minimum concentration of 1.71mmol/L by measuring urea whole blood samples at different concentrations, fitting a trend line with a correlation coefficient squared of greater than 0.99, and a current value CV of less than 5% each.
Experiment (II) test 40 hospital collected urea whole blood samples at different concentrations
Collecting 40 urea whole blood samples with different concentrations in the kidney department of a hospital, and recording the concentration measured by a large-scale full-automatic biochemical analyzer as a biochemical analyzer urea value; and then testing 40 whole blood samples by using urea nitrogen test paper, recording the urea value tested by the test paper, simultaneously comparing the urea value of the test paper with the urea value of a biochemical analyzer, and calculating to obtain relative deviation, wherein the statistical related data of the examples 1-5 are shown in tables 6-7.
TABLE 6
Figure SMS_10
TABLE 7
Figure SMS_11
As can be seen from tables 6-7, the deviation of the test is small and no false positive phenomenon appears in the example 1, while samples with deviations exceeding 10% from the biochemical analyzer appear in the tests in the examples 2, 4 and 5, and the false positive phenomenon appears in the tests of the samples No. 17 and No. 18 in the example 3, and according to the results, the invention has better test results by controlling the proportion of each component in the reaction enzyme solution within a specific range.
Comparative example 1
The comparative example was conducted in the same manner as in example 1 except that the mass concentration of urease in the reaction enzyme solution was 3%.
The test method is shown in examples 1-7, and the result shows that the concentration of urease in comparative example 1 is insufficient, the reaction signal is weak, the sample current value with the concentration of the urea above 25.8mmol/L is basically unchanged, namely the high-concentration urea detection data is unreliable, and the test accuracy is far lower than that of example 1.
Comparative example 2
The comparative example was conducted in the same manner as in example 1 except that the mass concentration of urease in the reaction enzyme solution was 10%.
Test methods referring to the test methods in examples 1-7, the results show that the square of the correlation coefficient of the overall trend line in comparative example 1 is only 0.72, and that a higher concentration of urease adversely affects the reaction, resulting in lower accuracy of the test than in example 1.
When the mass percentages of the alpha-ketoglutarate and the reduced coenzyme I are adjusted to be too high or too low, the detection accuracy and the detection range are also reduced, and the details are not repeated.
Comparative example 3
In this comparative example, peroxidase was not added, and the other components were the same as in example 1.
The test method is shown in examples 1-7, and the result shows that the reaction speed of the test of comparative example 3 is very slow, more than 15 minutes are needed for response, the square of the correlation coefficient of the trend line is only 0.89, and the test accuracy is far lower than that of example 1.
It can be seen from comprehensive examples 1 and comparative examples 1-3 that the reaction enzyme solution for electrochemical urea nitrogen detection provided by the invention can obtain better test accuracy and sensitivity through controlling the mass concentration of urease and the proportion of each component, and the mass composition and the mutual proportion of each component are critical to the accuracy of the final test.
In conclusion, the test paper for electrochemical urea nitrogen detection belongs to a current type sensor, has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like, and can realize the aim of accurately detecting the urea nitrogen content in a whole blood sample within 2-10 seconds.
The detailed structural features of the present invention are described in the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope of the present invention and the scope of the disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims (10)

1. The reaction enzyme solution for electrochemical urea nitrogen detection is characterized by comprising urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I, glutamate oxidase, peroxidase and an electron mediator;
the mass concentration of the urease in the reaction enzyme liquid is 4-6%, and the mass ratio of the urease, the glutamate dehydrogenase, the alpha-ketoglutaric acid and the reduced coenzyme I in the reaction enzyme liquid is 1 (0.5-1): 1-1.5): 0.5-1.
2. The reaction enzyme solution for electrochemical urea nitrogen detection according to claim 1, wherein the mass concentration of urease in the reaction enzyme solution is 5%.
3. The reaction enzyme solution for electrochemical urea nitrogen detection according to claim 1, wherein the mass concentration of glutamate oxidase in the reaction enzyme solution is 4-10%.
4. The reaction enzyme solution for electrochemical urea nitrogen detection according to claim 1, wherein the mass ratio of glutamate oxidase, peroxidase and electron mediator in the reaction enzyme solution is 1 (0.4-1): 0.4-1;
the mass ratio of the glutamate oxidase to the urease in the reaction enzyme liquid is (1.5-2.5): 1.
5. The reactive enzyme solution for electrochemical urea nitrogen detection according to claim 1, further comprising a thickener, a protectant and a surfactant;
the mass ratio of the urease to the thickener to the protective agent to the surfactant is 1 (0.1-2): 0.5-1.
6. The test paper for electrochemical urea nitrogen detection is characterized by comprising the reactive enzyme solution for electrochemical urea nitrogen detection according to any one of claims 1-5.
7. The test strip for electrochemical urea nitrogen detection according to claim 6, wherein the test strip comprises an insulating substrate, an electrode layer and a hydrophilic film layer on the insulating substrate; one end of the hydrophilic film layer is provided with a reagent window; the hydrophilic film layer is adhered to one side of the electrode layer, so that one end part of the electrode layer is exposed along the direction away from the reagent window; a reaction area is arranged on the electrode layer at a position corresponding to the reagent window; wherein the reaction zone is provided with the reaction enzyme solution and a sample to be tested;
the volume ratio of the sample to be detected to the reaction enzyme solution is 1 (1-4).
8. The test paper for electrochemical urea nitrogen detection according to claim 7, wherein the insulating substrate is made of a high polymer material, the high polymer material comprises any one of a PET sheet, a PVC sheet or a PP sheet, and the thickness of the insulating substrate is 1-1.5mm;
the electrode layer is a metal thin layer or printing carbon paste attached to the insulating substrate; the metal material corresponding to the metal thin layer comprises any one or alloy formed by at least two of gold, platinum, palladium, nickel or titanium, and the thickness of the metal thin layer is 5-20 nm.
9. The test strip for electrochemical urea nitrogen detection according to claim 7, wherein the reaction zone comprises a first reaction zone and a second reaction zone, the first reaction zone is disposed at the front end of the reagent window, and the second reaction zone is disposed at the rear end of the reagent window and in contact with the second reaction zone;
the first reaction zone is provided with urease, glutamate dehydrogenase, alpha-ketoglutarate and reduced coenzyme I in a reaction enzyme solution;
the second reaction zone is provided with glutamate oxidase, peroxidase and an electron mediator in a reaction enzyme solution;
the sample to be measured is dripped into the first reaction zone and is infiltrated and extended to the second reaction zone.
10. An electrochemical urea nitrogen detection device system, characterized in that the device system comprises the electrochemical urea nitrogen detection test paper according to any one of claims 7-9.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0135092A2 (en) * 1983-08-12 1985-03-27 Yamasa Shoyu Kabushiki Kaisha Method for determination of ammonia
CN102928468A (en) * 2012-09-14 2013-02-13 北京体育大学 Whole blood urea biosensing test paper
CN113957121A (en) * 2021-10-11 2022-01-21 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) Urea determination kit
CN114235925A (en) * 2021-12-17 2022-03-25 南京晶捷生物科技有限公司 Anti-interference electrochemical uric acid test paper and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0135092A2 (en) * 1983-08-12 1985-03-27 Yamasa Shoyu Kabushiki Kaisha Method for determination of ammonia
CN102928468A (en) * 2012-09-14 2013-02-13 北京体育大学 Whole blood urea biosensing test paper
CN113957121A (en) * 2021-10-11 2022-01-21 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) Urea determination kit
CN114235925A (en) * 2021-12-17 2022-03-25 南京晶捷生物科技有限公司 Anti-interference electrochemical uric acid test paper and preparation method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
冯德荣,李智红,周万里,朱思荣,史建国: "尿素氮-葡萄糖双功能分析仪的研究", 生物工程学报, vol. 12, no. 02, pages 5 - 13 *
宋耀虹, 李学仁, 马德均, 林其燧: "酶偶联法尿素氮测定最适条件的探讨", 生物化学与生物物理进展, no. 03 *
缪慧,尹立志,张国蓉,邢克礼: "过氧化氢生物传感器的研制", 分析化学, no. 06, pages 734 *

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