CN101639444B - Method for nano particle reinforced fluorescence polarization analysis - Google Patents
Method for nano particle reinforced fluorescence polarization analysis Download PDFInfo
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- CN101639444B CN101639444B CN2008100411662A CN200810041166A CN101639444B CN 101639444 B CN101639444 B CN 101639444B CN 2008100411662 A CN2008100411662 A CN 2008100411662A CN 200810041166 A CN200810041166 A CN 200810041166A CN 101639444 B CN101639444 B CN 101639444B
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Abstract
The invention provides a fluorescence polarization analysis method with high sensitivity and specificity and wider range. The method comprises the following steps that: a sample to be tested enters a molecular recognition element by diffusion action, undergoes molecular recognition and specially combines with the molecular recognition element, so that the signal generated by the biological or chemical reaction of the sample to be tested can amplify the fluorescence polarization signal by using the nano particles, and the aim of high-sensitivity detection is achieved. Compared with the conventional fluorescence polarization method, the method of the invention has the characteristics of high sensitivity, accurate and reliable result, low detection limit, wideapplication range, real-time on-line detection, a few interference factors and wide linear range, so that the method is the simple and practicable analysis technology.
Description
Technical field
The present invention relates to the check and analysis field of medical treatment, environment, food, relate in particular to a kind of nano particle reinforced fluorescence polarization assay method.
Background technology
The fluorescence polarization principle is widely used in intermolecular Study of Interaction in 1920 foundation with this fluorescence polarization technology that develops out, comprises that DNA-albumen, protein-protein, Ag-Ab combine.Fluorescence polarization detection is a kind of fluorescence labeling detection technique; It tagged at the predetermined substance molecule; Make originally that faint reaction signal changes into stronger fluorescence signal, add respectively that at the correct position of light path the polarizer and analyzer just can measure the polarized fluorescence intensity of determinand simultaneously.In the method that analyzing molecules combines; Fluorescence polarization method is the method for a uniqueness, because this method does not need the tracer of separated free and combination, all mensuration are all carried out in solution; Can reach real balance, have characteristics such as quick, simple to operate and accurate.Detection sensitivity is one of the most basic detection performance index, and high detection sensitivity also just means pin-point accuracy, is determining whether it finally can be widely used.
No. 988012847 patents of invention of Chinese patent mandate ZL provide the fluorescence polarization method of detected object in a kind of analytic sample.Said fluorescence polarization method may further comprise the steps: (a) provide albumen and fluorescent dye covalently bound fluorescence labeling albumen, wherein said albumen can the said detected object of specific bond; (b) said fluorescence labeling albumen and said detected object are combined; (c) detect in said fluorescence labeling albumen the variation of the fluorescence polarization degree that combines with said detected object to produce owing to it.
But above-mentioned patent spells out it and is suitable for detecting high molecular weight material, so there is the not high shortcoming of sensitivity in existing fluorescence polarization assay method when measuring the micromolecule interphase interaction.
Summary of the invention
The object of the present invention is to provide that a kind of sensitivity is higher, specificity is stronger and the wider fluorescence polarization assay method of usable range; Testing sample gets into molecular recognition elements through diffusion in this method; Through molecular recognition; Combine with molecular recognition elements generation specificity then, the signal that its biological or chemical reaction produces amplifies the fluorescence polarization signal through nano particle, reaches the purpose of high-sensitivity detection with this.Technical scheme of the present invention is described below.
A kind of nano particle reinforced fluorescence polarization assay method, it comprises following steps:
(1) a kind of nano particle is provided, assembles first recognition component that first recognition component obtains nanometer particle to mark to the said nano particle;
(2) a kind of indicating dye is provided, said indicating dye mark is obtained second recognition component of indicating dye mark to second recognition component;
(3) standard solution that contains target molecules to be measured is provided; Second recognition component of first recognition component of said nanometer particle to mark and said indicating dye mark is inserted in the said standard solution that contains target molecules to be measured; Under first recognition component and the second recognition component acting in conjunction, cause the specific recognition reaction; The fluorescence polarization intensity that causes solution system changes, then according to the concentration of target molecules to be measured in the said standard solution and corresponding fluorescence polarization intensity production standard working curve;
(4) sample solution that contains target molecules to be measured is provided; Second recognition component of first recognition component of said nanometer particle to mark and said indicating dye mark is inserted in the said sample solution that contains target molecules to be measured; Under first recognition component and the second recognition component acting in conjunction, cause the specific recognition reaction; The fluorescence polarization intensity that causes solution system changes, and infers the concentration that target molecules to be measured according to the fluorescence polarization intensity of target molecules to be measured in the said sample solution then.
Wherein, Said nano particle is a good dispersion, all once high nano particle, and the grain size scope of this nano particle is 1-100nm; Said nano particle comprises metal nanoparticle, semi-conductor nano particles or composite nano particle; Said metal nanoparticle comprises nm of gold, Nano Silver etc., and said semi-conductor nano particles comprises zinc sulphide, cadmium sulfide or vulcanized lead etc., and said composite nano particle is meant that macromolecular material processes.
Wherein, said first recognition component is assembled on the said nanoparticle surface through modes such as physisorption, Electrostatic Absorption, specific recognition or covalent bond couplings.Said first recognition component and second recognition component are a kind of in the biomolecule such as sugar, nucleic acid, enzyme, fat, polypeptide, antigen, antibody or protein, and said first recognition component can be identical with second recognition component, also can be different.
Wherein, said biomolecule is assembled on the nano particle and can change its biologically active.
Wherein, said indicating dye is a kind of among plain Cy3 of fluorescein isothiocynate FITC, red fluorescence or the plain Cy5 of blue-fluorescence.
Wherein, described target molecules is chemical micromolecule or sugar, nucleic acid, metallic ion such as biomolecule such as polypeptide, enzyme, fat, antigen, antibody or protein or mercury, lead, cadmium.
The present invention select for use nano particle reinforced fluorescence polarization detection signal to be because: nano particle has characteristics such as big specific surface area, quantum size effect, chemical reactivity and biological capacitive altogether, can fix recognition component of different nature through modes such as physisorption, Electrostatic Absorption, specific recognition and covalent bond couplings.Because fluorescence polarization technology is not high to interactional precision and sensitivity between the research micromolecule, so the fluorescence polarization technology range of application is wideless.But through introducing nano particle, even the interaction between the research micromolecule also can obtain fine detection sensitivity.This is because the huge surface area and the biological character of capacitive altogether of nano particle; Can greatly improve the supported quantity of recognition component; And increase the volume of recognition component and fluorescein-labeled recognition component and target molecules complex effectively; Thereby reach amplification, thereby significantly improved sensitivity and the degree of stability that detects, have simple to operate, sensitivity characteristics rapidly detection signal.Because according to different experiment purposes, its recognition component can be different biological elements, has very wide applicability.
Nano particle reinforced fluorescence polarization assay method provided by the present invention compares with traditional fluorescence polarization method, and it is higher that this method has sensitivity; The result accurately and reliably; Detection limit is lower, and the scope of application is wider, and real-time online detects; The characteristics of the few and range of linearity broad of disturbing factor are a kind of simple and practical analytical technologies.
Description of drawings
Fig. 1 is the synoptic diagram of testing process in the embodiment of the invention 1;
Fig. 2 is the synoptic diagram of testing process in the embodiment of the invention 2.
Embodiment
Existing according to accompanying drawing and combine embodiment, the present invention is done further description.
Embodiment 1The mensuration of mercury ion in the river
Please refer to shown in Figure 1ly, at first adopt trisodium citrate reduction gold chloride to prepare good dispersion, all once high solution that contains nano Au particle 1, (sequence is: 5 '-SH-(CH2) with sulfydryl modification nucleic acid probe 2 then
6-A
10-GCTTC TGTTC TCTAC-3 ') through forming the S-Au covalent bond, the surface that is assembled into nano Au particle 1 forms the sulfydryl modification nucleic acid probe 3 of nano Au particle mark.And the nucleic acid probe 4 modified of preparation fluorescein isothiocynate (sequence is: 5 '-FAM-(CH2)
6-GTTGT GTTCA GTTGC), use fluorescein isothiocynate 5 in the preparation process.
Get 10 parts in the river sample that contains mercury ion 6, every part of 5-10 milliliter adopts 0.22 micrometer fibers cellulose ester film to filter respectively.In order to reduce error, adopt same ELISA Plate that standard mercury solution and water sample are detected simultaneously in the ensuing checkout procedure.The 0.1M NaNO that standard mercury solution and water sample is added the nucleic acid probe 4 that the sulfydryl modification nucleic acid probe 3 that contained the nano Au particle mark and fluorescein isothiocynate modify respectively
3In the ELISA Plate of (pH 7.2) solution, mixing, low-speed centrifugal room temperature reaction 10-15 minute, carries out fluorescence polarization then and detects.If adopt 96 orifice plates, the reaction cumulative volume is 200 microlitres, if adopt 384 orifice plates, the reaction cumulative volume is 20 microlitres.In the absence that mercury ion 6 exists, not complementary between the nucleic acid probe because of sequence, and can not form the hybridization dimer, the fluorescence polarization intensity of solution system can not change.Under the situation that has mercury ion 6 to exist, because nucleic acid probe contains the thymidine (T) of some separately, can specificity combine mercury ion, form T-Hg
2+-T complex 7, thus the hybridization dimer formed between the probe, cause that the fluorescence polarization intensity of solution system changes.Make mercury ion fluorescent polarization strength criterion working curve according to the standard mercury solution, compare the fluorescence polarization intensity of sample then, can calculate the ion concentration of mercury in the river sample.
Embodiment 2The mensuration of nucleic acid molecules
At first adopt trisodium citrate reduction gold chloride to prepare good dispersion, equal once high solution that contains nano Au particle 10, (sequence is: 5 '-SH-(CH2) with sulfydryl modification nucleic acid probe 11 then
6-A
10-ATGATCACCATAAG-3 ') surface that is assembled into nano Au particle 10 through the Au-S covalent bond forms the sulfydryl modification nucleic acid probe 12 of nano Au particle mark.And the nucleic acid probe 13 of preparation fluorescein isothiocynate modification (sequence is: 3 "-FAM-(CH2)
6-TCAGCAGCG GCAGCA), use fluorescein isothiocynate 14 in the preparation process.
The sample to be detected that will contain nucleic acid molecules 15 joins in the mixed solution of the nucleic acid probe 13 that the sulfydryl modification nucleic acid probe 12 that contains the nano Au particle mark and fluorescein isothiocynate modify; Also contain 25mM TrisCl (pH 7.2) in this mixed solution; 0.3M NaCl; Room temperature reaction 10-15 minute, carry out fluorescence polarization then and detect.In sample to be tested, do not contain target nucleic acids molecule 15, then can not form the hybridization dimer, the fluorescence polarization intensity of solution system can not change.(sequence of this target nucleic acids molecule 15 is: in the time of 5 '-TGCTGCCGCTGCTGAGAGTACGCAAGCGTCTTATGGTGATCAT-3 ') when containing target nucleic acids molecule 15 in the solution system; It can make the sulfydryl modification nucleic acid probe 12 of nano Au particle mark and the nucleic acid probe 13 of fluorescein isothiocynate modification that hybridization reactions take place; Form the hybridization dimer, cause that the fluorescence polarization intensity of solution system changes.Make nucleic acid molecules fluorescence polarization strength criterion working curve according to standard nucleic acid solution, compare the fluorescence polarization intensity of sample then, can calculate the nucleic acid molecules concentration in the river sample.
Claims (7)
1. nano particle reinforced fluorescence polarization assay method, it comprises following steps:
(1) a kind of nano particle is provided, assembles first recognition component that first recognition component obtains nanometer particle to mark to the said nano particle;
(2) a kind of indicating dye is provided, said indicating dye mark is obtained second recognition component of indicating dye mark to second recognition component;
Said first recognition component and second recognition component are a kind of in sugar, nucleic acid, enzyme, fat, polypeptide, antigen or the antibody, and said indicating dye is a kind of among plain Cy3 of fluorescein isothiocynate FITC, red fluorescence or the plain Cy5 of blue-fluorescence;
(3) standard solution that contains target molecules to be measured is provided; Second recognition component of first recognition component of said nanometer particle to mark and said indicating dye mark is inserted in the said standard solution that contains target molecules to be measured; Under first recognition component and the second recognition component acting in conjunction, cause the specific recognition reaction; The fluorescence polarization intensity that causes solution system changes, then according to the concentration of target molecules to be measured in the said standard solution and corresponding fluorescence polarization intensity production standard working curve;
(4) sample solution that contains target molecules to be measured is provided; Second recognition component of first recognition component of said nanometer particle to mark and said indicating dye mark inserted in the said sample solution that contains target molecules to be measured cause the specific recognition reaction; The fluorescence polarization intensity that causes solution system changes, and infers the concentration that target molecules to be measured according to the fluorescence polarization intensity and the standard working curve of target molecules to be measured in the said sample solution then.
2. fluorescence polarization assay method as claimed in claim 1 is characterized in that said nano particle is a good dispersion, all once high nano particle, and the range of size of this nano particle is the 1-100 nanometer.
3. fluorescence polarization assay method as claimed in claim 1 is characterized in that, said first recognition component is assembled on the said nanoparticle surface through physisorption, Electrostatic Absorption, specific recognition or covalent bond coupling scheme.
4. fluorescence polarization assay method as claimed in claim 1 is characterized in that, said first recognition component is identical with second recognition component, and perhaps first recognition component and second recognition component are inequality.
5. fluorescence polarization assay method as claimed in claim 1 is characterized in that, said first recognition component and second recognition component are assembled on the nano particle and can change its biologically active.
6. according to claim 1 or claim 2 fluorescence polarization assay method is characterized in that said nano particle is a metal nanoparticle.
7. fluorescence polarization assay method as claimed in claim 6 is characterized in that, said metal nanoparticle is nm of gold or Nano Silver.
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| CN103342999A (en) * | 2013-05-23 | 2013-10-09 | 厦门大学 | Bio-functionalized gold nano fluorescent probe and preparation method thereof |
| CN105548086A (en) * | 2015-12-09 | 2016-05-04 | 上海大学 | Method for testing microalgae protein through nano-gold |
| CN111454947B (en) * | 2020-03-03 | 2022-04-22 | 华南师范大学 | Mesenchymal stem cell osteogenic differentiation inducer and preparation method thereof |
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