CN102539742B - Method for detecting heavy metals in carbohydrate agricultural products and application thereof - Google Patents
Method for detecting heavy metals in carbohydrate agricultural products and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting heavy metals in carbohydrate agricultural products and application thereof. The method of the invention comprises the following concrete steps: homogenizing a carbohydrate agricultural product, and leaching with a diluted acid; removing insoluble substances, centrifuging, and collecting supernatant to obtain a solution containing heave metal; and directly using the to-be-tested sample solution to enzyme linked immunosorbent assay, and obtaining heavy metal content in the to-be-tested sample. The method of the invention can be used for leaching heavy metal ions copper, lead and cadmium in vegetables, fruits and foodstuff agricultural products, which not only quickens the time of leaching heave metal from a sample, but also reduces environmental pollution brought by the conventional sample pretreatment technology. The method has mild reaction conditions, and achieves rapid, efficient and convenient field detection of agricultural products.
Description
Technical field
The present invention relates to a kind of method of extracting heavy metal, particularly a kind of method and application that detects heavy metals in carbohydrate agricultural products.The method be a kind of easy, efficiently, detection method accurately.
Background technology
In recent years, the economy of China has had development at full speed, but the environmental pollution that meanwhile caused is also further obvious, particularly the heavy metal pollution of industry and the atmosphere that brings of processing manufacturing industry, water body, soil.The heavy metal pollution in water body, soil and farmland is to cause the main reason that in the agricultural product such as vegetables, fruit and grain, content of beary metal exceeds standard.
Heavy metal copper, lead, cadmium are mainly to enter human body by food and breathing.Although heavy metal generally can not cause people's acute poisoning in agricultural product, heavy metal at people's body accumulation, and then affects health by food chain.The safety problem of the food, particularly agricultural product causing in the face of more and more severeer environmental pollution, has produced adverse effect and potential risks to the people's health.The detection technique of setting up heavy metal in a kind of quick, simple and direct, efficient and safe food is extremely urgent.
In recent years, immunology detection technology has been widely used in the detection in the fields such as toxin, pesticide, explosive.Along with the day by day maturation of immunology detection technology on heavy metal detects, ELISA kit has become a kind of quick, easy and reliable detection means, and its application has solved that traditional detection technology for detection needs that large-sized analytic instrument, instrument are expensive, complex operation, require testing staff to possess the drawbacks such as certain professional knowledge.It can replace traditional detection technology to adapt to enterprise's Quality Control and the examination and test of products, the spot check of product and the requirement speeding passage through customs that product is imported and exported.
This seminar has successfully made the enzyme linked immunological kit of heavy metal lead, the lead ion in can testing environment water sample.Applied for a patent, application number is that " 201010539824.8 ", application name are called " a kind of immunological method and kit of fast detecting heavy metal lead ion ".The colloid gold reagent bar of cadmium ion and lead ion is successfully prepared, can successfully detect liquid heavy metal ion.Existing achievement in research shows, ELISA heavy metal detection method can only detect the heavy metal of liquid ionic state, cannot detect the heavy metal in conjunction with state.Therefore, ELISA heavy metal detection method is applied in real work efficiently, rapidly, also need to solve the problem of important but loaded down with trivial details Sample Pretreatment Technique.At present, in agricultural product and food, the pretreatment technology of heavy metal has wet digestion method, dry ashing method and Microwave Digestion, is all destructions of organic material, changes sample into liquid settled solution and measures.Although these pretreatment technologies can accurately extract the heavy metal in sample, but time-consuming, require great effort and produce a large amount of harmful gases, the digestion solution obtaining has highly acid, and enzyme in ELISA is exempted from reaction needed and carried out, so cannot directly applying to ELISA, the digestion solution that traditional sample-pretreating method obtains detects heavy metal under a gentle environment (neutral to alkalescence on the weak side).
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides a kind of method that detects heavy metals in carbohydrate agricultural products.
Another object of the present invention is to the application of the method that described detection heavy metals in carbohydrate agricultural products is provided.
Object of the present invention is achieved through the following technical solutions: a kind of method that detects heavy metals in carbohydrate agricultural products, comprises the following steps:
(1) pre-treatment of sample: by clean carbohydrates agricultural products, homogenate, add diluted acid lixiviate; Then remove insoluble substance, obtain the testing sample solution that contains heavy metal ion;
(2) testing sample solution is directly used in to Enzyme-linked Immunosorbent Assay reaction (ELISA), obtains content of beary metal in testing sample.
Carbohydrates agricultural products described in step (1) is greengrocery agricultural product, fruits agricultural product or grain agricultural products;
Described greengrocery agricultural product are preferably cabbage heart, carrot and pumpkin;
Described fruits agricultural product are preferably apple, grape and banana;
Described grain agricultural products is preferably rice, wheat and brown rice;
Diluted acid described in step (1) refers to the acid that concentration is less than 6mol/L; Preferred concentration is the acid solution of 1～4mol/L; More preferably 2～4mol/L;
Described acid solution is preferably HCl solution, HNO
3solution or H
2sO
4solution;
The condition of the lixiviate described in step (1) is preferably 4～70 DEG C of lixiviate 10～20min;
The mode of described removal insoluble substance is preferably centrifugal;
Described centrifugal condition is preferably the centrifugal 15min of 4000～8000rpm; The more preferably centrifugal 10min of 10000～13000rpm;
Heavy metal ion described in step (1) is preferably copper ion, lead ion or cadmium ion;
Enzyme-linked Immunosorbent Assay reaction described in step (2) is preferably racing ELISA detecting method;
The process of described racing ELISA detecting method is as follows:
1. coated: to dissolve heavy metal-sequestrant-protein complex with carbonate buffer solution, be coated in ELISA Plate micropore;
2. wash plate: dry liquid in plate hole, then in hole, add PBS-T cleansing solution, place, then rock gently, discard washing lotion, pat dry, repeat to wash plate three times;
3. sealing: with the PBST damping fluid sealing that contains skimmed milk power; As front method is washed plate three times;
4. application of sample: add successively heavy metal ion standard items and the aforesaid testing sample solution that contains heavy metal ion in ELISA ELISA Plate hole, then add heavy metal ion antibody working fluid, with cover plate film shrouding, hatch; Take out ELISA Plate, as front method is washed plate three times;
5. add enzyme labeling two anti-: add HRP-sheep anti-mouse igg antibody working fluid, hatch; Take out ELISA Plate, wash plate five times;
6. add nitrite ion: add TMB nitrite ion, vibration mixes gently, the reaction of room temperature lucifuge;
7. cessation reaction: add H
2sO
4cessation reaction; Measure every hole absorbance in microplate reader 450nm place;
8. result is judged: the each concentration standard solution obtaining and the mean value (B) of sample absorbance are multiplied by 100% again divided by the absorbance (B0) of first standard (0 standard), i.e. percentage absorbance;
Percentage absorbance (%)=B/B0 × 100%;
In formula: the mean light absorbency value of B-standard solution or sample solution;
The mean light absorbency value of B0-0ng/ml standard solution;
Taking standard items percentage absorptance as ordinate, taking the logarithm of heavy metal standard items concentration (ng/ml) as horizontal ordinate, drawing standard curve map, by in the percentage absorptance substitution typical curve of sample, read the corresponding concentration of sample from typical curve, be multiplied by its corresponding extension rate and be heavy metal actual concentrations in sample.
The concentration of the carbonate buffer solution of step described in is 1. 0.05M, and pH value is 9.6;
Heavy metal-sequestrant-the protein complex of step described in is 1. preferably Cu
2+-DTPA-OVA, Pb
2+-DTPA-OVA or Cd
2+-DTPA-OVA;
The consumption of described heavy metal-sequestrant-protein complex is preferably every hole in 96 orifice plates and uses 16ng;
The coated condition of step described in is 1. preferably in 4 DEG C of coated 16h;
The PBST of step described in is 2. the PBS solution that contains percent by volume 0.05%Tween-20; The concentration of PBS is 0.015M, and pH value is 7.4;
The time of the placement of step described in is 2. preferably 2～4min;
In the PBST damping fluid that contains skimmed milk power of step described in 3., the concentration of skimmed milk power is 0.05g/ml; PBST is the PBS solution that contains percent by volume 0.05%Tween-20; The concentration of PBS is 0.015M, and pH value is 7.4;
The condition of the sealing of step described in is 3. preferably 37 DEG C of reaction 1h;
The condition of hatching of step described in is 4. preferably 37 DEG C and hatches 1h;
The condition of hatching of step described in is 5. preferably 37 DEG C and hatches 30min.
The application of the method for aforementioned detection heavy metals in carbohydrate agricultural products in the heavy metal of carbohydrates agricultural products detects.
The present invention has following advantage and effect with respect to prior art:
(1) pre-treatment step in the method for detection heavy metals in carbohydrate agricultural products of the present invention can be for the lixiviate of heavy metal ion copper, lead, cadmium in greengrocery, fruits, grain agricultural products, not only accelerate heavy metal lixiviate time out from sample, also reduced the environmental pollution that traditional Sample Pretreatment Technique brings simultaneously.The reaction conditions gentleness of this step.For cabbage heart, carrot and apple, this step can replace traditional sample-pretreating method completely and carry out the lixiviate of heavy metal.
(2) scene that the method for detection heavy metals in carbohydrate agricultural products provided by the present invention realizes agricultural product is detected fast, efficiently and easily.
Brief description of the drawings
Fig. 1 is the comparative result figure that the present invention and two kinds of methods of GF-AAS coupling pre-treatment step are measured respectively heavy metals in carbohydrate agricultural products copper.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) preparation of sample:
First with tap water, sample (vegetables, fruit) is rinsed well, then used ultrapure water rinsing 3 times, dry moisture.Carry out homogenate with food refiner, be stored in the beaker of 250ml, freezing saving backup at-4 DEG C.
Directly dry grinding is extremely Powdered for grain samples, is stored in seal box and saves backup under normal temperature.
(2) pre-treatment of sample (diluted acid lixiviate sample)
1. greengrocery agricultural product
A, take the cabbage heart 2.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HCl solution 2.0ml of 2.0M, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, get supernatant and regulate pH value to 6.0～8.0, be settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures its heavy metal copper, plumbous, the content of cadmium, (pre-treating method is Microwave Digestion for result and National Standard Method sample-pretreating method, can be with reference to GB/T5009.13-1996, GB/T 5009.15-2003 and GB/T5009.12-2010) the ratio of measurement result be followed successively by 86.95%, 72.39%, 89.78%.
Sampling Graphite Furnace Atomic spectrophotometer uses C type coating graphite tube, light temperature control type of heating, and polarization Zeeman method is proofreaied and correct background signal.The major parameter of instrument and condition of work are respectively in table 1 and 2.
Table 1 Instrument working parameter
Table 2
B, take the cabbage heart 2.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HNO of 2.0M
3solution 2.0ml, under normal temperature, react 15min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 98.84%, 94.57%, 92.28%.
C, take the cabbage heart 2.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the H of 2.0M
2sO
4solution 2.0ml, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 81.06%, 76.11%, 80.45%.
D, take the carrot 2.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HCl solution 2.0ml of 2.0M, under normal temperature, react 15min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, get supernatant and regulate pH value to 6.0～8.0, be settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 72.85%, 63.55%, 75.99%.
E, take the carrot 2.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HNO of 2.0M
3solution 2.0ml, under normal temperature, react 20min, centrifugal 10min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 74.97%, 65.72%, 75.77%.
F, take the pumpkin 2.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HCl solution 2.0ml of 2.0M, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, get supernatant and regulate pH value to 6.0～8.0, be settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 97.77%, 73.11%, 75.35%.
G, take the pumpkin 2.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HNO of 2.0M
3solution 2.0ml, under normal temperature, react 10min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 100.31%, 61.15%, 90.09%.
2. fruits agricultural product
A, take the apple 1.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HCl solution 2.0ml of 2.0M, under normal temperature, react 15min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, get supernatant and regulate pH value to 6.0～8.0, be settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 82.98%, 73.30%, 94.06%.
B, take the apple 1.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the HNO of 4.0M
3solution 2.0ml, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 83.67%, 73.90%, 99.42%.
C, take the apple 1.0g of preparation in step (1), be placed in 10ml centrifuge tube, add the H of 2.0M
2sO
4solution 2.0ml, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 80.74%, 85.47%, 95.54%.
3. grain agricultural products
A, take the rice 0.5g of preparation in step (1), be placed in 10ml centrifuge tube, add the HCl solution 3.0ml of 2.0M, under normal temperature, react 20min, centrifugal 20min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, get supernatant and regulate pH value to 6.0～8.0, be settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 50.75%, 56.67%, 57.02%.
B, take the rice 0.5g of preparation in step (1), be placed in 10ml centrifuge tube, add the H of 4.0M
2sO
4solution 3.0ml, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 50.60%, 52.93%, 63.14%.
C, take the brown rice 0.5g of preparation in step (1), be placed in 10ml centrifuge tube, add the HCl solution 3.0ml of 2.0M, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, get supernatant and regulate pH value to 6.0～8.0, be settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 52.35%, 56.45%, 56.29%.
D, take the brown rice 0.5g of preparation in step (1), be placed in 10ml centrifuge tube, add the HNO of 2.0M
3solution 3.0ml, under normal temperature, react 20min, centrifugal 15min under 4000rpm, get the centrifuge tube that supernatant is placed in 2ml, centrifugal 10min under 13000rpm, gets supernatant and regulates pH value to 6.0～8.0, is settled to 4ml, GF-AAS (sampling Graphite Furnace Atomic spectrophotometer) measures the content of its heavy metal copper, lead, cadmium, and the ratio of the measurement result of result and National Standard Method sample-pretreating method is followed successively by 53.49%, 63.06%, 71.76%.
Embodiment 2
The precision of the pre-treatment step of sample in the inventive method:
(1) take many group samples, carry out, after micro-wave digestion, recording the content of heavy metal copper, lead, cadmium in every group of sample with GF-AAS according to National Standard Method, average and obtain the content of beary metal before sample mark-on, specific as follows:
1. the micro-wave digestion to greengrocery agricultural product:
Take each 3 parts of cabbage heart, carrot, pumpkin sample with electronic balance respectively, every part of 2.000g left and right, puts into teflon counteracting tank, adds respectively the dense HNO that 5ml top grade is pure
3, the 30%H of 2ml
2o
2, room temperature is placed 8～12 hours, then puts into microwave dissolver and clears up, and the condition of clearing up is as table 3.
The micro-wave digestion condition of table 3 greengrocery sample
End to be cleared up, waits the cooling rear transfer of digestion solution, catches up with acid, is settled to 8ml with ultrapure water afterwards, shakes up, then measures the content of each target heavy metal with GF-AAS, the measured value that the mean value of 3 duplicate samples is this sample.Sample blank is processed equally.
2. the micro-wave digestion to fruits agricultural product
Take each 3 parts of apple, grape, banana sample with electronic balance respectively, every part of 2.000g left and right, puts into teflon counteracting tank, adds the dense HNO that 6ml top grade is pure
3after pre-service, add the dense HNO of 2ml
3, the 30%H of 2ml
2o
2, room temperature is placed 8～12 hours, then puts into microwave dissolver and clears up, and the condition of clearing up is as table 4.
The micro-wave digestion condition of table 4 fruits sample
End to be cleared up, waits the cooling rear transfer of digestion solution, catches up with acid, is settled to 8ml with ultrapure water afterwards, shakes up, then measures the content of each target heavy metal with GF-AAS, the measured value that the mean value of 3 duplicate samples is this sample.Sample blank is processed equally.
3. the micro-wave digestion to grain agricultural products
Take each 3 parts of rice, wheat, brown rice sample with electronic balance respectively, every part of 2.000g left and right, puts into teflon counteracting tank, adds the dense HNO that 6ml top grade is pure
3after pre-service, 20min adds the dense HNO of 2ml
3, the 30%H of 2ml
2o
2, room temperature is placed 12～16 hours, then puts into microwave dissolver and clears up.The condition of clearing up is as table 5.
The micro-wave digestion condition of table 5 grain class sample
End to be cleared up, waits the cooling rear transfer of digestion solution, catches up with acid, is settled to 8ml with ultrapure water afterwards, shakes up, then measures the content of each target heavy metal with GF-AAS, the measured value that the mean value of 3 duplicate samples is this sample.Sample blank is processed equally.
(2) get the sample 2.0g preparing in embodiment 1 and add 200 μ g/L copper mark liquid 1.0ml, the plumbous mark of 20 μ g/L liquid 1.0ml, 20 μ g/L cadmium mark liquid 1.0ml, use diluted acid extraction of the present invention lixiviate, the condition of lixiviate is to use respectively the HCl solution of 2.0M, 2.0M HNO
3the H of solution and 2.0M
2sO
4solution lixiviate, reaction 10min, the centrifugal 15min of 4000rpm, gets supernatant (13000rpm, 10min) in the centrifuge tube of 2ml centrifugal.Get supernatant and regulate pH value to 6.0～8.0, be settled to 4ml.Thereby the concentration that records solution obtains the content of beary metal after sample mark-on, try to achieve the recovery of standard addition (table 6, table 7 and table 8) of sample according to formula 1.
W (%)=(C
1-C
0)/C × 100% (formula 1)
In formula: the recovery of standard addition (%) of W-sample;
C
0content of beary metal (μ g/kg) before-sample mark-on;
C
1content of beary metal (μ g/kg) after-sample mark-on;
C-reality add scalar (μ g/kg).
The recovery of standard addition of the National Standard Method of table 6 sample pre-treatments and rare HCl extraction
The National Standard Method of table 7 sample pre-treatments and rare HNO
3the recovery of standard addition of extraction
The National Standard Method of table 8 sample pre-treatments and rare H
2sO
4the recovery of standard addition of extraction
The result of table 6, table 7 and table 8 shows, the recovery of standard addition of the pre-treatment of National Standard Method to greengrocery agricultural product is 93～108%, and the recovery of standard addition of rare HCl extraction is 76～106%, lower than National Standard Method; Rare HNO
3the recovery of standard addition of extraction is 87～109%, approaches with National Standard Method; Rare H
2sO
4the recovery of standard addition of extraction is 83～114%, also approaches National Standard Method, thus the recovery of standard addition of three kinds of diluted acids compare with National Standard Method, rare HNO
3the rare H of ≈
2sO
4the rare HCl of >.The recovery of standard addition of the pre-treatment of National Standard Method to fruits agricultural product is 90～113%, and the recovery of standard addition of rare HCl extraction is 69～104%, lower than National Standard Method; Rare HNO
3the recovery of standard addition of extraction is 87～111%, approaches with National Standard Method; Rare H
2sO
4the recovery of standard addition of extraction is 87～102%, also approaches National Standard Method, thus the recovery of standard addition of three kinds of diluted acids compare with National Standard Method, rare HNO
3the rare H of >
2sO
4the rare HCl of >.The recovery of standard addition of the pre-treatment of National Standard Method to grain agricultural products is 92～114%, and the recovery of standard addition of rare HCl extraction is 78～100%, lower than National Standard Method; Rare HNO
3the recovery of standard addition of extraction is 82～113%, a little less than National Standard Method; Rare H
2sO
4the recovery of standard addition of extraction is 86～121%, approaches National Standard Method, thus the recovery of standard addition of three kinds of diluted acids compare with National Standard Method, rare H
2sO
4the rare HNO of >
3the rare HCl of >.Diluted acid extraction, not only the recovery of lixiviate sample heavy metal is higher, and comparatively approaching with the recovery of standard addition of National Standard Method, and therefore this method is a kind of efficient, reliable landfill method.
Embodiment 3
Utilize the inventive method to detect the heavy metal of carbohydrates agricultural products:
(1) coated: with 0.05M pH9.6 carbonate buffer solution dissolve Cu-DTPA-OVA (refer to: the foundation [J] of the preparation of heavy metal copper ion monoclonal antibody and racing ELISA detecting method thereof. Products in China magazine, 2011,24 (6): 720-723.), concentration is 160ng/ml, every hole 100 μ l, are coated in ELISA Plate micropore in 4 DEG C of 16h.
(2) dry liquid in plate hole, then in hole, add PBS-T (0.015M, pH 7.4, containing 0.05%Tween-20) cleansing solution, every hole 300 μ l, place 3min, rock gently, discard washing lotion, pat dry, and repeat to wash plate three times;
(3) sealing: with the PBST damping fluid sealing that contains 0.05g/ml skimmed milk power, every hole 200 μ l, 37 DEG C of 1h; As front method is washed plate three times.
(4) application of sample: add successively in ELISA ELISA Plate hole that concentration is 0,2,5,10,20,50,100ng/ml Cu
2+sample prepared by standard items and step (1), (dilution in 1: 160000 by volume obtains to add copper antibody working fluid again, referring to: the foundation [J] of the preparation of heavy metal copper ion monoclonal antibody and racing ELISA detecting method thereof. Products in China is learned magazine, 2011,24 (6): 720-723) 50 μ l/ holes, with cover plate film shrouding.Hatch 1h for 37 DEG C; Take out ELISA Plate, as front method is washed plate three times;
5) adding enzyme labeling two resists: add HRP-sheep anti-mouse igg antibody working fluid (times dilution in 1: 8000 by volume, precious Imtech) 100 μ l/ holes, hatch 30min for 37 DEG C; Take out ELISA Plate, wash plate five times;
6) add nitrite ion: in every hole, add A, the each 50 μ l of B substrate solution (TMB nitrite ion), vibration mixes gently, room temperature lucifuge reaction 10min;
7) cessation reaction: the H that adds 2.0M in every hole
2sO
4stop buffer 50 μ l, vibration mixes gently; Measure every hole absorbance in microplate reader 450nm place.
8) result is judged: the each concentration standard solution obtaining and the mean value (B) of sample absorbance are multiplied by 100% again divided by the absorbance (B0) of first standard (0 standard), i.e. percentage absorbance.
Percentage absorbance (%)=B/B0 × 100%
In formula: the mean light absorbency value of B-standard solution or sample solution
The mean light absorbency value of B0-0ng/ml standard solution
Taking standard items percentage absorptance as ordinate, with Cu
2+the logarithm of standard items concentration (ng/m1) is horizontal ordinate, drawing standard curve map.By in the percentage absorptance substitution typical curve of sample, read the corresponding concentration of sample from typical curve, be multiplied by its corresponding extension rate and be Cu in sample
2+actual concentrations.
Vegetables (cabbage heart, carrot, pumpkin), fruit (apple, grape, banana) and grain (rice, wheat, brown rice) (prepare by embodiment 1) after dilute acid pretreatment is good, detect respectively the Cu of sample with ELISA and GF-AAS, measurement result is in table 9, as can be seen from the table, the coefficient of variation of testing result is all less than 13%, and 87～118%, illustrate that the ELISA method of setting up has good repeatability and precision with the coincidence rate of GF-AAS measurement result.
Table 9
Diluted acid lixiviate-ELISA detection method and diluted acid lixiviate-GFAAS (GFAAS (graphite furnace atomic absorption spectrometry)) detection method are carried out to statistical analysis, linear regression analysis shows, two kinds of detection methods have significant correlativity (P=5.9926E-18 < 0.001), and equation of linear regression is:
y＝1.0314x-1.0101(R
2＝0.9515)
In formula, y represents the concentration (μ g/L) of heavy metal Cu in diluted acid lixiviate-ELISA detection method, and x represents the concentration (μ g/L) of Cu in diluted acid lixiviate-GFAAS detection method, R
2for related coefficient.
Therefore, diluted acid lixiviate-ELISA detection technique can be measured the content of the heavy metal Cu in carbohydrates agricultural products effectively.By the comparison of diluted acid lixiviate-ELISA detection technique and diluted acid lixiviate-GFAAS detection technique, known diluted acid lixiviate-ELISA detection technique is not only feasible, and has good repeatability and accuracy; Than National Standard Method, not only easy (not needing large-scale instrument and equipment and professional and technical personnel), (pre-service and measure only need an about 1h) saves time, and environmentally friendly (do not need a large amount of acid solutions, do not produce toxic and harmful) is a kind of comparatively ideal heavy metal detection method.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (5)
1. detect a method for heavy metals in carbohydrate agricultural products, it is characterized in that comprising the following steps:
(1) pre-treatment of sample: by clean carbohydrates agricultural products, homogenate, add diluted acid lixiviate; Then remove insoluble substance, obtain the testing sample solution that contains heavy metal ion;
(2) testing sample solution is directly used in to Enzyme-linked Immunosorbent Assay reaction, obtains content of beary metal in testing sample;
Described carbohydrates agricultural products is greengrocery agricultural product, fruits agricultural product or grain agricultural products; Described greengrocery agricultural product are cabbage heart, carrot and pumpkin; Described fruits agricultural product are apple, grape and banana; Described grain agricultural products is rice, wheat and brown rice;
Described diluted acid refers to the acid that concentration is less than 6mol/L; Described diluted acid refers to the acid solution that concentration is 1～4mol/L; Described diluted acid refers to the acid solution that concentration is 2～4mol/L; Described acid solution is HNO
3solution or H
2sO
4solution;
The condition of described lixiviate is 4～70 DEG C of lixiviate 10～20min; The mode of described removal insoluble substance is centrifugal; Described centrifugal condition is the centrifugal 15min of 4000～8000rpm; Described heavy metal ion is lead ion.
2. the method for detection heavy metals in carbohydrate agricultural products according to claim 1, is characterized in that: described Enzyme-linked Immunosorbent Assay reaction is racing ELISA detecting method.
3. the method for detection heavy metals in carbohydrate agricultural products according to claim 2, is characterized in that: the process of described racing ELISA detecting method is as follows:
1. coated: to dissolve heavy metal-sequestrant-protein complex with carbonate buffer solution, be coated in ELISA Plate micropore;
2. wash plate: dry liquid in plate hole, then in hole, add PBS-T cleansing solution, place, then rock gently, discard washing lotion, pat dry, repeat to wash plate three times;
3. sealing: with the PBST damping fluid sealing that contains skimmed milk power; As front method is washed plate three times;
4. application of sample: add respectively heavy metal ion standard items and the aforesaid testing sample solution that contains heavy metal ion in ELISA ELISA Plate hole, then add heavy metal ion antibody working fluid, with cover plate film shrouding, hatch; Take out ELISA Plate, as front method is washed plate three times;
5. add enzyme labeling two anti-: add HRP-sheep anti-mouse igg antibody working fluid, hatch; Take out ELISA Plate, wash plate five times;
6. add nitrite ion: add TMB nitrite ion, vibration mixes gently, the reaction of room temperature lucifuge;
7. cessation reaction: add H
2sO
4cessation reaction; Measure every hole absorbance in microplate reader 450nm place;
8. result is judged: the each concentration standard solution obtaining and the mean value of sample absorbance are multiplied by 100% again divided by the absorbance of first standard, i.e. percentage absorbance;
Percentage absorbance (%)=B/B0 × 100%;
In formula: the mean light absorbency value of B-standard solution or sample solution;
The mean light absorbency value of B0-0ng/ml standard solution;
Taking standard items percentage absorptance as ordinate, taking the logarithm of heavy metal standard items concentration as horizontal ordinate, drawing standard curve map, by in the percentage absorptance substitution typical curve of sample, read the corresponding concentration of sample from typical curve, be multiplied by its corresponding extension rate and be heavy metal actual concentrations in sample.
4. the method for detection heavy metals in carbohydrate agricultural products according to claim 3, is characterized in that:
The concentration of the carbonate buffer solution of step described in is 1. 0.05M, and pH value is 9.6;
Heavy metal-sequestrant-the protein complex of step described in is 1. Pb
2+-DTPA-OVA;
The consumption of described heavy metal-sequestrant-protein complex is that in 96 orifice plates, every hole is used 16ng;
The coated condition of step described in is 1. in 4 DEG C of coated 16h;
The PBST of step described in is 2. the PBS solution that contains percent by volume 0.05%Tween-20; The concentration of PBS is 0.015M, and pH value is 7.4;
The time of the placement of step described in is 2. 2～4min;
In the PBST damping fluid that contains skimmed milk power of step described in 3., the concentration of skimmed milk power is 0.05g/ml; PBST is the PBS solution that contains percent by volume 0.05%Tween-20; The concentration of PBS is 0.015M, and pH value is 7.4;
The condition of the sealing of step described in is 3. 37 DEG C of reaction 1h;
The condition of hatching of step described in is 4. 37 DEG C and hatches 1h;
The condition of hatching of step described in is 5. 37 DEG C and hatches 30min.
5. the application of the method that detects heavy metals in carbohydrate agricultural products described in claim 1～4 any one in carbohydrates agricultural products heavy metal detects.
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Application publication date: 20120704 Assignee: Shenzhen City Triphil Bio-tech Co., Ltd. Assignor: Jinan University Contract record no.: 2015440020046 Denomination of invention: Method for detecting heavy metals in carbohydrate agricultural products and application thereof Granted publication date: 20141203 License type: Exclusive License Record date: 20150211 |