CN110987833A

CN110987833A - Glucose biosensor material, preparation method and application

Info

Publication number: CN110987833A
Application number: CN201911067874.8A
Authority: CN
Inventors: 卢玉超; 陈岚; 董贺楠; 申煜丽; 姜宝杰; 孙旭飞; 张伟; 张雪娇; 王琳琳; 卢鑫
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2020-04-10

Abstract

The invention discloses a glucose biosensor material, a preparation method and application, wherein 4-aminoantipyrine, dopamine and laccase are mixed according to a mass ratio of 1.5-1.6: 1:1, mixing the mixture into a phosphate buffer solution, and adjusting the pH value of the buffer solution to 6.0-6.8 to obtain a weakly acidic buffer solution; adding glucose oxidase and catalase into a weak acid buffer solution, and mixing to obtain the glucose biosensor material. Compared with the conventional glucose biosensor, the glucose sensor obtained by the invention has the advantages of less dosage, high detection precision and high detection efficiency when used for detecting glucose.

Description

Glucose biosensor material, preparation method and application

Technical Field

The invention belongs to the technical field of glucose biosensors, and particularly relates to an environment-friendly glucose biosensor material, a preparation method and application thereof.

Background

The rapid and efficient detection of glucose has very wide application in the fields of clinical medicine, biological analysis, biological medicine, agricultural production, food industry and the like. At present, methods for detecting glucose mainly include a colorimetric method, an enzymatic method, an electrochemical method, a fluorescence method and the like, wherein the glucose oxidase-colorimetric method for detecting glucose is widely concerned by researchers due to the simplicity, practicability and wide application range.

The principle of glucose oxidase-color method is to utilize Glucose Oxidase (GOD) to catalyze glucose to generate H₂O₂，H₂O₂The chromogens (4-aminoantipyrine and phenol) were oxidized by catalase (POD) to give a red quinoneimine. However, the chromogen substance in the current method comprises phenol, and the phenol has corrosiveness, high toxicity and teratogenicity; and the prepared glucose biosensor material is used in a large amount when detecting glucose, thereby greatly limiting the development of the glucose biosensor material.

Disclosure of Invention

Aiming at the technical problems, the invention provides a glucose biosensor, a preparation method and application thereof, and solves the problems of large using amount, low accuracy, corrosivity and toxicity of a glucose biosensor material prepared by the existing method in detection.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing a glucose biosensor material, comprising: 4-aminoantipyrine, dopamine and laccase are mixed according to the mass ratio of 1.5-1.6: 1:1, adding the buffer solution into a phosphate buffer solution, and adjusting the pH value of the buffer solution to 6.0-6.8 to obtain a weak acid buffer solution; adding glucose oxidase and catalase into a weak acid buffer solution, and mixing to obtain the glucose biosensor material.

Preferably, the phosphate buffer solution comprises disodium hydrogen phosphate and potassium dihydrogen phosphate, and the disodium hydrogen phosphate, the disodium hydrogen phosphate and the 4-aminoantipyrine are mixed in a molar ratio of 12:8: 0.15.

Preferably, the mass ratio of the glucose oxidase to the catalase is 1: 1.

Preferably, the pH of the dopamine phosphate buffer is 6.5.

Preferably, the dopamine is dopamine containing a catechol structure.

The invention also discloses a glucose biosensor material prepared by the preparation method of the glucose biosensor material.

The invention also discloses application of the glucose biosensor material in detecting glucose.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, toxic and harmful phenol is replaced by environment-friendly dopamine, and the dopamine is catalyzed by laccase to be self-polymerized in a weak acid buffer solution to form polydopamine immobilized enzyme and a chromogen compound, so that the obtained glucose sensor has the advantages of small dosage and high detection precision in the process of detecting glucose compared with the conventional glucose biosensor; meanwhile, the invention adopts a 96-well plate method to detect glucose, thereby obviously improving the detection effect.

Drawings

FIG. 1 is a schematic view of a process for producing a glucose biosensor according to the present invention.

FIG. 2 is a standard graph of the glucose biosensor versus the glucose solution in example 1.

Detailed Description

The invention discloses a preparation method of a glucose biosensor material, which comprises the following steps:

step 1, mixing disodium hydrogen phosphate, disodium hydrogen phosphate and 4-aminoantipyrine, wherein the molar ratio of the disodium hydrogen phosphate to the 4-aminoantipyrine is 12:8: 0.15.

Step 2, adding dopamine and laccase into the phosphate buffer solution to obtain a dopamine phosphate buffer solution, and adjusting the pH of the dopamine phosphate buffer solution to 6.0-6.8, preferably 6.5; generally, sodium hydroxide is used for adjusting the pH value to obtain a weak acid buffer solution; the dopamine in the present invention preferably contains a catechol structure.

Step 3, adding glucose oxidase and catalase into the weak acid buffer solution for mixing to obtain a glucose biosensor material; wherein the mass ratio of the glucose oxidase to the catalase is 1: 1.

The glucose biosensor material prepared by the method is applied to detecting glucose, and specifically comprises the following steps: the prepared glucose biosensor material was added to a 96-well plate at about 50. mu.l per well, mixed well, and then glucose was added thereto for colorimetric detection.

The following embodiments of the present invention are provided, and it should be noted that the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention are within the protection scope of the present invention.

Example 1

Step 1, preparing a phosphate buffer solution: weighing 17.34g of disodium hydrogen phosphate, 10.6g of disodium hydrogen phosphate and 0.313g of 4-aminoantipyrine in a 1000ml beaker, adding distilled water to about 1000ml, adjusting the pH of the solution to 6.5 by using 1mol/L sodium hydroxide solution, transferring the solution into a 1000ml volumetric flask, and fixing the volume to 1000ml to obtain a phosphate buffer solution for later use;

step 2, preparation of a weak acid buffer solution: weighing 200mg of dopamine containing a catechol structure and 200mg of laccase in a 100ml beaker, adding 100ml of phosphate buffer solution prepared in the step 1, controlling the pH to be 6.5, and preparing 2mg/ml of dopamine phosphate buffer solution;

and 3, weighing 200mg of Glucose Oxidase (GOD) and 200mg of catalase (POD), adding into 100ml of the dopamine phosphate buffer solution prepared in the step 2, shaking up, adding into a 96-pore plate, and uniformly mixing, thus obtaining the glucose biosensor, wherein each pore is 50 microliters.

The glucose biosensor prepared in the embodiment is used for detecting glucose, and the specific detection process is as follows:

firstly, preparing glucose standard solutions with different concentrations:

glucose 6.25mg, 12.5mg, 18.75mg, 25mg, 31.25mg, 37.5mg and 43.75mg were weighed out in a 10ml beaker, dissolved in a small amount of ultrapure water, transferred to a 25ml volumetric flask, and made up to 25ml with ultrapure water to obtain glucose standard solutions with concentrations of 0.25mg/ml, 0.50mg/ml, 0.75mg/ml, 1.00mg/ml, 1.25mg/ml, 1.50mg/ml and 1.75mg/ml, respectively, as shown in FIG. 1.

Then, glucose standard solutions with different concentrations are detected

And respectively adding 20 mul of glucose standard solution (each column is a concentration gradient) into 96 holes of the prepared glucose biosensor, shaking uniformly, reacting at room temperature for 30min, measuring the absorbance of the glucose biosensor at 505nm by using a microplate reader, and recording data. The result shows that the linear equation is that y is 0.0208x-0.0152, and the correlation coefficient R is²0.997 as shown in fig. 2. As can be seen from FIG. 2, a standard curve was established to verify the feasibility of the method by testing glucose standard solutions at different concentrations. The result shows that the detection method has good linear relation and can be successfully used for detecting the glucose; meanwhile, the method has good application prospect in the aspect of glucose detection due to simple preparation, environment friendliness and high detection efficiency.

Example 2

This example differs from example 1 in that: the pH of the phosphate buffer solution was 6.0.

The glucose biosensor prepared in this example was used to detect glucose in the same manner as in example 1, under the conditions of this example, the line of the linear equationThe sexual relationship is R²0.9907, slightly worse than example 1, indicating that the activity of the enzyme in this example is less stable than that of example 1.

Comparative example 1

This comparative example differs from example 1 in that: the buffer pH was 6.8. The prepared glucose biosensor material is used for detecting glucose, the detection process is the same as that of example 1, and the linear relation R of the linear equation of the glucose biosensor material²< 0.99, largely due to the instability of laccase activity at this pH.

Comparative example 2

This comparative example differs from example 1 in that: no laccase was added, and the other conditions were the same as in example 1.

The prepared glucose biosensor material was used for detecting glucose in the same manner as in example 1, and the results showed that the enzyme and the chromogen compound were not effectively immobilized in a 96-well plate because dopamine could not self-polymerize due to the lack of catalysis by laccase at pH 6.5.

Claims

1. A method for preparing a glucose biosensor material, comprising:

4-aminoantipyrine, dopamine and laccase are mixed according to the mass ratio of 1.5-1.6: 1:1, adding the buffer solution into a phosphate buffer solution, and adjusting the pH value of the buffer solution to 6.0-6.5 to obtain a weak acid buffer solution; adding glucose oxidase and catalase into a weak acid buffer solution, and mixing to obtain the glucose biosensor material.

2. The method of claim 1, wherein the phosphate buffer solution comprises disodium hydrogen phosphate and potassium dihydrogen phosphate, and the disodium hydrogen phosphate, the disodium hydrogen phosphate and the 4-aminoantipyrine are mixed in a molar ratio of 12:8: 0.15.

3. The method of claim 1 or 2, wherein the mass ratio of glucose oxidase to catalase is 1: 1.

4. The method of claim 1, wherein the dopamine phosphate buffer has a pH of 6.5.

5. The method of claim 1, wherein the dopamine is a dopamine containing catechol structure.

6. The glucose biosensor material produced by the method for producing a glucose biosensor material according to any one of claims 1 to 5.

7. Use of the glucose biosensor material according to claims 1 to 5 for the detection of glucose.