CN101255462A - Dendritic structure mark and preparation method thereof - Google Patents
Dendritic structure mark and preparation method thereof Download PDFInfo
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- CN101255462A CN101255462A CNA2007100641015A CN200710064101A CN101255462A CN 101255462 A CN101255462 A CN 101255462A CN A2007100641015 A CNA2007100641015 A CN A2007100641015A CN 200710064101 A CN200710064101 A CN 200710064101A CN 101255462 A CN101255462 A CN 101255462A
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Abstract
The invention discloses a tree-like marker including nucleic acid, fluorescence molecule and protein/compound; a method for preparing the tree-like molecular; and an immunity detection reagent kit for forming the tree-like marker. The fluorescence molecule and the nucleic acid used as vector are combined into a signal marker in non-embedded manner, the signal marker and the protein or compound are bonded in non-covalent bond to from a tree-like structure; the label method is used for all immunity tests (tumor marker, communicable disease, hormone, heart disease, food contaminant, environmental pollutant, etc.), can obviously increase signal molecular number in immunity detection, and greatly improve detection sensitivity.
Description
Technical field
The present invention relates to marker that uses in the antigen/antibody immunoassay and preparation method thereof, especially, relate to the method for immunodetection being carried out multiple labeling with the binding substances of nucleic acid and fluorescence molecule.
Technical background
Immune analysis method has been widely used in life science, pharmaceutical analysis, clinical diagnosis and environmental monitoring field owing to have the specificity of height, advantage such as quick, easy.Because the physical change that immune response self produces is very faint, be difficult to detect, mark produces the material or the molecule of special signal on antigen or antibody usually, is called marker.According to the difference that produces signal, marker can be divided into radio isotope, fluorescence dye, electrochemistry molecule, enzyme, chemiluminescent substance, electrochemiluminescence thing etc., and wherein the fluoroscopic examination application is very extensive, and is the standard detecting method of biochip.Yet along with the demand that low-abundance protein is detected is urgent day by day, the sensitivity that further improves fluroimmunoassay becomes the focus of research.In immune analysis method, two kinds of means are mainly taked in the raising of detection sensitivity: the one, and the amplification in vitro low-abundance protein; The one, the quantity of increase signaling molecule.The former is difficult to realize in practice, because present technical qualification are difficult to make protein as nucleic acid (for example: DNA), adopt polymerase chain reaction,PCR at amplification in vitro.Therefore, the quantity of increase signaling molecule just becomes the key breakthrough mouth that improves protein detection sensitivity.In the immunoassay process, signaling molecule normally is marked on antibody or the antigen, how more signaling molecule is marked on same antigen or the antibody, and detection signal is enhanced, not influencing simultaneously the recognition performance of antigen or antibody, is one of maximum difficult point.According to marker be labeled the difference of bind mode between the albumen, marking method can be divided into covalent labeling and non-covalent mark two classes.And, can be divided into a plurality of sites mark and single site mark again according to the number of marker and albumen linked loci.Generally speaking, the preparation difficulty of covalent labeling is greater than non-covalent mark, and single site mark is difficult to a plurality of sites mark.The protein labeling method of wherein single site multiple labeling can improve detection signal significantly owing to introduced a plurality of markers on a protein molecular.Owing on target protein, only relate to single linked loci, can reduce marker to greatest extent simultaneously, therefore take into account the strength of signal and the specificity of immunoassay the immunocompetent interference of target protein.
Adopt nucleic acid to carry out the immunoassay mark, the existing description or report in patent and scientific research article.In U.S. Pat 4748111 (open day: 1988-05-31), people such as Dattagupta have described one and as the signaling molecule carrier immunoassay have been carried out the method for multiple labeling with nucleic acid, and 3 ' end of its amplifying nucleic acid is connected by covalent linkage with the protein of immunoassay.In U.S. Pat 4921788 (open day: 1990-05-01), Deutsch has described the single-chain nucleic acid analogue of an immunoassay analyte, after this analogue and the antibodies, lost the hybridization ability with complementary nucleic acid, caused descending with the signal of nucleic acid double chain specificity bonded signaling molecule.(open day: 2001-08-28), people such as Glazer described one and as the fluorescence molecule carrier immunoassay have been carried out the method for multiple labeling with double-strandednucleic acid, and fluorescence molecule wherein is the nucleic acid intercalator and is with two positive charges in U.S. Pat 6280933.(open day: 2000-09-12), Nilsen described a specific dendroid nucleic acid (DNA dendrimer) carries out multiple labeling to immunoassay method in U.S. Pat 6117631.In addition, in U.S. Pat 5665539 (open day: 1997-09-09), Sano etc. have described a method that is called immuno-PCR (immuno-PCR), with nucleic acid immunoassay is carried out mark, by PCR nucleic acid marking is increased then, carry out immunoassay by nucleic acid quantification at last.The domestic patent that does not also have this respect at present.
Summary of the invention
The present invention improves at the deficiency of prior art the following aspects: 1) in order to be reduced in the difficulty of mark signaling molecule on the antibody, adopt non-covalent mode to introduce the signaling molecule carrier; 2) in order to realize the strategy of single site multiple labeling, with nucleic acid the signaling molecule carrier; 3) in order to obtain signaling molecule marking method easy and that subject range is wider, adopt non-embedded nucleic acid binding agent.In brief, promptly be to be the signaling molecule carrier with nucleic acid, introduce a large amount of fluorescent signal molecules by a plurality of action sites of nucleic acid, build a kind of chain marker that constitutes by nucleic acid/fluorescence molecule combination, be used for immunodetection.With the test of the immunofluorescence of routine relatively, therefore not only one of the fluorescent signal molecule in the marker that present method provides, but a plurality of even tens can increase substantially the intensity of detection signal, obviously improves detection sensitivity.Compare with organic polymer, dendroid organic molecule (dendrimer), inorganic complex and inorganic nano marker by the chemical synthesis process preparation, present method adopts routine biochemistry and chemical reagent, is easy to obtain, and is cheap, simple to operate, good reproducibility.
One aspect of the present invention provides a kind of dendritic structure mark, it comprises nucleic acid, fluorescence molecule, and protein/compound, wherein said fluorescence molecule forms signal tracer with combining by non-embedded mode as the nucleic acid of carrier, described signal tracer is connected in the non covalent bond mode with protein or compound, forms tree structure.
Second aspect of the present invention provides the preparation method of described dendritic structure mark, and it comprises the following steps:
(a) provide the carrier of nucleic acid as fluorescence molecule;
(b) with non-embedded mode a plurality of fluorescence molecules are combined with described nucleic acid, form the binding substances of high-bonding-ratio; With
(c) with described binding substances as signal tracer, connect in the non covalent bond mode with protein or compound, be used for immunoassay.
The 3rd aspect of the present invention provides a kind of immune analysis method of quick, easy, efficient and cost-effective, and it also comprises the following steps: except the step of second aspect present invention
(d) fluorescent signal of measurement fluorescence molecule carries out qualitative or quantitative analysis according to the intensity of signal to sample.
Its representative operation steps is a (see figure 2):
(a) according to routine operation, finish immune response, wherein the reactant of final step has biotin labeling,
(b) after immune response is finished, adding avidin or its analogue (avidin, streptavidin, neutravidin),
(c) nucleic acid of adding biotin modification by the non covalent bond effect between the biotin/avidin, is finished the nucleic acid marking to immunoassay,
(d) add the fluorescent core acid binding agent, as SYBR Green I, make it with the nucleic acid height ratio combine, finish highly fluorescent markers to immunoassay.
(e) detect fluorescent signal,, the sample of immunoassay is carried out qualitative or quantitative analysis according to the variation of strength of signal.
It is the immunoassay kits of multiple labeling with nucleic acid/fluorescence molecule binding substances that the 4th aspect of the present invention provides a kind of, and representative test kit comprises:
(a) biotin labeled antibody or compound;
(b) avidin or its analogue (avidin, streptavidin, neutravidin);
(c) nucleic acid of biotin modification;
(d) fluorescent core acid binding agent; With
(e) instructions
In one aspect of the invention, the tree-shaped immunodetection marker of being made up of nucleic acid/fluorescence molecule binding substances that is provided has following feature:
Nucleic acid as the fluorescent signal molecular vehicle is the thymus nucleic acid (ssDNA) that process is modified, double stranded DNA (dsDNA), singlestranded RNA (ssRNA), double stranded RNA (dsRNA), double stranded DNA/Yeast Nucleic Acid (dsRNA/DNA), oligonucleotide (oligonucleotide), aptamer (aptamer), or peptide ribonucleic acid (peptidenucleic acid, PNA).They are connected by the non covalent bond mode with protein that is labeled or compound.With the covalently bound comparison that United States Patent (USP) 4748111 is described, present method has more universality and operability.
In another aspect of the present invention, the mode of connection of described binding substances and protein or compound is biological the connection.Such as, (biotin/neutravidin) the biological keying action of the high specific between and high-affinity is connected nucleic acid with protein or compound for biotin/avidin, biotin/streptavidin can to pass through vitamin H/affinity element.The vitamin H (biotin) of various molecules (comprising nucleic acid, protein and compound) is modified has become routine techniques, processing ease, and expense is cheap, and the result repeats reliably.
In another aspect of the present invention, described protein is antigen, haptens, antibody and or as the protein of small molecules antigen vectors.
In another aspect of the present invention, described compound is organic compound, hormone, steroid, carbohydrate, VITAMIN, monose, oligosaccharides, lipid, amino acid, peptide, nucleosides, Nucleotide, oligonucleotide or its mixture.
In another aspect of the present invention, described fluorescence molecule is inorganic molecule, organic molecule, inorganic/organic compound molecule, inorganic polymer, organic polymer, inorganic/the organic composite polymeric thing, inorganic particulate matter, organic particulate matter or inorganic/organic composite granulated thing.
In one embodiment of the invention, described fluorescence molecule is a nucleic acid binding agent.In another embodiment of the invention, described fluorescence molecule is a fluorescence dye, and wherein said fluorescence dye is DAPI dyestuff, Pico-Green dyestuff, SYBR Green dyestuff, SYBR Gold dyestuff.Fluorescent signal molecule among the present invention all is a nucleic acid binding agent, and the mode of they and nucleic acid effect is non-embedded, comprising ditch in conjunction with, major groove combination and static in conjunction with and other mode combinations.The fluorescent signal molecule can be an organic fluorescent dye, as DAPI dyestuff, Pico-Green dyestuff, SYBRGreen dyestuff, SYBR Gold dyestuff (referring to document: Cosa, G., et al.Photochem.Photobio.2001.73:585-599.) etc.Also can be complex compound, such as the complex compound (referring to document: Barton, JK.et al.J.Am.Chem.Soc.1990.112:4960-4962.) of ruthenium or osmium and the formation of nitrogenous heteroaromatic with metal/organic ligand of fluorescence property.The embedded fluorescence dye of describing with United States Patent (USP) 6280933 relatively, present method has more universality, but both mark single-chain nucleic acids, but also mark double-strandednucleic acid.
In one aspect of the invention, described fluorescence molecule is the complex compound of metal/organic ligand.In one embodiment, the metal in described metal/organic ligand complex compound is ruthenium or osmium.In another embodiment, the organic ligand in described metal/organic ligand complex compound is nitrogenous heteroaromatic.In one embodiment of the invention, described organic ligand be terpyridyl, the replacement of connection pyrazine, the replacement of dipyridyl, the replacement of dipyridyl, connection pyrazine, terpyridyl, phenanthrol, phthalein cyanogen dyestuff, replacement phenanthrol, two pyridos [3,2-a; 2 ', 3 '-c] azophenlyene or adjacent two single aza-phenanthrenes.
In one aspect of the invention, described fluorescence molecule and the combination of nucleic acid effect be that ditch combines, major groove in conjunction with or electrostatic interaction.
Nucleic acid of the present invention/fluorescence molecule binding substances can obtain by ordinary method, representative synthetic method example following (as shown in Figure 1): the end at one section nucleic acid connects 9 successive deoxythymidines, connects vitamin H again.Add the fluorescent core acid binding agent, as SYBR Green I, make it by non-embedded mode and nucleic acid height ratio combine, formation is the protrude mark thing of carrier with nucleic acid.
Utilize the advantage of method labelled immune test of the present invention to be: (1) is the carrier of fluorescence molecule with nucleic acid, can introduce signaling molecule in a large number, greatly strengthens the sensitivity that detects; (2) nucleic acid is connected by the non covalent bond mode with protein that is labeled or compound, has universality and operability; (3) mark in the single site of protein can reduce its bioactive influence, reduces nonspecific reaction greatly; (4) mode of fluorescent signal molecule and nucleic acid effect is non-embedded, but both mark single-chain nucleic acids, but also mark double-strandednucleic acid; (5) preparation method of nucleic acid markers is simple, and experimental cost is low; (6) all labelled reagents can be preserved separately, now join before the use, can prolong the quality guaranteed period of reagent.
Description of drawings
Fig. 1 is nucleic acid/fluorescence molecule binding substances.
Fig. 2 is that nucleic acid/fluorescence molecule binding substances thing that serves as a mark is used for the synoptic diagram of immunoassay.
Fig. 3 uses nucleic acid/fluorescence molecule binding substances as signal tracer with the direct mark of fluorescence molecule, resulting immunoassay result respectively.
Embodiment
Material is prepared before the experiment
1, the preparation of biotin labeling double chain oligonucleotide
With 2 * SSC (0.3M Trisodium Citrate, 30mM sodium-chlor, pH7.0) the plain mark single stranded deoxyribonucleic acid of damping fluid equal proportion mixed biologic (Invitrogen, Carlsbad, CA) and complementary nucleic acid chain (Invitrogen, Carlsbad, CA), for example two chains are respectively: chain A:biotin-5 '-TTT TTT TTT GCG GGT AAC GTC AAT ATT AAC TTT ACT CCC-3 ', chain B:5 '-GGG AGT AAA GTT AAT ATT GAC GTT ACC CGC-3 '.95 ℃ of sex change 5 minutes naturally cool to room temperature, determine the concentration of hybridization back nucleic acid with spectrophotometer.
2, fluorescein isothiocyanate (Fluorescein isothiocyanate, FITC) traget antibody or protein molecular
Be dissolved in (50mM yellow soda ash in the carbonate buffer solution, the 50mM sodium bicarbonate, pH9.15) mouse immuning ball protein G (mIgG), goat-anti mouse immuning ball protein G and streptavidin (SA) respectively according to 1: 5.6 mol ratio be dissolved in dimethyl sulfoxide (DMSO) in the FITC (5mg FITC/mLDMSO) of (DMSO) mix, room temperature reaction 4 hours adopts polyacrylamide gel desalting column (Pierce, Rockford, IL) purifying traget antibody, spectrophotometer detects the mark rate of antibody concentration and FITC.
3, activatory biotin labeling antibody
Taking by weighing an amount of activatory vitamin H is that vitamin H-N-carboxyl succimide ester (BNHS) is dissolved in anhydrous N, and in the dinethylformamide (DMF), final concentration is 20mg BNHS/ml DMF.Goat-anti mouse immuning ball protein G mixes with BNHS according to 1: 5.6 mol ratio and is dissolved in (50mM yellow soda ash in the carbonate buffer solution, the 50mM sodium bicarbonate, pH9.15), room temperature reaction 4 hours adopts polyacrylamide gel desalting column (Pierce, Rockford, IL) purifying traget antibody, spectrophotometer detects antibody concentration, HABA/Avidin test kit (Sigma, St.Louis, MO) the biotin labeling rate of detection antibody.
The bag of embodiment 1, mouse immuning ball protein G is by condition optimizing
The mouse immuning ball protein G (FITC-mIgG) that gets 100 μ l concentration respectively and be the FITC mark of 0,0.3,1,3,10,30,100 μ g/mL is dissolved in and contains the 50mM sodium bicarbonate, the pH value is in 9.6 the damping fluid, by 96 orifice plates, 4 ℃ are spent the night according to the consumption bag of every hole 100 μ l.With containing 50mM 3-methylol-aminomethane, 50mM sodium-chlor and 0.1% polysorbas20, the pH value is after 8.0 the damping fluid flushing 3 times, best fluoroscopic examination condition with FITC (is best fluorescence exciting wavelength, excites slit width, the fluorescent emission wavelength, the emission slit width), the variation of fluorescence intensity, with the saturation concentration of indication FITC-mIgG, and with this saturation concentration as the bag of unlabelled mIgG by concentration.
The experiment condition optimization of embodiment 2, biotin labeled goat-anti mouse immuning ball protein G
With the mIgG bag optimized by the condition bag by mIgG, 4 ℃ are spent the night.After washing 3 times, add 300 μ l and contain in the phosphoric acid buffer of 1% bovine serum albumin, 4 ℃ are spent the night.After washing 3 times, add the goat anti-mouse igg that 100 μ l concentration are respectively 0,1.0,3.2,9.7,29.0,87.1,261.3,784.0,2352.0 μ g/mLFITC marks, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; After washing 3 times, with the best fluoroscopic examination condition of FITC, the variation of fluorescence intensity, with the saturation concentration of indication FITC-mIgG, and with the working concentration of this saturation concentration as biotin labeled goat anti-mouse igg (BT-Ab).
The experiment condition optimization of embodiment 3, streptavidin
With the optimal conditions bag by mIgG and BT-Ab after, add the streptavidin (FITC-SA) that 100 μ l concentration are the FITC mark of 0,0.01,0.03,0.09,0.27,0.82,2.47,7.40,22.2 μ g/mL respectively, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; After washing 3 times, with the best fluoroscopic examination condition of FITC, the variation of fluorescence intensity, with the saturation concentration of indication FITC-SA, and with the working concentration of this saturation concentration as unlabelled streptavidin.
The experiment condition optimization of embodiment 4, biotinylation double chain oligonucleotide and fluorescence molecule
With the optimal conditions bag by mIgG, BT-Ab and streptavidin after, adding 100 μ l concentration respectively is the biotinylation double chain oligonucleotide of 0,0.04,0.12,0.37,1.11,3.33,10 μ M, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; Wash 3 times, add 100 μ l concentration respectively and be 0,14,27,55,110,219, nucleotide fluorescent dye such as the SYBR Green I of 438nM, room temperature, 200rpm vibration lucifuge was hatched 10 minutes.Perhaps be pre-mixed the double-stranded DNA and the nucleotide fluorescent dye of biotin modification according to a certain percentage, again with avidin at 37 ℃, airbath constant temperature 200rpm oscillatory reaction 2 hours.Do not clean unnecessary dyestuff or clean unnecessary dyestuff, with nucleotide fluorescent dye and the best fluoroscopic examination condition of nucleic acid bonded (is best fluorescence exciting wavelength, excite slit width, the fluorescent emission wavelength, the emission slit width), the variation of fluorescence intensity, with the saturation concentration of indication double chain oligonucleotide and fluorescence dye, and with this saturation concentration as working concentration.
Embodiment 5, build the immune response system
According to optimal conditions, to get 30 μ g/mL mouse immuning ball protein G (mIgG) and be dissolved in and contain the 50mM sodium bicarbonate, the pH value is in 9.6 the damping fluid, by 96 orifice plates, 4 ℃ are spent the night according to the consumption bag of every hole 100 μ l.With containing 50mM 3-methylol-aminomethane, 50mM sodium-chlor and 0.1% polysorbas20, the pH value is after 8.0 the damping fluid flushing 3 times, to add 300 μ l and contain in the phosphoric acid buffer of 1% bovine serum albumin, 4 ℃ are spent the night.After washing 3 times, add the biotin labeled goat anti-mouse igg of 100 μ l, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; After washing 3 times, add 100 μ l avidins, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; Wash 3 times.
Embodiment 6, the marker that contains nucleotide fluorescent dye and analyte response
After the washing, add the double-stranded DNA of 100 μ l biotin modifications, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; Wash 3 times, add 100 μ l nucleotide fluorescent dyes such as SYBR Green I, room temperature, 200rpm vibration lucifuge was hatched 10 minutes.Perhaps be pre-mixed the double-stranded DNA and the nucleotide fluorescent dye of biotin modification according to a certain percentage, again with avidin at 37 ℃, airbath constant temperature 200rpm oscillatory reaction 2 hours.
Embodiment 7, contrast as experimental system with the goat anti-mouse igg of the streptavidin detection of biological elementization of the direct mark of fluorescence molecule
According to the experimental procedure of embodiment 5, to get 30 μ g/mL mouse immuning ball protein G (mIgG) and be dissolved in and contain the 50mM sodium bicarbonate, the pH value is in 9.6 the damping fluid, by 96 orifice plates, 4 ℃ are spent the night according to the consumption bag of every hole 100 μ l.With containing 50mM 3-methylol-aminomethane, 50mM sodium-chlor and 0.1% polysorbas20, the pH value is after 8.0 the damping fluid flushing 3 times, to add 300 μ l and contain in the phosphoric acid buffer of 1% bovine serum albumin, 4 ℃ are spent the night.After washing 3 times, add the biotin labeled goat anti-mouse igg of 100 μ l, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; After washing 3 times, add the streptavidin of 100 μ l marked by fluorescein isothiocyanate, 37 ℃, airbath constant temperature 200rpm vibration 2 hours; Wash 3 times.
Embodiment 8, fluorescence intensity detect
Do not clean unnecessary dyestuff or clean unnecessary dyestuff, with the best fluoroscopic examination condition of nucleotide fluorescent dye and nucleic acid bonded, the variation of fluorescence intensity is with the concentration of the goat anti-mouse igg of the plain mark of indicator organism.By figure three as seen, there is corresponding relation between the fluorescence signal intensity that detects and the concentration of goat anti-mouse igg, and with nucleic acid/fluorescence molecule binding substances as signal tracer, compare with the direct mark of fluorescence molecule (being the streptavidin of marked by fluorescein isothiocyanate), strength of signal exceeds about 10 times.
Claims (14)
1. dendritic structure mark, comprise nucleic acid, fluorescence molecule, and protein/compound, wherein said fluorescence molecule forms signal tracer with combining by non-embedded mode as the nucleic acid of carrier, described signal tracer is connected in the non covalent bond mode with protein or compound, forms tree structure.
2. dendritic structure mark according to claim 1, wherein said nucleic acid are single stranded deoxyribonucleic acid, double stranded DNA, singlestranded RNA, double stranded RNA, double stranded DNA/Yeast Nucleic Acid, oligonucleotide, aptamer, or peptide ribonucleic acid.
3. dendritic structure mark according to claim 1, wherein said protein is antigen, haptens, antibody and or as the protein of small molecules antigen vectors; Described compound is organic compound, hormone, steroid, carbohydrate, VITAMIN, monose, oligosaccharides, lipid, amino acid, peptide, nucleosides, Nucleotide, oligonucleotide or its mixture.
4. dendritic structure mark according to claim 1, the mode of connection of wherein said binding substances and protein or compound are biological the connection.
5. dendritic structure mark according to claim 4 is characterized in that, the mode of connection of described binding substances and protein or compound links for the biology by vitamin H/affinity element.
6. according to any one dendritic structure mark among the claim 1-5, wherein said fluorescence molecule is inorganic molecule, organic molecule, inorganic/organic compound molecule, inorganic polymer, organic polymer, inorganic/the organic composite polymeric thing, inorganic particulate matter, organic particulate matter or inorganic/organic composite granulated thing.
7. dendritic structure mark according to claim 6, wherein said fluorescence molecule are nucleic acid binding agent.
8. dendritic structure mark according to claim 7, wherein said fluorescence molecule are fluorescence dye.
9. dendritic structure mark according to claim 8, wherein said fluorescence dye are DAPI dyestuff, Pico-Green dyestuff, SYBR Green dyestuff, SYBR Gold dyestuff.
10. dendritic structure mark according to claim 7, wherein said fluorescence molecule are the complex compound of metal/organic ligand.
11. according to any one dendritic structure mark among claim 1-5 and the 7-10, wherein said fluorescence molecule and the combination of nucleic acid effect are that ditch combines, major groove in conjunction with or electrostatic interaction.
12. a method for preparing the dendritic structure mark of claim 1-10 or 11 in any one, it comprises the following steps:
(a) provide the carrier of nucleic acid as fluorescence molecule;
(b) with non-embedded mode a plurality of fluorescence molecules are combined with described nucleic acid, form the binding substances of high-bonding-ratio; With
(c) with described binding substances as signal tracer, connect in the non covalent bond mode with protein or compound, be used for immunoassay.
13. one kind is carried out the method for multiple labeling to immunodetection, it comprises the fluorescent signal by fluorescence molecule in the dendritic structure mark of measuring claim 12, the step of sample being carried out qualitative or quantitative analysis according to the intensity of signal.
14. a test kit that is used for immunoassay, it comprises
(a) antibody of biotin modification or compound;
(b) avidin or its analogue;
(c) nucleic acid of biotin modification;
(d) fluorescent core acid binding agent; With
(e) instructions.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435730A (en) * | 2011-09-22 | 2012-05-02 | 江阴天瑞生物科技有限公司 | High flux detection method and biochip based on nucleic acid address coding |
| CN105372418A (en) * | 2014-08-25 | 2016-03-02 | 常州博闻迪医药科技有限公司 | Signal amplification immunodetection method |
| CN107356572A (en) * | 2017-06-28 | 2017-11-17 | 成都理工大学 | The FRET method of carrier is used as by the use of nucleic acid |
| CN110568175A (en) * | 2019-09-06 | 2019-12-13 | 成都理工大学 | Method for detecting DNA (deoxyribonucleic acid) based on rapid aggregation of nanogold induced by nucleic acid dye |
-
2007
- 2007-02-28 CN CNA2007100641015A patent/CN101255462A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435730A (en) * | 2011-09-22 | 2012-05-02 | 江阴天瑞生物科技有限公司 | High flux detection method and biochip based on nucleic acid address coding |
| CN105372418A (en) * | 2014-08-25 | 2016-03-02 | 常州博闻迪医药科技有限公司 | Signal amplification immunodetection method |
| CN107356572A (en) * | 2017-06-28 | 2017-11-17 | 成都理工大学 | The FRET method of carrier is used as by the use of nucleic acid |
| CN110568175A (en) * | 2019-09-06 | 2019-12-13 | 成都理工大学 | Method for detecting DNA (deoxyribonucleic acid) based on rapid aggregation of nanogold induced by nucleic acid dye |
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