CN116083523A - Reactive enzyme liquid, test paper and device system for electrochemical urea nitrogen detection - Google Patents

Abstract

The invention relates to a reactive enzyme solution, test paper and a device system for electrochemical urea nitrogen detection, wherein the reactive enzyme solution comprises a first reactive enzyme solution and a second reactive enzyme solution; the first reaction enzyme solution comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I and beta-hydroxybutyric acid; the second reaction enzyme solution comprises D-3-hydroxybutyrate dehydrogenase, diaphorase, aniline and ruthenium hexammoniate. The device system is directly proportional to the urea nitrogen concentration based on the electron transfer rate, so that the urea nitrogen concentration in the sample to be detected can be obtained. The invention has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like.

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 instruments, in particular to a urea nitrogen detection method, and especially relates to a reactive enzyme solution, test paper and device system for electrochemical urea nitrogen detection.

Background

Urea is one of the end products of protein metabolism in the body. Deamination of amino acids in humans produces ammonia and carbon dioxide, both of which can synthesize urea in the liver, approximately 0.3g per gram of protein is metabolized to urea, which is excreted from urine primarily by glomerular filtration. The normal adult fasting urea nitrogen content (BUN) is 3.2-7.1 mmol/L (9-20 mg/dL). Renal diseases such as advanced renal disease, renal failure, acute glomerulonephritis, chronic pyelonephritis, and toxic nephritis, which affect glomerular filtration, all cause an increase in serum urea nitrogen content. Many extra-renal factors may also cause elevated serum urea nitrogen, and BUN concentrations up to 21.4mmol/L (60 mg/dL) are one of the diagnostic indicators of uremia after the exclusion of extra-renal factors.

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 ammoniumRoot and carbon dioxide. Sodium nitrosoferricyanide catalyzed NH under alkaline conditions₄⁺ 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 CuSO₄ 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, the main methods for detecting urea nitrogen by a large-scale biochemical analyzer are a chemiluminescence method, a photochemistry colorimetric method and an electrochemical method. The chemiluminescence method cannot test whole blood, because red blood cells can influence the test result, the blood consumption is large, the cost is high, the test process is complex, and the test time is long. The photochemical colorimetric method needs a large sample size, has long detection time, is easily influenced by reduction interfering substances, and consumes glutamate dehydrogenase and NADH at the same time under the condition of high blood ammonia content, so that the detection result is inaccurate. The electrochemical method has the advantages of stable amplification of current signals, high sensitivity, short detection time, simple and convenient operation, easy mass production and low cost. However, most of the electrodes in the conventional electrochemical method are of a potential type, but the current type performance of the urea sensor is better than that of the potential type due to the existence of interference, and although the current type urea sensor has been developed, no prior art has proposed direct detection by the current method.

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, the design and development of the test strip which is simple and convenient to operate, small and portable, small in blood consumption, suitable for fingertip blood, low in price, free from endogenous substance interference and capable of accurately measuring urea nitrogen concentration has important significance.

Disclosure of Invention

In view of the problems existing in the prior art, the invention provides a reactive enzyme solution, test paper and a device system for electrochemical urea nitrogen detection, wherein the reactive enzyme solution is divided into a first reactive enzyme solution and a second reactive enzyme solution, so that urea in a sample to be detected can generate oxidized coenzyme I under the action of urease and glutamate dehydrogenase during detection, then the oxidized coenzyme I generates reduced coenzyme I under the action of D-3-hydroxybutyrate dehydrogenase, further oxidized ruthenium hexammoniate chloride is reduced under the action of diaphorase, and an excitation potential is applied to obtain a current signal.

To achieve the purpose, the invention adopts the following technical scheme:

the invention aims to provide a reactive enzyme solution for electrochemical urea nitrogen detection, which comprises a first reactive enzyme solution and a second reactive enzyme solution; the first reaction enzyme solution comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I and beta-hydroxybutyric acid; the second reaction enzyme solution comprises D-3-hydroxybutyrate dehydrogenase, diaphorase, aniline and ruthenium hexammoniate.

According to the invention, the reaction enzyme solution is divided into the first reaction enzyme solution and the second reaction enzyme solution, so that the serial reaction can be applied to the electrochemical detection of urea, and in the detection process, a sample to be detected firstly reacts under the action of substances in the first reaction enzyme solution, namely urea generates oxidized coenzyme I under the action of urease and glutamate dehydrogenase; and then the oxidized coenzyme I is generated under the action of D-3-hydroxybutyrate dehydrogenase in the second reaction enzyme solution to generate reduced coenzyme I, further under the action of diaphorase, oxidized ruthenium hexammoniate chloride is reduced, the excitation potential is applied to obtain current signal excitation potential, and the transfer rate of electrons is proportional to the concentration of urea nitrogen, so that the concentration of urea nitrogen in a sample to be detected can be obtained. Aniline can act as a bridge, allowing reduction of oxidized ruthenium hexammoniate chloride.

Compared with the traditional reaction enzyme solution, the urea nitrogen is detected by adopting an oxidation method, the first reaction enzyme solution and the second reaction enzyme solution are arranged for the two reaction areas, the sensitivity of the two-stage reaction is obviously improved, and compared with the process of mixing all substances together, the two-stage reaction is not affected each other, so that the detection accuracy is improved; in addition, the reaction enzyme solution provided by the invention has the advantages that the reaction raw materials do not contain substances such as glucose and the like in application, and the negative influence of the existence of glucose in a human body on the detection of urea nitrogen can be effectively avoided.

The reactive enzyme liquid provided by the invention has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like after being applied to urea nitrogen detection.

As a preferable technical scheme of the invention, the mass concentration of the urease in the first reaction enzyme solution is 4-6%, and the mass ratio of the urease, the glutamate dehydrogenase, the alpha-ketoglutarate, the reduced coenzyme I and the beta-hydroxybutyric acid is 1 (0.5-1.5): 0.1-0.5): 0.1-0.6): 0.1-0.3.

Compared with the traditional test paper which only controls the loading amount of the urease on the test paper, the invention strictly controls the mass concentration of the urease in the reaction enzyme liquid, is favorable for improving the uniformity of the loading amount of the urease on the subsequent test paper, and greatly improves the detection sensitivity; meanwhile, the proportion of each component in the first reaction enzyme solution is further preferably controlled, and compared with the traditional reaction enzyme solution in which a large amount of alpha-ketoglutaric acid, reduced coenzyme I and other substances are required to be added, the method disclosed by the invention has the advantages that the quantity of the alpha-ketoglutaric acid and the reduced coenzyme I is controlled to be smaller than that of urease, a better detection effect is obtained, and a system taking the urease as a dominant component in the reaction enzyme solution is formed, so that the system has a more sensitive response effect with urea in a sample to be tested after the system contacts the sample to be tested.

Wherein the mass concentration of urease in the first reaction enzyme solution is 4-6%, for example, 4%, 4.2%, 4.5%, 4.8%, 5.0%, 5.2%, 5.5%, 5.8% or 6%, etc.

The mass ratio of the urease, the glutamate dehydrogenase, the alpha-ketoglutarate, the reduced coenzyme I and the beta-hydroxybutyric acid is 1 (0.5-1.5) (0.1-0.5) (0.2-0.6) (0.1-0.3), wherein the numerical value of the glutamate dehydrogenase in the mass ratio can be, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5; the value of α -ketoglutarate may be, for example, 0.1, 0.2, 0.3, 0.4, or 0.5, etc.; the value of reduced coenzyme I may be, for example, 0.2, 0.3, 0.4, 0.5 or 0.6; the value of β -hydroxybutyrate may be, for example, 0.1, 0.2 or 0.3.

As a preferable technical scheme of the invention, the mass concentration of the D-3-hydroxybutyrate dehydrogenase in the second reaction enzyme solution is 2-4%, and the mass ratio of the D-3-hydroxybutyrate dehydrogenase, the diaphorase, the aniline and the ruthenium hexammoniate chloride in the second reaction enzyme solution is 1 (0.3-0.5): 0.1-0.2): 0.3-0.5.

The invention further preferably has the mass concentration of the D-3-hydroxybutyrate dehydrogenase in the second reaction enzyme solution of 2-4%, and similar to the mass concentration control of the urease in the first reaction enzyme solution, the invention controls the mass concentration of the D-3-hydroxybutyrate dehydrogenase in the reaction enzyme solution, so that the D-3-hydroxybutyrate dehydrogenase on the subsequent test paper is distributed more uniformly, reacted more sensitively and detected more accurately. Meanwhile, the mass ratio of each component in the second reaction enzyme solution is controlled, so that the detection speed is high, the sensitivity is high, and the accuracy is better.

As a preferred embodiment of the present invention, the solvent of the first reaction enzyme solution and the solvent of the second reaction enzyme solution each independently include any one or a combination of at least two of Tris buffer, phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, ACES buffer, sodium acetate buffer, MES buffer, good's buffer, and glycine buffer, and the pH of the solvent is controlled to be 5 to 9, and may be, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, or 9.

As a preferable technical scheme of the invention, the first reaction enzyme solution and the second reaction enzyme solution respectively and independently further comprise a thickening agent, a protective agent and a surfactant; the mass ratio of the urease, the thickening agent and the protective agent in the first reaction enzyme solution is 1 (0.5-1.5) (0.1-2), for example, 1:0.6:0.1, 1:0.8:0.1, 1:1.0:0.1, 1:0.5:0.5, 1:0.8:0.5, 1:0.9:1, 1:0.5:1.5, 1:0.7:2 and the like; the mass ratio of the D-3-hydroxybutyrate dehydrogenase, the thickener and the protective agent in the second reaction enzyme solution is 1 (0.5-1.5) (0.1-2), and can be 1:0.6:0.1, 1:0.8:0.1, 1:1.0:0.1, 1:0.5:0.5, 1:0.8:0.5, 1:0.9:1, 1:0.5:1.5, 1:0.7:2 or the like.

Illustratively, the thickener is any one or a combination of at least two of methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, sodium starch phosphate, propylene glycol alginate, or carboxymethyl cellulose, wherein typical but non-limiting combinations are combinations of methylcellulose and hydroxypropyl methylcellulose, combinations of hydroxyethyl cellulose and hydroxypropyl methylcellulose, combinations of sodium starch phosphate and hydroxypropyl methylcellulose, and combinations of methylcellulose and sodium starch phosphate.

Illustratively, 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.

Illustratively, 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 thickening agent can enable the components in the first reaction enzyme liquid and the second reaction enzyme liquid to be uniformly distributed, especially in the drying process of preparing the electrochemical urea nitrogen test paper, and the thickening agent 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 electrochemical urea nitrogen test paper, the protective agent can protect the activity of the enzyme for a longer time under the drying condition, so that the stability of the electrochemical urea nitrogen test paper capable of being stored for a long time is enhanced; the surfactant has the functions of reducing interfacial tension between solid and liquid, increasing adsorption between the solid and the liquid, and enabling the enzyme layer to be uniformly dispersed on the surface of the electrode.

The second purpose of the invention is to provide a test paper for electrochemical urea nitrogen detection, wherein the reaction area of the test paper for electrochemical urea nitrogen detection is formed by adding the reaction enzyme solution for electrochemical urea nitrogen detection according to one of the purposes of the invention and drying.

The test paper for electrochemical urea nitrogen detection provided by the invention has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like because the test paper is formed by adopting the reaction enzyme solution of the first purpose.

Specifically, the reaction of the test strip of the second object of the present invention at the time of detection is as follows:

（i）

（ii）

（iii）

（iv）

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 first reaction zone and a second reaction zone are arranged on the electrode layer at positions corresponding to the reagent window, and the first reaction zone and the second reaction zone are arranged front and back and are adjacent or connected front and back along the direction away from the reagent window;

Wherein the first reaction zone is formed by dripping the first reaction enzyme solution and drying, and comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I and beta-hydroxybutyric acid; the second reaction zone is formed by dropwise adding the second reaction enzyme solution and drying, and comprises D-3-hydroxybutyrate dehydrogenase, diaphorase, aniline and ruthenium hexammoniate.

The test paper for electrochemical urea nitrogen detection can enable a sample to be tested to sequentially pass through a first reaction zone and a second reaction zone under the siphon action, and specifically comprises the following steps: urea in a sample to be detected firstly generates oxidized coenzyme I under the action of urease and glutamate dehydrogenase in a first reaction zone, then based on the fact that the first reaction zone and a second reaction zone are both water-soluble, the sample after reaction from the first reaction zone easily flows from the first reaction zone to the second reaction zone, oxidized coenzyme I generates reduced coenzyme I under the action of D-3-hydroxybutyrate dehydrogenase, further under the action of diaphorase, oxidized ruthenium hexammoniate chloride is reduced, excitation potential is applied to obtain a current signal, and the transfer rate of electrons is proportional to the concentration of urea nitrogen, so that the current value corresponding to the sample to be detected can be detected by utilizing electrodes in an electrode layer, and the concentration of urea nitrogen in the sample to be detected is calculated. The electrochemical urea nitrogen test paper belongs to a current type sensor and has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding endogenous substance interference and the like.

The reaction is preferably carried out in two reaction areas, so that the mutual interference between the two reaction areas can be avoided, and if the two reaction areas are combined into one reaction area, the reaction cannot be carried out completely, because the first reaction enzyme solution and the second reaction enzyme solution are respectively in a reduction reaction and an oxidation reaction, and after the two reaction areas are combined into one reaction area, reactants between the two reaction areas can react with each other, so that the detection cannot be carried out.

As a preferable technical scheme of the invention, the level of the second reaction zone is lower than that of the first reaction zone.

The invention further preferably has the level of the second reaction zone lower than that of the first reaction zone, so that the sample to be measured can be smoothly infiltrated and extended into the second reaction zone to carry out the tandem reaction after being added.

The insulating substrate is made of a polymer material, the polymer material comprises any one of PET sheets, PVC sheets or PP sheets, and the thickness of the insulating substrate is 1-1.5mm, for example, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm or 1.5mm, etc.

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 1-50nm, for example, 1nm, 2nm, 3nm, 4nm, 5nm, 10nm, 12nm, 15nm, 18nm, 20nm, 22nm, 25nm, 30nm, 35nm, 40nm, 45nm or 50nm and the like.

The electrode layer comprises a working electrode, a reference electrode, a liquid flowing in-place electrode and a connecting electrode.

It is worth to describe 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.

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 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 first reaction enzyme liquid on the position of the first reaction zone on the electrode layer in a liquid dropping mode, controlling the load to be 1-2mg, and drying at 40-60 ℃ for 20-120min; similarly, covering a layer of second reaction enzyme liquid on the position of the second reaction zone on the electrode layer in a liquid dropping mode, controlling the load to be 1-2mg, and drying at 40-60 ℃ for 20-120min;

(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 siphon pool capable of sucking a sample to be detected is formed by the first reaction area and the second reaction area at the reagent window and the hydrophilic film layer between the first reaction area and the second reaction area, and finally, after lamination and cutting, the obtained electrochemical urea nitrogen test paper is stored 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 an electrochemical urea nitrogen test paper into a socket of a detection device matched with the electrode, dripping a sample to be tested from an inlet of a siphon pool of the electrochemical urea nitrogen test paper, enabling urea in the sample to be tested to firstly flow into a first reaction zone through siphoning, generating oxidized coenzyme I under the action of urease and glutamate dehydrogenase in the first reaction zone, then flowing into a second reaction zone, enabling the oxidized coenzyme I to generate reduced coenzyme I under the action of D-3-hydroxybutyrate dehydrogenase, further reducing oxidized ruthenium hexamine chloride under the action of diaphorase, applying an excitation potential to the detection device matched with the detection device, enabling the excitation potential to react with enzyme liquid electrochemically to generate a current value, and obtaining urea nitrogen content in the sample to be tested through conversion of software in the detection device matched with the detection device.

It is a further object of the present invention to provide a device system for electrochemical urea nitrogen detection, said device system comprising: the second purpose of the invention is the test paper for electrochemical urea nitrogen detection; a measuring unit connected to the test paper for electrochemical urea nitrogen detection and measuring the electrochemical reaction current value; and a calculation display 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 test paper for electrochemical urea nitrogen detection is used for receiving a sample to be detected and generating electrochemical reaction in a reaction zone under the action of excitation potential to form an electric signal.

The device system provided by the invention can efficiently realize the detection of urea nitrogen, and has the advantages of small blood sampling amount, high detection speed, high sensitivity, good accuracy, capability of avoiding the interference of endogenous substances and the like.

As a preferable technical scheme of the invention, the volume ratio of the sample to be detected to the first reaction enzyme solution is 1 (1-3), for example, 1:1, 1:1.2, 1:1.3, 1:1.5, 1:1.8, 1:2.0, 1:2.2, 1:2.5, 1:2.8 or 1:3.0, etc.

Preferably, the excitation potential is 100-500mV, which may be, for example, 100mV, 105mV, 110mV, 150mV, 180mV, 200mV, 250mV, 300mV, 350mV, 400mV, 500mV, or the like.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) According to the invention, the specific substances are selected and the mass concentration and the mass ratio of the specific substances are prioritized, so that the detection deduction of the interferents is realized through a multienzyme and mediator system, the reagent cost is lower, and the reaction sensitivity is high;

(2) 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, and can obtain a test result only by 3-5s, thereby achieving the purpose of instant detection;

(3) The electrochemical urea nitrogen device system has simple reaction, is directly proportional to the urea nitrogen concentration based on the transfer rate of electrons, can obtain the urea nitrogen concentration in a sample to be detected, realizes the aim of detecting the urea nitrogen by utilizing the principle of an electrochemical current type, and is beneficial to industrialized popularization and application.

Drawings

Fig. 1 is a schematic structural exploded view of the test paper for electrochemical urea nitrogen detection provided in application example 1 of the present invention.

Fig. 2 is a graph showing a linear relationship between current and concentration using oxidation current as a detection signal in application example 1 of the present invention.

In the figure: 1-an insulating substrate; 2-electrode layers; 3-a hydrophilic membrane layer; 4-siphoning holes; 5-switching on the electrode; 6-working electrode; 7-a reference electrode; 8-flowing liquid in-place electrode; 9-a first reaction zone; 10-a second reaction zone.

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:

example 1 preparation method of reactive enzyme solution for electrochemical urea Nitrogen detection

The embodiment provides a reactive enzyme solution for electrochemical urea nitrogen detection, and the preparation method of the reactive enzyme solution comprises the following steps:

preparing a first reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; then weighing 2% of trehalose, 4% of TritonX-1001% of hydroxyethyl cellulose, 1% of sodium chloride and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the mixture is completely dissolved; finally weighing 5% of urease, 3% of glutamate dehydrogenase, 0.5% of alpha-ketoglutarate, 0.5% of reduced coenzyme I and 1% of beta-hydroxybutyric acid according to mass percentage, mixing and stirring for 20min until the solution is completely dissolved, and obtaining a first reaction enzyme solution.

Preparing a second reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; then weighing 2% of trehalose, 4% of TritonX-1001% of hydroxyethyl cellulose, 1% of sodium chloride and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the mixture is completely dissolved; and finally, weighing 3% of D-3-hydroxybutyrate dehydrogenase, 1% of diaphorase, 0.5% of aniline and 1% of ruthenium hexammoniate chloride according to the mass percentage, mixing and stirring for 20min until the mixture is completely dissolved, and obtaining a second reaction enzyme solution.

Example 2 preparation method of reactive enzyme solution for electrochemical urea Nitrogen detection

The embodiment provides a reactive enzyme solution for electrochemical urea nitrogen detection, and the preparation method of the reactive enzyme solution comprises the following steps:

preparing a first reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; then weighing 2% of trehalose, 4% of TritonX-1001% of hydroxyethyl cellulose, 1% of sodium chloride and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the mixture is completely dissolved; finally, weighing 4% of urease, 2% of glutamate dehydrogenase, 0.4% of alpha-ketoglutarate, 0.8% of reduced coenzyme I and 0.4% of beta-hydroxybutyric acid according to mass percentage, mixing and stirring for 10min until the components are completely dissolved, and obtaining a first reaction enzyme solution.

Preparing a second reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; then weighing 2% of trehalose, 4% of TritonX-1001% of hydroxyethyl cellulose, 1% of sodium chloride and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the mixture is completely dissolved; and finally, weighing 4% of D-3-hydroxybutyrate dehydrogenase, 2% of diaphorase, 0.4% of aniline and 1.2% of hexammoniate ruthenium chloride according to the mass percentage, mixing and stirring for 10min until the mixture is completely dissolved, and obtaining a second reaction enzyme solution.

Example 3 preparation method of reactive enzyme solution for electrochemical urea Nitrogen detection

The embodiment provides a reactive enzyme solution for electrochemical urea nitrogen detection, and the preparation method of the reactive enzyme solution comprises the following steps:

preparing a first reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; then weighing 2% of trehalose, 4% of TritonX-1001% of hydroxyethyl cellulose, 1% of sodium chloride and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the mixture is completely dissolved; finally, weighing 4.5 mass percent of urease, 2.5 mass percent of glutamate dehydrogenase, 1 mass percent of alpha-ketoglutarate, 2.5 mass percent of reduced coenzyme I and 0.35 mass percent of beta-hydroxybutyric acid, mixing and stirring for 15 minutes until the urease, the glutamate dehydrogenase and the alpha-ketoglutarate are completely dissolved, and obtaining a first reaction enzyme solution.

Preparing a second reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; then weighing 2% of trehalose, 4% of TritonX-1001% of hydroxyethyl cellulose, 1% of sodium chloride and the balance of the Tris-HCl buffer solution obtained above according to mass percentage, and mixing until the mixture is completely dissolved; and finally, weighing 2.5% of D-3-hydroxybutyrate dehydrogenase, 1% of diaphorase, 0.25% of aniline and 0.75% of ruthenium hexammoniate chloride according to the mass percentage, mixing and stirring for 5min until the mixture is completely dissolved, and obtaining a second reaction enzyme solution.

Example 4 preparation method of reactive enzyme solution for electrochemical urea Nitrogen detection

The embodiment provides a reactive enzyme solution for electrochemical urea nitrogen detection, and the preparation method of the reactive enzyme solution comprises the following steps:

preparing a first reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.5 as a solvent; then weighing 1.8% of trehalose, 3.8% of TritonX-1001.2% of hydroxyethyl cellulose, 1.5% of sodium chloride 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, 1 percent of urease, 1 percent of glutamate dehydrogenase, 0.3 percent of alpha-ketoglutarate, 0.25 percent of reduced coenzyme I and 0.2 percent of beta-hydroxybutyric acid are weighed according to the mass percent, and are mixed and stirred for 5 minutes until the solution is completely dissolved, so as to obtain a first reaction enzyme solution.

Preparing a second reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; weighing 1.8% of trehalose, 4.3% of TritonX-1001.5% of hydroxyethyl cellulose, 1.2% of sodium chloride 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; and finally, weighing 3.5% of D-3-hydroxybutyrate dehydrogenase, 1.1% of diaphorase, 0.4% of aniline and 4% of ruthenium hexammine chloride according to the mass percentage, mixing and stirring for 5min until the mixture is completely dissolved, and obtaining a second reaction enzyme solution.

Example 5 preparation method of reactive enzyme solution for electrochemical urea Nitrogen detection

The embodiment provides a reactive enzyme solution for electrochemical urea nitrogen detection, and the preparation method of the reactive enzyme solution comprises the following steps:

preparing a first reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; weighing 2.5% of trehalose, 3.5% of TritonX-1001.5% of hydroxyethyl cellulose, 1.5% of sodium chloride 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 6% of urease, 3% of glutamate dehydrogenase, 2.5% of alpha-ketoglutarate, 1.5% of reduced coenzyme I and 0.7% of beta-hydroxybutyric acid according to mass percent, mixing and stirring for 15min until the components are completely dissolved, and obtaining a first reaction enzyme solution.

Preparing a second reaction enzyme solution: firstly, preparing Tris buffer solution with the concentration of 0.1mol/L and the pH of 7.4 as a solvent; weighing 2.5% of trehalose, 3.5% of TritonX-1001.5% of hydroxyethyl cellulose, 1.5% of sodium chloride 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; and finally, weighing 4% of D-3-hydroxybutyrate dehydrogenase, 1.5% of diaphorase, 0.8% of aniline and 10% of ruthenium hexammoniate according to the mass percentage, mixing and stirring for 20min until the mixture is completely dissolved, and obtaining a second reaction enzyme solution.

Example 6 preparation method of reactive enzyme solution for electrochemical Urea Nitrogen detection

This example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of urease in the first reaction enzyme solution is 8%, and the mass ratio of other substances to urease is kept unchanged.

Example 7 preparation method of reactive enzyme solution for electrochemical Urea Nitrogen detection

This example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of urease in the first reaction enzyme solution is 3%, and the mass ratio of other substances to urease is kept unchanged.

Example 8 preparation method of reactive enzyme solution for electrochemical Urea Nitrogen detection

The present example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of α -ketoglutaric acid in the first reaction enzyme solution is 5%, so that the mass ratio of urease to α -ketoglutaric acid is 1:1, and the mass concentrations of other substances are kept unchanged.

Example 9 preparation method of reactive enzyme solution for electrochemical urea Nitrogen detection

The present example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of α -ketoglutaric acid in the first reaction enzyme solution is 0.1%, so that the mass ratio of urease to α -ketoglutaric acid is 1:0.02, and the mass concentrations of other substances are kept unchanged.

Example 10 preparation method of reactive enzyme solution for electrochemical Urea Nitrogen detection

This example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of reduced coenzyme I in the first reaction enzyme solution is 0.3%, the mass ratio of urease to reduced coenzyme I is 1:0.06, and the mass concentrations of other substances are kept unchanged.

Example 11 preparation method of reactive enzyme solution for electrochemical Urea Nitrogen detection

This example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of reduced coenzyme I in the first reaction enzyme solution is 6%, the mass ratio of urease to reduced coenzyme I is 1:1.2, and the mass concentrations of other substances are kept unchanged.

Example 12 preparation method of reactive enzyme solution for electrochemical Urea Nitrogen detection

This example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of D-3-hydroxybutyrate dehydrogenase in the second reaction enzyme solution is 6%, and the mass ratio of other substances to D-3-hydroxybutyrate dehydrogenase is kept unchanged.

Example 13 preparation method of reactive enzyme solution for electrochemical Urea Nitrogen detection

This example provides a reaction enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the mass concentration of D-3-hydroxybutyrate dehydrogenase in the second reaction enzyme solution is 1%, and the mass ratio of other substances to D-3-hydroxybutyrate dehydrogenase is kept unchanged.

Comparative example 1

This comparative example provides a reactive enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1, except that the first reactive enzyme solution and the second reactive enzyme solution are mixed together.

In this comparative example, the first reaction enzyme solution and the second reaction enzyme solution are mixed together, so that substances in the two reaction enzyme solutions are mutually influenced, and the electrochemical urea nitrogen test cannot be performed.

Comparative example 2

This comparative example provides a reactive enzyme solution for electrochemical urea nitrogen detection, which is the same as example 1 except that the ruthenium hexammoniate chloride in the second reactive enzyme solution is replaced by potassium ferricyanide.

Application example 1

The application example provides a test paper for electrochemical urea nitrogen detection, as shown in fig. 1, the test paper for electrochemical urea nitrogen detection comprises an insulatingsubstrate 1, anelectrode layer 2 and ahydrophilic film layer 3, wherein theelectrode layer 2 and thehydrophilic film layer 3 are positioned on the insulatingsubstrate 1; one end of thehydrophilic membrane layer 3 is provided with a reagent window; thehydrophilic film layer 3 is adhered to one side of theelectrode layer 2, so that one end part of theelectrode layer 2 is exposed along the direction away from the reagent window; a first reaction zone 9 and asecond reaction zone 10 are arranged on theelectrode layer 2 at positions corresponding to the reagent window, and the first reaction zone 9 and thesecond reaction zone 10 are arranged front and back along the direction away from the reagent window and are adjacent or connected front and back;

wherein the first reaction zone 9 is formed by dropping the first reaction enzyme solution in example 1 and drying, and the first reaction zone 9 comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I and beta-hydroxybutyric acid; thesecond reaction zone 10 was formed by dropping the second reaction enzyme solution described in example 1 and drying, and thesecond reaction zone 10 included D-3-hydroxybutyrate dehydrogenase, diaphorase, aniline and ruthenium hexammoniate.

Theelectrode layer 2 comprises a workingelectrode 6, areference electrode 7, a liquid flowing in-place electrode 8 and a connectingelectrode 5.

And the hydrophilic film layer is provided with siphonholes 4.

Wherein the preparation of the metal electrode layer comprises the following steps: 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 electrochemical urea nitrogen test paper: and (3) covering a layer of first reaction enzyme liquid on the obtained metal electrode layer in a liquid-dropping mode corresponding to the position of the first reaction zone, controlling the load amount to be 1-2mg, covering a layer of second reaction enzyme liquid in a liquid-dropping mode corresponding to the position of the second reaction zone, controlling the load amount to be 1-2mg, and performing heat treatment and drying for 30min at a drying channel section at 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 siphon pool capable of sucking a sample to be tested is formed by the first reaction zone and the second reaction zone at the reagent window and the hydrophilic film layer between the first reaction zone and the second reaction zone, and finally, after lamination and cutting, the obtained electrochemical urea nitrogen test paper is stored in a sealed plastic cylinder with a molecular sieve drying agent.

The application example also provides a device system for electrochemical urea nitrogen detection, the device system comprises: the test paper for electrochemical urea nitrogen detection is described in the application example; a measuring unit connected to the test paper for electrochemical urea nitrogen detection and measuring the electrochemical reaction current value; and a calculation display 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 test paper for electrochemical urea nitrogen detection is used for receiving a sample to be detected and generating electrochemical reaction in a reaction zone under the action of excitation potential to form an electric signal.

Application examples 2 to 13

Application examples 2 to 13 provide test paper for electrochemical urea nitrogen detection, and the rest are the same as application example 1 except that the reaction enzyme solution is replaced with the first reaction enzyme solution and the second reaction enzyme solution in examples 2 to 13, respectively. The preparation method is described in application example 1.

Comparative example 1 was used

Application comparative example 1 provides an electrochemical urea nitrogen test strip having only one reaction zone and the reaction zone is covered with a layer of the reaction enzyme solution of comparative example 1, the rest being the same as in application example 1. The preparation method is described in application example 1.

Comparative example 2 was used

Application comparative example 2 provides an electrochemical urea nitrogen test strip, which is identical to application example 1 except that the reaction enzyme solutions are replaced with the first reaction enzyme solution and the second reaction enzyme solution in comparative example 2, respectively. The preparation method is described in application example 1.

The application method of the test paper for electrochemical urea nitrogen detection comprises the following steps:

the method comprises the steps of inserting a part of exposed electrode of the electrochemical urea nitrogen test paper into a socket of detection equipment matched with the electrode, dripping a sample to be tested from an inlet of a siphon pool of the electrochemical urea nitrogen test paper, enabling urea in the sample to be tested to flow into a first reaction area through siphoning, enabling urea in the sample to generate oxidized coenzyme I under the action of urease and glutamate dehydrogenase in the first reaction area, enabling the urea to flow into a second reaction area, enabling excitation voltage to generate electrochemical reaction with enzyme liquid and ruthenium hexammine chloride to generate a current value under the action of excitation potential 200mV, and obtaining urea nitrogen content in the sample to be tested through conversion of software in detection equipment matched with the excitation voltage.

According to the application method of the electrochemical urea nitrogen test paper, the test paper in the application example and the application comparative example is subjected to the following performance test:

(one) obtaining a 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 theoretical concentrations are respectively: 1mmol/L, 1.5mmol/L, 2mmo/L, 2.5mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 7mmol/L, 9mmol/L; and then according to the using method of the electrochemical urea nitrogen test paper, controlling the excitation potential to be 200mV, respectively testing current responses to urea whole blood samples with 9 different concentrations, taking current values when the reaction time is 5s, testing each urea whole blood sample in parallel for 5 times, and taking an average value, thereby judging the repeatability, the relative sensitivity and the linear range. Raw data of the relevant test of application example 1 are listed in table 1, with urea concentration as abscissa and current value as ordinate, and figure 2 is obtained by fitting a trend line.

TABLE 1

As can be seen from Table 1 and FIG. 2, a calibration curve was established using the theoretical concentration of urea nitrogen and the average value of the current values obtained by the test in example 1, and the square of the correlation coefficient (R² ) 0.9979, the method has good fitting degree, can be used as a urea nitrogen concentration-current value standard curve, and the CV of the obtained current value is below 5.6% in the parallel test process of urea whole blood samples of each concentration section, and the detection response time is only 4s.

(II) specific test of 40 urea Whole blood samples at different concentrations

40 urea whole blood samples distributed at different concentrations were randomly selected for testing and then evaluated in comparison with the results of the hospital biochemical analyzer, wherein the results of the application example 1 and the linear correlation are shown in table 2. For simplicity, other application examples and comparative examples only give the results of the maximum deviation, as shown in table 2 above.

TABLE 2

For simplicity, other application examples and comparative examples only give the calculated results, as shown in table 3.

TABLE 3 Table 3

From tables 1 to 3, the following points can be found:

(1) Comprehensive application examples 1-5 show that the reactive enzyme solution for electrochemical urea nitrogen detection provided by the invention has good linear relation in the electrochemical detection process, wherein the square of the correlation coefficient of linear fitting is larger than 0.99, CV repeated 10 times of current value is within 6.1%, and the maximum deviation absolute value in 40 samples is within 15.2%;

(2) As can be seen from the comprehensive application examples 1 and 6-7, compared with the application example 1 in which the mass concentration of the urease is 5% and the application examples 6-7 in which the mass concentration of the urease is 8% and 3% respectively, the current response effect of the application examples 6-7 is obviously reduced from 617 of the application example 1 to only 492 and 443 of the difference value of the average value of the currents, and the linear correlation coefficients are reduced, which indicates that the accuracy of the test is reduced, wherein the main reason is that the higher concentration of the urease in the application example 6 causes the urease in the first reaction zone to flow into the second reaction zone so as to react with the substances in the reaction enzyme liquid of the second reaction zone, thereby affecting the samming star of the second reaction zone, and the lower concentration of the urease in the first reaction zone causes the sensitivity of the first reaction zone to be reduced, so that the invention obviously improves the sensitivity and accuracy of the detection by controlling the mass concentration of the urease in a specific range;

(3) As can be seen from the combination of application examples 1 and application examples 8 to 9, the mass concentration of α -ketoglutaric acid in application example 1 was 0.5%, and CV of the current value in application example 1 was 5.6% or less, and the square of the correlation coefficient (R² ) 0.9979, the square of the correlation coefficient of the linear fitting in application examples 8-9 is only 0.87 and 0.95, respectively, and the sensitivity of the current response is also reduced, mainly due to the higher concentration of the alpha-ketoglutaric acid in application example 8, resulting in an overall The pH value of the system is lower, the isoelectric point of the enzyme is easy to be reached, the activity of the enzyme is reduced, the concentration of the alpha-ketoglutaric acid in application example 9 is lower, and the current response is reduced, so that the invention obviously improves the sensitivity and the accuracy of detection by controlling the concentration of the alpha-ketoglutaric acid in a specific range; likewise, as can be seen from the comprehensive application examples 1 and 10-13, the addition amounts of the D-3-hydroxybutyrate dehydrogenase and the reduced coenzyme I are critical to the final detection result, and the high-efficiency detection of electrochemical urea nitrogen is realized through the synergistic combination of the components;

(4) As can be seen from the comprehensive application example 1 and the application comparative example 2, after the electron mediator is replaced, the linear correlation coefficient in the application comparative example 2 is reduced to 0.63, and the detection result of the application comparative example 2 is different from that of a hospital biochemical analyzer by as high as 38.2%, so that the detection accuracy and sensitivity are improved by selecting the hexaammine ruthenium chloride as the electron mediator to be combined with other enzymes.

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 method is characterized in that the reaction enzyme liquid for electrochemical urea nitrogen detection comprises a first reaction enzyme liquid and a second reaction enzyme liquid;

the first reaction enzyme solution comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I and beta-hydroxybutyric acid;

the second reaction enzyme solution comprises D-3-hydroxybutyrate dehydrogenase, diaphorase, aniline and ruthenium hexammoniate.

2. The reaction enzyme solution according to claim 1, wherein the mass concentration of urease in the first reaction enzyme solution is 4 to 6% and the mass ratio of urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I and beta-hydroxybutyric acid is 1 (0.5 to 1.5): (0.1 to 0.5): (0.1 to 0.6): (0.1 to 0.3).

3. The reaction enzyme solution according to claim 1, wherein the mass concentration of D-3-hydroxybutyrate dehydrogenase in the second reaction enzyme solution is 2-4% and the mass ratio of D-3-hydroxybutyrate dehydrogenase, diaphorase, aniline and ruthenium hexammoniate chloride in the second reaction enzyme solution is 1 (0.3-0.5): 0.1-0.2): 0.3-0.5.

4. The reaction enzyme solution according to claim 1, wherein the solvent of the first reaction enzyme solution and the solvent of the second reaction enzyme solution each independently comprise any one or a combination of at least two of Tris buffer, phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, ACES buffer, sodium acetate buffer, MES buffer, good's buffer, or glycine buffer, and the pH of the solvents is controlled to 5 to 9.

5. The reactive enzyme solution of claim 1, wherein the first reactive enzyme solution and the second reactive enzyme solution each independently further comprise a thickener, a protectant, and a surfactant; the mass ratio of the urease, the thickening agent and the protective agent in the first reaction enzyme solution is 1 (0.5-1.5) (0.1-2); the mass ratio of the D-3-hydroxybutyrate dehydrogenase, the thickening agent and the protective agent in the second reaction enzyme solution is 1 (0.5-1.5) to 0.1-2.

6. The test paper for electrochemical urea nitrogen detection is characterized in that a reaction zone of the test paper for electrochemical urea nitrogen detection is formed by adding the reaction enzyme solution for electrochemical urea nitrogen detection according to any one of claims 1-5 and drying.

7. The test strip for electrochemical urea nitrogen detection according to claim 6, wherein the test strip for electrochemical urea nitrogen detection 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 first reaction zone and a second reaction zone are arranged on the electrode layer at positions corresponding to the reagent window, and the first reaction zone and the second reaction zone are arranged front and back and are adjacent or connected front and back along the direction away from the reagent window;

wherein the first reaction zone is formed by dripping the first reaction enzyme solution and drying, and comprises urease, glutamate dehydrogenase, alpha-ketoglutarate, reduced coenzyme I and beta-hydroxybutyric acid; the second reaction zone is formed by dropwise adding the second reaction enzyme solution and drying, and comprises D-3-hydroxybutyrate dehydrogenase, diaphorase, aniline and ruthenium hexammoniate.

8. The test strip of claim 7, wherein the level of the second reaction zone is lower than the level of the first reaction zone;

the insulating substrate is made of a high polymer material, the high polymer material comprises any one of PET (polyethylene terephthalate) sheets, PVC (polyvinyl chloride) sheets or PP (polypropylene) sheets, 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 1-50nm;

the electrode layer comprises a working electrode, a reference electrode, a liquid flowing in-place electrode and a connecting electrode.

9. 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 6-8.

10. The apparatus system for electrochemical urea nitrogen detection according to claim 9, further comprising a measuring means connected to the test paper for electrochemical urea nitrogen detection and measuring the electrochemical reaction current value;

And a calculation display 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 test paper for electrochemical urea nitrogen detection is used for receiving a sample to be detected and generating electrochemical reaction in a reaction zone under the action of excitation potential to form an electric signal.

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