CN102095873A - Protein chip for rapidly detecting mycotoxin in agricultural products and food and preparation method thereof - Google Patents
Protein chip for rapidly detecting mycotoxin in agricultural products and food and preparation method thereof Download PDFInfo
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- CN102095873A CN102095873A CN2010105700825A CN201010570082A CN102095873A CN 102095873 A CN102095873 A CN 102095873A CN 2010105700825 A CN2010105700825 A CN 2010105700825A CN 201010570082 A CN201010570082 A CN 201010570082A CN 102095873 A CN102095873 A CN 102095873A
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Abstract
The invention discloses a protein chip for rapidly detecting mycotoxin in agricultural products and food and a preparation method thereof. The protein chip comprises a chip body, a mycotoxin artificial antigen, a mycotoxin-resisting monoclonal antibody and a fluorescence labeling secondary antibody, wherein the mycotoxin artificial antigen is dripped on a solid phase vector according to a microarray. The preparation method comprises the following steps of: fixing a plurality of target mycotoxin antigens to be detected which are prepared in advance onto the solid phase vector in a matrix form by a microarray technology; detecting the content of the mycotoxin in detected samples by competition law principles; and identifying, reading and determining results by using a fluorescence scanning microscope. The protein chip has the functions of continuity, integration, miniaturization, high throughput, rapid detection and the like of the detection and analysis process.
Description
Technical field
The present invention relates to the biochip technology field, relate generally to a kind of protein chip that is used for fast detecting agricultural product, food mycotoxin and preparation method thereof.
Background technology
China is agricultural product production and consumption big country, and the detection of eumycetin element is directly connected to agricultural products in China production, outlet and resident's food consumption safety in the agricultural product.Mycotoxin is mycetogenetic secondary metabolite, usually be present in the cereal that goes mouldy, in case after entering human body by food chain, may cause teratogenesis, mutagenesis and serious harm such as carcinogenic, because the parasitism of fungi and the generation of mycotoxin, have a strong impact on the output of crops, reduce agricultural product and feed quality, cause the tremendous economic loss.Therefore in global food-safety problem, receive much concern.At present, food-safety problem has become one of key content that country pays close attention to, and the Fast Detection Technique and the product that have started various objectionable impuritiess in non-polluted farm product and the food continuously become an important demand.
Biochip technology becomes one of present life science field technology with fastest developing speed rapidly.Biochip has two kinds of main types: genetic chip and protein chip.Protein-chip (proteinchip) is the another biochip that human health is had major application value after genetic chip.Protein-chip is made up of the antigen or the antibody microarray that are fixed on the different supporting dielectrics, the position of fixed member and composition are known in the array, react with the antibody of mark (fluorescent material, enzyme or chemiluminescent substance etc.) or the probe on antigen and the chip, detect by specific scanister then, the result is handled by Computer Analysis.The application of protein-chip comprises the detection of protein expression; Interactional evaluation between protein one protein, protein one DNA and protein one micromolecule etc.; The evaluation of antibody.This new technology makes the researchist can compare the relative abundance of hundreds of protein in the biological sample in once testing.Protein-chip is compared its superiority with traditional research method and is mainly reflected in high flux, high sensitivity and high accuracy.Existing protein chip is mainly used in medical domain, and the applied research in agricultural product check field still is in space state.
Summary of the invention
Problem at the prior art existence, the objective of the invention is to solve the used protein chip of prior art and have that specificity is weak, sensitivity is low, range of application is narrow, the shortcoming of complicated operation, a kind of simple in structure, easy to operate, low price, the serialization of check and analysis process, integrated, microminiaturized, the high-throughout protein chip that is used for fast detecting agricultural product, food mycotoxin are provided; Another object of the present invention provides a kind of above-mentioned methods of making protein chips.
For achieving the above object, a kind of protein chip that is used for fast detecting agricultural product, food mycotoxin of the present invention, comprise that chip, mycotoxin artificial antigen, antimycotic toxin monoclone antibody, fluorescence labeling two resist, wherein, the mycotoxin artificial antigen according to microarray point on solid phase carrier.
Further, described antimycotic toxin monoclone antibody diluted is concentration 0.3-0.9 μ g/ml.
Further, described dilution is the PBS solution of 0.01mol, and its PH is 7.4.
Further, the described mycotoxin artificial antigen of main points system is dissolved in the CBS damping fluid on the described solid phase carrier, then with the automatic point sample of chip system with these protein point samples on solid phase carrier, point sample density is 16 points/cm
2, the point sample amount is the 50nl/ point.
Further, described solid phase carrier is the slide of surperficial aldehyde radicalization.
A kind of above-mentioned methods of making protein chips is specially:
1) utilize the chip point sample instrument with the antigen liquid of damping fluid dilution and blank dilution by the microarray point on chip;
2) the dried chip of point sample places BSA solution to seal, and cleans with PBST afterwards;
3) the selected number micromatrix of wherein setting, the citrinin, deoxynivalenol and the fumonisins B1 standard items mixed liquor that in each micromatrix, add the PBS solution dilution respectively, in the other sub-micro array, three kinds of mycotoxin sample mix dilutions that each adds the good feed negative sample dilution of advanced processing and adds variable concentrations respectively; To resist citrinin antibody, anti-deoxynivalenol antibody, anti-fumonisins B1 antibody to dilute, and the antibody dilution mixed liquor joined in each inferior chip react with PBS;
4) carry out lucifuge in each the inferior matrix with fluorescently-labeled two anti-adding chips and hatch, clean with the PBST lucifuge afterwards;
5) chip fluorescent scanning instrument reads signal, adopt PerkinElmer fluorescent scanning microscope to carry out data processing to gathering image, be that ordinate, citrinin, deoxynivalenol, fumonisins standard items concentration are horizontal ordinate with the combination rate, adopt RIDAWIN software to draw competition respectively and suppress curve, utilizing three kinds of mycotoxin combination rate of gained regression equation calculation is 50% o'clock concentration;
6) suppress the content of mycotoxin in the curve calculation sample according to competition: the content that the fluorescent value substitution competition that records sample is suppressed contained mycotoxin in interpolation concentration in the sample that curve is or the sample.
Further, the damping fluid dilution is provided with 12 micromatrixs altogether for the CBS damping fluid dilution of 0.05mol PH9.6 in the described step 1) on the described chip, 16 points of each micromatrix point, and the point sample amount is the 50nl/ point, it is fixing that normal temperature spends the night; Wherein first three row is respectively citrinin antigen, deoxynivalenol antigen, fumonisins B1 antigen, and last row is blank dilution.
Further, described step 2) the BSA solution concentration is 3% in, and be 30min off-period in BSA solution, and the PBST solution concentration is 0.1%, gives a baby a bath on the third day after its birth time each 5min with it.
Further, anti-citrinin antibody described in the described step 3), anti-deoxynivalenol antibody, anti-fumonisins B1 antibody dilute with the PBS of 0.01mol PH7.4, and get 25 μ L antibody dilution mixed liquors and join in each inferior chip, 37 ℃ of reaction 30min, and with 0.01mol PH7.4 PBST washing lotion flushing 3 times, each 5min.
Further, the anti-PBS dilution of two in the described step 4) with 0.01mol, PH7.4.
The present invention adopts microarray technology, previously prepared several purpose mycotoxin antigens to be detected are fixed on the solid phase carrier with matrix form, adopt mycotoxin levels in the competition law principle test sample, by the identification of fluorescence surface sweeping microscope, reading and result of determination, have functions such as the serialization of check and analysis process, integrated, microminiaturized, high flux and fast detecting.
Description of drawings
Accompanying drawing 1 for bag in the protein chip of the present invention by the citrinin antigen concentration at 0.35 μ g/ml by the matched curve between RIDAWIN software analysis variable concentrations mycotoxin standard items and the fluorescent value;
Accompanying drawing 2 for for bag in the protein chip of the present invention by the deoxynivalenol antigen concentration at 0.8 μ g/ml by the matched curve between RIDAWIN software analysis variable concentrations mycotoxin standard items and the fluorescent value;
Accompanying drawing 3 for bag in the protein chip of the present invention by the fumonisins antigen concentration at 0.6 μ g/ml by the matched curve between RIDAWIN software analysis variable concentrations mycotoxin standard items and the fluorescent value;
Accompanying drawing 4 is the microarray point master drawing that three kinds of mycotoxins detect simultaneously.
Embodiment
Following embodiment further specifies the present invention, but described embodiment only is used to describe content of the present invention and does not limit the present invention.
As shown in Figures 1 to 4, a kind of protein chip that is used for fast detecting food, agricultural byproducts mycotoxin of the present invention comprises that chip, mycotoxin artificial antigen, antimycotic toxin monoclone antibody, fluorescence labeling two resist, wherein, the mycotoxin artificial antigen according to microarray point on solid phase carrier.
The present invention adopts microarray technology, previously prepared several purpose mycotoxin antigens to be detected are fixed on the solid phase carrier with matrix form, adopt mycotoxin levels in the competition law principle test sample, by the identification of fluorescence surface sweeping microscope, reading and result of determination.
The present invention adopts the competition ratio juris: adopt immune competition law to detect multiple mycotoxin, its principle of work is to utilize the mycotoxin that pollutes in the sample and be fixed on the immune response that is at war with of the common antagonist of mycotoxin antigen on the carrier, add that fluorescence labeling two is anti-can the quantitative test sample in the content of mycotoxin.Amount one timing at antimycotic toxin antibody, be fixed on on-chip mycotoxin and tested mycotoxin and depend on the two concentration ratio with the amount that combines of its antibody, tested mycotoxin is printed and is fixed on combining of on-chip mycotoxin antigen and antibody, two anti-mark fluorescents, obtain corresponding fluorescent value by the fluorescent scanning microscope, just the content of mycotoxin is just low more in the high more then testing sample of fluorescent value.Otherwise, then high more.
Below be application index of the present invention:
Stability: the holding time of chip and reagent was greater than 6 months
The recovery: 85%-96%
Specificity: do not have any cross reaction
Accuracy: CV＜10%
1. sample-pretreating method
(1) adds the citrinin sample treatment
1. take by weighing 4 parts in feed, every part of 2g, a as feminine gender, other three parts are added 10ng/ml, 100ng/ml, 300ng/ml citrinin standard items respectively.
2. add 10ml in every duplicate samples and contain 10% methyl alcohol PBS (0.01mol PH7.4), vibrated 30 minutes.
3. after vibrating, centrifugal 20 minutes of 1500g.
4. get supernatant after centrifugal, supernatant dilutes with PBS (0.02mol PH7.4) at 1: 4.Dilute it
After promptly can be used for detecting.
(2) add the deoxynivalenol sample treatment
1. take by weighing 4 parts in feed, every part of 2g, a as feminine gender, other three parts are added 10ng/ml, 100ng/ml, 300ng/ml deoxynivalenol standard items respectively.
2. add 10ml water in every duplicate samples, vibrated 3 minutes.
3. after vibrating, centrifugal 20 minutes of 1500g.
4. get supernatant after centrifugal, supernatant dilutes with PBS (0.02mol PH7.4) at 1: 4.Promptly can be used for after the dilution detecting.
(3) add fumonisins B1 sample treatment
1. take by weighing 4 parts in feed, every part of 2g, a as feminine gender, other three parts are added 10ng/ml, 100ng/ml, 300ng/ml fumonisins standard items respectively.
2. add 10ml in every duplicate samples and contain 70% methyl alcohol sodium chloride solution, vibrated 30 minutes.The concentration of sodium chloride is 0.1g/ml.
3. after vibrating, centrifugal 20 minutes of 1500g.
4. get supernatant after centrifugal, supernatant dilutes with PBS (0.02mol PH7.4) at 1: 4.Promptly can be used for after the dilution detecting.
2. the preparation of protein chip
1. utilize the chip point sample instrument with the antigen liquid of the CBS damping fluid of 0.05mol PH9.6 dilution and blank dilution by the microarray point on chip, totally 12 micromatrixs, 16 points of each micromatrix point.Be 4 rows, 4 points of every row, the point sample amount is the 50nl/ point, normal temperature spends the night fixing.Wherein first three row is respectively citrinin antigen, deoxynivalenol antigen, fumonisins B1 antigen, and last row is blank dilution.
2. the dried chip of point sample places 3% BSA solution to seal 30min, gives a baby a bath on the third day after its birth time each 5min afterwards with 0.1% PBST.
3. selected wherein 6 micromatrixs, the citrinin, deoxynivalenol and the fumonisins B1 standard items mixed liquor (0/0/0 that in each micromatrix, add the PBS solution dilution of 25 μ L 0.02molPH7.4 respectively, 10/10/1.9,30/30/7.6,90/90/31,270/270/62,810/810/125ng/ml), other six sub-micro arrays, three kinds of mycotoxin sample mix dilutions that each adds the good feed negative sample dilution of 25 μ L advanced processing and adds variable concentrations respectively.Anti-citrinin antibody, anti-deoxynivalenol antibody, anti-fumonisins B1 antibody are diluted with the PBS of 0.01mol PH7.4, getting 25 μ L antibody dilution mixed liquors joins in each inferior chip, 37 ℃ of reaction 30min, with 0.01molPH7.4PBST washing lotion flushing 3 times, each 5min
4. be 0.1mg/ml with fluorescently-labeled two anti-PBS dilutions, add in each inferior matrix of chip each inferior matrix 50 μ L with 0.01mol PH7.4.37 ℃, lucifuge is hatched 30min.It is inferior to give a baby a bath on the third day after its birth with 0.1% PBST lucifuge afterwards, each 5min.
5. chip fluorescent scanning instrument reads signal, adopt PerkinElmer fluorescent scanning microscope to carry out data processing to gathering image, with combination rate (%) is that ordinate, citrinin, deoxynivalenol, fumonisins standard items concentration are horizontal ordinate, adopt RIDAWIN software to draw competition respectively and suppress curve, utilizing three kinds of mycotoxin combination rate of gained regression equation calculation is 50% o'clock concentration (IC50).
6. the content that suppresses mycotoxin in the curve calculation sample according to competition: the fluorescent value substitution competition that records sample is suppressed the content that curve promptly gets contained mycotoxin in interpolation concentration in the sample or the sample.
The solution of above-mentioned use is as follows:
Confining liquid: 3%BSA solution
The PBS damping fluid: the 0.01mol/L phosphate buffer, pH 7.4
NaCl 8.0g
KCl 0.2g
KH
2PO
4 0.2g
Na
2HPO
4·12H
2O 2.9g
Distilled water (ddH
2O) 1000mL
The PBS damping fluid: the 0.02mol/L phosphate buffer, pH 7.4
NaCl 8.5g
Na
2HPO
4·12H
2O 5.16g
NaH
2PO
4·2H
2O 0.87g
Distilled water (ddH
2O) 1000mL
The CBS damping fluid: the 0.05mol/L carbonate buffer solution, pH 9.6
Na
2CO
3 10g
NaHCO
3 10g
ddH
2O 1000mL
The PBST washing fluid: 1 times of phosphate buffer, pH 7.4
NaCl 8.0g
KCl 0.2g
KH
2PO
4 0.2g
Na
2HPO
412H
2O 2.9g
Polysorbas20 1mL
NaN
3 0.2g
Distilled water (ddH
2O) 1000mL
Claims (10)
1. protein chip that is used for fast detecting agricultural product, food mycotoxin, it is characterized in that, this protein chip comprises that chip, mycotoxin artificial antigen, antimycotic toxin monoclone antibody, fluorescence labeling two is anti-, the mycotoxin artificial antigen according to microarray point on solid phase carrier.
2. the protein chip that is used for fast detecting agricultural product, food mycotoxin as claimed in claim 1 is characterized in that, described antimycotic toxin monoclone antibody diluted is concentration 0.3-0.9 μ g/ml.
3. the protein chip that is used for fast detecting agricultural product, food mycotoxin as claimed in claim 1 is characterized in that, described dilution is the PBS solution of 0.01mol, and its PH is 7.4.
4. the protein chip that is used for fast detecting agricultural product, food mycotoxin as claimed in claim 1, it is characterized in that, the described mycotoxin artificial antigen of main points system is dissolved in the CBS damping fluid on the described solid phase carrier, then with the automatic point sample of chip system with these protein point samples on solid phase carrier, point sample density is 16 points/cm
2, the point sample amount is the 50nl/ point.
5. the protein chip that is used for fast detecting agricultural product, food mycotoxin as claimed in claim 1 is characterized in that described solid phase carrier is the slide of surperficial aldehyde radicalization.
6. an above-mentioned methods of making protein chips is characterized in that, this method is specially:
1) utilize the chip point sample instrument with the antigen liquid of damping fluid dilution and blank dilution by the microarray point on chip;
2) the dried chip of point sample places BSA solution to seal, and cleans with PBST afterwards;
3) the selected number micromatrix of wherein setting, the citrinin, deoxynivalenol and the fumonisins B1 standard items mixed liquor that in each micromatrix, add the PBS solution dilution respectively, in the other sub-micro array, three kinds of mycotoxin sample mix dilutions that each adds the good feed negative sample dilution of advanced processing and adds variable concentrations respectively; To resist citrinin antibody, anti-deoxynivalenol antibody, anti-fumonisins B1 antibody to dilute, and the antibody dilution mixed liquor joined in each inferior chip react with PBS;
4) carry out lucifuge in each the inferior matrix with fluorescently-labeled two anti-adding chips and hatch, clean with the PBST lucifuge afterwards;
5) chip fluorescent scanning instrument reads signal, adopt PerkinElmer fluorescent scanning microscope to carry out data processing to gathering image, be that ordinate, citrinin, deoxynivalenol, fumonisins standard items concentration are horizontal ordinate with the combination rate, adopt RIDAWIN software to draw competition respectively and suppress curve, utilizing three kinds of mycotoxin combination rate of gained regression equation calculation is 50% o'clock concentration;
6) suppress the content of mycotoxin in the curve calculation sample according to competition: the content that the fluorescent value substitution competition that records sample is suppressed contained mycotoxin in interpolation concentration in the sample that curve is or the sample.
7. preparation method as claimed in claim 6 is characterized in that, the damping fluid dilution is the CBS damping fluid dilution of 0.05mol PH9.6 in the described step 1), be provided with 12 micromatrixs on the described chip altogether, 16 points of each micromatrix point, the point sample amount is the 50nl/ point, normal temperature spends the night fixing; Wherein first three row is respectively citrinin antigen, deoxynivalenol antigen, fumonisins B1 antigen, and last row is blank dilution.
8. preparation method as claimed in claim 6 is characterized in that, described step 2) in the BSA solution concentration be 3%, be 30min off-period in BSA solution, the PBST solution concentration is 0.1%, gives a baby a bath on the third day after its birth time with it, each 5min.
9. preparation method as claimed in claim 6, it is characterized in that, anti-citrinin antibody described in the described step 3), anti-deoxynivalenol antibody, anti-fumonisins B1 antibody dilute with the PBS of 0.01mol PH7.4, and get 25 μ L antibody dilution mixed liquors and join in each inferior chip, 37 ℃ of reaction 30min, and with 0.01mol PH7.4PBST washing lotion flushing 3 times, each 5min.
10. preparation method as claimed in claim 6 is characterized in that, the anti-PBS dilution with 0.01mol, PH7.4 of two in the described step 4).
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|---|---|---|---|
| CN2010105700825A CN102095873A (en) | 2010-12-02 | 2010-12-02 | Protein chip for rapidly detecting mycotoxin in agricultural products and food and preparation method thereof |
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| CN102788877A (en) * | 2012-09-04 | 2012-11-21 | 南京祥中生物科技有限公司 | Chip detection method of visual protein in residual gentamicin in animal derived food |
| CN105548576A (en) * | 2016-02-03 | 2016-05-04 | 中国疾病预防控制中心营养与健康所 | Protein chip capable of simultaneously detecting plurality of types of proteins and nutritive values of cow milk and kit of protein chip |
| CN110865071A (en) * | 2019-11-05 | 2020-03-06 | 天津大学 | Visual mycotoxin detection method with multi-chamber hydrogel particles as capture carrier |
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| CN102788877B (en) * | 2012-09-04 | 2015-04-22 | 南京祥中生物科技有限公司 | Chip detection method of visual protein in residual gentamicin in animal derived food |
| CN105548576A (en) * | 2016-02-03 | 2016-05-04 | 中国疾病预防控制中心营养与健康所 | Protein chip capable of simultaneously detecting plurality of types of proteins and nutritive values of cow milk and kit of protein chip |
| CN110865071A (en) * | 2019-11-05 | 2020-03-06 | 天津大学 | Visual mycotoxin detection method with multi-chamber hydrogel particles as capture carrier |
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Application publication date: 20110615 |