CN101285833A - Electrochemical luminescent dendritical structure marker and method for making same - Google Patents
Electrochemical luminescent dendritical structure marker and method for making same Download PDFInfo
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- CN101285833A CN101285833A CNA2007101007046A CN200710100704A CN101285833A CN 101285833 A CN101285833 A CN 101285833A CN A2007101007046 A CNA2007101007046 A CN A2007101007046A CN 200710100704 A CN200710100704 A CN 200710100704A CN 101285833 A CN101285833 A CN 101285833A
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Abstract
The invention discloses a tree-structure marker comprising nucleic acid, electro-chemiluminescent molecule and protein/compound, a method for preparing the tree-structure molecule and an immunity detection reagent box for forming the tree-structure marker, wherein the electro-chemiluminescent molecule is combined with nucleic acid acted as carrier at a plurality of sites to form signal marker of high mark rate; the signal marker is connected with protein or compound in a non-covalent bond mode to form a tree structure; and the marking method can be used in all immunity tests (tumor marker, infective disease, hormone, heart disease, food contaminant and environment pollutants, etc.) to remarkably increase the number of signal molecule during immunity detection and substantially improve detection sensitivity.
Description
Technical field
The present invention relates to label that uses in the antigen/antibody immunoassay and preparation method thereof, especially, relate to the method for immune detection being carried out multiple labeling with the bond of nucleic acid and electrochemiluminescence 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 label.According to the difference that produces signal, label can be divided into radioactive isotope, fluorescent dye, galvanochemistry molecule, enzyme, chemiluminescent substance, electrochemiluminescence thing etc., and the immunoassay of wherein last two class label correspondences has the highest detection sensitivity at present.Yet along with the demand that low-abundance protein is detected is urgent day by day, the sensitivity that further improves immunity test 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 conditions 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 label be labeled the difference of bind mode between the albumen, labeling 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 label 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 labels on a protein molecular.Owing on target protein, only relate to single linked loci, can reduce label to greatest extent simultaneously, therefore take into account the signal intensity 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 bond with the protein of immunoassay.In U.S. Pat 4921788 (open day: 1990-05-01), Deutsch has described the single-chain nucleic acid analog of an immunoassay analyte, after this analog and the antibodies, lose the hybridization ability with complementary nucleic acid, caused the signal of the signaling molecule that combines with the nucleic acid double chain specificity to descend.(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.In brief, promptly be to be the signaling molecule carrier with nucleic acid, introduce a large amount of electrochemiluminescence signaling molecules by a plurality of action sites of nucleic acid, build a kind ofly by the chain label that nucleic acid/electrochemiluminescence molecule combination constitutes, be used for immune detection.With the immunoassay of routine relatively, therefore not only one of the signaling molecule in the label that this 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 composite and inorganic nano label by the chemical synthesis process preparation, this 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, electrochemiluminescence molecule and protein/compound, wherein said electrochemiluminescence molecule combines in a plurality of sites with nucleic acid as carrier and forms signal tracer, 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 the electrochemiluminescence molecule;
(b) a plurality of electrochemiluminescence molecules are combined with described nucleic acid, form the bond of high-bonding-ratio; With
(c) with described bond 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) the electrochemiluminescence signal of measurement markers thing carries out qualitative or quantitative test 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 analog (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 electrochemiluminescence nucleic acid binding agent, as Ru (bpy)
2(dppz), make it with the nucleic acid height ratio combine, finish protrude mark to immunoassay.
(e) detect the electrochemiluminescence signal,, the sample of immunoassay is carried out qualitative or quantitative test according to the variation of signal intensity.
It is the immunoassay kits of multiple labeling with nucleic acid/electrochemiluminescence molecule bond that the 4th aspect of the present invention provides a kind of, and representative kit comprises:
(a) biotin labeled antibody or compound;
(b) Avidin or its analog (avidin, streptavidin, neutravidin);
(c) nucleic acid of biotin modification;
(d) electrochemiluminescence nucleic acid binding agent; With
(e) instructions
In one aspect of the invention, the tree-shaped immune detection label of being made up of nucleic acid/electrochemiluminescence molecule bond that is provided has following feature:
Nucleic acid as electrochemiluminescence signaling molecule carrier is the DNA (deoxyribonucleic acid) (ssDNA) that process is modified, double stranded DNA (dsDNA), singlestranded RNA (ssRNA), double stranded RNA (dsRNA), double stranded DNA/RNA (ribonucleic acid) (dsRNA/DNA), oligonucleotides (oligonucleotide), aptamer (aptamer), or peptide ribonucleic acid (peptide nucleic 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, this method has more universality and operability.
In another aspect of the present invention, the mode of connection of described bond and protein or compound is biological the connection.Such as, (biotin/neutravidin) the biological combination 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 biotin/affinity element.The biotin (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 micromolecule antigen vectors.
In another aspect of the present invention, described compound is organic compound, hormone, steroids, carbohydrates, vitamin, monose, oligosaccharides, lipid, amino acid, peptide, nucleosides, nucleotide, oligonucleotides or its compound.
In another aspect of the present invention, described electrochemiluminescence 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 aspect of the invention, the described electrochemiluminescence molecule complex compound that is 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 (referring to document: Barton, JK.et al.J.Am.Chem.Soc.1990.112:4960-4962.).In one embodiment of the invention, described organic ligand be terpyridyl, the replacement of connection pyrazine, the replacement of dipyridine, the replacement of dipyridine, 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, the combination of described electrochemiluminescence molecule and nucleic acid effect for embed, ditch combine, major groove combination or electrostatic interaction.
Nucleic acid of the present invention/electrochemiluminescence molecule bond can obtain by conventional method, representative synthetic method example following (as shown in Figure 1): the end at one section nucleic acid connects 9 continuous deoxythymidines, connects biotin again.Add the electrochemiluminescence nucleic acid binding agent, as Ru (bpy)
2(dppz), make it with the nucleic acid height ratio combine, forming with nucleic acid is the protrude mark thing of carrier.
Utilize the advantage of method labelled immune test of the present invention to be: (1) is the carrier of electrochemiluminescence 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, greatly reduces nonspecific reaction; (4) preparation method of nucleic acid markers is simple, and experimental cost is low; (5) all labelled reagents can be preserved separately, now join before the use, can prolong the shelf-life of reagent.
Description of drawings
Fig. 1 is nucleic acid/electrochemiluminescence molecule bond.
Fig. 2 is that nucleic acid/electrochemiluminescence molecule bond thing that serves as a mark is used for the synoptic diagram of immunoassay.
Fig. 3 uses nucleic acid/electrochemiluminescence molecule bond as signal tracer and the direct mark of electricity consumption chemiluminescent 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 sodium citrate, 30mM sodium chloride, 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 ACTCCC-3 ', chain B:5 '-GGG AGT AAA GTT AAT ATT GAC GTT ACCCGC-3 '.95 ℃ of sex change 5 minutes naturally cool to room temperature, determine the concentration of hybridization back nucleic acid with spectrophotometer.
2, direct labelled antibody of electrochemiluminescence molecule (Ru-NHS) or protein molecular
Be dissolved in (50mM sodium carbonate 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 Ru-NHS (5mg Ru-NHS/mL DMSO) of (DMSO) mix, room temperature reaction 4 hours, adopt polyacrylamide gel desalting column (Pierce, Rockford, IL) purifying labelled antibody, spectrophotometer detects the mark rate of antibody concentration and Ru-NHS.
3, Huo Hua biotin labeling antibody
The biotin that takes by weighing an amount of activation is that biotin-N-carboxyl succimide ester (BNHS) is dissolved in anhydrous N, and in the dinethylformamide (DMF), final concentration is 20mg BNHS/mlDMF.Goat-anti mouse immuning ball protein G mixes with BNHS according to 1: 5.6 mol ratio and is dissolved in (50mM sodium carbonate in the carbonate buffer solution, the 50mM sodium bicarbonate, pH 9.15), room temperature reaction 4 hours adopts polyacrylamide gel desalting column (Pierce, Rockford, IL) purifying labelled antibody, spectrophotometer detects antibody concentration, HABA/Avidin 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 (Ru-mIgG) that gets 100 μ l concentration respectively and be the Ru-NHS 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 chloride and 0.1% polysorbas20, the pH value is after 8.0 the damping fluid flushing 3 times, best electrochemiluminescence testing conditions with Ru, detect the variation of electrochemiluminescence intensity, with the saturation concentration of indication Ru-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 phosphate buffer of 1% bovine serum albumin(BSA), 4 ℃ are spent the night.After washing 3 times, add the goat anti-mouse igg (Ru-Ab) 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/mL Ru-NHS marks, 37 ℃, air bath constant temperature 200rpm vibration 2 hours; After washing 3 times,, detect the variation of electrochemiluminescence intensity with the best electrochemiluminescence testing conditions of Ru, with the saturation concentration of indication Ru-Ab, 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 (Ru-SA) that 100 μ l concentration are the Ru-NHS mark of 0,0.01,0.03,0.09,0.27,0.82,2.47,7.40,22.2 μ g/mL respectively, 37 ℃, air bath constant temperature 200rpm vibration 2 hours; After washing 3 times,, detect the variation of electrochemiluminescence intensity with the best electrochemiluminescence testing conditions of Ru, with the saturation concentration of indication Ru-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 electrochemiluminescence 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 ℃, air bath constant temperature 200rpm vibration 2 hours; Wash 3 times, add 100 μ l concentration respectively and be 0,14,27,55,110,219, electrochemiluminescence nucleic acid binding agent such as the Ru (bpy) of 438nM
2(dppz), room temperature, 200rpm vibration lucifuge was hatched 10 minutes.Perhaps be pre-mixed the double-stranded DNA and the nucleic acid binding agent of biotin modification according to a certain percentage, again with Avidin at 37 ℃, air bath constant temperature 200rpm oscillating reactions 2 hours.Clean unnecessary bond.Detect the variation of electrochemiluminescence intensity, with the saturation concentration of indication double chain oligonucleotide and electrochemiluminescence bond, 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 chloride 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 phosphate buffer of 1% bovine serum albumin(BSA), 4 ℃ are spent the night.After washing 3 times, add the biotin labeled goat anti-mouse igg of 100 μ l, 37 ℃, air bath constant temperature 200rpm vibration 2 hours; After washing 3 times, add 100 μ l Avidins, 37 ℃, air bath constant temperature 200rpm vibration 2 hours; Wash 3 times.
Embodiment 6, contain the label and the analyte response of nucleic acid/electrochemiluminescence molecule bond
After the washing, add the double-stranded DNA of 100 μ l biotin modifications, 37 ℃, air bath constant temperature 200rpm vibration 2 hours; Wash 3 times, add 100 μ l electrochemiluminescence nucleic acid binding agents such as Ru (bpy)
2(dppz), room temperature, 200rpm vibration lucifuge was hatched 10 minutes.Perhaps be pre-mixed the double-stranded DNA and the electrochemiluminescence nucleic acid binding agent of biotin modification according to a certain percentage, again with Avidin at 37 ℃, air bath constant temperature 200rpm oscillating reactions 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 electrochemiluminescence 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 chloride 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 phosphate buffer of 1% bovine serum albumin(BSA), 4 ℃ are spent the night.After washing 3 times, add the biotin labeled goat anti-mouse igg of 100 μ l, 37 ℃, air bath constant temperature 200rpm vibration 2 hours; After washing 3 times, add the streptavidin of the direct mark of 100 μ l electrochemiluminescence molecules, 37 ℃, air bath constant temperature 200rpm vibration 2 hours; Wash 3 times.
Embodiment 8, electrochemiluminescence intensity detection
Clean unnecessary electrochemiluminescence bond, detect the variation of electrochemiluminescence intensity, 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 electrochemiluminescence signal intensity that detects and the concentration of goat anti-mouse igg, and with nucleic acid/electrochemiluminescence molecule bond as signal tracer, than the direct mark of electricity consumption chemiluminescent molecule, signal intensity exceeds about 10 times.
Claims (10)
1. dendritic structure mark, comprise nucleic acid, electrochemiluminescence molecule and protein/compound, wherein said electrochemiluminescence molecule combines in a plurality of sites with nucleic acid as carrier and forms signal tracer, 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/RNA (ribonucleic acid), oligonucleotides, 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 micromolecule antigen vectors; Described compound is organic compound, hormone, steroids, carbohydrates, vitamin, monose, oligosaccharides, lipid, amino acid, peptide, nucleosides, nucleotide, oligonucleotides or its compound.
4. dendritic structure mark according to claim 1, the mode of connection of wherein said bond and protein or compound are biological the binding.
5. dendritic structure mark according to claim 4 is characterized in that, the mode of connection of described bond and protein or compound links for the biology by biotin/affinity element.
6. dendritic structure mark according to claim 1, wherein said electrochemiluminescence molecule are the complex compound of metal/organic ligand.
7. dendritic structure mark according to claim 6 is characterized in that, the metal in described metal/organic ligand complex compound is ruthenium or osmium.
8. dendritic structure mark according to claim 1, the combination of wherein said electrochemiluminescence molecule and nucleic acid effect for embed, ditch combine, major groove combination or electrostatic interaction.
9. method for preparing the dendritic structure mark among any one of the claim 1-8, it comprises the following steps:
(a) provide the carrier of nucleic acid as the electrochemiluminescence molecule;
(b) a plurality of electrochemiluminescence molecules are combined with described nucleic acid, form the bond of high-bonding-ratio; With
(c) with described bond as signal tracer, connect in the non-covalent bond mode with protein or compound, be used for immunoassay.
10. kit that is used for immunoassay, it comprises
(a) antibody of biotin modification or compound;
(b) Avidin or its analog;
(c) nucleic acid of biotin modification
(d) electrochemiluminescence nucleic acid binding agent; With
(e) instructions.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109477834A (en) * | 2016-05-19 | 2019-03-15 | 凸版印刷株式会社 | The detection method and target molecule detection kit of target molecule |
| CN112505023A (en) * | 2019-10-31 | 2021-03-16 | 联吡啶钌科学公司 | Method for improving electrochemiluminescence signal in bioanalytical assays |
-
2007
- 2007-04-14 CN CNA2007101007046A patent/CN101285833A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109477834A (en) * | 2016-05-19 | 2019-03-15 | 凸版印刷株式会社 | The detection method and target molecule detection kit of target molecule |
| CN112505023A (en) * | 2019-10-31 | 2021-03-16 | 联吡啶钌科学公司 | Method for improving electrochemiluminescence signal in bioanalytical assays |
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Application publication date: 20081015 |