CN100351392C - Marking probe of nano microparticle and affinity element and its preparation method as well as application - Google Patents
Marking probe of nano microparticle and affinity element and its preparation method as well as application Download PDFInfo
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- CN100351392C CN100351392C CNB02137418XA CN02137418A CN100351392C CN 100351392 C CN100351392 C CN 100351392C CN B02137418X A CNB02137418X A CN B02137418XA CN 02137418 A CN02137418 A CN 02137418A CN 100351392 C CN100351392 C CN 100351392C
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Abstract
The present invention provides a marking probe of nanometer fine particles, an affinity element., a preparation method and the application thereof. The probe comprises the nanometer fine particles or colloid fine particles and the affinity element or antibiotics. The preparation method of the gene probe comprises: (1) colloidal gold or nanometer particle preparation and (2) compound preparation. The probe can be used for mutation and non-mutation detection of DNA, and a detection signal is enhanced by adding a silver reinforcing agent or a gold reinforcing agent during the detection. The method has the advantages of high sensibility, good specificity, convenience, rapidness and low price, the filtration can be carried out in high flux and low cost, the method is used for monogenic detection or polygene integration detection, such as clinic detection of many infective microbial pathogens, cancer early diagnosis, prenatal diagnosis of hereditary diseases, conventional physical examination in vitro, gene expression regulation and control researches, characteristic gene expression spectrum detection, gene library filtration, etc., and the probe is particularly suitable for detecting gene chips with low integrity and has better clinic use value.
Description
Technical field
The present invention relates to a kind of technique of gene detection, more particularly, relate to nanometer or colloidal particle and avidin mixture label probe and preparation method thereof, and this probe associating silver enhancing/golden enhanced detects the method for DNA.
Background technology
Gene studies has driven developing rapidly of whole life science of 20th century; In 21 century, gene studies will promote life science and develop in depth, and gene test is not only most important to biological study, and fields such as clinical medicine, pharmacy, agrotechnique, environmental monitoring, science of law evaluation also are extremely important.
Gene test is general adopts directly order-checking or hybridization to detect.Hybridization detects radioactive method, enzyme linked immunosorbent assay, chemiluminescent labeling etc.Radioactive method is highly sensitive, but because of it has pollution to environment, is replaced by the non-radioactive method just gradually.Enzyme linked immunosorbent assay is owing to the reaction process complexity, and the substrate for enzymatic activity reaction is too fast, and color development area is not concentrated, and has limited its use, particularly is not suitable for the application in biochip field.Fluoroscopic examination exists process complexity, detecting instrument costliness, operator and needs through training, thereby is difficult to shortcoming such as promotion and application.
Other marking method of chemiluminescent labeling, common argentation and some then makes its application be very limited owing to sensitivity is lower.So far, the gene chip detection method of comparative maturity is the laser scanning co-focusing microscope method, but its program complexity, the test set costliness is difficult to promotion and application.
Chinese patent CN1339609A discloses a kind of gene probe of nanoparticle mark and the application in gene test thereof, this technology is because the sodium rice particle instability for preparing, reasons such as easy gathering exist cost of manufacture height, poor stability and awkward defective.
At present, some biomolecules is widely used in different diagnosis and screening chip by medicine, biotechnology personnel with other intermolecular specific binding abilities.When a kind of biomolecules had specificity bonding force with another kind of biomolecules, these two molecules just can form effective ligand-receptor connection, as betided non-covalent combination the between avidin and small molecules vitamin H.Effect between this vitamin H and the avidin is widely used in DNA, RNA and proteic quantitative and purifying.These technology are applied to antigenic location, immunodetection, DNA hybridization technique in cell, the tissue.But, do not solve because connectivity problem between nano particle and DNA and preservation problem thereof are also basic, so it promotes the use of the also actual difficulty of existence.As the disclosed technology of CN1339609A patent.
Summary of the invention
The technical issues that need to address of the present invention one of are to provide a kind of nanoparticle and avidin label probe, have avoided nano particle that prior art exists and the connectivity problem between DNA;
Two of the technical issues that need to address of the present invention are the preparation methods that disclose above-mentioned probe;
Three of the technical issues that need to address of the present invention are to disclose the application of above-mentioned probe in gene test, to overcome the problem and shortage that existing detection technique exists.
Technical conceive of the present invention is such:
Streptavidin is a kind of tetramer albumen, and its each monomer is by hydrophilic bond and Van der Waals force and the stable and difficult fracture of chain that forms between the two after a vitamin H combines.Therefore, utilize the avidity between vitamin H and Streptavidin,, utilize the detection probes reaction of good avidin of mark or Streptavidin again with a biotin labeled probe and example reaction,
The present invention is with surperficial synthetic chemistry, biological chemistry and the Measurement for Biochemistry preparation avidin/Streptavidin/antibiotin-nanometer/colloidal particle mixture that combines, utilize the more stable key of the formation between vitamin H and avidin, to solve the connectivity problem between nano particle and DNA.Provide the painted minim DNA that carries out of silver particles/gold particle to detect the most up-to-date techniques of amplifying, solve the key issue that visual method detects with signal.According to T.Andrew Taton etc. at Scanmetric DNAArray Detection with nanoparticle Probes (science, 289, when mentioning employing nanoparticle-probe complex 2000), great variety takes place at one in than the narrow temperature scope in the specificity combination rate of DNA and the ratio of single base mismatch rate.Therefore, can utilize DNA-biotin-avidin/Streptavidin/antibiotin-nanometer/distinctive physical properties of colloidal particle mixture to be used for the detection of mutant.
Technical scheme of the present invention:
Nanoparticle of the present invention and avidin label probe are a kind of mixture, be made of nanometer or colloidal particle and avidin, Streptavidin or antibiotin, avidin, Streptavidin or antibiotin engage with nanometer or colloidal particle by the CNHO group.
The nanometer of being addressed or the chemical composition of colloidal particle comprise gold and silver, iron etc., and its particle diameter is 5-160nm.
The preparation method of gene probe of the present invention comprises the steps:
(1) preparation of Radioactive colloidal gold or nanoparticle: the preparation of Radioactive colloidal gold or nanoparticle can adopt conventional chlorauric acid solution to boil reduction method, controls particle diameter by adding reductive agent and stirring velocity, also can take additive method to prepare.As adopting the disclosed method of CNI1339609A patent, this technology is conventional technology, and the present invention repeats no more.
(2) preparation of mixture: the self-assembly of mixture and sealing have only strict control reaction conditions, and avidin/Streptavidin/antibiotin is attached on nanometer gold or the nanoparticle firmly, and use the bovine serum albumin branch to close unnecessary avtive spot.
Use K
2CO
3(salt of wormwood) or Tris alkali (Tutofusin tris alkali) are regulated the pH of Radioactive colloidal gold or nano-particle solution to set point value 6-8, centrifugal removal throw out, avidin, Streptavidin or antibiotin are joined in Radioactive colloidal gold or the nano-particle solution, and the weight ratio of avidin/Streptavidin/antibiotin and Radioactive colloidal gold or nanoparticle is:
Avidin/Streptavidin/antibiotin: Radioactive colloidal gold or nanoparticle=1: 1～1: 3;
The room temperature placement adds the bovine serum albumin branch and closes unnecessary avtive spot after 10-40 minute, room temperature was placed after 2～6 hours again, left the heart 20～60 minutes in 16000～25000/ minutes, get supernatant, it is resuspended to add the solution that contains PBS (phosphoric acid buffer) and stablizer such as glycerine, can repeat for several times, to remove unsettled nanometer gold/Radioactive colloidal gold, impurity such as free protein, it is added concentration is the sodium-chlor of 0.1～0.2M, 20～60mM phosphoric acid buffer, 0.1 the BSA of～0.5wt% (bovine serum albumin), 0.05 in the solution system of the sodium azide of～0.15wt%, the pH value of this system is 6～8, promptly obtains nanoparticle of the present invention and avidin label probe, 4 ℃ of preservations.
Nanoparticle of the present invention and avidin label probe can be used for dna mutation and nonmutationed detection, and detection method comprises the steps:
(1) contains the oligonucleotide of biotin (vitamin H) mark, and be fixed on the solid phase carrier detection probes and hybridize.This step is a conventional steps, and the present invention repeats no more.
(2) mixture and hybrid reaction:
1. for detecting T
mValue differs less mutant, hybridization back PBS, 0.1%Tween-20 washing back adds with 1xPBS (phosphoric acid buffer), 0.1 the mixture of the Avidin/streptavidin-Au of～0.5wt%BSA (bovine serum albumin) solution dilution, room temperature temperature are bathed after 0.5～1 hour washing lotion (as SSC or the SSPE solution) wash-out under 30～60 temperature with high tight degree.Again with after the PBS washing, add silver-colored toughener or golden toughener strengthens detection signal, repeatedly wash seasoning at last with distilled water.
2. detect for other hybridization, then can be after hybridization direct wash-out, bathe with mixture temperature again, after PBS washs, add silver-colored toughener or golden toughener strengthens detection signal,
(3) result judges:
Every painted very dark, be spot, the powerhouse is judged to strong positive with background reflectance; Painted darker, the powerhouse is judged to the positive with background reflectance; Painted lighter, little person is judged to the weak positive with background reflectance; The tinter is not judged to feminine gender.
Detection method provided by the invention has following advantage:
1. highly sensitive, specificity is good, easy, quick, cheap.
2. can carry out high-throughput, screening cheaply, fully need not expensive fluid operated mechanical manipulator with manual finishing.Can detect mutant, only need visual differentiation result, need not to utilize expensive detection equipment.
3. both having can be used for single-gene detects, also can be used for the integrated detection of polygene, as the clinical detection of multiple infectious microorganism pathogenic agent, early diagnosis of cancer, inherited disease antenatal diagnosis with at external routine physical examination, gene expression regulation research, the characterizing gene express spectra detects, gene library screenings etc. are particularly useful for low integrated level gene chip and detect, and good clinical use value is arranged.
Description of drawings
Fig. 1 is the good Streptavidin nano-Au composite synoptic diagram of mark.
Embodiment
Embodiment 1
The preparation (100ml) of nanometer gold (Radioactive colloidal gold):
With 0.01%HAuClO
4(hydrochloro-auric acid) is heated to and boils.Stir and add 4ml, the trisodium citrate (Na of 1wt% down
3C
6H
5O
72H
2O), continued heated and boiled 15 minutes, be cooled to room temperature under stirring, return to original volume with distilled water;
The preparation of Streptavidin-Radioactive colloidal gold:
K with 0.2M
2CO
3(salt of wormwood) or 1M Tris alkali are regulated the pH to 6.5 of colloidal gold solution;
It is centrifugal to get the nanometer or the Radioactive colloidal gold that prepare, 10000 rev/mins, centrifugal 5 minutes, remove throw out, the Streptavidin of 60ug is added in the colloidal gold solution of 10ml, room temperature was placed after 4 hours, again 20000 rev/mins centrifugal 40 minutes, remove supernatant, it is resuspended to add the solution contain PBS and glycerine stablizer, repeat 3 times, to remove impurity such as unsettled nanometer gold/Radioactive colloidal gold, free protein.
Adding final concentration at last is 0.15M NaCl, the 20mM phosphoric acid buffer, and 0.1%BSA, in the solution system of 0.05% sodium azide, 4 ℃ of preservations.As shown in Figure 1.
Embodiment 2
Mycobacterium tuberculosis detects the Rifampin resistance
The solid phase carrier that fixes is placed hybrid pipe, add 45～60 ℃ of hybridization of 6x SSPE/0.5%SDS hybridization solution 2 hours contain the pcr amplification product behind the alkaline denaturation.Solid phase carrier after the hybridization is taken out, be placed in 10 minutes with 1x PBS, tween 20 room temperature washing in the solution of the embodiment 1 that uses 200 times of dilutions of 1xPBS/0.1%BSA solution, the room temperature temperature was bathed 1-2 hour.Again solid phase carrier is placed hybrid pipe, with 50-65 ℃ of wash-out 10-20 of 1 * SSPE elutriant minute.
Take out solid phase carrier and wash 3 times with 1xPBS, each 5 minutes, add the silver-colored toughener (mixture that Silver Nitrate of now joining and reductive agent constitute) that configures, reacted 10 minutes, repeatedly wash the back seasoning with distilled water at last.
The result judges
Every painted very dark, be spot, the powerhouse is judged to strong positive with background reflectance; Painted darker, the powerhouse is judged to the positive with background reflectance; Painted lighter, little person is judged to the weak positive with background reflectance; The tinter is not judged to feminine gender.
Claims (6)
1. nanoparticle and avidin label probe is characterized in that being made of nanometer or colloidal particle and avidin, Streptavidin or antibiotin, and avidin, Streptavidin or antibiotin engage with nanometer or colloidal particle by the CNHO group.
2. probe according to claim 1 is characterized in that the nanometer addressed or the chemical composition of colloidal particle comprise gold and silver or iron.
3. probe according to claim 2, the particle diameter that it is characterized in that nanometer or colloidal particle is 5-160nm.
4. according to the preparation method of claim 1,2 or 3 described probes, it is characterized in that comprising the steps:
(1) preparation of colloid or nanoparticle;
(2) preparation of mixture: use K
2CO
3Or Tutofusin tris alkali is regulated the pH of colloid or nanoparticle solution to set point value 6-8, centrifugal removal throw out, avidin, Streptavidin or antibiotin are joined in colloid or the nanoparticle solution, and the weight ratio of avidin/Streptavidin/antibiotin and colloid or nanoparticle is:
Avidin/Streptavidin/antibiotin: colloid or nanoparticle=1: 1～1: 3;
Add the unnecessary avtive spot of bovine serum albumin sealing, after room temperature leaves standstill 2～6 hours again, 16000～25000 rev/mins of centrifugations 20～60 minutes, get supernatant, the solution that adds phosphoric acid damping fluid and stablizer is resuspended, remove impurity, its adding is contained in the solution system of sodium azide of bovine serum albumin, 0.05～0.15wt% of sodium-chlor that concentration is 0.1～0.2M, 20～60mM phosphoric acid buffer, 0.1～0.5wt%, the pH value of this system is 6～8, promptly obtains nanoparticle of the present invention and avidin label probe.
5. preparation method according to claim 4 is characterized in that, room temperature is placed and added the unnecessary avtive spot of bovine serum albumin sealing after 10-40 minute.
6. according to the application of claim 1,2 or 3 described probes, it is characterized in that being used for dna mutation and nonmutationed detection.
7. application according to claim 6 is characterized in that detection method comprises the steps:
(1) contains biotin labeled oligonucleotide, and be fixed on the solid phase carrier detection probes and hybridize;
(2) mixture and hybrid reaction:
1. for detecting T
mValue differs less mutant, hybridization back phosphoric acid buffer, 0.1%Tween-20 washing back adds uses the 1x phosphoric acid buffer, 0.1～0.5wt% bovine serum albumin solution is diluted to the mixture of avidin/antibiotin one gold medal of working concentration, working concentration is to count 0.1ng/ml-20ng/ml with bonded avidin/antibiotin amount on the nanometer gold, the room temperature temperature is bathed after 0.5～2 hour with SSC or SSPE solution wash-out under the temperature of room temperature～60 ℃, again with after the phosphoric acid buffer washing, add silver-colored toughener or golden toughener strengthens detection signal, repeatedly wash seasoning at last with distilled water;
2. detect for other hybridization, then can be after hybridization direct wash-out, bathe with mixture temperature again, after phosphoric acid buffer washs, add silver-colored toughener or golden toughener strengthens detection signal;
(3) result judges:
Every painted very dark, be spot, the powerhouse is judged to strong positive with background reflectance; Painted darker, the powerhouse is judged to the positive with background reflectance; Painted lighter, little person is judged to the weak positive with background reflectance; The tinter is not judged to feminine gender.
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|---|---|---|---|
| CNB02137418XA CN100351392C (en) | 2002-10-14 | 2002-10-14 | Marking probe of nano microparticle and affinity element and its preparation method as well as application |
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| CNB02137418XA CN100351392C (en) | 2002-10-14 | 2002-10-14 | Marking probe of nano microparticle and affinity element and its preparation method as well as application |
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| CN100351392C true CN100351392C (en) | 2007-11-28 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101929997B (en) * | 2008-12-26 | 2014-03-19 | 上海透景生命科技有限公司 | Method for improving sensitivity of immune detection |
| CN102565382B (en) * | 2012-01-06 | 2013-12-04 | 苏州浩欧博生物医药有限公司 | Immunochromatography method for detecting allergen-specific IgE antibodies in blood samples |
| CN103267854B (en) * | 2013-05-03 | 2015-08-05 | 西安交通大学 | A kind of method strengthening detection paper signal |
| CN103389382B (en) * | 2013-08-07 | 2015-02-25 | 中国科学院广州生物医药与健康研究院 | Signal-enhanced test strip biosensor for detecting histone methylation |
| CN103868914A (en) * | 2014-02-17 | 2014-06-18 | 南昌大学 | Aptamer-based method for detecting adenosine through nanogold and hydroxylamine amplification chemiluminiscence |
| CN107058584A (en) * | 2017-06-07 | 2017-08-18 | 浙江殷欣生物技术有限公司 | A kind of nucleic acid hybridizes chemical luminescence detection method |
| CN116930481A (en) * | 2023-09-12 | 2023-10-24 | 重庆医科大学绍兴柯桥医学检验技术研究中心 | Cross-molecule detection method for magnetic field driven micro-nano motor |
| CN117368464A (en) * | 2023-10-12 | 2024-01-09 | 上海领检科技有限公司 | Nano gold microsphere compound and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1392269A (en) * | 2002-06-11 | 2003-01-22 | 刘全俊 | Nano gold mark silver dyeing detection method of gene chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1392269A (en) * | 2002-06-11 | 2003-01-22 | 刘全俊 | Nano gold mark silver dyeing detection method of gene chip |
Non-Patent Citations (2)
| Title |
|---|
| 纳米磁性粒子在DNA分离与纯化中的应用进展 孙敏莉.生物工程学报，第17卷第6期 2001 * |
| 链霉亲和素-胶体金探针的制备及应用 丁杰.中华微生物学和免疫学杂志，第12卷第6期 1992 * |
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