CN1850989A - High sensitivity biosensor gene chip and clinical diagnosis technology - Google Patents
High sensitivity biosensor gene chip and clinical diagnosis technology Download PDFInfo
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Abstract
The invention relates to the sensitive biosensor gene chip and the clinic diagnosing technic. The testing signal can be magnified to be distinguished by the naked eye using the nanometer material probe technic and the optics sensor technic. So the bottle-neck of the traditional gene chip technic that the large model instrument such as the laser scanner can be needed in the testing is break. The gene chip can be applied widely without the especial instrument.
Description
Technical field
The invention belongs to biological gene technology and field of biosensors, relate to a kind of biochip technology and clinical disease diagnosis technology and deutero-range gene diagnostic products thereof.
Background technology
Biochip technology is that a high-new biotechnology that grows up nineteen nineties (can be referring to Collins, F.S., Ahead of schedule and under budget:the Genome Project passes itsfifth birthday.Proc Natl Acad Sci U S A, 1995.92 (24): p.10821-3; Pease, A.C., etc., Light-generated oligonucleotide arrays for rapid DNA sequence analysis.Proc NatlAcad Sci U S A, 1994.91 (11): p.5022-6; And Shalon, D., S.J.Smith and P.O.Brown, A DNA microarray system for analyzing complex DNA samples using two-colorfluorescent probe hybridization.Genome Res, 1996.6 (7): p.639-45), its ultimate principle is with a large amount of target fragments in an orderly manner, be arranged in specific carrier matrix (as silicon higher density, glass, on fiber or the nylon membrane), be called gene chip (micro-matrix approach, Microarray) or at slide, or application conductor etching technology, the direct in-situ synthetic oligonucleotide probe.By the P.Brown of U.S. Stanford university invention (nineteen ninety-five), the latter has its patent by U.S. Affymetrix company the earliest for the former.Because it has flux height (detecting tens of to tens thousand of genes on demand), parallel analysis (carrying out under identical conditions) and the high degree of specificity advantages such as (can differentiate the change of single Nucleotide) that nucleic acid hybridization had are once finding to be the countries in the world widespread use.Biochip technology has become the basic fundamental in genomics and the back genomics research.
Early stage biochip technology is mainly used in high-throughput, the demand of broad scale research gene function and producing, but progress along with biochip technology, the pleasantly surprised discovery of people, except that original effect, gene chip is diseases related at range gene, has unique application prospect as the diagnosis of transmissible disease and heredopathia, Polymorphism Analysis etc.:
1. its detection fragment is all kinds of Disease-causing gene fragments, because the probe amount is big, the flux height significantly improves detection efficiency; Can once finish multiple disease and the multiple different states of same morbid state or/and the detection of genetic background;
2. use the nucleic acid hybridization technique with high degree of specificity in detecting, its specificity can be told the change of a nucleic acid, therefore, has the specificity of full accuracy.
3. detect and directly use nucleic acid, only need the sample of trace can finish diagnosis,, need not the immune response stage, can finish diagnosis in initial infection as to pathogen detection;
4. level of automation height, favorable repeatability can be adapted to detect and use on a large scale.
Yet, the detection of traditional biochip technology, the fluorescence radiation that is based on fluorescence labeling probe carries out relevant detection.A little less than its signal, must be through exciting, ability is luminous, needs expensive laser scanning equipment.In the clinical detection application process, need invest the large-scale laser scanning equipment of purchasing owing to detect unit, on the one hand, significantly increased the cost of investment of carrying out correlation detection, on the other hand, also directly increased the cost of using, limited this The Application of Technology scope significantly, and become the bottleneck problem of this technology in clinical expansion.Make this a pair of medical diagnosis on disease have high degree of specificity and high-throughout detection technique, the restriction of examined cost can't be used widely in clinical practice.But its potential social progress and huge industrial value impel people constantly to invent new technology and method.
Summary of the invention
One of purpose of the present invention is, the applying nano materials labeling technique replaces traditional fluorescent probe, change the physical optics characteristic of probe, probe is detected in visible spectrum range, solve the deficiency that fluoroscopic examination need be used main equipment, thereby in the gene chip application process, need not the large laser scanner, be convenient to the biochip technology widespread use.
Two of purpose of the present invention is, the applying biological sensor technology, visible light detection signal that will be more weak amplifies, and realizes chip detection signal naked eyes or by simple optical device interpretation, and can realize that the computer automation of chip handles by simply quantize equipment such as digital camera.
Three of purpose of the present invention is to use special positive signal treating processes, the check signal of acquisition high specific.
Four of purpose of the present invention is to use special genes probe and corresponding micromatrix, the specific diagnosis of realization disease.
For this reason, first aspect present invention relates to a kind of biochip, and this chip comprises: silicon dioxide substrates; Be present in the optical coating on this silicon dioxide substrates; With the Poly(Phe)-Methionin coating that is present on this optical coating.
In one embodiment of the present invention, the thickness of the silicon dioxide substrates of biochip of the present invention is 2-4mm, and optical coating comprises the silicon nitride of 350-550 dust and the poly-ammonia dimethyl-silicon alkane of T structure of 80-200 dust.
In another embodiment of the present invention, thereby this chip also comprises linking to each other with its optical coating by chemical bond and is fixed in the nucleic acid probe of chip surface.
In one embodiment of the present invention, the thickness of the Poly(Phe) of this chip-Methionin coating is the 20-200 dust.
The present invention also relates to a kind of method for preparing biochip of the present invention on the other hand, and this method comprises:
(1) on silicon dioxide substrates, plates with optical coating;
(2) on the coating of step (1) gained, plate again with Poly(Phe)-Methionin coating, obtain to have plated the silicon dioxide substrates of optical coating and Poly(Phe)-Methionin coating; (3) with the silicon dioxide substrates of hydrochloric acid succinimido hydrazine nicotinate treatment step (2) gained, clean, obtain described chip.
In one embodiment of the present invention, the concentration of this hydrochloric acid succinimido hydrazine nicotinate is 1-10 μ M, and the treatment time is 10-30 minute.
In another embodiment of the present invention, this method also comprises step (4): the point sample stationary probe, and make its chemical coating carry out the ammonium aldehyde combined reaction through room temperature and chip surface, form covalently bound compound, be fixed on the surface of chip.
The present invention also relates to the method for the nucleic acid to be checked in a kind of test sample on the other hand, and this method comprises:
(1) provides chip of the present invention, fixing in its surface and nucleic acid specificity bonded nucleic acid probe to be checked;
(2) make the described chip of step (1) with sample, contact with the label probe of nucleic acid specificity hybridization to be checked, wherein, this label probe is marked with vitamin H;
(3) carry out wash-out with NaOH solution, obtain not eluted product;
(4) the chain parent albumen with the Nano-Au probe mark contacts with the not eluted product that step (3) obtains, and forms nucleic acid probe-label probe-vitamin H-chain parent albumen-nano-Au composite; With
(5) detection chip signal is determined the existence of nucleic acid to be checked.
In one embodiment of the present invention, the concentration of NaOH is 0.0001-0.1M in the aforesaid method step (3).
Technical scheme of the present invention has following advantage:
(1) applying nano materials technology with traditional gene chip detection signal, by fluorescent probe technique, is converted to the visible light probe technique, has realized the visible light interpretation of gene signal, thereby not needing to have realized the difficult problem of main equipments such as fluorescent scanning instrument technically.
(2) Chang Gui nano material visible light probe technique is in process of clinical application, it is on the low side to exist sensitivity, during the interpretation of ordinary optical equipment, exist insufficient sensitivity and picked up signal is understable, for this reason, we have introduced biosensor technology, once more nano material probe signals specificity is amplified, significantly improve the detection signal specificity and the detection sensitivity of gene chip, more conventional chip detection technology for detection sensitivity improves more than 1000 to 1,000,000 times, realize the specific detection of gene chip signal fully, until the level of sensitivity that reaches the naked eyes interpretation.
(3) can realize the simple deciphering of gene chip signal.Range of application is wider, and being convenient to basic unit's clinical detection and suitable simple and crude field condition etc. is one of its maximum characteristics.Because using visible light spectrum and high-sensitive biosensor technology make chip signal reach naked eyes interpretation level, and can realize that automatization and mass detect by simple optical device.Therefore, in application, need not main equipment, very easily apply.
(4) adopt special high temperature interconnection technique, significantly improve the specificity of hybridization signal, reduce background signal, Signal-to-Noise significantly improves, and has realized the clear accurate interpretation that the signal of the single nucleic acid molecule difference of chip has and do not have.
(5) with low cost.Owing to need not main equipment during detection, use cost significantly descends and is easy to and promotes, and will promote popularizing and promoting of this new and high technology of gene chip, and promotes along with popularizing of technology reaches, and will further reduce chip cost.
(6) operating process is simple.Simplify and use, the clinical detection process time is significantly shortened, the signal processing of the uniqueness of passing through is realized the detection of specific signals; The testing process time short (except that external pcr amplification, the hybridization testing process only needs 20 minutes).
Description of drawings
Fig. 1 shows the yin and yang attribute signal.Figure 1A is negative, and 1B is positive.
Fig. 2 shows actual chips and detection signal thereof.
Fig. 3 display chip result's naked eyes interpretation.Fig. 3 A is the digital photograph of actual result, and 3B is the chip signal design diagram, appears at location in the chip by its positive signal, the mutation type of its positive signal representative of interpretation.
Fig. 4 shows the realization principle of special visible light signal on the thin film bio sensor chip.Wherein, Fig. 4 A capture probe is fixed on the biologic sensor chip of preparation by-NH-key; Fig. 4 B forms target fragment-capture probe-label probe double-stranded DNA by capture probe, and is on the basis of bridge in target fragment, and capture probe links with biotinylated probe; The label probe that can finish the link reaction behind Fig. 4 C wash-out and form links with capture probe and forms special strand, and one end and biosensor are connected and fixed by covalent linkage, and the other end has biotin labeling; The general probe that Fig. 4 D contains nanometer gold combines with vitamin H, finishes being connected of nucleic acid and nanometer gold, and the latter forms visible light signal, and amplifies and be naked eyes or the interpretation of simple optical device institute through biosensor.
Embodiment
One, detects the preparation of using the thin film bio sensor chip
1. the surface treatment of thin film bio transmitter:
At the about 10-20cm silicon chip of diameter (SO
2) go up plating with certain thickness silicon nitride (Si
4N
3) press the vaccum gas phase sedimentation method plated film, and (the T structure is gathered ammonia dimethyl-silicon alkane, UCT, Bristol, film PA) (Jenison, R., La, H., Haeberli, A., Ostroff, R.﹠amp with TSPS to rotate vacuum plating unit; Polisky, B. (2001) Clin.Chem. (Washington, D.C.) 47,1894-1900; Jenison, R., Yang, S., Haeberli, A.﹠amp; Polisky, B. (2001) Nat.Biotechnol.19,62-65), prepare corresponding biosensor and more routinely method plating with Poly(Phe)-Methionin coating, after 1-10 μ M hydrochloric acid succinimido hydrazine nicotinate (succinimidyl hydrazinium nicotinate hydrochloride) is handled about 10-30 minute (as 15-25 minute, preferable about 20 minutes), clear water is cleaned for chip manufacturing.
The thickness of this silicon chip can be the 1-5 millimeter, preferably the 2-4 millimeter; The thickness of this silicon nitride can be as 350-550 dust, preferred 400-500 dust; The thickness of this TSPS can be the 80-200 dust, as the 100-150 dust; The thickness of this Poly(Phe)-Methionin coating can be the 20-200 dust, as being 50-150 dust, 80-120 dust.
2. the design of gene chip micromatrix and realization
Free nucleic acid to be checked, (A-T C-G), is fixed in chip surface by combining with the specificity capture probe that is fixed in chip surface to the base complementrity principle of process Nucleotide.Nonspecific nucleic acid, fixing because of not catching, through elution process and by wash-out, thereby realize nucleic acid to be checked and other separate nucleic acid.
The design of gene trap probe C1: analyze all hepatitis B viruses (Hepatitis B virus in the existing National Library of Medicine gene pool by retrieval, HBV) gene order, the present invention selects the HBV fragment of high conservative, the synthetic corresponding probe of design and preparation, synthetic through the DNA of standard building-up process (AppliedBiosystems) or commercialization, high-efficient liquid phase chromatogram purification obtains chromatographically pure dna probe.And be the working fluid of 1 μ M concentration, and, design corresponding matrix and carry out point sample and making by the demand of medical diagnosis on disease with 0.1M PBS pH 7.8 damping fluids dilutions.
The matrix of forming by different capture probe in specific zone, and is fixed on the nucleic acid molecule at different physics position by detection with different purpose separate nucleic acid, realizes the interpretation of the molecule of specific nucleic acid.
3. the gene probe point sample and the chemically crosslinked of thin film bio sensor chip surface
Capture probe after point sample is fixing carries out the ammonium aldehyde combined reaction with the chemical coating of thin film bio sensor chip surface under room temperature, form covalently bound compound, stably is fixed in the surface of chip, and keeps good biologic activity; Again through clear water rinsing, drying; Can for preserved 1 year under the room temperature or more than.Through above technology, finished the making processes of thin film bio sensor chip.
Two, the realization of special visible light signal and reading on the thin film bio sensor chip
1. specificity detection probe preparation
(1) general gold nano-material probe and preparation thereof
What the label probe system of traditional gene chip adopted is that cy3 cy5 is fluorescein-labelled, and cy3, visible light must could take place through exciting in cy5, also is to detect the basic reason that needs expensive laser scanner in gene chip.Therefore, as obtaining visible light signal, must change the tagged molecule of probe in the detection, be the detection signal of suitable highly sensitive and high resolution, the gold nano-material molecule that serves as a mark is the most sophisticated existing tagged molecule, its preparation technology is simple, and the nano material diameter is easily controlled.For this reason, the application selects this molecule molecule that serves as a mark for use.
The preparation method is a lot of for the special-purpose nanometer Au probe, and the direct marking nano technology for gold of nucleic acid has been used in some invention, and specificity is better, but owing to mark gold nano molecule on the chain is less, sensitivity is relatively low; In the gene chip, detection probes quantity is huge, marking nano Au probe one by one, and quantities is big, and versatility is low.The present invention has adopted the method with gold nano-material mark chain parent's albumen (streptavidin), prepared chain parent protein nano gold general probe, this probe has and arbitrary specific nucleic acid chain high-bond affinity that is marked with biotin molecule, thereby obtains the high general detection probes of versatility.By utilizing general nano material probe, improve the versatility of nano-probe, reduce the preparation technology of nano-probe, reduce the chip system cost.
The chain parent protein nano gold general probe that makes thus, can with arbitrary specific nucleic acid chain high-bond affinity that is marked with biotin molecule, thereby by combining with the arbitrary special nucleic acid probe that has the biotin labeling molecule, form probe-vitamin H-chain parent albumen-nano-Au composite, the special nucleic acid nano Au probe of the specific nucleic acid detecting variation that rapid batch preparations quantity is huge.
(2) specific marker probe and preparation thereof
The versatility nano-probe has good versatility and suitable batch preparation, but does not possess the needed specificity of chip detection, for this reason, realizes the detection of chip specific signals, also needs to prepare a large amount of specific label probes.For this reason, we also need the difference according to the goal gene that is detected, utilize nucleic acid A-TC-G complementary characteristic, use general chemical synthesis process (can entrust domestic and international commercialization company synthetic), synthetic specific complementary probe, and on its 3 ' end mark biotin molecule (biotin), be beneficial to it and be connected with the combination of universal nano-probe.
2. the realization of naked eyes interpretation signal on the thin film bio sensor chip
(1) single stranded of the pcr amplification of gene fragment to be checked and product is handled
Nucleic acid molecule to be checked through pcr amplification, obtains nucleic acid DNA fragment to be checked, (for example handles 2 minutes at 95 ℃) after latter's sex change, places rapidly on ice, forms the nucleic acid fragment of strand.
(2) with the formation of the complete double-stranded DNA of nucleic acid to be checked and the mark of the plain geochemical exploration pin of specific biological
Nucleic acid molecule to be checked after the single stranded, by nucleic acid A-G C-T complementary principle, at nucleic acid to be checked is that bridge hybridization forms double chain DNA molecule, and at high temperature (55-65 ℃, preferable 60 ℃), 3 ' end-OH the base of capture probe links reaction with the label probe that contains vitamin H, forms the complete dna double chain of 60-100bp length, and finishes biotin molecule on the mark on this two strands.
(3) contain the fixing removing that reaches non-specific vitamin H specific probe on chip of vitamin H specific probe
Be marked with the nucleic acid to be checked of the complete two strands of vitamin H, in rigorous NaOH solution under (0.001-0.1M) or the hot conditions (as 65-80 ℃, be generally 70-75 ℃, as 70 ℃) unwind once more, form strand, the label probe that is marked with vitamin H that links fully with capture probe because there is capture probe to be connected with the chemistry that chip forms, so under this rigorous wash conditions not by wash-out.And be under the condition of bridge at the nucleic acid to be checked of non-purpose probe, label probe can not link fully with the capture probe on the chip, therefore can not form the nucleic acid that is marked with vitamin H of long-chain, thereby with this understanding by wash-out.Thereby finish chip to the segmental specific isolation of purpose, and specific nucleic acid to be checked kept and be fixed on the gene chip surface.
3. the realization of specificity signal to be checked on the thin film bio transmitter
Aforementioned wash-out with separate after chip, on certain location, remain with and have biotin labeled specific nucleic acid, and on other site, because of no longer contained the DNA chain of biotin molecule by wash-out.The former combines with general gold nano-probe again, has realized the gold nano-probe mark to specificity probe to be checked.
General gold nano-probe and the not label probe that has biotin molecule of wash-out-capture probe chain generation affinity reaction form capture probe-label probe-vitamin H-chain parent albumen-nano-Au composite, and form precipitation.Conventional glass-chip, owing to losing of optical signalling, formed gold nano optics precipitation under this experiment condition, both having made is the further amplification (about 10000 times) of process silver ions, still is difficult to realize the strength of signal or the sensitivity of stable naked eyes interpretation.And the optical characteristics that the thin film bio sensor chip is possessed can significantly be amplified this visible optical precipitation signal, reaches the interpretation of naked eyes visible light signal.
4. the reading of naked eyes interpretation signal on the thin film bio sensor chip
Computer batch processing after the naked eyes interpretation of signal or simple digital imaging device (digital camera, CCD imaging device or the scanner) imaging.
Concrete detection step is as follows:
The thin film bio sensor chip is put 60 ℃ of preheatings;
Connect liquid mixture 50-100 μ l and place on the thin film bio sensor chip, 60 ℃ were reacted 10 minutes;
60 ℃ with the 0.01N NaOH of preheating washing 3 times, each 5-10 second;
Again chip is put among 0.1 * SSC and washed 3 times under the room temperature, each 1-2 minute;
Chain parent albumen-Au solution by 1: 1000 in 1 * hybridization buffer (5XSSC) dilution, and get 50-100 μ l and join chip surface;
Reaction is 5 minutes under the room temperature;
Wash chip 3 times with 0.1 * SSC, each 1-2 minute; And in the end dry up chip surface with net air;
Naked eyes or by the magnifying glass interpretation; Or the application digital camera is made a video recording, the computer interpretation signal.
Hereinafter will set forth the present invention in the mode of specific embodiment.Should be understood that these embodiment only are illustrative and nonrestrictive, the present invention is not subjected to the restriction of these specific embodiments.Scope of the present invention is limited by the content of claims.
Embodiment 1: the preparation of thin film bio transmitter
Press the vaccum gas phase sedimentation method plated film, use the rotation vacuum plating unit to be about 1-3mm, the about 15cm silicon chip of diameter (SO at thickness
2) the last silicon nitride (Si that plates with 475 dusts
3N
4) and the film of the TSPS of 135 dusts, prepare corresponding biosensor, and cover the Poly(Phe)-Methionin coating of 150 dusts, after 1-10 μ M hydrochloric acid succinimido hydrazine nicotinate was handled about 20 minutes, clear water is cleaned for chip manufacturing.
Embodiment 2: the preparation of capture probe
Adopt the nucleic acid chemistry synthetic method of this area routine to make following capture probe (can entrust domestic or specialized company in the world, be prepared) by following chemical structure:
| L528M P1-T SEQ ID NO：1 | ALD-AAAAAAAAAACGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCT |
| L528M P1-A SEQ ID NO：2 | ALD-AAAAAAAAAACGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCA |
| M539V P1-A SEQ ID NO：3 | ALD-AAAAAAAAAAGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATA |
| M539V P1-G SEQ ID NO：4 | ALD-AAAAAAAAAAGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATG |
| M539I P1-G SEQ ID NO：5 | ALD-AAAAAAAAAATCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATG |
| M539I P1-T SEQ ID NO：6 | ALD-AAAAAAAAAATCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATT |
(ALD=aldehyde radical)
Embodiment 3: the preparation of thin film bio sensor chip
The fixing capture probe that makes of embodiment 2 of point sample on the chip of embodiment 1, make its chemical coating carry out the ammonium aldehyde combined reaction through room temperature and chip surface, form covalently bound compound, be fixed on the surface of chip, make and have fixed thin film bio sensor chip.
Embodiment 4: the preparation of universal gold nano-material label probe
Configuration is used for 25 batches following solution:
1.0mM chlorauric acid solution: 0.1g HAuCl is dissolved in the 500mL distilled water gets final product.Can put the medium-term and long-term preservation of brown bottle.
1% sodium citrate (Na
3C
6H
5O
72H
2O): with 0.5g Na
3C
6H
5O
72H
2O dissolves in the 50mL distilled water.
1M NaCl: 0.5g of NaCl is dissolved in the 10mL distilled water.
With 20mL 1.0mM HAuCl
4Join in the 50mL Erlenmeyer flask, insert magnetic stirring bar and place on the magnetic stirrer and heat, be stirred to boiling.1% sodium citrate that slowly adds 2mL, visible gold nano forms (about 10 minutes) gradually, when transferring scarlet to, stops stirring heating.
With nanometer gold pH regulator to 8.0, magnetic stirs a small amount of distilled water dissolved chain parent of adding down albumen, and (Sigma-Eldrich USA), acts on 10 minutes, can obtain being marked with the chain parent albumen of nanometer gold; Add 5% bovine serum albumin (BSA again; Boehringer Co. Gereman), makes its final concentration to 1%.Chain parent albumen-Au that the reaction back generates is centrifugal with 15000g, draws supernatant, crosses Sephacryl S-400 post (Pharmacia), collects the dark red purification part of interlude, and 4 ℃ of preservations are standby.
Embodiment 5: the detection of hepatitis B virus YMDD sudden change
1. sample is prepared
(1) anti-freezing or not anticoagulation 2ml, 1000rpm is centrifugal 5 minutes under the normal temperature, gets serum or blood plasma 100 μ l with micro suction dispenser;
(2) add isopyknic reagent and boil 5min for 1,100 ℃ with following composition:
| HBV forward primer (SEQ ID NO:7) | TGCACCTGTATTCCCATCC |
| HBV reverse primer (SEQ ID NO:8) | GCGGTATAAAGGGACTCACG |
(3) centrifugal 5 minutes of 5000g is standby.
2. the segmental pcr amplification of purpose
(1) gets pcr amplification pipe numbering under the room temperature, place in order;
(2) get the sample supernatant 5 μ l of above-mentioned preparation;
(3) add deionized water 19 μ l;
(4) add Taq enzyme (Zhuhai Sinochips Biotechnology Co., Ltd.) 1 μ l;
(5) 4 ℃ centrifugal 1 minute;
(6) increase by following condition: 94 ℃ of sex change 5 minutes; Again by 94 ℃, 30 seconds--56 ℃, 30 seconds--72 ℃, carried out 30 in 30 seconds to take turns circulation, 72 ℃ are extended after 10 minutes 4 ℃ of sealings and preserve standby.
3. adopt the nucleic acid chemistry synthetic method of this area routine to make following capture probe (can entrust domestic or specialized company in the world, be prepared) by following chemical structure:
| L528M P2-vitamin H (SEQ ID NO:9) | Phosphate-TGGCTCAGTTTACTAGTGCC-biotin |
| M539V P2-vitamin H (SEQ ID NO:10) | Phosphate-TGGATGATGTGGTATTGGGG-biotin |
| M539I P2-vitamin H (SEQ ID NO:11) | Phosphate-GATGATGTGGTATTGGGGGC-biotin |
(wherein, " Phosphate " refers to phosphate radical, and " biotin " refers to vitamin H)
4. gene chip detects
(1) with 95 ℃ of heating of pcr amplification product 3 minutes, places rapidly on ice;
(2) get on the optical pickocff gene chip that the 10ml pcr amplification product makes to embodiment 3;
(3) getting 1 μ l ligase enzyme (Zhuhai Sinochips Biotechnology Co., Ltd.) adds on the chip;
(4) get 60 ℃ of pre-hot tie-in liquid 3 (link enzyme 5u, link enzyme buffer liquid, 5mg/mlATC, acidifying) (Zhuhai Sinochips Biotechnology Co., Ltd.) and drip on chip, 60 ℃ of reactions 10 minutes;
(5) with the 0.01-0.1N NaOH of 60 ℃ of preheatings washing 3 times, each 5-10 second;
(6) again chip is put among 0.1 * SSC washing 3 times under the room temperature, each 1-2 minute; Air drying then;
(7) every 3 of the gold nano-material probes that embodiment 4 is made drip in chip surface, and react at normal temperatures 5 minutes;
(8) wash chip 3 times with 0.1 * SSC, each 1-2 minute; And in the end dry up chip surface with cleaned air;
(9) chip of water flushing promptly is 3 times, and cleaned air dries up chip surface, carries out the chip interpretation.
5. the result judges
Take a picture or scanning input computer with ordinary digital camera, according to the position that signal occurs, the type of certain sudden change.The result is presented among Fig. 3.Fig. 3 display chip result's naked eyes interpretation.Fig. 3 A is the digital photograph of actual result, and 3B is the chip signal design diagram, appears at location in the chip by its positive signal, the mutation type of its positive signal representative of interpretation.
The result proves, adopt the P gene of round pcr amplification hepatitis B virus (HBV), utilize nucleic acid hybridization technique and biochip technology again, the variation of the single Nucleotide of the P gene of detection HBV, and applying nano materials probe technique and optical sensor technology, realized the amplification of detection signal, reach the level of naked eyes interpretation, thereby break through traditional biochip technology needs to use main equipments such as laser scanner in detection bottleneck restriction, gene chip need not specific equipment and can be extensive use of.
This money chip is primarily aimed in the anti-HBV therapeutic process of ucleosides, induce HBV to produce the YMDD region mutation, thereby cause the drug-fast genetics characteristic of virus to lamivudine, the applying gene chip technology, detect the gene mutation type in the YMDD district of HBV, thereby the medication foundation of science is provided for clinical antiviral therapy.
Sequence table
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＜120〉a kind of high sensitivity biosensor gene chip and clinical diagnosis technology
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Claims (9)
1. a biochip is characterized in that, this chip comprises:
Silicon dioxide substrates;
Be present in the optical coating on this silicon dioxide substrates; With
Be present in the Poly(Phe)-Methionin coating on this optical coating.
2. biochip as claimed in claim 1 is characterized in that, the thickness of described silicon dioxide substrates is 2-4mm, and optical coating comprises the poly-ammonia dimethyl-silicon alkane of T structure that silicon nitride that thickness is the 350-550 dust and thickness are the 80-200 dust.
3. biochip as claimed in claim 1 is characterized in that, thereby this chip also comprises linking to each other with its optical coating by chemical bond and is fixed in the nucleic acid probe of chip surface.
4. biochip as claimed in claim 1 is characterized in that, the thickness of described Poly(Phe)-Methionin coating is the 20-200 dust.
5. a method for preparing the described biochip of claim 1 is characterized in that, this method comprises:
(1) on silicon dioxide substrates, plates with optical coating;
(2) on the coating of step (1) gained, plate again with Poly(Phe)-Methionin coating, obtain to have plated the silicon dioxide substrates of optical coating and Poly(Phe)-Methionin coating;
(3) with the silicon dioxide substrates of hydrochloric acid succinimido hydrazine nicotinate treatment step (2) gained, clean, obtain described chip.
6. method as claimed in claim 5 is characterized in that, the concentration of described hydrochloric acid succinimido hydrazine nicotinate is 1-10 μ M, and the treatment time is 10-30 minute.
7. method as claimed in claim 5 is characterized in that, this method also comprises:
(4) point sample stationary probe makes its chemical coating through room temperature and chip surface carry out the ammonium aldehyde combined reaction, forms covalently bound compound, is fixed on the surface of chip.
8. the method for the nucleic acid to be checked in the test sample is characterized in that this method comprises:
(1) provide claim 1 described chip, fixing in its surface and nucleic acid specificity bonded nucleic acid probe to be checked;
(2) make the described chip of step (1) with sample, contact with the label probe of nucleic acid specificity hybridization to be checked, wherein, this label probe is marked with vitamin H;
(3) carry out wash-out with NaOH solution, obtain not eluted product;
(4) the chain parent albumen with the Nano-Au probe mark contacts with the not eluted product that step (3) obtains, and forms nucleic acid probe-label probe-vitamin H-chain parent albumen-nano-Au composite; With
(5) detection chip signal is determined the existence of nucleic acid to be checked.
9. method as claimed in claim 8 is characterised in that, the concentration of NaOH is 0.001-0.1M in the step (3).
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| CN1184331C (en) * | 2001-02-09 | 2005-01-12 | 中国科学院电子学研究所 | DNA-PCR biochip and miniature heat circulator using it |
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