CN109540882A - Organic phosphorus detection kit and its detection method in a kind of food - Google Patents
Organic phosphorus detection kit and its detection method in a kind of food Download PDFInfo
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Abstract
The invention discloses detection kits and its detection method organic phosphorus in a kind of food, the detection kit includes the first reagent and the second reagent, first reagent includes following components: buffer, gold chloride, reducing agent and pH adjusting agent, the second reagent includes following components: enzyme solution, color developing agent and substrate；Detection method includes the following steps: the pre-treatment of sample for this；The qualitative detection of sample；The production of standard curve；The quantitative detection of sample；The present invention is measured by qualitative detection continues quantitative detection containing organic phosphorus sample, and the detection efficiency of sample can be improved in this way, saves time cost；It is reduced costs under the precursor for meeting testing result accuracy using content organic phosphorus in spectrophotometry measurement sample; the stronger organic solvent of toxicity is not used, protects environment, and provide the experimental place of a safety for experimenter; easy to operate, the used time is short, high-efficient.
Description
Technical field
The present invention relates to technical field of food safety detection, in specifically a kind of food organic phosphorus detection kit and its
Detection method.
Background technique
In process of production, in order to improve yield, peasant household can largely be prevented and treated using chemical fertilizer and pesticide agricultural and sideline product at present
Disease and insect, organophosphorus pesticide is because it is with wide spectrum, efficient, lower-price characteristic, it has also become the big pillar of our times pesticide three
One of, wherein group thiophosphate is more common a kind of in organophosphorus pesticide, the toxicity such as Rogor, thimet, malathion
Relatively by force, the pesticide of good disinsection effect belongs to such, plays an important role in terms of guaranteeing stable and high agricultural yields.However, by
Lack the knowledge of scientifical use pesticide in user, covet yield, causes all different journeys of the agricultural and sideline product to circulate in the market
Degree there are Pesticide Residue, become the hidden danger for threatening people's life and health so that remain a large amount of pesticide in agricultural and sideline product,
The pollution for causing agricultural and sideline product causes serious influence to the health of people when people eat.In order to guarantee people's lives water
It is flat, before agricultural and sideline product enters market, need organic phosphorus to detect to contained therein, it is ensured that it meets the edible mark of health
It is quasi-.
Since the active constituent of organophosphorus pesticide is small molecular organic compounds mostly, thus mostly using gas-chromatography (GC),
High performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC-MS) and high performance liquid chromatography-mass spectrometry (HPLC-MS) and
Multi-stage ms joint technology, wherein research application it is most be GC-MS, be suitable for most of Detecting Pesticide, it is above-mentioned these
Although instrument has many advantages, such as quantitative accurate, high sensitivity, technical requirements are high, and detection cycle is long, at the scene quickly detection and
There is limitation in batch samples screening, it is difficult to meet prevention and control emergency event, and above-mentioned instrument price is expensive, at
This height.
Summary of the invention
The purpose of the present invention is to provide detection kits and its detection method organic phosphorus in a kind of food, existing to solve
There is the problems in technology.
To achieve the above object, the invention provides the following technical scheme:
Organic phosphorus detection kit in a kind of food, the detection kit include the first reagent and the second reagent, the first reagent
Including following components: buffer, gold chloride, reducing agent and pH adjusting agent, the second reagent includes following components: enzyme solution, color developing agent
And substrate.
As optimization, buffer is acetic acid-sodium acetate buffer solution, dipotassium hydrogen phosphate-potassium phosphate buffer, lemon
Any one in acid-sodium citrate buffer solution, oligosaccharide-acetate buffer or phosphate-citrate buffer, buffer
PH value be 8-9.
As optimization, reducing agent is sodium dithionite, trisodium citrate, sodium borohydride, ascorbic acid or stannous octoate
In any one, the concentration of reducing agent is 1-10g/L.
As optimization, enzyme solution is acetylcholinesterase, and the concentration of enzyme solution is 0.3-1 μ g/ml, and substrate is thioacetyl gallbladder
Alkali, the concentration of substrate are 5-8mg/ml, and pH adjusting agent is sulfuric acid or sodium hydrate aqueous solution, and the mass fraction of aqueous sulfuric acid is
5-10%, the mass fraction of sodium hydrate aqueous solution are 10-20%.
As optimization, color developing agent is dissolved in 1L by 8-16g dithiobis-nitrobenzoic acid and 0.8-1.6g sodium bicarbonate and delays
It rushes in solution and is formulated.
Testing principle of the invention are as follows: the gold ion in gold chloride can be reduced into nanogold, nanogold dispersion by reducing agent
In solution, solution be in pink colour, red or claret, when in sample to be tested containing it is organic phosphorus when, it is organic phosphorus in phosphorus methylthio group
Golden sulfide linkage can be passed through with nanogold to interact, under the action of multiple golden sulfide linkage, nanogold is easy to assemble, and leads to solution face
Color changes, and using the variation of the color, qualitative detection organic phosphorus in sample may be implemented.
It is quantitative for detecting then to carry out quantitative detection containing organic phosphorus sample by organic phosphorus qualitative detection
The principle of detection are as follows: organic phosphorus inhibited to acetylcholinesterase, inhibiting rate is directly proportional to organic phosphorus content, when
There is no it is organic phosphorus when, acetylcholine ester substrate for enzymatic activity acetylthiocholine hydrolysis, acetylthiocholine hydrolysis product with
Color developing agent reaction, generates yellow substance, and when there are organic phosphorus, acetylcholinesterase is suppressed, and is unable to the thio second of catalysis substrate
Phatidylcholine hydrolysis, measures the absorbance value amount of changing with time with ultraviolet-uisible spectrophotometer at 412nm, calculates inhibition
Rate calculates content organic phosphorus in sample by inhibiting rate.
Organic phosphorus detection method in a kind of food, should detection method includes the following steps:
(1) pre-treatment of sample；
(2) qualitative detection of sample；
(3) production of standard curve；
(4) quantitative detection of sample.
As optimization, organic phosphorus detection method in a kind of food should detection method includes the following steps:
(1) pre-treatment of sample: extracting sample using buffer, and centrifugation obtains extract liquor, spare；
(2) it the qualitative detection of sample: takes step (1) resulting extract liquor to be placed in test tube, chlorine is then sequentially added into test tube
Auric acid, reducing agent and pH adjusting agent are vortexed, and mix, and stand, and observe color change；
(3) production of standard curve:
Buffer is added into colorimetric cylinder, then enzyme solution and color developing agent are added into colorimetric cylinder, shakes up, when placing one section in water-bath
Between, the absorbance value of above-mentioned solution is measured with ultraviolet-uisible spectrophotometer, then adds substrate, is shaken up, and is opened in colorimetric cylinder
Beginning chromogenic reaction after a period of time, with the absorbance value of solution after ultraviolet-uisible spectrophotometer measurement reaction, calculates reaction
Preceding absorbance value with react after absorbance value variable quantity △ A0；
Several colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, constant volume is configured to
The standard curve serial solution of various concentration is separately added into enzyme solution and color developing agent in Xiang Shangshu standard curve serial solution, shakes up,
A period of time is placed in water-bath, and the absorbance value A of above-mentioned solution is measured with ultraviolet-uisible spectrophotometer1, then add bottom
Object shakes up, and starts chromogenic reaction in colorimetric cylinder, after a period of time, with solution after ultraviolet-uisible spectrophotometer measurement reaction
Absorbance value, calculate reaction before absorbance value with react after absorbance value variable quantity △ A1, calculate various concentration
Standard curve serial solution to the inhibiting rate of enzyme, the concentration value of the standard curve serial solution of various concentration is taken respectively
log10, log is taken with the concentration value of the standard curve serial solution to various concentration10The logarithm obtained the bottom of for is abscissa, with
Its corresponding inhibiting rate is ordinate, makes standard curve, and find out the regression equation of the standard curve；
(4) quantitative detection of sample:
It takes the obtained sample extraction liquid of step (1) that colorimetric cylinder is added, then enzyme solution and color developing agent is added into colorimetric cylinder, shake up,
A period of time is placed in water-bath, and the absorbance value A of above-mentioned solution is measured with ultraviolet-uisible spectrophotometer2, then add bottom
Object shakes up, and starts chromogenic reaction in colorimetric cylinder, after a period of time, with solution after ultraviolet-uisible spectrophotometer measurement reaction
Absorbance value, calculate reaction before absorbance value with react after absorbance value variable quantity △ A2, according to the meter of inhibiting rate
It calculates formula and inhibiting rate is calculated, which is brought into the regression equation of standard curve, calculates organic phosphorus in sample contain
Amount.
As optimization, organic phosphorus detection method in a kind of food should detection method includes the following steps:
(1) pre-treatment of sample: 5-15g sample is accurately weighed, is placed in the centrifuge tube of 50-250ml, is added into centrifuge tube
Then 50-200ml buffer, the ultrasonic extraction 15-30min at 50-100KHz are centrifuged in the case where revolving speed is 3000-5000r/min
5-10min takes upper layer of extraction liquid, spare；The purpose of ultrasound is to make buffer to the more abundant of the organic phosphorus extraction in sample, is mentioned
The accuracy of high assay result；
(2) qualitative detection of sample: taking 3-5ml step (1) resulting extract liquor to be placed in test tube, then successively adds into test tube
Enter 3-5ml gold chloride, 3-5ml reducing agent and 1-2mlpH regulator, cap, vortex 30-60s is uniformly mixed, and stands 5-
10min observes color change, if the aobvious blue of the color of solution in test tube, remain in sample it is organic phosphorus, if solution in test tube
The aobvious red of color, then without organophosphorus residue in sample；It is measured by qualitative detection and continues to determine containing organic phosphorus sample
Amount detection, for organic phosphorus sample is not detected, need not carry out quantitative detection again, and the detection effect of sample can be improved in this way
Rate saves time cost；
(3) production of standard curve:
1 colorimetric cylinder is taken, 50ml buffer is added into colorimetric cylinder, then 2ml enzyme solution and 2ml color developing agent are added into colorimetric cylinder,
15min is placed in 35-40 DEG C of water-bath after shaking up, measures above-mentioned solution at 412nm with ultraviolet-uisible spectrophotometer
Absorbance value A0, 2ml substrate is then added, is shaken up, starts chromogenic reaction in colorimetric cylinder, after 3-5min, with UV, visible light point
The absorbance value of light photometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after suction
The variable quantity △ A of shading value0；
6 colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, is settled to buffer
50ml is configured to the standard curve series that concentration is respectively 5 μ g/L, 10 μ g/L, 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L
Solution is separately added into 2ml enzyme solution and 2ml color developing agent in Xiang Shangshu standard curve serial solution, in 35-40 DEG C of water-bath after shaking up
Middle placement 15min measures the absorbance value A of above-mentioned solution with ultraviolet-uisible spectrophotometer at 412nm1, then add
2ml substrate, shakes up, beginning chromogenic reaction in colorimetric cylinder, after 3-5min, is measured at 412nm with ultraviolet-uisible spectrophotometer
The absorbance value of solution after reaction, calculate reaction before absorbance value with react after absorbance value variable quantity △ A1, meter
Inhibiting rate of the standard curve serial solution to enzyme for calculating various concentration, respectively to the standard curve serial solution of various concentration
Concentration value takes log10, log is taken with the concentration value of the standard curve serial solution to various concentration10The logarithm obtained the bottom of for is
Abscissa makes standard curve using its corresponding inhibiting rate as ordinate, and finds out the regression equation of the standard curve；It is organic
Solute contained in phosphorus mixed standard solution has DDVP, Rogor, parathion-methyl, malathion, Hostathion and thimet.
Table 1 is the equation of linear regression, related coefficient and detection limit of the method for the present invention.
Table 1
Note: the concentration value and analyte that y and x respectively represents analyte are to the inhibiting rate of enzyme.
From table 1 it follows that this 6 kinds organic phosphorus in the corresponding range of linearity, there is good linear relationship, meet
Analysis method requirement.
(4) quantitative detection of sample:
Take the obtained sample extraction liquid of 50ml step (1) that colorimetric cylinder is added, then addition 2ml enzyme solution and 2ml are aobvious into colorimetric cylinder
Toner places 15min in 35-40 DEG C of water-bath after shaking up, and is measured at 412nm with ultraviolet-uisible spectrophotometer above-mentioned molten
The absorbance value A of liquid2, then add 2ml substrate, shake up, start chromogenic reaction in colorimetric cylinder, after 3-5min, with it is ultraviolet can
The absorbance value for seeing spectrophotometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after
Absorbance value variable quantity △ A2, inhibiting rate is calculated according to the calculation formula of inhibiting rate, brings the inhibiting rate into standard
The regression equation of curve calculates content organic phosphorus in sample.
As optimization, the concentration of organic phosphorus mixed standard solution is 10mg/ml in step (3).
As optimization, the calculation formula of inhibiting rate in step (3) are as follows:
；
Wherein, △ A0For the variable quantity of the absorbance before and after blank sample solution reaction；△A1For the suction of sample solution reaction front and back
The variable quantity of luminosity.
Using content organic phosphorus in spectrophotometry measurement sample, under the precursor for meeting testing result accuracy, not
It using as large-scale instruments such as gas chromatograph, gas chromatograph-mass spectrometer (GC-MS)s, reduces costs, in the processing and detection of sample
The stronger organic solvent of some toxicity is not used in the process, protects environment, and provide the experiment of a safety for experimenter
Place, the pre-treatment of sample and detection process are easy to operate, and the used time is short, high-efficient.
Compared with prior art, the beneficial effects of the present invention are:
First is that in detection kit and its detection method organic phosphorus in a kind of food of the present invention, reducing agent can will be in gold chloride
Gold ion is reduced into nanogold, and nanogold is scattered in solution, and solution is in pink colour, red or claret, when containing in sample to be tested
When having organic phosphorus, it is organic phosphorus in phosphorus methylthio group can pass through golden sulfide linkage with nanogold and interact, in the work of multiple golden sulfide linkage
Under, nanogold is easy to assemble, and solution colour is caused to change, and using the variation of the color, may be implemented organic in sample
The qualitative detection of phosphorus；By organic phosphorus qualitative detection, for detecting then to carry out quantitative detection containing organic phosphorus sample,
Organic phosphorus inhibited to acetylcholinesterase, inhibiting rate is directly proportional to organic phosphorus content, when there is no organic phosphorus
When, the product of the hydrolysis of acetylcholine ester substrate for enzymatic activity acetylthiocholine, acetylthiocholine hydrolysis is reacted with color developing agent, raw
Yellowly substance, when there are organic phosphorus, acetylcholinesterase is suppressed, and is unable to the hydrolysis of catalysis substrate acetylthiocholine, is used
Ultraviolet-uisible spectrophotometer measures the absorbance value amount of changing with time at 412nm, calculates inhibiting rate, passes through inhibiting rate
Calculate content organic phosphorus in sample；
Second is that in detection method organic phosphorus in a kind of food of the present invention, by qualitative detection measure containing organic phosphorus sample after
The continuous quantitative detection that carries out need not carry out quantitative detection again, sample can be improved in this way for organic phosphorus sample is not detected
Detection efficiency saves time cost；
In a kind of three food of the present invention in organic phosphorus detection method, contained using organic phosphorus in spectrophotometry measurement sample
Amount is not used under the precursor for meeting testing result accuracy as the large sizes such as gas chromatograph, gas chromatograph-mass spectrometer (GC-MS)
Instrument reduces costs, and the stronger organic solvent of some toxicity is not used in the processing and detection process of sample, protects ring
Border, and the experimental place of a safety is provided for experimenter, the pre-treatment of sample and detection process are easy to operate, and the used time is short,
It is high-efficient.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1: organic phosphorus detection in spinach
Organic phosphorus detection kit in a kind of food, the detection kit include the first reagent and the second reagent, the first reagent
Including following components: the pH value of acetic acid-sodium acetate buffer solution, gold chloride, sodium dithionite and aqueous sulfuric acid, buffer is
8, the concentration of hydrogensulfite solution is 1g/L, and the mass fraction of aqueous sulfuric acid is 5%, and the second reagent includes following components:
The concentration of acetylcholinesterase enzyme solution, color developing agent and substrate acetylthiocholine, enzyme solution is 0.3 μ g/ml, and the concentration of substrate is
5mg/ml, color developing agent are dissolved in 1L buffer solution by 8g dithiobis-nitrobenzoic acid and 0.8g sodium bicarbonate and are formulated.
Organic phosphorus detection method in a kind of food, should detection method includes the following steps:
(1) it the pre-treatment of sample: after spinach samples are dried, crushes, accurately weighs 5g spinach samples, be placed in the centrifuge tube of 50ml
In, 50ml acetic acid-sodium acetate buffer solution is added into centrifuge tube, then the ultrasonic extraction 15min at 50KHz is in revolving speed
It is centrifuged 5min under 3000r/min, takes upper layer of extraction liquid, it is spare；
(2) it the qualitative detection of sample: takes 3ml step (1) resulting extract liquor to be placed in test tube, is then sequentially added into test tube
3ml gold chloride, 3ml sodium dithionite and 1ml aqueous sulfuric acid, cap, vortex 30s are uniformly mixed, and stand 5min,
Observe color change, if the aobvious blue of the color of solution in test tube, remain in spinach samples it is organic phosphorus, if solution in test tube
The aobvious red of color, then without organophosphorus residue in spinach samples；
(3) production of standard curve:
1 colorimetric cylinder is taken, 50ml acetic acid-sodium acetate buffer solution is added into colorimetric cylinder, then 2ml acetyl gallbladder is added into colorimetric cylinder
Alkali esterase enzyme solution and 2ml color developing agent, place 15min in 35 DEG C of water-bath after shaking up, are existed with ultraviolet-uisible spectrophotometer
The absorbance value A of above-mentioned solution is measured at 412nm0, 2ml acetylthiocholine substrate is then added, shakes up, is opened in colorimetric cylinder
Beginning chromogenic reaction after 3min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, calculates
Out react before absorbance value with react after absorbance value variable quantity △ A0；
6 colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, is settled to buffer
50ml is configured to the standard curve series that concentration is respectively 5 μ g/L, 10 μ g/L, 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L
Solution is separately added into 2ml acetylcholinesterase enzyme solution and 2ml color developing agent in Xiang Shangshu standard curve serial solution, after shaking up
15min is placed in 35 DEG C of water-bath, measures the absorbance value A of above-mentioned solution at 412nm with ultraviolet-uisible spectrophotometer1,
Then 2ml acetylthiocholine substrate is added, is shaken up, starts chromogenic reaction in colorimetric cylinder, after 3min, is divided with UV, visible light
The absorbance value of photometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after extinction
The variable quantity △ A of angle value1, inhibiting rate of the standard curve serial solution to enzyme of various concentration is calculated, respectively to various concentration
The concentration value of standard curve serial solution take log10, taken with the concentration value of the standard curve serial solution to various concentration
log10The logarithm obtained the bottom of for is abscissa, using its corresponding inhibiting rate as ordinate, makes standard curve, and find out the mark
The regression equation of directrix curve；Solute contained in organic phosphorus mixed standard solution has DDVP, Rogor, parathion-methyl, horse traction
Sulphur phosphorus, Hostathion and thimet.
Table 1 is the equation of linear regression, related coefficient and detection limit of the method for the present invention.
Table 1
Note: the concentration value and analyte that y and x respectively represents analyte are to the inhibiting rate of enzyme.
From table 1 it follows that this 6 kinds organic phosphorus in the corresponding range of linearity, there is good linear relationship, meet
Analysis method requirement.
(4) quantitative detection of sample:
It takes the obtained spinach samples extract liquor of 50ml step (1) that colorimetric cylinder is added, then 2ml acetylcholine is added into colorimetric cylinder
Esterase enzyme solution and 2ml color developing agent, place 15min in 35-40 DEG C of water-bath after shaking up, are existed with ultraviolet-uisible spectrophotometer
The absorbance value A of above-mentioned solution is measured at 412nm2, 2ml acetylthiocholine substrate is then added, shakes up, is opened in colorimetric cylinder
Beginning chromogenic reaction, after 3-5min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, meter
Calculate reaction before absorbance value with react after absorbance value variable quantity △ A2, calculated according to the calculation formula of inhibiting rate
The inhibiting rate is brought into the regression equation of standard curve by inhibiting rate out, calculates content organic phosphorus in spinach samples.
The concentration of organic phosphorus mixed standard solution is 10mg/ml in step (3).
As optimization, the calculation formula of inhibiting rate in step (3) are as follows:
；
Wherein, △ A0For the variable quantity of the absorbance before and after blank sample solution reaction；△A1Before and after spinach samples solution reaction
Absorbance variable quantity.
Be not detected through detecting, in spinach samples 6 kinds it is organic phosphorus, 6 kinds of organic phosphorus different mark-ons are dense in mark-on spinach samples
The recovery testu of degree level the results are shown in Table 2, and every mark-on spinach samples are measured in parallel 5 times.
Table 2
Note: ND is to be not detected.
From Table 2, it can be seen that the recovery of standard addition of mark-on spinach samples is 96-104%, relative deviation (n=5) is 2.7-
4.1%, meet analysis method to the rate of recovery and reproducibility requirement.
Embodiment 2: organic phosphorus detection in apple
Organic phosphorus detection kit in a kind of food, the detection kit include the first reagent and the second reagent, the first reagent
Including following components: dipotassium hydrogen phosphate-potassium phosphate buffer, gold chloride, trisodium citrate and sodium hydrate aqueous solution delay
The pH value of fliud flushing is 8.2, and the concentration of trisodium citrate is 3g/L, and the mass fraction of sodium hydrate aqueous solution is 12%, the second reagent
Including following components: acetylcholinesterase enzyme solution, color developing agent and substrate acetylthiocholine, the concentration of enzyme solution are 0.4 μ g/ml,
The concentration of substrate is 6mg/ml, and color developing agent is dissolved in 1L buffer solution by 9g dithiobis-nitrobenzoic acid and 0.9g sodium bicarbonate
In be formulated.
Organic phosphorus detection method in a kind of food, should detection method includes the following steps:
(1) pre-treatment of sample: being cut into powder for apple sample, accurately weighs 8g apple sample, is placed in the centrifuge tube of 100ml
In, addition 80ml dipotassium hydrogen phosphate-potassium phosphate buffer into centrifuge tube, the ultrasonic extraction 18min at 60KHz, then
It is centrifuged 6min in the case where revolving speed is 3500r/min, takes upper layer of extraction liquid, it is spare；
(2) it the qualitative detection of sample: takes 3ml step (1) resulting extract liquor to be placed in test tube, is then sequentially added into test tube
3ml gold chloride, 3ml trisodium citrate and 1ml sodium hydrate aqueous solution, cap, vortex 35s are uniformly mixed, and are stood
6min observes color change, if the aobvious blue of the color of solution in test tube, remain in apple sample it is organic phosphorus, if in test tube
The aobvious red of the color of solution, then without organophosphorus residue in apple sample；
(3) production of standard curve:
1 colorimetric cylinder is taken, 50ml dipotassium hydrogen phosphate-potassium phosphate buffer is added into colorimetric cylinder, then is added into colorimetric cylinder
Enter 2ml acetylcholinesterase enzyme solution and 2ml color developing agent, places 15min in 36 DEG C of water-bath after shaking up, be divided with UV, visible light
Photometer measures the absorbance value A of above-mentioned solution at 412nm0, 2ml acetylthiocholine substrate is then added, is shaken up, than
Start chromogenic reaction in colour tube, after 3min, with the absorbance of ultraviolet-uisible spectrophotometer solution after the measurement of the place 412nm is reacted
Value, calculate reaction before absorbance value with react after absorbance value variable quantity △ A0；
6 colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, is settled to buffer
50ml is configured to the standard curve series that concentration is respectively 5 μ g/L, 10 μ g/L, 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L
Solution is separately added into 2ml acetylcholinesterase enzyme solution and 2ml color developing agent in Xiang Shangshu standard curve serial solution, after shaking up
15min is placed in 36 DEG C of water-bath, measures the absorbance value A of above-mentioned solution at 412nm with ultraviolet-uisible spectrophotometer1,
Then 2ml acetylthiocholine substrate is added, is shaken up, starts chromogenic reaction in colorimetric cylinder, after 3min, is divided with UV, visible light
The absorbance value of photometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after extinction
The variable quantity △ A of angle value1, inhibiting rate of the standard curve serial solution to enzyme of various concentration is calculated, respectively to various concentration
The concentration value of standard curve serial solution take log10, taken with the concentration value of the standard curve serial solution to various concentration
log10The logarithm obtained the bottom of for is abscissa, using its corresponding inhibiting rate as ordinate, makes standard curve, and find out the mark
The regression equation of directrix curve；Solute contained in organic phosphorus mixed standard solution has DDVP, Rogor, parathion-methyl, horse traction
Sulphur phosphorus, Hostathion and thimet.
Table 1 is the equation of linear regression, related coefficient and detection limit of the method for the present invention.
Table 1
Note: the concentration value and analyte that y and x respectively represents analyte are to the inhibiting rate of enzyme.
From table 1 it follows that this 6 kinds organic phosphorus in the corresponding range of linearity, there is good linear relationship, meet
Analysis method requirement.
(4) quantitative detection of sample:
It takes the obtained apple sample extraction liquid of 50ml step (1) that colorimetric cylinder is added, then 2ml acetylcholine is added into colorimetric cylinder
Esterase enzyme solution and 2ml color developing agent, place 15min in 36 DEG C of water-bath after shaking up, with ultraviolet-uisible spectrophotometer in 412nm
Place measures the absorbance value A of above-mentioned solution2, 2ml acetylthiocholine substrate is then added, is shaken up, starts to show in colorimetric cylinder
Colour response after 3min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, calculates anti-
Absorbance value before answering with react after absorbance value variable quantity △ A2, inhibition is calculated according to the calculation formula of inhibiting rate
The inhibiting rate is brought into the regression equation of standard curve by rate, calculates content organic phosphorus in apple sample.
The concentration of organic phosphorus mixed standard solution is 10mg/ml in step (3).
As optimization, the calculation formula of inhibiting rate in step (3) are as follows:
；
Wherein, △ A0For the variable quantity of the absorbance before and after blank sample solution reaction；△A1Front and back is reacted for apple sample solution
Absorbance variable quantity.
Through detecting, be not detected in apple sample 6 kinds it is organic phosphorus, 6 kinds of organic phosphorus different mark-ons are dense in mark-on apple sample
The recovery testu of degree level the results are shown in Table 3, and every mark-on apple sample is measured in parallel 5 times.
Table 3
Note: ND is to be not detected.
From table 3 it is observed that the recovery of standard addition of mark-on spinach samples is 93-104%, relative deviation (n=5) is 2.7-
4.5%, meet analysis method to the rate of recovery and reproducibility requirement.
Embodiment 3: organic phosphorus detection in wheat
Organic phosphorus detection kit in a kind of food, the detection kit include the first reagent and the second reagent, the first reagent
Including following components: the pH value of citric acid-sodium citrate buffer solution, gold chloride, sodium borohydride and aqueous sulfuric acid, buffer is
9, the concentration of sodium borohydride is 5g/L, and the mass fraction of aqueous sulfuric acid is 8%, and the second reagent includes following components: acetylcholine
Esterase enzyme solution, color developing agent and acetylthiocholine substrate, the concentration of enzyme solution are 0.7 μ g/ml, and the concentration of substrate is 7mg/ml, are shown
Toner is dissolved in 1L buffer solution by 12g dithiobis-nitrobenzoic acid and 1.2g sodium bicarbonate and is formulated.
Organic phosphorus detection method in a kind of food, should detection method includes the following steps:
(1) pre-treatment of sample: by wheat crushing, 10g wheat samples is accurately weighed, are placed in the centrifuge tube of 150ml, to centrifugation
120ml citric acid-sodium citrate buffer solution is added in pipe, then the ultrasonic extraction 25min at 80KHz is 4500r/ in revolving speed
It is centrifuged 8min under min, takes upper layer of extraction liquid, it is spare；
(2) it the qualitative detection of sample: takes 4ml step (1) resulting extract liquor to be placed in test tube, is then sequentially added into test tube
4ml gold chloride, 4ml sodium borohydride and 2ml aqueous sulfuric acid, cap, vortex 55s are uniformly mixed, and stand 8min, observation
Color change, if the aobvious blue of the color of solution in test tube, remain in wheat samples it is organic phosphorus, if in test tube solution color
Aobvious red, then without organophosphorus residue in wheat samples；
(3) production of standard curve:
1 colorimetric cylinder is taken, 50ml citric acid-sodium citrate buffer solution is added into colorimetric cylinder, then 2ml second is added into colorimetric cylinder
Acetylcholinesterase enzyme solution and 2ml color developing agent, place 15min in 38 DEG C of water-bath after shaking up, use ultraviolet-uisible spectrophotometer
The absorbance value A of above-mentioned solution is measured at 412nm0, 2ml acetylthiocholine substrate is then added, is shaken up, in colorimetric cylinder
Start chromogenic reaction, after 4min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after 412nm locates measurement reaction, counts
Calculate reaction before absorbance value with react after absorbance value variable quantity △ A0；
6 colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, is settled to buffer
50ml is configured to the standard curve series that concentration is respectively 5 μ g/L, 10 μ g/L, 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L
Solution is separately added into 2ml acetylcholinesterase enzyme solution and 2ml color developing agent in Xiang Shangshu standard curve serial solution, after shaking up
15min is placed in 38 DEG C of water-bath, measures the absorbance value A of above-mentioned solution at 412nm with ultraviolet-uisible spectrophotometer1,
Then 2ml acetylthiocholine substrate is added, is shaken up, starts chromogenic reaction in colorimetric cylinder, after 4min, is divided with UV, visible light
The absorbance value of photometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after extinction
The variable quantity △ A of angle value1, inhibiting rate of the standard curve serial solution to enzyme of various concentration is calculated, respectively to various concentration
The concentration value of standard curve serial solution take log10, taken with the concentration value of the standard curve serial solution to various concentration
log10The logarithm obtained the bottom of for is abscissa, using its corresponding inhibiting rate as ordinate, makes standard curve, and find out the mark
The regression equation of directrix curve；Solute contained in organic phosphorus mixed standard solution has DDVP, Rogor, parathion-methyl, horse traction
Sulphur phosphorus, Hostathion and thimet.
Table 1 is the equation of linear regression, related coefficient and detection limit of the method for the present invention.
Table 1
Note: the concentration value and analyte that y and x respectively represents analyte are to the inhibiting rate of enzyme.
From table 1 it follows that this 6 kinds organic phosphorus in the corresponding range of linearity, there is good linear relationship, meet
Analysis method requirement.
(4) quantitative detection of sample:
It takes the obtained wheat samples extract liquor of 50ml step (1) that colorimetric cylinder is added, then 2ml acetylcholine is added into colorimetric cylinder
Esterase enzyme solution and 2ml color developing agent, place 15min in 38 DEG C of water-bath after shaking up, with ultraviolet-uisible spectrophotometer in 412nm
Place measures the absorbance value A of above-mentioned solution2, 2ml acetylthiocholine substrate is then added, is shaken up, starts to show in colorimetric cylinder
Colour response after 4min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, calculates anti-
Absorbance value before answering with react after absorbance value variable quantity △ A2, inhibition is calculated according to the calculation formula of inhibiting rate
The inhibiting rate is brought into the regression equation of standard curve by rate, calculates content organic phosphorus in wheat samples.
The concentration of organic phosphorus mixed standard solution is 10mg/ml in step (3).
As optimization, the calculation formula of inhibiting rate in step (3) are as follows:
；
Wherein, △ A0For the variable quantity of the absorbance before and after blank sample solution reaction；△A1Before and after wheat samples solution reaction
Absorbance variable quantity.
Be not detected through detecting, in wheat samples 6 kinds it is organic phosphorus, 6 kinds of organic phosphorus different mark-ons are dense in mark-on wheat samples
The recovery testu of degree level the results are shown in Table 4, and every mark-on wheat samples are measured in parallel 5 times.
Table 4
Note: ND is to be not detected.
As can be seen from Table 4, the recovery of standard addition of mark-on wheat samples is 95-103%, and relative deviation (n=5) is 2.7-
4.3%, meet analysis method to the rate of recovery and reproducibility requirement.
Embodiment 4: organic phosphorus detection in cucumber
Organic phosphorus detection kit in a kind of food, the detection kit include the first reagent and the second reagent, the first reagent
Including following components: oligosaccharide-acetate buffer, gold chloride, ascorbic acid and sodium hydrate aqueous solution, the pH value of buffer
It is 8, the concentration of ascorbic acid is 9g/L, and the mass fraction of sodium hydrate aqueous solution is 19%, and the second reagent includes following components:
The concentration of acetylcholinesterase enzyme solution, color developing agent and acetylthiocholine substrate, enzyme solution is 0.9 μ g/ml, and the concentration of substrate is
7mg/ml, color developing agent are dissolved in 1L buffer solution by 15g dithiobis-nitrobenzoic acid and 1.5g sodium bicarbonate and are formulated.
Organic phosphorus detection method in a kind of food, should detection method includes the following steps:
(1) pre-treatment of sample: being cut into powder for cucumber sample, accurately weighs 14g cucumber sample, is placed in the centrifuge tube of 200ml
In, 180ml oligosaccharide-acetate buffer is added into centrifuge tube, then the ultrasonic extraction 29min at 90KHz is in revolving speed
It is centrifuged 9min under 4500r/min, takes upper layer of extraction liquid, it is spare；
(2) it the qualitative detection of sample: takes 3ml step (1) resulting extract liquor to be placed in test tube, is then sequentially added into test tube
3ml gold chloride, 3ml ascorbic acid and 1ml sodium hydrate aqueous solution, cap, vortex 55s are uniformly mixed, and stand 9min,
Observe color change, if the aobvious blue of the color of solution in test tube, remain in cucumber sample it is organic phosphorus, if solution in test tube
The aobvious red of color, then without organophosphorus residue in cucumber sample；
(3) production of standard curve:
1 colorimetric cylinder is taken, 50ml oligosaccharide-acetate buffer is added into colorimetric cylinder, then 2ml acetyl is added into colorimetric cylinder
Cholinesterase enzyme solution and 2ml color developing agent, place 15min in 39 DEG C of water-bath after shaking up, are existed with ultraviolet-uisible spectrophotometer
The absorbance value A of above-mentioned solution is measured at 412nm0, 2ml acetylthiocholine substrate is then added, shakes up, is opened in colorimetric cylinder
Beginning chromogenic reaction after 5min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, calculates
Out react before absorbance value with react after absorbance value variable quantity △ A0；
6 colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, is settled to buffer
50ml is configured to the standard curve series that concentration is respectively 5 μ g/L, 10 μ g/L, 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L
Solution is separately added into 2ml acetylcholinesterase enzyme solution and 2ml color developing agent in Xiang Shangshu standard curve serial solution, after shaking up
15min is placed in 39 DEG C of water-bath, measures the absorbance value A of above-mentioned solution at 412nm with ultraviolet-uisible spectrophotometer1,
Then 2ml acetylthiocholine substrate is added, is shaken up, starts chromogenic reaction in colorimetric cylinder, after 5min, is divided with UV, visible light
The absorbance value of photometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after extinction
The variable quantity △ A of angle value1, inhibiting rate of the standard curve serial solution to enzyme of various concentration is calculated, respectively to various concentration
The concentration value of standard curve serial solution take log10, taken with the concentration value of the standard curve serial solution to various concentration
log10The logarithm obtained the bottom of for is abscissa, using its corresponding inhibiting rate as ordinate, makes standard curve, and find out the mark
The regression equation of directrix curve；Solute contained in organic phosphorus mixed standard solution has DDVP, Rogor, parathion-methyl, horse traction
Sulphur phosphorus, Hostathion and thimet.
Table 1 is the equation of linear regression, related coefficient and detection limit of the method for the present invention.
Table 1
Note: the concentration value and analyte that y and x respectively represents analyte are to the inhibiting rate of enzyme.
From table 1 it follows that this 6 kinds organic phosphorus in the corresponding range of linearity, there is good linear relationship, meet
Analysis method requirement.
(4) quantitative detection of sample:
It takes the obtained cucumber sample extraction liquid of 50ml step (1) that colorimetric cylinder is added, then 2ml acetylcholine is added into colorimetric cylinder
Esterase enzyme solution and 2ml color developing agent, place 15min in 39 DEG C of water-bath after shaking up, with ultraviolet-uisible spectrophotometer in 412nm
Place measures the absorbance value A of above-mentioned solution2, 2ml acetylthiocholine substrate is then added, is shaken up, starts to show in colorimetric cylinder
Colour response after 5min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, calculates anti-
Absorbance value before answering with react after absorbance value variable quantity △ A2, inhibition is calculated according to the calculation formula of inhibiting rate
The inhibiting rate is brought into the regression equation of standard curve by rate, is calculated cucumber and is gone out content organic phosphorus in sample.
The concentration of organic phosphorus mixed standard solution is 10mg/ml in step (3).
As optimization, the calculation formula of inhibiting rate in step (3) are as follows:
；
Wherein, △ A0For the variable quantity of the absorbance before and after blank sample solution reaction；△A1Front and back is reacted for cucumber sample solution
Absorbance variable quantity.
Through detecting, be not detected in cucumber sample 6 kinds it is organic phosphorus, 6 kinds of organic phosphorus different mark-ons are dense in mark-on cucumber sample
The recovery testu of degree level the results are shown in Table 5, and every mark-on cucumber sample is measured in parallel 5 times.
Table 5
Note: ND is to be not detected.
As can be seen from Table 5, the recovery of standard addition of mark-on cucumber sample is 95-103%, and relative deviation (n=5) is 3.2-
4.1%, meet analysis method to the rate of recovery and reproducibility requirement.
Embodiment 5: organic phosphorus detection in soybean
Organic phosphorus detection kit in a kind of food, the detection kit include the first reagent and the second reagent, the first reagent
Including following components: phosphate-citrate buffer, gold chloride, stannous octoate and aqueous sulfuric acid, the pH value of buffer are 9,
The concentration of stannous octoate is 10g/L, and the mass fraction of aqueous sulfuric acid is 10%, and the second reagent includes following components: acetylcholine
Esterase enzyme solution, color developing agent and acetylthiocholine substrate, the concentration of enzyme solution are 1 μ g/ml, and the concentration of substrate is 8mg/ml, colour developing
Agent is dissolved in 1L buffer solution by 16g dithiobis-nitrobenzoic acid and 1.6g sodium bicarbonate and is formulated.
Organic phosphorus detection method in a kind of food, should detection method includes the following steps:
(1) pre-treatment of sample: soybean sample is crushed, and is accurately weighed 15g soybean sample, is placed in the centrifuge tube of 250ml, to
200ml phosphate-citrate buffer is added in centrifuge tube, then the ultrasonic extraction 30min at 100KHz is in revolving speed
It is centrifuged 10min under 5000r/min, takes upper layer of extraction liquid, it is spare；
(2) it the qualitative detection of sample: takes 5ml step (1) resulting extract liquor to be placed in test tube, is then sequentially added into test tube
5ml gold chloride, 5ml stannous octoate and 2ml aqueous sulfuric acid, cap, vortex 60s are uniformly mixed, and stand 10min, observation
Color change, if the aobvious blue of the color of solution in test tube, remain in soybean sample it is organic phosphorus, if in test tube solution color
Aobvious red, then without organophosphorus residue in soybean sample；
(3) production of standard curve:
1 colorimetric cylinder is taken, 50ml phosphate-citrate buffer is added into colorimetric cylinder, then 2ml acetyl is added into colorimetric cylinder
Cholinesterase enzyme solution and 2ml color developing agent, place 15min in 40 DEG C of water-bath after shaking up, are existed with ultraviolet-uisible spectrophotometer
The absorbance value A of above-mentioned solution is measured at 412nm0, 2ml acetylthiocholine substrate is then added, shakes up, is opened in colorimetric cylinder
Beginning chromogenic reaction after 5min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, calculates
Out react before absorbance value with react after absorbance value variable quantity △ A0；
6 colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, is settled to buffer
50ml is configured to the standard curve series that concentration is respectively 5 μ g/L, 10 μ g/L, 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L
Solution is separately added into 2ml acetylcholinesterase enzyme solution and 2ml color developing agent in Xiang Shangshu standard curve serial solution, after shaking up
15min is placed in 40 DEG C of water-bath, measures the absorbance value A of above-mentioned solution at 412nm with ultraviolet-uisible spectrophotometer1,
Then 2ml acetylthiocholine substrate is added, is shaken up, starts chromogenic reaction in colorimetric cylinder, after 5min, is divided with UV, visible light
The absorbance value of photometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after extinction
The variable quantity △ A of angle value1, inhibiting rate of the standard curve serial solution to enzyme of various concentration is calculated, respectively to various concentration
The concentration value of standard curve serial solution take log10, taken with the concentration value of the standard curve serial solution to various concentration
log10The logarithm obtained the bottom of for is abscissa, using its corresponding inhibiting rate as ordinate, makes standard curve, and find out the mark
The regression equation of directrix curve；Solute contained in organic phosphorus mixed standard solution has DDVP, Rogor, parathion-methyl, horse traction
Sulphur phosphorus, Hostathion and thimet.
Table 1 is the equation of linear regression, related coefficient and detection limit of the method for the present invention.
Table 1
Note: the concentration value and analyte that y and x respectively represents analyte are to the inhibiting rate of enzyme.
From table 1 it follows that this 6 kinds organic phosphorus in the corresponding range of linearity, there is good linear relationship, meet
Analysis method requirement.
(4) quantitative detection of sample:
It takes the obtained soybean sample extract liquor of 50ml step (1) that colorimetric cylinder is added, then 2ml acetylcholine is added into colorimetric cylinder
Esterase enzyme solution and 2ml color developing agent, place 15min in 40 DEG C of water-bath after shaking up, with ultraviolet-uisible spectrophotometer in 412nm
Place measures the absorbance value A of above-mentioned solution2, 2ml acetylthiocholine substrate is then added, is shaken up, starts to show in colorimetric cylinder
Colour response after 5min, with the absorbance value of ultraviolet-uisible spectrophotometer solution after measurement reaction at 412nm, calculates anti-
Absorbance value before answering with react after absorbance value variable quantity △ A2, inhibition is calculated according to the calculation formula of inhibiting rate
The inhibiting rate is brought into the regression equation of standard curve by rate, calculates content organic phosphorus in soybean sample.
The concentration of organic phosphorus mixed standard solution is 10mg/ml in step (3).
As optimization, the calculation formula of inhibiting rate in step (3) are as follows:
；
Wherein, △ A0For the variable quantity of the absorbance before and after blank sample solution reaction；△A1Before and after soybean sample solution reaction
Absorbance variable quantity.
Be not detected through detecting, in soybean sample 6 kinds it is organic phosphorus, 6 kinds of organic phosphorus different mark-ons are dense in mark-on soybean sample
The recovery testu of degree level the results are shown in Table 6, and every mark-on soybean sample is measured in parallel 5 times.
Table 6
Note: ND is to be not detected.
As can be seen from Table 6, the recovery of standard addition of mark-on soybean sample is 94-103%, and relative deviation (n=5) is 2.4-
4.3%, meet analysis method to the rate of recovery and reproducibility requirement.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any label in claim should not be construed as limiting the claims involved.
Claims (10)
1. organic phosphorus detection kit in a kind of food, which is characterized in that the detection kit includes the first reagent and second
Reagent, first reagent includes following components: buffer, gold chloride, reducing agent and pH adjusting agent, and second reagent includes
Following components: enzyme solution, color developing agent and substrate.
2. organic phosphorus detection kit in a kind of food according to claim 1, it is characterised in that: the buffer is
Acetic acid-sodium acetate buffer solution, dipotassium hydrogen phosphate-potassium phosphate buffer, citric acid-sodium citrate buffer solution, oligosaccharide-vinegar
Any one in phthalate buffer or phosphate-citrate buffer, the pH value of the buffer are 8-9.
3. organic phosphorus detection kit in a kind of food according to claim 2, it is characterised in that: the reducing agent is
Any one in sodium dithionite, trisodium citrate, sodium borohydride, ascorbic acid or stannous octoate, the reducing agent
Concentration is 1-10g/L.
4. organic phosphorus detection kit in a kind of food according to claim 3, it is characterised in that: the enzyme solution is second
Acetylcholinesterase, the concentration of the enzyme solution are 0.3-1 μ g/ml, and the substrate is acetylthiocholine, and the concentration of the substrate is
5-8mg/ml, the pH adjusting agent are sulfuric acid or sodium hydrate aqueous solution, and the mass fraction of the aqueous sulfuric acid is 5-10%,
The mass fraction of the sodium hydrate aqueous solution is 10-20%.
5. organic phosphorus detection kit in a kind of food according to any one of claim 1 to 4, it is characterised in that:
The color developing agent be dissolved in 1L buffer solution by 8-16g dithiobis-nitrobenzoic acid and 0.8-1.6g sodium bicarbonate prepare and
At.
6. organic phosphorus detection method in a kind of food, which is characterized in that detection method includes the following steps for this:
(1) pre-treatment of sample；
(2) qualitative detection of sample；
(3) production of standard curve；
(4) quantitative detection of sample.
7. organic phosphorus detection method in a kind of food according to claim 6, which is characterized in that the detection method includes
Following steps:
(1) pre-treatment of sample: extracting sample using buffer, and centrifugation obtains extract liquor, spare；
(2) it the qualitative detection of sample: takes step (1) resulting extract liquor to be placed in test tube, chlorine is then sequentially added into test tube
Auric acid, reducing agent and pH adjusting agent are vortexed, and mix, and stand, and observe color change；
(3) production of standard curve:
Buffer is added into colorimetric cylinder, then enzyme solution and color developing agent are added into colorimetric cylinder, shakes up, when placing one section in water-bath
Between, the absorbance value of above-mentioned solution is measured with ultraviolet-uisible spectrophotometer, then adds substrate, is shaken up, and is opened in colorimetric cylinder
Beginning chromogenic reaction after a period of time, with the absorbance value of solution after ultraviolet-uisible spectrophotometer measurement reaction, calculates reaction
Preceding absorbance value with react after absorbance value variable quantity △ A0；
Several colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, constant volume is configured to
The standard curve serial solution of various concentration is separately added into enzyme solution and color developing agent in Xiang Shangshu standard curve serial solution, shakes up,
A period of time is placed in water-bath, and the absorbance value A of above-mentioned solution is measured with ultraviolet-uisible spectrophotometer1, then add bottom
Object shakes up, and starts chromogenic reaction in colorimetric cylinder, after a period of time, with solution after ultraviolet-uisible spectrophotometer measurement reaction
Absorbance value, calculate reaction before absorbance value with react after absorbance value variable quantity △ A1, calculate various concentration
Standard curve serial solution to the inhibiting rate of enzyme, the concentration value of the standard curve serial solution of various concentration is taken respectively
log10, log is taken with the concentration value of the standard curve serial solution to various concentration10The logarithm obtained the bottom of for is abscissa, with
Its corresponding inhibiting rate is ordinate, makes standard curve, and find out the regression equation of the standard curve；
(4) quantitative detection of sample:
It takes the obtained sample extraction liquid of step (1) that colorimetric cylinder is added, then enzyme solution and color developing agent is added into colorimetric cylinder, shake up,
A period of time is placed in water-bath, and the absorbance value A of above-mentioned solution is measured with ultraviolet-uisible spectrophotometer2, then add bottom
Object shakes up, and starts chromogenic reaction in colorimetric cylinder, after a period of time, with solution after ultraviolet-uisible spectrophotometer measurement reaction
Absorbance value, calculate reaction before absorbance value with react after absorbance value variable quantity △ A2, according to the meter of inhibiting rate
It calculates formula and inhibiting rate is calculated, which is brought into the regression equation of standard curve, calculates organic phosphorus in sample contain
Amount.
8. organic phosphorus detection method in a kind of food according to claim 7, which is characterized in that the detection method includes
Following steps:
(1) pre-treatment of sample: 5-15g sample is accurately weighed, is placed in the centrifuge tube of 50-250ml, is added into centrifuge tube
Then 50-200ml buffer, the ultrasonic extraction 15-30min at 50-100KHz are centrifuged in the case where revolving speed is 3000-5000r/min
5-10min takes upper layer of extraction liquid, spare；
(2) qualitative detection of sample: taking 3-5ml step (1) resulting extract liquor to be placed in test tube, then successively adds into test tube
Enter 3-5ml gold chloride, 3-5ml reducing agent and 1-2mlpH regulator, cap, vortex 30-60s is uniformly mixed, and stands 5-
10min observes color change, if the aobvious blue of the color of solution in test tube, remain in sample it is organic phosphorus, if solution in test tube
The aobvious red of color, then without organophosphorus residue in sample；
(3) production of standard curve:
1 colorimetric cylinder is taken, 50ml buffer is added into colorimetric cylinder, then 2ml enzyme solution and 2ml color developing agent are added into colorimetric cylinder,
15min is placed in 35-40 DEG C of water-bath after shaking up, measures above-mentioned solution at 412nm with ultraviolet-uisible spectrophotometer
Absorbance value A0, 2ml substrate is then added, is shaken up, starts chromogenic reaction in colorimetric cylinder, after 3-5min, with UV, visible light point
The absorbance value of light photometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after suction
The variable quantity △ A of shading value0；
6 colorimetric cylinders are taken, different amounts of organic phosphorus mixed standard solution is added into colorimetric cylinder respectively, is settled to buffer
50ml is configured to the standard curve series that concentration is respectively 5 μ g/L, 10 μ g/L, 20 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L
Solution is separately added into 2ml enzyme solution and 2ml color developing agent in Xiang Shangshu standard curve serial solution, in 35-40 DEG C of water-bath after shaking up
Middle placement 15min measures the absorbance value A of above-mentioned solution with ultraviolet-uisible spectrophotometer at 412nm1, then add
2ml substrate, shakes up, beginning chromogenic reaction in colorimetric cylinder, after 3-5min, is measured at 412nm with ultraviolet-uisible spectrophotometer
The absorbance value of solution after reaction, calculate reaction before absorbance value with react after absorbance value variable quantity △ A1, meter
Inhibiting rate of the standard curve serial solution to enzyme for calculating various concentration, respectively to the standard curve serial solution of various concentration
Concentration value takes log10, log is taken with the concentration value of the standard curve serial solution to various concentration10The logarithm obtained the bottom of for is
Abscissa makes standard curve using its corresponding inhibiting rate as ordinate, and finds out the regression equation of the standard curve；
(4) quantitative detection of sample:
Take the obtained sample extraction liquid of 50ml step (1) that colorimetric cylinder is added, then addition 2ml enzyme solution and 2ml are aobvious into colorimetric cylinder
Toner places 15min in 35-40 DEG C of water-bath after shaking up, and is measured at 412nm with ultraviolet-uisible spectrophotometer above-mentioned molten
The absorbance value A of liquid2, then add 2ml substrate, shake up, start chromogenic reaction in colorimetric cylinder, after 3-5min, with it is ultraviolet can
The absorbance value for seeing spectrophotometer solution after measurement reaction at the 412nm, absorbance value before calculating reaction with react after
Absorbance value variable quantity △ A2, inhibiting rate is calculated according to the calculation formula of inhibiting rate, brings the inhibiting rate into standard
The regression equation of curve calculates content organic phosphorus in sample.
9. organic phosphorus detection method in a kind of food according to claim 8, it is characterised in that: in the step (3)
The concentration of organic phosphorus mixed standard solution is 10mg/ml.
10. organic phosphorus detection method in a kind of food according to claim 8 or claim 9, it is characterised in that: the step
(3) calculation formula of inhibiting rate in are as follows:
；
Wherein, △ A0For the variable quantity of the absorbance before and after blank sample solution reaction；△A1For the extinction of sample solution reaction front and back
The variable quantity of degree.
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| CN110793929A (en) * | 2019-11-06 | 2020-02-14 | 吉林大学 | Pesticide residue detection and distinguishing method based on multienzyme inhibition |
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