CN109632732B - Near-infrared fluorescence sensitization method for determining glucose - Google Patents
Near-infrared fluorescence sensitization method for determining glucose Download PDFInfo
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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
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- 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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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/1717—Systems in which incident light is modified in accordance with the properties of the material investigated with a modulation of one or more physical properties of the sample during the optical investigation, e.g. electro-reflectance
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/1717—Systems in which incident light is modified in accordance with the properties of the material investigated with a modulation of one or more physical properties of the sample during the optical investigation, e.g. electro-reflectance
- G01N2021/1725—Modulation of properties by light, e.g. photoreflectance
Abstract
The invention belongs to the technical field of functional material preparation and application, and provides a near-infrared fluorescence sensitization method for measuring glucose in order to solve the problems of false positive and serious interference of a conventional fluorescence quenching method. According to the invention, a gold nano-glucose oxidase hybrid material is used as a near-infrared fluorescent probe, after glucose is added, the near-infrared fluorescence of gold nanoparticles is enhanced, and the enhancement degree of the fluorescence is in direct proportion to the concentration of the glucose, so that the detection of the glucose is realized. The detection method is simple and convenient to operate and quick in response, the maximum emission of the fluorescent probe is in a near infrared region, the interference of autofluorescence and Raman Rayleigh scattering and the like of coexisting substances, particularly biological samples, can be effectively avoided, and meanwhile, compared with a fluorescence quenching method, the fluorescence sensitization method has higher sensitivity and selectivity.
Description
Technical Field
The invention belongs to the technical field of preparation and application of functional materials, and particularly relates to a near-infrared fluorescence sensitization method for determining glucose.
Background
Glucose is an essential nutrient substance in the basic life activity process of organisms, can directly participate in metabolism, and has important significance for ensuring the energy supply of various tissues and organs of human bodies and keeping the health of the human bodies. In addition to physiological requirements, glucose has found widespread use in the food industry, in pharmaceutical manufacturing, in environmental monitoring, and in bioprocess monitoring.
In recent years, various methods have been developed for detecting the glucose content, such as high performance liquid chromatography, optical rotation, spectrophotometry, electrochemical method, and fluorescence method. However, these methods have certain disadvantages, such as the chromatography usually requires sample pretreatment in the detection process, and the operation is complicated; the optical rotation law is only suitable as an auxiliary method; adding a color developing agent in the detection by a spectrophotometry; electrochemical methods have high sensitivity but are less reproducible. Compared with other detection methods, the fluorescence method generally has the advantages of high sensitivity, good selectivity, simple and quick operation, capability of realizing on-line real-time analysis and the like. However, the conventional fluorescence method is a quantitative detection method based on the concentration relationship between the quenching degree of fluorescence and the object to be detected, and in practical application, impurities in the sample may greatly cause the quenching of the fluorescence of the probe, cause false positive signals, and finally affect the detection result. The fluorescence sensitization method is used for analyzing and detecting through the correlation between the enhancement degree of fluorescence intensity and the concentration of an object to be detected, so that the interference of false positive signals can be greatly reduced, and the sensitivity and the selectivity of detection are improved.
Disclosure of Invention
The invention provides a near-infrared fluorescence sensitization method for measuring glucose, aiming at solving the problems of false positive and serious interference of the conventional fluorescence quenching method, and the method takes gold nano-glucose oxidase hybrid material as a fluorescence probe, and has the advantages of simple operation, quick response, high sensitivity and good selectivity.
The invention adopts the following technical scheme: a near-infrared fluorescence sensitization method for determining glucose is characterized in that gold nanoparticles with the particle size less than 2 nm are prepared by taking a sulfhydryl compound as a modifier, and are adsorbed on glucose oxidase through electrostatic action to prepare a gold nano-glucose oxidase hybrid material; the gold nano-glucose oxidase hybrid material is used as a near-infrared fluorescent probe, after glucose is added, the near-infrared fluorescence of the gold nano particles is enhanced, and the enhancement degree of the fluorescence is in direct proportion to the concentration of the glucose, so that the detection of the glucose is realized.
The preparation method of the gold nano-glucose oxidase near-infrared fluorescent probe comprises the following steps:
(1) preparing gold nanoparticles: with mercapto compoundsN-acetyl-L-cysteine as ligand, by NaBH4Reduction of HAuCl4∙3H2O to synthesize gold nanoparticles; detailed description of the inventionThe method comprises the following steps: respectively weighing chloroauric acid and N-acetyl-L-cysteine in a molar ratio of 1:2.5-1:4, dissolving the chloroauric acid and the N-acetyl-L-cysteine in a methanol/glacial acetic acid solvent in a volume ratio of 6:1 to fully dissolve the chloroauric acid and the N-acetyl-L-cysteine, then placing the solution in an ice bath, adding a sodium borohydride ethanol solution under stirring, reacting for 30-50 min, adding acetone to stop the reaction, centrifuging to remove the solvent to obtain a crude gold nanoparticle product; re-dissolving the crude product with secondary water, then putting the crude product into a dialysis bag for purification, and carrying out vacuum drying to obtain powdery gold nanoparticles;
(2) preparing a gold nano-glucose oxidase hybrid material: respectively weighing gold nanoparticles and glucose oxidase in a mass ratio of 1:5-1:25, dissolving the gold nanoparticles and the glucose oxidase in secondary water, and stirring for 5 hours; adding a proper amount of methanol, ethanol or acetone into the reaction solution, centrifuging at 13000 rpm and 10 ℃ for 15 min, removing supernatant, and performing vacuum drying on the precipitate to obtain the gold nano-glucose oxidase hybrid material.
The standard curve of the glucose detection is obtained by the following steps:
(1) weighing the prepared gold nano-glucose oxidase hybrid material, dissolving the gold nano-glucose oxidase hybrid material in secondary water to prepare 0.05 mg/ml solution, putting 2 ml of the solution in a cuvette, placing the cuvette on a sample rack of a fluorescence photometer, and scanning the fluorescence spectrum of the cuvette, wherein the excitation wavelength is 520 nm, and the scanning range of the emission wavelength is 550-850 nm;
(2) preparing glucose standard solutions with a plurality of concentration gradients;
(3) respectively adding the prepared glucose standard solution into the gold nano-glucose oxidase solution, incubating for 10 min at normal temperature, and sequentially scanning fluorescence spectrum;
(4) processing the obtained data, and taking the concentration of glucose as an abscissa, wherein the fluorescence intensity of the gold nano-glucose oxidase hybrid material is enhancedF/F 0 Plotted as ordinate, whereinF 0 Is the fluorescence intensity at 680 nm of the maximum emission peak of the fluorescent probe when no glucose is added,Fis a corresponding fluorescent probe after adding glucoseAnd performing linear fitting on the dot diagram to obtain a standard curve equation for detecting the glucose.
The detection steps of the glucose in the actual sample are as follows:
(1) preparing a gold nano-glucose oxidase hybrid material with the concentration of 0.05 mg/ml, and putting 2 ml in a cuvette;
(2) after centrifugal filtration or centrifugal ultrafiltration is carried out on an actual sample, 50 mu l of the actual sample is added into the cuvette, the actual sample is incubated for 10 min at normal temperature, and then the fluorescence spectrum is scanned, wherein the excitation wavelength is 520 nm, and the scanning range of the emission wavelength is 550-850 nm;
(3) the intensity of the maximum emission peak can be obtained from the fluorescence spectrumFFurther calculated to obtainF/F 0 The value of (c) is substituted into a known standard curve equation to calculate the glucose concentration.
The invention has the following beneficial technical effects: according to the invention, the gold nanoparticles with the particle size of less than 2 nm are prepared by taking a sulfhydryl compound as a modifier, and the nanoparticles have excellent near-infrared fluorescence property; then adsorbing the hybrid material to glucose oxidase through electrostatic interaction to prepare a gold nano-glucose oxidase hybrid material; the hybrid material can be used as a near-infrared fluorescence probe, and glucose is determined through the correlation between the sensitization degree of near-infrared fluorescence of gold nanoparticles and the concentration of glucose.
(1) The method established by the invention is based on the fluorescence sensitization principle, can effectively avoid false positive interference caused by the conventional fluorescence quenching method, and well improves the detection sensitivity.
(2) The fluorescence emission of the gold nanoparticles prepared by the method is positioned in a near infrared region, so that the interference of background fluorescence and Rayleigh Raman scattering can be greatly reduced, and the gold nanoparticles have the advantages of simple preparation steps, mild synthesis conditions, and good water solubility, dispersibility, stability and biocompatibility.
(3) The invention uses the gold nano-glucose oxidase hybrid material as the fluorescent probe, and has good specificity for the identification of glucose, thereby improving the selectivity of the method and widening the linear range of detection. The method established by the method does not need a complex sample pretreatment process, is simple and convenient to operate, and can increase the stability of the enzyme by combining the gold nanoparticles and the glucose oxidase.
The invention can be used for measuring glucose in food, industry, environment and life systems.
Drawings
Fig. 1 is a TEM image of the gold nano-glucose oxidase hybrid material prepared in example 1. FIG. 2 is a fluorescence spectrum of the near-infrared fluorescent probe prepared in example 1. FIG. 3 is a graph showing the effect of glucose on the fluorescence spectrum of the prepared fluorescent probe in example 1. FIG. 4 is a standard curve for glucose measurement in example 1. FIG. 5 is a graph showing the effect of glucose on the fluorescence spectrum of the prepared fluorescent probe in example 2. FIG. 6 is a standard curve for glucose measurement in example 2.
Detailed Description
Example 1: a near-infrared fluorescence sensitization method for determining glucose is characterized in that a gold nano-glucose oxidase hybrid material is used as a near-infrared fluorescence probe, after glucose is added, the near-infrared fluorescence of gold nano particles is enhanced, and the enhancement degree of the fluorescence is in direct proportion to the concentration of the glucose, so that the glucose is detected.
(1) And (3) synthesis of gold nanoparticles: 0.0394g of HAuCl was weighed out separately4∙3H2O and 0.0490g of N-acetyl-L-cysteine, mixing and dissolving in 5 ml of methanol/glacial acetic acid (volume ratio is 6: 1) organic solvent, placing in an ice bath, rapidly adding an ethanol solution containing 0.0775g of sodium borohydride while stirring, continuously stirring for controlling the reaction time to be 30 min, then adding 10 ml of acetone, and removing the solvent by centrifugation to obtain a crude product of the gold nanoparticles. Dissolving the obtained crude product in 1 ml of secondary water, transferring the crude product into a dialysis bag, purifying for 3 days, and then carrying out vacuum drying to obtain powdery gold nanoparticles.
(2) Preparing a gold nano-glucose oxidase hybrid material: weighing 10 mg of gold nanoparticles, dissolving the gold nanoparticles in 0.5 ml of secondary water, adding 10 ml of glucose oxidase solution with the concentration of 20.0 mg/ml under stirring, and reacting for 5 hours under slow stirring. Adding 10 ml methanol into the reacted solution, centrifuging for 15 min at 10 ℃ by a centrifuge with 13000 rpm, discarding the supernatant, and drying the precipitate in vacuum to obtain the gold nano-glucose oxidase hybrid material, wherein the attached figure 1 is a TEM image thereof.
(3) Drawing a standard curve for measuring glucose: weighing 0.1 mg of prepared gold nano-glucose oxidase hybrid material, dissolving the gold nano-glucose oxidase hybrid material in 2 ml of secondary water to prepare 0.05 mg/ml solution, putting the solution into a quartz cup, and placing the quartz cup in a sample groove of a fluorescence spectrometer to scan a fluorescence spectrogram (see attached figure 2). The excitation wavelength was set at 520 nm and the emission wavelength was swept over a range of 550-850 nm. As can be seen from FIG. 2, the maximum emission peak of gold nanoparticles is at 680 nm, located in the near infrared region.
To the above-mentioned gold nano-glucose oxidase solution, glucose solutions with concentrations of 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60 mmol/L were added, respectively. After incubation for 10 min at room temperature, the fluorescence spectra were recorded, respectively, and the results are shown in FIG. 3. Processing the obtained data, and taking the concentration of glucose as an abscissa, wherein the fluorescence intensity of the gold nano-glucose oxidase hybrid material is enhancedF/F 0 Plotted as ordinate, whereinF 0 Is the fluorescence intensity at 680 nm of the maximum emission peak of the fluorescent probe when no glucose is added,Fis the corresponding fluorescence intensity after adding glucose with a certain concentration, and finally, the standard curve graph is obtained by linear fitting of the dot diagram, as shown in figure 4. The linear range of the measured glucose obtained by the method is 5.0 multiplied by 10-6 ~ 6.0×10-4mol/L, regression equation is F/F0=1.0604c +1.3842 (c is 10)-4 mol/L) with a detection limit of 1.0X 10-7 mol/L，R2=0.9906。
(4) Detection of glucose in fresh human blood: taking fresh human blood, carrying out centrifugal ultrafiltration (the molecular weight cutoff is 3000) by using an ultrafiltration tube, transferring 50 mu l of filtrate by using a pipettor, adding the filtrate into 2 ml of 0.05 mg/ml gold nano-glucose oxidase hybrid material solution, incubating for 10 min at normal temperature, and scanning the fluorescence spectrum to obtain the intensity of the maximum emission peakFCalculatingF/ F 0 Into (a) value of3) The concentration of glucose in the test solution was found to be 1.42X 10 in the standard curve equation of (1)-4mol/L, and the human blood is diluted by 40 times after being added into the detection solution, so that the content of the glucose in the obtained human blood is 5.68 multiplied by 10-3mol/L. The results measured by the invention have no obvious difference compared with the standard method.
Example 2: a near-infrared fluorescence sensitization method for determining glucose is characterized in that a gold nano-glucose oxidase hybrid material is used as a near-infrared fluorescence probe, after glucose is added, the near-infrared fluorescence of gold nano particles is enhanced, and the enhancement degree of the fluorescence is in direct proportion to the concentration of the glucose, so that the glucose is detected.
(1) And (3) synthesis of gold nanoparticles: 0.0394g of HAuCl was weighed out separately4∙3H2O and 0.0572g of N-acetyl-L-cysteine, mixing and dissolving in 6 ml of methanol/glacial acetic acid (volume ratio is 6: 1) organic solvent, placing in an ice bath, rapidly adding an ethanol solution containing 0.0775g of sodium borohydride while stirring, continuously stirring and controlling the reaction time to be 40 min, then adding 10 ml of acetone, and removing the solvent by centrifugation to obtain a crude product of the gold nanoparticles. Dissolving the obtained crude product in 1 ml of secondary water, transferring the crude product into a dialysis bag, purifying for 3 days, and then carrying out vacuum drying to obtain powdery gold nanoparticles.
(2) Preparing a gold nano-glucose oxidase hybrid material: 10 mg of gold nanoparticles were weighed and dissolved in 0.5 ml of secondary water, 5 ml of glucose oxidase solution with a concentration of 20.0 mg/ml was added under stirring, and the reaction was carried out for 4.5 hours under slow stirring. To the reacted solution was added 10 ml of methanol, passed through a 13000 rpm centrifuge at 10oAnd C, centrifuging for 15 min, discarding supernatant, and performing vacuum drying on the precipitate to obtain the gold nano-glucose oxidase hybrid material.
(3) Drawing a standard curve for measuring glucose: weighing the prepared gold nano-glucose oxidase hybrid material, dissolving the gold nano-glucose oxidase hybrid material in secondary water to prepare 0.02 mg/ml solution, putting 0.5 ml in a quartz cup, and placing the quartz cup in a sample groove of a fluorescence spectrometer to scan a fluorescence spectrogram of the quartz cup. The excitation wavelength was set at 520 nm and the emission wavelength was swept over a range of 550-850 nm.
Glucose solutions with concentrations of 0, 0.04, 0.15, 0.30, 0.40, 0.50 and 0.55 mmol/L are respectively added into the gold nano-glucose oxidase solution. After incubation for 10 min at room temperature, the fluorescence spectra were recorded, respectively, and the results are shown in FIG. 5. Processing the obtained data, and taking the concentration of glucose as an abscissa, wherein the fluorescence intensity of the gold nano-glucose oxidase hybrid material is enhancedF/F 0 Plotted as ordinate, whereinF 0 Is the fluorescence intensity at 660 nm of the maximum emission peak of the fluorescent probe without adding glucose,Fis the corresponding fluorescence intensity after adding glucose with a certain concentration, and finally, the standard curve graph is obtained by linear fitting of the dot diagram, as shown in fig. 6. The linear range of the measured glucose obtained by the method is 4.0 multiplied by 10-6 ~ 5.5×10-4mol/L, regression equation ofF/F 0 = 0.4060c + 0.9679 (c is 10)-4 mol/L) with a detection limit of 1.0X 10-7 mol/L，R2=0.9965。
(4) Detection of glucose in great wall red wine: transferring 10 mu l of great wall dry red wine by using a pipettor, adding the great wall dry red wine into 2 ml of 0.05 mg/ml gold nano-glucose oxidase hybrid material solution, incubating for 10 min at normal temperature, and scanning the fluorescence spectrum to obtain the intensity of the maximum emission peakFCalculatingF/F 0 The value of (3) was substituted into the standard curve equation of (3), to obtain a glucose concentration of 9.9X 10 in the mixed solution-5mol/L, and the concentration of the wine is diluted by 200 times after the wine is added into the solution, so that the content of the glucose in the great wall dry red wine is 1.98 multiplied by 10-2mol/L. The results measured by the invention have no obvious difference compared with the standard method.
Claims (3)
1. A near-infrared fluorescence sensitization method for determining glucose is characterized in that: gold nanoparticles with the particle size less than 2 nm are prepared by taking a sulfhydryl compound as a modifier, and are adsorbed on glucose oxidase through electrostatic action to prepare a gold nano-glucose oxidase hybrid material; the gold nano-glucose oxidase hybrid material is used as a near-infrared fluorescent probe, after glucose is added, the near-infrared fluorescence of the gold nano particles is enhanced, and the enhancement degree of the fluorescence is in direct proportion to the concentration of the glucose, so that the detection of the glucose is realized;
the preparation method of the gold nano-glucose oxidase near-infrared fluorescent probe comprises the following steps:
1) preparing gold nanoparticles: with mercapto compoundsN-acetyl-L-cysteine as ligand, by NaBH4Reduction of HAuCl4∙3H2O to synthesize gold nanoparticles; the specific synthesis steps are as follows: respectively weighing chloroauric acid and N-acetyl-L-cysteine in a molar ratio of 1:2.5-1:4, dissolving the chloroauric acid and the N-acetyl-L-cysteine in a methanol/glacial acetic acid solvent in a volume ratio of 6:1 to fully dissolve the chloroauric acid and the N-acetyl-L-cysteine, then placing the solution in an ice bath, adding a sodium borohydride ethanol solution under stirring, reacting for 30-50 min, adding acetone to stop the reaction, centrifuging to remove the solvent to obtain a crude gold nanoparticle product; re-dissolving the crude product with secondary water, then putting the crude product into a dialysis bag for purification, and carrying out vacuum drying to obtain powdery gold nanoparticles;
2) preparing a gold nano-glucose oxidase hybrid material: respectively weighing gold nanoparticles and glucose oxidase in a mass ratio of 1:5-1:25, dissolving the gold nanoparticles and the glucose oxidase in secondary water, and stirring for 5 hours; adding a proper amount of methanol, ethanol or acetone into the reaction solution, centrifuging at 13000 rpm and 10 ℃ for 15 min, removing supernatant, and performing vacuum drying on the precipitate to obtain the gold nano-glucose oxidase hybrid material.
2. The method for detecting glucose according to claim 1, wherein the standard curve chart of glucose detection is obtained by the following steps:
1) weighing the prepared gold nano-glucose oxidase hybrid material, dissolving the gold nano-glucose oxidase hybrid material in secondary water to prepare 0.05 mg/ml solution, putting 2 ml of the solution in a cuvette, placing the cuvette on a sample rack of a fluorescence photometer, and scanning the fluorescence spectrum of the cuvette, wherein the excitation wavelength is 520 nm, and the scanning range of the emission wavelength is 550-850 nm;
2) preparing glucose standard solutions with a plurality of concentration gradients;
3) respectively adding the prepared glucose standard solution into the gold nano-glucose oxidase solution, incubating for 10 min at normal temperature, and sequentially scanning fluorescence spectrum;
4) processing the obtained data, and taking the concentration of glucose as an abscissa, wherein the fluorescence intensity of the gold nano-glucose oxidase hybrid material is enhancedF/F 0 Plotted as ordinate, whereinF 0 Is the fluorescence intensity at 680 nm of the maximum emission peak of the fluorescent probe when no glucose is added,Fthe standard curve equation for detecting the glucose is obtained by linearly fitting the dot diagram according to the fluorescence intensity of the maximum emission peak of the corresponding fluorescent probe after the glucose is added.
3. The method for detecting glucose by using near-infrared fluorescence sensitization according to claim 2, wherein the standard curve equation of the detected glucose is used for detecting the glucose in the actual sample, and the method comprises the following steps:
1) preparing a gold nano-glucose oxidase hybrid material with the concentration of 0.05 mg/ml, and putting 2 ml in a cuvette;
2) after centrifugal filtration or centrifugal ultrafiltration is carried out on an actual sample, 50 mu l of the actual sample is added into the cuvette, the actual sample is incubated for 10 min at normal temperature, and then the fluorescence spectrum is scanned, wherein the excitation wavelength is 520 nm, and the scanning range of the emission wavelength is 550-850 nm;
3) the intensity of the maximum emission peak can be obtained from the fluorescence spectrumFFurther calculated to obtainF/F 0 The value of (3) is substituted into the obtained standard curve equation to calculate the concentration of glucose.
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| CN102512181A (en) * | 2011-11-17 | 2012-06-27 | 东南大学 | Minimally invasive blood glucose monitoring microneedle and preparation method thereof |
| CN103509552B (en) * | 2012-06-29 | 2015-06-17 | 中国科学院大连化学物理研究所 | Functional near-infrared fluorescence nanoparticles and preparation and application thereof |
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