A glucose detection test strip and a preparation method and application thereof

Abstract

The application provides a glucose detection test strip and a preparation method and application thereof. The preparation method of the glucose detection test strip comprises the following steps: taking copper sulfate pentahydrate, a phosphate buffer solution, glucose oxidase and horseradish peroxidase, mixing uniformly to obtain an enzyme-containing mixed solution; immersing a test strip in the enzyme-containing mixed solution, and obtaining an organic-inorganic hybrid nanoflower loaded test strip after reaction; immersing the organic-inorganic hybrid nanoflower loaded test strip in a mixed solution containing a chromogenic agent, a gel, bovine serum albumin and polyethylene glycol to obtain a glucose detection test strip. The preparation method of the glucose detection test strip has higher sensitivity and shorter response time.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of in vitro diagnostic reagents, in particular to a glucose detection test strip and a preparation method and application thereof. BACKGROUND

[0002] The glucose content is a commonly used index in biochemical analysis and is often used for the diagnosis of various diseases, such as the diagnosis of diabetes. The colorimetric sensing technology based on test strips has the advantages of low cost, simple operation and rapid detection, and occupies a large proportion in the field of on-site instant detection. However, the concentration of salivary glucose is often very low, about 5-10 mg / L for healthy people, and more than 10 mg / L for diabetic patients. This is a major challenge to the sensitivity of the colorimetric sensor based on test strips.

[0003] Effective immobilization of enzymes is a key factor determining the performance of colorimetric sensors based on test strips. Currently, enzyme immobilization is mainly achieved by pure adsorption, covalent coupling and polymer entrapment. However, during the immobilization process, the conformation of the enzyme is prone to change, which reduces the stability and activity of the enzyme; in addition, the mass transfer hindrance on the solid support greatly reduces the catalytic efficiency of the enzyme, limiting its application in low-concentration salivary glucose.

[0004] Currently, organic-inorganic hybrid nanoflowers (HNFs) have been reported to attract widespread attention due to their non-toxicity, ease of preparation and good biocompatibility. For example, a multi-enzyme co-coated organic-inorganic HNF was synthesized with glucose oxidase (GOx) and horseradish peroxidase (HRP) as organic components and copper phosphate (Cu3(PO4)2) as inorganic components, and further used as a colorimetric sensor for visual detection of glucose. However, traditional colorimetric sensors for detecting glucose usually use HNFs in solution, which requires multiple steps of centrifugation, washing, drying and other steps, which may cause the enzyme to leak from the solid support; in addition, the large size of HNFs (usually > 4 μm) immobilized on the test strip by polymer entrapment still faces the problem of low immobilization efficiency, which limits its application in low salivary glucose monitoring. By loading HNFs on test paper, the obtained HNFs are difficult to have a complete structure, and the loading amount is low, resulting in a relatively long response time (5 min) and an unsatisfactory detection limit (18 mg / L), which limits its rapid detection of low-concentration salivary glucose.

[0005] Therefore, there is an urgent need to develop a glucose test strip with high sensitivity and simple preparation process. SUMMARY

[0006] The purpose of the present application is to provide a glucose detection test strip and a preparation method and application thereof, which solve the technical problems of low sensitivity and complicated preparation process of the glucose test strip in the prior art.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A method for preparing a glucose test strip includes the following steps:

[0009] (1) Take copper sulfate pentahydrate, phosphate buffer solution, glucose oxidase and horseradish peroxidase, stir to dissolve them completely, and obtain a mixed solution containing enzymes; immerse the test strip in the mixed solution containing enzymes, and react at 25-37℃ for 48-72h, so that organic-inorganic hybrid nanoflowers grow in situ on the test strip, and obtain test strips loaded with organic-inorganic hybrid nanoflowers;

[0010] (2) The test strips loaded with organic-inorganic hybrid nanoflowers obtained in step (1) are stored at -20°C for 1-8 hours, dried at 30°C for 1-4 hours, and then soaked in a mixed solution containing colorimetric reagent, gel, bovine serum albumin and polyethylene glycol for 12-24 hours to obtain glucose test strips; the mass ratio of bovine serum albumin and polyethylene glycol is 1:1.

[0011] Preferably, in step (1), the pH range of the phosphate buffer solution is 7.0 to 7.5.

[0012] Preferably, in step (1), the mass ratio of horseradish peroxidase, glucose oxidase, copper sulfate pentahydrate, and phosphate buffer solution is 1:(1-10):(150-200):(6000-7000).

[0013] Preferably, in step (1), the activity of glucose oxidase is greater than 180 U / mg, and the activity of horseradish peroxidase is greater than 300 U / mg.

[0014] Preferably, in step (2), the mass ratio of the gel, bovine serum albumin, and chromogenic agent is 1:(0.5-2.5):(5-10).

[0015] Preferably, in step (2), the color developer is one or more of 3,3,5,5-tetramethylbenzidine and 2,4,6-tribromo-3-hydroxybenzoic acid.

[0016] Preferably, in step (2), the gel is one or more of gelatin, sodium carboxymethyl cellulose, and polyvinyl alcohol.

[0017] Preferably, in step (2), the mass fraction of the gel is 20wt% to 40wt%.

[0018] The present invention also provides a glucose test strip prepared by the method described above.

[0019] The application further provides application of the glucose detection test strip in the field of in-vitro diagnosis.

[0020] The above scheme of the application at least has the following beneficial effects:

[0021] The preparation method of the glucose detection test strip comprises the following steps: taking copper sulfate pentahydrate, phosphate buffer solution, glucose oxidase and horseradish peroxidase, stirring to fully dissolve, and obtaining an enzyme-containing mixed solution; soaking the test strip in the enzyme-containing mixed solution, reacting for 48-72h under the condition of 25-37 DEG C, so that the organic-inorganic hybrid nanoflower grows in situ on the test strip, and an organic-inorganic hybrid nanoflower loaded test strip is obtained; the organic-inorganic hybrid nanoflower loaded test strip is sequentially stored at-20 DEG C for 1-8h, dried at 30 DEG C for 1-4h, and then soaked in a mixed solution containing a chromogenic agent, a gel, bovine serum albumin and polyethylene glycol for 12-24h, and a glucose detection test strip is obtained; the mass ratio of bovine serum albumin to polyethylene glycol is 1:1. Attached Figure Description

[0022] Figure 1 This is a scanning electron microscope image of the test strip loaded with the organic-inorganic hybrid nanoflowers. Detailed Implementation

[0023] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0024] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0025] Example 1

[0026] The preparation method of the glucose test strip in this embodiment includes the following steps:

[0027] (1) Copper sulfate pentahydrate and phosphate buffer solution were stirred at 800 rpm to mix them evenly. Glucose oxidase and horseradish peroxidase were added and stirred at 1000 rpm to dissolve them completely, resulting in an enzyme-containing mixed solution. The test strip was immersed in the enzyme-containing mixed solution and reacted at 30°C for 72 h to obtain an organic-inorganic hybrid nanoflower-loaded test strip. The pH value of the phosphate buffer solution was 7.4; the mass ratio of horseradish peroxidase, glucose oxidase, copper sulfate pentahydrate, and phosphate buffer solution was 1:5:175:6500; the activity of glucose oxidase was 200 U / mg, and the activity of horseradish peroxidase was 320 U / mg.

[0028] (2) The test strips loaded with the organic-inorganic hybrid nanoflowers obtained in step (1) were stored at -20°C for 2 hours, dried at 30°C for 2 hours, and then soaked in a mixed solution containing a chromogenic agent, gel, bovine serum albumin, and polyethylene glycol for 12 hours to obtain glucose test strips; the mass ratio of bovine serum albumin to polyethylene glycol was 1:1. The mass ratio of the gel, bovine serum albumin, and chromogenic agent was 1:1.5:7.5; the chromogenic agent was 3,3,5,5-tetramethylbenzidine; and the gel was 30 wt% gelatin.

[0029] Example 2

[0030] The preparation method of the glucose detection test strip of the embodiment comprises the following steps:

[0031] (1) Take copper sulfate pentahydrate and phosphate buffer solution and stir them at a speed of 800 rpm to mix them uniformly, add glucose oxidase and horseradish peroxidase, and stir them at a speed of 1000 rpm to dissolve them fully, to obtain a mixed solution containing enzymes; immerse the test strip in the mixed solution containing enzymes, and react at 37°C for 48 h to obtain an organic-inorganic hybrid nanoflower loaded test strip. The pH value of the phosphate buffer solution is 7.5; the mass ratio of the horseradish peroxidase, glucose oxidase, copper sulfate pentahydrate, and phosphate buffer solution is 1:1:150:7000; the activity of the glucose oxidase is 200 U / mg, and the activity of the horseradish peroxidase is 320 U / mg.

[0032] (2) Store the organic-inorganic hybrid nanoflower loaded test strip obtained in step (1) successively at -20°C for 8 h and at 30°C for 4 h, then immerse it in a mixed solution containing a chromogenic agent, a gel, bovine serum albumin, and polyethylene glycol for 12 h to obtain a glucose detection test strip; the mass ratio of the bovine serum albumin and polyethylene glycol is 1:1. The mass ratio of the gel, bovine serum albumin, and chromogenic agent is 1:2.5:5; the chromogenic agent is 3,3,5,5-tetramethylbenzidine; and the gel is polyvinyl alcohol with a mass fraction of 40 wt%.

[0033] Embodiment 3

[0034] The preparation method of the glucose detection test strip of the embodiment comprises the following steps:

[0035] (1) Take copper sulfate pentahydrate and phosphate buffer solution and stir them at a speed of 800 rpm to mix them uniformly, add glucose oxidase and horseradish peroxidase, and stir them at a speed of 1000 rpm to dissolve them fully, to obtain a mixed solution containing enzymes; immerse the test strip in the mixed solution containing enzymes, and react at 37°C for 48 h to obtain an organic-inorganic hybrid nanoflower loaded test strip. The pH value of the phosphate buffer solution is 7.0; the mass ratio of the horseradish peroxidase, glucose oxidase, copper sulfate pentahydrate, and phosphate buffer solution is 1:1:200:6000; the activity of the glucose oxidase is 200 U / mg, and the activity of the horseradish peroxidase is 320 U / mg.

[0036] (2) The organic-inorganic hybrid nanoflower loaded test strip obtained in step (1) is sequentially stored at -20°C for 1 h, dried at 30°C for 1 h, and then soaked in a mixed solution containing a color developing agent, a gel, bovine serum albumin, and polyethylene glycol for 12 h to obtain a glucose detection test strip; the mass ratio of the bovine serum albumin to the polyethylene glycol is 1:1. The mass ratio of the gel, the bovine serum albumin, and the color developing agent is 1:2.5:10; the color developing agent is 3,3,5,5-tetramethylbenzidine; and the gel is carboxymethylcellulose sodium with a mass fraction of 35 wt%.

[0037] Example 4

[0038] The preparation method of the glucose detection test strip of the present example includes the following steps:

[0039] (1) Copper sulfate pentahydrate and a phosphate buffer solution are stirred at a rotation speed of 800 rpm to uniformly mix them, and glucose oxidase and horseradish peroxidase are added and stirred at a rotation speed of 1000 rpm to fully dissolve them, to obtain an enzyme-containing mixed solution; a test strip is soaked in the enzyme-containing mixed solution and reacted at 28°C for 50 h to obtain an organic-inorganic hybrid nanoflower loaded test strip. The pH value of the phosphate buffer solution is 7.4; the mass ratio of the horseradish peroxidase, the glucose oxidase, the copper sulfate pentahydrate, and the phosphate buffer solution is 1:10:150:7000; the activity of the glucose oxidase is 200 U / mg, and the activity of the horseradish peroxidase is 320 U / mg.

[0040] (2) The organic-inorganic hybrid nanoflower loaded test strip obtained in step (1) is sequentially stored at -20°C for 4 h, dried at 30°C for 2 h, and then soaked in a mixed solution containing a color developing agent, a gel, bovine serum albumin, and polyethylene glycol for 18 h to obtain a glucose detection test strip; the mass ratio of the bovine serum albumin to the polyethylene glycol is 1:1. The mass ratio of the gel, the bovine serum albumin, and the color developing agent is 1:0.5:5; the color developing agent is 3,3,5,5-tetramethylbenzidine; and the gel is gelatin and polyvinyl alcohol with a mass fraction of 35 wt%, and the mass ratio of the gelatin to the polyvinyl alcohol is 1:1.

[0041] Example 5

[0042] The preparation method of the glucose detection test strip of the present example includes the following steps:

[0043] (1) Take copper sulfate pentahydrate, phosphate buffer solution, and stir at a speed of 800 rpm to make them uniformly mixed, add glucose oxidase and horseradish peroxidase, and stir at a speed of 1000 rpm to make them fully dissolved, to obtain a mixed solution containing enzymes; immerse the test strip in the mixed solution containing enzymes, and react at 35°C for 55h to obtain an organic-inorganic hybrid nanoflower loaded test strip. Among them, the pH value of the phosphate buffer solution is 7.1; the mass ratio of the horseradish peroxidase, glucose oxidase, copper sulfate pentahydrate, and phosphate buffer solution is 1:10:200:6000; the activity of the glucose oxidase is 200U / mg, and the activity of the horseradish peroxidase is 320U / mg.

[0044] (2) The organic-inorganic hybrid nanoflower loaded test strip obtained in step (1) is sequentially stored at -20°C for 5h, dried at 30°C for 2h, and then immersed in a mixed solution containing a chromogenic agent, a gel, bovine serum albumin, and polyethylene glycol for 22h to obtain a glucose detection test strip; the mass ratio of the bovine serum albumin and polyethylene glycol is 1:1. Among them, the mass ratio of the gel, bovine serum albumin, and chromogenic agent is 1:0.5:10; the chromogenic agent is 2,4,6-tribromo-3-hydroxybenzoic acid; the gel is 33wt% gelatin and sodium carboxymethyl cellulose, and the mass ratio of the gelatin and sodium carboxymethyl cellulose is 1:1.

[0045] Example 6

[0046] The preparation method of the glucose detection test strip of the present embodiment comprises the following steps:

[0047] (1) Take copper sulfate pentahydrate, phosphate buffer solution, and stir at a speed of 800 rpm to make them uniformly mixed, add glucose oxidase and horseradish peroxidase, and stir at a speed of 1000 rpm to make them fully dissolved, to obtain a mixed solution containing enzymes; immerse the test strip in the mixed solution containing enzymes, and react at 35°C for 55h to obtain an organic-inorganic hybrid nanoflower loaded test strip. Among them, the pH value of the phosphate buffer solution is 7.1; the mass ratio of the horseradish peroxidase, glucose oxidase, copper sulfate pentahydrate, and phosphate buffer solution is 1:10:200:6000; the activity of the glucose oxidase is 200U / mg, and the activity of the horseradish peroxidase is 320U / mg.

[0048] (2) The organic-inorganic hybrid nanoflower loaded test strip obtained in step (1) is sequentially stored at -20°C for 4h, dried at 30°C for 4h, and then soaked in a mixed solution containing a color developing agent, a gel, bovine serum albumin, and polyethylene glycol for 20h to obtain a glucose detection test strip; the mass ratio of the bovine serum albumin to the polyethylene glycol is 1:1. The mass ratio of the gel, the bovine serum albumin, and the color developing agent is 1:1.5:10; the color developing agent is 2,4,6-tribromo-3-hydroxybenzoic acid; and the gel is polyvinyl alcohol and sodium carboxymethyl cellulose with a mass fraction of 37wt%, and the mass ratio of the polyvinyl alcohol to the sodium carboxymethyl cellulose is 1:1.

[0049] Example 7

[0050] The preparation method of the glucose detection test strip of the present example comprises the following steps:

[0051] (1) Copper sulfate pentahydrate and a phosphate buffer solution are stirred at a rotation speed of 800rpm to uniformly mix them, and glucose oxidase and horseradish peroxidase are added and stirred at a rotation speed of 1000rpm to fully dissolve them, to obtain an enzyme-containing mixed solution; a test strip is soaked in the enzyme-containing mixed solution and reacted at 32°C for 70h to obtain an organic-inorganic hybrid nanoflower loaded test strip. The pH value of the phosphate buffer solution is 7.4; the mass ratio of the horseradish peroxidase, the glucose oxidase, the copper sulfate pentahydrate, and the phosphate buffer solution is 1:5:200:6000; the activity of the glucose oxidase is 200U / mg, and the activity of the horseradish peroxidase is 320U / mg.

[0052] (2) The organic-inorganic hybrid nanoflower loaded test strip obtained in step (1) is sequentially stored at -20°C for 6h, dried at 30°C for 3h, and then soaked in a mixed solution containing a color developing agent, a gel, bovine serum albumin, and polyethylene glycol for 16h to obtain a glucose detection test strip; the mass ratio of the bovine serum albumin to the polyethylene glycol is 1:1. The mass ratio of the gel, the bovine serum albumin, and the color developing agent is 1:2.5:7; the color developing agent is 2,4,6-tribromo-3-hydroxybenzoic acid; and the gel is gelatin, polyvinyl alcohol, and sodium carboxymethyl cellulose with a mass fraction of 35wt%, and the mass ratio of the gelatin, the polyvinyl alcohol, and the sodium carboxymethyl cellulose is 1:1:1.

[0053] Example 8

[0054] The preparation method of the glucose detection test strip of the present example is the same as that of Example 1, except that the gel is gelatin, polyvinyl alcohol, and sodium carboxymethyl cellulose with a mass fraction of 35wt%, and the mass ratio of the gelatin, the polyvinyl alcohol, and the sodium carboxymethyl cellulose is 1:1:1.

[0055] Comparative Example 1

[0056] The preparation method of the glucose test strip in this comparative example is the same as that in Example 1, except that the enzyme-containing mixed solution is cast onto the test strip to obtain an organic-inorganic hybrid nanoflower-loaded test strip.

[0057] Comparative Example 2

[0058] The preparation method of the glucose test strip in this comparative example is the same as that in Example 1, except that the step of storing at -20°C is omitted and the strip is dried directly.

[0059] Comparative Example 3

[0060] The preparation method of the glucose test strip in this comparative example is the same as that in Example 1, except that the mixed solution does not contain bovine serum albumin and polyethylene glycol.

[0061] Comparative Example 4

[0062] The preparation method of the glucose test strip in this comparative example is the same as that in Example 1, except that the mass ratio of bovine serum albumin to polyethylene glycol is 2:1.

[0063] Comparative Example 5

[0064] The preparation method of the glucose test strip in this comparative example is the same as that in Example 1, except that the mass ratio of bovine serum albumin to polyethylene glycol is 1:2.

[0065] Effect Comparison

[0066] To verify the technical effectiveness of the glucose test strip described in this invention, the following experiments were conducted:

[0067] The test strip loaded with the organic-inorganic hybrid nanoflowers obtained in Example 1 was observed under a scanning electron microscope, and the results are as follows. Figure 1 As shown, the organic-inorganic hybrid nanoflowers prepared by the method of this embodiment have a size of about 4 μm and a porous, dense spherical structure. At the same time, thanks to the close interaction between the organic-inorganic hybrid nanoflowers and the moistened test strip, the organic-inorganic hybrid nanoflowers have a high loading amount on the test strip loaded with organic-inorganic hybrid nanoflowers.

[0068] The glucose test strips prepared in Examples 1-8 and Comparative Examples 1-5 were dripped with 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, and so on of glucose solution in sequence, and the color change on the test strips was observed, the concentration of the glucose solution corresponding to the color change of the glucose test strip was recorded to obtain the detection limit, and the time from dripping the glucose solution to the color change was recorded to obtain the response time.

[0069] Through experiments, the results are as follows:

[0070] Group Detection limit Response time Example 1 10 mg / L 60s Example 2 13 mg / L 65s Example 3 11 mg / L 60s Example 4 12 mg / L 60s Example 5 12 mg / L 60s Example 6 10 mg / L 66s Example 7 10 mg / L 65s Example 8 10 mg / L 67s Comparative Example 1 1260 mg / L 70s Comparative Example 2 50 mg / L 65s Comparative Example 3 50 mg / L 66s Comparative Example 4 15 mg / L 66s Comparative Example 5 14 mg / L 68s

[0071] From the above results, it can be seen that the glucose test strips of Examples 1-8 have lower detection limits and shorter response times compared with Comparative Examples 1-5.

[0072] According to the comparison between Example 1 and Comparative Example 1, compared with casting the enzyme-containing mixed solution on the test strip, immersing the test strip in the enzyme-containing mixed solution, and reacting for 48-72 h at 25-37 ℃, in-situ growth of the organic-inorganic hybrid nanoflower on the test strip can make the organic-inorganic hybrid nanoflower have an integrated and dense porous structure, and can increase the loading amount of the organic-inorganic hybrid nanoflower on the test strip, thereby improving the sensitivity of the glucose test strip and shortening the response time.

[0073] According to the comparison between Example 1 and Comparative Example 2, it can be seen that storage of the organic-inorganic hybrid nanoflower at-20 ℃ for 1-8 h can make the organic-inorganic hybrid nanoflower and the test strip tightly combined, thereby obtaining a lower detection limit and a shorter response time.

[0074] According to the comparison between Example 1 and Comparative Examples 4 and 5, it can be seen that when the mass ratio of the bovine serum albumin and the polyethylene glycol is 1:1, the glucose test strip obtained has a lower detection limit and a shorter response time.

[0075] From the technical common sense, the present application can be realized by other embodiments without departing from the spirit or essential characteristics thereof. Therefore, the above-mentioned embodiments of the present application are only illustrative in all aspects, and are not the only ones. All changes within the scope of the present application or within the scope equivalent to the present application are included in the present application.

Claims

1. A method for preparing a glucose test strip, characterized in that, Includes the following steps: (1) Take copper sulfate pentahydrate, phosphate buffer solution, glucose oxidase and horseradish peroxidase, stir to dissolve them completely to obtain a mixed solution containing enzymes; immerse the test strip in the mixed solution containing enzymes and react at 25-37℃ for 48-72h to allow organic-inorganic hybrid nanoflowers to grow in situ on the test strip to obtain a test strip loaded with organic-inorganic hybrid nanoflowers; (2) The test strips loaded with organic-inorganic hybrid nanoflowers obtained in step (1) are stored at -20°C for 1-8 hours, dried at 30°C for 1-4 hours, and then soaked in a mixed solution containing color reagent, gel, bovine serum albumin and polyethylene glycol for 12-24 hours to obtain glucose test strips; the mass ratio of bovine serum albumin and polyethylene glycol is 1:

1.

2. The method for preparing the glucose test strip according to claim 1, characterized in that, In step (1), the pH range of the phosphate buffer solution is 7.0~7.

5.

3. The method for preparing the glucose test strip according to claim 1, characterized in that, In step (1), the mass ratio of horseradish peroxidase, glucose oxidase, copper sulfate pentahydrate, and phosphate buffer solution is 1:(1~10):(150~200):(6000~7000).

4. The method for preparing the glucose test strip according to claim 1, characterized in that, In step (1), the activity of glucose oxidase is greater than 180 U / mg, and the activity of horseradish peroxidase is greater than 300 U / mg.

5. The method for preparing the glucose test strip according to claim 1, characterized in that, In step (2), the mass ratio of the gel, bovine serum albumin, and chromogenic agent is 1:(0.5~2.5):(5~10).

6. The method for preparing the glucose test strip according to claim 1, characterized in that, In step (2), the colorimetric agent is one or more of 3,3,5,5-tetramethylbenzidine and 2,4,6-tribromo-3-hydroxybenzoic acid.

7. The method for preparing the glucose test strip according to claim 1, characterized in that, In step (2), the gel is one or more of gelatin, sodium carboxymethyl cellulose, and polyvinyl alcohol.

8. The method for preparing the glucose test strip according to claim 1, characterized in that, In step (2), the mass fraction of the gel is 20wt%~40wt%.

9. A glucose test strip prepared by the method of any one of claims 1-8.

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