CN1908622A - Analysis method of enzyme inhibiting agent and module thereof - Google Patents
Analysis method of enzyme inhibiting agent and module thereof Download PDFInfo
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- CN1908622A CN1908622A CN 200510089748 CN200510089748A CN1908622A CN 1908622 A CN1908622 A CN 1908622A CN 200510089748 CN200510089748 CN 200510089748 CN 200510089748 A CN200510089748 A CN 200510089748A CN 1908622 A CN1908622 A CN 1908622A
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Abstract
The provided method to detect the enzyme inhibitor content in aqueous solution comprises: a cast-type test block, a capsule, and multifunctional container for the chemical reaction and detection. It is also fit to detect the pesticide residue.
Description
Technical field
The present invention relates to a kind of method and module of enzyme inhibitor quantitative test, relate in particular to the quantitative test of agricultural chemicals with inhibitory enzyme activity.
Background technology
The method of agricultural chemicals residual volume has spectrophotometric method, polarography, atomic absorption spectrography (AAS), thin layer chromatography, vapor-phase chromatography, liquid phase chromatography, isotope-labelling method, nuclear magnetic resonance spectroscopy, application of gas chromatorgraphy/mass method, fluorometric method, immunoassay or the like in the known analysis vegetables and fruits.At present general adopted still based on vapor-phase chromatography and liquid phase chromatography, because of it has good repeatability, sensitivity, and can determine advantage such as pesticide species.
Yet the aforementioned approaches method all needs the professional and technical personnel to carry out the preparation of a large amount of test solutions in the laboratory, can't be easily in the crops place of production, ordinary consumer buying places such as market, amount dealer supermarket, by analyzing fast without the personnel that screw up discipline.Therefore, need a kind of quantitative analysis method that general personnel use of being convenient to, the work of analysis can be carried out in arbitrary site, exempt the configuration of multiple a large amount of test solutions.
Summary of the invention
The present invention is a characteristic of utilizing general agricultural chemicals tool inhibitory enzyme activity, provides a kind of easy enzyme inhibitor quantitative analysis method and module, with the content of agricultural chemicals in the analytic sample.
The present invention comprises provides enzyme, in order to the chemical reaction of catalysis first compound, therefore the catalytic capability of this enzyme can be suppressed by enzyme inhibitor, is suppressed the content that degree change (being the slack-off degree of this chemical reaction) can be calculated enzyme inhibitor from enzyme.The present invention measures enzyme, and to be suppressed degree methods be to utilize second compound.This second compound therewith chemical reaction the product rapid reaction and generate the 3rd compound.Because the optics color comparator can be detected the light of the specific wavelength that the 3rd compound absorbed, so can change in the light absorption value of this specific wavelength by the 3rd compound in this hydrolysis reaction of measurement, record the slack-off degree of this chemical reaction, and calculate the content of enzyme inhibitor.
The present invention more comprises a kind of constituent in order to quantitatively analyzing enzyme inhibitors, contains above-mentioned enzyme, first compound and second compound.
The present invention more comprises, when each analysis, and all ingredients that uses dried forms to present, and all only contain single analyses dosage, can exempt the configuration of multiple a large amount of test solutions.
The present invention more comprises multifunction container, for splendid attire solution to be measured, also in this container, carry out hybrid reaction for various dried reagents, this container also can directly be inserted in the portable type optics color comparator, measure the light absorption value of solution to be measured, analytical work can be carried out in arbitrary site easily.
Description of drawings
Fig. 1 to Fig. 4 shows various demonstration module of the present invention respectively.
Fig. 5 shows the relation of Mei Wensong content and its extinction value difference.
The main element symbol description
11 first test pieces
12 second test pieces
13 test pieces
21,22,23,24 containers
30 optics color comparators
31 microprocessors
32 calibration cells
41 first loam cakes
42 second loam cakes
43 loam cakes
51 first capsules
52 second capsules
S control group solution
B cushions salt
Embodiment
As described above, the present invention is a characteristic of utilizing general agricultural chemicals tool inhibitory enzyme activity, provides a kind of easy enzyme inhibitor quantitative analysis method, with the content of agricultural chemicals in the analytic sample.The agricultural chemicals that the present invention can analyze to be to have the enzyme rejection characteristic, comprise but be not limited only to organophosphorus, the sulfo-organophosphorus handled with bromine water, and carbamate.
Following illustration reagent of the present invention and chemical reaction thereof should be appreciated that this only illustrates, but not in order to limit the scope of the invention:
First compound: acetyl thio cholinester
(Acetylthiocholine is called for short ACTC).
Enzyme: acetyl thio cholinesterase (being called for short AChE).
Second compound: 5,5 '-two sulphur two (2-nitrobenzoic acid) (5,5 '-Dithiobis (2-nitrobenzoic acid), be called for short DTNB).
ACTC produces acetate (Acetate) and thiocholine (Thiocholine) through the hydrolysis meeting.Thiocholine can form 5-sulphur-2-nitrobenzoyl acid esters (5-Thio-2-nitrobenzoate is called for short TNBZ) with the DTNB reaction, is the 3rd above-mentioned compound, and it has one to absorb spike in visible region 412nm.The principle of relevant above-mentioned reaction can see the Pharmacology in Biochemical for details, and 1961, Vol.7, page88-95, it is for reference to include the present invention at this.
The present invention is the multi-functional container of design, and for splendid attire solution to be measured, solution to be measured can contain the agricultural chemicals with enzyme inhibitor composition.This container also carries out hybrid reaction therein for above-mentioned all ingredients, more this container directly can be inserted in the optics color comparator to measure the light absorption value of solution to be measured.It should be noted that this container is the sign that containing mark is arranged, control the volume of solution to be measured to make things convenient for the user.And the material of container needs the light of measuring specific wavelength (as 412nm) is penetrated, and solution to be measured just can receive this light, and this material is generally glass or clear plastic, but is not limited thereto.So should understand container of the present invention except that can be used as reactor, more can directly insert in the optics color comparator and analyze, exempt the known inconvenience that needs to change multiple container.
Now the step that describes quantitative analysis method of the present invention in detail is as follows:
(a) provide first compound (as ACTC); Enzyme (as AChE); And second compound (as DTNB).
Should note first compound used in the present invention, enzyme, and second compound all present dried forms, and only contain single analyses dosage respectively.For example use water-fast base material,, be fixed with the reagent of single analyses dosage on it, can easily base material directly be inserted in the solution to be measured during use, make the agent dissolves of fixing on it as the plastic cement test piece.Immobilizing indicator simultaneously on the base material is a water soluble and have naked eyes can debate the color of knowledge.When base material inserted in the solution to be measured, whether whether the indicator color of observing on the base material removed fully, dissolve in fully in the solution to be measured to judge reagent.Whether the use of indicator, decided by the variation of other reagent character.For example, the second Compound D TNB itself promptly has color, and can have the function of indicator concurrently, thereby need not additionally to use another indicator.
Mentioned reagent on being fixed on base material, also can be fixed on container bottom, or the inboard of the loam cake of container, indicator also can be contained simultaneously in the inboard of loam cake.Perhaps, also can use capsule, with the packing all ingredients.Should notice that the enzyme and first compound before dissolving in aqueous solution, should separate preferable mutually.
In addition, it is above-mentioned several to notice that agents useful for same of the present invention is not limited in, and those who are familiar with this art can optionally add other necessary reagent, for example cushion salt, to regulate the pH-value of aqueous solution.
(b) provide first sample, the enzyme inhibitor with known content, this kind are the control group.Should notice that this known content also comprises zero.
(c) mix first sample, enzyme, first compound, and second compound, to form aqueous solution.Its order by merging is that first compound is dissolved in the water at last is preferable, yet should notice that scope of the present invention does not limit its order.
(d) utilize the optics color comparator, under specific wavelength (as 412nm), measure the extinction value difference of this aqueous solution in a set time, be defined as the first extinction value difference (Δ Abs,
Control).Can the different control group of a plurality of concentration carry out above-mentioned analytical procedure, be zero skip test to obtain the relation of enzyme inhibitor concentration and extinction value difference, wherein also to comprise enzyme inhibitor concentration.The example of measurement result can be with reference to shown in Figure 5: the graph of a relation of Mei Wensong pesticide concentration and its extinction value difference.
(e) provide second sample, the enzyme inhibitor of the unknown content of tool, this is called the test group.Should notice that this unknown content also may be zero.
(f) replace this first sample with this second sample and carry out (a) step, measure the extinction value difference of this aqueous solution in the identical set time, be defined as second extinction value difference (the Δ Abs to (c)
Test).
(g) relatively this second extinction value difference and this first extinction value difference are to calculate the content of this enzyme inhibitor in this second sample.For example, according to Fig. 5, can obtain the function of enzyme inhibitor concentration, with microprocessor processes Δ Abs to the extinction value difference
TestThe relation of function therewith can obtain the concentration of second sample institute enzyme-containing inhibitor.Perhaps, can utilize measured extinction value difference (the Δ Abs of above-mentioned skip test
Blank), the direct extinction value difference of surveying with second sample relatively can calculate the degree of contained enzyme inhibitory enzyme activity in second sample, is called inhibition number percent, and calculating formula is as follows:
Suppress number percent=1-Δ Abs
Test/ Δ Abs
Blank) * 100%
Above-mentioned calculating all can utilize the microprocessor that is arranged in the optics color comparator to handle automatically.
By following example is the operation that describes method of the present invention and module in detail, should be appreciated that the present invention is not limited to this example of Denging.
First module
As shown in Figure 1, get first test piece 11 (containing the AChE of about 0.03mg and the DTNB of about 0.132mg); Get second test piece 12 (containing the ACTC of about 0.288mg and the DTNB of about 0.132mg), first test piece 11 and second test piece 12 are all not moisture.In room temperature, in regular turn control to be organized solution S and inserted in the container 21, the bottom of container 21 scribbles an amount of buffering salt B in advance.Then, successively first test piece 11 and second test piece 12 are inserted in the container 21, treat to take out these test pieces after the agent dissolves on it.Then container 21 is inserted in the portable type optics color comparator 30, measuring with 2 minutes is extinction value difference (the Δ Abs of spacing
Control).Use test group solution then instead, repeat above-mentioned steps and record extinction value difference (Δ Abs
Test).Microprocessor 31 with portable type optics color comparator 30 calculates the concentration of test group automatically or suppresses number percent, and will the results are shown on the screen.
Second module
As shown in Figure 2, provide container 22 and can be in order to first loam cake 41 and second loam cake 42 of covering container 22.Container 22 bottoms are coated with the AChE of the 0.03mg that has an appointment and an amount of buffering salt B, and are all not moisture; First loam cake, 41 inboards are coated with the ACTC of the 0.288mg that has an appointment and the DTNB of about 0.264mg, and are all not moisture.Second loam cake 42 does not contain any ACTC or BTNB.Solution S is organized in control inserted in the container 22, the reagent of container 22 bottoms is dissolved fully, then container 22 is placed 37 ℃ of calibration cells 32 with second loam cake, 42 covering containers 22 and violent the mixing.After arriving design temperature, take out container 22, remove second loam cake 42, change, the reagent of first loam cake, 41 inboards is dissolved fully, then container 22 is put constant temperature in the calibration cell 32 with first loam cake, 41 covering containers 22 and violent the mixing.After arriving design temperature, measure with the extinction value difference that 2 minutes was spacing (Δ Abs,
Control).Then use test group solution instead, repeat above-mentioned steps and record extinction value difference (Δ Abs
Test).At last, with the microprocessor 31 of portable type optics color comparator 30, calculate the concentration of test group automatically or suppress number percent, and will the results are shown on the screen.
Three module
As shown in Figure 3, provide container 23, can be in order to the loam cake 43 that covers this container, and test piece 13.Container 23 bottoms scribble an amount of buffering salt B, and loam cake 43 inboards scribble the AChE of 0.03mg and the DTNB of 0.264mg, are coated with the ACTC of the 0.288mg that has an appointment in the test piece 13.Solution S is organized in control inserted in the container 23, it is dissolved fully with loam cake 43 covering containers 23 and violent the mixing.Subsequently test piece 13 is inserted in the container 23, treat to take out test piece 13 after the agent dissolves on it.Then container 23 is inserted in the portable type optics color comparator 30, measure with the extinction value difference that 2 minutes was spacing (Δ Abs,
Control).Use test group solution at last instead, repeat above-mentioned steps and record extinction value difference (Δ Abs
Test), and calculate the concentration of test groups automatically or suppress number percents with the microprocessor 31 of portable type optics color comparator 30, and will the results are shown on the screen.
Four module
As shown in Figure 4, get first capsule 51 (containing the DTNB of the AChE of about 0.03mg, about 0.264mg and the buffering salt B of appropriate amount) and second capsule 52 (ACTC that contains about 0.288mg), first capsule 51 and second capsule 52 are all not moisture.In room temperature, in regular turn control to be organized solution S and inserted in the container 24, buffering salt B is contained in the bottom of container 24.Then, successively the reagent in first capsule 51 and second capsule 52 is poured in the container 24, treat its dissolving after, container 24 is inserted in the portable type optics color comparator 30, measure with the extinction value difference that 2 minutes was spacing (Δ Abs,
Control).Use test group solution at last instead, repeat above-mentioned steps and record extinction value difference (Δ Abs
Test), and calculate the concentration of test groups automatically or suppress number percents with the microprocessor 31 of portable type optics color comparator 30, and will the results are shown on the screen.
Claims (18)
1. the quantitative analysis method of an enzyme inhibitor comprises:
(a) provide first compound; Enzyme is in order to the chemical reaction of this first compound of catalysis; And second compound, reacting in order to product, and generate the 3rd compound with this chemical reaction, the 3rd compound is the light that can absorb specific wavelength; Wherein this first compound, this enzyme, and this second compound all present dried forms, and only contain single analyses dosage respectively;
(b) provide first sample, the enzyme inhibitor with known content, in order to suppress the catalytic capability of this enzyme, this known content comprises zero;
(c) mix this first sample, this enzyme, this first compound, and this second compound, to form aqueous solution;
(d) utilize the optics color comparator, under this specific wavelength, measure the extinction value difference of this aqueous solution in the set time, be defined as the first extinction value difference;
(e) provide second sample, this enzyme inhibitor of the unknown content of tool;
(f) replace this first sample with this second sample, carry out above-mentioned (a) step to (c), and measure the extinction value difference of the aqueous solution that forms in this set time, be defined as the second extinction value difference;
(g) ratio of this second extinction value difference and this first extinction value difference relatively is to calculate the content of this enzyme inhibitor in this aqueous solution.
2. the method for claim 1 wherein should (a) step more comprise indicator, had the cognizable color of naked eyes.
3. method as claimed in claim 2, wherein this indicator of this first compound and part content is to be fixed in first test piece; This indicator of this enzyme and another part content then is fixed in second test piece.
4. the method for claim 1 more comprises container, in order to this aqueous solution of splendid attire, makes and uses the reactor that this first compound is hydrolyzed and reacts as; This container is that the transparent material of the light of this specific wavelength is made, can place this optics color comparator to carry out this step (d) or (f).
5. method as claimed in claim 4 more comprises the bottom of this enzyme or this first compound being fixed in this container.
6. method as claimed in claim 4 more comprises the loam cake of this container, and indicator is contained in the inboard of this loam cake, the cognizable color of tool naked eyes.
7. method as claimed in claim 6 more comprises the inboard of this enzyme or this first compound being fixed in this loam cake.
8. the method for claim 1, wherein this enzyme inhibitor is to be selected from organic phosphorus compound and Carbamates thing.
9. the method for claim 1 wherein should (a) step more comprise the buffering salt.
10. the method for claim 1, wherein this optics color comparator more comprises calibration cell or thermometer.
11. an enzyme inhibitor quantitative test module comprises:
Container, for the splendid attire aqueous solution, this aqueous solution has the enzyme inhibitor that content is X; Wherein, this container is useed the reactor that first compound carries out chemical reaction as, this chemical reaction can be by the catalysis of enzyme institute, the catalytic capability of this enzyme can be suppressed by this enzyme inhibitor, the product of this chemical reaction can react to generate the 3rd compound with second compound, be the light that can absorb the tool specific wavelength, and this container is that the transparent material of light of this specific wavelength is made; And
The optics color comparator can hold this container, and sees through this aqueous solution of this container measurement in the light absorption value of this specific wavelength.
12. module as claimed in claim 11 more comprises the bottom that this first compound or this enzyme is fixed on this container.
13. module as claimed in claim 11 more comprises test piece, this test piece comprises indicator, is the cognizable colors of tool naked eyes.
14. module as claimed in claim 13, wherein this test piece more comprises this first compound or this enzyme.
15. module as claimed in claim 11, wherein this container more comprises loam cake, and this loam cake comprises indicator, is the cognizable colors of tool naked eyes.
16. module as claimed in claim 15, wherein this loam cake more comprises this first compound or this enzyme.
17. module as claimed in claim 11, wherein this X comprises zero.
18. the constituent in order to quantitatively analyzing enzyme inhibitors comprises:
First compound;
Enzyme is in order to the chemical reaction of this first compound of catalysis; And
Second compound reacts in order to the product with this chemical reaction, and generates the 3rd compound, and the 3rd compound is the light that can absorb specific wavelength; Wherein, this first compound, this enzyme, and this second compound all present dried forms, and only contain single analyses dosage respectively.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2005100897484A CN1908622B (en) | 2005-08-05 | 2005-08-05 | Analysis method of enzyme inhibiting agent and module thereof |
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|---|---|---|---|
| CN2005100897484A CN1908622B (en) | 2005-08-05 | 2005-08-05 | Analysis method of enzyme inhibiting agent and module thereof |
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| CN1908622B CN1908622B (en) | 2010-07-07 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304558A (en) * | 2011-05-25 | 2012-01-04 | 青岛大学 | Method for analyzing inhibition of immobilized flow injection enzyme |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1234503A (en) * | 1998-05-04 | 1999-11-10 | 北京市计量科学研究所 | Composite for quickly detecting pesticide of organic phosphorus and amino formate and detection method thereof |
| CN1118704C (en) * | 2000-12-08 | 2003-08-20 | 中国科学院水生生物研究所 | Reagent kit for diagnosing white-spot baculovirus of prawn and its test method |
| CN100500862C (en) * | 2002-10-29 | 2009-06-17 | 上海理工大学 | Pesticide residue enzymatic detection method using same kind deactivated enzyme as reference and its device |
| CN1414371A (en) * | 2002-12-13 | 2003-04-30 | 江苏省农业科学院植物保护研究所 | Conventional pesticide residue quick detecting method |
| MY137266A (en) * | 2002-12-18 | 2009-01-30 | Inst Medical Res | Acetylcholinesterase assay |
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| CN102304558A (en) * | 2011-05-25 | 2012-01-04 | 青岛大学 | Method for analyzing inhibition of immobilized flow injection enzyme |
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