CN111983235A

CN111983235A - Test paper for quantitatively determining urea and preparation method thereof

Info

Publication number: CN111983235A
Application number: CN202010679404.3A
Authority: CN
Inventors: 熊攀; 李强
Original assignee: Hubei Jinjian Biology Co ltd
Current assignee: Hubei Jinjian Biology Co ltd
Priority date: 2020-07-15
Filing date: 2020-07-15
Publication date: 2020-11-24

Abstract

The invention discloses test paper for quantitatively determining urea and a preparation method thereof, belonging to the field of in vitro diagnosis. The test paper comprises a diffusion layer, a blood filtering layer, a reaction layer and a hydrophobic breathable color development layer; the blood filtering layer contains buffer salt, divalent metal ions, polyvinylpyrrolidone, hemagglutinin or anti-erythrocyte antibodies, and the reaction layer contains buffer salt, surfactant, divalent metal ions, urease, polysaccharide, ascorbic acid oxidase, polyalcohol, polyvinylpyrrolidone and enzyme protective agent; the color developing layer contains an alcohol and a developer. The test paper is prepared by preparing a blood filtering solution, a reaction solution and a color developing agent solution containing related components, soaking a blood filtering layer and a reaction layer material in the blood filtering solution and the reaction solution respectively, spraying the color developing agent solution on a hydrophobic air-permeable color developing layer material, drying to obtain the blood filtering layer, the reaction layer and the hydrophobic air-permeable color developing layer, and assembling the blood filtering layer, the reaction layer and the hydrophobic air-permeable color developing layer with other components. The test paper can simply and quickly detect the urea content in whole blood, blood plasma and blood serum in a quantitative manner.

Description

Test paper for quantitatively determining urea and preparation method thereof

Technical Field

The invention belongs to the field of in-vitro diagnosis, relates to detection of urea, and particularly relates to test paper for quantitatively determining urea and a preparation method thereof.

Background

Urea (Urea) is the end product of amino acid catabolism in vivo. The liver cells produce urea from ammonia, and the liver is the most important organ for urea production. Urea is the major component of non-protein nitrogen in the blood and normally accounts for about 50% of the total non-protein nitrogen. Urea is mainly excreted by the kidney, urea in blood is completely filtered by the glomerulus, 30-40% of urea is reabsorbed by the renal tubules, a small amount of urea is also secreted by the renal tubules, and the secretion amount is increased when renal failure is serious. The measurement of urea is one of the commonly used indicators for measuring the function of the glomerulus.

The most common methods for measuring urea in clinical laboratories at present are diacetyl-oxime chromogenic method and enzyme coupling rate method (urease method coupled with glutamate dehydrogenase). The diacetyl-oxime method is based on the chromogenic reaction of diacetyl with urea to form a colored complex of diazine derivatives. Since diacetyl itself is unstable and corrosive reagents need to be added to the reaction, clinical testing has been rarely used. The enzyme coupling rate method can only detect serum and plasma samples and needs special equipment for detection, and the operation is also complicated.

In view of the above, it is very necessary to develop a product for quantitative detection of urea, which is simple to operate and suitable for various sample types.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the test paper for quantitatively detecting urea, which can simply and quickly quantitatively detect the content of urea without centrifugation and large-scale detection equipment. The invention also aims to provide a preparation method of the test paper for quantitatively determining urea.

The purpose of the invention is realized by the following technical scheme:

a test paper for quantitatively determining urea comprises a diffusion layer, a blood filtering layer, a reaction layer and a hydrophobic breathable color development layer which are sequentially bonded from top to bottom.

The diffusion layer is made of hydrophilic mesh materials, such as hydrophilic gauze, non-woven fabric, polyester fiber and the like.

The blood filtering layer is made of glass fiber membrane materials, such as commercially available Pall A/D glass fiber membrane, Pall A/E glass fiber membrane, WHATMAN FUSON5 glass fiber membrane, etc. The blood filtering layer comprises the following components: buffer salts, divalent metal ions, polyvinylpyrrolidone, hemagglutinin, or anti-erythrocyte antibodies. The pH value of the blood filtering layer is preferably 9.0-12.0.

The reaction layer is made of filter paper material, and the thickness of the filter paper material is preferably 0.3-0.6 mm. The reaction layer comprises the following components: buffer salts, divalent metal ions, surfactants, urease, polysaccharides, ascorbic acid oxidase, polyols, polyvinylpyrrolidone, and enzyme protectants. The pH value of the reaction layer is preferably 9.0-12.0.

The hydrophobic air-permeable color development layer is an acrylic copolymer film, such as Versapor-200R, Versapor-450R, Versapor-800R and the like which are commercially available, and the aperture of the hydrophobic air-permeable color development layer is preferably 0.2-1.2 mu m. The upper surface of the hydrophobic breathable color development layer is a hydrophobic breathable layer, the lower surface of the hydrophobic breathable color development layer is a color development layer, and the color development layer comprises the following components: alcohols and color developers. The pH value of the color development layer is preferably 3.0-6.0.

The buffer salt is preferably one or more of N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine, trimethylol methylaminopropanesulfonic acid (TAPS) and Tris-hydrochloric acid.

The divalent metal ion is preferably any one of magnesium ion, zinc ion, ferrous ion, tin ion, lead ion, calcium ion and manganese ion.

The surfactant is preferably one or more of Tween-20, Tween-80, Triton-100, Brij-35, Brij-58 and LS-114.

The polyols and polysaccharides may be selected from those commonly used in the art, for example, sorbitol, xylitol, etc. may be selected as the polyol, trehalose, sucrose, etc. may be selected as the polysaccharide, and the specific types of the polyols and polysaccharides are not particularly limited in the present invention.

The enzyme protective agent may be selected from protective agents commonly used in the art, such as bovine serum albumin, casein, sorbitol, polyethylene glycol, and the like, and the specific type of the enzyme protective agent is not particularly limited in the present invention.

The alcohol is preferably one or more of methanol, ethanol, n-butanol and n-pentanol.

The color developing agent is preferably one or more of bromophenol blue, bromocresol green and bromothymol blue.

Further, the test paper for quantitatively determining urea further comprises an upper plate and a lower plate, wherein the upper plate is provided with a sample adding hole, and the lower plate is provided with a light hole. The test paper comprises an upper plate, a diffusion layer, a blood filtering layer, a reaction layer, a hydrophobic and breathable color development layer and a lower plate from top to bottom in sequence. The upper plate and the lower plate are made of ABS plastic.

The preparation method of the test paper for quantitatively determining urea comprises the following steps:

(1) the filtered blood solution, the reaction solution and the color developing agent solution are prepared respectively according to the following compositions.

Blood filtration solution: 50-200mmol/L of buffer salt, 0.2-2 percent (mass ratio) of divalent metal salt, 0.1-0.8mg/mL of hemagglutinin or anti-erythrocyte antibody, 0.2-2 percent (mass ratio) of polyvinylpyrrolidone, purified water as a solvent, and 9.0-12.0 of pH;

reaction solution: 500mmol/L of buffer salt 100-;

developer solution: 98-99.5 percent of alcohol (mass ratio), 0.05-2 percent of color developing agent (mass ratio) and 3.0-6.0 of pH.

(2) Soaking a hemofiltration layer material in a hemofiltration solution, soaking a reaction layer material in a reaction solution, spraying a color developing agent solution on one surface of a hydrophobic air-permeable color development layer material (the surface sprayed with the color developing agent solution is a color development layer, and the other surface is a hydrophobic air-permeable layer), drying, sequentially obtaining a hemofiltration layer, a reaction layer and a hydrophobic air-permeable color development layer, and assembling the hemofiltration layer, the reaction layer and the hydrophobic air-permeable color development layer with other components to obtain a finished product.

In the step (2), the drying conditions of the hemofiltration layer material are preferably as follows: the drying temperature is 37-60 ℃, and the drying time is 10-24 h; the conditions for drying the reaction layer material are preferably as follows: the drying temperature is 45-65 ℃, and the drying time is 10-60 min; the conditions for drying the hydrophobic breathable chromogenic layer material are preferably as follows: the drying temperature is 45-75 ℃, and the drying time is 5-20 min.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the invention combines the hydrophobic breathable layer and the color development layer into a whole by using a special material, so that the test paper has a simpler structure, and the detection time is greatly shortened.

(2) The test paper provided by the invention is simple in structure, is suitable for whole blood, plasma and serum samples, is convenient to operate, can obtain a urea detection result within 3 minutes, and is suitable for hospital emergency and family detection.

Drawings

FIG. 1 is a schematic structural view of a test paper for quantitatively determining urea according to the present invention; in the figure, 100-sample adding holes, 200-upper plate, 300-diffusion layer, 400-blood filtering layer, 500-reaction layer, 600-hydrophobic air-permeable layer and color development layer, 610-hydrophobic air-permeable layer, 620-color development layer, 700-lower plate and 800-light-transmitting holes.

FIG. 2 is a graph of the linear correlation of the test results of the present invention and hospital test results.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments described herein are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention. The experimental methods in the present invention are conventional methods unless otherwise specified. The experimental materials used in the present invention were all purchased from the market unless otherwise specified.

The test paper for quantitatively determining urea is shown in figure 1 and comprises an upper plate 200, a diffusion layer 300, a blood filtering layer 400, a reaction layer 500, a hydrophobic and breathable color development layer 600 and a lower plate 700. The upper plate 200 is provided with a sample adding hole 100; the upper surface of the hydrophobic breathable color development layer 600 is a hydrophobic breathable layer 610, and the lower surface is a color development layer; the lower plate 700 is provided with a light-transmitting hole 800.

The diffusion layer 300, the blood filter layer 400, the reaction layer 500, and the hydrophobic air-permeable color developing layer 600 are sequentially bonded from top to bottom, and then assembled with the upper plate 200 and the lower plate 700.

Example 1

The test paper for quantitatively determining urea of the embodiment comprises an upper plate, a diffusion layer, a blood filtering layer, a reaction layer, a hydrophobic air-permeable color development layer and a lower plate, wherein:

the upper plate is made of ABS plastic, and a sampling hole is formed in the middle of the upper plate;

the diffusion layer is made of commercially available hydrophilic gauze (hydrophilic gauze series of commercially available gauze), and the gauze is cut into 4mm × 8mm size for later use;

the blood filtering layer is prepared by soaking glass fiber material (PALL series glass fiber material) in blood filtering solution at a soaking amount of 495g/m²(namely, 495g of hemofiltration solution is used for soaking each square meter of glass fiber material), the glass fiber material is placed into an air-blast drying oven to be dried, the drying temperature is 37 ℃, the drying time is 12 hours, and the glass fiber is cut into 4mm by 4mm for standby after being dried, wherein the hemofiltration solution comprises the following components:

purified water 90% (mass ratio)

Magnesium chloride 1% (mass ratio)

CHES 100mmol/L

Anti-erythrocyte antibody 0.2mg/mL

Polyvinylpyrrolidone 1% (mass ratio)

The pH of the hemofilter solution was adjusted to 9.0. + -. 0.05 with 6M NaOH and made up to 100% with purified water.

The reaction layer was a filter paper having a thickness of 0.5mm and immersed in the reaction solution at an immersion treatment amount of 50g/m²Drying in a blast drying oven at 55 deg.C for 30min, cutting the filter paper into 4mm by 4mm,the reaction solution comprises the following components:

purified water 90% (mass ratio)

CHES 100mmol/L

Tween-200.6% (mass ratio)

Magnesium chloride 0.3% (mass ratio)

Trehalose 0.5% (mass ratio)

Polyvinylpyrrolidone 0.75% (mass ratio)

Urease 100U/g

Ascorbic acid oxidase 200U/g

Sorbitol 1.5% (mass ratio)

Bovine serum albumin 0.3% (mass ratio)

The pH of the reaction solution was adjusted to 9.0. + -. 0.05 with 6M NaOH and made up to 100% with purified water.

The hydrophobic air-permeable color developing layer is prepared by coating a developer solution on one surface of an acrylic copolymer film (color developing layer on the surface coated with the developer solution, and hydrophobic air-permeable layer on the other surface) with a thickness of 15g/m²Drying in a blast drying oven at 50 deg.C for 5min, cutting the acrylic copolymer film into 4mm, wherein the color developing agent solution comprises the following components:

bromophenol blue 0.1% (mass ratio)

Ethanol 90% (mass ratio)

Adjusting pH of the developer solution to 3.0 + -0.05 with 1M NaOH, and adding ethanol to make up to 100%.

The lower plate is made of ABS plastic and is provided with light holes.

The prepared diffusion layer, the blood filtering layer, the reaction layer and the hydrophobic breathable color development layer are bonded from top to bottom, and the combined test strip is assembled with the upper plate and the lower plate to prepare the test paper for quantitatively determining urea.

Example 2

The procedure of this example was repeated except that the composition of the hemofiltration solution was 0.5% magnesium chloride, 150mmol/L TAPS, 0.3mg/mL PHA (PHA) and 1% polyvinylpyrrolidone, to prepare test strips, as described in example 1.

Example 3

The procedure of this example was carried out in the same manner as in example 1 except that the reaction mixture was 120mmol/L CHES, 0.8% Triton-100, 0.6% zinc chloride, 150U/g urease, 2% sucrose, 300U/g ascorbate oxidase, 2% sorbitol, 1% polyvinylpyrrolidone, and 0.5% casein.

Example 4

The procedure of this example was carried out in the same manner as in example 1 except that the reaction mixture was 150mmol/L CHES, 1% Triton-100, 1% zinc chloride, 180U/g urease, 2.4% sucrose, 400U/g ascorbate oxidase, 2% sorbitol, 1.2% polyvinylpyrrolidone, and 0.8% casein.

Example 5

The procedure of this example was repeated except that the reaction mixture was composed of 200mmol/L CHES, 1.2% Triton-100, 1.2% zinc chloride, 200U/g urease, 2% sucrose, 500U/g ascorbate oxidase, 2.3% sorbitol, 1.8% polyvinylpyrrolidone, and 1.2% casein.

Example 6

The procedure of this example was repeated except that the reaction mixture was composed of 300mmol/L CHES, 1.5% Triton-100, 1.6% zinc chloride, 300U/g urease, 2.5% sucrose, 600U/g ascorbate oxidase, 3% sorbitol, 2.5% polyvinylpyrrolidone, and 1.5% casein.

Example 7

The test paper of this example was prepared in the same manner as in example 1 except that the developer solution was composed of 0.2% bromocresol green and 99.8% methanol.

Example 8

The test paper of this example was prepared in the same manner as in example 1 except that the drying temperature of the blood filtration layer was 55 ℃ and the drying time was 10 hours.

Example 9

The test paper of this example was prepared in the same manner as in example 1 except that the drying temperature of the reaction layer was 65 ℃ and the drying time was 15 min.

Example 10

The test paper of this example was prepared in the same manner as in example 1 except that the drying temperature of the hydrophobic air-permeable color developing layer was 55 ℃ and the drying time was 6 min.

Example 11

The test paper of this example was prepared in the same manner as in example 1 except that 6M NaOH was used to adjust the pH of the hemofilter solution to 10.0. + -. 0.05.

Example 12

The test paper of this example was prepared in the same manner as in example 1 except that 6M NaOH was used to adjust the pH of the reaction solution to 10.0. + -. 0.05.

Example 13

The test paper of this example was prepared in the same manner as in example 1 except that the pH of the developing solution was adjusted to 4.0. + -. 0.05 with 1M NaOH.

Example 14

The test paper of this example was prepared in the same manner as in example 1 except that the hydrophobic air-permeable color developing layer was an acrylic copolymer film having a pore size of 0.45. mu.m.

Example 15

The test paper of this example was prepared in the same manner as in example 1 except that the hydrophobic air-permeable color developing layer was an acrylic copolymer film having a pore size of 0.8. mu.m.

Example 16

The test paper of this example was prepared in the same manner as in example 1 except that the hydrophobic air-permeable color developing layer was an acrylic copolymer film having a pore size of 1.2. mu.m.

Comparative example 1

In the preparation of the test paper of this example, the hydrophobic air-permeable layer and the color-developing layer were separated, and the hydrophobic air-permeable layer was cut into 4mm × 4mm pieces by using a commercially available gauze.

The color development layer adopts transparent PET with the thickness of 0.2mm as a base material, and the color development agent solution is coated on the transparent PET with the coating thickness of 200 μm. After coating, the obtained product is placed into a blast drying oven for drying at the drying temperature of 55 ℃ for 2h, a color development layer is cut into 4mm by 4mm in size for later use after drying, and a color development agent solution comprises the following components:

91.3 percent of purified water (mass ratio)

Cellulose acetate 5% (mass ratio)

Titanium dioxide 2% (mass ratio)

Triton-1000.5% (mass ratio)

Glycerol 1% (mass ratio)

Bromophenol blue 0.2% (mass ratio)

The other steps were the same as in example 1.

Comparative example 2

The developing layer selects transparent PET with the thickness of 0.2mm as a base material, and the developing solution is coated on the transparent PET with the coating thickness of 200 μm. After coating, the obtained product is placed into a blast drying oven for drying at the drying temperature of 55 ℃ for 2h, a color development layer is cut into 4mm by 4mm in size for later use after drying, and a color development agent solution comprises the following components:

purified water 87.1% by mass

Hydroxypropyl methylcellulose 6% (mass ratio)

Titanium dioxide 4% (mass ratio)

Triton-1001% (mass ratio)

Glycerol 1.5% (mass ratio)

Bromophenol blue 0.4% (mass ratio)

The other steps were the same as in example 1.

Comparative example 3

In the preparation of the test paper of this example, the hydrophobic air-permeable layer and the color-developing layer were separated, and the hydrophobic air-permeable layer was a hydrophobic series gauze of commercially available gauze, and was cut to 4mm × 4mm for use.

The developing layer selects transparent PET with the thickness of 0.3mm as a base material, and the developing solution is coated on the transparent PET with the coating thickness of 300 mu m. After coating, the obtained product is placed into a blast drying oven for drying at the drying temperature of 55 ℃ for 2h, a color development layer is cut into 4mm by 4mm in size for later use after drying, and a color development agent solution comprises the following components:

87.1 percent (mass ratio) of purified water

Hydroxypropyl methylcellulose 6% (mass ratio)

Titanium dioxide 4% (mass ratio)

Triton-1001% (mass ratio)

Glycerol 1.5% (mass ratio)

Bromophenol blue 0.4% (mass ratio)

The other steps were the same as in example 1.

When the test paper for quantitatively determining urea of the present invention is used, 10. mu.L of a whole blood, serum or plasma sample is added from the well of the upper plateThen, the whole blood, serum or plasma sample is uniformly diffused to the blood filtering layer through the diffusion layer, reacts with the reagent in the blood filtering layer, the red blood cells stay on the blood filtering layer, the sample continuously diffuses into the reaction layer, and the target substance urea in the sample is catalyzed by urease to generate NH₃，NH₃The blue compound is produced by the reaction of the hydrophobic breathable layer and the yellow color developing agent on the color developing layer, and the shade of the color of the blue compound is in direct proportion to the concentration of the urea. After sample adding, the test paper is directly inserted into a dry chemical analyzer, and after 3 minutes, the result can be displayed on the analyzer.

The test paper for quantitatively determining urea in examples 1 to 16 and comparative examples 1 to 3 was tested for its performance by the following method: whole blood, serum and plasma samples with urea concentrations of 5mmol/L, 50mmol/L and 100mmol/L were prepared, respectively. The samples were tested using the test strips of examples 1-16 and comparative examples 1-3, and the samples with the same concentration were tested 5 times, and the test results are shown in tables 1, 2 and 3.

TABLE 1 Whole blood sample test results

TABLE 2 plasma sample test results

TABLE 3 serum sample test results

As can be seen from tables 1, 2 and 3, the test results of the test paper in examples 1 to 16 are good in deviation and consistency, and the deviation of the results can be kept within 10%, which indicates that the test paper of the invention can detect the urea content in whole blood, plasma and serum. The test results of comparative example 1, comparative example 2 and comparative example 3 were all large in variation and were all negative in variation. In the comparative examples, the hydrophobic air-permeable layer and the color-developing layer may be separated from each other, and the reaction time may be insufficient, resulting in a large deviation of the results. Therefore, the reaction time in the comparative example was extended to 6 minutes, and the test was conducted. The results of the measurements are shown in tables 4, 5 and 6 below.

TABLE 4 Whole blood sample test results

TABLE 5 plasma sample test results

TABLE 6 serum sample test results

As can be seen from tables 4, 5 and 6, after the reaction time of the test paper in the comparative example is prolonged, the consistency and deviation of the detection result are obviously improved, and the deviation of the result can be kept within 15%, which indicates that a longer reaction time is required after the hydrophobic breathable layer and the color development layer are separated, so that a more ideal result can be obtained. The invention combines the hydrophobic breathable layer and the color development layer into a whole, and the prepared test paper can greatly shorten the detection time and has better detection accuracy.

Using the test paper of example 1, 50 urea whole blood samples distributed at different concentrations were randomly selected for testing, and then compared with the test results of a hospital full-automatic biochemical analyzer for evaluation, and the test results and linear correlation thereof are shown in table 7 and fig. 2 below.

TABLE 7 comparison of the test results of the present invention with those of hospitals

From the above, the test paper for quantitatively determining urea of the present invention has the following advantages: the test paper has a simple structure, combines the hydrophobic breathable layer and the color development layer into a whole, can obtain the detection result of urea within 3 minutes, has higher accuracy compared with the traditional method, is simultaneously suitable for whole blood, plasma and serum samples, is used together with a portable dry chemical analyzer, and is more suitable for emergency treatment in hospitals and family users.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A test paper for quantitatively determining urea, characterized in that: comprises a diffusion layer, a blood filtering layer, a reaction layer and a hydrophobic breathable color development layer which are sequentially bonded from top to bottom; the hydrophobic breathable color development layer is an acrylic copolymer film, the upper surface of the hydrophobic breathable color development layer is a hydrophobic breathable layer, and the lower surface of the hydrophobic breathable color development layer is a color development layer;

the blood filtering layer comprises the following components: buffer salts, divalent metal ions, polyvinylpyrrolidone, hemagglutinin, or anti-erythrocyte antibodies;

the reaction layer comprises the following components: buffer salt, divalent metal ions, a surfactant, urease, polysaccharide, ascorbic acid oxidase, polyol, polyvinylpyrrolidone and an enzyme protectant;

the color development layer comprises the following components: alcohols and color developers.

2. The reagent paper for quantitatively determining urea according to claim 1, characterized in that: the buffer salt is one or more of N-cyclohexyl-2-aminoethanesulfonic acid, N-cyclohexyl-3-aminopropanesulfonic acid, glycine, trihydroxymethyl methylaminopropanesulfonic acid and Tris-hydrochloric acid.

3. The reagent paper for quantitatively determining urea according to claim 1, characterized in that: the divalent metal ion is any one of magnesium ion, zinc ion, ferrous ion, tin ion, lead ion, calcium ion and manganese ion.

4. The reagent paper for quantitatively determining urea according to claim 1, characterized in that: in the reaction layer, the surfactant is one or more of Tween-20, Tween-80, Triton-100, Brij-35, Brij-58 and LS-114; the enzyme protective agent is one or more of bovine serum albumin, casein, sorbitol and polyethylene glycol.

5. The reagent paper for quantitatively determining urea according to claim 1, characterized in that: in the color development layer, the alcohol is one or more of methanol, ethanol, n-butanol and n-pentanol; the color developing agent is one or more of bromophenol blue, bromocresol green and bromothymol blue.

6. The reagent paper for quantitatively determining urea according to claim 1, characterized in that: the pH value of the blood filtering layer is 9.0-12.0; the pH value of the reaction layer is 9.0-12.0; the pH value of the color development layer is 3.0-6.0.

7. The reagent paper for quantitatively determining urea according to claim 1, characterized in that: the diffusion layer is made of hydrophilic mesh fabric material; the blood filtering layer is made of glass fiber membrane material; the reaction layer is made of filter paper materials.

8. The reagent paper for quantitatively determining urea according to claim 1, characterized in that: the sample adding device also comprises an upper plate and a lower plate, wherein the upper plate is provided with a sample adding hole, and the lower plate is provided with a light transmitting hole; the test paper comprises an upper plate, a diffusion layer, a blood filtering layer, a reaction layer, a hydrophobic and breathable color development layer and a lower plate from top to bottom in sequence.

9. The method for preparing a test paper for the quantitative determination of urea according to any one of claims 1 to 8, wherein: the method comprises the following steps:

(1) preparing a blood filtering solution, a reaction solution and a color developing agent solution respectively according to the following compositions;

blood filtration solution: 50-200mmol/L of buffer salt, 0.2-2% of divalent metal salt, 0.1-0.8mg/mL of hemagglutinin or anti-erythrocyte antibody, 0.2-2% of polyvinylpyrrolidone, purified water as a solvent, and the pH value of 9.0-12.0;

reaction solution: 500mmol/L of buffer salt, 0.2 to 1.5 percent of surfactant, 0.2 to 2 percent of divalent metal salt, 50 to 500U/g of urease, 0.2 to 4 percent of polysaccharide, 800U/g of ascorbic acid oxidase, 0.5 to 5 percent of polyalcohol, 0.1 to 5 percent of polyvinylpyrrolidone, 0.1 to 5 percent of enzyme protective agent, purified water as solvent and pH of 9.0 to 12.0;

developer solution: 98-99.5% of alcohol, 0.05-2% of color developing agent and 3.0-6.0 of pH;

(2) soaking a hemofiltration layer material in a hemofiltration solution, soaking a reaction layer material in a reaction solution, spraying a color developing agent solution on one surface of a hydrophobic air-permeable color developing layer material, drying to obtain a hemofiltration layer, a reaction layer and a hydrophobic air-permeable color developing layer in sequence, and assembling the hemofiltration layer, the reaction layer and the hydrophobic air-permeable color developing layer with other components to obtain a finished product.

10. The method of claim 9, wherein: in the step (2), the drying conditions of the hemofiltration layer material are as follows: the drying temperature is 37-60 ℃, and the drying time is 10-24 h; the drying conditions of the reaction layer material are as follows: the drying temperature is 45-65 ℃, and the drying time is 10-60 min; the drying conditions of the hydrophobic breathable chromogenic layer material are as follows: the drying temperature is 45-75 ℃, and the drying time is 5-20 min.