CN110006885A - It is a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method - Google Patents
It is a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method Download PDFInfo
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- CN110006885A CN110006885A CN201910312277.0A CN201910312277A CN110006885A CN 110006885 A CN110006885 A CN 110006885A CN 201910312277 A CN201910312277 A CN 201910312277A CN 110006885 A CN110006885 A CN 110006885A
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000002131 composite material Substances 0.000 title claims abstract description 27
- 239000002057 nanoflower Substances 0.000 title claims abstract description 26
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 20
- 239000000243 solution Substances 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 238000002835 absorbance Methods 0.000 claims abstract description 12
- 108010025188 Alcohol oxidase Proteins 0.000 claims abstract description 11
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 10
- 239000001110 calcium chloride Substances 0.000 claims abstract description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000011088 calibration curve Methods 0.000 claims description 2
- 230000003595 spectral effect Effects 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims 1
- 239000012488 sample solution Substances 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 8
- 239000000463 material Substances 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000006555 catalytic reaction Methods 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 2
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000007254 oxidation reaction Methods 0.000 abstract description 2
- 238000004064 recycling Methods 0.000 abstract 1
- 235000019441 ethanol Nutrition 0.000 description 52
- 102000004190 Enzymes Human genes 0.000 description 22
- 108090000790 Enzymes Proteins 0.000 description 22
- 238000001514 detection method Methods 0.000 description 16
- 238000005259 measurement Methods 0.000 description 6
- 230000001476 alcoholic effect Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000002086 nanomaterial Substances 0.000 description 5
- 210000002700 urine Anatomy 0.000 description 4
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 240000003291 Armoracia rusticana Species 0.000 description 2
- 235000011330 Armoracia rusticana Nutrition 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- -1 alcohol compound Chemical class 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 1
- 241000549556 Nanos Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229920001131 Pulp (paper) Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 229910001430 chromium ion Inorganic materials 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940077449 dichromate ion Drugs 0.000 description 1
- SOCTUWSJJQCPFX-UHFFFAOYSA-N dichromate(2-) Chemical compound [O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O SOCTUWSJJQCPFX-UHFFFAOYSA-N 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004374 forensic analysis Methods 0.000 description 1
- 238000003988 headspace gas chromatography Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
The invention discloses a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method.The method includes by alcohol oxidase (AOX), horseradish peroxidase (HRP), CaCl2Solution is added in phosphate buffer solution, is stored at room temperature reaction, obtains double enzyme-inorganic nano flower composite materials (AOHNF).Reaction condition is simple, and obtained material enzymatic activity and stability significantly increases.Alcohol, which reacts, in recycling AOX catalysis oxidation solution generates H2O2, HRP catalysis TMB and H2O2Reaction makes solution that blue be presented, and absorbance of the absorbing wavelength at 650nm is measured, to obtain the size of alcohol concentration in solution.Analysis method is easy to operate, testing result high sensitivity.Of the present invention to have the characteristics that easy to operate, high sensitivity, stability are good based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method, development prospect is good.
Description
Technical field
The invention belongs to quantitative analysis fields, and in particular to a kind of based on double enzymes-inorganic nano flower composite material alcohol
Quantitative analysis method.
Background technique
With the development of the times, the detection of highly sensitive, highly selective and high accuracy alcohol compound it is many not
With field be widely used, it is quick, sensitive, select and quantitatively detect alcohols material for law enforcement, chemical industry, pharmacy and fermentation etc.
Industry is also of great significance, wherein accurately and quickly measurement ethanol content is extremely important in clinical and forensic analysis.In addition,
Simply, quick and economic alcohol analysis method be also used in paper pulp, food and beverage industries control fermentation process and its
The quality of product.Durings traffic administration, legal medical expert, clinical analysis etc., common alcohol detection technique is expiration detection method, saliva
Liquid detection method, urine blood testing method.Expiration method is a kind of non-intrusion type, convenient and quick diagnosis method, generally using exhaling
Gas formula alcohol content tester is measured, and the alcohol content in usual implication is unstable, is remained vulnerable to measured's expiration amount, oral cavity
There is the influence for not absorbing alcohol etc., measurement result is caused mistake occur；Saliva rule is mainly measured using enzyme test peper item,
This method is quick, cheap and easy to operate, the relatively often application generally in emergency treatment department；Urine detection method, which refers to, works as thing by extracting
The urine of people carries out urine Alcohol mental disorders, and blood testing method is mainly the ethyl alcohol in extraction blood to detect its content, leads to
Often in this way when handling some accidents and as foundation.The detection for carrying out blood using enzyme process, urinating alcohol concentration
With good accuracy, repeatability and specificity, but can not on-site test, professional institution's indoor test can only be transferred to, if sending
Inspection will appear biggish error not in time.
In recent years, it has been reported that many methods for being used for alcoholometry, such as colorimetric method, chromatography, redox titration
Method, index method etc., wherein colorimetric method and chromatography are using relatively large number of method.Conventional colorimetric method is mainly using weight chromium
Sour potassium is measured, but it has one disadvantage in that it is not meet " Beer law ", and one such oxidation product chromium ion is green,
It is mixed with different concentration with the yellow dichromate ion measured, and Colorimetric results is made error occur, and measurement is caused to be tied
Fruit loss of stability.Detection of the chromatography for alcohol in blood or body fluid, now commonly uses Head-space sampling and direct sample injection two
Item technology is come the method for replacing pervious solvent extraction or distillation, and automation is high, and measurement result is reliable and stable, but the survey of this method
It is more complex to determine process, is easy to pollute injection port, pillar etc., and the consuming time is longer, is unable to satisfy and alcohol is carried out fastly
The requirement of speed quantitative determination.In order to overcome these disadvantages, occur based on NAD+Dependence alcohol dehydrogenase or alcohol oxidase it is each
Kind alcohol biosensor carries out analysis test to alcohol, these methods are mainly to utilize biological enzyme living to the catalysis of alcohols material
Property, also successfully it has been used in the alcohol criminal in monitoring criminal justice, alcohol in medical monitoring and fermentation process at present
Measurement.This kind of sensor has highly selective, but its relevant cost of use is very high, and is unable to fully solve alcohol detection
The shortcomings that, such as muting sensitivity, low stability and narrow linear operating range in practical applications.Biological enzyme stability
It is low, it is not easy to which that long-term preservation, easy in inactivation, this gives the method for carrying out alcohol detection above by biological enzyme to bring certain office
It is sex-limited.
Enzyme is as multi-purpose tool, due to its unique property, such as high atopic, high catalytic activity and hypotoxicity
It is widely studied, but natural resolvase haves the shortcomings that some intrinsic, such as prepares and purifies that at high cost, operational stability is low, urges
Change active sensitive to environmental condition and is difficult to recycle.In order to overcome these disadvantages, it has been reported that propose enzyme immobilization
Enzyme insertion nano material is become the hot spot of Recent study by concept.Studies have shown that the enzyme being fixed in nano material have than
The characteristics of broader pH of native enzyme and Acclimation temperature range and higher thermal stability.Since the nano material of three-dimensional structure exists
Surface area, reactivity, stability etc. are better than other nano materials, therefore three-dimensional nanometer material becomes Recent study enzyme
Fixed hot spot.
Summary of the invention
In order to solve the above problems existing in the present technology, the object of the present invention is to provide one kind, and enzyme-is inorganic receives based on double
The alcohol quantitative analysis method of popped rice composite material.This method mainly forms flower-shaped nano junction using enzyme and inorganic material
Structure constructs double enzyme-inorganic nanos and spends to be measured to alcohol.The reaction system has been related to alcohol oxidase and horseradish peroxidating
Two kinds of enzymes of object enzyme belong to multienzyme cascade reaction, and major advantage is that the active site of enzyme is close to each other in nano material, contracting
The distance between small enzyme and reactant minimize the diffusion of enzyme intermediate in reaction, increase the reactant concentration around enzyme, by
This enhances total reaction efficiency and specificity.The present invention selects inorganic phosphate to fix alcohol oxidase AOX and horseradish for carrier
Peroxidase HRP, to realize that multienzyme fixes reaction system altogether.This double enzymes-inorganic nano flower is compared with resolvase, enzyme activity
Property and stability significantly increase.To improve the sensitivity of alcohol detection.
The purpose of the present invention is achieved through the following technical solutions:
It is a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method, comprising the following steps:
Step 1) first by alcohol oxidase solution, horseradish peroxidase solution be added phosphate buffer solution in, then plus
Enter CaCl2Solution mixes, and stands, is centrifuged after reaction, removes supernatant, adds phosphate buffer solution, and mixing is equal
It is even, obtain double enzyme-inorganic nano flower composite materials；
Step 2) is by a series of sample of double enzyme-inorganic nano flower composite material and different alcohol concentrations made from step 1)
Solution mixing, is added color developing agent, carries out UV-vis spectroscopy spectral scan, records absorbance value, with alcohol concentration for horizontal seat
Mark, absorbance value is ordinate, obtains standard curve；
The alcohol concentration of institute's sample can be obtained according to calibration curve method in step 3).
Preferably, the concentration of the step 1) phosphate buffer solution is 10mM.
Preferably, the step 1) CaCl2The concentration of solution is 100mM.
Preferably, the temperature of the step 1) reaction is 30 DEG C, reacts duration 48h.
Preferably, the step 2) color developing agent is TMB solution.
It is provided by the invention based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method, by by alcohol oxygen
Change enzyme and horseradish peroxidase is fixed on formation nano flower-like structure on inorganic phosphate skeleton altogether, effectively increases reaction
Contact area, prevent the loss of intermediate product to greatest extent, while maintaining biggish enzymatic activity again, be used for alcohol quantitative
Analysis detection improves the sensitivity of detection, meanwhile, operating method is simple, quick, and stability is high.More specifically, of the invention
Have the beneficial effect that:
(1) the united inorganic nano flower composite material preparation of multienzyme at normal temperatures and pressures, is not required to any organic reagent, operates
Simply, material morphology is special, has high specific surface area, substantially increases the activity and stability of enzyme.
(2) detection alcohol need to only be can be completed by ultraviolet-visible spectrophotometer, do not needed complex operations, do not needed
Large-scale instrument, it is more convenient and practical.
(3) range of linearity that experiment measures alcohol quantitative analysis is 0.1~2.5mg/mL, in China, internal alcohol concentration
It is judged as drunk driving when >=0.2mg/mL, when internal alcohol concentration >=0.8mg/mL is judged as drunk driving, and the method can be well
Accurate quantitative analysis applied to drunk driving judges.
Detailed description of the invention
Fig. 1 is the preparation method figure of double enzyme-inorganic nano flower composite materials (AOHNF)；
Fig. 2 is the SEM figure of double enzyme-inorganic nano flower composite materials；
Fig. 3 is the vis spectroscopy spectrogram that alcohol quantitative analysis detection obtains；
Fig. 4 is absorbance value at 650nm measured by alcohol quantitative analysis method with the variation relation figure of alcohol concentration.
Specific embodiment
Technical solutions according to the invention are further described in detail below by specific embodiment combination attached drawing,
But it is necessary to note that following embodiment is served only for the description to summary of the invention, does not constitute a limitation on the scope of protection of the present invention.
Embodiment 1
It is a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method, comprising the following steps:
(1) double enzyme-inorganic nano flower composite materials are prepared: as shown in Figure 1, by alcohol oxidase (AOX) solution 1.0mg/mL,
Each 15 μ L of horseradish peroxidase (HRP) 1.0mg/mL is added in the phosphate buffer solution (PBS) of 1.41mL, then by 168 μ
The CaCl of L 100mM2Solution is added in above solution, stands after mixing, takes out after incubating 48h at 30 DEG C, then with
The revolving speed of 10000r/min is centrifuged 15min, removes supernatant, adds the PBS of 150 μ L, is uniformly mixed, and experiment institute can be obtained
Double enzymes-inorganic nano the flower composite material (AOHNF) needed.
As shown in Fig. 2, double enzymes-inorganic nano flower composite material is characterized by SEM, double enzymes-inorganic nano flower composite material
For uniform nano flower-like ball, average diameter is about 6 μm, is evenly distributed, and pattern is uniform, has good dispersibility and multilayer three
Tie up structure.
(2) alcohol quantitative detect: first preparation various concentration alcoholic solution (0,0.1,0.2,0.3,0.5,0.7,0.8,
1.0,1.2,1.5,1.75,2.0,2.2,2.5, unit is mg/mL), by the AOHNF of 5 μ L respectively with 100 μ L various concentrations
Alcoholic solution is uniformly mixed, and is placed in constant incubator, after 37 DEG C of incubation 5min, the TMB solution of 100 μ L is added, is again placed in
In constant incubator, 37 DEG C of incubation 5min.By ultraviolet-visible spectrophotometer, measured using the microcolorimetric ware of 350 μ L anti-
The absorption peak spectrogram of solution after answering.
During alcohol oxidase oxygenolysis alcohol, H is produced2O2, in double enzymes-inorganic nano flower composite material
Another enzyme --- HRP utilizes H2O2And catalysis reaction is carried out using TMB as its substrate, ultimately generate blue material, and solution face
Color is gradually deepened with the raising of alcohol concentration.And the alcoholic solution of various concentration reacted with AOHNF after wavelength be 650nm at
Absorbance it is also different, Fig. 3 is the relationship of alcohol concentration and the wavelength absorbance at 650nm, as alcohol concentration is from 0mg/mL
It is increased to 2.5mg/mL, absorbance is also gradually increased, and increasing degree is the most obvious between 0.1~2.0mg/mL in alcohol concentration.
Therefore the detection range of the detection method is 0.1~2.5mg/mL.
The alcohol of various concentration is directlyed proportional for the absorbance at 650nm to alcohol concentration after reacting to AOHNF in wavelength, such as
Shown in Fig. 4, equation of linear regression Y=0.08974X+0.06762, X indicate the concentration (mg/mL) of alcohol, and Y indicates to correspond to
Absorbance.Therefore when we obtain the alcoholic solution of a unknown concentration, it is only necessary to the absorbance after measuring its reaction, so
Its corresponding alcohol concentration is found out from linear relationship afterwards, it can be learnt that the concentration of former alcoholic solution.It is provided with national sector standard
Headspace gas chromatography method compare, the alcohol sample in standard need in head-space sampler or constent temperature heater 70 DEG C heating
15min is to be measured, and this method needs 10min only to prepare sample to be tested, more time saving compared with the time required to standard method, and
In standard method, the measuring samples prepared also need to enter gas chromatograph, by record sample and add the second in sample
Pure and mild tert-butyl alcohol peak face area, to carry out final quantitative test to sample to be tested, and this method only needs measurement to prepare
The absorbance of measuring samples can measure the alcohol concentration of original solution, and testing process is more convenient.
Claims (5)
1. a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method, which is characterized in that including following step
It is rapid:
First alcohol oxidase solution, horseradish peroxidase solution are added in phosphate buffer solution for step 1), are then added
CaCl2Solution mixes, and stands, is centrifuged after reaction, removes supernatant, adds phosphate buffer solution, is uniformly mixed,
Obtain double enzyme-inorganic nano flower composite materials；
Step 2) is by a series of sample solution of double enzyme-inorganic nano flower composite material and different alcohol concentrations made from step 1)
Color developing agent is added in mixing, carries out UV-vis spectroscopy spectral scan, records absorbance value, using alcohol concentration as abscissa, inhales
Shading value is ordinate, obtains standard curve；
The alcohol concentration of institute's sample can be obtained according to calibration curve method in step 3).
2. it is according to claim 1 a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method,
It is characterized in that, the concentration of the step 1) phosphate buffer solution is 10mM.
3. it is according to claim 1 a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method,
It is characterized in that, the step 1) CaCl2The concentration of solution is 100mM.
4. it is according to claim 1 a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method,
It is characterized in that, the temperature of the step 1) reaction is 30 DEG C, reacts duration 48h.
5. it is according to claim 1 a kind of based on double enzymes-inorganic nano flower composite material alcohol quantitative analysis method,
It is characterized in that, the step 2) color developing agent is TMB solution.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656104A (en) * | 2019-09-26 | 2020-01-07 | 江苏大学 | GA @ GOx hybrid nanoflower and preparation method and application thereof |
| CN111504986A (en) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | Method for rapidly detecting diamine biogenic amine |
| CN111504987A (en) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | Method for rapidly detecting diamine biogenic amine by using inorganic hybrid nano-anthocyanidin |
| CN112683890A (en) * | 2020-12-03 | 2021-04-20 | 南京师范大学 | Kit for rapidly detecting glucose based on double-enzyme-hydrogel composite material and detection method thereof |
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