CN1148457C - Nanometer particle mark gene probe and its preparing method and use - Google Patents
Nanometer particle mark gene probe and its preparing method and use Download PDFInfo
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- CN1148457C CN1148457C CNB011335270A CN01133527A CN1148457C CN 1148457 C CN1148457 C CN 1148457C CN B011335270 A CNB011335270 A CN B011335270A CN 01133527 A CN01133527 A CN 01133527A CN 1148457 C CN1148457 C CN 1148457C
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Abstract
The present invention relates to a gene detecting technology, more specifically a nanometer particle mark gene probe, a preparing method and the application thereof. The nanometer particle mark gene probe replaces traditional mark probes to respectively overcome the problems and the defects of the traditional mark probes, so the nanometer particle mark gene probe can trend to the practical application from laboratories. The nanometer particle mark gene probe has the preparation method: the preparation of colloidal gold or nanometer particles, the design, synthesis and retouching of DNA probe molecules, and the self-assembling synthesis and sealing of a gold mark probe. The probe is suitable for hybridization detection, nanometer amplification and visual color display of various genes and is particularly suitable for the application field of gene chips in a low integrity diagnosis type.
Description
One, technical field
The present invention relates to a kind of technique of gene detection, more particularly, relate to nanometer particle mark gene probe and preparation method thereof and the application in gene recombination.
Two, 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.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.After directly order-checking is exactly direct separation, amplification, purified genes fragment, adopts terminal chain termination reaction principle to measure each Nucleotide in the nucleic acid and form and put in order.Sequence measurement has that isotropic substance, silver dye, four look fluorescent mark laser co-focusing order-checkings etc., and these methods all need complicated program, the expensive reagent and the equipment of supporting costliness.By contrast, hybridization detects carries out gene tester simply, conveniently, and therefore, gene sensor and gene chip etc. is a kind of method that detects based on hybridization.
The marking method that gene probe is carried out in the tradition gene test has radioactive method, enzyme linked immunosorbent assay, chemiluminescent labeling etc.; In recent years, argentation also is used widely in dna sequence analysis.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.Other marking method of chemiluminescent labeling, common argentation and some then makes its application be very limited owing to sensitivity is lower.
When the appearance of gene chip makes extensive bioinformation storage be treated as reality.So far, the gene chip detection method of comparative maturity is the laser scanning co-focusing microscopy, but its program complexity, the test set costliness is difficult to promotion and application.
Three, summary of the invention
The objective of the invention is to overcome the problem and shortage that existing technique of gene detection exists, a kind of nanometer particle mark gene probe that is made of nano-particle surface covalent attachment oligonucleotide strand gene probe molecule is provided; Another object of the present invention provides a kind of preparation method of nanometer particle mark gene probe of the present invention; A further object of the invention is that gene probe of the present invention is detected as gene recombination, is particularly suitable for the application of low integrated level diagnosis type gene chip.
The object of the present invention is achieved like this, is about to surperficial synthetic chemistry, biological chemistry and physical chemistry technology and combines, the gordian technique in solving the design of visual method technique of gene detection and making; Provide the painted minim DNA that carries out of two probe hybridization silver particles to detect the most up-to-date techniques of amplifying with information, and then research has the visual method gene chip detecting system that several kinds of target gene detects that can be used in of practical value, clinical as hepatitis communicable diseases such as (seven kinds of hepatitis viruss) or genetic diseases with visual method gene chip diagnosis system, promote the gene diagnosis development of technology.The present invention includes the content of three aspects: the preparation method of nano gene probe, this kind probe, the application of this kind probe.Its innovative point is the probe preparation, and nanometer is amplified, and visual colour developing major technique is applicable to the gene test of all kinds of purposes, is particularly suitable for low integrated level gene chip field.
1, probe preparation, i.e. the preparation of gene probe.This probe is to be made of 100～1000 oligonucleotide strands of nano-particle surface covalent attachment gene probe molecule, the chemical constitution of nanoparticle comprises the gold and silver atom, Cadmium Sulfide, ferric oxide, silicon oxide equimolecular, particulate combines by disulfide linkage, ehter bond, thiamines ester bond with oligonucleotide, there is the aliphatic hydrocarbon arm junction of oligonucleotide and particulate, and oligonucleotide is single stranded DNA or RNA.
The preparation method of probe and precaution comprise as follows:
1. the preparation of Radioactive colloidal gold or nanoparticle by chlorauric acid solution is boiled reduction method, is controlled particle diameter by adding reductive agent and stirring velocity at present.Also can adopt method preparations such as reversed micelle method.General 8～160 nanometers of using of particle diameter.
2. the synthetic and modification of dna probe molecule design and trust, to design different DNA base sequence and connect different groups according to different purposes, as be used for the probe of commissure to the slide, except adding the amino isoreactivity group, also should increase sterically hindered when overcoming hybridization of fatty acid chain or non-specific Nucleotide at 5 ' end.The dna probe that is used for the nanoparticle mark sterically hindered when considering hybridization, the more important thing is to consider to use to be easy to and nanoparticle commissure fixed active group, (SH) as sulfydryl.
3. the synthetic and sealing of the self-assembly of nanometer label probe, this is the key that whether realizes, has only strict control reaction conditions, just can make and reach hundreds of gene probe firmly to be covalently bound on the nanometer gold, after using the unnecessary avtive spot of bovine serum albumin or polyoxyethylene glycol or salmon sperm dna sealing, hybridization through follow-up just can make the silver particles specific enrichment.
2, nanometer is amplified, main by " golden labeled oligonucleotide probe " or nanoparticle probe be fixed to the complementation specifically of on-chip pair of probe such as wave plate, nylon membrane, nitrocellulose filter and purpose or dna fragmentation to be checked and hybridize, form a kind of multistage three-dimensional accumulative three-dimensional arrangement by hydrogen bond, rely on for support with substrate on the fixed probe form chemical bond and will hybridize aggregate and be anchored on the solid support, realize the molecular aggregates amplification of hybridization signal.
3, visual colour developing, be on the basis that above-mentioned specific hybridization aggregate forms, silver salt (as Silver Nitrate) and reductive agent are added in the hybridization system, at first be attracted to around the electronegative nucleic acid fragment positively charged silver ions and the nano gold spherical surface by electrostatic interaction, be reduced into silver atoms again,, around the hybridization aggregate, demonstrate silver gray, thereby hybridization signal is amplified more than 1,000,000 times in nucleation theory effect deposit.On optical effect, realize visual colour developing, observed the gene recombination result.
The present invention has the following advantages and positively effect:
1. have highly sensitive, specificity good, easy, quick, cheap characteristics.
2. both having can be used for single-gene detects, also can be applicable to the integrated detection of polygene, as the clinical detection of multiple infectious microorganism cause of disease, early diagnosis of cancer, inherited disease antenatal diagnosis with at the body 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.
3. nanoparticle of the present invention is applicable to that also the immunohistochemical methods that adopts immunology principle detects, as the protein chip detection etc.
Four, description of drawings
Fig. 1 is a invention process step skeleton diagram;
Fig. 2 is a silanization processing reaction molecule mechanism synoptic diagram.
Wherein: the synthetic and modification of 1-gene probe, 2-nanoparticle prepare and characterize, golden probe preparation and the sign marked of 3-silanization processing 4-, 5-carrier surface probe stationary, 6-hybridization is amplified with nanometer, and 7-pattern recognition system is supporting, 8-gene visual detection, the 9-interpretation of result.
As shown in Figure 1, realization of the present invention and application comprise 9 big steps: the skill of each step Art requires all higher, must strictly control technical process by the professional and technical personnel, can reach The standard of the described genetic test of new method.
1. the synthetic and modification 1 of gene probe mainly is to perform MOLECULE DESIGN, generally relies on molecule for support The biology design software is finished. As mentioned above, be according to the different DNA alkali of different purposes designs Base sequence also connects different groups. Total requirement is that to be convenient to probe and solid support commissure solid Fixed and be convenient to abundant hybridization.
2. nano particle preparation and sign 2, we use the heat reduction method, also can use oppositely Other method such as micella. Prepare receiving of different-grain diameter and external form according to different genetic test needs The rice particulate. The sign of nano particle can be used transmission electron microscope, Rayleigh (Rayleigh) Scattering spectrum, X-ray diffraction, now existing special nano measurement instrument is sold.
3. silanization processes 3, uses the slide processing method in the existing biochip technology, slightly Add improvement.
4. the preparation of Nanoparticle labeling probe and sign 4 refers to the preparation of gold mark gene probe. Select The nm of gold of suitable particle size, generally select 8～160nm (resolution requires more high particle diameter more little, Concentration is 10～20nM) and designs, synthesizes and use alkyl thiol for the genes of interest fragment in advance Modifying good concentration is the DNA oligonucleotide aqueous solution of 1-10 μ M, is 15-40 in temperature ℃ reaction, the pH value of control reaction is between the 6-8, the time is 10-20 hour, continuation At 0.1M NaCl, place after 30-50 hour 12000-in the 10mM phosphate buffer The centrifugal supernatant that goes of 16000rpm adds 0.1M NaCl, 10mM phosphate buffer cyclic washing Three times, add at last 0.3M NaCl, 10mM phosphate buffer (pH7), 0.01% Azide Sodium, 4 ℃ of preservations. Product is stored refrigerated. Characterizing method adopt at present ultraviolet absorption method and Rayleigh scattering method (XShe Xianfenxi).
5. the carrier surface probe fixes 5, adopts direct point sample method, concentration and probe concentration 10-5～10
-11Mole. To keep wetting and germ-free condition in the course of reaction.
6. hybridization amplifies 6 with nanometer, as previously mentioned.
7. pattern-recognition system is supporting 7, good as a result decision method, the mark of self-explanatory characters' elder generation's design code Accurate; Join with the identification, scanning and the analytical equipment that detect corresponding signal identification software and necessity Cover.
8. the gene visual detection 8, dye to see whether show by amplifying silver through nanometer behind the Probe Hybridization Look shows the dun gene recombination signal that has been, and can detect by an unaided eye directly to judge inspection Survey the result.
9. interpretation of result 9, with above-mentioned colour developing result, and after pattern-recognition and analyzing and processing, With testing result arrangement output.
As shown in Figure 2, surface of glass slide has been passed through a series of chemical changes in processing procedure, by Silanization coupling agent and/or arm molecule are covalently bound to oligonucleotide probe on the slide, are convenient to The steady implementation of later step. If solid phase carrier is with nitrocellulose filter, nylon membrane, nanometer Grain etc. can be processed without silanization, only needs activation process.
Flow process of the present invention is as follows:
1, substrate surface treatment
Detection chip take slide as substrate is example:
Boil with concentrated nitric acid and to wash slide 10 minutes, with distillation washing 3 times, put into 40 ℃ of oven dry of baking oven again.
Get 1.5 ml waters, 28 milliliters of acetone and 600 microlitre silanization coupling agent PDITC (benzene 2 lsothiocyanates) add in the container, slide are immersed wherein 40 minutes again.
With acetone (analytical pure) washing slide 5 times (5 minutes/time), again slide is dried.
Slide is put into 0.2%PDITC solution, placed baking oven 2 hours (37 ℃ of holding temperatures).
From PDITC, take out slide, with methanol wash 5 times (5 minutes/time), use washing with acetone again 2 times earlier, with the distilled water washing once, dry at last.
With optical density value is that 5 ' of 1OD (absorbance)-amino oligonucleotide is dissolved in 100 μ l (microlitres, volume unit) TE (1 * Tris-EDTA damping fluid, the molecular biology common agents) in, then with single stranded DNA point on slide, slide was placed 5 hours at 4 ℃, changed over to moist environment and placed 2 hours for following 40 ℃.
2, the preparation (100ml) of nanometer gold particulate (Radioactive colloidal gold)
HAuClO with 0.01%
4(hydrochloro-auric acid) is heated to and boils.Stir and to add 2-4ml, 1% trisodium citrate (Na down
3C
6H
5O
72H
2O), continued heated and boiled 15 minutes.Stop heating, be cooled to room temperature under stirring, return to original volume with distilled water, particle diameter 8～140 nanometers.
3, the preparation of nanometer gold microparticle probes
Use 0.1M Na
2CO
3(sodium carbonate) or 1M Tris alkali are regulated the pH of colloidal gold solution to set point value, and selecting pH7 ± 0.5 is pH value in reaction.Can select nucleic acid to be marked or isoelectric points of proteins in principle, also can inclined to one side slightly alkali.But optimal reaction pH often needs could determine through test of many times usually.
Get the nano gold spherical 1ml (14nm) for preparing, centrifugal (10000 turn left the right side) remove throw out.16000-25000 leaves heart 30-50 minute again, removes supernatant, adds 1ml (0.1M NaCl, 10mM phosphoric acid buffer, 0.2% PEG (polyoxyethylene glycol) or BSA (bovine serum albumin)) solution again, and ultrasonic spin-up repeats 3 times, to remove foreign material.
1OD DNA is dissolved among the 100 μ lTE (1TE), add in the above-mentioned 1ml nano gold spherical solution, room temperature was placed 1 hour, spend the night 4 ℃ of placements then, it is 0.3M NaCl that centrifuge washing goes to add final concentration behind the supernatant, the 10mM phosphoric acid buffer, in the solution system of 0.01% sodiumazide, 4 ℃ of preservations are standby.
4, hybridization
The target sequence of getting PCR (polymerase chain reaction) amplified production or directly extracting is dissolved in 100 μ lTE (in 1 * TE), getting 1-10 μ l is dissolved among 1-50ml 8 * PBS (phosphoric acid buffer) (concentration is greater than 100nM), the slide that fixes DNA is hybridized with it (50 ℃ 1 hour, 4 ℃ of placements are spent the night), then, add Nano-Au probe 1-50 μ l (concentration is greater than 5nM), continue hybridization 2-4 hour.
5, dyeing
(1) solution preparation:
A, silver-colored developing solution:
* silver lactate developing solution
A.10% the gum arabic aqueous solution or 1% gelatin 60ml
B. citrate buffer (pH3.5) 10ml
C. Resorcinol 0.85g adds water 15ml dissolving
D. silver lactate 0.11g adds water 15ml dissolving
The preceding hybrid filtering of above a～c adds d. at last and notes lucifuge.
* Silver Nitrate developing solution:
A.10% the gum arabic aqueous solution or 1% gelatin 60ml
B. citrate buffer (pH3.5) 10ml
C. Resorcinol 1.7g adds water 30ml dissolving
D. Silver Nitrate 50mg adds water 2ml dissolving
The preceding hybrid filtering of above a～c adds d. at last and notes lucifuge.
More than in two kinds of developing solutions, as replacing gum arabic or gelatin with distilled water, then color speed is obviously accelerated, and control the colour developing degree under mirror.
B, citrate buffer
Citric Acid (C
6H
8O
7H
2O) 2.55g
Citric Acid trisodium (C
6H
5Na
3O
7H
2O) 2.35g
Adding distil water 100ml dissolving.
C, stop bath
20% Sulfothiorine
(2) development step:
Slide after the hybridization is taken out, use 8 * PBN rinsing respectively 3 times, each 2 minutes.Silver staining reaction: gene chip is used deionized water wash 3 * 6 minutes, remove Cl
-(chlorion) then soaked 3 minutes in 0.2M pH3.5 citrate buffer solution, then slide put into the plate effect 10～15 minutes that developing solution is housed, and slide put into stop bath 3 minutes again, took out with behind a large amount of deionized water wash seasoning.
Judge: every painted very dark, grey black spot of being uniformity is judged to strong positive (+++) with the background reflectance powerhouse; Painted darker, be judged to the positive (++) with background reflectance than the powerhouse; Painted light, little person is judged to the weak positive (+) with background reflectance; The tinter is not judged to feminine gender (-).
Five, embodiment
Example 1, nanometer gold silver are amplified visual development process and are detected people's second, hepatitis C virus pathogenic genes, and its flow process is as follows:
(1) PDITC (1, the 4-benzene diisothio-cyanate) solution of preparation 0.2%
1. mixed solvent
Pyridine: DMF (N, dinethylformamide)=1: 9 (noting it being anhydrous solvent), example: 0.19gPDC+10ml pyridine+90mlDMF is the PDITC solution of 100ml0.2%.
2. the processing of slide
Boil with concentrated nitric acid and to wash slide 10 minutes, with the distillation washing, put into oven for drying again.
3. the preparation of oligonucleotide solution
With optical density value is that the 5 '-amino oligonucleotide of 1.0 (about 33ug) is dissolved in 100ulTE (in 1 * TE), concentration is about 3.35 * 10-5mol/L.5 '-amino oligonucleotide sequence: 5 '-NH
2-GGA CTT CTC TCA ATT TTC TAG GG-3 '
(2) silanization of slide is handled, capture dna and slide crosslinked
1. activation
Get 1.5 ml waters, 28 milliliters of acetone and 600 microlitre silanization coupling agents add in the culture dish, slide are immersed wherein 40 minutes again.
With washing with acetone slide 5 times (5 minutes/time), again slide is dried.
2. PDC is crosslinked
Slide is put into 0.2%PDITC solution, placed baking oven 2 hours (keeping 37 degrees centigrade of humidity).
From PDITC, take out slide, with methanol wash 5 times (5 minutes/time), use washing with acetone again 2 times earlier, with the distilled water washing once, dry at last.
3. dna probe is fixing
With strand hepatitis B and C dna probe point different site on slide, then slide to be placed 5 hours at 4 ℃, moist environment was placed 2～4 hours for following 40 ℃.
(3) the nanoparticle gold and silver amplifies visual development process detection gene
1. the preparation of Radioactive colloidal gold is the same.
2. on nano gold spherical, modify HS-DNA.Technology is the same, and the second hepatitis C virus specific gene probe of band sulfydryl modification is by entrusting the synthetic HS-DNA of specialized company after the specialized designs software design sequence.
(4) goal gene obtains and prepares.Extract viral nucleic acid or carry out pcr amplification as masterplate with conventional Protocols in Molecular Biology, make pathogenic genes reach the detection minimum concentration with this.
(5) hybridization.
Getting the 1OD target sequence is dissolved among the 100ulTE, get 1-10 μ l and be dissolved into (concentration is greater than 100nM) among 1-50ml8 * PBS, the slide that fixes DNA is hybridized with it (50 ℃ 2 hours, 4 ℃ of placements are spent the night), then, add Nano-Au probe 1-50 μ l (concentration is greater than 5nM), continue hybridization 2 hours.
(6) dyeing
1. silver-colored developing solution: Silver Nitrate developing solution, the same preparation of citrate buffer.
2. stop bath: 20% Sulfothiorine
3. colour developing
Slide after the hybridization is taken out, use 8 * PBN rinsing respectively 3 times, each 2 minutes.Silver staining reaction: gene chip is used deionized water wash 3 * 6 minutes, remove Cl
-, then in 0.2M pH3.5 citrate buffer solution, soaked 3 minutes, then slide is put into the plate effect 10～15 minutes that developing solution is housed, slide was put into stop bath 3 minutes again, take out with behind a large amount of deionized water wash seasoning.
Judge: criterion is the same.
(7) result.
The point that is fixed with the hepatitis B virus gene probe on the chip is hybridized the grey black spot that after stain is very dark, be uniformity with corresponding hepatitis B virus detection gold mark probe and hepatitis B positive serum pcr amplification product, strong with background reflectance. colour developing rate 100%, detect coincidence rate 100% with sepharose.Make the capture dna probe and contrast used fourth liver specificity cDNA, detect hepatitis B positive serum pcr amplification product, be blank spot, not painted, be judged to feminine gender.
Claims (1)
1, a kind of method for preparing nanometer particle mark gene probe comprises:
1. the preparation of Radioactive colloidal gold or nanoparticle;
2. dna probe molecule design and synthetic and modification;
3. the synthetic and sealing of the self-assembly of gold nano genetic marker probe;
It is characterized in that:
The synthetic nanometer gold of selecting granularity 8-160nm for use of the self-assembly of gold nano genetic marker probe, with design at target gene fragment in advance, synthetic and be that the DNA oligonucleotide aqueous solution of 1-10 μ M is 15-40 ℃ of reaction in temperature with the concentration that alkyl thiol is modified, the control reaction pH value is 6-8, time is 10-20 hour, continuation is at 0.1M NaCl, place after 30-50 hour in the 10mM phosphoric acid buffer, the centrifugal supernatant that goes of 12000-16000rpm, add 0.1M NaCl, 10mM phosphoric acid buffer repetitive scrubbing three times adds 0.3M NaCl at last, the 10mM phosphoric acid buffer, 0.01% sodiumazide, 4 ℃ of preservations.
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| CNB011335270A CN1148457C (en) | 2001-09-30 | 2001-09-30 | Nanometer particle mark gene probe and its preparing method and use |
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|---|---|---|---|
| CNB011335270A CN1148457C (en) | 2001-09-30 | 2001-09-30 | Nanometer particle mark gene probe and its preparing method and use |
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| Publication Number | Publication Date |
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| CN1339609A CN1339609A (en) | 2002-03-13 |
| CN1148457C true CN1148457C (en) | 2004-05-05 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100400677C (en) * | 2006-02-28 | 2008-07-09 | 武汉大学 | Fluorescent quantum dot marking DNA bioprobe, and its preparing method |
| CN101178362B (en) * | 2006-11-08 | 2011-03-09 | 北京金迪克生物技术研究所 | Establishment and application of novel E-LAMP |
| CN104384523B (en) * | 2014-09-23 | 2017-02-08 | 南京农业大学 | Preparing method of three-dimensional chiral silver nanometer material |
| CN105505755A (en) * | 2015-12-23 | 2016-04-20 | 杭州谷禾信息技术有限公司 | Space transcriptome database building and sequencing method and device adopted for same |
| CN105861646A (en) * | 2016-03-02 | 2016-08-17 | 成都市妇女儿童中心医院 | Electrochemical biological chip for detecting urinary tract pathogen 16SrRNA and technical application thereof |
| CN106198952A (en) * | 2016-07-01 | 2016-12-07 | 清华大学 | A kind of suppress the nucleic acid molecules enclosure method to sensing interface non-specific adsorption |
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2001
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