CN102495205B - Quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay method for small-molecule organic matter - Google Patents
Quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay method for small-molecule organic matter Download PDFInfo
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- CN102495205B CN102495205B CN201110399385.XA CN201110399385A CN102495205B CN 102495205 B CN102495205 B CN 102495205B CN 201110399385 A CN201110399385 A CN 201110399385A CN 102495205 B CN102495205 B CN 102495205B
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Abstract
The invention discloses a quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay method for a small-molecule organic matter, and relates to a quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay technology for the small-molecule organic matter. Fluorescent light emits high-efficiency water-soluble quantum dots with different wavelengths to label antibodies resisting different target analytes. Different quantum dot labeled antibodies are dispersed into a phosphate buffering solution, and a sample solution containing the corresponding target analytes is added into the phosphate buffering solution and uniformly mixed with the phosphate buffering solution, so that non-competitive homogeneous immunoassay reaction is performed; respective labeled samples, samples and a blank contrast system which are subjected to reaction and balanced are directly subjected to fluorescent scanning or multi-wavelength detection under the excitation of ultraviolet light with the same wavelength; and the high-sensitivity quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay technology for quickly detecting various target small-molecule organic matters is constructed according to a rule that a ratio of the characteristic fluorescent light intensity of the blank contrast system to the characteristic fluorescent light intensity of a sample system is in direct proportion to the concentration of the corresponding target analytes in a certain range.
Description
Technical field
The present invention relates to the non-competing homogeneous immunoassay technology of quantum dot-labeled antibody polycomponent of small organic molecule (agricultural chemicals, medicine, environment incretion interferent).
Background technology
Environment and agricultural and animal products Pesticides, medicine, the residual of environment incretion interferent exceed standard, and environment and environmental organism are formed to certain threat, affect food security and human health.Environmental enhancement and food safety monitoring, need efficiently analytical technology fast.
The how residual detection method of agricultural chemicals, medicine, environment incretion interferent of having reported at present mainly contains chromatography and application of gas chromatorgraphy/mass method.Adopt these methods to need expensive instrument and equipment, specialized laboratory and well-trained specialized personnel, sample pre-treatments requirement is high, process is complicated, speed is slow, testing cost is high, and specificity is not strong, is difficult to adapt to the requirement of a large amount of samples and field quick detection.Existing small organic molecule multi-component immunity analytical, conventionally adopt at the different parts of polystyrene micropore plate fixedly different antibodies or antigen, by spatial discrimination, carry out different immune responses, react and carry out in solid phase and liquid interface, by mode of washing, realize Separation of Solid and Liquid, complicated operation; Or adopt different labels, as mark different antibodies or antigens such as enzyme, fluoresceins, reaction conditions and detection means are difficult to compatibility.Therefore, small organic molecule multi-component immunity analytical technology truly waits to set up.
Summary of the invention
The efficient water soluble quantum dot that the object of the invention is to using different fluorescent emission wavelength is analyzed the antibody of small organic molecule (agricultural chemicals, medicine, environment incretion interferent) as the anti-different target of namo fluorescence probe mark, set up a kind of high specificity, highly sensitive, simple and effective, for the non-competing polycomponent homogeneous immunoassay of the quantum dot-labeled antibody technology of plurality of target analyte fast detecting.
Technical scheme of the present invention is: using excitation wave length and width, fluorescent emission wavelength is narrow and do not interfere with each other, fluorescence quantum efficiency is high, the different water-soluble quantum dots of good stability, surperficial tool pendant carboxylic group are as namo fluorescence probe, adopts the carbodlimide method anti-different target analyte of mark (agricultural chemicals, medicine, environment incretion interferent) antibody respectively; Different quantum dot-labeled antibody is diluted to the mixed solution of debita spissitudo with phosphate buffer, add the mixed sample containing corresponding target analytes, in homogeneous system, there is panimmunity reaction simultaneously, under similarity condition, with testing sample and target analytes blank, replace standard specimen, sample and blank reaction system are set, quantum dot-labeled antibody with corresponding target analytes in conjunction with rear generation fluorescent quenching; The reaction system that reaches balance is directly carried out to fluorescent scanning or multi-wavelength detection under same excitation wavelength, calculate respectively the Characteristic fluorescence intensity F of each quantum dot-labeled antibody in blank system
0characteristic fluorescence intensity F with each corresponding quantum dot labelled antibody in Standard Sample System
sratio F
0/ F
s; On the basis of condition optimizing, according to F
0/ F
sthe concentration of (the cancellation degree of characteristic fluorescence) and corresponding target analytes is proportional rule and the F of testing sample system within the specific limits
0/ F
s, in calculating testing sample, the content of each target analytes, sets up the non-competing homogeneous immunoassay method of quantum dot-labeled antibody polycomponent that high sensitivity fast detecting plurality of target is analyzed small organic molecule (agricultural chemicals, medicine, environment incretion interferent).
The present invention fully utilizes that immunoassay high specificity, quantum dot fluorescence excellent performance, homogeneous phase immune response speed are fast, panimmunity reaction is carried out in homogeneous system simultaneously, equilibration time is short, directly carry out the advantages such as fluoroscopic examination after molecular balance.Compare with routine immunization analytical technology, the non-competing homogeneous immunoassay technology of quantum dot-labeled antibody polycomponent that the present invention sets up, can to plurality of target, analyze small organic molecule (agricultural chemicals simultaneously, medicine, environment incretion interferent) thing carries out qualitative and quantitative detection, Separation of Solid and Liquid and chromogenic reaction step have been saved, high specificity not only, highly sensitive, favorable reproducibility, and simple and effective more, be about 1/4 of conventional single component ELISA detection time, to the development of existing small organic molecule immuno analytical method and innovation, there is not yet so far similar report, there is very high development and application values.
Separately, efficient water soluble quantum dot of the present invention is used Same Wavelength ultraviolet excitation within the scope of 200～350nm, transmitting half-peak breadth is less than 30nm, non-interfering characteristic fluorescence, the interval >50nm of the maximum fluorescence wavelength of different quantum dots, and fluorescence quantum efficiency is higher than 60%.
Accompanying drawing explanation
Fig. 1 is the non-competing homogeneous immunoassay fluorescence spectrum of quantum dot-labeled anti-pesticide antibody polycomponent figure.
Fig. 2 is the non-competing homogeneous immunoassay fluorescence spectrum of quantum dot-labeled anti-drug antibodies polycomponent figure.
Fig. 3 is the relation curve of variable concentrations chloromycetin and quantum dot-labeled chloramphenicol resistance antibody fluorescence intensity change.
Embodiment
One, the non-competing homogeneous immunoassay technology of quantum dot-labeled anti-pesticide antibody polycomponent embodiment 1
By fluorescent emission wavelength, be 545nm, 625nm, the efficient water soluble quantum dot of 705nm adopts carbodlimide method (Greg T. Hermanson, Bioconjugate Techniques, 2ed 2008, p495-496) with anti-uniconazole P antibody, anti-cypermethrin antibody, anti-carbofuran antibody covalent coupling, prepare respectively the quantum dot-labeled anti-uniconazole P antibody that fluorescent emission wavelength is about 545nm, fluorescent emission wavelength is about the quantum dot-labeled anti-cypermethrin antibody of 625nm and the quantum dot-labeled anti-carbofuran antibody that fluorescent emission wavelength is about 705nm, after ultrafiltration purification, use pH8.3, 0.05mol/L borate buffer solution dissolving containing 0.5g/L sodium azide and 1mol/L betaine is settled to 1 μ moL/L, 4 ℃ save backup, before use respectively by described quantum dot-labeled anti-uniconazole P antibody, quantum dot-labeled anti-cypermethrin antibody and quantum dot-labeled anti-carbofuran antibody 0.02mol/L, 100 times of the PBS dilutions of pH7.0, after 200 times and 50 times, equal-volume mixes.
First with methyl alcohol configuration, contain the mother liquor of uniconazole P, cypermethrin and each 1mg/mL of carbofuran standard specimen, then be diluted to respectively containing uniconazole P, cypermethrin and carbofuran standard specimen standard specimen 10～10 with 0.02mol/L, pH7.0 PBS
-4the mixed sample series of μ g/mL, with testing sample and the blank standard specimen that replaces of target analytes, arranges sample and blank under similarity condition.
In 100 μ L standard specimens, testing sample and blank solution, add respectively isopyknic described quantum dot-labeled antibody mixed liquor, at room temperature the about 10～15min of hybrid reaction.After molecular balance, under 235nm excitation wavelength, scan (see figure 1) or detect respectively near maximum Characteristic fluorescence intensity 545nm, 625nm and 705nm.
Optimize the correlated conditions such as reaction medium, reactant concentration ratio, temperature of reaction and time, excitation wavelength and fluorescent emission wavelength, select that quantum dot labelled antibody is fast with corresponding target analytes immune response speed, susceptibility by force, few, the blank system fluorescence intensity (F of good stability, quantum dot-labeled antibody consumption
0) with containing the fluorescence intensity (F of corresponding target analysis objects system
s) ratio is high, background interference is few conditional combination.
On this basis, according to F
0/ F
sthe rule that (degree of quantum dot-labeled antibody fluorescent quenching) is directly proportional within the specific limits with corresponding target analyte concentration and the F of each sample
0/ F
sthe content of uniconazole P, cypermethrin and carbofuran in calculation sample, uniconazole P in testing sample, cypermethrin and carbofuran are carried out to qualitative, quantitative fast detecting, thereby set up the non-competing homogeneous immunoassay technology of quantum dot-labeled antibody polycomponent of uniconazole P, cypermethrin and carbofuran.
Two, the non-competing homogeneous immunoassay technology of quantum dot-labeled anti-drug antibodies polycomponent embodiment 2
By fluorescent emission wavelength, be 545nm, 625nm, the efficient water soluble quantum dot of 705nm adopts carbodlimide method (Greg T. Hermanson, Bioconjugate Techniques, 2ed 2008, p495-496) with chloramphenicol resistance antibody, anti-diethylstilbestrol antibody, anti-clenbuterol antibody covalent coupling, prepare respectively the quantum dot-labeled chloramphenicol resistance antibody that fluorescent emission wavelength is about 545nm, fluorescent emission wavelength is about the quantum dot-labeled anti-diethylstilbestrol antibody of 625nm and the quantum dot-labeled anti-clenbuterol antibody that fluorescent emission wavelength is about 705nm, quantum dot-labeled antibody pH8.3 after ultrafiltration purification, 0.05mol/L borate buffer solution dissolving containing 0.5g/L sodium azide and 1mol/L betaine is settled to 1 μ moL/L, 4 ℃ save backup.After respectively the PBS of described quantum dot-labeled chloramphenicol resistance antibody, quantum dot-labeled anti-diethylstilbestrol antibody and quantum dot-labeled anti-clenbuterol 0.02mol/L, pH7.0 for antibody being diluted to 100 times, 200 times and 50 times before use, equal-volume mixes.
First with methyl alcohol configuration, contain the mother liquor of chloromycetin, diethylstilbestrol and each 1mg/mL of Clenbuterol standard specimen, then be diluted to chloride mycin, diethylstilbestrol and Clenbuterol standard specimen 1～10 respectively with 0.02mol/L, pH7.0 PBS
-5the mixed sample series of μ g/mL, with testing sample and the blank standard specimen that replaces of target analytes, arranges sample and blank under similarity condition.
In 100 μ L standard specimens, testing sample and blank solution, add respectively isopyknic described quantum dot-labeled antibody mixed liquor, at room temperature the about 10～15min of hybrid reaction.Scanning (as shown in Figure 2) or detect respectively near maximum Characteristic fluorescence intensity 545nm, 625nm and 705nm under 235nm excitation wavelength after molecular balance.
Optimize the correlated conditions such as reaction medium, reactant concentration ratio, temperature of reaction and time, excitation wavelength and fluorescent emission wavelength, select that quantum dot labelled antibody is fast with corresponding target analytes immune response speed, susceptibility by force, few, the blank system fluorescence intensity (F of good stability, quantum dot-labeled antibody consumption
0) with containing the fluorescence intensity (F of corresponding target analytes reaction system
s) ratio is high, background interference is few conditional combination.
On this basis, according to F
0/ F
sthe rule that (degree that reflects quantum dot-labeled antibody fluorescent quenching) is directly proportional within the specific limits with corresponding target analyte concentration and the F of each sample
0/ F
sthe content of chloromycetin, diethylstilbestrol and Clenbuterol in calculation sample, chloromycetin in testing sample, diethylstilbestrol and Clenbuterol are carried out to qualitative, quantitative fast detecting, thereby set up the non-competing homogeneous immunoassay technology of quantum dot-labeled antibody polycomponent of chloromycetin, diethylstilbestrol and Clenbuterol.
The relation curve of variable concentrations chloromycetin and quantum dot-labeled chloramphenicol resistance antibody fluorescence intensity change, as shown in Figure 3, F
0/ F
sbe directly proportional within the specific limits to the concentration of chloromycetin.
Claims (1)
1. the non-competing homogeneous immunoassay method of quantum dot-labeled antibody polycomponent of a small organic molecule, it is characterized in that: using that excitation wave length and width, fluorescence quantum efficiency are high, fluorescent emission wavelength different and do not interfere with each other, different IPs shell structure water-soluble quantum dot that good stability, surface have pendant carboxylic group is as namo fluorescence probe, adopts the anti-different target of carbodlimide method mark to analyze the antibody of small organic molecule; By phosphate buffer dilution configuration mixed solution for the quantum dot-labeled antibody of difference, add the mixed sample containing corresponding target analytes, in homogeneous system, there is panimmunity reaction simultaneously, under similarity condition, with testing sample and the blank standard specimen that replaces of target analytes, sample and blank reaction system are set; The reaction system that reaches balance is directly carried out to fluorescent scanning or multi-wavelength detection under same excitation wavelength, calculate respectively the Characteristic fluorescence intensity F of each quantum dot-labeled antibody in blank system
0characteristic fluorescence intensity F with each corresponding quantum dot labelled antibody in Standard Sample System
sratio; On the basis of condition optimizing, according to F
0/ F
swith the concentration of corresponding target analytes proportional rule and the F of testing sample system within the specific limits
0/ F
s, in calculating testing sample, the content of each target analytes, sets up the non-competing polycomponent homogeneous immunoassay method of quantum dot-labeled antibody that high sensitivity fast detecting plurality of target is analyzed small organic molecule; Described efficient water soluble quantum dot is used Same Wavelength excitation within the scope of 200～350nm, transmitting half-peak breadth is less than 30nm, non-interfering characteristic fluorescence, the interval >50nm of the maximum fluorescence wavelength of different quantum dots, fluorescence quantum efficiency is higher than 60%.
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| CN1945331A (en) * | 2006-10-20 | 2007-04-11 | 邹明强 | Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds |
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| CdTe量子点的合成及其基荧光淬灭作用的分析应用;伊魁宇;《工程科技Ⅰ辑》;20100608;B014-46 * |
| 伊魁宇.CdTe量子点的合成及其基荧光淬灭作用的分析应用.《工程科技Ⅰ辑》.2010,B014-46. |
| 糖皮质激素多残留免疫研究与量子点在免疫分析中的应用;袁媛;《医药卫生科技辑》;20091222;E055-46 * |
| 袁媛.糖皮质激素多残留免疫研究与量子点在免疫分析中的应用.《医药卫生科技辑》.2009,E055-46. |
| 邵君.量子点在免疫分析中的应用.《医药卫生科技辑》.2007,E060-3. |
| 量子点在免疫分析中的应用;邵君;《医药卫生科技辑》;20070605;E060-3 * |
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