CN107383871B - Preparation and application of fluorescein isothiocyanate modified poly-dopamine composite material loaded with glucose oxidase - Google Patents
Preparation and application of fluorescein isothiocyanate modified poly-dopamine composite material loaded with glucose oxidase Download PDFInfo
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- 239000002131 composite material Substances 0.000 title claims abstract description 61
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 235000019420 glucose oxidase Nutrition 0.000 title claims abstract description 25
- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 16
- 239000004366 Glucose oxidase Substances 0.000 title claims abstract description 16
- 229940116332 glucose oxidase Drugs 0.000 title claims abstract description 16
- 229920001690 polydopamine Polymers 0.000 title claims abstract description 16
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- 239000008103 glucose Substances 0.000 claims abstract description 73
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 16
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- 229960001149 dopamine hydrochloride Drugs 0.000 claims description 10
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- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
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- C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
- C08L79/04—Polycondensates having nitrogen-containing heterocyclic rings in the main chain; Polyhydrazides; Polyamide acids or similar polyimide precursors
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention provides a preparation method of a fluorescein isothiocyanate functionalized polydopamine composite material capable of loading glucose oxidase. The method solves the problems of high operation requirement on glucose detection, complex detection process, high detection cost, long detection time, large background interference and the like in the prior art, and has the advantages of low cost, simple and convenient operation and capability of quickly detecting the glucose content in the sample.
Description
Technical Field
The invention belongs to the technical field of glucose detection, and particularly relates to a preparation method and application of a fluorescein isothiocyanate modified poly-dopamine composite material loaded with glucose oxidase.
Background
Direct blood detection is commonly used in clinical and home blood glucose monitors, and frequent blood collection can cause discomfort to patients. Urine glucose examination is the simplest method for noninvasive primary screening of diabetes mellitus, urine of healthy people almost has no glucose, only when the blood glucose concentration is higher than a renal glucose threshold (8.88-9.99 mmol/L), the urine has glucose, and the concentration and the blood glucose concentration have good correlation, but the current screening test paper is influenced by various factors and cannot accurately reflect the blood glucose concentration, so that the reference value of the method is limited. As proved by researches, for individuals, the glucose concentration in body fluids such as saliva and sweat before meals of human bodies has good correlation with the blood glucose concentration, can be used for indirectly reflecting the blood glucose concentration, and has huge application value in the fields of early screening of diabetes, blood glucose detection and the like if the glucose content in the body fluids can be accurately measuredThe value, however, is generally 2X 10 due to its low glucose concentration-2～2.4×10-1mmo/L), it has not been possible to detect accurately with commercially available blood glucose monitors.
Among the patents related to glucose content measurement that have been published so far, electrochemical methods are mainly used (patent No. 201310087177.5; patent No. 201310141232.4; patent No. 201310290148.9; patent No. 201310354574.4). The electrochemical detection has high sensitivity, but is easily interfered by blood oxygen and other electroactive substances in blood, certain energy is consumed in the detection process, and the preparation of the electrode is complex. Other methods for detecting glucose, such as resonance scattering spectroscopy (patent No. 200810073470.5), near infrared spectroscopy (patent No. 98100787.2), chemiluminescence (patent No. 200910236714.1; patent No. 201210509888.2), fluorescence spectroscopy (patent No. 200680029822.6; patent No. 201310250093.9), etc., require expensive professional instruments and are performed by a professional technician. However, the related reagent kit (patent No. 201210459910.7), test paper (patent No. 200410033020.5; patent No. 201210579953.9), etc. for rapid determination of glucose are greatly limited in practical application due to their complicated structure, complicated manufacturing process, and high price.
The colorimetric detection method based on an enzyme catalysis system is a popular detection method in recent years, realizes the detection of a target object by utilizing the change of system color and chromaticity in the reaction process, has the advantages of low detection limit and high sensitivity, but often needs peroxidase (HRP) which has high price, is easy to inactivate and is difficult to store. Therefore, a method for detecting serum glucose based on a mimic enzyme has been proposed. Since Yanxinyu topic group of China academy of sciences, discovered that nano-material ferroferric oxide has peroxidase-like catalytic activity (Nature Nanotechnology, 2007, 2, 577-. In the patent application, a method for colorimetric detection of glucose based on a mimetic enzyme is also proposed (patent No. 201110275358.1; patent No. 201310244132.4; patent No. 201310260524. X). However, these methods are costly, relatively single in color change, and not easy to store and carry.
The invention provides a preparation method of a fluorescein isothiocyanate modified poly-dopamine composite material loaded with glucose oxidase.
Disclosure of Invention
Aiming at the defects, the invention provides a method for detecting glucose, which solves the problems of high operation requirement, complex detection process, high detection cost, long detection time, large background interference and the like in the prior art, and the method for preparing the material is simple and environment-friendly, has low cost, simple and convenient operation and high sensitivity, and can quickly detect the content of the glucose in a sample.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of fluorescein isothiocyanate modified poly-dopamine composite material loaded with glucose oxidase comprises the following steps:
A. preparation of PDA-FITC composite material: adding ammonia water into a beaker filled with ethanol water solution, adjusting the pH value to 8.4-8.6 to obtain mixed solution, then adding dopamine hydrochloride and Fluorescein Isothiocyanate (FITC), placing the mixture in a water bath at 30 ℃ and stirring the mixture for 28-30h to obtain suspension, adding HCl dilute solution into the suspension, adjusting the pH value to 4.8-5.0, placing the suspension in a centrifuge for centrifugation to obtain lower-layer precipitate, dispersing the centrifuged lower-layer precipitate into distilled water to obtain the PDA-FITC composite material water solution, wherein the volume ratio of the ammonia water to the ethanol water solution is 1: (48-52), the mass ratio of the dopamine hydrochloride to the Fluorescein Isothiocyanate (FITC) is (50-200): (2.5-10), the solid content of the dopamine hydrochloride in an ethanol aqueous solution is (1.5-7) mg/mL, and the volume ratio of distilled water to the ethanol aqueous solution is 1: 2;
B. preparation of PDA-FITC-GOx composite material: adding PBS buffer solution, glucose oxidase GOx and distilled water into the PDA-FITC composite material aqueous solution prepared in the step A, carrying out constant temperature oscillation reaction for 24 hours, adding HCl dilute solution to adjust the pH value to 4.8-5.0, centrifuging the suspension with the adjusted pH value by using a centrifugal machine to obtain lower-layer precipitate, dispersing the centrifuged lower-layer precipitate into the distilled water, adding NaOH dilute solution to adjust the pH value to 7.3-7.5, and obtaining the PDA-FITC-GOx composite material aqueous solution; wherein the volume ratio of the PDA-FITC composite material aqueous solution to the PBS buffer solution to the distilled water is 5:1:14, the volume ratio of the first distilled water to the second distilled water is 4:10, and the solid content of the glucose oxidase GOx in the PDA-FITC composite material aqueous solution is (0.5-2) mg/mL.
Preferably, the mass ratio of the dopamine hydrochloride to fluorescein isothiocyanate FITC is 20: 1.
The volume percentage concentration of the ethanol water solution is 25-35%.
The mass percentage concentration of the ammonia water is 28%, the concentration of the HCl dilute solution is 1mol/L, and the concentration of the NaOH dilute solution is 1 mol/L.
The pH of the PBS buffer solution is 6.3-6.6.
The constant temperature was 25 ℃.
The centrifugal rotating speed is 6000-.
The application of the PDA-FITC-GOx composite material prepared by the preparation method of the fluorescein isothiocyanate modified poly-dopamine oxidase-loaded composite material in the aspect of detecting the concentration of glucose adopts the following steps:
taking a series of 5mL colorimetric tubes with plug scales, respectively adding 100 mu L of the prepared PDA-FITC-GOx composite material aqueous solution into each colorimetric tube, respectively adding 0-80 mu L of glucose solutions with different volumes into each colorimetric tube, diluting each colorimetric tube to 1mL, carrying out constant temperature oscillation reaction for 1h, placing in a fluorescence cuvette, and measuring fluorescence intensity by using a fluorescence spectrometer at an excitation wavelength of 470nm and an emission wavelength of 523 nm; and drawing in a rectangular coordinate system by taking the glucose concentration as an abscissa and the fluorescence intensity value of the reaction solution at the wavelength of 523nm as an ordinate to obtain a linear relation graph between the glucose concentration and the fluorescence intensity, and detecting the glucose concentration according to the linear relation between the glucose concentration and the fluorescence intensity.
The working curve in the linear relation graph is that y is 200.25+42.93x, and the linear correlation coefficient R is2Is 0.98.
The final concentration of the glucose solution is 0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M, 60. mu.M, 70. mu.M and 80. mu.M respectively.
The application of the PDA-FITC-GOx composite material in the aspect of detecting the concentration of glucose comprises the following specific steps:
A. preparation of PDA-FITC composite material: adding 0.6mL of ammonia water into 30mL of ethanol water solution to adjust the pH value to 8.4-8.6, then adding 50-200 mg of dopamine hydrochloride and 2.5-10 mg of Fluorescein Isothiocyanate (FITC), placing the mixture into a water bath at 30 ℃, stirring the mixture for 30 hours, adding a dilute HCl solution to adjust the pH value to 5.0, centrifuging the suspension with the adjusted pH value by using a centrifuge to obtain a lower-layer precipitate, and re-dispersing the centrifuged lower-layer precipitate into 60mL of distilled water to obtain a PDA-FITC composite material water solution;
B. preparation of PDA-FITC-GOx composite material: adding 1mL of PBS buffer solution with the pH value of 6.5, 5-40mg of glucose oxidase GOx and 4mL of distilled water into 5mL of the PDA-FITC composite material aqueous solution prepared in the step A, carrying out constant-temperature oscillation reaction for 24 hours, adding a dilute HCl solution to adjust the pH value to 4.8-5.2, centrifuging the solution with the adjusted pH value by using a centrifuge to obtain a lower-layer precipitate, re-dispersing the centrifuged lower-layer precipitate into 10mL of distilled water, adding a dilute NaOH solution to adjust the pH value to 7.4, and obtaining the PDA-FITC-GOx composite material aqueous solution;
C. and (3) detection of glucose: taking 11 5mL colorimetric tubes with plug scales, respectively adding 100 μ L of the PDA-FITC-GOx composite material aqueous solution prepared in the step B into each colorimetric tube, and then sequentially adding 0 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 3 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L and 80 μ L into each colorimetric tube respectively, wherein the concentration of the mixture is 1 × 10-3Diluting each colorimetric tube of the M glucose solution to 1mL, carrying out constant-temperature oscillation reaction for 1h, placing the obtained product in a fluorescence cuvette, and measuring the fluorescence intensity by using a fluorescence spectrometer at an excitation wavelength of 470nm and an emission wavelength of 523 nm; plotting the glucose concentration as abscissa and the fluorescence intensity value of the reaction solution at 523nm wavelength as ordinate in a rectangular coordinate system to obtainThe linear relation graph between the glucose concentration and the fluorescence intensity has a working curve of y being 200.25+42.93x and a linear correlation coefficient R2And 0.98, and the concentration of glucose was measured based on the linear relationship between them.
The detection method can eliminate the interference of other substances; the other substances are as follows: NaCl, KCl, CaCl2D-sorbose, D-fructose, D-galactose, D-mannose and D-maltose.
The invention has the beneficial effects that: the method adopts Fluorescein Isothiocyanate (FITC) modified polydopamine PDA composite material PDA-FITC-GOx loaded with glucose oxidase GOx, utilizes glucose to generate gluconic acid under the action of the glucose oxidase, the gluconic acid can quench the FITC, other saccharides do not influence the fluorescence intensity, and the detection of the concentration of the glucose is realized by the fluorescence spectrometry. The method solves the problems of high operation requirement on glucose detection, complex detection process, high detection cost, long detection time, large background interference and the like in the prior art, and has the advantages of low cost, simple and convenient operation and capability of quickly detecting the glucose content in the sample.
Drawings
FIG. 1 is a TEM representation of a PDA-FITC composite;
FIG. 2 is a chart of infrared spectra of PDA, FITC, and PDA-FITC composites;
FIG. 3 is a fluorescence spectrum diagram of a PDA-FITC-GOx composite material for detecting glucose;
FIG. 4 is a graph of the linear relationship between glucose concentration and fluorescence intensity;
FIG. 5 is a histogram of fluorescence intensity of glucose and other substances at 523nm in the PDA-FITC-GOx detection system, in which 0 is a glucose-added blank control, and 1-8 are added 1. mu.L of NaCl, KCl and CaCl at a concentration of 1mol/L2D-sorbose, D-fructose, D-galactose, D-mannose and D-maltose;
FIG. 6 is a graph showing an interference test in which other substances are added to a glucose test system, wherein 0 is a blank control, and 1 to 8 are 1. mu.L of NaCl, KCl, and,CaCl2D-sorbose, D-fructose, D-galactose, D-mannose and D-maltose.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the invention, are given by way of illustration and explanation only. In the examples, all raw materials were purchased from the gard chemical network.
Example 1
Preparation of PDA-FITC-GOx composite material
1. Preparation of PDA-FITC composite material:
adding 0.6mL of ammonia water into 30mL of ethanol water solution to adjust the pH value to 8.5, then adding 100mg of dopamine hydrochloride and 5mg of Fluorescein Isothiocyanate (FITC), placing the mixture into a water bath at 30 ℃ to stir for 30h, then adding a dilute HCl solution to adjust the pH value to 5.0, centrifuging the suspension with the adjusted pH value for 30min at a rotation speed rate of 6000 rpm by using a centrifuge to obtain a lower-layer precipitate, and re-dispersing the centrifuged lower-layer precipitate into 60mL of distilled water to obtain the PDA-FITC composite water solution. The obtained PDA-FITC composite material is uniform in size and has particle size distribution of 150-200nm as shown in figure 1, and the infrared spectrum of figure 2 shows that fluorescein isothiocyanate FITC is successfully loaded on the polydopamine microspheres.
2. Preparation of PDA-FITC-GOx composite material: adding 1mL of PBS buffer solution with the pH value of 6.5, 20mg of glucose oxidase GOx and 4mL of distilled water into 5mL of the PDA-FITC composite material aqueous solution prepared above, carrying out constant-temperature oscillation reaction at 25 ℃ for 24 hours, adding a dilute HCl solution to adjust the pH value to 5.0, centrifuging the suspension with the adjusted pH value by using a centrifuge to obtain a lower-layer precipitate, re-dispersing the centrifuged lower-layer precipitate into 10mL of distilled water, and adding a dilute NaOH solution to adjust the pH value to 7.4; the PDA-FITC-GOx composite material is calibrated by a 3,3 ', 5, 5' -tetramethylbenzidine colorimetric method (TMB), and the loading amount of GOx in the composite material is measured to be 1.52 mu g/mL.
Example 2
PDA-FITC-GOx was used for glucose detection, comprising the following steps:
taking 11 5mL colorimetric tubes with plug scales, and adding the colorimetric tubes into each colorimetric tube respectively100 mu L of the aqueous solution of the PDA-FITC-GOx composite material prepared above is added into each colorimetric tube in sequence, and the concentration is 1 multiplied by 10, 5, 10, 15, 20, 3, 40, 50, 60, 70 and 80 mu L-3Diluting each colorimetric tube of the M glucose solution to 1mL, carrying out constant-temperature oscillation reaction at 25 ℃ for 1h, placing the obtained product in a fluorescence cuvette, and measuring fluorescence intensity by using a fluorescence spectrometer at an excitation wavelength of 470nm and an emission wavelength of 523 nm; as shown in FIG. 3, it can be seen that the fluorescence intensity decreased with increasing concentration of added glucose, indicating that the synthesized PDA-FITC-GOx composite can be used for the determination of glucose.
As shown in FIG. 4, the fluorescence intensity of the reaction solution at 523nm is plotted in a rectangular coordinate system with the glucose concentration as the abscissa and the fluorescence intensity value as the ordinate, it can be seen that there is a good linear relationship between the glucose concentration and the fluorescence intensity, the linear correlation coefficient is 0.98, the glucose concentration is good in the range of 0-80 μ M, the operating curve in the linear relationship is y 200.25+42.93x, and the glucose concentration is detected according to the linear relationship.
Example 3
Interference experiment
Taking 95 mL colorimetric tubes with plug scales, marking as 0-8, respectively adding 100 μ L of the above prepared PDA-FITC-GOx composite aqueous solution, and then adding 50 μ L of 1 × 10 concentration colorimetric tube marked as 0-3M glucose solution, and sequentially adding 1 μ L of 1mol/L (NaCl, KCl, CaCl) solution into a colorimetric tube labeled as 1-82D-sorbose, D-fructose, D-galactose, D-mannose and D-maltose) interference substances, diluting each colorimetric tube to 1mL, carrying out constant temperature oscillation reaction at 25 ℃ for 1h, placing the colorimetric tube in a fluorescence cuvette, and measuring the fluorescence intensity by using a fluorescence spectrometer at an excitation wavelength of 470nm and an emission wavelength of 523 nm; taking the added substance as the abscissa and the fluorescence intensity of the system at 523nm as the ordinate as the histogram, as shown in FIG. 5, it can be seen from the graph that the fluorescence intensity value of the system at 523nm after adding different substances, except the glucose solution, the fluorescence intensity is not obviously reduced by adding other substances, which indicates that the system dissolves glucoseThe liquid has better selectivity.
Marking 95 mL colorimetric tubes with plug scales as 0-8, respectively adding 100 μ L of the above prepared PDA-FITC-GOx composite aqueous solution, and sequentially adding 50 μ L of 1 × 10 concentration colorimetric tubes-3M glucose solution, and sequentially adding 1 μ L of 1mol/L (NaCl, KCl, CaCl) solution into a colorimetric tube labeled as 1-82D-sorbose, D-fructose, D-galactose, D-mannose and D-maltose) interference substances, diluting each colorimetric tube to 1mL, carrying out constant temperature oscillation reaction at 25 ℃ for 1h, placing the colorimetric tube in a fluorescence cuvette, and measuring the fluorescence intensity by using a fluorescence spectrometer at an excitation wavelength of 470nm and an emission wavelength of 523 nm; the added substance is used as the abscissa, the fluorescence intensity of the system at 523nm is used as the ordinate to make a histogram, as shown in fig. 6, it can be seen from the graph that other interfering substances have little interference to the detection experiment of the glucose solution, which indicates that the PDA-FITC-GOx system has good interference resistance to the glucose detection.
Claims (9)
1. A preparation method of fluorescein isothiocyanate modified poly-dopamine composite material loaded with glucose oxidase is characterized by comprising the following steps:
A. preparation of PDA-FITC composite material: adding ammonia water into a beaker filled with ethanol water solution, adjusting the pH value to 8.4-8.6 to obtain mixed solution, then adding dopamine hydrochloride and Fluorescein Isothiocyanate (FITC), placing the mixture in a water bath at 30 ℃ and stirring the mixture for 28-30h to obtain suspension, adding HCl dilute solution into the suspension, adjusting the pH value to 4.8-5.0, placing the suspension in a centrifuge for centrifugation to obtain lower-layer precipitate, dispersing the centrifuged lower-layer precipitate into distilled water to obtain the PDA-FITC composite material water solution, wherein the volume ratio of the ammonia water to the ethanol water solution is 1: (48-52), the mass ratio of the dopamine hydrochloride to the Fluorescein Isothiocyanate (FITC) is (50-200): (2.5-10), the solid content of the dopamine hydrochloride in an ethanol aqueous solution is (1.5-7) mg/mL, and the volume ratio of distilled water to the ethanol aqueous solution is 1: 2;
B. preparation of PDA-FITC-GOx composite material: adding PBS buffer solution, glucose oxidase GOx and distilled water into the PDA-FITC composite material aqueous solution prepared in the step A, carrying out constant temperature oscillation reaction for 24 hours, adding HCl dilute solution to adjust the pH value to 4.8-5.0, centrifuging the suspension with the adjusted pH value by using a centrifugal machine to obtain lower-layer precipitate, dispersing the centrifuged lower-layer precipitate into the distilled water, adding NaOH dilute solution to adjust the pH value to 7.3-7.5, and obtaining the PDA-FITC-GOx composite material aqueous solution; wherein the volume ratio of the PDA-FITC composite material aqueous solution to the PBS buffer solution to the total distilled water in the step B is 5:1:14, the volume ratio of the first distilled water to the second distilled water is 4:10, and the solid content of the glucose oxidase GOx in the PDA-FITC composite material aqueous solution is (0.5-2) mg/mL.
2. The preparation method of the fluorescein isothiocyanate modified poly-dopamine oxidase-loaded composite material according to claim 1, wherein the preparation method comprises the following steps: the volume percentage concentration of the ethanol water solution is 25-35%.
3. The preparation method of the fluorescein isothiocyanate modified poly-dopamine oxidase-loaded composite material according to claim 1, wherein the preparation method comprises the following steps: the mass percentage concentration of the ammonia water is 28%, the concentration of the HCl dilute solution is 1mol/L, and the concentration of the NaOH dilute solution is 1 mol/L.
4. The preparation method of the fluorescein isothiocyanate modified poly-dopamine oxidase-loaded composite material according to claim 1, wherein the preparation method comprises the following steps: the pH of the PBS buffer solution is 6.3-6.6.
5. The preparation method of the fluorescein isothiocyanate modified poly-dopamine oxidase-loaded composite material according to claim 1, wherein the preparation method comprises the following steps: the constant temperature was 25 ℃.
6. PDA-FITC-GOx composite material obtained by the preparation method of any one of claims 1-5.
7. Use of the PDA-FITC-GOx composite of claim 6 for detecting glucose concentration.
8. Use of PDA-FITC-GOx composite according to claim 7 for the detection of glucose concentration, characterized in that the following method steps are used:
taking a series of 5mL colorimetric tubes with scales and plugs, respectively adding 100 mu L of the prepared PDA-FITC-GOx composite material aqueous solution into each colorimetric tube, sequentially adding 0-80 mu M of glucose solutions with different concentrations into each colorimetric tube, adding distilled water into each colorimetric tube to dilute to 1mL, carrying out constant temperature oscillation reaction for 1h, and placing the colorimetric tubes into a fluorescence cuvette to measure fluorescence intensity by using a fluorescence spectrometer at an excitation wavelength of 470nm and an emission wavelength of 523 nm; and drawing in a rectangular coordinate system by taking the glucose concentration as an abscissa and the fluorescence intensity value of the reaction solution at the wavelength of 523nm as an ordinate to obtain a linear relation graph between the glucose concentration and the fluorescence intensity, and detecting the glucose concentration according to the linear relation between the glucose concentration and the fluorescence intensity.
9. The use of PDA-FITC-GOx composite material in the detection of glucose concentration according to claim 7, wherein the steps are specifically as follows:
A. preparation of PDA-FITC composite material: adding 0.6mL of ammonia water into 30mL of ethanol water solution to adjust the pH value to 8.4-8.6, then adding 50-200 mg of dopamine hydrochloride and 2.5-10 mg of Fluorescein Isothiocyanate (FITC), placing the mixture into a water bath at 30 ℃, stirring the mixture for 30 hours, adding a dilute HCl solution to adjust the pH value to 5.0, centrifuging the suspension with the adjusted pH value by using a centrifuge to obtain a lower-layer precipitate, and re-dispersing the centrifuged lower-layer precipitate into 60mL of distilled water to obtain a PDA-FITC composite material water solution;
B. preparation of PDA-FITC-GOx composite material: adding 1mL of PBS buffer solution with the pH value of 6.5, 5-40mg of glucose oxidase GOx and 4mL of distilled water into 5mL of the PDA-FITC composite material aqueous solution prepared in the step A, carrying out constant-temperature oscillation reaction for 24 hours, adding a dilute HCl solution to adjust the pH value to 4.8-5.2, centrifuging the solution with the adjusted pH value by using a centrifuge to obtain a lower-layer precipitate, re-dispersing the centrifuged lower-layer precipitate into 10mL of distilled water, adding a dilute NaOH solution to adjust the pH value to 7.4, and obtaining the PDA-FITC-GOx composite material aqueous solution;
C. and (3) detection of glucose: taking 11 5ml colorimetric tubes with plug scales, respectively adding 100 μ L of the PDA-FITC-GOx composite material aqueous solution prepared in the step B into each colorimetric tube, and then sequentially adding 0 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 3 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L and 80 μ L into each colorimetric tube, wherein the concentration of the mixture is 1 × 10-3Adding distilled water into each colorimetric tube of the glucose solution of M to dilute to 1mL, carrying out constant-temperature oscillation reaction for 1h, placing the colorimetric tube in a fluorescence cuvette, and measuring fluorescence intensity by using a fluorescence spectrometer at an excitation wavelength of 470nm and an emission wavelength of 523 nm; and drawing in a rectangular coordinate system by taking the glucose concentration as an abscissa and the fluorescence intensity value of the reaction solution at the wavelength of 523nm as an ordinate to obtain a linear relation graph between the glucose concentration and the fluorescence intensity, and detecting the glucose concentration according to the linear relation between the glucose concentration and the fluorescence intensity.
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