CN106399457A - Method based on nano mimic enzyme for visually and quickly detecting bio-enzyme, protein and its inhibitor - Google Patents
Method based on nano mimic enzyme for visually and quickly detecting bio-enzyme, protein and its inhibitor Download PDFInfo
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- CN106399457A CN106399457A CN201610852201.3A CN201610852201A CN106399457A CN 106399457 A CN106399457 A CN 106399457A CN 201610852201 A CN201610852201 A CN 201610852201A CN 106399457 A CN106399457 A CN 106399457A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
- C12Q1/46—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase involving cholinesterase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
Abstract
The invention discloses a detection reagent containing nano mimic enzyme. The invention further discloses a visual detection method of bio-enzyme or protein and its inhibitor on the basis of nano mimic enzyme technology. The method shows good pH dependence for the oxidization reaction of many developing agents on the basis of some nano materials capable of simulating natural oxidase, and many oxidase or proteins can release/consume H+ characteristics in the biological catalysis process, and uses the in-site change of pH of reaction system to influence the catalytic oxidization ability of the nano material, thus the detection reagent has color change at different levels, and the visual detection of bio-enzyme or protein activity is realized, and further the visual detection of corresponding inhibitor is realized. The physical signal output by the detection method can show a very good linear relationship for the bio-enzyme or protein activity within a certain concentration scale, and thereby realizing the high-sensitivity detection of a target to be tested.
Description
Technical field
The invention belongs to field of bioanalysis and in particular to visualization quick detection biology enzyme based on nanometer analogue enztme,
Protein and its method for inhibitor.
Background technology
There is in biological a series of in vivo biochemical reaction the participation of corresponding biology enzyme invariably.Exactly these have Gao Xuan
Selecting property, the regulation of the biology enzyme of high catalytic activity, organism just can complete the Advanced Life activity of complexity.For example, acetylcholine
Esterase (acetylcholinesterase, AChE) is very important a kind of biology enzyme in central nervous system, can be catalyzed
The acetylcholine hydrolyzation of intracerebral generates acetic acid and choline.Research shows, the active mistake of acetylcholinesterase in central nervous system
The low a large amount of accumulations that can lead to acetylcholine, thus cause organ damage even dead.And if the activity of acetylcholinesterase
Too high, can cause that in central nervous system, acetyl choline content is too low again, easily cause Alzheimer disease and cerebrosclerosis.Cause
This, detection acetylcholinesterase has particularly important meaning in a physiologically and medically.Additionally, the agricultural chemicals of organic phosphates is
In modern agriculture, conventional a kind of highly toxic agricultural chemicals reagent, although they can effectively kill agricultural pests, equally right
Human body has violent toxicity, therefore detects that the agricultural chemicals of the organic phosphates remaining in fruits and vegetables is particularly important for health of people.
Research shows, organophosphorus insecticide is the inhibitor of acetylcholinesterase, and they can efficiently suppress its activity, therefore, right
The detection of acetylcholine esterase active also can be used for the detection of organophosphorus insecticide.Except H can be produced+Biology enzyme, also
There are some enzymes can consume H during living things catalysis+.Such as urase (urease), it is hydrolyzed by catalyzing urea and produces ammonia
Gas and carbon dioxide.Clinically, bacterium leads to urease activity rise be often numerous disease pathogenesis, such as hepatitis,
Hepatic coma, Infective calculus and peptic ulcer etc..Therefore, detect that the activity of urase clinically equally has particularly important
Physiological significance.And mostly many inhibitor of urase are the materials with environmental toxicity, also there is injury to health, therefore to urea
The detection method of enzymatic activity can be equally used for the detection of these high toxic materials.In sum, the inspection to biology enzyme/protein
Survey not only can help people to explore some pathological processes, signal transduction process and some diseases for from molecular level
Pathogenesis, and for therapy-related disease drug research and development also great instruct and reference value, also for some
The detection with the material of environmental toxicity provides new method and platform.
The method of traditional detection biology enzyme/protein active is concentrated mainly on high performance liquid chromatography (High
Performance liquid chromatography, HPLC), Capillary Electrophoresis (Capillary electrophoresis,
CE), mass spectrum (mass spectrum), fluorescence imaging (Fluorescence imaging, FI) etc..But these classical ways
There are many weak points when detecting biology enzyme/protein active.First, these methods need the instrument of costliness and complexity mostly
Device, needs specialized instrumentation person to be operated, therefore, it is difficult to realizing field assay.Secondly, biological sample needs complexity
Pre-treatment, preenrichment and separate etc. step, analysis time length it is impossible to realize the quick analysis of determinand in biological sample, when
Between resolution ratio also poor.Additionally, physiological environment complex, many biology enzyme/protein concentration in vivo and very low, many
Conventional detection means are difficult to it and quickly measure.Therefore, highly desirable development a kind of new detection technique be used for biology enzyme/
Quick, highly sensitive, the high selectivity detection of protein active.
Content of the invention
Goal of the invention：The present invention is directed to complex operation, the consumption that current some biology enzymes/protein active detection method exists
The present situation such as duration, sensitivity is poor, by using some nano materials that can simulate native enzyme activity, develops new principle and realizes
Quick, the detection of high selectivity, high sensitivity to associated biomolecule enzyme/protein active.
The invention provides a kind of nano material that can simulate Native Oxide enzymatic activity.This nano material can be as some
Native enzyme is the same, and oxidant (as hydrogen peroxide etc.) that need not be extra directly is realized some conventional developers are urged using oxygen
Change oxidation, and produce the change of color.Therefore, this kind of nano material is also referred to as nanometer analogue enztme by us.Received using this kind of
Rice analogue enztme its catalytic capability characteristic that pH is relied on when simulating natural enzymatic oxidation developer, by biology enzyme to be measured/
The change in protein catalytic substrate course of reaction, the pH value of detection reagent being produced, the catalysis activity of impact nanometer analogue enztme,
Thus obtaining corresponding output signal, and by rational means, this output signal is read, thus realizing to determinand
Analysis.It has been found that if adding some reaction promoters in this detection process, the carrying out of chromogenic reaction can be accelerated, and
Improve the sensitivity of detection further, lower test limit.
Technical scheme：In order to solve above-mentioned technical problem, the invention provides a kind of examination of the detection containing nanometer analogue enztme
Agent, includes nano material, developer and reaction promoter in this detection reagent.
Wherein, above-mentioned nano material contains one of metal or metal oxide or two kinds.
Wherein, above-mentioned developer is the compound with redox characteristic.
Wherein, above-mentioned reaction promoter is the biomolecule containing energy-rich phosphate bond, including adenosine triphyosphate, bird
Purine nucleosides triphosphoric acid, cytidine triphosphate, thymidine triphosphate, uridine diphosphate guanosine triphosphate and its analog
One or more of, preferably ATP.
A kind of visualization quick detection biology enzyme based on nanometer analogue enztme, the method for protein active, walk including following
Suddenly：
1) preparation of nanometer analogue enztme：Aoxidizing containing metal or metal of native enzyme can be simulated using wet chemistry preparation
The nano material of thing, separated, purify and measure standby after its concentration；
2) preparation of detection reagent buffer solution：Prepare suitable pH value and the cushioning liquid of concentration is stand-by；
3) preparation of the detection reagent of detection biology enzyme/protein active：Take a certain amount of developer, reaction promoter with
And the reaction substrate of tested biology enzyme/protein is added in the buffer solution preparing, stand-by after mixing；For can release
Put H+Biology enzyme/protein, the phosphate buffer being 7.0 from pH, and for H can be consumed+Biology enzyme/protein, then
The phosphate buffer being 4.5 from pH；
4) Visual retrieval of biology enzyme/protein active：Above-mentioned detection reagent is detected biology enzyme/egg with containing to be measured
After the sample of white matter and the mixing of a certain amount of nanometer of analogue enztme at 37 DEG C incubation reaction, then according to detection reagent color
Change or the activity observing by the naked eye to biology enzyme/protein to be measured carry out semiquantitative judgement；Or pass through corresponding instrument
The activity of biology enzyme/protein to be measured is accurately measured and quantitative.
Wherein, above-mentioned steps 2) in cushioning liquid for pH be 7.0 or 4.5, concentration be 5mM phosphate buffer solution.
Wherein, above-mentioned steps 3) in developer be 3,3,5,5- tetramethyl benzidines.
Wherein, above-mentioned biology enzyme is one of acetylcholinesterase and urase.
Wherein, above-mentioned reaction promoter is ATP.
A kind of method visualizing biology enzyme/protein inhibitor activity based on nano oxidized enzyme, methods described includes walking
Rapid 1)～3), also include step 4) Visual retrieval of the corresponding inhibitor of biology enzyme/protein：By the biology enzyme of known activity/
Protein obtains mixture with effect a period of time after corresponding inhibitor mixed, then by this mixture and above-mentioned detection reagent
And incubation reaction, the change finally according to detection reagent color or pass through at 37 DEG C after the mixing of a certain amount of nanometer of analogue enztme
Visually observe the inhibitor to biology enzyme/protein to be measured and carry out semiquantitative judgement；Or by corresponding instrument to life to be measured
The inhibitor of thing enzyme/protein is accurately measured and quantitative.
The inhibitor of the present invention is acetylcholinesteraseinhibitors inhibitors methyl to phosphine oxide and Tacrine and urease inhibitor F-.
The quantitative determination principle to physiologically active small molecule to be measured for the present invention：This nano material is that nanometer analogue enztme can profit
It is directly realized by the catalysis oxidation to developer with oxygen, produce the change of color.But the ability of this catalysis oxidation is in certain pH
In the range of present pH dependence characteristic.In general, in pH partial neutral, catalytic capability is weaker, the color of generation is shallower, and
In pH slant acidity, catalytic capability is stronger, and the color of generation is deeper.In some reaction promoters as in the presence of ATP, this urge
Change ability can effectively be strengthened, and the therefore catalytic reaction time can greatly shorten.Using this characteristic, by biology to be measured
The impact to solution ph during catalytic phase answers substrate reactions of enzyme/protein, enters to nanometer analogue enztme activity original position used
Row regulation and control, carry out producing different degrees of color change as signal output, finally realize biology enzyme/protein to be measured and its suppression
The analysis of preparation.
Beneficial effect：The physics letter that should be exported based on the detection method of the nanometer analogue enztme simulating native enzyme in the present invention
Number extraordinary linear relationship is assumed in the range of finite concentration to biology enzyme/protein active in system to be measured, thus realizing
Highly sensitive detection to target to be measured.This detection architecture is hardly subject to disturbing of other common biology enzyme/protein, therefore right
The detection of target determinand has high selectivity.In addition the method instrument is simple, easy and simple to handle, with low cost, need sample amount few,
Both the quick visualization detection of biological internal associated biomolecule enzyme/protein can have been realized, can be used in again some to health
There is the quick detection of the poisonous and harmful substances of threat, be therefore of very high actual application value.This technology is used for these and gives birth to
Reason material detection has advantages below：1) this detection method has very high sensitivity to the detection of corresponding biology enzyme/protein,
The detection that biological internal associated biomolecule enzyme/protein active can be met requires；2) selective good, other biological enzyme/protein
Activity does not interfere with；3) react quickly it is only necessary to 5-10 minute can observe by the naked eye the change of detection reagent color；4) grasp
Make easy, only just can realize the semi-quantitative analysis to determinand with naked eyes, rely on instrument then can realize more fixed
Amount；5) with low cost, on the one hand, need not specially to design and synthesize to the selective response of biology enzyme/protein active to be measured
Fluorescent probe molecule or other biological recognition unit, on the other hand, the nano material being used instead of conventional native enzyme
Reagent, makes testing cost obtain great reduction；6) need sample amount very little, detection is easy and simple to handle, and analyze speed is very fast, Ke Yishi
Detection and detection when participating in the cintest by existing bed.
Brief description
The working curve that Fig. 1 nanometer analogue enztme nanoceria detects to acetylcholinesterase；
The working curve that Fig. 2 nanometer analogue enztme nanoceria detects to phosphine oxide to acetylcholinesteraseinhibitors inhibitors methyl；
The working curve that Fig. 3 nanometer analogue enztme nanoceria detects to acetylcholinesteraseinhibitors inhibitors Tacrine；
The working curve that Fig. 4 nanometer analogue enztme nanoceria detects to urase；
Fig. 5 nanometer analogue enztme nanoceria is to urease inhibitor F-The working curve of detection.
Specific embodiment
Below technical solution of the present invention is described in detail, but protection scope of the present invention is not limited to described enforcement
Example.
The example being described nanometer analogue enztme with nano-cerium oxide (nanoceria), its chemical formula is CeO2.
With acetylcholinesterase and its inhibitor methyl to phosphine oxide (methyl-paraoxon), Tacrine (tacrine) and
Urase and its inhibitor fluorine ion (F-) described biology enzyme/protein and its inhibitor example.Agents useful for same of the present invention
By commercially available acquisition.
Developer used is 3,3,5,5- tetramethyl benzidines (TMB), and reaction promoter used is preferably ATP.
U in the embodiment of the present invention is enzyme activity unit, mU=10-3U.
Embodiment 1
1) nanometer analogue enztme nanoceria preparation：
By 2.52g Ce (NO3)3It is dissolved in the 1 of 100mL water and ethylene glycol:In 1 mixed solution, and heating stirring is to 60 DEG C.
Then rapidly join the fresh ammoniacal liquor of 16mL under conditions of stirring, continue stirring 3 hours at 60 DEG C.Product is centrifuged, and according to
Secondary wash with water and obtain nanoceria crude product after being centrifuged 3 times.This nanoceria crude product is scattered in 100mL concentration is
In the sodium citrate solution of 15mg/mL, ultrasonic 30 minutes, after treating that nanoceria and sodium citrate fully act on, add
100mL ethanol, centrifugation after stirring, products therefrom is successively with 1:After the mixed liquor of 1 water and ethanol washs and is centrifuged 3 times, point
Dissipate in pure water.The concentration that to be calculated, measurement obtains nanoceria in this dispersion liquid is 14mg/mL, and the concentration obtaining is
14mg/mL nanoceria dispersion liquid preserves after being diluted obtaining the nanoceria storing solution that concentration is 2.5mg/mL, treats
With.
2) prepare phosphate buffer：Prepare pH and be respectively 7.0 and 4.5, concentration is that the phosphate buffer solution of 5mM is stand-by.
3) prepare the detection reagent of detection biology enzyme/protein active：By 4 μ L TMB solution (25mM), 2 μ L ATP solution
(100mM) phosphoric acid buffer that, 10 μ L acetylcholine solution (1.0M) add the concentration that the pH prepared by 180 μ L is 7.0 to be 5mM is molten
In liquid, after mixing add variable concentrations acetylcholinesterase (0,7,14,35,70,175,350,700,1400mU) with
And 1.5 μ L nanoceria storing solution (14mg/mL), obtain detection reagent after mixing, this detection reagent is placed in 37 DEG C
Water-bath in react, and the change using absorbance at ultraviolet-visible spectrophotometer real time record 652nm.
As seen from Figure 1, in the case of there is acetylcholinesterase, the absorbance at 652nm constantly rises, and institute is described
Constantly aoxidized with TMB developer and developed the color.Its color speed with acetylcholinesterase concentration rise and rise, illustrate with
The raising of acetylcholine ester enzyme concentration, the change of pH value of solution is accelerated, and the speed that TMB is aoxidized by nanometer mimetic enzyme catalysis is also therewith
Accelerate.During with detection time for 600s, absorbance at 652nm for the solution responds output (A as signal652), and it is right
The concentration mapping of acetylcholinesterase, its result is as shown in Figure 1B.Absorbance at 652nm for this detection reagent with added
Acetylcholine ester enzyme concentration present extraordinary linear relationship (A in the range of finite concentration652=0.0024 × [AChE]
mU-1+ 0.137), the range of linearity is 7mU～135mU, and its lowest detection is limited to 3.5mU, and (under this concentration, its signal responds as making an uproar
3 times of sound).Additionally, we similarly see that the method is to acetylcholine in the case of being added without ATP by control experiment
The detection sensitivity of esterase have dropped half (0.0012vs.0.0024), and test limit also higher (25mU) is it was demonstrated that reaction promotes
The addition of agent ATP can significantly improve the detection performance of the method.
2 nanometers of simulation zymotechnics of embodiment are in acetylcholinesteraseinhibitors inhibitors methyl to answering in phosphine oxide and Tacrine detection
With
First by the methyl of 1.5U acetylcholinesterase and variable concentrations to phosphine oxide (0,0.01,0.02,0.05,0.1,0.2,
0.5th, 1,2,5,10 μM) or the mixing of (0,0.5,5,50 μM) of Tacrine after be 5mM with concentration phosphate buffer (pH 7.0) fixed
Hold to 50 μ L, react 30 minutes at room temperature.After enzymatic activity is adequately suppressed, the phosphate buffer (pH being 5mM with concentration
7.0) dilute this mixed liquor to 180 μ L, be subsequently added 4 μ L TMB solution (25mM), 2 μ L ATP solution (100mM), 10 μ L acetyl
Choline solution (1.0M) and 1.5 μ L nanoceria storing solution (14mg/mL).After mixing, this reagent is placed in 37 DEG C
React in water-bath, and the change using absorbance at ultraviolet-visible spectrophotometer real time record 652nm.
It will be seen that the rising to phosphine oxide concentration with organophosphorus pesticide methyl, the color of solution from Fig. 2A
More shallow, illustrate that the activity of acetylcholinesterase is suppressed to phosphine oxide by methyl it is difficult to be catalyzed the hydrolysis of acetylcholine.During detecting
Between for 600s when, absorbance 652nm at for the solution as signal response export (A652), and during by itself and without inhibitor
Recorded the A obtaining652Ratio methyl is mapped to the concentration of phosphine oxide, its result is as shown in Figure 2 B.By A652Absorbance
Record, we can obtain the actual concentrations to phosphine oxide for the methyl.Using the method, as shown in Figure 2 B, even if the methyl of 10nM
Phosphine oxide can also be produced with the change of obvious absorbance, illustrate that the method has very high spirit to methyl to the detection of phosphine oxide
Sensitivity.
Tacrine is the inhibitor of another kind of acetylcholinesterase, for the clinical treatment of Alzheimer disease.From Fig. 3
It will again be seen that rising with Tacrine concentration, the color of detection reagent shoals successively, the absorbance at 652nm for we
Value also declines therewith, illustrates that the method can be used for the screening of relevant disease medicine.
Application in urase detection for the 3 nanometers of simulation zymotechnics of embodiment
4 μ L TMB solution (25mM), 2 μ L ATP solution (100mM), 10 μ L urea (1.0M) is added prepared by 180 μ L
Concentration be 5mM pH be in 4.5 phosphate buffer solution, add after mixing different amounts of urase (0,7.5,15,37,
5th, 75,187.5,375,750,1500mU) and 3 μ L nanoceria storing solution (2.5mg/mL), after mixing, this is tried
Agent is placed in 37 DEG C of water-bath reacts, and the change using absorbance at ultraviolet-visible spectrophotometer real time record 652nm
Change.
It can be seen from figure 4 that in the case of there is urase, the absorbance at detection solution 652nm constantly declines.
This is because urase hydrolysis urea produces ammonia and consumes H+, solution system pH gradually rises, and nanometer analogue enztme nanoceria urges
Change oxidability gradually to be suppressed, TMB is also weakened therewith by the speed of catalysis oxidation.Equally, the absorbance at 652nm and institute
The urase concentration adding presents extraordinary linear relationship in the range of finite concentration.During with detection time for 600s, solution
Absorbance at 652nm responds output (A as signal652), and concentration mapping to urase, its result such as Fig. 4 B by it
Shown, its range of linearity is 0～750mU.Additionally, comparison diagram 4B and Fig. 4 D is it was also found that recorded when not using ATP
The A arriving652It is only 1/10th when using ATP, display the method drops to original ten to the detection sensitivity of urase/
One, the range of linearity also very narrow (0～75mU) is it was demonstrated that the addition of reaction promoter ATP can significantly improve the detection of the method
Performance.
4 nanometers of simulation zymotechnics of embodiment are in urease inhibitor F-Application in detection
First by the F of 1.5U urase and variable concentrations-(0,0.01,0.02,0.05,0.1,0.2,0.5,1,2,5mM) mixing
The phosphate buffer being 5mM with concentration afterwards (pH 4.5) is settled to 50 μ L, reacts 30 minutes at room temperature.Treat that enzymatic activity is abundant
After suppression, the phosphate buffer being 5mM with concentration (pH 4.5) dilutes this mixed liquor to 180 μ L, is subsequently added 4 μ L TMB solution
(25mM), 2 μ L ATP solution (100mM), 10 μ L urea (1.0M) and 3 μ L nanoceria storing solution (2.5mg/mL).Mixed
This reagent is placed in 37 DEG C of water-bath after closing uniformly and reacts, and using at ultraviolet-visible spectrophotometer real time record 652nm
The change of absorbance.
It will be seen that with there being F from Fig. 5-The rising of concentration, the color of detection solution is gradually increasing, and urea is described
The activity of enzyme is by F-Suppressed the hydrolysis it is difficult to catalyzing urea, the pH change of detection architecture also weakens certainly therewith.During detecting
Between for 600s when, absorbance 652nm at for the solution as signal response export (A652), and by its be not added with F-When A652
Ratio to F-Concentration mapping, its result is as shown in Figure 5 B.Using this ratio, we can obtain F-Actual concentrations.By scheming
Shown in 5B, we obtain F at detection-The vigor of 1.5U urase can be suppressed the concentration of half be about 750 μM.This method also may be used
Screening for other urease inhibitors.
Claims (10)
1. a kind of detection reagent containing nanometer analogue enztme is it is characterised in that include nano material, colour developing in this detection reagent
Agent and reaction promoter.
2. the detection reagent containing nanometer analogue enztme according to claim 1 is it is characterised in that this nano material contains gold
Belong to or one of metal oxide or two kinds.
3. the detection reagent containing nanometer analogue enztme according to claim 1 is it is characterised in that described developer is to have
The compound of redox characteristic.
4. the detection reagent containing nanometer analogue enztme according to claim 1 is it is characterised in that described reaction promoter is
Biomolecule containing energy-rich phosphate bond, including adenosine triphyosphate, guanopterin nucleoside triphosphate, cytidine three phosphorus
One or more of acid, thymidine triphosphate, uridine diphosphate guanosine triphosphate and its analog, preferably ATP.
5. a kind of method of visualization quick detection biology enzyme based on nanometer analogue enztme, protein active is it is characterised in that bag
Include following steps：
1) preparation of nanometer analogue enztme：Using wet chemistry preparation can simulate native enzyme containing metal or metal oxide
Nano material, separated, purify and measure standby after its concentration；
2) preparation of detection reagent buffer solution：Prepare suitable pH value and the cushioning liquid of concentration is stand-by；
3) preparation of the detection reagent of detection biology enzyme/protein active：Take a certain amount of developer, reaction promoter and quilt
The reaction substrate surveying biology enzyme/protein is added in the buffer solution preparing, stand-by after mixing；
4) Visual retrieval of biology enzyme/protein active：Above-mentioned detection reagent is detected biology enzyme/protein with containing to be measured
Sample and the mixing of a certain amount of nanometer of analogue enztme after incubation reaction, the then change according to detection reagent color at 37 DEG C
Or the activity observing by the naked eye to biology enzyme/protein to be measured carries out semiquantitative judgement；Or treated by corresponding instrument
The activity surveying biology enzyme/protein is accurately measured and quantitative.
6. a kind of visualization quick detection biology enzyme based on nanometer analogue enztme according to claim 5, protein active
Method it is characterised in that described step 2) in cushioning liquid be 7.0 or 4.5 for pH, concentration is the phosphate buffer solution of 5mM.
7. a kind of visualization quick detection biology enzyme based on nanometer analogue enztme according to claim 5, protein active
Method it is characterised in that described step 3) in developer be 3,3,5,5- tetramethyl benzidines.
8. a kind of visualization quick detection biology enzyme based on nanometer analogue enztme according to claim 5, protein active
Method it is characterised in that described biology enzyme be one of acetylcholinesterase and urase.
9. a kind of visualization quick detection biology enzyme based on nanometer analogue enztme according to claim 5, protein active
Method it is characterised in that described reaction promoter be ATP.
10. a kind of the method for biology enzyme/protein inhibitor activity is visualized based on nano oxidized enzyme it is characterised in that described
The step 1 that method includes any one of claim 5～8)～3), also include step 4) biology enzyme/protein correspondence inhibitor
Visual retrieval：Biology enzyme/the protein of known activity was mixed with effect a period of time after corresponding inhibitor mixed
Compound, is incubated anti-at 37 DEG C after then mixing this mixture with above-mentioned detection reagent and a certain amount of nanometer of analogue enztme
Answer, the change finally according to detection reagent color or the inhibitor observing by the naked eye to biology enzyme/protein to be measured carry out half
Quantitative judgement；Or the inhibitor of biology enzyme/protein to be measured is accurately measured and quantitative by corresponding instrument.
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| CN109781719A (en) * | 2019-02-27 | 2019-05-21 | 福建医科大学 | A kind of kit detecting trypsase and its inhibitor based on platinum cluster |
| CN114053473A (en) * | 2021-11-10 | 2022-02-18 | 昆明理工大学 | Preparation method and application of ferroferric oxide composite nano-enzyme antibacterial agent |
| CN114441458A (en) * | 2021-05-24 | 2022-05-06 | 中国科学院海洋研究所 | Application of ZIF material in inhibition of mimic enzyme |
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| CN107817241A (en) * | 2017-10-31 | 2018-03-20 | 安徽师范大学 | A kind of biology sensor, the application of preparation method and detection acetylcholinesterase, a kind of method for detecting organophosphorus pesticide |
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| CN109781719A (en) * | 2019-02-27 | 2019-05-21 | 福建医科大学 | A kind of kit detecting trypsase and its inhibitor based on platinum cluster |
| CN114441458A (en) * | 2021-05-24 | 2022-05-06 | 中国科学院海洋研究所 | Application of ZIF material in inhibition of mimic enzyme |
| CN114441458B (en) * | 2021-05-24 | 2023-06-09 | 中国科学院海洋研究所 | Application of ZIF material in inhibition of mimic enzyme |
| CN114053473A (en) * | 2021-11-10 | 2022-02-18 | 昆明理工大学 | Preparation method and application of ferroferric oxide composite nano-enzyme antibacterial agent |
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