A blood ammonia sensor and preparation method thereof

Abstract

The present invention discloses a blood ammonia sensor, comprising an insulating substrate layer, an electrode layer, a biosensitive layer, an insulating layer, a hydrophilic cover plate layer and a masking tape layer; wherein the electrode layer is screen-printed on the surface of the insulating substrate layer, and the electrode layer comprises a counter electrode, a working electrode and a reference electrode; the insulating layer is arranged above the biosensitive layer, the insulating layer covers the electrode layer, and a blank window is left on the counter electrode, the working electrode and the reference electrode to form a reaction tank, and a sampling opening is provided on one side of the reaction tank; the biosensitive layer covers the electrode layer and the working electrode in the reaction tank, and is fixed on the layer surface of the working electrode by film formation; the biosensitive layer is prepared by drying the prepared reaction solution at 25°C-50°C for 10-30min; the hydrophilic cover plate layer covers the insulating layer; a drainage device is provided on the hydrophilic cover plate layer; a vent is provided on the hydrophilic cover plate layer; and the masking tape layer covers the insulating layer and the hydrophilic cover plate layer. A preparation method thereof is also disclosed.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, enzyme electrodes and blood ammonia sensors, and in particular to a blood ammonia sensor and a preparation method thereof. Background Art

[0002] Blood ammonia is formed when amino acids in the body are broken down and metabolized and ammonia absorbed from the intestines enters the blood. Under normal circumstances, this ammonia combines with glutamate to form glutamine, which is then transported to the liver for further metabolism. Elevated ammonia levels, or hyperammonemia, are a potentially fatal symptom that is closely related to a variety of diseases. Diseases such as cirrhosis and neonatal urea cycle disorders can lead to the occurrence of hyperammonemia. If not treated in time, hyperammonemia may cause cognitive development disorders, epileptic seizures, other neurological problems, and even death. Therefore, it is of great significance to be able to accurately and effectively detect blood ammonia concentrations in a short period of time.

[0003] At present, the main in vitro detection methods of blood ammonia include chromatography, spectrophotometry, ion-selective electrode method, and micro-diffusion dry method technology. These methods usually have problems such as large equipment size, high price, long time required for the determination process, complex operation, high reagent cost, and large sample volume required, which are limited in the process of instant blood ammonia detection. Therefore, it is required to further shorten the determination time, simplify the operation process, and reduce the required sample volume to realize instant blood ammonia detection.

[0004] In clinical testing of blood ammonia, biochemical analyzers and blood ammonia detectors are often used to measure blood ammonia, including:

[0005] 1. Biochemical Analyzer

[0006] Measurement principle: The principle of biochemical analyzer to detect blood ammonia is mainly based on enzyme kinetics. This method uses a specific enzyme reaction to measure the concentration of blood ammonia. In this process, blood ammonia reacts with α-ketoglutarate under the catalysis of glutamate dehydrogenase, as well as β-NADPH (reduced coenzyme II) and H + With the participation of , a reaction occurs to generate glutamate. This reaction will lead to the oxidation of β-NADPH, and its absorbance at 340nm will change. This change is proportional to the concentration of blood ammonia, so the concentration of blood ammonia can be determined by measuring the change in absorbance.

[0007] Test process: The sample processing process of the biochemical analyzer to test blood ammonia mainly includes the following steps:

[0008] (1) Sample collection: Select a suitable blood collection needle and blood collection tube (usually ammonia-free blood collection tube), and collect a blood sample from the patient according to the standard blood collection procedure.

[0009] (2) Sample processing: The collected blood samples should be sent for testing immediately to avoid long-term storage that may cause changes in blood ammonia concentration. During the testing process, the samples should be kept on ice.

[0010] (3) Sample preparation: In order to avoid the release of ammonia from red blood cells causing an increase in ammonia in the sample, the sample needs to be centrifuged as quickly as possible.

[0011] Minimum sample volume: 1mL;

[0012] Test time: within 1 hour.

[0013] Disadvantages: The determination process takes a long time, the operation is complicated, the reagent cost is high, and a large amount of sample is required. If the sample is not handled properly, the ammonia concentration in the sample will increase.

[0014] 2. Blood ammonia detector:

[0015] like Figure 1 As shown, the measurement principle: using micro-diffusion dry method technology, can perform high-precision detection on whole blood

[0016] (1) Add the sample to the sample diffusion layer;

[0017] (2) NH4 in the spacer layer samples + becomes NH3;

[0018] (3) Ammonia reacts with the reagent layer, causing a color change reaction;

[0019] (4) Measure the chromaticity of reflected light.

[0020] Sample volume: 20 μL of whole blood is required, blood is collected from the ear;

[0021] Test time: about 3 minutes and 20 seconds / each test;

[0022] Disadvantages: The measurement process takes a long time, the operation is complicated, the reagent cost is high, and the reagent layer has poor anti-interference ability.

[0023] After searching the prior art, we found that:

[0024] CN109324099A discloses a voltammetric ammonium ion electrochemical biosensor and its preparation method and application. The preparation method is as follows: first, porous ferroferric oxide nano-microspheres are prepared by a solvothermal method, and then the ferroferric oxide nano-microspheres and graphene are ultrasonically dispersed in a chitosan solution for modifying a glassy carbon electrode, and then glutamate dehydrogenase is modified to obtain the sensor; the present invention also includes a voltammetric ammonium ion electrochemical biosensor prepared by the above preparation method and its application. The present invention is simple, fast, sensitive and accurate in detecting ammonium ions, and the instrument is simple, low in cost and portable. It can overcome the shortcomings of low detection limit, complex sample pretreatment, and expensive instrumentation of traditional detection methods, and has very important application value. However, it is difficult to commercialize the modified glassy carbon electrode in the technical solution; the electrode only modifies glutamate dehydrogenase, and it is necessary to add dielectric substances, coenzymes, and α-ketoglutaric acid to the electrolyte before a reaction may occur, and the sample volume is required to be several mL.

[0025] In summary, the existing technology has problems such as bulky equipment, high price, long time required for the determination process, complicated operation, high reagent cost, and large sample volume required. Therefore, it is required to further shorten the determination time, simplify the operation process, reduce the required sample volume, and realize instant blood ammonia detection. Summary of the invention

[0026] The purpose of the present invention is to overcome the defects of the prior art and provide a blood ammonia sensor and a preparation method thereof. The blood ammonia sensor belongs to a blood ammonia electrochemical sensor based on screen-printed electrodes. The measurement principle is mainly based on enzyme catalytic reaction and electrochemical detection technology. Specifically, under the catalysis of glutamate dehydrogenase, β-NADPH (reduced coenzyme II) and H + With the participation of, blood ammonia reacts with α-ketoglutarate to generate glutamate, and the electrons generated by the enzymatic reaction are captured by the dielectric substance. This process can be detected by the electrodes in the electrochemical sensor. In the blood ammonia sensor, glutamate dehydrogenase and α-ketoglutarate are fixed on the electrode surface by film formation to form a biosensitive layer. When the ammonia in the sample comes into contact with the biological activity, the enzyme catalyzes ammonia and α-ketoglutarate to generate glutamate. The generated electrons are transmitted through the electrode to the electronic receiver in the electrochemical sensor, and then converted into an electrical signal output. The electrical signal is proportional to the concentration of blood ammonia in the sample, so the concentration of blood ammonia can be quantitatively determined by measuring the size of the electrical signal.

[0027] A first aspect of the present invention provides a blood ammonia sensor, comprising an insulating substrate layer (1), an electrode layer (2), a biosensitive layer (3), an insulating layer (4), a hydrophilic cover layer (5) and a masking tape layer (6); wherein:

[0028] The electrode layer (2) is screen-printed onto the surface of the insulating substrate layer (1), and the electrode layer (2) comprises a counter electrode (11), a working electrode (12), and a reference electrode;

[0029] The insulating layer (4) is arranged above the biosensitive layer (3), the insulating layer (4) covers the electrode layer (2), and blank windows are left on the counter electrode (11), the working electrode (12) and the reference electrode to form a reaction groove (9), so that parts of the working electrode (12), the reference electrode and the counter electrode (11) are exposed in the reaction groove (9), and a sample injection opening (10) is provided on one side of the reaction groove (9);

[0030] The biosensitive layer (3) covers the electrode layer (2); the biosensitive layer (3) covers the working electrode (12) in the reaction tank and is fixed on the surface of the working electrode (12) by film formation; the biosensitive layer (3) is prepared by drying the prepared reaction solution at 25°C-50°C for 10-30 minutes;

[0031] The hydrophilic cover layer (5) covers the insulating layer (4); a drainage device is provided on the hydrophilic cover layer (5); and a ventilation hole is provided above the hydrophilic cover layer (5);

[0032] The masking tape layer (6) covers the insulating layer (4) and the hydrophilic cover plate layer (5).

[0033] Preferably, the material of the insulating substrate layer (1) is selected from any one of polyethylene terephthalate (PET), polystyrene (PVC) and polystyrene (PS).

[0034] Preferably, the materials constituting the working electrode (12) and the counter electrode (11) are both selected from at least one of graphene, carbon nanotubes, graphite carbon paste, gold, platinum and palladium; and the material of the reference electrode is a mixture of silver / silver chloride particles.

[0035] Preferably, the material of the insulating layer (4) is at least one of polypropylene and polyethylene.

[0036] Preferably, the reaction solution comprises a buffer, a biological enzyme reagent, a conductive medium and α-ketoglutaric acid; the buffer is at least one of a phosphate buffer solution with a concentration of 50-200 mmol / L and a pH of 6.5 to 7.8, a Tris-HCl buffer solution with a concentration of 100-300 mmol / L and a pH of 6-8, or a triethanolamine buffer solution with a concentration of 50-300 mmol / L and a pH of 7.2-8.3; the biological enzyme reagent comprises glutamate dehydrogenase and a coenzyme; wherein the coenzyme is at least one of β-NADPH or NADH; the content of glutamate dehydrogenase in each piece of the blood ammonia sensor is 0.5U-1.2U; the conductive medium is at least one of potassium ferrocyanide, a ruthenium compound or polyacrylamide.

[0037] Preferably, the reaction solution further comprises monosaccharides or polysaccharides as enzyme stabilizers and film-forming materials, and the monosaccharides or polysaccharides include at least one or more of chitosan oligosaccharides, chitosan, dextran, trehalose, cyclodextrin, sucrose or maltose.

[0038] Preferably, the reaction solution further comprises a surfactant, and the surfactant is at least one or more of EDTA, polyethylene glycol, SDS, PVP or Triton.

[0039] Preferably, the hydrophilic cover plate layer (5) is made of a polyester material with a hydrophilic layer attached to one side.

[0040] Preferably, the masking tape layer (6) is made of polyolefin or polyester material.

[0041] The second aspect of the present invention is to provide a method for preparing the blood ammonia sensor of the first aspect, comprising the following steps:

[0042] S1, screen-printing the electrode layer (2) on the insulating substrate layer (1) according to a preset shape and position and drying the same, thereby sequentially obtaining an electrode system consisting of the working electrode (12), the counter electrode (11) and the reference electrode;

[0043] S2, dissolving the material constituting the insulating layer (4) in a solvent, and sequentially inkjet printing the material on the electrode system obtained in S1 according to a preset shape and position, and ensuring that blank windows are left on the counter electrode (11), the working electrode (12) and the reference electrode to form a reaction tank; curing the insulating layer (4) under ultraviolet light for 5-15 seconds;

[0044] S3, in the reaction tank, sequentially solidifying the material constituting the biosensitive layer (3) onto the working electrode (12) of the electrode system obtained in S1 according to a preset shape and position and drying at 30-60° C. for 10-60 min;

[0045] S4, laying the hydrophilic cover layer (5) provided with a drainage device on top of the insulating layer (4), so that the reaction tank is formed below the hydrophilic cover layer (5) to form a siphon channel, and opening an air permeable hole on the hydrophilic cover layer (5) corresponding to the top of the siphon channel;

[0046] S5, covering the masking tape layer (6) on the insulating layer (4) and the hydrophilic cover layer (5) to protect the blood ammonia sensor.

[0047] Beneficial effects of the sensor and preparation method of the present invention:

[0048] The measurement principle of the blood ammonia sensor of the present invention is mainly based on enzyme catalysis reaction and electrochemical detection technology. Specifically, under the catalysis of glutamate dehydrogenase, β-NADPH (reduced coenzyme II) and H + With the participation of, blood ammonia reacts with α-ketoglutarate to generate glutamate, and the electrons generated by the enzymatic reaction are captured by the dielectric substance. This process can be detected by the electrode in the electrochemical sensor. In the blood ammonia sensor, glutamate dehydrogenase and α-ketoglutarate are fixed on the electrode surface by film formation to form a biosensitive layer. When the ammonia in the sample contacts the biological activity, the enzyme catalyzes ammonia and α-ketoglutarate to generate glutamate, and the generated electrons are transmitted to the electronic receiver in the electrochemical sensor through the electrode, and then converted into an electrical signal output. The electrical signal is proportional to the concentration of blood ammonia in the sample, so the concentration of blood ammonia can be quantitatively determined by measuring the size of the electrical signal. Specifically, when the tested sample reaches the reaction tank and undergoes a biochemical reaction with the bioactive substance on the electrode and generates an electrical signal, the electrical signal can be collected by the electrochemical analyzer. A linear relationship is established between the signal intensity and the blood ammonia in the blood sample to quantitatively analyze the blood ammonia content in the blood. The sensor only needs 3-5ul of fingertip blood in the in vitro detection of blood ammonia, and the sample blood ammonia test results can be obtained in 10s-15s. The sensor has the characteristics of low cost, convenient production, fast response speed, good selectivity, small sample volume, etc., which can solve the problems of large equipment volume, high price, long time required for determination, complicated operation, high reagent cost, large sample volume required, etc. in the prior art, thereby realizing the instant detection of blood ammonia. In addition, the present invention uses screen-printed electrodes, which are easy to commercialize; and all the required substances are dripped on the surface of the electrode, and the electrode can directly test the whole blood sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 A schematic diagram of the measurement principle of a blood ammonia detector provided according to the prior art;

[0051] Figure 2 The figure is a schematic diagram of the structural principle of a blood ammonia sensor provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0054] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0055] like Figure 2 As shown, the first aspect of this embodiment is to provide a blood ammonia sensor, including an insulating substrate layer 1, an electrode layer 2, a biosensitive layer 3, an insulating layer 4, a hydrophilic cover layer 5 and a masking tape layer 6; wherein,

[0056] The electrode layer 2 is screen-printed onto the surface of the insulating substrate layer 1, and the electrode layer 2 includes a counter electrode 11, a working electrode 12, and a reference electrode (not shown in the figure);

[0057] The insulating layer 4 is arranged above the biosensitive layer 3, and the insulating layer 4 covers the electrode layer 2, and blank windows are left on the counter electrode 11, the working electrode 12 and the reference electrode to form a reaction tank 9, so that part of the working electrode 12, the reference electrode and the counter electrode 11 are exposed in the reaction tank 9, and a sample injection opening 10 is provided on one side of the reaction tank 9;

[0058] The biosensitive layer 3 covers the electrode layer 2; the biosensitive layer 3 covers the working electrode 12 in the reaction tank and is fixed on the surface of the working electrode 12 by film formation;

[0059] The hydrophilic cover layer 5 covers the insulating layer 4; a drainage device is provided on the hydrophilic cover layer 5; and a vent hole is provided on the hydrophilic cover layer 5 to ensure that the sample enters the reaction tank smoothly.

[0060] The masking tape layer 6 covers the insulating layer 4 and the hydrophilic cover plate layer 5 .

[0061] As a preferred embodiment, the insulating substrate layer 1 is made of any one of polyethylene terephthalate (PET), polystyrene (PVC) and polystyrene (PS). As a more preferred embodiment, the insulating substrate layer 1 is made of polyethylene terephthalate (PET).

[0062] As a preferred embodiment, the materials constituting the working electrode 12 and the counter electrode 11 can be selected from at least one of graphene, carbon nanotubes, graphite carbon paste, gold, platinum and palladium; the material of the reference electrode is a mixture of silver / silver chloride particles.

[0063] As a preferred embodiment, the material of the insulating layer 4 is selected from at least one of polypropylene and polyethylene.

[0064] As a preferred embodiment, the biosensitive layer is prepared by drying the prepared reaction solution at 25°C-50°C for 10-30 minutes; more preferably, it is prepared by drying the prepared reaction solution at 45°C for 20 minutes; wherein: the reaction solution comprises a buffer, a biological enzyme reagent, a conductive medium and α-ketoglutaric acid; the buffer is at least one of a phosphate buffer solution with a concentration of 50-200mmol / L and a pH of 6.5 to 7.8, a Tris-HCl buffer solution with a concentration of 100-300mmol / L and a pH of 6-8, or a triethanolamine buffer solution with a concentration of 50-300mmol / L and a pH of 7.2-8.3;

[0065] The bio-enzyme reagent comprises glutamate dehydrogenase and a coenzyme; wherein the coenzyme is at least one of β-NADPH or NADH; in order to meet the detection sensitivity while controlling the cost, the content of the glutamate dehydrogenase in each sensor is preferably 0.5U-1.2U; the conductive medium is at least one of potassium ferrocyanide, a ruthenium compound or polyacrylamide.

[0066] As a preferred embodiment, the reaction solution further comprises monosaccharides or polysaccharides as enzyme stabilizers and film-forming materials, and the monosaccharides or polysaccharides include at least one or more of chitosan oligosaccharides, chitosan, dextran, trehalose, cyclodextrin, sucrose or maltose.

[0067] As a preferred embodiment, in order to improve the reaction speed and sensitivity, the reaction solution further comprises a surfactant, and the surfactant is at least one or more of EDTA, polyethylene glycol, SDS, PVP or Triton.

[0068] This embodiment is the most preferred implementation mode, using a Tris-HCl buffer solution with a concentration of 200 mmol / L and a pH of 7.3 as a buffer; using β-NADPH as a coenzyme; using potassium ferrocyanide as a conductive medium; using chitosan oligosaccharides, cyclodextrin and trehalose as carbohydrates; and using EDTA as a surfactant.

[0069] As a preferred embodiment, the hydrophilic cover layer 5 is made of a polyester material with a hydrophilic layer attached to one side.

[0070] As a preferred embodiment, the masking tape layer 6 is made of polyolefin or polyester material.

[0071] The second aspect of this embodiment is to provide a method for preparing the blood ammonia sensor of the first aspect, comprising the following steps:

[0072] S1, screen-printing the electrode layer 2 on the insulating substrate layer 1 according to a preset shape and position and drying the same to obtain an electrode system consisting of the working electrode 12, the counter electrode 11 and the reference electrode in sequence;

[0073] S2, dissolving the material constituting the insulating layer 4 in a solvent, and sequentially inkjet printing the material on the electrode system obtained in S1 according to a preset shape and position, and ensuring that blank windows are left on the counter electrode 11, the working electrode 12 and the reference electrode to form a reaction tank; curing the insulating layer 4 under ultraviolet light for 5-15 seconds;

[0074] S3, in the reaction tank, sequentially solidify the materials constituting the biosensitive layer 3 onto the working electrode 12 of the electrode system obtained in S1 according to a preset shape and position and dry at 30-60° C. for 10-60 min;

[0075] In this embodiment, the best drying effect is achieved by drying at 45° C. for 20 minutes.

[0076] S4, laying the hydrophilic cover layer 5 provided with the drainage device on the top of the insulating layer 4, so that the reaction tank is formed below the hydrophilic cover layer 5 to form a siphon channel, and opening an air hole on the hydrophilic cover layer 5 corresponding to the top of the siphon channel;

[0077] S5, covering the insulating layer 4 and the hydrophilic cover layer 5 with the masking tape layer 6 to protect the blood ammonia sensor.

[0078] Application Examples Example 1

[0079] The blood ammonia sensor structure includes: an insulating substrate layer 1, an electrode layer 2, a biosensitive layer 3, an insulating layer 4, a hydrophilic cover layer 5 and a masking tape layer 6, as shown in FIG. Figure 1 .

[0080] First, a PET substrate (50 mm × 6 mm × 250 mm in length) is prepared as the insulating substrate layer 1 of the blood ammonia sensor. The electrode layer 2 is screen-printed on the PET substrate, and the electrode layer is provided with a counter electrode 11, a working electrode 12, and a reference electrode. The printing material constituting the working electrode and the counter electrode is carbon black with a particle size of 50 nm, and the mass percentage concentration of the nano carbon black is 8%; the ink constituting the reference electrode is silver / silver chloride, and the mass percentage concentration of the silver and silver chloride is 15%.

[0081] Then, the material constituting the insulating layer 4 is dissolved in ethylene glycol and is screen-printed on the top of the electrode layer according to a preset shape and position. The insulating layer 4 is provided with a reaction tank 9, so that part of the working electrode 12, the reference electrode and the counter electrode 11 are exposed in the reaction tank, and a sampling opening 10 is provided on one side of the reaction tank 9. The material constituting the insulating layer is polystyrene with a mass percentage concentration of 1.5% in the corresponding solution, and is cured under ultraviolet light for 10-20s.

[0082] In a reaction tank controlled by an insulating layer, the bioactive material is drop-coated on the surface of the working electrode 12 according to the formula in Table 1, and dried at 45° C. for 20 minutes to form a biosensitive layer 3 .

[0083] Table 1

[0084] Substance name content Glutamate dehydrogenase 1U / Piece Sensor Potassium ferrocyanide 100mg / mL β-NADPH 2mmoL / L α-Ketoglutarate 200mmol / L Trehalose 6% Cyclodextrin 0.5% Tris-HCl (200mmol / L, pH7.3) 89% EDTA 0.1%

[0085] The hydrophilic cover plate layer 5 is pasted on the top of the insulating layer and forms a siphon channel 8 with the reaction tank. A vent hole 7 is opened on the hydrophilic cover plate 5 corresponding to the top of the siphon channel.

[0086] During the test, the sample enters the reaction tank through the siphon channel 8, and reacts on the biosensitive layer formed on the surface of the working electrode in the reaction tank to generate an electrical signal. The electrical signal can be collected by the electrochemical analyzer. A linear relationship is established between the signal intensity and the blood ammonia in the blood sample to quantitatively analyze the blood ammonia content in the blood.

[0087] Finally, the masking tape layer 6 is covered on the insulating layer 4 and the hydrophilic cover plate 5 to protect the blood ammonia sensor.

[0088] Example 2

[0089] The blood ammonia sensor of Example 1 was used to measure the blood ammonia of the sample. As the sample, venous whole blood with adjusted ammonium ion concentration (ammonium ion concentration: 10, 20, 30, 50, 100, 200, 300, 400, 500umol / L) was used. Under a 25°C environment, 5uL of the sample was supplied to the blood ammonia sensor, a voltage of 300mV was applied, and the current was recorded 10s after the spot detection.

[0090] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A blood ammonia sensor, characterized in that: It comprises an insulating substrate layer (1), an electrode layer (2), a biosensitive layer (3), an insulating layer (4), a hydrophilic cover layer (5) and a masking tape layer (6); wherein: The electrode layer (2) is screen-printed onto the surface of the insulating substrate layer (1), and the electrode layer (2) comprises a counter electrode (11), a working electrode (12), and a reference electrode; The insulating layer (4) is arranged above the biosensitive layer (3), the insulating layer (4) covers the electrode layer (2), and blank windows are left on the counter electrode (11), the working electrode (12) and the reference electrode to form a reaction groove (9), so that parts of the working electrode (12), the reference electrode and the counter electrode (11) are exposed in the reaction groove (9), and a sample injection opening (10) is provided on one side of the reaction groove (9); The biosensitive layer (3) covers the electrode layer (2); the biosensitive layer (3) covers the working electrode (12) in the reaction tank and is fixed on the surface of the working electrode (12) by film formation; the biosensitive layer (3) is prepared by drying the prepared reaction solution at 25°C-50°C for 10-30 minutes; The hydrophilic cover layer (5) covers the insulating layer (4); a drainage device is provided on the hydrophilic cover layer (5); and a ventilation hole is provided above the hydrophilic cover layer (5); The masking tape layer (6) covers the insulating layer (4) and the hydrophilic cover plate layer (5); The material of the insulating substrate layer (1) is selected from any one of polyethylene terephthalate and polystyrene; The reaction solution comprises a buffer, a biological enzyme reagent, a conductive medium and α-ketoglutaric acid; the buffer is at least one of a phosphate buffer solution with a concentration of 50-200 mmol / L and a pH of 6.5 to 7.8, a Tris-HCl buffer solution with a concentration of 100-300 mmol / L and a pH of 6-8, or a triethanolamine buffer solution with a concentration of 50-300 mmol / L and a pH of 7.2-8.3; the biological enzyme reagent comprises glutamate dehydrogenase and a coenzyme; wherein the coenzyme is at least one of β-NADPH or NADH; the content of glutamate dehydrogenase in each piece of the blood ammonia sensor is 0.5U-1.2U; the conductive medium is at least one of potassium ferrocyanide, a ruthenium compound or polyacrylamide; The reaction solution also contains monosaccharides or polysaccharides as enzyme stabilizers and film-forming materials; The reaction solution further comprises a surfactant, and the surfactant is at least one or more of EDTA, polyethylene glycol, SDS, PVP or Triton.

2. A blood ammonia sensor according to claim 1, characterized in that: The materials constituting the working electrode (12) and the counter electrode (11) are both selected from at least one of graphene, carbon nanotubes, graphite carbon paste, gold, platinum and palladium; the material of the reference electrode is a mixture of silver / silver chloride particles.

3. A blood ammonia sensor according to claim 2, characterized in that: The material of the insulating layer (4) is at least one of polypropylene and polyethylene.

4. A blood ammonia sensor according to claim 3, characterized in that: The hydrophilic cover plate layer (5) is made of a polyester material with a hydrophilic layer attached to one side.

5. A blood ammonia sensor according to claim 4, characterized in that: The masking tape layer (6) is made of polyolefin or polyester material.

6. The method for preparing the blood ammonia sensor according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, screen-printing the electrode layer (2) on the insulating substrate layer (1) according to a preset shape and position and drying the same, thereby sequentially obtaining an electrode system consisting of the working electrode (12), the counter electrode (11) and the reference electrode; S2, dissolving the material constituting the insulating layer (4) in a solvent, and sequentially inkjet printing the material on the electrode system obtained in S1 according to a preset shape and position, and ensuring that blank windows are left on the counter electrode (11), the working electrode (12) and the reference electrode to form a reaction tank; curing the insulating layer (4) under ultraviolet light for 5-15 seconds; S3, in the reaction tank, sequentially solidifying the material constituting the biosensitive layer (3) onto the working electrode (12) of the electrode system obtained in S1 according to a preset shape and position and drying at 45° C. for 20 minutes; S4, laying the hydrophilic cover layer (5) provided with a drainage device on top of the insulating layer (4), so that the reaction tank is formed below the hydrophilic cover layer (5) to form a siphon channel, and opening an air permeable hole on the hydrophilic cover layer (5) corresponding to the top of the siphon channel; S5, covering the masking tape layer (6) on the insulating layer (4) and the hydrophilic cover layer (5) to protect the blood ammonia sensor.

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