CN104422686A - Method for determining aflatoxin - Google Patents
Method for determining aflatoxin Download PDFInfo
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- CN104422686A CN104422686A CN201310397266.XA CN201310397266A CN104422686A CN 104422686 A CN104422686 A CN 104422686A CN 201310397266 A CN201310397266 A CN 201310397266A CN 104422686 A CN104422686 A CN 104422686A
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Abstract
The invention discloses a method for determining aflatoxin. The method is characterized by comprising the following steps: (1) preparing a solution 1 for later use; (2) preparing a solution 2 for later use; (3) preparing R1 modified by HRP (horseradish peroxidase); (4) preparing R2 modified by HRP; and (5) detecting aflatoxin. Red rhodamine B can neither cause concentration quenching, nor cause insufficient light intensity. By adding mica powder, the luminescence lifetime of an oxalate luminescence system is significantly prolonged. Ruthenium terpyridine and chemiluminescence have the advantages of high detection sensitivity, wide linear range and the like, and aflatoxin can be detected more effectively. The method disclosed by the invention has the excellent properties of lower toxicity, higher flash point and lower vapor pressure. By detecting the content of aflatoxin in liquid milk and using a standard addition recovery method, the recovery yield of 98.4%-100.4% is obtained, indicating that the method has good accuracy in determination of aflatoxin.
Description
Technical field
The invention belongs to analytical chemistry and chemiluminescence field, specifically relate to a kind of method measuring aflatoxin.
Background technology
Aflatoxin (AFT) is the compound that a class chemical constitution is similar, is the derivant of dihydrofuran cumarin.Aflatoxin is the secondary metabolite produced primarily of aspergillus flavus aspergillus parasiticus, occurs that the probability of aflatoxin is the highest in the food and feed of damp-heat area.
100,000 turkey sudden death events that the sixties in 20th century occurs in Britain are identified relevant with from the peanut meal of Brazilian import. and further investigate proof, these peanut meals are polluted by a kind of noxious material from fungi the toxic metabolite material that these research work finally make to have it is found that aspergillus flavus produces.Aflatoxin is the metabolic product of aspergillus flavus and aspergillus parasiticus.Tequ is mould also can produce aflatoxin, but output is less. and the aflatoxin of generation mainly contains B1, B2, G1, G2 and other two kinds of metabolic product M1, M2. wherein M1 and M2 are the .B1 separated from milk, B2, G1, G2, M1 and M2 are very close on molecular structure.
Aflatoxin is far away higher than the toxicity of prussiate, arsenide and organic agricultural chemicals, wherein maximum with B1 toxicity.When people's intake is large, can acute poisoning be there is, occur oxyhepatitis, hemorrhagic necrosis, hepatic cell fattydegeneration and bile duct proliferation.When trace continues to take in, can slow poisoning be caused, growth disorder, cause fibrous lesions, cause proliferation of fibrous tissue.The carcinogenicity of AFT also ranks first, and is one of known most strong carcinogen at present.
Chemiluminescent oxalate system should be able to produce efficient chemical light, has good bin stability and is safe." hydrogen peroxide component " wherein containing the tertiary alcohol, is considered to optimum formula all the time and is widely used.But this contains, and " hydrogen peroxide component " bin stability of the tertiary alcohol is lower, light output density is not high, the defect especially also existing that component flash-point is low, vapor pressure is high, security is not good etc. and should improve.
The detection time of current a sample aflatoxin composition, usually need at least three to four hours, the fastest method also needs two hours.And lowest detectable limit can not meet the demands, sensing range is wide not, testing process complicated operation, and instrument generally costly.
The advantages such as tris (bipyridine) ruthenium and chemiluminescence have original position response, detection sensitivity is high, the range of linearity is wide and instrument is simple, are widely applied in Pharmaceutical Analysis, amino acid analysis, DNA probe analysis, enzyme biologic sensor field.But at present also without any relevant report chemiluminescence being used for aflatoxin detection.The object of the invention is for existing Determination Methods of Aflatoxins, propose a kind of method that brand-new detectability is lower, detect the chemiluminescence detection aflatoxin that the range of linearity is wider, sensitivity is higher.
After the energy excitation that fluorescent material molecule is discharged by chemical reaction the light that sends be referred to as chemiluminescence.Chemiluminescence is a kind of is at room temperature the course of reaction of luminous energy by chemical reaction Conversion of Energy.With the chemical luminous system that oxalate, hydrogen peroxide and fluorescer are main composition, there is the advantage of many uniquenesses such as luminescence efficiency is high, intensity is large, the life-span is long, environmental protection, get the attention, have huge application prospect.
But with the addition of high molecular polymer in oxalate luminescence system after, luminescent lifetime extends to some extent, but luminous intensity obviously reduces.With the addition of catalyzer or inhibitor in system after, luminescent lifetime or luminous intensity decline to a great extent, and luminescence efficiency is not high.Therefore, be necessary to develop new oxalate luminescence system, there is good luminous intensity and good luminescent lifetime simultaneously.
The shortcoming of the chemical luminescence substrate system in chemiluminescence detection kit commercially available is at present: sensitivity and the specificity of detection are not high enough, reagent stability not, cost is higher, it is simple and efficient not etc. to detect.So must develop a kind of highly sensitive, simple novel detection method.
Summary of the invention
When there being object aflatoxin to exist, by the magnetic bead effect of functionalization, chain type hybridization reaction is carried out under HRP-R1 and HRP-R2 modifying HRP exists, there is magnetic resolution, recycling HRP produces chemiluminescence to the catalytic action of chemical luminous system, by measuring the mensuration of chemiluminescence intensity realization to aflatoxin.
The present invention is achieved by the following technical solutions: a kind of method measuring aflatoxin, is characterized in that, said method comprising the steps of:
(1) preparation of stand-by liquid 1: just after the washing of Magnetic gold nanoparticle phosphate buffer, get 0.5 × 10
-5the tris (bipyridine) ruthenium of M, adding 250 μ l pH values is 0.1 × 10 of 9-10
-5in the sodium borate buffer solution of M, under 37 ° of C, react 15h; The functional magnetic golden nanometer particle phosphate buffer of gained is washed, is then dispersed in phosphate buffer, for subsequent use under being kept at 5 ° of C;
(2) preparation of stand-by liquid 2: after being washed by Magnetic gold nanoparticle phosphate buffer, add 150 μ l 2.0 × 10
-5two (2,4-dinitrophenyl) oxalates, 1.0 × 10 of M
-5two (2, the 5-dinitrophenyl) oxalate of M, to add 50 μ l ratios be the female powder of 0.06mg/ μ l 350 object and 50 μ l 0.5 × 10
-5the red rhodamine B of M.15h is reacted under 37 ° of C; The functional magnetic golden nanometer particle phosphate buffer of gained is washed, is then dispersed in phosphate buffer, for subsequent use under being kept at 5 ° of C;
(3) HRP(horseradish peroxidase is prepared) R1 that modifies: at 100 μ l 1.5 × 10
-5the ammonium molybdate solution of M, add 15 mg NHS(N-N-Hydroxysuccinimide in the EDTA of 80 μ l 0.005M and the quinol solution of 100 μ l 0.008M), stir 2 hours under 37 ° of C, then add 150 μ l HRP, under 37 ° of C, stir 24h;
(4) R2 that HRP modifies is prepared: at 100 μ l 1.5 × 10
-5chloro-4 hydroxyacetanilides of 3-of M, 130 μ l 0.5 × 10
-515 mg NHS and 30 μ l 0.5 × 10 are added in M sodium sulphite
-5the catalyzer of M, stirs 1.5h under 37 ° of C, then adds 100 μ l HRP, under 37 ° of C, stirs 24h;
(5) detection of aflatoxin: the standard solution 100 μ L got containing aflatoxin joins stand-by liquid 1 solution of 200 μ L, 37 ° of C react 40min, and then potpourri is separated on magnetic frame, is transferred to by supernatant in stand-by liquid 2 solution; Then add containing 2.5 × 10-7 M HRP-R1 and 2.0 × 10-7 M HRP-R2 solution 200 μ L, Magneto separate is carried out after 37 DEG C of reaction 2 h, removing supernatant, by magnetic bead with after 120 μ L phosphate buffer solution cleaning twice, be scattered in 1.0 ml phosphate buffer solutions, then carry out chemiluminescence detection.
A kind of above-mentioned method measuring aflatoxin, it is characterized in that, the catalyzer in described step (4) is one or more in salicylic acid TBuA, pentachloro-phenol potassium, potassium salicylate, benzoic acid TBuA, 5-chloro-salicylic acid lithium, sodium salicylate, TBAP, sodium trifluoroacetate.
Sample preparation of the present invention comprises the following steps: take 30 g fresh milks, adds 30 mL Cl
3cCOOH and 60 mL CH
3in OH mixed solution, ultrasonic 20 min, get supernatant after centrifugal 20 min under 10000 r/min rotating speeds, filter with the screen membrane of 0.4 μm, carry out analysis and measure, and carry out recovery testu to gained filtrate.
The beneficial effect of the invention:
The present invention selects the red rhodamine B commonly used as fluorescer, because its fluorescence quantum yield is higher in the conventional fluorescer of oxalate ester peroxide system, rhodamine B is also soluble in the solvent of above-mentioned oxalate component in addition.Both can not cause concentration quenching, light intensity also can not be caused not enough.By in chemical luminous system, add the mica powder of inorganic additive, thus obtain luminous intensity not significantly reduce but the oxalate luminescence system of luminescent lifetime significant prolongation.Tris (bipyridine) ruthenium and chemiluminescence have the advantages such as detection sensitivity is high, the range of linearity is wide, and more effectively can detect aflatoxin, detectability is lower, and detect the range of linearity wider, highly sensitive, favorable reproducibility, test can complete instantaneously.Compare with traditional chemical luminous system, there are lower toxicity, higher flash-point and these good characteristics of lower vapor pressure.The reliability of the method for inspection is carried out by the content detecting aflatoxin in liquid milk.And utilizing using standard samples recovery to investigate the method, the recovery, between 98.4%-100.4%, shows that the method measures aflatoxin and has good accuracy in complicated substrate.Accuracy of the present invention and high specificity, sensitivity and stability high, pollute little, cost is low, it is simple and efficient etc. to detect, and application prospect is extensive.
Embodiment
(1) preparation of stand-by liquid 1: just after the washing of Magnetic gold nanoparticle phosphate buffer, get 0.5 × 10
-5the tris (bipyridine) ruthenium of M, adding 250 μ l pH values is 0.1 × 10 of 9-10
-5in the sodium borate buffer solution of M, under 37 ° of C, react 15h; The functional magnetic golden nanometer particle phosphate buffer of gained is washed, is then dispersed in phosphate buffer, for subsequent use under being kept at 5 ° of C;
(2) preparation of stand-by liquid 2: after being washed by Magnetic gold nanoparticle phosphate buffer, add 150 μ l 2.0 × 10
-5two (2,4-dinitrophenyl) oxalates, 1.0 × 10 of M
-5two (2, the 5-dinitrophenyl) oxalate of M, to add 50 μ l ratios be the female powder of 0.06mg/ μ l 350 object and 50 μ l 0.5 × 10
-5the red rhodamine B of M.15h is reacted under 37 ° of C; The functional magnetic golden nanometer particle phosphate buffer of gained is washed, is then dispersed in phosphate buffer, for subsequent use under being kept at 5 ° of C;
(3) HRP(horseradish peroxidase is prepared) R1 that modifies: at 100 μ l 1.5 × 10
-5the ammonium molybdate solution of M, add 15 mg NHS(N-N-Hydroxysuccinimide in the EDTA of 80 μ l 0.005M and the quinol solution of 100 μ l 0.008M), stir 2 hours under 37 ° of C, then add 150 μ l HRP, under 37 ° of C, stir 24h;
(4) R2 that HRP modifies is prepared: at 100 μ l 1.5 × 10
-5chloro-4 hydroxyacetanilides of 3-of M, 130 μ l 0.5 × 10
-515 mg NHS and 30 μ l 0.5 × 10 are added in M sodium sulphite
-5the catalyzer of M, stirs 1.5h under 37 ° of C, then adds 100 μ l HRP, under 37 ° of C, stirs 24h;
(5) detection of aflatoxin: the standard solution 100 μ L got containing aflatoxin joins stand-by liquid 1 solution of 200 μ L, 37 ° of C react 40min, and then potpourri is separated on magnetic frame, is transferred to by supernatant in stand-by liquid 2 solution; Then add containing 2.5 × 10-7 M HRP-R1 and 2.0 × 10-7 M HRP-R2 solution 200 μ L, Magneto separate is carried out after 37 DEG C of reaction 2 h, removing supernatant, by magnetic bead with after 120 μ L phosphate buffer solution cleaning twice, be scattered in 1.0 ml phosphate buffer solutions, then carry out chemiluminescence detection.
2, a kind of method measuring aflatoxin according to claim 1, it is characterized in that, the catalyzer in described step (4) is one or more in salicylic acid TBuA, pentachloro-phenol potassium, potassium salicylate, benzoic acid TBuA, 5-chloro-salicylic acid lithium, sodium salicylate, TBAP, sodium trifluoroacetate.
The preparation of sample
Sample preparation of the present invention comprises the following steps: take 30 g fresh milks, adds 30 mL Cl
3cCOOH and 60 mL CH
3in OH mixed solution, ultrasonic 20 min, get supernatant after centrifugal 20 min under 10000 r/min rotating speeds, filter with the screen membrane of 0.4 μm, carry out analysis and measure, and carry out recovery testu to gained filtrate.
Result and discussion
Along with the reaction time increases, chemiluminescence intensity increases gradually.But the increasing degree of chemiluminescence intensity slows down after reaction proceeds to 60 min.Experimental selection 60 min is as the hybridization chain reaction reaction time of the best.
Investigate the composition such as ion, amino acid, vitamin often contained in food measures aflatoxin selectivity to the method.Experimental result shows, when the concentration of aflatoxin is 10
-7during mol/L, error K of 1000 times in the scope of 5%
+, Na
+, Mg
2+, Ca
2+, Fe
3+, NH
4 +, Ba
2+, Zn
2+, Cl
-, NO
3 -, SO
4 2-, PO
4 3-, CO
3 2-, C
2o
4 -, glucose, tryptophane, lysine, histidine, arginine, vitamin B2 and vitamin C; The Fe of 500 times
2+, ascorbic acid, adenine, guanine, uric acid, ATP, methionine, halfcystine; The Cd of 100 times
2+, Sn
2+and Pb
2+all do not disturb the method to the response of aflatoxin.Show that method has high selectivity, this method has the feasibility being applied to authentic sample and detecting.
Investigate the relation of aflatoxin concentration and chemiluminescence intensity.Result shows along with aflatoxin concentration increases, chemiluminescence intensity is larger, and chemiluminescence intensity becomes nonlinear relationship with aflatoxin concentration within the scope of 1 nM ~ 5000 nM: y=547143.47*exp (x/4223147)-4286.23*exp (-x/669.24)-542818.62, R
2=0.9986.Chemiluminescence intensity becomes internal linear relation with aflatoxin concentration in 1 nM ~ 100 nM scope: y=6.9027x+29.317, R
2=0.9987, detect and be limited to 0.3 nM.The RSD aflatoxin of 50 nM being carried out to 7 replicate determinations is 3.8%.
The chain growth of hybridization chain reaction is alternately formed by connecting by R1 and R2 sequence, and therefore two kinds of sequences are for indispensable hybridization chain reaction system.In order to study in reaction the growth efficiency of hybridizing chain reaction further, in contrast experiment, only adding HRP-R2, ensureing the corresponding HRP of an object aflatoxin, according to HRP-luminol-R2O
2chemiluminescence intensity, carries out quantitatively object.Determine to hybridize the enlargement factor of chain reaction according to rolling off the production line of the range of linearity.Along with the increase of aflatoxin concentration, chemiluminescence intensity is larger, and chemiluminescence intensity and aflatoxin concentration linear within the scope of 70 nM ~ 1000 nM: y=0.8891x-38.652, R
2=0.9994, detect and be limited to 45 nM.As with the lower limit of the range of linearity for object compares sensitivity, then hybridize 70 times that chain reaction sensitivity is non-hybridization chain reaction.
The reliability of the method for inspection is carried out by the content detecting aflatoxin in liquid milk.And utilize using standard samples recovery to investigate the method, the results are shown in table 1.The recovery, between 98.4%-100.4%, shows that the method measures aflatoxin and has good accuracy in complicated substrate.
The measurement result of aflatoxin in table 1 fresh milk sample.
| Numbering | Content a, b | Addition | Measured amount | The recovery (%) |
| 1 | ND c | 10.00 | 10.03 | 100.4 |
| 2 | ND | 5.02 | 5.03 | 100.1 |
| 3 | ND | 5.03 | 4.93 | 98.4 |
a7 measurement results
bunit: nM
cnD: do not detect.
Claims (2)
1. measure a method for aflatoxin, it is characterized in that, said method comprising the steps of:
(1) preparation of stand-by liquid 1: just after the washing of Magnetic gold nanoparticle phosphate buffer, get 0.5 × 10
-5the tris (bipyridine) ruthenium of M, adding 250 μ l pH values is 0.1 × 10 of 9-10
-5in the sodium borate buffer solution of M, under 37 ° of C, react 15h; The functional magnetic golden nanometer particle phosphate buffer of gained is washed, is then dispersed in phosphate buffer, for subsequent use under being kept at 5 ° of C;
(2) preparation of stand-by liquid 2: after being washed by Magnetic gold nanoparticle phosphate buffer, add 150 μ l 2.0 × 10
-5two (2,4-dinitrophenyl) oxalates, 1.0 × 10 of M
-5two (2, the 5-dinitrophenyl) oxalate of M, to add 50 μ l ratios be the female powder of 0.06mg/ μ l 350 object and 50 μ l 0.5 × 10
-5the red rhodamine B of M, reacts 15h under 37 ° of C; The functional magnetic golden nanometer particle phosphate buffer of gained is washed, is then dispersed in phosphate buffer, for subsequent use under being kept at 5 ° of C;
(3) HRP(horseradish peroxidase is prepared) R1 that modifies: at 100 μ l 1.5 × 10
-5the ammonium molybdate solution of M, add 15 mg NHS(N-N-Hydroxysuccinimide in the EDTA of 80 μ l 0.005M and the quinol solution of 100 μ l 0.008M), stir 2 hours under 37 ° of C, then add 150 μ l HRP, under 37 ° of C, stir 24h;
(4) R2 that HRP modifies is prepared: at 100 μ l 1.5 × 10
-5chloro-4 hydroxyacetanilides of 3-of M, 130 μ l 0.5 × 10
-515 mg NHS and 30 μ l 0.5 × 10 are added in M sodium sulphite
-5the catalyzer of M, stirs 1.5h under 37 ° of C, then adds 100 μ l HRP, under 37 ° of C, stirs 24h;
(5) detection of aflatoxin: the standard solution 100 μ L got containing aflatoxin joins stand-by liquid 1 solution of 200 μ L, 37 ° of C react 40min, and then potpourri is separated on magnetic frame, is transferred to by supernatant in stand-by liquid 2 solution; Then add containing 2.5 × 10-7 M HRP-R1 and 2.0 × 10-7 M HRP-R2 solution 200 μ L, Magneto separate is carried out after 37 DEG C of reaction 2 h, removing supernatant, by magnetic bead with after 120 μ L phosphate buffer solution cleaning twice, be scattered in 1.0 ml phosphate buffer solutions, then carry out chemiluminescence detection.
2. a kind of method measuring aflatoxin according to claim 1, it is characterized in that, the catalyzer in described step (4) is one or more in salicylic acid TBuA, pentachloro-phenol potassium, potassium salicylate, benzoic acid TBuA, 5-chloro-salicylic acid lithium, sodium salicylate, TBAP, sodium trifluoroacetate.
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Cited By (7)
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| CN104964969A (en) * | 2015-05-28 | 2015-10-07 | 广东省生态环境与土壤研究所 | Label-free visualization detection method and label-free visualization detection kit of aflatoxin B1 |
| CN107219216A (en) * | 2017-06-26 | 2017-09-29 | 中国热带农业科学院农产品加工研究所 | A kind of method of aflatoxin B1 in chemiluminescence immunoassay detection baby rice powder |
| CN107764784A (en) * | 2017-09-01 | 2018-03-06 | 杨蕾 | A kind of fluorescent method based on copper nano-cluster detection aflatoxin B1 |
| CN108088838A (en) * | 2017-12-13 | 2018-05-29 | 西南大学 | Application and method of double [2,4,6- trichlorophenyls] oxalates in the mould toxin of rod method is measured |
| CN109387503A (en) * | 2018-12-14 | 2019-02-26 | 中国科学院合肥物质科学研究院 | A kind of chemiluminescence nano-sensor and its application for detecting remains of pesticide thiram |
| US20220291208A1 (en) | 2021-06-07 | 2022-09-15 | Jiangnan University | Method for Detecting Aflatoxin B1 Based on Fluorescent Copper Nanoparticles |
| WO2022257295A1 (en) * | 2021-06-07 | 2022-12-15 | 江南大学 | Method for detecting aflatoxin b1 on basis of fluorescent copper nanoparticles |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104964969A (en) * | 2015-05-28 | 2015-10-07 | 广东省生态环境与土壤研究所 | Label-free visualization detection method and label-free visualization detection kit of aflatoxin B1 |
| CN107219216A (en) * | 2017-06-26 | 2017-09-29 | 中国热带农业科学院农产品加工研究所 | A kind of method of aflatoxin B1 in chemiluminescence immunoassay detection baby rice powder |
| CN107764784A (en) * | 2017-09-01 | 2018-03-06 | 杨蕾 | A kind of fluorescent method based on copper nano-cluster detection aflatoxin B1 |
| CN108088838A (en) * | 2017-12-13 | 2018-05-29 | 西南大学 | Application and method of double [2,4,6- trichlorophenyls] oxalates in the mould toxin of rod method is measured |
| CN109387503A (en) * | 2018-12-14 | 2019-02-26 | 中国科学院合肥物质科学研究院 | A kind of chemiluminescence nano-sensor and its application for detecting remains of pesticide thiram |
| US20220291208A1 (en) | 2021-06-07 | 2022-09-15 | Jiangnan University | Method for Detecting Aflatoxin B1 Based on Fluorescent Copper Nanoparticles |
| WO2022257295A1 (en) * | 2021-06-07 | 2022-12-15 | 江南大学 | Method for detecting aflatoxin b1 on basis of fluorescent copper nanoparticles |
| US11828761B2 (en) | 2021-06-07 | 2023-11-28 | Jiangnan University | Method for detecting aflatoxin B1 based on fluorescent copper nanoparticles |
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Application publication date: 20150318 |