CN107561070B - A kind of glucose quick visualization detection method and its preparation and application - Google Patents
A kind of glucose quick visualization detection method and its preparation and application Download PDFInfo
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Abstract
The invention discloses a kind of glucose quick visualization detection methods and its application, reaction step to include：1. generating H using glucose in glucose oxidase (GOD) oxidation sample2O2；2. under the catalytic action of ultraviolet lighting (abbreviation hv) and disodium ethylene diamine tetraacetate (ethylenediaminetetraacetic acid), by the H of 3,3', 5,5'- tetramethyl benzidines (abbreviation TMB) and generation2O2Reaction；3. changing the concentration of glucose of judgement sample by solution absorbance.It can make visually classification and the quantitative analysis in a short time of glucose sample to be tested by adjusting photic catalysis.
Description
Technical field
The present invention relates to trace detection fields, and in particular to a kind of glucose quick visualization detection method and its prepare and
Using.
Background technology
Glucose (Glucose, chemical formula C6H12O6), it is the basic element of human body and most basic medical material, makees
With very extensive with purposes.It can be used for fermentation industry, be the major ingredient of fermentation medium as the carbon source of microorganism, such as anti-
Raw element, monosodium glutamate, vitamin, amino acid, organic acid, enzyme preparation etc. all need largely to use glucose, while also being used as microorganism
The raw material of fermentation polysaccharide and organic solvent.With numerous people improvement of living standard, glucose is also as the replacement of sucrose
It is applied to food service industry with sugar, such as candy industry, bakes industry, vegetables top grade is fresh-keeping, beverage and meat products, is the application of glucose
Broader field is opened up.The application of glucose industrially is also very wide, makees reducing agent in leather industry of printing and dyeing, in mirror processed
Industry, the glass liner of a thermos flask is silver-plated and the chemical silverings industry such as glass fibre is silver-plated also commonly use glucose and make reducing agent.Glucose can be into
Row hydrogenation, oxidation, isomery, alkaline bleach liquor degradation, esterification, acetalization reaction etc., synthesize or are converted into other products.Therefore glucose
Content detection is increasingly becoming important industrial detection project.
Its main application pharmaceutically can be divided into oral and two kinds of injection, and oral glucose individually can take or be made multidimensional
Glucose is taken.In addition, glucose blood of human body, urine either hydrops content increase or reduce can indicate some diseases
Generation.Especially for non-ketone stupor of some diabetes acute disease such as ketoacidosis and hypertonicity etc., need to examine in a short time
Patient blood glucose is surveyed, and other detections is coordinated to take corresponding emergency treatment.Therefore the quick detection of glucose is in industrial production and people
Body health aspect has great meaning.
The method of detection glucose mainly has at present：Titration, Plasmon Resonance, electrochemical methods, efficient liquid phase
Chromatography and gas chromatography.Wherein titration principle is simple, but sensitivity is not high, cannot detect micro-example；Electrochemistry
Analytic approach is easy to operate, apparatus structure is simple, easy to carry and accuracy is high, but selectivity is poor, detects easily by other objects
Matter is interfered, and practicability needs to be further increased；High performance liquid chromatography result is more accurate, high sensitivity, but the preceding place of sample
The reason time is long, is unsuitable for quickly measuring, and instrument and equipment is at high price, and promotion and popularization have certain difficulty；Gas-chromatography
Method has good linear relationship, but glucose is needed by silicon etherification process, and operation is more complicated.So current traditional detection
The more existence times of method are long, and poor sensitivity, specificity is not strong, and false positive rate is high, the low disadvantage of testing result repetitive rate.
Invention content
For the above-mentioned prior art the problem of, the purpose of the present invention is to provide a kind of glucose quick visualization detection sides
Method can make visually classification and the quantitative analysis in a short time of glucose sample to be tested by adjusting photic catalysis.
A kind of glucose quick visualization detection method is provided, reaction step includes：1. using glucose oxidase (GOD)
Glucose generates H in oxidation sample2O2；2. in urging for ultraviolet lighting (abbreviation hv) and disodium ethylene diamine tetraacetate (ethylenediaminetetraacetic acid)
Under change effect, by the H of 3,3', 5,5'- tetramethyl benzidines (abbreviation TMB) and generation2O2Reaction；3. being become by solution absorbance
Change the concentration of glucose of judgement sample.
The present invention generates H by GOD oxidizing glucoses2O2And glucose, in pair of special spectrum ultraviolet lighting and EDTA
Again under catalytic action, make TMB and the H of generation2O2Chromogenic reaction occurs, solution is changed from colourless to blue-green, the depth of color
That is the height of absorbance and the concentration of glucose in sample is proportional.Reaction mechanism of the present invention such as formula 1) shown in.
Preferably, step 2. in, the ultraviolet lighting spectrum be 254nm and/or 365nm.It is furthermore preferred that described ultraviolet
The photocatalytic reaction time is 2-8min.
Preferably, step 2. in, when ultraviolet light, EDTA, which receives 254nm and 365nm dual wavelength lights, can generate freedom
Electronics, the free electron is by H2O2Capture generates OH, and the OH generates the Ox-TMB of blue with TMB immediate responses.
Preferably, step 3. in, solution absorbance by visual observation colorimetric method or spectrophotometry judgement Ox-TMB discoloration
Concentration, and then judge the concentration of glucose of the sample.Preferably, the spectrophotometry is inhaled by detecting at 625nm wavelength
It receives peak judgement Ox-TMB and becomes colour saturation.It is furthermore preferred that by ultraviolet-visible spectrophotometry, to different concentration of glucose
Standard solution system carries out spectrum analysis, establishes the linear relationship of concentration of glucose and absorbance, and then to glucose in sample
Concentration carries out quantitative analysis.
Preferably, the Ox-TMB becomes 1. H that colour saturation is generated with step2O2Concentration is proportionate.
A kind of ultraviolet light induced application being catalyzed in the detection of glucose quick visualization, detecting step include：
A. glucose standards solution is configured：Glucose is quantitatively weighed to be completely dissolved in water；B. TMB standard solution is configured：
TMB is quantitatively weighed to be completely dissolved in dimethylformamide (abbreviation DMF)；C. EDTA standard solution is configured：Quantitatively weigh EDTA
It is completely dissolved in water；
Wherein, glucose standards solution:TMB standard solution:Concentration ratio is 1 between EDTA standard solution:100：100.
Resultant effect caused by the present invention is as described below：
1, electric in the enzymatic reaction of peroxide conversion enzyme in conventional glucose detection reaction according to kinetics
Sub- transfer time restricts whole reaction rate；And most peroxide conversion enzyme is of high cost, and preparation process is cumbersome, the period
It is long.For technical solution of the present invention using ultraviolet lighting and EDTA as dual inducing catalysis agent, rapid reaction is effective.
2, existing reaction is heated at high temperature especially in free electron and free radical generation phase, when needing long to reduce reaction more
Threshold energy, which greatly limits W-response rates.Chromogenic reaction of the present invention passes through the specific purple that mutually agrees with EDTA absorbing wavelengths
Outside line activated reactant improves hydrogen peroxide chemical potential indirectly.Therefore, the present invention can be achieved with visualization inspection in minutes
It surveys, and can further quantify, be better than half an hour in the even more long reaction time of other technologies scheme, and without heating.
3, the reagent cost that needs is cheap in present invention reaction, and preparation is simple and efficient, and property is stablized, be easy to long-term preservation and
Room temperature carries, and industrial prospect is wide.
4, the absorbance after TMB colour developings and the concentration of glucose are in 0.02~0.20mmolL-1In the range of have it is good
Linear relationship, detection precision is high, and the rate of recovery is strong, and sxemiquantitative and quantitative total detection time are short, have the characteristics that simple and effective.
Description of the drawings
Fig. 1 is the photic mechanism of catalytic reaction schematic diagram of detection method of the embodiment of the present invention.
Fig. 2 is that detection method of the embodiment of the present invention obtains different glucose sample colour developing schematic diagram.
Fig. 3 is that detection method of the embodiment of the present invention obtains each glucose sample UV, visible light spectrophotometric spectra figure.
Fig. 4 is that detection method of the embodiment of the present invention obtains each glucose sample concentration and its fitting of absorbance (Abs) is straight
Line chart.
Specific implementation mode
Explanation is further explained to the present invention with reference to specific embodiment and attached drawing, it should be appreciated that model of the invention
It encloses without being limited thereto.In the present embodiment, spectrophotometer selects the ultraviolet specrophotometer of Hitachi Hitachis, model U-
3900.Specifically, spectrophotometry used by solution glucose content is quantitatively detected, the state issued according to State General Administration for Quality Supervision
Mark GB/T9721-2006 is implemented.
Embodiment
A kind of glucose quick visualization detection method, as shown in Figure 1, reaction step includes：
1. the preparation of glucose standards solution：Precise 0.1000g glucose samples are placed in 50mL distilled water, fully
Dissolving, is made 10mmolL-1Glucose standards solution, it is spare.
The preparation of 2.TMB standard solution：The 3,3' of precise 0.0375g, 5,5'- tetramethyl benzidine are placed in 1mL's
In n,N-Dimethylformamide, fully dissolves, 0.1mmolL is made-1TMB standard solution, it is spare.
The preparation of 3.EDTA standard solution：Precise 0.1862g disodium ethylene diamine tetraacetates are placed in 50mL distilled water,
Fully dissolving, is made 10mmolL-1EDTA standard solution, it is spare.
The preparation of 4.GOD standard solution：The accurate glucose oxidase for weighing 0.0100g is dissolved in 1mL distilled water, is filled
Divide dissolving, it is spare.
5. the preparation of buffer solution：0.2M phosphate buffer solutions are configured, it is 6 to adjust pH.
6. pipetting 2 μ L TMB standard solution, 2 μ L EDTA standard solution, 3 μ L GOD standard solution respectively with liquid-transfering gun to add
Enter into 11 centrifuge tubes, embodiment number is 1,2,3,4,5,6,7,8,9,10 and 11.Then as shown in table 1, at 1~No. 11
In centrifuge tube, the 1mmolL of different volumes is added-1Glucose solution adds buffer solution, by each embodiment centrifuge tube Portugal
Grape sugar juice is made into a concentration of 0.02~0.20mmolL respectively-1, volume is the solution of 1000 μ L.In hand-held ultraviolet lamp
Illumination 5min under 254nm+365nm wavelength.
7. color change is observed, as shown in Fig. 2, colorimetric carries out semi-quantitative analysis by visual observation.
8. as shown in Figure 3-4, measuring absorbance at 625nm wavelength using ultraviolet-uisible spectrophotometer, being quantified
Analysis.
The result that glucose is detected using the present embodiment method is as in Figure 2-4：
From Fig. 2, it can be seen that gradually deepening with the increase blue-green of concentration of glucose from left to right, illustrate color change
The depth and concentration of glucose it is directly proportional.Thus, it is possible to the depths according to color, and sample concentration of glucose is observed with the naked eye out, side
Method is easy, visual and clear, total half-quantitative detection time≤8min.
As shown in Figure 3-4, each embodiment sample is detected using ultraviolet-visible spectrophotometry, the results showed that after TMB colour developings
Absorbance and glucose concentration in 0.02~0.20mmolL-1In the range of have good linear relationship, linear regression
Equation is A=1.8532C-0.0339, coefficient R2It is 0.9902, detection is limited to 0.01mmolL-1, the rate of recovery is
98.70%~103.35%, it always quantifies detection time and is less than or equal to≤20min.
In 1 Visual retrieval of table each embodiment centrifuge tube be added glucose amount and centrifuge tube in concentration of glucose
Although the present invention has been described in detail, it will be understood by those skilled in the art that in spirit and scope of the invention
Modification will be apparent.However, it should be understood that various aspects, different specific implementation mode that the present invention records
Each section and the various features enumerated can be combined or all or part of exchange.In above-mentioned each specific implementation mode, that
A little embodiments with reference to another embodiment can be combined suitably with other embodiment, this is will be by this field skill
Art personnel are to understand.In addition, it will be understood to those of skill in the art that the description of front is only exemplary mode, not purport
In the limitation present invention.
Claims (7)
1. a kind of application by the ultraviolet detection method for carrying out photic catalysis in the detection of glucose quick visualization, feature
It is, reaction step includes：1. generating H using glucose in glucose oxidase sample2O2；2. in ultraviolet lighting and second
Under the catalytic action of edetate disodium, by the H of 3,3', 5,5'- tetramethyl benzidines and generation2O2Reaction；3. passing through solution
Absorbance change judges the concentration of glucose of sample；
Step 2. in, the ultraviolet lighting wavelength is 254nm and 365nm, when ultraviolet light, EDTA receive 254nm and
365nm dual wavelength lights can generate free electron, and the free electron is by H2O2Capture generates OH, and the OH and TMB is anti-immediately
The Ox-TMB of blue should be generated.
2. application according to claim 1, which is characterized in that the ultraviolet lighting catalysis reaction time is 2-8min.
3. application according to claim 1, which is characterized in that step 3. in, solution absorbance colorimetric method by visual observation
Or spectrophotometry judgement Ox-TMB becomes colour saturation, and then judge the concentration of glucose of the sample.
4. application according to claim 3, which is characterized in that the spectrophotometry is inhaled by detecting at 625nm wavelength
It receives peak judgement Ox-TMB and becomes colour saturation, spectrum analysis is carried out to the standard solution system with different concentration of glucose, establishes Portugal
The linear relationship of grape sugar concentration and absorbance, and then quantitative analysis is carried out to concentration of glucose in sample.
5. application according to claim 4, the spectrophotometry is ultraviolet-visible spectrophotometry.
6. application according to claim 5, which is characterized in that the Ox-TMB becomes 1. H that colour saturation is generated with step2O2
Concentration is proportionate.
7. application according to claim 1, which is characterized in that detecting step includes：
A. glucose standards solution is configured：Glucose is quantitatively weighed to be completely dissolved in water；B. TMB standard solution is configured：It is quantitative
TMB is weighed to be completely dissolved in dimethylformamide；C. EDTA standard solution is configured：It quantitatively weighs EDTA and is completely dissolved in water
In,
Wherein, glucose standards solution:TMB standard solution:Concentration ratio is 1 between EDTA standard solution:100：100.
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