CN100396790C - 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
- Publication number
- CN100396790C CN100396790C CNB2004100095741A CN200410009574A CN100396790C CN 100396790 C CN100396790 C CN 100396790C CN B2004100095741 A CNB2004100095741 A CN B2004100095741A CN 200410009574 A CN200410009574 A CN 200410009574A CN 100396790 C CN100396790 C CN 100396790C
- Authority
- CN
- China
- Prior art keywords
- protein
- oligonucleotide
- chip
- molecule
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The present invention provides a protein chip for solution recognition and surface addressing. The protein chip of the present invention comprises a chip substrate and card oligonucleotide solidified on the chip substrate, and thus, a probe molecule comprises a protein, oligonucleotide connected with the protein, a target protein molecule labelled by fluorescence and a dye molecule which is specifically embedded in a double strand of the oligonucleotide to emit light. An organic film whose end has reaction active functional group is covered on the surface of the chip substrate, and the oligonucleotide is fixed on the surface of the substrate through covalent bonding. The present invention also provides a method capable of fixing and detecting a protein. The method uses card DNA and a potential control technique to couple a protein molecule so as to realize solution recognition and surface addressing, the surface denaturation of the protein is avoided to a maximum extent, and the defect that a protein sensor and the protein chip can not be stored for a long time is overcome.
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 (structure gene group) epoch.Genetic 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, as interaction between proteinic modification processing, transhipment, location, structural changes, protein-protein, protein and other biological macromole etc., all can't on genomic level, obtain.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 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 and Actuators, 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.Journal 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 2 probes, 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 testing sample D N A 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 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 transmitter and protein chip can't prolonged preservation on the surface.And introduce fluorescence resonance energy transmission (FRET) and realize high specific and highly sensitive 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 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.
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.But this step is not must step.The concrete grammar of surface potential scanning is referring to another part patent application 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 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.
Embedded dyestuff 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.
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.
This protein chip various piece specifically describes as follows:
1, chip base and curing hair fastener shape oligonucleotide or analogue thereon
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 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 (6)
1. the protein chip of a solution identification, surface addressing comprises the chip base and the protein soln that are solidified with oligonucleotide, it is characterized in that:
Described protein soln contains the combined probe molecule, and this combined probe molecule comprises probe albumen and the coupled known single strain oligonucleotide of a class sequence;
The described chip base that is solidified with oligonucleotide is to cover the last layer end at substrate surface to be the organic film of reactive functional, then the known hair fastener shape of another kind of sequence single strain oligonucleotide is fixed on it;
There is one section complete complementary sequence in the two above-mentioned class single strain oligonucleotide sequences.
2. the protein chip of solution identification according to claim 1, surface addressing is characterized in that: chip base is 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 probe albumen in the described combined probe molecule is the protein macromolecule or the polypeptide fragment that can carry out specific recognition with target protein.
4. the protein chip of solution identification according to claim 3, surface addressing is characterized in that: the probe albumen in the described combined probe molecule is antibody or acceptor.
5. the protein chip of solution identification according to claim 1, surface addressing, it is characterized in that: described oligonucleotide is oligodeoxynucleotide, oligomerization ribonucleotide, or the analogue of oligonucleotide, wherein, described oligonucleotide analogue comprises polypeptide nucleotide.
6. the preparation 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 hair fastener shape single strain oligonucleotide fragment be fixed on substrate surface by covalent bonding;
(2) albumen becomes the combined probe molecule with oligonucleotide coupling joint group
Probe albumen and single strain oligonucleotide are passed through covalently or non-covalently mode coupling connection, and there is one section complete complementary sequence in single strain oligonucleotide wherein at least with in a kind of hair fastener shape single strain oligonucleotide sequence that is solidificated in substrate surface.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100095741A CN100396790C (en) | 2004-09-17 | 2004-09-17 | Solution identification and surface addressing protein chip and its preparing and detecting method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100095741A CN100396790C (en) | 2004-09-17 | 2004-09-17 | Solution identification and surface addressing protein chip and its preparing and detecting method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1749752A CN1749752A (en) | 2006-03-22 |
| CN100396790C true CN100396790C (en) | 2008-06-25 |
Family
ID=36605313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100095741A Expired - Fee Related CN100396790C (en) | 2004-09-17 | 2004-09-17 | Solution identification and surface addressing protein chip and its preparing and detecting method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100396790C (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009545747A (en) | 2006-08-02 | 2009-12-24 | カリフォルニア インスティチュート オブ テクノロジー | Method and system for detecting and / or sorting targets |
| CN101666805A (en) * | 2009-07-15 | 2010-03-10 | 苏州纳米技术与纳米仿生研究所 | Method for preparing specific protein detection chip |
| US20190300945A1 (en) | 2010-04-05 | 2019-10-03 | Prognosys Biosciences, Inc. | Spatially Encoded Biological Assays |
| US10787701B2 (en) | 2010-04-05 | 2020-09-29 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
| GB201106254D0 (en) | 2011-04-13 | 2011-05-25 | Frisen Jonas | Method and product |
| CN103149179A (en) * | 2013-01-15 | 2013-06-12 | 华北电力大学（保定） | Method of quantitatively detecting proteins by Co(NH3)6)3+-DNA resonance light scattering |
| WO2014210225A1 (en) | 2013-06-25 | 2014-12-31 | Prognosys Biosciences, Inc. | Methods and systems for determining spatial patterns of biological targets in a sample |
| CN104020198B (en) * | 2014-06-18 | 2016-05-18 | 青岛科技大学 | A kind of signal amplification technique electrochemical sensor detects the method for DNA |
| US10774374B2 (en) | 2015-04-10 | 2020-09-15 | Spatial Transcriptomics AB and Illumina, Inc. | Spatially distinguished, multiplex nucleic acid analysis of biological specimens |
| WO2020123316A2 (en) | 2018-12-10 | 2020-06-18 | 10X Genomics, Inc. | Methods for determining a location of a biological analyte in a biological sample |
| US11649485B2 (en) | 2019-01-06 | 2023-05-16 | 10X Genomics, Inc. | Generating capture probes for spatial analysis |
| US11926867B2 (en) | 2019-01-06 | 2024-03-12 | 10X Genomics, Inc. | Generating capture probes for spatial analysis |
| WO2021092433A2 (en) | 2019-11-08 | 2021-05-14 | 10X Genomics, Inc. | Enhancing specificity of analyte binding |
| FI3891300T3 (en) | 2019-12-23 | 2023-05-10 | 10X Genomics Inc | Methods for spatial analysis using rna-templated ligation |
| US11732299B2 (en) | 2020-01-21 | 2023-08-22 | 10X Genomics, Inc. | Spatial assays with perturbed cells |
| US11702693B2 (en) | 2020-01-21 | 2023-07-18 | 10X Genomics, Inc. | Methods for printing cells and generating arrays of barcoded cells |
| US11898205B2 (en) | 2020-02-03 | 2024-02-13 | 10X Genomics, Inc. | Increasing capture efficiency of spatial assays |
| US11732300B2 (en) | 2020-02-05 | 2023-08-22 | 10X Genomics, Inc. | Increasing efficiency of spatial analysis in a biological sample |
| US11891654B2 (en) | 2020-02-24 | 2024-02-06 | 10X Genomics, Inc. | Methods of making gene expression libraries |
| EP4242325A3 (en) | 2020-04-22 | 2023-10-04 | 10X Genomics, Inc. | Methods for spatial analysis using targeted rna depletion |
| EP4153775A1 (en) | 2020-05-22 | 2023-03-29 | 10X Genomics, Inc. | Simultaneous spatio-temporal measurement of gene expression and cellular activity |
| WO2021252499A1 (en) | 2020-06-08 | 2021-12-16 | 10X Genomics, Inc. | Methods of determining a surgical margin and methods of use thereof |
| AU2021294334A1 (en) | 2020-06-25 | 2023-02-02 | 10X Genomics, Inc. | Spatial analysis of DNA methylation |
| US11761038B1 (en) | 2020-07-06 | 2023-09-19 | 10X Genomics, Inc. | Methods for identifying a location of an RNA in a biological sample |
| US11926822B1 (en) | 2020-09-23 | 2024-03-12 | 10X Genomics, Inc. | Three-dimensional spatial analysis |
| US11827935B1 (en) | 2020-11-19 | 2023-11-28 | 10X Genomics, Inc. | Methods for spatial analysis using rolling circle amplification and detection probes |
| WO2022140028A1 (en) | 2020-12-21 | 2022-06-30 | 10X Genomics, Inc. | Methods, compositions, and systems for capturing probes and/or barcodes |
| WO2023034489A1 (en) | 2021-09-01 | 2023-03-09 | 10X Genomics, Inc. | Methods, compositions, and kits for blocking a capture probe on a spatial array |
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 |
| CN1379114A (en) * | 2002-03-20 | 2002-11-13 | 东南大学 | Process for preparing microarray chip of double-stranded nucleic acid |
| WO2003040679A2 (en) * | 2001-11-09 | 2003-05-15 | Friz Biochem Gmbh | Reversibly binding a fluorophore to a surface for the purpose of detecting ligate/ligand association events by fluorescence quenching |
| 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 |
| US20040158051A1 (en) * | 2002-11-19 | 2004-08-12 | Mihri Ozkan | Mono and dual conjugation of nanostructures and methods of making and using thereof |
-
2004
- 2004-09-17 CN CNB2004100095741A patent/CN100396790C/en not_active Expired - Fee Related
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 |
| WO2003040679A2 (en) * | 2001-11-09 | 2003-05-15 | Friz Biochem Gmbh | Reversibly binding a fluorophore to a surface for the purpose of detecting ligate/ligand association events by fluorescence quenching |
| 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 |
| US20040158051A1 (en) * | 2002-11-19 | 2004-08-12 | Mihri Ozkan | Mono and dual conjugation of nanostructures and methods of making and using thereof |
| CN1435492A (en) * | 2003-03-05 | 2003-08-13 | 东南大学 | Chip for non-label detecting DNA bindin, preparation and use method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1749752A (en) | 2006-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100396790C (en) | Solution identification and surface addressing protein chip and its preparing and detecting method | |
| CN109321635B (en) | Nucleic acid detection method based on multiple hybrid chain reactions and application | |
| EP2129805B1 (en) | Bioassay system including optical detection apparatuses, and method for detecting biomolecules | |
| Pejcic et al. | The role of biosensors in the detection of emerging infectious diseases | |
| Wang | Survey and summary: From DNA biosensors to gene chips | |
| EP2430428B1 (en) | Single-molecule detection system and methods | |
| US7811810B2 (en) | Bioassay system including optical detection apparatuses, and method for detecting biomolecules | |
| Raymond et al. | Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support | |
| JP2005501248A (en) | Biosensing platform for detecting and quantifying biological molecules | |
| KR20170036659A (en) | Improved assay methods | |
| KR100823684B1 (en) | Method for detecting a biological target material using barcode dna | |
| WO2005108609A1 (en) | Method for identification and analysis of certain molecules using the dual function of single strand nucleic acid | |
| US20130065780A1 (en) | Label-Free Multiplexing Bioassays Using Fluorescent Conjugated Polymers and Barcoded Nanoparticles | |
| Homs | DNA sensors | |
| Lee et al. | DNA-based nanostructures for molecular sensing | |
| KR101048429B1 (en) | Target material detection and quantification method using biochip | |
| Wang et al. | Silica nanoparticle assisted DNA assays for optical signal amplification of conjugated polymer based fluorescent sensors | |
| CN100396791C (en) | Method for detecting oligomeric nucleotide through intercalating dye fluorescent resonant energy transfer molecule | |
| US20050266449A1 (en) | Method of detecting a hybrid, formed of at least two species, on a substrate | |
| CA2686537A1 (en) | Nucleic acid chip for obtaining bind profile of single strand nucleic acid and unknown biomolecule, manufacturing method thereof and analysis method of unknown biomolecule using nucleic acid chip | |
| Wang | PNA biosensors for nucleic acid detection | |
| CN115698318A (en) | Control for proximity detection assay | |
| Bosch et al. | Optical chemical biosensors for high throughput screening of drugs | |
| Davis et al. | General introduction to biosensors and recognition receptors | |
| WO2011084168A1 (en) | Bioassays based on polymeric sequence probes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080625 Termination date: 20100917 |