CN100396791C - Method for detecting oligomeric nucleotide through intercalating dye fluorescent resonant energy transfer molecule - Google Patents
Method for detecting oligomeric nucleotide through intercalating dye fluorescent resonant energy transfer molecule Download PDFInfo
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Abstract
The present invention introduces a fluorescence resonance energy transmission (FRET) technique into the preparation and the detection of a protein chip. A dye molecule which is specifically embedded into an oligonucleotide double strand to emit light and a fluorescent molecule labeled on a target molecule are designed to form an FRET donator and a receptor. By introducing FRET, the specificity of the protein chip is greatly enhanced, and the present invention is the extension and the development of a recognizing and fixing method. One aim of the present invention is to provide a protein chip on the basis of fluorescence resonance energy transmission. The protein chip of the present invention comprises a chip substrate and card oligonucleotide solidified on the chip substrate, and thus, a compound probe molecule comprises a protein, oligonucleotide connected with the protein, a target protein molecule labeled by fluorescence and the dye molecule which is specifically embedded into an oligonucleotide double strand to emit light. The other aim of the present invention is to provide a method capable of fixing and detecting a protein.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of oligonucleotide intercalative dye fluorescence resonance energy and transmit molecular detecting method.
Background technology
Along with carrying out of back genome and protein group work, researchists press for a kind of not only efficient but also stable high-throughput analysis of protein technology.Protein chip a kind of technology that comes to this, it is inheritance and development on the basis of gene chip.Protein is by the folding three-dimensional three-dimensional arrangement that forms of amino acid long chain coiling, just can realize function with complexity such as other molecular recognition by this three-dimensional arrangement 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 specified 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-throughput, high-sensitivity analysis method.Gene chip provides the successful example of a high-throughput, high-sensitivity analysis gene order.Especially our two patent applications (publication number is respectively 1373228 and 1477210), make highly sensitive cheaply gene chip become possibility.Protein chip can be regarded the continuity and the development of gene chip as, becomes very important integral part 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 biological activity as much as possible.Substrate also should have the characteristic that is fit to certain detection means.The substrate of generally adopting now has polymer (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 andActuators, 101:361 (2004)) etc.
Protein: the main undertaker who is the protein chip function embodiment.Also can substitute with proteinic aggregate (cell, virus, bacterium, tissue etc.) or analogue (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 carries out proteinic multiple class, high-throughput, parallel detection.
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 method has two kinds: the one, and biotechnological means through plasmid transfection, is expressed, and separates, and steps such as purification take time and effort, and can only carry out the proteic preparation of minority; The 2nd, utilize chemical polypeptide synthetic method, this method is difficult to synthetic bigger albumen, only is suitable for synthetic short polypeptide fragment.
The high reactivity 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 direct fixed method, with protein molecule by physical adsorption, 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 link molecule of one deck, to keep the separate state and the activity of protein molecule, usually the method that adopts is ProteinG-IgG, vitamin H-microbiotic, special interacting molecules such as DNA-oligonucleotide are right, and these class methods need extra step, and fixed efficiency low (referring to document Baumann S.etal.Jornal of Immunological Methods, 221:95 (1998)).
High-sensitivity detecting method: with respect to traditional biosensor since protein chip on proteinic amount still less, so require proofing unit that higher sensitivity is arranged.The most normally used is the method for fluorescent mark laser scanning imaging.
The gene 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 gene 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(Tm) of hair fastener shape oligonucleotide probe, in the melting temperature(Tm)-5 of single-point mismatch hybridization state ℃ between the scope of the melting temperature(Tm) of mating the hybridization state fully+5 ℃.The present invention can further optimize hybridization conditions by this gene chip is carried out control of Electric potentials.Gene 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 gene 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 gene chip electric potential scanning, adopt gene chip with hairpin-shaped probes, at first use the target preparing standard solution, with standardized solution and probe hybridization, and add embedded fluorescence dye, chip base is applied current potential and carries out electric potential scanning, the fluorescent signal typical curve of probe under the writing scan and standardized solution hybridization; The fluorescent method of then the testing sample dna solution being packed into is measured the device of chip hybridization, with probe hybridization, and adds embedded fluorescence dye; Equally chip base is applied current potential and carries out electric potential scanning, the curve that the fluorescent 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 the identification of single nucleotide polymorphism (SNP) 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.
Protein is easy of hydrophobic interaction causes the destruction of its three-dimensional structure at solid surface, thereby causes the forfeiture of its biological function.So albumen is a great bottleneck of restriction protein biochip technology at the inactivation of solid surface.We innovate from preparation and detection thinking, invented first identification back fixed novel protein chip, be that probe albumen and target protein are discerned in solution earlier, be associated in the oligonucleotide hybridization on oligonucleotide on the probe albumen and chip base surface then by coupling, mixture after the identification is fixed on specified location on the chip, has so just fundamentally solved the problem of albumen effectively at surperficial inactivation.
Such protein chip has been introduced a new problem again, and promptly oligonucleotide is hybridized specific problem.Non-specific hybridization will be introduced bigger error to protein chip.The present invention can address this problem effectively.
Summary of the invention
In the present invention, we are incorporated into the technology of fluorescence resonance energy transmission (FRET) in the preparation and detection of protein chip.
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 emmission 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.
By design, we make the double-stranded and luminous dye molecule of special embedding oligonucleotide, give body and acceptor with the fluorescence molecule of mark on the target molecule forms FRET.Like this, have only when the oligonucleotide two strands and hybridize fully and probe target molecule specific recognition, under the situation that these two conditions satisfy simultaneously, protein chip could produce the FRET signal.By introducing FRET, improved the specificity of protein chip greatly, be the continuity and the development of identification back fixing means earlier.
An object of the present invention is, a kind of protein chip that transmits based on fluorescence resonance energy 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.After introducing the fluorescence resonance energy transmission, realize high specific and highly sensitive Protein Detection.
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, adopt 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 transmitter and protein chip can't prolonged preservation on the surface.
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 mixture with gained in the step (2) mixes with fluorescent mark target protein solution to be measured.
(4) mixture is solidificated in the surface.
With resulting mixing solutions in the step (3), carry out hybridization with resulting oligonucleotide solidified surface in the step (1).
(5) fluorescent signal detects.
The chip of having caught target protein is placed buffered soln, and add the double-stranded luminous fluorescence 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.
Compare with traditional protein chip detection, embedded dyestuff of the present invention and fluorescent tag molecule produce fluorescent 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 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 random 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 be in the Protein Detection process, introduce the factor of electric potential scanning, and replace single absolute strength with fluorescence intensity with the variation spectral line of current potential, come special and non-specific responding are distinguished.Like this, can obtain gem-pure difference spectrum, greatly eliminate the influence of reaction conditions and fluorescence intensity.
In addition, traditional protein chip detects takes to fix earlier the method for afterwards discerning, and the present invention takes identification back fixed method earlier, 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, the existing protein-denatured one-tenth side of body, the steric factor of also having living space), the mixture after the identification again by the specific recognition effect of oligonucleotide in the substrate surface addressing.Different with traditional method is, do not need when preserving protein chip to preserve to be solidified with proteic surface, and just passable as long as preserve the substrate and the protein soln that are solidified with oligonucleotide.
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 analogue 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 one deck at these material surfaces and help oligonucleotide solidified film, both can adopt covalently bound method, can adopt the method for physical adsorption to be cured again.Nucleic acid oligomer probe after design is synthetic has following structure: have the complete complementary nucleic acid oligomer of the sequence 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 analogue 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 protein macromolecule or the polypeptide fragment that can discern with albumen.The fragment that the oligonucleotide that links to each other with albumen 4 exists the detecting area of one section energy and the hair fastener shape oligonucleotide that is solidificated in substrate surface to match each other for the design synthetic.Protein can be taked covalent attachment with being connected of oligonucleotide 4, promptly utilizes the active group of protein surface to be connected with the active group formation covalent linkage of oligonucleotide 4 ends; Also can adopt non-covalent mode of connection, promptly albumen and oligonucleotide all be carried out the small molecules mark, the macromole by specific recognition couples together the two then, utilization be special interaction between macromole and the small molecules.
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 small molecules and macromole.Target protein molecule 5 is present in the solution to be measured that the user provides, i.e. the material of required detection.Fluorescent mark is classical marking 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 surperficial fixed hair fastener shape oligonucleotide 2 hybridization of the probe protein molecular 3 that has joined the oligonucleotide chain when coupling and chip base 1, 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 fluorescent 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 natural oxidation silicon layer pick up reagent (70% vitriol oil: 95 degree high temperature oxidations 30% hydrogen peroxide), pass through NH then
4The corrosion of F, the silicon face of formation hydrogen termination under UV-light causes, has the unsaturated alkane chain and the Si-H surface reaction of ester group functional group, forms fine and close ester group terminated organic membrane.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 vitamin H and microbiotic (nucleophilic nuclein) couples together albumen and oligonucleotide in non-covalent mode with albumen and oligonucleotide vitamin H on the mark all.In patent application, 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 mixture 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 fluorescent mark target protein molecule 5 solution to be measured with the probe albumen-oligonucleotide mixture of gained in the step 2.Wherein, the probe albumen of specific recognition and target protein will form oligonucleotide-probe albumen-target protein mixture.
4, mixture is solidificated in the surface
With resulting mixing solutions in the step 3, carry out hybridization with resulting oligonucleotide solidified surface in the step 1.Oligonucleotide in the mixing solutions-oligonucleotide of probe albumen-target protein mixture and the oligonucleotide specific hybridization of surface cure are gone up (as among Fig. 2 10) thereby oligonucleotide-probe albumen-target protein mixture is solidificated in chip base 1 surface.
5, fluorescent signal detects
Fluorescent signal detects 11, and the chip of having caught target protein is placed buffered soln, and adds the double-stranded luminous fluorescence 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 emmission 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 variation, 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 carboxyl terminated silicon face 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 buffered soln, adds embedded dye molecule PicoGreen during detection in the buffered soln.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 carboxyl terminated silicon face 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 buffered soln, add embedded dye molecule PicoGreen during detection in the buffered soln, 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 prolonged preservation.The surface fixed is an oligonucleotide, and oligonucleotide is more stable much than protein at substrate surface, so chip can prolonged 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 (4)
1. an oligonucleotide intercalative dye fluorescence resonance energy transmits molecular detecting method, comprises the following steps:
(1) is the organic film of reactive functional at chip base surface coverage last layer end,, known first single strain oligonucleotide with hair fastener shape structure of a class sequence is fixed on substrate surface by covalent bonding;
(2) known second single strain oligonucleotide of probe albumen and another kind of sequence is linked to each other make the combined probe molecule, wherein: this second single strain oligonucleotide and be fixed on and have one section complete complementary sequence in the first single strain oligonucleotide sequence on the chip base, and probe albumen can combine with target protein specificity to be measured;
(3) the target protein molecule is carried out fluorescent mark;
(4) combined probe molecule and fluorescently-labeled target protein to be measured carry out molecular recognition in solution, form oligonucleotide-probe albumen-target protein mixture;
(5) described oligonucleotide-probe albumen-target protein mixture and the resulting oligonucleotide solidified of step (1) surface are carried out hybridization;
(6) in the oligonucleotide two strands that hybridization forms, embed special luminous dye molecule, give body and acceptor with the fluorescence molecule of mark on the target protein forms that fluorescence resonance energy transmits;
(7) detect because the fluorescent signal that the fluorescence resonance energy transmission produces.
2. oligonucleotide intercalative dye fluorescence resonance energy according to claim 1 transmits molecular detecting method, and it is characterized in that: described probe albumen is the protein macromolecule or the polypeptide fragment that can carry out specific recognition with target protein.
3. oligonucleotide intercalative dye fluorescence resonance energy according to claim 2 transmits molecular detecting method, and it is characterized in that: described probe albumen is antibody or acceptor.
4. oligonucleotide intercalative dye fluorescence resonance energy according to claim 1 transmits molecular detecting method, it is characterized in that: described oligonucleotide is oligodeoxynucleotide, oligomerization ribonucleotide, or the analogue of oligonucleotide, wherein, described oligonucleotide analogue comprises polypeptide nucleotide.
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| JP5518450B2 (en) * | 2008-12-04 | 2014-06-11 | 富士フイルム株式会社 | Fragmented antibody-immobilized carrier and method for producing the same |
| CN102890155B (en) * | 2012-09-12 | 2015-04-29 | 暨南大学 | Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5556752A (en) * | 1994-10-24 | 1996-09-17 | Affymetrix, Inc. | Surface-bound, unimolecular, double-stranded DNA |
| EP1179363A2 (en) * | 2000-08-09 | 2002-02-13 | Fuji Photo Film Co., Ltd. | Fixation of nucleotide derivatives to solid carrier |
| CN1379114A (en) * | 2002-03-20 | 2002-11-13 | 东南大学 | Process for preparing microarray chip of double-stranded nucleic acid |
| WO2003019147A2 (en) * | 2001-08-30 | 2003-03-06 | Combimatrix Corporation | Enzyme-amplified redox microarray detection process |
| CN1435492A (en) * | 2003-03-05 | 2003-08-13 | 东南大学 | Chip for non-label detecting DNA bindin, preparation and use method thereof |
| US6713262B2 (en) * | 2002-06-25 | 2004-03-30 | Agilent Technologies, Inc. | Methods and compositions for high throughput identification of protein/nucleic acid binding pairs |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5556752A (en) * | 1994-10-24 | 1996-09-17 | Affymetrix, Inc. | Surface-bound, unimolecular, double-stranded DNA |
| EP1179363A2 (en) * | 2000-08-09 | 2002-02-13 | Fuji Photo Film Co., Ltd. | Fixation of nucleotide derivatives to solid carrier |
| WO2003019147A2 (en) * | 2001-08-30 | 2003-03-06 | Combimatrix Corporation | Enzyme-amplified redox microarray detection process |
| CN1379114A (en) * | 2002-03-20 | 2002-11-13 | 东南大学 | Process for preparing microarray chip of double-stranded nucleic acid |
| US6713262B2 (en) * | 2002-06-25 | 2004-03-30 | Agilent Technologies, Inc. | Methods and compositions for high throughput identification of protein/nucleic acid binding pairs |
| CN1435492A (en) * | 2003-03-05 | 2003-08-13 | 东南大学 | Chip for non-label detecting DNA bindin, preparation and use method thereof |
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