CN1749752A - Solution identification and surface addressing protein chip and its preparing and detecting method - Google Patents
Solution identification and surface addressing protein chip and its preparing and detecting method Download PDFInfo
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Abstract
The protein chip for solution identification and surface addressing includes substrate, cured hairpin type oligonucleotide, and probe including protein, connected oligonucleotide, fluorescently labeled target protein molecule and dye molecule specifically embedded into the oligonucleotide double strand to emit light. On the surface of the substrate, one layer of organic film with reaction active functional group in the end is covered, and oligonucleotide is fixed through covalent bonding. The present invention also provides protein fixing and detecting method, in which protein molecule is coupled by means of hairpin DNA and potential controlling technology and identified in solution before surface addressing. The present invention avoids the denaturation of protein and makes it possible for preserve protein sensor and protein chip for long period.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of protein sensing device and protein-chip, and its preparation method and utilize this chip to carry out the method for protein or other Molecular Detection.
Background technology
Along with finishing of human genome examining order, life science progressively carries out the transition to back genome (functional genome) and protein group epoch by genome (structural gene group) epoch.Hereditary information is transferred to expression of gene to the research emphasis of genome times afterwards comprehensively and regulation and control come up from disclosing.The main agent of biological function is a protein, and protein has himself distinctive rule, modification processing, transhipment, location, structural change, protein-protein, protein and the big intermolecular interaction of other biological etc. as protein all can't obtain on genomic level.Just because of the limitation of genomics, impel people to have to from the composition and the characteristics of motion thereof of integral level discussion cell protein.
1994, the notion (seeing document Alan Dove, Nature Biotechnology, 17:233 (1999)) that the Wilkins of Australian Macquarie university and Williams at first propose protein group (Proteome).Be defined as at first: specific cells expressed all proteins in special time.Research about protein group then is called proteomics.In order to study the thousands of protein interactions and the characteristics of motion, need a kind of high flux, high-sensitivity analysis method.Genetic chip provides the successful example of a high flux, high-sensitivity analysis gene order.Especially our two patented claims (publication number is respectively 1373228 and 1477210), make high sensitivity cheaply genetic chip become possibility.Protein-chip can be regarded the continuity and the development of genetic chip as, becomes very important ingredient in the analyzing biochips method.
The notion of protein-chip:
Protein-chip (Protein Chip) is called protein microarray (ProteinMicroarray) (referring to document MacBeath, G.and S.L.Schreiber, Science, 2000.289:1760 (2000)) again.Be new ideas that propose late 1990s, refer to protein molecule is solidificated in substrate surface with the form of microarray, utilize the evident characteristics of protein molecule to finish the integrating device of certain function.
The protein-chip key element:
Substrate: be the carrier of protein-chip, it must fix the protein as function body, and keeps its biologically active as much as possible.Substrate also should have the characteristic that is fit to certain detection means.The substrate of generally adopting now has macromolecule (referring to document Zhang M.Q.et al.Biomaterials, 19:953 (1998)), gold (referring to document Smith A.M.et al.Biosensors﹠amp; Bioelectronics, 15:183 (2000)), glass is (referring to document Boyle M.D.et al.Journal of Microbiological Methods, 46:87 (2001)), semiconductor material (referring to document Liao W et al.Sensors and Actuators, 101:361 (2004)) etc.
Protein: the main undertaker who is the protein-chip function embodiment.Also can substitute with the aggregate of protein (cell, virus, bacterium, tissue etc.) or analog (small peptide fragment, fit, zinc fingers).
Function: obviously, protein-chip must be finished certain function, and function can be synthetic, and screening separates, and detects or theirs comprehensive.
The protein-chip feature:
Protein molecule is arranged in array in substrate, and such being placed with is beneficial to multiple class, high flux, the parallel detection of carrying out protein.
Be fixed in the substrate and system to be measured in the amount of desired protein all very low, according to the unusual μ g-ng magnitude that reaches of detection means, to adapt to the requirement of low dose, high-sensitivity detection.
The protein biochip technology difficult point:
The protein probe preparation: how obtaining a large amount of highly purified protein probe molecules, is the biology bottleneck of protein-chip preparation.Usual way has two kinds: the one, and biological technique method through plasmid transfection, is expressed, and separates, and steps such as purification take time and effort, and can only carry out the preparation of minority albumen; The 2nd, utilize the synthetic method of chemical polypeptide, this method is difficult to synthetic bigger albumen, only is suitable for synthetic short polypeptide fragment.
The high activity of protein molecule is fixed: protein molecule is easy to cause owing to surface and protein-interacting the loss of protein molecule sex change and function at solid surface.So the activity that how to be maintained fixed the back protein molecule is the chemical bottleneck of protein-chip preparation.Normally used fixing means has two classes: a class is directly fixing method, with protein molecule by physisorption, methods such as chemical bond coupling connection directly are fixed on solid substrate surface, in these class methods, protein molecule directly contacts with substrate, and easily sex change and inactivation are (referring to document SmithA.M.et al.Biosensors ﹠amp; Bioelectronics, 15:183 (2000)); Second class is the method for indirect securement, promptly between protein molecule and substrate, add the biological molecule that is connected of one deck, to keep the separate state and the activity of protein molecule, usually the method that adopts is ProteinG-IgG, biotin-microbiotic, special interacting molecules such as DNA-oligonucleotides are right, and these class methods need extra step, and fixed efficiency low (referring to document Baumann S.etal.Journal of Immunological Methods, 221:95 (1998)).
High-sensitivity detecting method: with respect to traditional biology sensor since protein-chip on protein amount still less, so require pick-up unit that higher sensitivity is arranged.The most normally used is the method for fluorescence labeling laser scanning imaging.
The genetic chip that we have applied for the patent of being correlated with
1, gene chip with hairpin-shaped probes and preparation method thereof and detection method
Application number: 02116302.2
Publication number: 1373228
Open date: 2002-10-09
This invention relates to a kind of genetic chip, comprises chip base and curing oligonucleotide probe thereon, and probe comprises detecting area and cane district; Oligonucleotide sequence in the cane district and form hair fastener shape duplex structure with the oligonucleotide sequence of its coupling, the matching way of its nucleotide and base number satisfy following condition: the melting temperature of hair fastener shape oligonucleotide 2 probes, in the melting temperature-5 of single-point mismatch hybridization state ℃ between the scope of the melting temperature of mating the hybridization state fully+5 ℃.The present invention can further optimize hybridization conditions by this genetic chip is carried out control of Electric potentials.Genetic chip of the present invention and sample all need not be made any mark, and chip can be reused, and make and use cost reduces greatly, and have identification and mate ability with the oligonucleotide sequence of single-point mispairing fully.Can be widely used in biological technical field.
2, the unmarked fluorescence detection method of genetic chip electric potential scanning
Application number: 03142612
Publication number: 1477210
Open date: 2004-02-25
This invention relates to the unmarked fluorescence detection method of a kind of genetic chip electric potential scanning, adopt gene chip with hairpin-shaped probes, at first use the target preparing standard solution, with standard solution and probe hybridization, and add embedded fluorescent dye, chip base is applied current potential and carries out electric potential scanning, the fluorescence signal typical curve of probe under the writing scan and standard solution hybridization; The fluorescence method of then the testing sample dna solution being packed into is measured the device of chip hybridization, with probe hybridization, and adds embedded fluorescent dye; Equally chip base is applied current potential and carries out electric potential scanning, the curve that the fluorescence signal of probe under the writing scan and testing sample hybridization changes; This curve and typical curve are compared, provide recognition result.The present invention can be definitely and is realized SNP identification reliably, and probe and sample all need not to carry out special mark, greatly reduce cost.Can be widely used in the biochip technology field.
Summary of the invention
An object of the present invention is, the protein-chip of a kind of solution identification, surface addressing is provided, protein-chip according to the present invention comprises chip base and curing hair fastener shape oligonucleotide thereon, the combined probe molecule comprises protein and coupled oligonucleotide, double-stranded and the luminous dye molecule of fluorescently-labeled target protein molecule and special embedding oligonucleotide.
Chip base surface coverage last layer end is the organic film of reactive functional, by covalent bonding, oligonucleotide is fixed on substrate surface.
Another object of the present invention is, provides a kind of and can fix and detect method of protein.According to fixing and detection method of protein of the present invention, utilize hair fastener shape oligonucleotide and control of Electric potentials technology coupled protein matter molecule, realize that the identification back is to surface addressing in the solution, farthest avoid the sex change of protein, and overcome the difficulty that albumen sensor and protein chip can't long preservation on the surface.And introduce fluorescence resonance energy transmission (FRET) and realize high specific and high sensitivity Protein Detection.
FRET is meant that excited energy gives the transmission of body (donor) to acceptor (acceptor) from what initially excite.(referring to document Joseph R.Lakowicz, Principles ofFluorescence Spectroscopy, Kluwer Academic/Plenum Publisher, 1999) in general has overlapping for the emission spectrum of body and the absorption spectrum of acceptor.When giving distance between body and the acceptor in 10 nanometer scale, FRET just can take place, and will pass to acceptor for the energy that body absorbed, and launches photon by acceptor again, produces fluorescence.
According to fixing and detection method of protein of the present invention, specifically may further comprise the steps:
(1) solidifies oligonucleotide fragment.
Be that chip base surface coverage last layer is terminal for the organic film of reactive functional,, the known oligonucleotide fragment of at least one class sequence be fixed on substrate surface by covalent bonding.
(2) the known oligonucleotide coupling connection of probe albumen and another kind of sequence forms the combined probe molecule.
Probe albumen and oligonucleotide coupling connection can be taked covalency and non-covalent dual mode.
(3) combined probe and target protein molecular recognition.
Probe albumen-oligonucleotide compound with gained in the step (2) mixes with fluorescence labeling target protein solution to be measured.
(4) compound is solidificated in the surface.
With resulting mixed solution in the step (3), carry out hybridization reaction with the surface that resulting oligonucleotide in the step (1) solidifies.
(5) fluorescence signal detects.
The chip of having caught target protein is placed buffer solution, and add the double-stranded luminous fluorescent dye of special embedding oligonucleotide.The fluorescence molecule generation fluorescence resonance energy of mark transmits on intercalative dye and the target protein, can detect reading or imaging by LASER Excited Fluorescence.
(6) surface potential scanning
By surface potential scanning, can further improve the specificity and the recognition capability of detection.But this step is not must step.The concrete grammar of surface potential scanning is referring to another part patented claim CN 1477210.
With fix the method for afterwards discerning earlier during traditional protein chip detects and compare, the present invention has fundamentally avoided the influence of protein surface sex change as much as possible, has protected activity of proteins to greatest extent.In the method for the invention, probe and target molecule at first in solution, carry out specific recognition reaction (before method in, this process takes place on the surface, existing protein-denatured threat, the steric factor of also having living space), the compound after the identification again by the specific recognition effect of oligonucleotide in the substrate surface addressing.Different with classic method is, does not need to preserve the surface that is solidified with albumen when preserving protein chip, and just passable as long as preserve the substrate and the protein solution that are solidified with oligonucleotide.
Embedded dyestuff and fluorescent tag molecule produce fluorescence signal by the fluorescence resonance energy transmission, because embedded dyestuff is to embed specifically among the two strands, add the physical condition that FRET takes place, greatly reduce the false positive background that non-specific recognition and non-specific hybridization are brought, thereby improved the accuracy of signal to noise ratio (S/N ratio) and detection.
In traditional method of protein detection based on fluorescence, all be the quantity of coming profiling protein with the absolute figure of fluorescence intensity, the easy like this accidental error that is subjected to, Effect of Environmental, and all being reflected at carries out under the identical conditions guaranteeing that all identification systems all are in the best identified condition in the protein chip.This invention can be adopted traditional intensity method, also can introduce the factor of electric potential scanning in the Protein Detection process, with the variation spectral line replacement single absolute strength of fluorescence intensity with current potential, comes to distinguish with non-specific responding special.Like this, can obtain gem-pure difference spectrum, greatly eliminate the influence of reaction conditions and fluorescence intensity.
Description of drawings
Below in conjunction with accompanying drawing the present invention is illustrated in further detail:
Fig. 1 is the protein chip structural representation;
Fig. 2 is protein chip preparation and reactions steps schematic flow sheet;
Fig. 3 is the fluorescence spectrum comparing result that does not have under the electric potential scanning;
Fig. 4 is the change curve of fluorescence intensity with electric potential scanning.
Most preferred embodiment is described in detail
Below with reference to accompanying drawing of the present invention, more detailed description goes out most preferred embodiment of the present invention.
Figure 1 shows that according to protein chip structural representation of the present invention wherein A is the state before the chip detection, B is the state after the chip detection.This protein-chip comprises chip base 1 and curing hair fastener shape oligonucleotide 2 thereon, the oligonucleotide 4 that combined probe molecule 3 comprises protein and is coupled with it, double-stranded and the luminous dye molecule 6 of fluorescently-labeled target protein molecule 5 to be measured and special embedding oligonucleotide.
Chip base 1 surface coverage last layer end is the organic film of reactive functional, by covalent bonding, hair fastener shape oligonucleotide 2 is fixed on substrate surface.
This protein-chip various piece specifically describes as follows:
1, chip base and curing hair fastener shape oligonucleotide or analog thereon
Chip base 1 is the solid support plane of chip, can be made of multiple material, comprises metal, glass, macromolecular material, and our present employed silicon.Can cover the film that one deck helps oligonucleotide to solidify at these material surfaces, both can adopt covalently bound method, can adopt the method for physisorption to be cured again.Nucleic acid oligomer probe after design is synthetic has following structure: has the nucleic acid oligomer sequence of the complete complementation of sequence in a section or plurality of sections and the combined probe molecule, be called detecting area; Exist one section or plurality of sections nucleic acid oligomer sequence and with the nucleic acid oligomer sequence that this sequence is complementary, be called the cane district; Nucleic acid oligomer sequence in the cane district and form " hair fastener shape " duplex structure with the nucleic acid oligomer sequence of its coupling.Oligonucleotide can be an oligodeoxynucleotide, can be the oligomerization ribonucleotide, also can be the analog of oligonucleotide, as polypeptide nucleotide (PNA).
2, combined probe molecule
Combined probe molecule 3 comprises protein and coupled oligonucleotide 4.Wherein, protein can be antibody, acceptor, and other protein macromolecule or the polypeptide fragment that can discern with albumen.The fragment that the oligonucleotide 4 that links to each other with albumen matches each other for the detecting area that designs synthetic one section energy of existence and the hair fastener shape oligonucleotide that is solidificated in substrate surface.Protein can be taked covalent bond with being connected of oligonucleotide 4, promptly utilizes the reactive group of protein surface to be connected with the reactive group formation covalent bond of oligonucleotide 4 ends; Also can adopt non-covalent connected mode, promptly albumen and oligonucleotide all be carried out the micromolecule mark, the big molecule by specific recognition couples together the two then, utilization be special interaction between big molecule and the micromolecule.
3, fluorescently-labeled target protein molecule
Fluorescently-labeled target protein molecule 5 to be measured for all can with probe protein molecular 3 special interactional micromolecule and big molecule.Target protein molecule 5 is present in the solution to be measured that the user provides, i.e. the material of required detection.Fluorescence labeling is classical labeling method, does not belong to the preparation process of protein chip of the present invention.The fluorescence molecule of institute's mark must satisfy and embed double-stranded dye molecule 6 condition that fluorescence resonance energy transmits takes place.
4, the double-stranded and luminous dye molecule of special embedding oligonucleotide
During the probe protein molecular 3 that has joined the oligonucleotide chain when coupling and chip base 1 surface fixing hair fastener shape oligonucleotide 2 hybridization, this dye molecule 6 will be embedded in the two strands, can send fluorescence after the laser excitation, and this dye molecule 6 is under non-embedding state, and Stimulated Light excites has only very weak fluorescence.
5, potential controlling apparatus and fluorescence detecting system
Among the present invention the potential controlling apparatus that uses identical with employed device among the patent of invention CN1477210, promptly chip base is applied current potential and carries out electric potential scanning, the curve of the fluorescence signal variation that the probe writing scan under and testing sample are hybridized; This curve and typical curve are compared, provide recognition result.But be not limited thereto device, the device that all energy control chip substrate 1 surface potentials change all can use.In our experiment, the fluoroscopic examination that we use RenishawRaman 1000 systems to carry out, other fluorescent microscope and biochip scanner all can be used for the fluoroscopic examination of this protein chip.
Figure 2 shows that protein chip preparation and reactions steps schematic flow sheet, specify below with reference to Fig. 2, according to the preparation of protein-chip of the present invention and the concrete steps of detection method:
1, solidifies oligonucleotide fragment
The step of solidifying oligonucleotide fragment is (among Fig. 2 7), and chip base 1 surface coverage last layer end is the organic film of reactive functional, by covalent bonding, at least a oligonucleotide fragment is fixed on substrate surface.Substrate can be adopted different materials such as silicon, glass, metal, macromolecular material, and curing also can be selected and the corresponding method of substrate.What we adopted is silicon base, and basic solidification process is as follows.The monocrystalline silicon piece that at first will be coated with the autoxidation silicon layer pick up reagent (70% concentrated sulphuric acid: 95 degree high-temperature oxydations 30% hydrogen peroxide), pass through NH then
4The corrosion of F, the silicon face of formation hydrogen termination under ultraviolet light causes, has the unsaturated alkane chain and the Si-H surface reaction of ester group functional group, forms the organic membrane that fine and close ester group stops.After the ester group acidifying became carboxyl, with the oligonucleotide solution reaction that contains activator (NHS and EDC), behind the wash-out, oligonucleotide just was fixed on the surface of silicon base 1.
2, probe albumen and oligonucleotide coupling connection
Probe protein molecular 3 and oligonucleotide 4 couplings connection (among Fig. 2 8) can be taked covalency and non-covalent dual mode.Non-covalent mode is that the specific recognition effect by biotin and microbiotic (nucleophilic nuclein) couples together albumen and oligonucleotide in non-covalent mode with albumen and oligonucleotide biotin on the mark all.In patented claim, what we adopted is covalently bound mode.Under the catalysis of activator (NHS and EDC), the probe protein molecular 3 and the oligonucleotide 4 of purifying reacted in solution.Solution behind the coupling connection is collected the probe albumen-oligonucleotide compound on the filter membrane through ultrafiltration and high speed centrifugation.
3, probe and target protein molecular recognition
As 9 being probe and target protein molecular recognition among Fig. 2,, mix with fluorescence labeling target protein molecule 5 solution to be measured with the probe albumen-oligonucleotide compound of gained in the step 2.Wherein, the probe albumen of specific recognition and target protein will form oligonucleotide-probe albumen-target protein compound.
4, compound is solidificated in the surface
With resulting mixed solution in the step 3, carry out hybridization reaction with the surface that resulting oligonucleotide in the step 1 solidifies.Oligonucleotide in the mixed solution-oligonucleotide of probe albumen-target protein compound and the oligonucleotide specific hybridization of surface cure are gone up (as among Fig. 2 10) thereby oligonucleotide-probe albumen-target protein compound is solidificated in chip base 1 surface.
5, fluorescence signal detects
Fluorescence signal detects 11, and the chip of having caught target protein is placed buffer solution, and adds the double-stranded luminous fluorescent dye of special embedding oligonucleotide.Because the fluorescence molecule generation fluorescence resonance energy of mark transmits on intercalative dye and the target protein, thereby can detect fluorescence resonance energy transmission fluorescence by laser excitation and come reading or imaging.
Fluorescence resonance energy transmits (FRET) and is meant that excited energy gives the transmission of body (donor) to acceptor (acceptor) from what initially excite, in general, has overlapping for the emission spectrum of body and the absorption spectrum of acceptor.When giving distance between body and the acceptor in 10 nanometer scale, FRET just can take place, and will pass to acceptor for the energy that body absorbed, and launches photon by acceptor again, produces fluorescence.
6, surface potential scanning
Can scan 12 by surface potential, analysis of fluorescence intensity is accurately distinguished the reaction of specific recognition and non-specific recognition with the curve of potential change, further improves specificity and the recognition capability that detects.
Figure 3 shows that the fluorescence spectrum comparing result that does not have under the electric potential scanning, in five contrast experiments, utilize the method Covalent Immobilization oligonucleotide sequence oligo-1 on the silicon face that carboxyl stops among the patent of invention CN1373228, at mouse IgG (mIgG, probe albumen) go up the coupling connection and go up oligonucleotide sequence oligo-2, detection is carried out in MES buffer solution, adds embedded dye molecule PicoGreen during detection in the buffer solution.Among the figure, curve 13 is that oligo-1 and oligo-2 are complementary fully, adds fluorescently-labeled goat anti-mouse IgG (AF-GAM, target protein molecule) and mIgG specific recognition, the i.e. result of positive control in the solution.All the other are the result of various negative controls.Curve 15 is not for oligo-1 and oligo-2 match, and AF-GAM and mIgG specific recognition, i.e. the negative control that identification is not hybridized.Curve 14 is oligo-1 and the complete complementary pairing of oligo-2, and AF-GAM and rIgG specific recognition not, promptly hybridizes the negative control of nonrecognition.Curve 17 does not add the target protein molecule in the solution, and the complete complementary pairing of oligo-1 and oligo-2 does not promptly have the negative control that FRET has only embedded dyestuff.Curve 16 is fixedly oligonucleotide, the i.e. negative control of the non-special absorption of target protein molecule of substrate.Can find out obviously that from the result positive findings is apparently higher than other negative control, but the background that some negative findings brought is bigger.
Figure 4 shows that the change curve of fluorescence intensity with electric potential scanning.In this group experiment, we contrast the change curve of three individual system under electric potential scanning.Covalent Immobilization oligonucleotide sequence oligo-1 on the silicon face that carboxyl stops respectively, at mouse IgG (mIgG, probe albumen) or rabbit IgG (rIgG, probe albumen) go up the coupling connection and go up oligonucleotide sequence oligo-2, detection is carried out in MES buffer solution, add embedded dye molecule PicoGreen during detection in the buffer solution, carry out the electric potential scanning fluorescence intensity.Among the figure, curve 18 positive contrasts, curve 19 is the negative control of hybridization nonrecognition, the negative control that curve 20 is not hybridized for identification.We are with half noble potential Δ E
1/2Come scanning curve is characterized, be followed successively by-0.71V-0.01V, 0.15V, to compare with embodiment 1, the differentiation degree improves greatly, and half noble potential is subjected to such environmental effects very little as the performance of system inherent attribute, can be used as to distinguish positive negative important parameter.
Protein probe of the present invention does not need to fix from the teeth outwards earlier, but can highly active method long preservation.What the surface was fixing is oligonucleotide, and oligonucleotide is more stable much than protein at substrate surface, so chip can long preservation and do not lose efficacy.
Although disclose most preferred embodiment of the present invention and accompanying drawing for the purpose of illustration, it will be appreciated by those skilled in the art that: without departing from the spirit and scope of the invention and the appended claims, various replacements, variation and modification all are possible.Therefore, the present invention should not be limited to most preferred embodiment and the disclosed content of accompanying drawing.
Claims (6)
1. the protein-chip of a solution identification, surface addressing is characterized in that:
The combined probe molecule comprises protein and the coupled known oligonucleotide of a class sequence;
Chip base surface coverage last layer end is the organic film of reactive functional, so that the oligonucleotide that another kind of sequence is known is fixed on substrate surface;
There is one section complementary fully sequence in the two class oligonucleotide sequences, has at least a class oligonucleotide can form " hair fastener shape " structure.
2. the protein-chip of solution identification according to claim 1, surface addressing is characterized in that: chip base can be made of metal, glass, silicon or macromolecular material.
3. the protein-chip of solution identification according to claim 1, surface addressing is characterized in that: the protein in the combined probe molecule can be antibody, acceptor, perhaps protein macromolecule or the polypeptide fragment that can discern with albumen.
4. the protein-chip of solution identification according to claim 1, surface addressing, it is characterized in that: oligonucleotide can be an oligodeoxynucleotide, can be the oligomerization ribonucleotide, can be the analog of oligonucleotide also, as polypeptide nucleotide.
5. the preparation and the detection method of a solution identification, surface addressing protein-chip specifically may further comprise the steps:
(1) solidifies oligonucleotide fragment
Be that chip base surface coverage last layer is terminal for the organic film of reactive functional,, at least a oligonucleotide fragment be fixed on substrate surface by covalent bonding;
(2) albumen becomes the combined probe molecule with oligonucleotide coupling joint group
Albumen and oligonucleotide coupling connection can be taked covalency and non-covalent dual mode;
(3) combined probe molecule and target protein molecular recognition
Probe albumen-oligonucleotide compound with gained in the step (2) mixes with fluorescence labeling target protein solution to be measured;
(4) compound is in the surface cure addressing
With resulting mixed solution in the step (3), carry out hybridization reaction with the surface that resulting oligonucleotide in the step (1) solidifies;
(5) fluorescence signal detects
The chip of having caught target protein is placed buffer solution, and add the double-stranded luminous fluorescent dye of special embedding oligonucleotide, carry out fluoroscopic examination.
6. the preparation and the detection method of solution identification according to claim 5, surface addressing protein-chip is characterized in that: further by surface potential scanning, improve the specificity and the recognition capability that detect.
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