CN103399152A - Method for quickly detecting aflatoxin B1 by PbS quantum dot - Google Patents
Method for quickly detecting aflatoxin B1 by PbS quantum dot Download PDFInfo
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Abstract
The invention discloses a method for quickly detecting aflatoxin B1 by PbS quantum dot. The method is characterized by comprising the steps of: adding Pb(NO3)2 solution into mercaptoacetic acid and Na2S solution to prepare PbS quantum dot; preparing 10mug/mL QD-Ab solution by coupling of PbS quantum dot and aflatoxin B1 monoclonal antibody; adding aflatoxin B1-bovine serum albumin conjugate in an elisa plate to coat the elisa plate, and simultaneously adding AFB1 solution and QD-Ab solution, after incubation digestion, adding Hg2Cl solution, getting constant volume by adding acetic acid buffer solution, then preparing the to-be-detected solution; deoxidizing the to-be-detected solution, adding the deoxidized to-be-detected solution into a pretreated glassy carbon electrode for detection, and calculating the content of the aflatoxin B1 according to a standard curve relation between the dissolving peak size of Pb<2+> and the concentration of aflatoxin B1 solution. The method has the advantages of high sensitivity, strong selectivity, excellent stability, high accuracy and quick detection speed.
Description
Technical field
The present invention relates to the food safety detection field, especially relate to a kind of method of the PbS of utilization quantum dot fast detecting aflatoxin B1.
Background technology
Aflatoxin be a class by aspergillus flavus (
asperillus flavus) and aspergillus parasiticus (
aspergillus parasiticus) infect the general name that oil plant, grain or other food produce the toxicity secondary metabolite, be the compound that a class has very strong mutagenesis, teratogenesis, carcinogenesis, be one of strong carcinogen of finding so far.Aflatoxin mainly pollutes peanut, corn, cereal, nut etc., and pollution is also arranged in addition in beans, cottonseed, meat, aquatic products, wherein with grain and oil agricultural product peanut and corn, pollutes the most serious.The aflatoxin kind is a lot, the kind more than 20 that has of having separated evaluation at present, wherein the toxicity of aflatoxin B1 (AFB1) is the strongest, 10 times of potassium cyanide, 68 times of arsenic, 416 times of melamine, can cause the animal acute poisoning, resistibility reduces even dead, and the Sensitivity animal half lethal dose is only the 0.294mg/kg body weight.Generally, in contaminated grain agricultural product, the content of AFB1 is maximum, therefore often usings AFB1 as the leading indicator of estimating aflatoxin contamination.Strict restriction has all been done to AFB1 allowance in food in countries in the world, and to assay method, sensitivity has high requirements for this.
At present, the detection means of AFB1 mainly contained: high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and euzymelinked immunosorbent assay (ELISA) etc.Although high performance liquid chromatography is highly sensitive, instrument is expensive, the program complexity, and the pre-treatment purification requires high, is not suitable for fast detecting.The thin layer chromatography operation is lengthy and tedious, and sensitivity is not high, and poor reproducibility is easily disturbed by other fluorescent material, large to the human and environment contamination factor.Euzymelinked immunosorbent assay (ELISA) is highly sensitive, high specificity, fast and convenient, but in test, the activity of enzyme is subject to the reaction conditions impact, and shorter particularly nitrite ion of reagent life-span needs low temperature to preserve.
Quantum dot (Quantum dots, QDs), mainly II-VI family or III-V compounds of group, particle diameter, at 1-100nm, is a kind of nano material of accurate zero dimension, not only has good optical characteristics, also there is good electrochemical properties, can be used as the carrier of metastatic electron, as in anodic stripping voltammetry, the QDs that different elements form has sensitive, stable different current potential stripping peak.At present, any method about the fast detecting aflatoxin B1 based on PbS quantum dot combined with electrochemical detection technique is not also disclosed both at home and abroad.
Summary of the invention
Technical matters to be solved by this invention be to provide a kind ofly have that highly sensitive, selectivity is strong, good stability, accuracy is high, detection speed the is fast method of utilizing PbS quantum dot fast detecting aflatoxin B1.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of method of the PbS of utilization quantum dot fast detecting aflatoxin B1 specifically comprises the following steps:
(1) preparation of PbS quantum dot
Get the Pb (NO of 10mmol/L
3)
2solution, in three-neck flask, under the condition of magnetic agitation, after slowly adding mercaptoacetic acid, is adjusted pH to 7, leads to nitrogen deoxygenation 25-35min, dropwise adds the Na of 25mol/L
2s solution, at N
2after the lower reaction 23-25h of protection, in rotating speed 10000-12000rpm, centrifugal 25-35min, get precipitation and alternately clean each 3-4 time with redistilled water and absolute ethyl alcohol, the gained solid matter is placed in to 45-55 ℃ of constant-temperature vacuum drying box dry, the gained material is the PbS quantum dot;
(2) PbS quantum dot coupling aflatoxin B1 monoclonal antibody
Getting the PbS quantum dot is dissolved in PBS solution, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS), ultrasonic mixing, under room temperature, lucifuge obtains mixed solution after swaying and cultivating 25-35min, the aflatoxin B1 monoclonal antibody is dissolved in to the aflatoxin B1 monoclonal antibody solution that is mixed with 1mg/mL in PBS solution, the aflatoxin B1 monoclonal antibody solution is added in mixed solution, after lucifuge concussion 1.5-2.5h, add again mercaptoethanol to stablize quantum dot, after dialysis 20-28h, in rotating speed 4000-6000rpm, centrifugal 2.5-3.5min, get precipitation with after PBS solution cleaning 2-4 time, obtain quantum dot-labeled monoclonal antibody (QD-Ab), quantum dot-labeled monoclonal antibody is dissolved in PBS solution, be mixed with the quantum dot-labeled monoclonal antibody solution of 10 μ g/mL, 4 ℃ keep in Dark Place stand-by,
(3) Immune competition based on quantum dot detects
In ELISA Plate, every hole adds 100 μ L 3mg/mL aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) as coating antigen, coated elisa plate, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 200 μ L 3% caseic aqueous solution sealings, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 50 μ L AFB1 solution and the quantum dot-labeled monoclonal antibody solution of 60 μ L10 μ g/mL simultaneously, hatch 1h for 37 ℃, the PBST damping fluid is washed plate 3 times, every hole adds the ultrasonic digestion of 100 μ L 1mol/L salpeter solution 30min, transfer to electrolytic cell through postdigestive sample, add 20 μ L 1mg/mL Hg in electrolytic cell
2cl solution, and be settled to 2mL with the acetate buffer solution of pH4.5, as solution to be measured, carry out next step Electrochemical Detection,
(4) electrode pre-service
By glass-carbon electrode successively at the α-Al of 1.0,0.3,0.05 μ m particle diameter
2o
3in burnishing powder, repeatedly polish to minute surface, then be placed in the respectively volume fraction ethanolic solution that is 50%, salpeter solution and the redistilled water ultrasonic cleaning 4-6min that volume fraction is 50%, dry up glass-carbon electrode standby with micro-nitrogen stream;
(5) assay method
To in solution to be measured, pass into high-purity N
2deoxygenation 5-15min, after static 5-15s, insert rotation electrode, start motor and rotate pretreated glass-carbon electrode, in take-off potential-1.0V place enrichment, the glass-carbon electrode that stops the rotation after enrichment 240s, standing 5-15s, the control square wave frequency is 30Hz, the current potential increment is 4mV/s, and square wave amplitude is 25mV, adopts square wave voltammetry from-0.8V reverse scan to-0.2V, the writing scan curve, record Pb in-0.52V
2+the stripping peak current, according to Pb
2+stripping peak current size and the typical curve relation between the aflatoxin B1 solution concentration, by the Pb of solution to be measured
2+stripping peak current substitution typical curve in, calculate the content of aflatoxin B1.
Pb (NO described in step (1)
3)
2solution, described mercaptoacetic acid and described Na
2the volume ratio of S solution is 50:0.25:30.
The ratio of the PbS quantum dot described in step (2), described PBS solution, described 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, described N-hydroxy-succinamide, described aflatoxin B1 monoclonal antibody solution and described mercaptoethanol is 1.0mg:200 μ L:1.0mg:1.0mg:20 μ L:100 μ L
The concentration of described PBS solution is 0.01M, and pH is 7.4.
The collocation method of the PBST damping fluid described in step (3) is as follows: take KH
2pO
40.2g, Na
2hPO
412H
2o 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5mL, add water and be settled to 1000mL and get final product.
What in step (5), adopt is three-electrode system: glass-carbon electrode is working electrode, and Ag/AgCl is contrast electrode, and platinum electrode is to electrode.
Pb described in step (5)
2+stripping peak current size and the typical curve between the aflatoxin B1 solution concentration be i=-0.69897log C+ 1.41537, R
2=0.99304, R
2=0.99304, wherein i is Pb
2+stripping peak current size, unit is μ A; C is the aflatoxin B1 solution concentration, and unit is ng/mL, and the range of linearity is 0.1-25ng/mL, detects and is limited to 0.03ng/mL.
Principle: coated AFB1 envelope antigen on microwell plate, utilize unconjugated site on BSA or HSA closed porosity, add testing sample and quantum dot-labeled AFB1 monoclonal antibody QD-Ab, AFB1 in sample will with microwell plate on AFB1 coating antigen competition in conjunction with limited QD-Ab, wash plate after reaction.By collect by immune response be combined on the solid phase microwell plate QD-Ab, content by the Pb element in label QD on Anodic Stripping Voltammetry Determination antibody, by the content of lead element and the relation between target AFB1 concentration, just can carry out quantitatively AFB1.Because what detection was taked is the Immune competition pattern, so in gained Pb constituent content and sample, the content of AFB1 to be measured is inversely proportional to, in sample, the content of AFB1 is higher, and the Pb constituent content is lower.
Compared with prior art, the invention has the advantages that: the method for a kind of PbS of utilization quantum dot of the present invention fast detecting aflatoxin B1, utilize the quantum dot-labeled aflatoxin B1 monoclonal antibody of PbS to substitute the enzyme labelled antibody of ELISA, set up the direct competitive immunization of measuring AFB1, can effectively overcome the activity of enzyme in euzymelinked immunosorbent assay (ELISA) and be subject to the reaction conditions impact, the reagent life-span is shorter, nitrite ion needs the shortcomings such as low temperature preservation, the signal enlarge-effect of while PbS quantum dot and the high sensitivity of anodic stripping voltammetry counterweight metal detection, all can greatly strengthen the sensitivity (detection is limited to 0.03ng/mL) of tested electrode, and the method stability, reappearance (RSDs<5%) and accuracy (recovery reaches 96.48%～104.0%) are good, reached quick to AFB1, the purpose of Sensitive Detection, method is simple, actual detection is easy and simple to handle.
The accompanying drawing explanation
Fig. 1 is the direct competitive immunization schematic diagram to AFB1 based on the PbS quantum dot;
Fig. 2 is the transmission electron microscope picture of the PbS quantum dot of preparation;
Fig. 3 is the X-ray diffractogram of the PbS quantum dot of preparation;
The anodic stripping voltammetry curve map that Fig. 4 is variable concentrations AFB1 gained; (a is that 0ng/mL, b are that 0.1ng/mL, c are that 0.5ng/mL, d are that 1ng/mL, e are that 2.5ng/mL, f are that 10ng/mL, g are 30ng/mL);
The typical curve that the current signal that Fig. 5 is object AFB1 and AFB1 concentration are set up.
Embodiment
Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.
Specific embodiment one
A kind of method of utilizing PbS quantum dot fast detecting aflatoxin B1 specifically comprises the following steps:
(1) preparation of PbS quantum dot
Normal temperature, under condition of normal pressure, get the Pb (NO of 50mL 10mmol/L
3)
2solution, in three-neck flask, under the condition of magnetic agitation, slowly adds the mercaptoacetic acid of 0.25mL, with the NaOH solution of 0.5mol/L, adjusts pH to 7, leads to nitrogen deoxygenation 30min, dropwise adds the Na of 30mL 25 mol/L
2s solution, at N
2after the lower reaction 24h of protection, use the supercentrifuge centrifuging, rotating speed is 11000rpm; time is 30min, and abandoning supernatant will precipitate with redistilled water and absolute ethyl alcohol and alternately clean each 3 times; the gained solid matter is placed in to 50 ℃ of vacuum drying chambers dry, the gained material is the PbS quantum dot.As shown in Figure 2, the PbS quantum dot obtained is uniformly dispersed the transmission electron microscope picture of PbS quantum dot, and profile is cube structure, and mean grain size is about 20-30nm.The X-ray diffractogram of PbS quantum dot as shown in Figure 3, in figure, but all diffraction peaks are distinguished (111), (200), (220), (311), (222), (400), (331), (420) and (422) crystal face of cubic system PbS crystal in reference standard card JCPDSNo.5-592, and there is no impurity peaks, show that gained PbS quantum dot belongs to centroid cubic crystal system.In Fig. 3, diffraction peak is very sharp-pointed, shows that product crystallinity is better;
(2) PbS quantum dot coupling aflatoxin B1 monoclonal antibody
Getting the above-mentioned PbS quantum dot of 1.0mg is dissolved in 200 μ L PBS solution (0.01M pH7.4), add 1.0mg1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and ultrasonic the mixing of 1.0mg N-hydroxy-succinamide (NHS), under room temperature, lucifuge is swayed and is cultivated 30min, by the aflatoxin B1 monoclonal antibody solution of 20 μ L 1mg/mL, (solvent is 0.01M subsequently, the PBS solution of pH7.4) join in this mixed solution, lucifuge concussion 2h, reaction adds 100 μ L mercaptoethanols to stablize quantum dot after finishing again, centrifugal after dialysis 24h, rotating speed is 5000rpm, time is 3min, remove supernatant, after precipitating and cleaning 3 times with PBS solution (0.01M pH7.4), obtain the quantum dot-labeled monoclonal antibody QD-Ab of PbS, QD-Ab is dissolved in PBS solution (0.01M pH7.4), be mixed with the QD-Ab solution of 10 μ g/mL, 4 ℃ keep in Dark Place stand-by,
(3) Immune competition based on quantum dot detects
In ELISA Plate, every hole adds 100 μ L 3mg/mL aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) as coating antigen, coated elisa plate, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 200 μ L 3% casein solution sealings, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 50 μ L AFB1 solution and 60 μ L 10 μ g/mL QD-Ab solution simultaneously, hatch 1h for 37 ℃, the PBST damping fluid is washed plate 3 times, every hole adds the ultrasonic digestion of 100 μ L 1mol/L salpeter solution 30min, transfer to electrolytic cell through postdigestive sample, add 20 μ L 1mg/mL Hg
2cl solution, and be settled to 2mL with the acetate buffer solution of pH 4.5, as solution to be measured, carry out next step Electrochemical Detection, wherein the collocation method of PBST damping fluid is as follows: take KH
2pO
40.2g, Na
2hPO
412H
2o 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5mL, add water and be settled to 1000mL and get final product,
(4) electrode pre-service
By glass-carbon electrode successively at the α-Al of 1.0,0.3,0.05 μ m particle diameter
2o
3in burnishing powder, repeatedly polish to minute surface, ultrasonic cleaning 5min in the ethanolic solution that then volume fraction is 50% respectively, the salpeter solution that volume fraction is 50% and redistilled water, dry up glass-carbon electrode standby with micro-nitrogen stream;
(5) assay method
Pass into high-purity N in solution to be measured
2deoxygenation 10min, after static 10s, insert rotation electrode, and start motor and rotate pretreated glass-carbon electrode, in take-off potential-1.0V place enrichment, the working electrode that stops the rotation after enrichment 240s, standing 10s.The control square wave frequency is 30Hz, and the current potential increment is 4mV/s, and square wave amplitude is 25mV, adopts square wave voltammetry from-0.8V reverse scan to-0.2V, and the writing scan curve records Pb in-0.52V
2+the stripping peak current, according to Pb
2+stripping peak current i size and the typical curve i (μ A) between aflatoxin B1 solution concentration C=-0.69897log C (ng/mL)+1.41537, R
2=0.99304, by the Pb of solution to be measured
2+stripping peak current substitution typical curve in, calculate the content of aflatoxin B1, said determination method three-electrode system: glass-carbon electrode is working electrode, Ag/AgCl is contrast electrode, platinum electrode is to electrode.
Embodiment 2
With above-mentioned embodiment 1, its difference is:
In step (1), logical nitrogen deoxygenation 25min, dropwise add Na
2s solution, at N
2after the lower reaction 23h of protection, with supercentrifuge, in rotating speed, be 10000rpm, centrifugal 25min, get precipitation and alternately clean each 4 times with redistilled water and absolute ethyl alcohol, the gained solid matter is placed in to 45 ℃ of constant-temperature vacuum drying boxes dry;
In step (2), under room temperature, lucifuge obtains mixed solution after swaying and cultivating 25min, the aflatoxin B1 monoclonal antibody solution is added in mixed solution, after lucifuge concussion 1.5h, then add mercaptoethanol to stablize quantum dot, after dialysis 20h, in rotating speed 4000rpm, centrifugal 2.5min, remove supernatant, will precipitate with after PBS solution cleaning 2 times, obtain quantum dot-labeled monoclonal antibody.
Embodiment 3
With above-mentioned embodiment 1, its difference is:
In step (1), logical nitrogen deoxygenation 35min, dropwise add Na
2s solution, at N
2after the lower reaction 25h of protection, with supercentrifuge, in rotating speed, be 12000rpm, centrifugal 35min, go precipitation alternately to clean each 4 times with redistilled water and absolute ethyl alcohol, the gained solid matter is placed in to 55 ℃ of constant-temperature vacuum drying boxes dry;
In step (2), under room temperature, lucifuge obtains mixed solution after swaying and cultivating 35min, the aflatoxin B1 monoclonal antibody solution is added in mixed solution, after lucifuge concussion 2.5h, add again mercaptoethanol to stablize quantum dot, centrifugal after dialysis 28h, in rotating speed, be 6000rpm, centrifugal 3.5min, get precipitation with after PBS solution cleaning 4 times, obtain quantum dot-labeled monoclonal antibody.
In addition to the implementation, Pb (NO
3)
2solution, mercaptoacetic acid and Na
2the volume ratio of S solution meets 50:0.25:30 and gets final product.The ratio of PbS quantum dot, PBS solution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxy-succinamide, aflatoxin B1 monoclonal antibody solution and mercaptoethanol meets 1.0mg:200 μ L:1.0mg:1.0mg:20 μ L:100 μ L and gets final product.
Specific embodiment two
Detection to the AFB1 standard solution
0,0.1,0.5,1,2.5,10, the standard solution of 30ng/mL with methyl alcohol, AFB1 is mixed with to 1mg/mL solution, then is followed successively by with containing the PBS solution (0.01M pH7.4) of 10% methyl alcohol, being diluted to concentration:.
(1) preparation of PbS quantum dot: with embodiment 1 in above-mentioned specific embodiment one.
(2) PbS quantum dot coupling aflatoxin B1 monoclonal antibody: with above-mentioned embodiment 1.
(3) Immune competition based on quantum dot detects
In ELISA Plate, every hole adds 100 μ L 3mg/mL aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) as coating antigen, coated elisa plate, 4 ℃ are spent the night, after the PBST damping fluid is washed plate 3 times, every hole adds 200 μ L 3% casein solution sealings, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 50 μ L AFB1 standard solutions and 60 μ L10 μ g/mL QD-Ab solution simultaneously, hatch 1h for 37 ℃, the PBST damping fluid is washed plate 3 times, every hole adds the ultrasonic digestion of 100 μ L 1mol/L salpeter solution 30min, transfer to electrolytic cell through postdigestive sample, add 20 μ L 1mg/mL Hg in electrolytic cell
2cl solution, and be settled to 2mL with the acetate buffer solution of pH4.5, as solution to be measured, carry out next step Electrochemical Detection.
(4) electrode pre-service: with above-mentioned embodiment 1.
(5) assay method: with above-mentioned embodiment 1.As shown in Figure 4, along with the AFB1 concentration C increases, corresponding Pb
2+stripping peak current i reduce.Criterion curve: i (μ A)=-0.69897logC (ng/mL)+1.41537, R
2=0.99304 (as shown in Figure 5), the range of linearity is: 0.1-25ng/mL, detect and be limited to 0.03ng/mL.
Specific embodiment three
The high selectivity of this detection method to AFB1.
The preparation of AFB1, AFB2, AFG1, AFG2, AFM1 solution:
Respectively AFB1, AFB2, AFG1, AFG2, AFM1 are mixed with to 1mg/mL solution with methyl alcohol, then are 10ng/mL by PBS solution dilution to the concentration containing 10% methyl alcohol.
(1) preparation of PbS quantum dot: with embodiment 1 in above-mentioned specific embodiment one.
(2) PbS quantum dot coupling aflatoxin B1 monoclonal antibody: with above-mentioned embodiment 1.
(3) Immune competition based on quantum dot detects
In ELISA Plate, every hole adds 100 μ L 3mg/mL aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) as coating antigen, coated elisa plate, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 200 μ L 3% casein solution sealings, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds respectively 50 μ L 10ng/mL AFB1 simultaneously, AFB2, AFG1, AFG2, AFM1 solution and 60 μ L 10 μ g/mL QD-Ab solution, hatch 1h for 37 ℃, the PBST damping fluid is washed plate 3 times, every hole adds the ultrasonic digestion of 100 μ L 1mol/L salpeter solution 30min, transfer to electrolytic cell through postdigestive sample, add 20 μ L 1mg/mL Hg in electrolytic cell
2cl solution, and be settled to 2mL with the acetate buffer solution of pH 4.5, as solution to be measured, carry out next step Electrochemical Detection.
(4) electrode pre-service: with above-mentioned embodiment 1.
(5) assay method: with above-mentioned embodiment 1.Obtain Pb separately
2+the stripping peak current, the electric current maximum that wherein AFB1 is corresponding, AFB2, AFG1, AFG2, the corresponding electric current of AFM1 gained all a little less than, account for respectively 11%, 26%, 5.7%, 3.5% of AFB1 corresponding current, the method shows good selectivity.
Specific embodiment four
Detection to peanut sample
Peanut sample is shelled to remove the peel and pulverized 20 mesh sieves, mix and take through levigate peanut sample 20.0g in 100mL tool plug conical flask, add 40.0mL methyl alcohol and 2g sodium chloride, the mechanical shaking extraction 30min that jumps a queue, standing 15min.The neutral qualitative filter paper of bilayer filters, and collects filtrate, with nitrogen, gently filtrate is evaporated to near doing under 30 ℃, and the PBS solution that contains 10% methyl alcohol is settled to 20mL.
(1) preparation of PbS quantum dot: with embodiment 1 in above-mentioned specific embodiment one.
(2) PbS quantum dot coupling aflatoxin B1 monoclonal antibody: with above-mentioned embodiment 1.
(3) Immune competition based on quantum dot detects
In ELISA Plate, every hole adds 100 μ L 3mg/mL aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) as coating antigen, coated elisa plate, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 200 μ L 3% casein solution sealings, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds respectively 50 μ L sample solutions and 60 μ L 10 μ g/mL QD-Ab solution simultaneously, hatch 1h for 37 ℃, the PBST damping fluid is washed plate 3 times, every hole adds the ultrasonic digestion of 100 μ L 1mol/L salpeter solution 30min, transfer to electrolytic cell through postdigestive sample, add 20 μ L 1mg/mL Hg
2cl solution, and be settled to 2mL with the acetate buffer solution of pH4.5, as solution to be measured, carry out next step Electrochemical Detection.
(4) electrode pre-service: with above-mentioned embodiment 1.
(5) assay method: with above-mentioned embodiment 1, testing result is as shown in table 1.
The testing result of AFB1 content in table 1 peanut sample
Specific embodiment five
Detection to powdered milk sample
Take powdered milk sample 20.0g in 100mL tool plug conical flask, add 40.0mL methyl alcohol and 2g sodium chloride, the mechanical shaking extraction 30min that jumps a queue, standing 15min.Double-deck neutral qualitative filter paper filters, and with nitrogen, gently filtrate is evaporated to near doing under 30 ℃, and the PBS solution that contains 10% methyl alcohol is settled to 2mL.
(1) preparation of PbS quantum dot: with embodiment 1 in above-mentioned specific embodiment one.
(2) PbS quantum dot coupling aflatoxin B1 monoclonal antibody: with above-mentioned embodiment 1.
(3) Immune competition based on quantum dot detects
In ELISA Plate, every hole adds 100 μ L 3mg/mL aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) as coating antigen, coated elisa plate, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 200 μ L 3% casein solution sealings, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds respectively 50 μ L sample solutions and 60 μ L 10 μ g/mL QD-Ab solution simultaneously, hatch 1h for 37 ℃, the PBST damping fluid is washed plate 3 times, and every hole adds the ultrasonic digestion of 100 μ L 1mol/L salpeter solution 30min.Transfer to electrolytic cell through postdigestive sample, add 20 μ L 1mg/mL Hg
2cl solution, and be settled to 2mL with the acetate buffer solution of pH4.5, as solution to be measured, carry out next step Electrochemical Detection.
(4) electrode pre-service: with above-mentioned embodiment 1.
(5) assay method: with above-mentioned embodiment 1, testing result is as shown in table 2.
The testing result of AFB1 content in table 2 powdered milk sample
Specific embodiment six
Detection to peanut sample
Peanut sample is shelled to remove the peel and pulverized 20 mesh sieves, mix the peanut sample 20.0g taken through levigate, repeat embodiment tetra-.Take 20.0g simultaneously, according to NYT 1286-2007, use high performance liquid chromatography to be detected.Testing result is as shown in table 3, and following two groups of the data SPSS softwares are carried out to the t check, and P>0.05 illustrates that this method and HPLC detection method are without significant difference.
The testing result of two kinds of method AFB1 content of table 3
| Specimen coding | This method result/μ g/g | This method RSD/%(n=5) | HPLC result/μ g/ |
| 1 | 0.255 | 4.1 | 0.235 |
| 2 | 0.247 | 3.4 | 0.246 |
| 3 | 0.234 | 2.2 | 0.242 |
| 4 | 0.238 | 4.5 | 0.233 |
| 5 | 0.227 | 3.7 | 0.261 |
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention also is not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (7)
1. a method of utilizing PbS quantum dot fast detecting aflatoxin B1 is characterized in that comprising the following steps:
(1) preparation of PbS quantum dot
Get the Pb (NO of 10mmol/L
3)
2solution, in three-neck flask, under the condition of magnetic agitation, after slowly adding mercaptoacetic acid, is adjusted pH to 7, leads to nitrogen deoxygenation 25-35min, dropwise adds the Na of 25mol/L
2s solution, at N
2after the lower reaction 23-25h of protection, in rotating speed 10000-12000rpm, centrifugal 25-35min, get precipitation and alternately clean each 3-4 time with redistilled water and absolute ethyl alcohol, the gained solid matter is placed in to 45-55 ℃ of constant-temperature vacuum drying box dry, obtains the PbS quantum dot;
(2) PbS quantum dot coupling aflatoxin B1 monoclonal antibody
Getting the PbS quantum dot is dissolved in PBS solution, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS), ultrasonic mixing, under room temperature, lucifuge obtains mixed solution after swaying and cultivating 25-35min, the aflatoxin B1 monoclonal antibody is dissolved in to the aflatoxin B1 monoclonal antibody solution that is mixed with 1mg/mL in PBS solution, the aflatoxin B1 monoclonal antibody solution is added in mixed solution, after lucifuge concussion 1.5-2.5h, add again mercaptoethanol to stablize quantum dot, after dialysis 20-28h, in rotating speed 4000-6000rpm, centrifugal 2.5-3.5min, get precipitation with after PBS solution cleaning 2-4 time, obtain quantum dot-labeled monoclonal antibody, quantum dot-labeled monoclonal antibody is dissolved in PBS solution, be mixed with the quantum dot-labeled monoclonal antibody solution of 10 μ g/mL, 4 ℃ keep in Dark Place stand-by,
(3) Immune competition based on quantum dot detects
In ELISA Plate, every hole adds 100 μ L 3mg/mL aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) as coating antigen, coated elisa plate, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 200 μ L 3% caseic aqueous solution sealings, 4 ℃ are spent the night, the PBST damping fluid is washed plate 3 times, every hole adds 50 μ L AFB1 solution and the quantum dot-labeled monoclonal antibody solution of 60 μ L10 μ g/mL simultaneously, hatch 1h for 37 ℃, the PBST damping fluid is washed plate 3 times, every hole adds the ultrasonic digestion of 100 μ L 1mol/L salpeter solution 30min, transfer to electrolytic cell through postdigestive sample, add 20 μ L 1mg/mL Hg in electrolytic cell
2cl solution, and be settled to 2mL with the acetate buffer solution of pH4.5, as solution to be measured, carry out next step Electrochemical Detection,
(4) electrode pre-service
By glass-carbon electrode successively at the α-Al of 1.0,0.3,0.05 μ m particle diameter
2o
3in burnishing powder, repeatedly polish to minute surface, then be placed in the respectively volume fraction ethanolic solution that is 50%, salpeter solution and the redistilled water ultrasonic cleaning 4-6min that volume fraction is 50%, dry up glass-carbon electrode standby with micro-nitrogen stream;
(5) assay method
To in solution to be measured, pass into high-purity N
2deoxygenation 5-15min, after static 5-15s, insert rotation electrode, start motor and rotate pretreated glass-carbon electrode, in take-off potential-1.0V place enrichment, the glass-carbon electrode that stops the rotation after enrichment 240s, standing 5-15s, the control square wave frequency is 30Hz, the current potential increment is 4mV/s, and square wave amplitude is 25mV, adopts square wave voltammetry from-0.8V reverse scan to-0.2V, the writing scan curve, record Pb in-0.52V
2+the stripping peak current, according to Pb
2+stripping peak current size and the typical curve relation between the aflatoxin B1 solution concentration, by the Pb of solution to be measured
2+stripping peak current substitution typical curve in, calculate the content of aflatoxin B1.
2. a kind of method of utilizing PbS quantum dot fast detecting aflatoxin B1 according to claim 1, is characterized in that: the Pb (NO described in step (1)
3)
2solution, described mercaptoacetic acid and described Na
2the volume ratio of S solution is 50:0.25:30.
3. a kind of method of utilizing PbS quantum dot fast detecting aflatoxin B1 according to claim 1, it is characterized in that: the ratio of the PbS quantum dot described in step (2), described PBS solution, described 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, described N-hydroxy-succinamide, described aflatoxin B1 monoclonal antibody solution and mercaptoethanol is 1.0mg:200 μ L:1.0mg:1.0mg:20 μ L:100 μ L.
4. a kind of method of utilizing PbS quantum dot fast detecting aflatoxin B1 according to claim 3, it is characterized in that: the concentration of described PBS solution is 0.01M, pH is 7.4.
5. a kind of method of utilizing PbS quantum dot fast detecting aflatoxin B1 according to claim 1, it is characterized in that: the collocation method of the PBST damping fluid described in step (3) is as follows: take KH
2pO
40.2g, Na
2hPO
412H
2o 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5mL, add water and be settled to 1000mL and get final product.
6. a kind of method of utilizing PbS quantum dot fast detecting aflatoxin B1 according to claim 1, it is characterized in that: what in step (5), adopt is three-electrode system: glass-carbon electrode is working electrode, Ag/AgCl is contrast electrode, and platinum electrode is to electrode.
7. a kind of method of utilizing PbS quantum dot fast detecting aflatoxin B1 according to claim 1, is characterized in that: the Pb described in step (5)
2+stripping peak current size and the typical curve between the aflatoxin B1 solution concentration be i=-0.69897log C+ 1.41537, R
2=0.99304, R
2=0.99304, wherein i is Pb
2+stripping peak current size, unit is μ A; C is the aflatoxin B1 solution concentration, and unit is ng/mL, and the range of linearity is 0.1-25ng/mL, detects and is limited to 0.03ng/mL.
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