CN103760161B - A kind of colorimetric detection method of glucose - Google Patents
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- CN103760161B CN103760161B CN201410035356.9A CN201410035356A CN103760161B CN 103760161 B CN103760161 B CN 103760161B CN 201410035356 A CN201410035356 A CN 201410035356A CN 103760161 B CN103760161 B CN 103760161B
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Abstract
The invention discloses a kind of colorimetric detection method of glucose, be by sample through pre-service, with damping fluid dilution after mix with glucose oxidase, reaction Hydrogen Peroxide, then molybdenum disulfide solution, developer and damping fluid is added, after hybrid reaction, visual colorimetry or ultraviolet-visible spectrophotometer is adopted to measure the content drawing glucose in sample.The present invention utilizes glucose oxidase glucose Hydrogen Peroxide, be the chromogenic reaction of catalyst hydrogen peroxide and developer again with molybdenum disulfide, adopt visual colorimetry semiquantitative determination concentration of glucose, or by ultraviolet spectrophotometer quantitative measurement concentration of glucose.The invention solves in prior art high to the operation requirements of glucose detection, testing process is complicated, and testing cost is high, and detection time is long, the problems such as background interference is large, method cost of the present invention is low, easy and simple to handle, can realize glucose content in visual quick detection sample.
Description
Technical field
The invention belongs to glucose detection technical field, be specifically related to a kind of colorimetric detection method of glucose.
Background technology
In recent years due to the change of dietary structure, be becoming tight the day of rhythm of life and the impact of the factors such as much few more dynamic life styles of sitting, the incidence of disease rapid development of whole world diabetes, makes diabetes become the chronic disease of the third-largest serious threat human health after tumour, cardiovascular pathological changes.The detection of glucose in serum content is the Main Means of clinical examination and monitoring diabetes.The state health standards reference method (standard No.: WS/T 350-2011) detected for serum glucose level is at present the method adopting hexokinase and glucose-6-phosphate dehydrogenase (G6PD) associating.These native enzyme have higher substrate specificity and catalytic efficiency in a mild condition, but the shortcoming such as native enzyme exists purification difficult, easy in inactivation, not easily preserves, price is higher.Detect in Patents at the glucose content announced at present, be mainly the electrochemical method (patent No.: 200710176028.0; The patent No.: 200910067282.6; The patent No.: 201010617684.1; The patent No.: 201110083906.0; The patent No.: 201110391001.X; The patent No.: 201110343243.1; The patent No.: 201110345081.5; The patent No.: 201220071992.3; The patent No.: 201210189645.5; The patent No.: 201210416267.X; The patent No.: 201310087177.5; The patent No.: 201310141232.4; The patent No.: 201310290148.9; The patent No.: 201310354574.4).Electrochemical Detection is highly sensitive, but be easily subject to the interference of other electroactive materials in blood oxygen and blood, and need to consume certain energy in testing process, in addition, the preparation of electrode is often comparatively complicated and cannot intuitive judgment serum glucose level, needs special instrument.200810073470.5), the near infrared spectroscopy (patent No.: 98100787.2), the chemoluminescence method (patent No.: 200910236714.1 other methods detecting glucose are as the resonance scattering spectrometry (patent No.:; 201210509888.2), the fluorescent spectrometry (patent No.: 200680029822.6 patent No.:; 201310250093.9) etc. the patent No.: all need to rely on expensive special instrument and the detection that just can complete under the operation of professional and technical personnel glucose.201210459910.7), the test paper (patent No.: 200410033020.5 and the related kit of the glucose Fast Measurement (patent No.:; 201210579953.9) etc. the patent No.: because of its complex structure, manufacturing process is loaded down with trivial details, expensive, is also very restricted in actual applications.Colorimetric detection method based on enzymatic system is detection method more popular in recent years, it utilizes the detection of change realization to object of system color and colourity in course of reaction, there is detection limit low, highly sensitive advantage, but often need to use peroxidase (HRP), and peroxidase price is higher, easy in inactivation, not easily preserve.Therefore, someone proposes the method detecting serum glucose based on analogue enztme again.Find that nano material tri-iron tetroxide has class Catalyzed Synthesis By Peroxidase activity (Nature Nanotechnology from Yan Xiyun seminar of the Chinese Academy of Sciences, 2007,2,577-583), the people such as Wang Erkang utilize this characteristic to achieve detection (Analytical Chemistry to hydrogen peroxide and glucose, 2008,80,2250-2254), graphene oxide is also found to have class peroxidase activity, and has been applied to detection (Advanced Materials, 2010 of hydrogen peroxide and glucose, 20,2255-2262).In the patent of application, people also propose the method (patent No.: 201110275358.1 based on analogue enztme colorimetric detection glucose; The patent No.: 201310244132.4; The patent No.: 201310260524.X).But these method costs are high, color change comparatively single, not easily preserve carry.
Summary of the invention
The object of the present invention is to provide a kind of colorimetric detection method of glucose, utilize glucose oxidase glucose Hydrogen Peroxide, be that the chromogenic reaction of catalyst hydrogen peroxide and developer is to detect glucose again with molybdenum disulfide, can solve in prior art high to the operation requirements of glucose detection, testing process is complicated, testing cost is high, detection time is long, the problems such as background interference is large, method cost of the present invention is low, easy and simple to handle, the content that can realize glucose in visual quick detection sample.
For achieving the above object, the present invention adopts following technical scheme:
A kind of colorimetric detection method of glucose, through pre-service by sample, mix with glucose oxidase with after damping fluid dilution, reaction Hydrogen Peroxide, then add molybdenum disulfide solution, developer and damping fluid and carry out chromogenic reaction, adopt visual colorimetry, reacted solution colour and color standards series are compared, or after chromogenic reaction, add sulfuric acid solution cessation reaction, then adopt the concentration of visual colorimetry semiquantitative determination glucose.
The preparation of described color standards series is diluted with damping fluid by dextrose standard sample, after the Glucose standards solution being mixed with known variable concentrations and glucose oxidase hybrid reaction, add molybdenum disulfide solution, developer and damping fluid to carry out chromogenic reaction and namely obtain color standards series, or after chromogenic reaction, add sulfuric acid solution cessation reaction obtain color standards series.
A kind of colorimetric detection method of glucose, through pre-service by sample, mix with glucose oxidase with after damping fluid dilution, reaction Hydrogen Peroxide, then add molybdenum disulfide solution, developer and damping fluid, adopt ultraviolet-visible spectrophotometer to measure the absorbance of solution after chromogenic reaction, or after chromogenic reaction, add sulfuric acid solution cessation reaction, adopt ultraviolet-visible spectrophotometer to measure the absorbance of solution again, and calculate the content of glucose according to typical curve equation.
The foundation of described typical curve equation is diluted with damping fluid by dextrose standard sample, after the Glucose standards solution being mixed with known variable concentrations and glucose oxidase hybrid reaction, add molybdenum disulfide solution, developer and damping fluid, ultraviolet-visible spectrophotometer is adopted to be determined at the absorbance at 652 nm wavelength places after chromogenic reaction, or after chromogenic reaction, add sulfuric acid solution cessation reaction, ultraviolet-visible spectrophotometer is adopted to measure absorbance at 450 nm wavelength places again, and using absorbance as ordinate, the concentration of glucose is as horizontal ordinate, drawing standard curve also draws typical curve equation.
The pH value of dilute sample and dextrose standard sample damping fluid used is 3-9.
In chromogenic reaction, the pH value of damping fluid used is 1-9.
The temperature of chromogenic reaction is 30 ~ 60 DEG C.
Developer used is TMB, 2,2-connection nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts or o-phenylenediamines.
remarkable advantage of the present invention is:
(1) the present invention is by glucose oxidase glucose Hydrogen Peroxide, then produces color by the agent of molybdenum disulfide catalyzed coloration and hydroperoxidation and change, to indicate the variable concentrations of glucose.Concentration of glucose is different, then solution colour and shade are all different; Can judge whether glucose exceeds standard by visual inspection, and need not by any instrument, therefore testing cost is low, easy and simple to handle.
(2) molybdenum disulfide used in the present invention is without the need to modifying the detection that namely can be used for glucose.
(3) in the present invention, the detectability of spectrophotometry glucose can reach 1 μM, and the range of linearity is 5 μMs to 150 μMs (R
2=0.9992).
(4) reaction conditions of the present invention is gentle, and detection speed is fast, favorable reproducibility, and without the need to the detecting instrument of costliness, easy and simple to handle, visual quick identification and the detection of glucose can be realized.
Embodiment
The detection of Standard glucose solution: the glucose solution of the variable concentrations prepared with 180 μ L damping fluids (pH 3-9) by 20 μ L glucose oxidase solutions mixes, 30 min are reacted at 30 ~ 60 DEG C, then the molybdenum disulfide solution of 0.05 mL, 0.05 mL developer and 0.2 mL buffer solution (pH 1-9) is added, mixing, after placing 30 min, visual colorimetry is utilized to carry out semi-quantitative analysis, or utilize UV-VIS spectrophotometry to measure absorbance at 652 nm wavelength places, carry out quantitative test; Or add 10 μ L 20%(V/V) after sulfuric acid solution cessation reaction, utilize visual colorimetry to carry out semi-quantitative analysis or utilize UV-VIS spectrophotometry to measure absorbances at 450 nm wavelength places, carrying out quantitative test.
Glucose detection in sample: get 30 μ L samples, be diluted with water to 50 μ L, then adds 500 μ L Ba (OH)
2solution, mixing, then add 500 μ L ZnSO
4solution, at centrifugal 10 min of 3880 rpm after mixing.Get 200 μ L supernatants, dilute 5 times with damping fluid (pH 3-9), as test sample.Other operations are with the mensuration of Standard glucose solution.
Developer used is TMB, 2,2-connection nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts or o-phenylenediamines.
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
The detection of Standard glucose solution: by 20 μ L glucose oxidase solutions (10 mg/mL) and 180 μ L Acetic acid-sodium acetate damping fluid (10 mM, pH 3) prepare variable concentrations glucose solution mixing, 30 min are reacted at 30 DEG C, then the molybdenum disulfide solution (18 mg/L) of 0.05 mL is added, 0.05 mL 2, 2-joins nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (20 mM) and 0.2 mL NaAc_HAc buffer solution (10 mM, pH 1), mixing, after placing 30 min, visual colorimetry is utilized to carry out semi-quantitative analysis, or utilize UV-VIS spectrophotometry to measure absorbance at 405 nm wavelength places, carry out quantitative test.
Serum glucose detects: get 30 μ L blood serum samples, be diluted with water to 50 μ L, then add 500 μ L Ba (OH)
2solution (0.11 M), mixing, then add 500 μ L ZnSO
4solution (0.0765 M), at centrifugal 10 min of 3880 rpm after mixing.Get 200 μ L supernatants, with Acetic acid-sodium acetate damping fluid (10mM, pH 3) dilution 5 times, as test sample.Other operations are with the mensuration of Standard glucose solution.
Embodiment 2
The detection of Standard glucose solution: by 20 μ L glucose oxidase solutions (10 mg/mL) and 180 μ L Tris-HCl damping fluid (10mM, pH 9) prepare variable concentrations glucose solution mixing, 30 min are reacted at 60 DEG C, then the molybdenum disulfide solution (18 mg/L) of 0.05 mL is added, 0.05 mL o-phenylenediamine (0.3M) and 0.2 mL Tris-HCl buffer solution (10mM, pH 9), mixing, after placing 30min, visual colorimetry is utilized to carry out semi-quantitative analysis, or utilize UV-VIS spectrophotometry to measure absorbance at 450 nm wavelength places, carry out quantitative test.
Serum glucose detects: get 30 μ L blood serum samples, be diluted with water to 50 μ L, then add 500 μ L Ba (OH)
2solution (0.11 M), mixing, then add 500 μ L ZnSO
4solution (0.0765 M), at centrifugal 10 min of 3880 rpm after mixing.Get 200 μ L supernatants, with Tris-HCl damping fluid (10mM, pH 9) dilution 5 times, as test sample.Other operations are with the mensuration of Standard glucose solution.
Embodiment 3
The detection of Standard glucose solution: by 20 μ L glucose oxidase solutions (10 mg/mL) and 180 μ L Tris-HCl damping fluid (10mM, pH 6.9) concentration prepared is followed successively by 0 μM, 5 μMs, 20 μMs, 40 μMs, 60 μMs, 80 μMs, 100 μMs, the Standard glucose solution of 150 μMs mixes respectively, 30 min are reacted under 37 C, then 0.05 mL molybdenum disulfide solution (18 mg/L) is added, 0.05 mL 3, 3 ', 5, 5 '-tetramethyl biphenyl amine aqueous solution (12 mM) and 0.2 mL Tris-HCl buffer solution (10mM, pH 6.9), mixing, place 30 min at 30 DEG C after, visual colorimetry is utilized to carry out semi-quantitative analysis, or utilize UV-VIS spectrophotometry to measure absorbance at 652 nm wavelength places, carry out quantitative test, or add 10 μ L 20%(V/V) after sulfuric acid solution cessation reaction, utilize visual colorimetry to carry out semi-quantitative analysis or utilize UV-VIS spectrophotometry to measure absorbances at 450 nm wavelength places, carrying out quantitative test.
Result shows, along with the increase of concentration of glucose, and molybdenum disulfide catalysis 3,3 ', 5, the product that 5 '-tetramethyl benzidine carries out chromogenic reaction increases gradually in the absorbance of about 652 nm, the color from yellow of corresponding solution gradually becomes light green color, then fades to blueness by blue-green.After adding sulfuric acid cessation reaction, along with the increase of concentration of glucose, molybdenum disulfide catalysis 3,3 ', 5, the product that 5 '-tetramethyl benzidine carries out chromogenic reaction increases gradually in the absorbance of about 450 nm, and the color from pale yellow look of corresponding solution gradually becomes buff, and linear equation is Y=0.234+0.0114X(R
2=0.9992).
Serum glucose detects: get 30 μ L blood serum samples and be diluted with water to 50 μ L, then add 500 μ L Ba (OH)
2solution (0.11 M), mixing, then add 500 μ L ZnSO
4solution (0.0765 M), at centrifugal 10 min of 3880 rpm after mixing.Get 200 μ L supernatants, with Tris-HCl damping fluid (10 mM, pH 6.9) dilution 5 times as test sample, other operations are with the mensuration of Standard glucose solution.
Result shows, and along with the rising of glucose in serum concentration, test sample solution colour (blue or buff) is deepened gradually.
The present invention utilizes glucose oxidase glucose Hydrogen Peroxide, be that the chromogenic reaction of catalyst hydrogen peroxide and developer is to detect glucose again with molybdenum disulfide, along with concentration of glucose is different, solution colour and shade are all different, can judge whether glucose exceeds standard by visual inspection, and any instrument need not be relied on, therefore testing cost is low, easy and simple to handle.And the detectability of spectrophotometry glucose can reach 1 μM, the range of linearity is 5 μMs to 150 μMs (R
2=0.9992).Reaction conditions of the present invention is gentle, and detection speed is fast, and favorable reproducibility, can realize visual quick identification and the detection of glucose.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (5)
1. the colorimetric detection method of a glucose, it is characterized in that: by sample through pre-service, mix with glucose oxidase with after damping fluid dilution, reaction Hydrogen Peroxide, then add molybdenum disulfide solution, developer and damping fluid and carry out chromogenic reaction, adopt visual colorimetry, reacted solution colour and color standards series are compared, or after chromogenic reaction, add sulfuric acid solution cessation reaction, then adopt the concentration of visual colorimetry semiquantitative determination glucose;
The preparation of described color standards series is diluted with damping fluid by dextrose standard sample, after the Glucose standards solution being mixed with known variable concentrations and glucose oxidase hybrid reaction, add molybdenum disulfide solution, developer and damping fluid to carry out chromogenic reaction and namely obtain color standards series, or after chromogenic reaction, add sulfuric acid solution cessation reaction obtain color standards series;
Developer used is TMB, 2,2-connection nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts or o-phenylenediamines.
2. the colorimetric detection method of a glucose, it is characterized in that: by sample through pre-service, with damping fluid dilution after mix with glucose oxidase, reaction Hydrogen Peroxide, then molybdenum disulfide solution, developer and damping fluid is added, ultraviolet-visible spectrophotometer is adopted to measure the absorbance of solution after chromogenic reaction, or after chromogenic reaction, add sulfuric acid solution cessation reaction, adopt ultraviolet-visible spectrophotometer to measure the absorbance of solution again, and calculate the content of glucose according to typical curve equation;
The foundation of described typical curve equation is diluted with damping fluid by dextrose standard sample, after the Glucose standards solution being mixed with known variable concentrations and glucose oxidase hybrid reaction, add molybdenum disulfide solution, developer and damping fluid, ultraviolet-visible spectrophotometer is adopted to measure the absorbance of solution after chromogenic reaction, or after chromogenic reaction, add sulfuric acid solution cessation reaction, ultraviolet-visible spectrophotometer is adopted to measure the absorbance of solution again, and using absorbance as ordinate, the concentration of glucose is as horizontal ordinate, drawing standard curve also draws typical curve equation,
Developer used is TMB, 2,2-connection nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts or o-phenylenediamines.
3., according to the colorimetric detection method of the arbitrary described glucose of claim 1 or 2, it is characterized in that: the pH value of dilute sample and dextrose standard sample damping fluid used is 3-9.
4., according to the colorimetric detection method of the arbitrary described glucose of claim 1 or 2, it is characterized in that: in chromogenic reaction, the pH value of damping fluid used is 1-9.
5., according to the colorimetric detection method of the arbitrary described glucose of claim 1 or 2, it is characterized in that: the temperature of chromogenic reaction is 30 ~ 60 DEG C.
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| JP2838333B2 (en) * | 1991-10-30 | 1998-12-16 | 日本特殊陶業株式会社 | Glucose concentration measuring device and glucose concentration measuring method |
| JP5188111B2 (en) * | 2006-07-10 | 2013-04-24 | 協和メデックス株式会社 | Aniline derivative, and method, quantitative reagent, and quantitative kit for component to be quantified in sample using the same |
| CN101571485A (en) * | 2008-04-28 | 2009-11-04 | 北京华大吉比爱生物技术有限公司 | Method and kit for measuring glucose |
| CN101387606A (en) * | 2008-08-01 | 2009-03-18 | 中国科学院长春应用化学研究所 | Method for detecting hydrogen peroxide or glucose based on enzyme simulation by ferroferric oxide magnetic nanometer particle |
| CN102042979B (en) * | 2010-10-28 | 2013-04-03 | 河南工业大学 | Method for quickly detecting content of glucose in starch fermentation liquor |
| CN103115919A (en) * | 2012-12-27 | 2013-05-22 | 中国科学院过程工程研究所 | Glucose detection test paper |
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