CN103323587A - Method for detecting imidacloprid by quantum-dot-marked sandwich fluorescence immunoassay - Google Patents
Method for detecting imidacloprid by quantum-dot-marked sandwich fluorescence immunoassay Download PDFInfo
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- CN103323587A CN103323587A CN2013102709427A CN201310270942A CN103323587A CN 103323587 A CN103323587 A CN 103323587A CN 2013102709427 A CN2013102709427 A CN 2013102709427A CN 201310270942 A CN201310270942 A CN 201310270942A CN 103323587 A CN103323587 A CN 103323587A
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Abstract
The invention discloses a method for detecting imidacloprid by quantum-dot-marked sandwich fluorescence immunoassay. According to the technical scheme, the method comprises the following steps of: 1, preparing a nuclear shell quantum dot and modifying the water solubility of a fat-soluble quantum dot by modification of different macromolecules; 2, preparing and passivating a quantum probe; and 3, constructing an imidacloprid antibody quantum dot immunoassay method. The method is applied to preparation, passivation and performance representation of the quantum dot and detection of precipitate imidacloprid residues and is a fluorescence immunoassay method for sensitively and quickly detecting the imidacloprid in vegetables based on the quantum dot and conjugates of IgG-HRP.
Description
Technical field
The invention belongs to the fluoroimmunoassay that detects Imidacloprid in the vegetables, particularly a kind of quantum dot-labeled sandwich fluorescence immunoassay detects the method for Imidacloprid.
The present invention mainly uses the quantum dot antagonist and carries out mark detection residues of pesticides, quantum dot is the nano material that in recent years new development is got up, have many good fluorescence properties, demonstrate wide application prospect in fields such as fluorescence immunoassay, genomics and medical science, the present invention reaches and uses in the pesticide imidacloprid residue detection at quantum dot preparation, purifying, performance characterization, be based on the conjugate that quantum dot and goat-anti mouse two resist, the fluoroimmunoassay of Imidacloprid in a kind of sensitivity of foundation, the fast detecting vegetables.
Background technology
At present, the food safety detection method mainly contains chemical analysis (CA), thin layer chromatography (TLC), vapor-phase chromatography (GC), high performance liquid chromatography (HPLC), GC-MS coupling method, enzyme linked immunosorbent assay (ELISA) etc., there are the restraining factors such as detection time is long, sensitivity is low, false positive is high, sample pre-treatments is complicated, sample substrate serious interference in these methods, are difficult to satisfy reality and detect the particularly needs of field quick detection.Present stage, the crucial detection technique of China's food security and developed country's gap are larger, bring hidden danger for China's food security to a great extent, have also limited the food trade of China simultaneously.Current, in the face of the International Food Safety Control new situations,, accurately and rapidly food safety detection new technology sensitive in the urgent need to development and exploitation.
Quantum dot claims again the semiconductor nanocrystals grain, and particle diameter mainly is comprised of II～VI family or III～group Ⅴ element between 1～100nm, and wherein, more with CdX (X=S, Se, Te) research, quantum dot is accepted can produce fluorescence behind the exciting light.Compare with traditional organic fluorescent dye, quantum dot has following characteristics: 1) exciting light spectrum width and continuous, emission spectrum is narrow, symmetrical, overlapping little.The exciting light spectrum width, can use a kind of exciting light to excite simultaneously multiple quantum dot continuously and obtain the fluorescence of different wave length; Emission peak is narrow, symmetrical, overlapping little, is conducive to improve selectivity and the sensitivity of mensuration; 2) size that can be by the control quantum dot and form tuning its emission wavelength is utilized these characteristics can select suitable quantum dot to reduce or is avoided background interference; 3) fluorescence intensity and stability are high, can realize the long period analyzing and testing.Just because of have the optical property of uniqueness like this, quantum dot can be used as a kind of good fluorescence probe and is applied in the biological study.But, directly preparing the quantum dot that particularly prepares in organic phase is difficult to biomacromolecule direct coupling occur, generally need with the functional group that contains sulfydryl, amino or carboxyl certain modification to be carried out on the quantum dot surface first, quanta point biological compatibility after the modification is better, can be by covalent bond and biomolecule coupling.At present, the most frequently used method is to introduce functional group with carboxyl at quantum dot, then take carbodiimide as coupling agent in conjunction with biomacromolecule such as enzyme, antibody etc., the quantum dot fluorescence probe of making can be used for analyzing and testing.In recent years, quantum dot as fluorescence labeling probe in bioanalysis especially widespread use in immunoassay, quick diagnosis.Goldman etc. are combined the CdSe/ZnS quantum dot of nucleocapsid structure with antibody, be used for fluoroimmunoassay.They at first are incorporated into recombinant protein on the quantum dot by electrostatic interaction, and then are connected with antibody, use this probe, utilize directly and indirect ELISA method to staphylococcal enterotoxin and 2,4,6-trinitro-toluene has carried out fluoroimmunoassay, and sensitivity is very high.Subsequently, quantum dot is modified with affinity element again by this seminar, biotin modification antibody, and Avidin and biotin specificity interact so that quantum dot easily is combined with antibody, and antibody still keeps original biologically active, and conjugate can be used for fluoroimmunoassay like this.Since a part Avidin can with the combination of four molecular biosciences elements, and adhesion is very strong, this ELISA method based on Quantum Dot Labeling is used for sample detection, the sensitivity of detection has had greatly and has improved.
Summary of the invention
Technical matters to be solved by this invention is: provide a kind of quantum dot-labeled sandwich fluorescence immunoassay to detect the method for Imidacloprid.
Technical scheme of the present invention is:
1, the preparation of core-shell quanta dots and different macromolecule modified water-soluble modified to fat-soluble quantum dot;
2, quantum dot-labeled Imidacloprid antibody preparation;
3, the foundation of Imidacloprid antibody quantum dot immune analytical approach.
A kind of quantum dot-labeled sandwich fluorescence immunoassay detects the method for Imidacloprid, it is characterized in that:
Semiconductor material commonly used is at present selected in the preparation of quantum dot: zinc, selenium, cadmium, silicon are raw material, adopt the colloidal chemistry method, prepare the quantum dot that has the stable nucleus shell structure, can present different fluorescence colors, and each quantum dot has all had a spectrum address; This method adopts respectively organic synthesis and two kinds of methods of Syntheses in water, organic synthesis CdX(X=Se, S, Te) the concrete technology path of quantum dot; Syntheses in water CdX(X=Se, S, Te), the fluorescence efficiency of raising quantum dot adopts the top-down quantum point making method to prepare TeS and CdS;
Quantum dot functional modification and antibody labeling are with above-mentioned quantum dot with stable nucleus shell structure, respectively with material with activity functional groups: mercaptopropionic acid, poly-hexanediol or amphiphilic macromolecular react, adopt ultrasonic emulsification and Chemical self-assembly method, prepare respectively band difference in functionality group: the water-soluble quantum dot of amino and carboxyl, and carry out chemical coupling with antibody etc., behind the gel chromatography separation purifying, prepare quantum dot-labeled antibody; Adopt enzyme linked immunological ELISA method that the activity of quantum dot-labeled antibody is detected, determine the top condition of quantum dot functional modification and antagonist mark; Make antibody and quantum dot covalently bound take NHS as auxiliary reagent, seal with BSA, OVA etc., stabilized processing makes quantum dot-labeled antibody;
Set up immuno-chromatographic test paper strip fast detecting analyzing and testing Imidacloprid.
Concrete grammar:
A kind of method of quantum dot-labeled immune detection Imidacloprid is characterized in that:
Semiconductor material commonly used is at present selected in the preparation of quantum dot: zinc, selenium, cadmium, silicon are raw material, adopt the colloidal chemistry method, prepare the quantum dot that has the stable nucleus shell structure, can present different fluorescence colors, and each quantum dot has all had a spectrum address; This method adopts respectively organic synthesis and two kinds of methods of Syntheses in water, organic synthesis CdX(X=Se, S, Te) the concrete technology path of quantum dot; Syntheses in water CdX(X=Se, S, Te), in order to improve the fluorescence efficiency of quantum dot, adopt the top-down quantum point making method to prepare TeS and CdS;
Taking by weighing 1gCdO(TeO) powder is in three neck round-bottomed flasks, and then adding 20mL glycerine slowly be heated to 160 ℃, and logical nitrogen protection in the process is retained to and forms red transparency liquid; Take by weighing the 0.1gSe powder and put into another three necks round-bottomed flask, add the 50mL whiteruss, then slowly be heated to 200 ℃, form transparency liquid; Then extract the 6mLCd precursor solution and be added in the Se precursor solution, then add the 2mL oleyl amine, continuous stirring, solution becomes is to orange.30mL quantum dot solution behind the 15min is added in 50mL4 ℃ the dimethylbenzene, namely obtains CdSe (TeSe).In the three neck round-bottomed flasks of 500mL, add the deionized water of 150mL, behind adding Cys and the Cdcl2 solution, the pH of mixed value transfers to 11.0 with NaOH solution, and under suitable stirring rate, add dimercaptosuccinic acid 30mL, finally keep L-cysteine:CdCl
2The mol ratio of=2:1 makes the CdTe/CdS core-shell type quantum point that dimercaptosuccinic acid coats.
1mL0.02mol/L phosphate buffer (PBS, pH7.6) in, add quantum dot and 120 μ L0.2g/LNHS that 300 μ L prepare, 25 ℃ of activation 10min, add again 100 μ L0.1g/L Imidacloprid polyclonal antibodies, then 25 ℃ of lower vortex mixing 25min behind the gel chromatography separation purifying, prepare quantum dot-labeled antibody; Adopt enzyme linked immunological ELISA method that the activity of quantum dot-labeled antibody is detected.
With a film instrument, Imidacloprid antigen (0.3g/L) is sprayed on the nitrocellulose membrane, goat-anti mouse two anti-(1.0g/L) is sprayed on glass fibre membrane forms detection band (T) and quality control band (C), T band and C band interval 5mm place fixedly 20min of 37 ℃ of constant temperature ovens.With nitrocellulose membrane, glass fibre membrane and water sucting plate successively sticking being posted on the water board, cut into test strips again, dry rear sealing is preserved.
Characteristics of the present invention and effect are:
The main quantum dot antagonist of using carries out mark detection residues of pesticides, quantum dot is the nano material that in recent years new development is got up, have many good fluorescence properties, in wide application in field such as fluorescence immunoassay, genomics and medical science, the present invention reaches and uses in the pesticide imidacloprid residue detection in quantum dot preparation, purifying, Imidacloprid antibody quantum dot immune analytical approach, be based on the conjugate that quantum dot and goat-anti mouse two resist, the fluoroimmunoassay of Imidacloprid in a kind of sensitivity of foundation, the fast detecting vegetables.
Embodiment
This quantum dot-labeled sandwich fluorescence immunoassay detects the method for Imidacloprid, and is as follows:
1, the preparation of core-shell quanta dots and different macromolecule modified water-soluble modified to fat-soluble quantum dot;
2, quantum dot-labeled Imidacloprid antibody preparation;
3, the foundation of Imidacloprid antibody quantum dot immune analytical approach.
1, a kind of quantum dot-labeled sandwich fluorescence immunoassay detects the method for Imidacloprid: semiconductor material commonly used is at present selected in the preparation of quantum dot: zinc, selenium, cadmium, silicon are raw material, adopt the colloidal chemistry method, prepare the quantum dot that has the stable nucleus shell structure, can present different fluorescence colors, each quantum dot has all had a spectrum address; This method adopts respectively organic synthesis and two kinds of methods of Syntheses in water, organic synthesis CdX(X=Se, S, Te) the concrete technology path of quantum dot; Syntheses in water CdX(X=Se, S, Te), the fluorescence efficiency of raising quantum dot adopts the top-down quantum point making method to prepare TeS and CdS;
Quantum dot functional modification and antibody labeling are with above-mentioned quantum dot with stable nucleus shell structure, respectively with material with activity functional groups: mercaptopropionic acid, poly-hexanediol or amphiphilic macromolecular react, adopt ultrasonic emulsification and Chemical self-assembly method, prepare respectively band difference in functionality group: the water-soluble quantum dot of amino and carboxyl, and carry out chemical coupling with antibody etc., behind the gel chromatography separation purifying, prepare quantum dot-labeled antibody; Adopt enzyme linked immunological ELISA method that the activity of quantum dot-labeled antibody is detected, determine the top condition of quantum dot functional modification and antagonist mark; Make antibody and quantum dot covalently bound take NHS as auxiliary reagent, seal with BSA, OVA etc., stabilized processing makes quantum dot-labeled antibody;
Set up immuno-chromatographic test paper strip fast detecting analyzing and testing Imidacloprid.
Concrete grammar:
1, semiconductor material commonly used is at present selected in the preparation of quantum dot: zinc, selenium, cadmium, silicon are raw material, adopt the colloidal chemistry method, prepare the quantum dot that has the stable nucleus shell structure, can present different fluorescence colors, each quantum dot has all had a spectrum address; This method adopts respectively organic synthesis and two kinds of methods of Syntheses in water, organic synthesis CdX(X=Se, S, Te) the concrete technology path of quantum dot; Syntheses in water CdX(X=Se, S, Te), in order to improve the fluorescence efficiency of quantum dot, adopt the top-down quantum point making method to prepare TeS and CdS;
2, taking by weighing 1gCdO(TeO) powder is in three neck round-bottomed flasks, and then adding 20mL glycerine slowly be heated to 160 ℃, and logical nitrogen protection in the process is retained to and forms red transparency liquid; Take by weighing the 0.1gSe powder and put into another three necks round-bottomed flask, add the 50mL whiteruss, then slowly be heated to 200 ℃, form transparency liquid; Then extract the 6mLCd precursor solution and be added in the Se precursor solution, then add the 2mL oleyl amine, continuous stirring, solution becomes is to orange.30mL quantum dot solution behind the 15min is added in 50mL4 ℃ the dimethylbenzene, namely obtains CdSe (TeSe).In the three neck round-bottomed flasks of 500mL, add the deionized water of 150mL, behind adding Cys and the Cdcl2 solution, the pH of mixed value transfers to 11.0 with NaOH solution, and under suitable stirring rate, add dimercaptosuccinic acid, finally keep L-cysteine:CdCl
2The mol ratio of=2:1 makes the CdTe/CdS core-shell type quantum point that dimercaptosuccinic acid coats.
3, at 1mL0.02mol/L phosphate buffer (PBS, pH7.6) in, add quantum dot and 120 μ L0.2g/LNHS that 200 μ L prepare, 25 ℃ of activation 10min, add again 100 μ L0.1g/L Imidacloprid polyclonal antibodies, then 25 ℃ of lower vortex mixing 25min behind the gel chromatography separation purifying, prepare quantum dot-labeled antibody; Adopt enzyme linked immunological ELISA method that the activity of quantum dot-labeled antibody is detected.4, with a film instrument, Imidacloprid antigen (0.3g/L) is sprayed on the nitrocellulose membrane, goat-anti mouse two anti-(1.0g/L) is sprayed on glass fibre membrane forms detection band (T) and quality control band (C), T band and C band interval 5mm place fixedly 20min of 37 ℃ of constant temperature ovens.With nitrocellulose membrane, glass fibre membrane and water sucting plate successively sticking being posted on the water board, cut into test strips again, dry rear sealing is preserved.
This method:
(1) preparation of core-shell quanta dots is the immunoassay system of carrier based on microballoon, utilize quantum dot excitation spectrum bandwidth, emission peak is narrow and symmetrical, color according to its size continuously the characteristic such as adjustable carry out, propose first to be used for the new method that Imidacloprid is analyzed.This method is the quantitative detection of quantum dot-labeled Imidacloprid antibody, so susceptibility is expected to increase substantially, and existing sensitive method (such as efficient liquid-phase chromatography method) of generally acknowledging can improve more than 1 order of magnitude at least.
(2) different macromolecule modified water-soluble modified to fat-soluble quantum dot, the preparation of quantum dot probe and purifying thereof utilize the total internal reflection high-resolution surface spectroscopic imaging means can be in the quantum dot probe characteristics of dynamic characterization different colours under the different wave length.
(3) set up Imidacloprid antibody quantum dot immune analysis test paper bar.
Claims (1)
1. a quantum dot-labeled sandwich fluorescence immunoassay detects the method for Imidacloprid, it is characterized in that:
Semiconductor material commonly used is at present selected in the preparation of quantum dot: zinc, selenium, cadmium, silicon are raw material, adopt the colloidal chemistry method, prepare the quantum dot that has the stable nucleus shell structure, can present different fluorescence colors, and each quantum dot has all had a spectrum address; This method adopts respectively organic synthesis and two kinds of methods of Syntheses in water, organic synthesis CdX(X=Se, S, Te) the concrete technology path of quantum dot; Syntheses in water CdX(X=Se, S, Te), the fluorescence efficiency of raising quantum dot adopts the top-down quantum point making method to prepare TeS and CdS;
Quantum dot functional modification and antibody labeling are with above-mentioned quantum dot with stable nucleus shell structure, respectively with material with activity functional groups: mercaptopropionic acid, poly-hexanediol or amphiphilic macromolecular react, adopt ultrasonic emulsification and Chemical self-assembly method, prepare respectively band difference in functionality group: the water-soluble quantum dot of amino and carboxyl, and carry out chemical coupling with antibody etc., behind the gel chromatography separation purifying, prepare quantum dot-labeled antibody; Adopt enzyme linked immunological ELISA method that the activity of quantum dot-labeled antibody is detected, determine the top condition of quantum dot functional modification and antagonist mark; Make antibody and quantum dot covalently bound take NHS as auxiliary reagent, seal with BSA, OVA etc., stabilized processing makes quantum dot-labeled antibody;
Set up immuno-chromatographic test paper strip fast detecting analyzing and testing Imidacloprid.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103760363A (en) * | 2014-01-08 | 2014-04-30 | 中国科学院长春光学精密机械与物理研究所 | Detection method for core-shell quantum dot-based flexible coupled marker immunochromatography test strip |
| CN104849234A (en) * | 2015-04-30 | 2015-08-19 | 江苏扬农化工集团有限公司 | Assay method for analyzing contents of principal components of imidacloprid based on near-infrared spectrum |
| CN104914084A (en) * | 2015-06-25 | 2015-09-16 | 浙江大学 | Method for detecting proteins toxin by quantum dot cation exchange signal amplification technology |
| CN105823876A (en) * | 2016-03-18 | 2016-08-03 | 南昌大学 | Salmonella detection method |
| CN106883840A (en) * | 2016-12-30 | 2017-06-23 | 锦州医科大学 | A kind of fluorescence/CT/MRI multi-modality imaging quantum dot probes and preparation method thereof |
| CN106914035A (en) * | 2015-12-28 | 2017-07-04 | 江南大学 | A kind of chromatographic separating process of soluble carbon point system |
| CN109799212A (en) * | 2018-11-22 | 2019-05-24 | 中南民族大学 | Method based on CdTe-ZnCdSe double quantum point paper chip substrate detection organophosphorus pesticide |
| CN111766388A (en) * | 2020-07-28 | 2020-10-13 | 江苏省农业科学院 | Fluorescent immunochromatographic test strip for detecting imidacloprid and preparation method and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103760363A (en) * | 2014-01-08 | 2014-04-30 | 中国科学院长春光学精密机械与物理研究所 | Detection method for core-shell quantum dot-based flexible coupled marker immunochromatography test strip |
| CN104849234A (en) * | 2015-04-30 | 2015-08-19 | 江苏扬农化工集团有限公司 | Assay method for analyzing contents of principal components of imidacloprid based on near-infrared spectrum |
| CN104914084A (en) * | 2015-06-25 | 2015-09-16 | 浙江大学 | Method for detecting proteins toxin by quantum dot cation exchange signal amplification technology |
| CN106914035A (en) * | 2015-12-28 | 2017-07-04 | 江南大学 | A kind of chromatographic separating process of soluble carbon point system |
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| CN106883840B (en) * | 2016-12-30 | 2018-03-30 | 锦州医科大学 | A kind of fluorescence/CT/MRI multi-modality imagings quantum dot probe and its application |
| CN109799212A (en) * | 2018-11-22 | 2019-05-24 | 中南民族大学 | Method based on CdTe-ZnCdSe double quantum point paper chip substrate detection organophosphorus pesticide |
| CN109799212B (en) * | 2018-11-22 | 2020-04-03 | 中南民族大学 | Method for detecting organophosphorus pesticide based on CdTe-ZnCdSe double-quantum dot paper chip substrate |
| CN111766388A (en) * | 2020-07-28 | 2020-10-13 | 江苏省农业科学院 | Fluorescent immunochromatographic test strip for detecting imidacloprid and preparation method and application thereof |
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Application publication date: 20130925 |