CN103760161A - Colorimetric detection method for glucose - Google Patents
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 83
- 239000008103 glucose Substances 0.000 title claims abstract description 77
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 239000000243 solution Substances 0.000 claims abstract description 59
- 238000006243 chemical reaction Methods 0.000 claims abstract description 49
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 20
- CWQXQMHSOZUFJS-UHFFFAOYSA-N molybdenum disulfide Chemical compound S=[Mo]=S CWQXQMHSOZUFJS-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229910052982 molybdenum disulfide Inorganic materials 0.000 claims abstract description 20
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 17
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 17
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 17
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 17
- 230000000007 visual effect Effects 0.000 claims abstract description 16
- 238000004737 colorimetric analysis Methods 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 238000013016 damping Methods 0.000 claims description 26
- 239000012530 fluid Substances 0.000 claims description 26
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 22
- 238000002835 absorbance Methods 0.000 claims description 18
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 14
- 238000010790 dilution Methods 0.000 claims description 7
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- 238000002360 preparation method Methods 0.000 claims description 7
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 4
- VPUSOBLQICITSB-UHFFFAOYSA-N [NH4+].[NH4+].C(C)N1CSC2=C1C=CC(=C2)S(=O)(=O)[O-].C(C)N2CSC1=C2C=CC(=C1)S(=O)(=O)[O-].[N] Chemical class [NH4+].[NH4+].C(C)N1CSC2=C1C=CC(=C2)S(=O)(=O)[O-].C(C)N2CSC1=C2C=CC(=C1)S(=O)(=O)[O-].[N] VPUSOBLQICITSB-UHFFFAOYSA-N 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 13
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 238000012113 quantitative test Methods 0.000 description 6
- 238000012764 semi-quantitative analysis Methods 0.000 description 6
- 102000003992 Peroxidases Human genes 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 description 4
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- 206010012601 diabetes mellitus Diseases 0.000 description 3
- CVIFVNPRIBETLU-BTVCFUMJSA-N hydrogen peroxide;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OO.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O CVIFVNPRIBETLU-BTVCFUMJSA-N 0.000 description 3
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- 238000002798 spectrophotometry method Methods 0.000 description 2
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- CMZYGFLOKOQMKF-UHFFFAOYSA-N 1-(3,5-dimethylphenyl)-3,5-dimethylbenzene Chemical group CC1=CC(C)=CC(C=2C=C(C)C=C(C)C=2)=C1 CMZYGFLOKOQMKF-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a colorimetric detection method for glucose. The colorimetric detection method for the glucose comprises the steps of pretreating a sample, adding a buffer solution to dilute the sample, mixing the sample with glucose oxidase, performing reaction to generate hydrogen peroxide, then adding a molybdenum disulfide solution, a developing agent and the buffer solution, and measuring the content of the glucose in the sample by a visual colorimetry method or through an ultraviolet-visible light spectrophotometer after mixing reaction is implemented. According to the colorimetric detection method, glucose oxidase is used for oxidizing the glucose to generate the hydrogen peroxide, and molybdenum disulfide serving as a catalyst is used for catalyzing the hydrogen peroxide to generate color development reaction with the developing agent; the glucose concentration is measured in a semi-quantified manner by the visual colorimetry method or is measured in a quantified manner through the ultraviolet-visible light spectrophotometer. According to the colorimetric detection method for the glucose, the problems of high operation requirement on glucose detection, complicated detection process, high detection cost, long detection time, high background interference and the like in the prior art are solved; the method disclosed by the invention is low in cost and easy to operate; the glucose content of the sample can be quickly detected in a visual manner.
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 because the day of the change of dietary structure, rhythm of life is becoming tight and the impact of the factors such as few moving life styles of sitting more, 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) detecting for serum glucose level is at present the method that adopts hexokinase and glucose-6-phosphate dehydrogenase (G6PD) associating.These native enzyme have higher substrate specificity and catalytic efficiency under temperate condition, but native enzyme exist purification difficult, easily inactivation, the shortcoming such as be difficult for preserving, price is higher.At the glucose content of having announced at present, detect in Patents, 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 in testing process, need to consume certain energy, 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 that detect 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 rely on expensive professional instrument and just can complete the detection to glucose under professional and technical personnel's operation.201210459910.7), the test paper (patent No.: 200410033020.5 the and 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 more popular in recent years detection method, it utilizes the variation of system color and colourity in course of reaction to realize the detection to object, there is detection limit low, highly sensitive advantage, but often need to use peroxidase (HRP), and higher, the easy inactivation of peroxidase price, be difficult for preserve.Therefore, someone proposes to detect based on analogue enztme the method for serum glucose again.From Yan Xiyun seminar of the Chinese Academy of Sciences, find that nano material tri-iron tetroxide has class Catalyzed Synthesis By Peroxidase activity (Nature Nanotechnology, 2007,2,577-583), the people such as Wang Erkang utilize this characteristic to realize detection (the 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, change color is comparatively single, be difficult for preservation, 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 oxidizing glucose Hydrogen Peroxide, take molybdenum disulfide as the chromogenic reaction of catalyst hydrogen peroxide and developer, detect glucose again, can solve in prior art the operation requirements to glucose detection high, testing process complexity, testing cost is high, detection time is long, the problems such as background interference is large, the content that method cost of the present invention is low, easy and simple to handle, can realize glucose in visual fast detecting 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, with mixing with glucose oxidase after damping fluid dilution, reaction Hydrogen Peroxide, then add molybdenum disulfide solution, developer and damping fluid to 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 that dextrose standard sample is diluted with damping fluid, be mixed with after the glucose standard solution and glucose oxidase hybrid reaction of known variable concentrations, add molybdenum disulfide solution, developer and damping fluid to carry out chromogenic reaction and obtain color standards series, or after chromogenic reaction, add sulfuric acid solution cessation reaction to obtain color standards series.
A kind of colorimetric detection method of glucose, through pre-service by sample, with mixing with glucose oxidase after damping fluid dilution, reaction Hydrogen Peroxide, then add molybdenum disulfide solution, developer and damping fluid, after chromogenic reaction, adopt ultraviolet-visible spectrophotometer to measure the absorbance of solution, or after chromogenic reaction, add sulfuric acid solution cessation reaction, adopt again ultraviolet-visible spectrophotometer to measure the absorbance of solution, and according to typical curve equation, calculate the content of glucose.
The foundation of described typical curve equation is that dextrose standard sample is diluted with damping fluid, be mixed with after the glucose standard solution and glucose oxidase hybrid reaction of known variable concentrations, add molybdenum disulfide solution, developer and damping fluid, after chromogenic reaction, adopt ultraviolet-visible spectrophotometer to be determined at the absorbance at 652 nm wavelength places, or after chromogenic reaction, add sulfuric acid solution cessation reaction, adopt again ultraviolet-visible spectrophotometer to measure absorbance at 450 nm wavelength places, 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 ℃.
Developer used is TMB, 2,2-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts or o-phenylenediamine.
remarkable advantage of the present invention is:
(1) the present invention passes through glucose oxidase oxidizing glucose Hydrogen Peroxide, then produces change color by molybdenum disulfide catalysis developer and hydroperoxidation, to indicate the variable concentrations of glucose.Concentration of glucose difference, solution colour and shade are all different; By visual inspection, can judge whether glucose exceeds standard, and needn't be by any instrument, therefore testing cost is low, easy and simple to handle.
(2) molybdenum disulfide used in the present invention is without modifying the detection that can be used for glucose.
(3) in the present invention, the detectability of spectrophotometry glucose can reach 1 μ M, and the range of linearity is that 5 μ M are to 150 μ M(R
2=0.9992).
(4) reaction conditions gentleness of the present invention, detection speed is fast, favorable reproducibility, and without expensive detecting instrument, easy and simple to handle, can realize visual quick identification and the detection of glucose.
Embodiment
The detection of standard glucose solution: 20 μ L glucose oxidase solutions are mixed with the glucose solution of 180 μ variable concentrations of damping fluid (pH 3-9) preparation for L, at 30 ~ 60 ℃, react 30 min, then add molybdenum disulfide solution, 0.05 mL developer and the 0.2 mL buffer solution (pH 1-9) of 0.05 mL, mix, place after 30 min, utilize visual colorimetry 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, carry out quantitative test.
Glucose detection in sample: get 30 μ L samples, be diluted with water to 50 μ L, then add 500 μ L Ba (OH)
2solution, mixes, then adds 500 μ L ZnSO
4solution, after mixing at centrifugal 10 min of 3880 rpm.Get 200 μ L supernatants, with 5 times of damping fluid (pH 3-9) dilutions, as test sample.Other operations are with the mensuration of standard glucose solution.
Developer used is TMB, 2,2-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts or o-phenylenediamine.
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 acetic acid-sodium-acetate buffer (10 mM for L, pH 3) preparation variable concentrations glucose solution mix, at 30 ℃, react 30 min, then add the molybdenum disulfide solution (18 mg/L) of 0.05 mL, 0.05 mL 2, 2-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (20 mM) and 0.2 mL NaAc_HAc buffer solution (10 mM, pH 1), mix, place after 30 min, utilize visual colorimetry 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), mixes, then adds 500 μ L ZnSO
4solution (0.0765 M), after mixing at centrifugal 10 min of 3880 rpm.Get 200 μ L supernatants, with 5 times of acetic acid-sodium-acetate buffer (10mM, pH 3) dilutions, 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 Tris-HCl damping fluid (10mM for 180 μ L, pH 9) preparation variable concentrations glucose solution mix, at 60 ℃, react 30 min, then add the molybdenum disulfide solution (18 mg/L) of 0.05 mL, 0.05 mL o-phenylenediamine (0.3M) and 0.2 mL Tris-HCl buffer solution (10mM, pH 9), mix, place after 30min, utilize visual colorimetry 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), mixes, then adds 500 μ L ZnSO
4solution (0.0765 M), after mixing at centrifugal 10 min of 3880 rpm.Get 200 μ L supernatants, with 5 times of Tris-HCl damping fluid (10mM, pH 9) dilutions, 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 Tris-HCl damping fluid (10mM for 180 μ L, pH 6.9) preparation concentration be followed successively by 0 μ M, 5 μ M, 20 μ M, 40 μ M, 60 μ M, 80 μ M, 100 μ M, the standard glucose solution of 150 μ M mixes respectively, under 37 C, react 30 min, then add 0.05 mL molybdenum disulfide solution (18 mg/L), 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), mix, at 30 ℃, place after 30 min, utilize visual colorimetry 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, carry out quantitative test.
Result demonstration, 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 652 nm left and right, the color of corresponding solution gradually becomes light green color by yellow, then fades to blueness by blue-green.Add after 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 450 nm left and right, and the color of corresponding solution is by the light yellow buff that gradually becomes, 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), mixes, then adds 500 μ L ZnSO
4solution (0.0765 M), after mixing at centrifugal 10 min of 3880 rpm.Get 200 μ L supernatants, with 5 times of conduct test samples of Tris-HCl damping fluid (10 mM, pH 6.9) dilution, other operations are with the mensuration of standard glucose solution.
Result shows, along with the rising of glucose in serum concentration, tests sample solution colour (blueness or buff) and deepens gradually.
The present invention utilizes glucose oxidase oxidizing glucose Hydrogen Peroxide, take molybdenum disulfide as the chromogenic reaction of catalyst hydrogen peroxide and developer, detect glucose again, along with concentration of glucose difference, solution colour and shade are all different, by visual inspection, can judge whether glucose exceeds standard, and needn't rely on any instrument, therefore testing cost is low, easy and simple to handle.And the detectability of spectrophotometry glucose can reach 1 μ M, the range of linearity is that 5 μ M are to 150 μ M(R
2=0.9992).Reaction conditions gentleness of the present invention, 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 of doing 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 (8)
1. the colorimetric detection method of a glucose, it is characterized in that: by sample through pre-service, with mixing with glucose oxidase after damping fluid dilution, reaction Hydrogen Peroxide, then add molybdenum disulfide solution, developer and damping fluid to 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.
2. the colorimetric detection method of glucose according to claim 1, it is characterized in that: the preparation of described color standards series is that dextrose standard sample is diluted with damping fluid, be mixed with after the glucose standard solution and glucose oxidase hybrid reaction of known variable concentrations, add molybdenum disulfide solution, developer and damping fluid to carry out chromogenic reaction and obtain color standards series, or after chromogenic reaction, add sulfuric acid solution cessation reaction to obtain color standards series.
3. the colorimetric detection method of a glucose, it is characterized in that: after sample is diluted through pre-service, with damping fluid, mix with glucose oxidase, reaction Hydrogen Peroxide, then add molybdenum disulfide solution, developer and damping fluid, after chromogenic reaction, adopt ultraviolet-visible spectrophotometer to measure the absorbance of solution, or after chromogenic reaction, add sulfuric acid solution cessation reaction, adopt again ultraviolet-visible spectrophotometer to measure the absorbance of solution, and according to typical curve equation, calculate the content of glucose.
4. the colorimetric detection method of glucose according to claim 3, it is characterized in that: the foundation of described typical curve equation is that dextrose standard sample is diluted with damping fluid, be mixed with after the glucose standard solution and glucose oxidase hybrid reaction of known variable concentrations, add molybdenum disulfide solution, developer and damping fluid, after chromogenic reaction, adopt ultraviolet-visible spectrophotometer to measure the absorbance of solution, or after chromogenic reaction, add sulfuric acid solution cessation reaction, adopt again ultraviolet-visible spectrophotometer to measure the absorbance of solution, and using absorbance as ordinate, the concentration of glucose is as horizontal ordinate, drawing standard curve also draws typical curve equation.
5. according to the colorimetric detection method of the arbitrary described glucose of claim 1-4, it is characterized in that: the pH value of dilute sample and dextrose standard sample damping fluid used is 3-9.
6. according to the colorimetric detection method of the arbitrary described glucose of claim 1-4, it is characterized in that: in chromogenic reaction, the pH value of damping fluid used is 1-9.
7. according to the colorimetric detection method of the arbitrary described glucose of claim 1-4, it is characterized in that: the temperature of chromogenic reaction is 30 ~ 60 ℃.
8. according to the colorimetric detection method of the arbitrary described glucose of claim 1-4, it is characterized in that: developer used is 3,3 ', 5,5 '-tetramethyl benzidine, 2,2-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts or o-phenylenediamine.
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Cited By (23)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103954618A (en) * | 2014-04-23 | 2014-07-30 | 叶伟荣 | Method for determining concentration of glucose by using colorimetric method |
| CN105572065A (en) * | 2016-01-29 | 2016-05-11 | 福建医科大学 | Glucose detection kit based on platinum-bismuth nano mimic peroxidase |
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