CN101001960A - Bio-barcode based detection of target analytes - Google Patents

Bio-barcode based detection of target analytes Download PDF

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Publication number
CN101001960A
CN101001960A CN 200480024522 CN200480024522A CN101001960A CN 101001960 A CN101001960 A CN 101001960A CN 200480024522 CN200480024522 CN 200480024522 CN 200480024522 A CN200480024522 A CN 200480024522A CN 101001960 A CN101001960 A CN 101001960A
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oligonucleotide
dna
probe
particle
specificity
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查德·A·米尔金
南载民
沙德·C·撤克斯顿
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Northwestern University
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Northwestern University
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Abstract

The present invention relates to screening methods, compositions, and kits for detecting for the presence or absence of one or more target analytes, e.g. biomolecules, in a sample. In particular, the present invention relates to a method that utilizes reporter oligonucleotides as biochemical barcodes for detecting multiple protein structures or other target analytes in a solution.

Description

Detect target analyte based on bio-barcode
The field of the invention:
The present invention relates to be used for test sample and whether have one or more target analytes, for example, protein, nucleic acid, or the screening method of other compound.Specifically, the present invention relates to use reporter oligonucleotides to detect the method for one or more analytes in the solution as biochemical barcode (barcode).
Cross reference:
The provisional application that the application applied on September 25th, 1 number 60/506,708; 60/482,979 of application on June 27th, 2003; 60/496,893 of application on August 21st, 2003; 60/515,243 of application on October 28th, 2003; 60/530 of application on December 18th, 2003,797 interests and it are the U.S. Patent Application Serial 10/108 of application on March 27th, 2002,211 part continuity, the U.S. Provisional Application of claimed 28 applications in the March, 2000 of quoting as a reference with its integral body of the latter number 60/192,699; With 60/350,560 interests of November 13 calendar year 2001 application, and it is the part continuity of the U.S. Patent Application Serial 09/820,279 of application on March 28 calendar year 2001.The work of being reported among the application is by NSF, ARO, and AFOSR, DARPA and NIH appropriation part are subsidized.Therefore, United States Government clearly enjoys the right of inventing described in the application.
Background of the present invention:
The detection of analyte all is important for molecular biology research and medical use.Now based on fluorescence, mass spectroscopy, gel electrophoresis, laser scanning and electrochemical diagnostic method can be used for identifying the range protein structure 1-4Be widely used in the genetics protein variants of identifying hemocyte based on the reaction of antibody, diagnose the illness, molecular probe in the position tissue and purifying molecule or finish sepn process 5For medical diagnostic applications (for example, malaria and HIV), such as enzyme-linked immunosorbent assay, the antibody test of Western trace and indirect fluorescent antibody test is for identifying that single target protein structure is exceedingly useful 6,7It all is useful that the multiple antibody that exists is carried out being selected in research and the clinical application with sample sifter simultaneously rapidly.Yet using above-mentioned methods involving to detect some protein structures simultaneously under test conditions is difficulties, costliness and consuming time.
The target amplification of polymerase chain reaction (PCR) and other form makes and is used for research that the exploitation of the effective tool of the detection of legal medical expert and clinical application and quantitative interested DNA target develops rapidly 26-32The exploitation of corresponding proteins matter target amplification method can significantly improve medical diagnosis and development proteinology (proteomics) field 33-36Although can't chemical replication protein target, can come this target of mark with the oligonucleotide mark, follow available PCR and increase, use the DNA detection method to identify this interested target then 37-45This method is commonly referred to immunity-PCR, and its allow to detect the protein (Fig. 5) that has various multi-form dna markers.So far, all immunity-PCR method all comprise heterogeneous assay method (heterogeneous assays), and promptly it comprises and begins target analyte is fixed to a surface, use the antibody of band dna marker (for example to detect subsequently, referring to U.S. Patent number 5,635,602, with 5,665,539).This dna marker generally is firmly bonded to (by covalent interaction or streptavidin-vitamin H combination) on the antibody.Although these methods have marked improvement in protein detection, they also have some shortcomings: 1) limited sensitivity owing to DNA recognition sequence and the ratio that detects antibody are low; 2) heterogeneous character owing to the target capture-process has delayed the target binding kinetics, has promptly increased minute and has reduced mensuration sensitivity (Fig. 5 step 3); 3) chemistry connect antibody and DNA-mark required combine chemical complexity (Fig. 5 step 4); With 4) need the pcr amplification step 45Therefore, needing can multipleization and a kind of sensitivity implemented easily and the method for test sample target test thing rapidly.
For the DNA detection method, use radio-labeling, molecular fluorescence group, chemiluminescence system, electrochemical label and the nearest mark based on nanostructure (nanostructure) have been developed multiple assay method 61-70Although some based on the method for nanostructure aspect the sensitivity near PCR, all can not reach the level of sensitivity of the 1-10 copy that PCR produces so far.Allow to carry out PCR-sample amplification of signal and do not have the complicacy relevant with PCR, costliness, the method for time and labour-intensive characteristics will provide remarkable advantages with respect to the method for PCR-based.
Summary of the present invention:
The present invention relates to use oligonucleotide to be used for detecting the method for the multiple analytes of solution, probe, composition, and test kit as biochemical barcode.This method utilize with nano particle (nanoparticles) directly or indirectly the specificity of functionalization (functionalized) in conjunction with right recognition component, with cause gold nano grain accumulative hybridisation events (for example obviously to change its physical property, optics, electricity, mechanics) former observations 8-12General thought be specificity in conjunction with each right recognition component can with have the different oligonucleotide sequences and a physical property relevant of separating and can cut out the hybridization of (tailorable) and the characteristic of unwinding and link with nano particle.The hybridization that should be separately and the characteristic of unwinding can be used for by the change of the physical property relevant with nano particle or by the hybridization/impurity elimination friendship or the event detection oligonucleotide sequence that unwinds/the anneal a series of analytes of multiple analyte in testing of decoding.
In one embodiment of the invention, provide a kind of method whether test sample exists one or more target analytes that is used for, this target analyte has at least two binding sites, and the method comprising the steps of:
A kind of matrix is provided; The particle probe of one or more types is provided, every type probe comprises one or more specificitys with concrete target analyte in conjunction with complement and one or more DNA barcodes and its bonded particle, wherein the specificity of every type particle probe has specificity in conjunction with complement to concrete target analyte, and the DNA barcode of every type particle probe is as the mark of this concrete target analyte;
This target analyte is fixed on this matrix;
Allow target analyte combine with the specificity of this analyte between the complement in conjunction with and under the condition for validity of formation mixture under the condition that target analyte exists, this fixed target analyte is contacted with the particle probe of one or more types;
Wash this matrix to remove unconjugated particle probe; With
The optional DNA barcode amplification that makes; With
Detect whether there is this DNA barcode, wherein whether having this mark is the index that whether has the specific target analyte in the sample.
Aspect of this embodiment of the present invention, this target analyte is that protein or haptens and its specificity are the antibody that comprises mono-clonal or polyclonal antibody in conjunction with complement.
In another aspect of this invention, the DNA barcode increases by PCR.
In another aspect of this invention, this particle carries out mark with at least two DNA barcodes.
In another aspect of this invention, arranged the capturing probe of one or more types of target analyte on this matrix.
In another embodiment of the present invention, the method that whether has one or more target analytes in a kind of test sample is provided, every kind of target analyte has at least two binding sites, and this method comprises:
The capturing probe that is incorporated into one or more types on the matrix is provided, and every type capturing probe comprises the specificity of first binding site of specific target analyte in conjunction with complement;
The detection probes of one or more types is provided, every type detection probes comprises the nano particle that has with its bonded oligonucleotide, one or more specificitys of second binding site of this specific target analyte are in conjunction with complement, with as one or more DNA barcodes of concrete target analyte mark, wherein at least a portion sequence of this DNA barcode and at least some oligonucleotide hybridizations that are attached on the nano particle;
When allowing that the specificity binding interactions takes place between target analyte and the probe and having target analyte, form under the condition for validity of assembling mixture, make sample, capturing probe, detection probes contact;
Washing matrix is to remove unconjugated detection probes;
Detect in the gathering mixture on the matrix whether have this DNA barcode, the detected result that wherein whether has this DNA barcode is the index that whether has target analyte in the sample.
Aspect of this embodiment of the present invention, this detection probes comprises one or more specificitys of second binding site of (i) specific target analyte in conjunction with complement, (ii) with the oligonucleotide of at least a type of nano particle bonded, with the DNA barcode that has with at least a portion complementary predetermined sequence of the oligonucleotide of at least a type, this DNA barcode combines the mark as concrete target analyte with all types of detection probes;
In this embodiment on the other hand, before described detection step, this method also comprises step:
Making mixture impurity elimination friendship and discharging under the condition for validity of this DNA barcode, handle this gathering mixture; With
Before described detection, make the DNA barcode amplification.
In this embodiment on the other hand, this DNA barcode passes through pcr amplification.
In this embodiment on the other hand, capturing probe is attached on the magnetic substrate such as magnetic-particle.
In this embodiment on the other hand, target analyte is the target nucleic acid with sequence of at least two parts, this detection probes comprises the nano particle that has with its bonded oligonucleotide, at least a portion of this oligonucleotide has and this DNA barcode complementary sequence, the specificity of this detection probes comprises first target identification oligonucleotide that has with first part's complementary sequence of target nucleic acid in conjunction with complement, and the specificity of capturing probe comprises second target identification oligonucleotide that has with the complementary of the second section at least sequence of target nucleic acid in conjunction with complement.
In this embodiment on the other hand, this target analyte is the target nucleic acid with sequence of at least two parts, this detection probes comprises the nano particle that combines oligonucleotide, this DNA barcode has and at least a portion complementary sequence that is attached to the oligonucleotide on this detection probes, this specificity comprises the target identification oligonucleotide of the sequence with at least the first and second parts in conjunction with complement, first part's complementation of this first part and target nucleic acid and second section and be attached at least a portion complementation of the oligonucleotide on the nano particle, the specificity of this matrix comprise the target identification oligonucleotide that has with second section complementary at least a portion of target nucleic acid in conjunction with complement.
In this embodiment on the other hand, this detection probes comprises SD (dendrimer).
In another embodiment of the present invention, the method that whether has one or more target analytes in the test sample is provided, every kind of target analyte has at least two binding sites, and this method comprises:
The capturing probe of one or more types is provided, and every type capturing probe comprises (i) magnetic-particle; (ii) be attached to first specificity on the magnetic-particle in conjunction with first right member, wherein first specificity is attached on first binding site of specific target analyte in conjunction with the first right member;
The detection probes of one or more types of each target analyte is provided, and every type detection probes comprises (i) nano particle; (ii) be attached to second specificity on the nano particle in conjunction with first right member, wherein this second specificity combines with second binding site of target analyte in conjunction with first right member; (iii) with the oligonucleotide of at least a type of nano particle bonded; The (iv) DNA barcode of at least a type, every type DNA barcode have with at least a portion complementary predetermined sequence of the oligonucleotide of particular type and as the mark of specific target analyte;
Form under the condition for validity of assembling mixture allowing to carry out the specificity binding interactions between target analyte and the probe and exist under the condition of target analyte to combine, make sample, capturing probe, detection probes contact with magnetic-particle;
Unconjugated detection probes on the flush away magnetic-particle; With
Detect in the mixture whether have the DNA barcode, wherein the detected result of this DNA barcode is the index that target analyte exists.
Aspect of this embodiment, this method also is included in the step before the described detection step:
Apply this gathering mixture of magnetic field separation;
Discharge under the condition for validity of this DNA barcode in impurity elimination friendship and gathering mixture, handle this gathering mixture;
The DNA barcode that separates this release.
In this embodiment on the other hand, this method also comprises the DNA barcode amplification that makes release.
In this embodiment on the other hand, this method also comprises:
The matrix that combines oligonucleotide is provided, and this oligonucleotide has at least a portion sequence complementary sequence with the DNA barcode;
The nano particle that comprises with its bonded oligonucleotide is provided, and at least a portion that wherein is attached to the oligonucleotide on the nano particle has at least a portion complementary sequence with the DNA barcode; With
Under the condition for validity of between the first part at least that allows the DNA barcode and the complementary oligonucleotide that is attached on the matrix and between the second section of DNA barcode and some oligonucleotide that are attached on the nano particle, hybridizing, make the DNA barcode, be attached to oligonucleotide on the matrix, reach nano particle and contact.
In this embodiment on the other hand, before detection, make the DNA barcode amplification by PCR.
In this embodiment on the other hand, this method also is included in to analyze and assembles preceding this gathering mixture that separates of mixture.
In this embodiment on the other hand, by being applied magnetic field, the gathering mixture separates this gathering mixture.
In this embodiment on the other hand, this nano particle is metal nanoparticle or the semiconductor nanoparticle such as gold nano grain.
In this embodiment on the other hand, the specificity combination is to being antibody and antigen; Acceptor and part; Enzyme and substrate; Medicine and target molecule; Antibody nucleic acid (aptamer) and antibody nucleic acid target; Article two, to small part complementary oligonucleotide chain.
In this embodiment on the other hand, this DNA barcode can be used biotin labeling, radio-labeling, or fluorescent mark.
In another embodiment of the present invention, the method that whether has one or more target analytes in the test sample is provided, this method comprises:
At least a or polytype particle composites probe is provided, every type probe comprises and its bonded oligonucleotide, one or more specificitys of specific target analyte are in conjunction with complement, be used as one or more DNA barcodes of concrete target analyte mark, wherein at least a portion sequence of this DNA barcode and at least some oligonucleotide hybridizations that are attached on the nano particle;
Under the condition for validity that allows formation gathering mixture under the condition that the specificity binding interactions takes place between target analyte and the particle composites probe and have target analyte, this sample is contacted with the particle composites probe; With
Observe and mixture formation whether occurs assembling.
Another embodiment of the present invention provides and has detected the method that whether has one or more target analytes, and every kind of target analyte has at least two binding sites.This method comprises for every kind of target analyte uses the capturing probe of at least a type and the detection probes of at least a type.These probes can produce before carrying out practical measurement or original position generation when measuring.Capturing probe comprises first specificity in conjunction with the first right member, and wherein this first specificity combines with first binding site of target analyte in conjunction with the first right member, and wherein first specificity combines with matrix in conjunction with the first right member is optional.In a preferred embodiment, this matrix comprises magnetic-particle.Detection probes comprises (1) nano particle; (2) be attached to second specificity on the nano particle in conjunction with the first right member, wherein second specificity combines with second binding site of target analyte in conjunction with the first right member; (3) with the oligonucleotide of at least a type of nano particle bonded; (4) the DNA barcode of at least a type, every type DNA barcode have at least a portion complementary predetermined sequence with the oligonucleotide of particular type.When using in the sample that is containing target analyte, first specificity on the capturing probe combines with first binding site of target analyte in conjunction with the first right member, and second specificity on the detection probes combines with second binding site of target analyte in conjunction with the first right member.Taking a time-out when capturing probe and detection probes to by target analyte occurs assembling.Separate this aggregation and carry out further melting analysis, wherein have aforesaid multiple target to identify concrete target analyte.In addition, but this aggregation impurity elimination hand over the released dna barcode.
Aspect of this embodiment, the DNA barcode in every type the particle composites probe has difference and is used as the sequence of concrete target analyte identifier.
In this embodiment on the other hand, this method also comprises following steps:
Separate and assemble mixture; With
Analyze this gathering mixture and have existing of not homotactic one or more DNA barcodes with mensuration.
In this embodiment on the other hand, this method also comprises the steps:
Separate this gathering mixture;
Assemble under the condition for validity of mixture impurity elimination friendship and released dna barcode at this, handle this gathering mixture;
Separate this DNA barcode; With
Detection has the existence of not homotactic one or more DNA barcodes, and wherein each DNA barcode is the index that has concrete target analyte in the sample.
In this embodiment on the other hand, this method also comprises the steps:
Separate and assemble mixture;
Assemble under the condition for validity of mixture impurity elimination friendship and released dna barcode at this, handle this gathering mixture;
Separate this DNA barcode;
Make the separated DNA barcode amplification; With
Detection has the existence of the DNA barcode of not homotactic one or more amplifications, and wherein each DNA barcode is the index that has concrete target analyte in the sample.
In this embodiment on the other hand, this target has plural binding site and at least two types particle composites probe is provided, first type probe has the specificity of first binding site on the target analyte in conjunction with complement, and second type probe has the specificity of second binding site on the probe in conjunction with complement.Multiple particle composites probe can be provided, and every type probe has a specific specificity of different binding sites on the target analyte in conjunction with complement.
In this embodiment on the other hand, exist the detection step of one or more DNA barcodes to comprise:
The matrix that combines oligonucleotide is provided, and this oligonucleotide has at least a portion sequence complementary sequence with the DNA barcode;
Provide and contain oligonucleotide and its bonded nano particle, wherein have at least a portion complementary sequence with the DNA barcode with at least a portion of nano particle bonded oligonucleotide; With
Under the condition for validity of between the first part at least that allows the DNA barcode and the complementary oligonucleotide that is attached on the matrix and between the second section of DNA barcode and some oligonucleotide that are attached on the nano particle, hybridizing, make the DNA barcode, be attached to oligonucleotide on the matrix, reach nano particle and contact; With
Observe detectable change.
In this embodiment on the other hand, this matrix comprises with array way and adheres to the polytype oligonucleotide on it so that allow to detect one or more dissimilar DNA barcodes.
In this embodiment on the other hand, detectable change is to form dark areas on matrix.
In this embodiment on the other hand, detectable change is observed with optical scanner.
In this embodiment on the other hand, matrix contacts to produce detectable change with silver-colored stain.
In this embodiment on the other hand, the DNA barcode is under the condition for validity that allows DNA barcode and the complementary oligonucleotide that is attached on the matrix to hybridize, contact with matrix, and will be attached to DNA barcode on the matrix subsequently under the condition for validity of the part hybridization that allows to be attached at least some oligonucleotide on the nano particle and the DNA bar code sequence on the matrix, contact with the nano particle that combines oligonucleotide.
In this embodiment on the other hand, the DNA barcode is allowing the DNA barcode and is being attached under the condition for validity of at least some oligonucleotide hybridizations on the nano particle, contacts with the nano particle that combines oligonucleotide; And will be attached to DNA barcode on the nano particle subsequently under at least a portion sequence that allows to be attached to the DNA barcode on the nano particle and the condition for validity that is attached to the complementary oligonucleotide hybridization on the matrix, contact with matrix.
In this embodiment on the other hand, DNA amplification barcode before contact procedure.
In this embodiment on the other hand, at least two types particle composites probe is provided, first type probe has the specificity of target analyte first binding site in conjunction with complement, and second type probe has the specificity of target analyte second binding site in conjunction with complement.
In another embodiment of the present invention, provide the particle composites probe.Therefore, aspect of this embodiment, the particle composites probe comprises the particle that combines oligonucleotide, one or more DNA barcodes, with have the oligonucleotide that combines complement with the specificity of its bonded specific target analyte, wherein (i) DNA barcode has the sequence of at least two parts; (ii) be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode; (iii) combine specificity and have second section complementary sequence with the DNA barcode in conjunction with the oligonucleotide of complement; (iv) the DNA barcode in every type the particle composites probe has difference and as the sequence of concrete target analyte identifier.
In this embodiment on the other hand, the particle composites probe comprises oligonucleotide and its bonded particle with at least two types, one or more DNA barcodes, and combine the oligonucleotide of the specificity of target analyte in conjunction with complement, wherein the oligonucleotide with the probe bonded first kind has and DNA barcode at least a portion complementary sequence, have such sequence with the oligonucleotide of second type of probe bonded, promptly it with have specificity and combine at least a portion sequence complementation of the oligonucleotide of complement.
In this embodiment on the other hand, the particle composites probe comprises the particle that combines oligonucleotide, one or more DNA barcodes, with the specificity of target analyte in conjunction with complement, wherein with at least a portion of particle bonded oligonucleotide have with at least a portion sequence complementary sequence of DNA barcode and wherein this DNA barcode as the identifier of concrete target analyte.
In another embodiment of the present invention, provide a kind of particle composites probe.Therefore in one embodiment of the invention, the particle composites probe that provides comprises the particle that combines oligonucleotide, the DNA barcode, and combine the oligonucleotide of the specificity of concrete target analyte in conjunction with complement, wherein (i) DNA barcode has the sequence of at least two parts; (ii) be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode; (iii) combine specificity and have second section complementary sequence with the DNA barcode in conjunction with the oligonucleotide of complement; (iv) the DNA barcode in every type the particle composites probe has difference and as the sequence of concrete target analyte identifier.
In another embodiment of the present invention, a kind of particle composites probe is provided, it comprises oligonucleotide and its bonded particle with at least two types, the DNA barcode, and combine the oligonucleotide of the specificity of target analyte in conjunction with complement, wherein the oligonucleotide with the probe bonded first kind has and DNA barcode at least a portion complementary sequence, have such sequence with the oligonucleotide of second type of probe bonded, promptly it with have specificity and combine at least a portion sequence complementation of the oligonucleotide of complement.
In another embodiment of the present invention, a kind of particle composites probe is provided, it comprises the particle that combines oligonucleotide, the DNA barcode, with the specificity of target analyte in conjunction with complement, wherein with at least a portion of particle bonded oligonucleotide have with at least a portion sequence complementary sequence of DNA barcode and wherein this DNA barcode as the identifier of concrete target analyte.
In another embodiment of the present invention, a kind of detection probes is provided, it comprises nano particle; Combine a right member with nano particle bonded specificity; Oligonucleotide with at least one type of nano particle bonded; The DNA barcode that has at least one type of predetermined sequence respectively, wherein at least a portion of every type DNA barcode and at least a type oligonucleotide hybridization.
In another embodiment of the present invention, provide the test kit that contains above-mentioned particle composites probe.
These and other embodiment of the present invention will become apparent according to following detailed.
Brief description of the drawings
Fig. 1 has shown the protein detection scheme based on the DNA/Au nano particle.(A) preparation of the nanoparticle probes of hapten transformation.(B) protein detection of the probe of use protein bound.Attention has 9 G in sequence A, C is right, and 2 G are only arranged in sequence B, and C is right.
Fig. 2 has shown the thermally denature figure of the Au nanoparticle aggregate thing that is connected with protein by DNA.The function (1 ℃/minute, 1 minute time length) that the delustring (extinction) of monitoring 260nm increases progressively as temperature.Measure each UV-Vis spectrum in constant agitation under with the condition of suspension aggregation.Carry out before the melting analysis all aggregations being suspended among the 0.3M PBS of 1ml.A) there are a kind of two kinds of probes (IgE (-), IgGl (---)) of target antibody; All data are through normalization; (B) two kinds of two kinds of probes that target antibody all exists.Illustration: first derivative of thermal denaturation curve (first derivative).
Fig. 3 has shown the protein detection scheme based on array of DNA as proteinic bio-barcode of using.
Fig. 4 has shown sweep measurement (scanometric) the DNA array detection method of DNA bio-barcode.Left column is the detection of the bio-barcode that links to each other with IgG1, and right row are detections of the bio-barcode that links to each other with IgE.The seizure oligonucleotide of IgG1 system is 5 '-ataactagaacttga (SEQ IDNO:1) of sulfydryl modification, the IgE system is 5 '-ttatctattatt (SEQ ID NO:2) of sulfydryl modification.Each spot diameter is approximately 250um and (Epson America, Longbeach California) read by gray scale-grade (gray-scale) with Epson Expression 1640XL flat-bed scanner.These assay methods and fairly good have been tested in 20nM effect in the target level scope of 700nM.
Fig. 5 has shown the detection of analytes method based on immunity-PCR of universal class.
Fig. 6 has described use barcode PCR (BPCR) scheme and has detected target analyte, i.e. prostate specific antigen (PSA).The A group has shown the design and the preparation of probe.The B group has shown that PSA detects and barcode DNA cloning and evaluation.
Fig. 7 has shown that the evaluation primer dimer forms, the control experiment of DNA barcode amplification and the influence that DMSO concentration increases progressively when using 25 thermal cyclings.Swimming lane 1 to 5 is the results that have the DNA barcode in the PCR reaction mixture, and no DNA barcode in the swimming lane 6 to 10.Attention increases (increment with 0.5% from 0 to 2%) from the DMSO of swimming lane 1 to 5 and from 6 to 10.
Fig. 8 has shown that PSA detects the gel electrophoresis photo and relative band strength figure of the barcode DNA of back pcr amplification.A group, swimming lane 1 and 2 are that (swimming lane 1: background albumen resists-dinitrobenzene (anti-dinitrophenyl) and beta-galactosidase enzymes, no PSA, swimming lane 2: no protein) in control experiment.From swimming lane 3 to 8, the PSA concentration in the sample (10 μ l) is respectively 300aM, 3fM, 30fM, 300fM, 3pM, and 30pM.The standard biological barcode DNA 40-aggressiveness of PSA other gel band behind electrophoresis and the PCR in swimming lane 9 compares.The B group, the relative gel electrophoresis band strength behind the BPCR.The C group, the lower concentration of PSA detects.From swimming lane 2 (3aM) to swimming lane 7 (300fM) with 10 times of weaker concns from 3aM to 300fM.Only contain the proteic negative control of background and show in swimming lane 1, standard biological barcode 40 aggressiveness (6 μ M bio-barcode duplex) show in article one swimming lane (swimming lane C).The D group, the relative gel electrophoresis band strength behind the BPCR.
Fig. 9 has shown that the sweep measurement of PSA-specificity barcode DNA detects.PSA concentration (10ml sample volume) from 300fM to 3aM between change and shown the negative control sample (contrast) that does not add PSA.In all 7 samples, add 2ml anti--dinitrobenzene (10pM) and 2ml beta-galactosidase enzymes (10pM) albumen as a setting.Shown that also not having PCR with 30nm NP probe detects PSA (30aM and contrast) (illustration).With Verigene ID system photography chip.
Figure 10 has shown the theoretical limit of detection of BPCR.Left side group, gel photograph has shown the band behind PCR when initial barcode DNA concentration is successively decreased.Swimming lane 1:3 * 10 9Copy, swimming lane 2:3 * 10 8Copy, swimming lane 3:3 * 10 7Copy, swimming lane 4:3 * 10 6Copy, swimming lane 5:3 * 10 5Copy, swimming lane 6:3 * 10 4Copy, swimming lane 7:3 * 10 3Copy, swimming lane 8:3 * 10 2Copy, swimming lane 9:3 * 10 1Copy and swimming lane 10: no barcode DNA.Right group, gel electrophoresis band strength figure relatively.
Figure 11 has shown the detection of PSA-specificity barcode DNA, and wherein PSA is dissolved in the compound sheep blood serum substratum.Each group has shown the signal that the analyte (3pM to 3aM) of various concentration produces by BPCR amplification barcode DNA.
Figure 12 has shown that not having PCR with 30nm NP probe detects PSA.Every group with column diagram on relevant relative intensity value shown in various concentration (30aM to 3pM, and contrast) signal by direct detector bar font code DNA (that is the amplification of nothings-BPCR) generation down.With Verigene ID system (Nanosphere, Inc., Northbrook, IL) photography chip.
Figure 13 has shown the DNA-BCA assay method.A. the preparation of nano particle and magnetic corpuscular probe.B. based on the DNA cloning scheme of the no PCR of nano particle.
Figure 14 has shown the anthrax barcode DNA detection that increases with Verigene ID system.A. carry out anthrax barcode DNA detection with 20nm NP probe.B. carry out anthrax barcode DNA detection with 30nm NP probe.
Figure 15 has shown that 20nm and 30nm NP probe silver dye the intensity pattern that strengthens back barcode DNA and the sandwich spot of NP probe.
Figure 16 has shown an embodiment of " general " nanoparticle probes detection scheme.A. synthetic have specific general probe to one or more target nucleic acid sequences.Target identification DNA can be used for controlling the specificity of this probe to target.This probe is used in the mensuration system that has single or multiple target nucleic acid sequence in the given solution to be measured.B. general probe can be used in combination with second type the identification oligonucleotide that is attached to such as on the matrix of magnetic corpuscular or sheet glass.Mix second type identification oligonucleotide, general probe and think the solution to be measured that contains target nucleic acid and react allowing hybridization and form under the condition of mixture.Separate this mixture and unreacted general probe and solution compolision to be measured, detect reporter oligonucleotides.
Figure 17 has shown another embodiment of general nano particle probe, comprises the SD probe, the SD probe of amplification, and SD-nano particle hybridization probe.First type SD probe comprise the identification oligonucleotide sequence and with reporter oligonucleotides complementary nucleotide sequence such as barcode DNA.Identification oligonucleotide sequence on first type SD probe can with second type SD probe hybridization.Under hybridization conditions, the SD probe complex that generation can be stretched on demand (or matrix (matrix)).Second type SD probe can combine and play the effect of amplification contained reporter oligonucleotides amount in the global matrix with multiple SD probe.Equally, can increase or reduce the amount of the identification oligonucleotide that exists on first type the SD,, perhaps provide more reporter oligonucleotides so that provide and second type SD compound multidigit point more.The SD probe of first kind or second type can be with using such as other particle probe of gold nano grain probe or magnetic-particle probe so that produce required hybridization probe mixture (or matrix) system of concrete assay method.
Detailed description of the present invention
Used herein have the nano particle that oligonucleotides adheres to it, coupled thing, particle, an emulsion Microsphere, " type " that waits refer to that the multiple oligonucleotides that this has same type adheres to it. " tool The nano particle that has oligonucleotides to adhere to it " or " having the nano particle that oligonucleotides adheres to it " Sometimes be also referred to as " nano particle oligonucleotides attachment " or, in detection method of the present invention, claim Be " nano particle oligonucleotide probe " " nanoparticle probes " or only be called " probe ".
" bar code " that the present invention uses, " biochemical bar code ", " bio-barcode ", " bar code DNA ", " DNA bar code ", " report bar code ", " report bar code DNA " waits all interchangeable And has identical implication. The DNA bar code can be nucleic acid, for example picodna or ribonucleic acid. Preferably, the DNA bar code is the oligonucleotides of predetermined sequence. If need this DNA bar shaped Code is for example available, biotin, and radioactive label, or fluorescence labeling carries out mark.
Term " nano-particle complex " or " nano-particle complex probe " refer to comprise nanometer Grain-oligonucleotides attachment, reporter oligonucleotides, and combine the specific binding complementation of target analyte The attachment of the oligonucleotides of body.
Term " analyte " or " target analyte " refer to testing compound or component, comprise medicine, Metabolite, pesticide, pollutant, etc. Analyte can comprise specific binding to (sbp) one Individual member and can be the part of unit price (single epi-position) or multivalence (multi-epitope), preferably antigen or haptens, And can be single compound or have at least one common epitope or the multiple chemical combination in determinant site Thing. Analyte can be such as the part of the cell of bacterium or carry such as A, B, and D is etc. blood group The cell of antigen or HLA antigen, or microorganism, for example, bacterium, fungi, protozoan, Or virus. If this analyte is single epi-position, can for example pass through, chemical mode, further modifying should Analyte is to provide one or more other binding sites. In practice of the present invention, this analyte Have at least two binding sites.
The multivalent ligand analyte generally is bigger organic compound, usually has the characteristic of polymer, Peptide and protein for example, polysaccharide, nucleic acid, and composition. Said composition comprises bacterium, virus, Chromosome, gene, mitochondria, nucleus, the composition of cell membrane etc.
Largely, the applicable multi-epitope ligand analysis of the present invention thing has at least about 5,000 Molecular weight, more commonly at least about 10,000. In the polymer molecule classification, interested Polymer generally is that 000 molecular weight is more commonly from about 20,000 from about 5,000 to 5,000 To 1,000,000 molecular weight; In interested hormone, molecular weight ranges is generally from about 5,000 To 60,000 molecular weight.
Polytype protein can think and belong to the protein families with analog structure feature to have The protein of special biological, with specified microorganisms, the relevant albumen of pathogenic microorganisms particularly Matter, etc. This protein comprises, for example, immunoglobulin (Ig), cell factor, enzyme, hormone, cancer is anti-Former, the nutrition mark, tissure specific antigen, etc.
Protein type of the present invention, clotting factor, proteohormone, antigenic polysaccharide, microorganism With interested other pathogen at U.S. Patent number 4,650, specifically open in 770, this paper is with its integral body Quote the disclosure content for your guidance.
Single epi-position ligand analysis thing is generally from about molecular weight of 100 to 2,000, more commonly from 125 to 1,000 molecular weight.
This analyte can be the molecule of directly finding in such as the sample of host body fluids. This sample can Directly but inspection or preliminary treatment are to cause this analyte to be easier to detect. In addition, work as by only detecting Can detect its existence, susceptible of proof interested analyte when having interested analyte in the sample Reagent, for example, with the specific binding of interested analyte complementation to the member, can measure this sense The analyte of interest. Therefore, the reagent of this analyte of susceptible of proof becomes the analyte that detects in test. Body fluid can be for example, to urinate blood, blood plasma, serum, saliva, seminal fluid, ight soil, phlegm, brain Spinal fluid, tears, mucus, etc.
Term " specific binding is to (sbp) member " refers to particular space and/or the polarity with another molecule Structure specific binding and may be defined as one of two complementary different moleculars. The one-tenth that specific binding is right The member can be described as part and acceptor (anti-ligandin (antiligand)). They are normally such as Ag-Ab Immunity is to the member, although other specific binding pair, biotin-avidin for example, enzyme-substrate, Enzyme-antagonist, enzyme-agonist, medicine-target molecule, hormone-hormone receptor, nucleic acid double helix, IgG-A-protein/protein G, such as DNA-DNA, the polynucleotides of DNA-RNA pair, protein-DNA, lipid-DNA, lipid-protein, polysaccharide-lipid, protein-polysaccharide, the antibody nuclear of nucleic acid Sour and relevant target ligands (for example, little organic compound, nucleic acid, protein, peptide, virus, cell, Deng) and analog be not the immunity right, but they are also included among the present invention and belong to sbp member's Definition. A right member of specific binding can be complete molecule, perhaps only is an one of this molecule Divide, but as long as the binding site of this member's specific binding target analyte is to form specific binding pair.
Term " part " refers to any organic compound that the natural existence of its acceptor maybe can prepare. Art The language part also comprises ligand analogs, and it is the part of modifying, normally organic free radical or analyte Analog has the molecular weight above 100 usually, it and similar Ligand Competition acceptor, and this modification is carried The means of linking ligand analog and another molecule have been supplied. This ligand analogs usually and the difference of part Be to replace hydrogen with the key that connects this ligand analogs and axle center thing (hub) or mark at least, but also needn't So. Ligand analogs can be by the mode similar to part and receptors bind. This analog can be, For example, the antibody of the idiotype antibody of anti-part.
Term " acceptor " or " anti-ligandin " refer to identify particular space and the polarity of molecule Structure, for example any compound or the composition in epi-position or determinant site. The acceptor of illustrative comprises Naturally occurring acceptor, for example, the globulin of thyroxine combination, antibody, enzyme, the Fab segment, solidifying Collection is plain, nucleic acid, the antibody nucleic acid of nucleic acid, avidin, a-protein, bacillus RNA Enzyme inhibitor (barstar), Complement Clq, etc. Avidin attempts to comprise white of an egg antibiosis The biotin in thing fibroin and other source is in conjunction with albumen, for example streptavidin.
Term " specific binding " refers to one of two different moleculars to the specific recognition of another molecule, Other molecule can not be identified basically by contrast. In general, this molecule is in its surface or hole Has the zone that causes specific recognition between two molecules. The example of specific binding is antibody-antigen Interact, enzyme-substrate interacts, and polynucleotides interact, etc.
Term " non-specific binding " refer to have relatively independent specific surfaces structure molecule it Between combination. Non-specific binding can be produced by some factors that comprise intermolecular hydrophobic interaction.
Term " antibody " refer to the particular space of another molecule and polar structure specific binding and because of This is defined as complementary immunoglobulin (Ig). Antibody can be monoclonal or polyclonal antibody and can pass through this The technology that the field is known prepares, for example immune host and collect serum (polyclonal antibody) or by the system Standby continuous hybrid clone is also collected secretory protein (monoclonal antibody), perhaps by cloning and expressing at least Nucleotide sequence or its mutagenesis type system of the amino acid sequence that coding natural antibody specific binding is required Standby. Antibody can comprise complete immunoglobulin (Ig) or its segment, this immunoglobulin (Ig) comprise all kinds and Isotype, IgA for example, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Its Segment can comprise Fab, Fv and F (ab ') .sub.2, and Fab ', etc. In addition, if suitable the use exempt from The aggregation of epidemic disease globulin or its segment, polymer, and attachment are as long as it can be kept specific branch The binding affinity of son.
The present invention relates to utilize oligonucleotides as the multiple analytes in the biochemical barcode detection sample Method. The method is utilized recognition component (for example, the protein with the direct or indirect functionalization of nano particle Or nucleic acid) and the hybridisation events that causes gold nano grain to be assembled can (for example, obviously change its physical characteristic Optics, electricity, mechanics) former observed result8-12 General thought be each recognition component can with have branch Open and different oligonucleotide sequences (DNA bar code) and a solution of tailorable hybridization and the characteristic of unwinding The physical characteristic relevant with nano particle that changes during chain links to decode in the multiple analyte test A series of analytes. Therefore, can use the melting temperature of the aggregation that DNA connects and taking place when unwinding The physical characteristic relevant with nano particle that the changes a series of analyses of multiple analyte in testing of decoding Thing. The bar code of this paper with based on physical diagnosis mark, for example nano strip23, the fluorogen marker beads24, and quantum dot25The difference of bar code be that decoded information is stored in the predetermined oligonucleotide sequence Semiochemical form.
The invention provides that some widely used methods be used for to use with the serious functionalization of oligonucleotides Nanoparticle probes (preferred gold nano grain probe), the various biomolecule in the test sample, for example, Single or multiple multivalence albumen. Specifically in the test sample known method of multiple proteins complicated and Usually need time-consuming and expensive assay method. In this respect, there is the people to use recently the peptide of fluorogen mark Nucleic acid and dna microarray are identified the multiple proteins target in the solution15-17 Yet the method relies on In the protein of using the oligonucleotides mark and the combination of microarray surface. The last step of methods described herein Rapid only take the surface chemistry of common DNA as the basis. Therefore, it can absorb receiving of state of the art The multiple high sensitivity factor of rice grain DNA detection method9，11, and permission detection such as protein Various biomolecule, rather than the DNA that no protein exists during the detection event. Survey for the surface Fixed, protein generally more is difficult to carry out than short oligonucleotide, because they tend to show and solid The non-specific binding that holder is bigger namely produces higher background signal usually. At last, for same Matter is measured, the unusual rapid solution chain pattern relevant with these nanoparticle structure and show normal state with The unwind probe of behavior of broad DNA is compared and is allowed the more bio-barcode of design.
The present invention relates to use any that oligonucleotides adheres to it that have who is suitable for for detection of test Suitable particles. Yet, when enforcement is of the present invention, preferred nano particle. The size of this particle, shape The characteristic of giving the gained probe that comprises the DNA bar code with chemical composition. These characteristics comprise the optics spy The property, photoelectronic property, electrochemical properties, characteristic electron, at various Stability in solutions, the hole and Channel sized changes, and separates the ability of bioactive molecule when serving as filter, etc. Expection can be used Have different sizes, the granulate mixture of shape and/or chemical composition, and use and have unified size, The nano particle of shape and chemical composition. The example of suitable particles includes, but not limited to nanometer and little Undersized core granule, aggregation particle, isotropism (for example spheric granules) and anisotropic particle (for example non-spherical bar, tetrahedron, prism) and core-shell particle, for example with its integral body as a reference The U.S. Patent Application No. 10/034,451 and 2002 of the application in 28 days December in 2002 of reference citation Particle described in the international application no PCT/US01/50825 of application on December 28, in. Implementing this During invention, detector probe preferably produces before carrying out practical measurement. In addition, detector probe can carried out Original position produces during mensuration.
Therefore, in one embodiment of the invention, provide nano particle attachment probe. With Nano particle in the present invention's practice comprises metal (for example, gold, silver, copper and platinum), semiconductor (example As, CdSe, CdS and the CdS or the CdSe that wrap up with ZnS) and magnetic (for example, ferromagnetism) colloid Material. Other nano particle that is used for the present invention's practice comprises ZnS, ZnO, TiO2，AgI，AgBr， HgI 2，PbS，PbSe，ZnTe，CdTe，In 2S 3，In 2Se 3，Cd 3P 2，Cd 3As 2, InAs and GaAs. The size of nano particle preferably from about 5nm to about 150nm (average diameter), more preferably From about 30 to about 100nm, most preferably from about 40 to about 80nm. The size of nano particle Can change with the needs of its concrete purposes or application. The variation of size can be advantageously used in optimization, and this is received Some physical characteristic of rice grain, for example, but the total surface area of optical characteristics or derivatization. Nanometer Grain also can be bar, prism, or tetrahedron.
The preparation metal, the method for semiconductor and magnetic nanoparticle is well known in the art. Referring to, example As, Schmid, G. (ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold:Principles, Methods, and Applications (Academic Press, San Diego, 1991); Massart, R., IEEE Taransactions On Magnetics, 17,1247 (1981); Ahmadi, T.S. etc., Science, 272,1924 (1996); Henglein, A. etc., J.Phys.Chem., 99,14129 (1995); Curtis, A.C., etc., Angew.Chem.Int.Ed.Engl., 27,1530 (1988).
Preparation ZnS, ZnO, TiO2，AgI，AgBr，HgI 2，PbS，PbSe，ZnTe，CdTe， In 2S 3，In 2Se 3，Cd 3P 2，Cd 3As 2, the method for InAs and GaAs nano particle be this area Know. Referring to, for example, Weller, Angew.Chem.Int.Ed.Engl., 32,41 (1993); Henglein, Top.Curr.Chem, 143,113 (1988); Henglein, Chem.Rev., 89,1861 (1989); Brus, Appl.phys.A., 53,465 (1991); Bahncmann, referring toPhotochemical Conversion and Storage of Solar Energy(Pelizetti and Schiavello compile, 1991), the 251st page; Wang And Herron, J.Phys.Chem., 95,525 (1991); Olshavsky etc., J.Am.Chem. Soc., 112,9438 (1990); Ushida etc., J.Phys.Chem., 95,5382 (1992).
Suitable nano particle also can obtain from commercial channels, for example, Ted Pella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc. (gold).
What be preferred at present detecting nucleic acid is gold nano grain. Gold colloid particles is for causing its beauty The band of color has high extinction coefficient. These strong colors are with granular size, concentration, particle Between distance and aggregation extent and aggregation shape (geometry) and change so that these materials are for colorimetric Determination method is especially attractive. For example, be attached to oligonucleotides and oligonucleotides on the gold nano grain With the hybridization of nucleic acid cause the visible direct color of bore hole change (referring to, for example, embodiment).
But the functionalized nano particle, oligonucleotides or both are in order to be attached to nano particle with oligonucleotides On. The method is known in the art. For example, with alkane thiol (alkanethiols) its 3 '-terminal or 5 '-oligonucleotides of end-functionalization is attached on the gold nano grain easily. Referring to Whitesides, Proceedings of the Robert A.Welch Foundation 39th Conferrence On Chemical Research Nanophase Chemistry, Houston, TX, 109-121 page or leaf (1995). Also can referring to, Mucic etc., Chem.Commun.555-557 (1996) (has described and has been attached to 3 ' sulfydryl DNA flat The method of gold surface; The method can be used for oligonucleotides is attached on the nano particle). This alkane thiol Method also can be used for oligonucleotides is attached to other metal, semiconductor and magnetic colloid and above listed Other nano particle on. Other functional group that oligonucleotides is attached to the surface of solids comprises sulfo-phosphorus Acid groups (referring to, for example, be used for oligonucleotides-D2EHDTPA is attached to the U.S. Patent number of gold surface 5,472,881), the alkylsiloxane of replacement (referring to, for example, be used for oligonucleotides be attached to silica and The Burwell of glass surface, Chemical Technology, 4,370-377 (1974) and Matteucci and Caruthers, J.Am.Chem.Soc., 103,3185-3191 (1981) and be used for the combination of aminoalkyl siloxanes Reach the Grabar of the similar combination that is used for the mercaptoalkyl siloxanes etc., Anal.Chem., 67,735-743). The oligonucleotides that end has 5 ' sulphur nucleosides (thionucleoside) or 3 ' sulphur nucleosides also can be used for few nucleosides Acid is attached on the surface of solids. Below list of references described to can be used for oligonucleotides is attached to and received Other method on the rice grain: Nuzzo etc., J.Am.Chem.Soc., 109,2358 (1987) (curing Thing is attached on the gold); Allara and Nuzzo, Langmuir, Isosorbide-5-Nitrae 5 (1985) (carboxylic acid is attached on the aluminium); Allara and Tompkins, J.Colloid Interface Sci., (carboxylic acid is attached to copper to 49,410-421 (1974) On); Iler,The Chemistry Of Silica, the 6th chapter, (Wiley 1979) (carboxylic acid is attached on the silica); Timmons and Zisman, J.Phys.Chem., 69,984-990 (1965) (carboxylic acid is attached on the platinum); Soriaga and Hubbard, J.Am.Chem.Soc., 104,3937 (1982) (aromatic compound is attached to platinum On); Hubbard, Acc .Chem.Res., 13,177 (1980) (sulfolane, sulfoxide and other functionalization solvents Be attached on the platinum); Hickman etc., J.Am.Chem.Soc., 111,7271 (1989) (isonitrile is attached to platinum On); Maoz and Sagiv, Langmuir, 3,1045 (1987) (silane attaches is to silicas); Maoz and Sagiv, Langmuir, 3,1034 (1987) (silane attaches is to silicas); Wasserman etc., Langmuir, 5,1074 (1989) (silane attaches is to silicas); Eltekova and Eltekov, Langmuir, 3,951 (1987) (pure and mild methoxyl group is attached on titanium dioxide and the silica for aromatic carboxylic acids, aldehyde); Lec etc., J.Phys.Chem., 92,2597 (1988) (rigidity phosphate is attached on the metal).
Aspect of this embodiment of the present invention, provide and widow's nuclear of using at least two types The nano particle that the SD of thuja acid mark connects. The SD molecule is to comprise a plurality of branch units monomer Structure, and can be used for various application. Referring to, for example, Barth etc., Bioconjugate Chemistry 5:58-66 (1994); Gitsov﹠Frechet, Macromolecules 26:6536-6546 (1993); Hawker﹠Frechet, J.Amer.Chem.Soc.112:7638-7647 (1990a); Hawker﹠ Frechet, Macromolecules 23:4726-4729 (1990b); Hawker etc., J.Chem.Soc. Perkin Trans.1:1287-1297 (1993); Lochmann etc., J.Amer.Chem.Soc.115: 7043-7044 (1993); Miller etc., J.Amer.Chem.Soc.114:1018-1025 (1992); Mousy Deng, Macromolecules 25:2401-2406 (1992); Naylor etc., J.Amer.Chem.Soc.111: 2339-2341 (1989); Spindler﹠Frechet, Macromolecules Macromolecules 26: 4809-4813 (1993); Turner etc., Macromolecules 26:4617-4623 (1993); Wiener Deng, Magnetic Resonance Med.31 (1): 1-8 (1994); Service, 267:458-459 (1995); Tomalia, Sci.Amer.62-66 (1995); U.S. Patent number 4,558,120 with Tomalia; 4,507,466; 4,568,737; 4,587,329; 4,857,599; 5,527,524; 5,338,532, and Nilsen United States Patent (USP) 6,274,723, all these documents are quoted with its integral body at this. Dendritic molecule is relative Supramolecular structure in other type provides important advantage, and for example the contact of minimal structure unit is maximum Volume, easier control size, the ability of weight and growth characteristics, but a plurality of ends of derivatization with Just be created in the molecule of the number of altitude that has void between the mark, or be other molecule, Or its mixture provides attachment site. Generally referring to United States Patent (USP) 6,274,723 and above synthetic method draw The list of references of card.
The nucleic acid SD that is used for the inventive method is available core acid functionalization or can be by nucleic acid/few nucleosides Known in the art any nucleic acid SD that acid produces. This SD can be according to such as Hudson etc., " nucleic acid SD: neoformation polymer architecture ", Am.Chem.Soc.115:2119-2124 (1993); The United States Patent (USP) 6,274,723 of Cantor; With U.S. Patent number 5,561,043 disclosure is synthetic.
In this embodiment of the present invention on the other hand, provide a kind of general nano particle-few nucleosides The acid attachment. General probe can be used for mensuration and comprises at least two-part any target nucleic acid. Should be " general Probe " comprise (i) that adhere to it with at least one section reporter oligonucleotides (for example, bar code DNA) Divide and (lead to the oligonucleotides of complementary single " seizures " sequence of the part of target identification oligonucleotides An embodiment with probe is described in Figure 16 A and 16B). Target identification oligonucleotides comprises and has At least two-part sequence; First comprises the complementary series of the seizure sequence of adhering to nano particle, Second portion comprises the complementary series of the first of particular target nucleotide sequence. Can use various types of Target identification oligonucleotides is with greatly convenient this general probe, so that can switch or exchange target identification widow The nucleotides storehouse is to select the particular target nucleotide sequence in the special test solution. Comprise and second of target nucleic acid The oligonucleotides of the second type of the sequence that part is complementary is attached to support surface, for example magnetic On grain or the sheet glass.
General probe and the solution that comprises reporter oligonucleotides (bar code DNA) and target identification oligonucleotides Contact generation " activation " general probe is used for and may contains target nucleic acid under the condition that allows hybridization Solution contact (Figure 16 A, top response diagram). Solution to be measured can and adhere under the condition that allows hybridization One of " activation " general probe to the holder or oligonucleotides of second type or both comply with Inferior or merging contacts. In case through the enough time that allows compound to form, that not compound test is molten The liquid composition separates with compound, detects this report oligonucleotides. An embodiment of this mensuration is at figure Describe among the 16B.
The concrete determination method that can implement as required operates to strengthen that it is excellent to these general probes Gesture. Can be so that this probe and various single target nucleic acid order by simple replacement or exchange target identification oligonucleotides Row " unanimously " are so that second portion comprises the complementary series of interested target nucleic acid. Equally, if Need to measure multiple target nucleic acid sequence in the single test solution, this report oligonucleotides can comprise each target The sequence of nucleic acid specificity. Therefore, to the inspection of reporter oligonucleotides with known and specific sequence Surveying the result can show have particular target nucleic acid in testing liquid.
In this embodiment of the present invention on the other hand, use sulfur-bearing functional group with the oligonucleotides combination To nano particle. U.S. Patent Application No. 09/760,500 and 09/820,279 and international application no PCT/US01/01190 and PCT/US01/10071 have described with cyclic disulfide (cyclic Disulfides) oligonucleotides of functionalization, it can be used for implementing the present invention. This cyclic disulfide is preferred In its ring, have 5 or 6 atoms, comprise two sulphur atoms. Suitable cyclic disulfide can lead to Cross that commodity approach obtains or can be synthetic by known method. Also can use the reduction of cyclic disulfide Form.
Preferably, this joint also comprises the alkyl that is connected on the cyclic disulfide. Suitable hydrocarbon can Obtain and be connected on the cyclic disulfide from commodity approach. Preferred alkyl is the steroids residue. Meaning The widow of the joint preparation that contains the steroids residue that is connected on the cyclic disulfide has been found to use in the other places Nucleotides-nano particle attachment is than the company that uses alkane thiol or acyclic disulphide as the joint preparation Connect thing to mercaptan (dithiothreitol (DTT) that for example, in polymerase chain reaction (PCR) solution, uses) obviously more Stable. In fact, found that oligonucleotides of the present invention-nano particle attachment stablizes 300 times. Should Beyond thought stability be likely due to the fact that, namely each oligonucleotides by two sulphur former Sub rather than single sulphur atom anchor on the nano particle. Specifically, think cyclic disulfide Two adjacent sulphur atoms may have chelation, and this may be conducive to stablize oligonucleotides-nano particle Attachment. The large-scale hydrophobic steroids residue of this joint is by the screen nano particle and near nano particle As if the water soluble molecules on surface also help the stability of this attachment.
In sum, two of cyclic disulfide sulphur atoms preferably close enough together so that two Individual sulphur atom can be attached on the nano particle simultaneously. Most preferably, two sulphur atoms are adjacent one another are. In addition, alkyl is should be enough big in order to the surface of bigger hydrophobic surface screen nano particle is provided.
Adopt the cyclic disulfide joint oligonucleotides-ring-type nano particle attachment can be by United States Patent (USP) Number 09/760,500 and 09/820,279 and international application no PCT/US01/01190 and PCT/US01/10071 is described to be used as probe in the diagnostic assay of test sample target test thing. Find that these attachments can improve the sensitivity of the diagnostic assay that uses them. Specifically, send out Now use to comprise the oligonucleotides of the joint preparation of the steroids residue that is attached on the cyclic disulfide-The determination method of nano particle attachment prepares as joint with alkane thiol or acyclic disulphide than adopting Sensitive about 10 times of the determination method of attachment.
Each nano particle has multiple oligonucleotides and adheres to it. As a result, each nano particle-oligonucleotides Attachment can be combined with multiple oligonucleotides or nucleic acid with complementary series.
The oligonucleotides of regulation sequence is used for various purposes in practice of the present invention. Preparation has predetermined The method of the oligonucleotides of sequence is known. Referring to, for example, Sambrook etc., Molecular Cloning:A Laboratory Manual (second edition in 1989) and F.Eckstein (volume) Oligonucleotides and Analogues, front page (Oxford University Press, New York, 1991). Preferred solid phase synthesis process is used for the oligomerization ribonucleotide and oligodeoxynucleotide (also can Use the synthetic RNA of well-known process of synthetic DNA). Also but enzymatic prepares oligomerization ribonucleotide and widow Poly-deoxyribonucleotide. For what have with the specific binding complement of the target analyte of its combination Oligonucleotides can use any suitable method to adhere to such as the specific binding complement of protein To oligonucleotides.
Can use any suitable method oligonucleotides to be attached to particle, nano particle, or nanosphere On the surface. Be used for oligonucleotides is attached to the particularly preferred method on surface based in June, 1999 09/603,830 of the U. S. application of application on the 25th number application on June 26th, 09/344,667,2000; 09/760,500 of application on January 12 calendar year 2001; 09/820,279 of application on March 28 calendar year 2001; 09/927,777 of application on August 10 calendar year 2001; International application with application on July 21st, 1 997 Number PCT/US97/12783; The PCT/USOO/17507 of application on June 26th, 2000; Calendar year 2001 1 The PCT/US01/01190 of the moon 12 application; The PCT/US01/10071 of application on March 28 calendar year 2001 Described in aging method (aging process), the disclosure of these documents is with its integral body as a reference Reference citation. Aging method provides the stability of unexpected enhancing for nano particle-oligonucleotides attachment Selectively.
The method comprises provides the oligonucleotides that preferably has half molecule covalently bound with it, this half point Attached bag contains the functional group that can be combined with nano particle. This half molecule and functional group be allow oligonucleotides with Nano particle is in conjunction with those half molecule and the functional groups of (that is, by chemisorbed or covalent bond). For example, Can use have with its 5 ' or 3 ' hold covalently bound alkane thiol, alkane disulphide or ring-type curing The oligonucleotides of thing is combined this oligonucleotides with the various nano particles that comprise gold nano grain.
Oligonucleotides is contacted time enough to allow at least some few nucleosides with nano particle in the water Acid and nano particle are by means of functional groups. Can measure by rule of thumb this time. For example, have been found that Approximately 12-24 hour time can produce preferably result. Other suitable condition of oligonucleotides combination Also can measure by rule of thumb. For example, the about concentrations of nanoparticles of 10-20nM and at room temperature incubation can Produce preferably result.
Then, Xiang Shuizhong adds at least a salt to form salting liquid. Salt can be any suitable water Soluble. For example, salt can be sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, second The acid ammonium, the combination of two or more these salt, or a kind of phosphate buffer of these salt. Preferably Be, this salt adds with concentrated solution, adds but also can be used as solid. Salt can all once be added to the water Perhaps can in a period of time, add gradually salt. " in a period of time gradually " refers to between a period of time What separate adds salt with two parts at least. The suitable time interval can be determined by rule of thumb.
The ionic strength of salting liquid must be enough to overcome oligonucleotides at least part of static row each other Scold and electronegative oligonucleotides to the electrostatic attraction of positively charged nano particle, or electronegative The oligonucleotides of lotus is to the Coulomb repulsion of electronegative nano particle. Have been found that by one section the time Between in add gradually salt and reduce gradually electrostatic attraction and repel and can produce highest face temperature at nano particle The oligonucleotides of density. Appropriate ions intensity for every kind of salt or combination salt can be measured by rule of thumb. Through finding the sodium chloride final concentration from about 0.1M to about 1.0M in phosphate buffer, preferred The concentration of sodium chloride increases in a period of time gradually, can produce preferably result.
After adding salt, with oligonucleotides and nano particle one section time enough of incubation again in salting liquid To allow other enough oligonucleotides to be attached to the stable nano particle of generation on the nano particle-few nucleosides The acid attachment. As will be described below, have been found that the surperficial close of oligonucleotides on the nano particle Degree increases can stablize attachment. The time of incubation can be determined by rule of thumb. Have been found that incubation is about altogether 24-48, (this is the total time of incubation can to produce preferably the result in preferred 40 hours; As mentioned above, exist This section can increase salinity in total time gradually). Second segment time this paper of incubation is called in salting liquid Step " wears out ". Other appropraite condition that is somebody's turn to do " wearing out " step also can be determined by rule of thumb. For example, At room temperature with pH7.0 in incubation can produce preferably result.
Have been found that the attachment that " wears out use " step produces is than without the generation of " wear out " step Attachment is much stable. As mentioned above, the stability increase is owing to realize by " wearing out " step The oligonucleotides density increase of nano grain surface causes. By " wearing out " step realize surperficial close Degree depends on the size of nano particle and length, sequence and the concentration of type and oligonucleotides. Be enough to So that the stable superficial density of nano particle and obtain for the required combination of nano particle and oligonucleotides The necessary condition of this superficial density can be determined by rule of thumb. In general, 10picomoles/cm at least2Superficial density be enough to the nano particle that provides stable-oligonucleotides attachment. Preferably, the surface is close Degree is 15picomoles/cm at least2 If because this superficial density can reduce too greatly widow's nuclear of attachment The ability of thuja acid and nucleic acid and the hybridization of oligonucleotides target, so superficial density preferably is not more than approximately 35-40picomoles/cm2
" stable " used herein refer to after attachment preparation at least 6 months during in, big Most oligonucleotides still be attached on the nano particle and oligonucleotides can the method that detects nucleic acid and Hybridize with nucleic acid and oligonucleotides target under the standard conditions that run in the method that nanometer makes up.
Have been found that comprise the identification oligonucleotides of identification division and spacer region part by use can be remarkable Increase the hybridization efficiency of nano particle-oligonucleotides attachment. " identification oligonucleotides " is to comprise and nucleic acid Or the oligonucleotides of the sequence of at least a portion sequence complementation of oligonucleotides target. In this embodiment, The identification oligonucleotides comprises identification division and spacer region part, and with the hybridization of nucleic acid or oligonucleotides target It is identification division. The spacer region that can design the identification oligonucleotides partly makes it be combined with nano particle. For example, this spacer region part can have half molecule covalently bound with it, this half point attached bag contain can with receive The functional group of rice grain combination. They are half molecule and functional groups same as described above. Because identification is few The compartment of nucleotides is combined with nano particle, so identification division and nano grain surface are separated And more accessible in order to hybridize with its target. The spacer region at identification division and the good interval of nano particle is provided Length and the sequence of part can be determined by rule of thumb. Have been found that to comprise at least about 10 nucleotides, excellent Select the spacer region part of 10-30 nucleotides can produce good effect. The spacer region part can not have to be done Any order of the ability of disturbing identification oligonucleotides and nano particle or being combined with nucleic acid or oligonucleotides target Row. For example, it is complimentary to one another that spacer region part should not have, with the complementation of identification oligonucleotides, or with knowledge The sequence of the nucleic acid of other oligonucleotides or the complementation of oligonucleotides target. Preferably, the nuclear of spacer region part The thuja acid base all is adenine, all is thymidine, all is cytimidine, all is guanine perhaps, removes Non-it can cause a just now described problem. More preferably, this base all is adenine, perhaps It all is thymidine. Most preferably this base all is thymidine.
Also have been found that except the identification oligonucleotides, use the dilution oligonucleotides to provide and cut out (tailoring) instrument of attachment is to produce the hybridization of desired level. Find dilution and identification oligonucleotides Attached with the ratio approximately identical with its ratio in solution with nano particle contact preparation attachment the time On the nano particle. Therefore, can control the dilution and identification oligonucleotides that is attached on the nano particle Ratio so that this attachment participates in requisite number purpose hybridisation events. The dilution oligonucleotides can not have to be done Disturb any of the identification oligonucleotides ability of being combined with nano particle or being combined with nucleic acid or oligonucleotides target Sequence. For example, the dilution oligonucleotides should not have with the identification oligonucleotides or with the identification oligonucleotides The sequence of nucleic acid or the complementation of oligonucleotides target. The dilution oligonucleotides is also preferably than the length of identifying oligonucleotides Spend shorter so that this identification oligonucleotides can be in conjunction with its nucleic acid or oligonucleotides target. If identify few nucleosides Acid comprises the spacer region part, and the dilution oligonucleotides is most preferably approximately the same long with the spacer region part. Like this, the dilution oligonucleotides just can not disturb identification division and nucleic acid or the few nucleosides of identification oligonucleotides The ability of acid target hybridization. Even more preferably, dilution oligonucleotides and identification oligonucleotides spacer region The sequence of part has identical sequence.
In another embodiment of the present invention, provide the particle composites probe. Every type Grain compound probe contains predetermined reporter oligonucleotides or the bar code of concrete target analyte. In the target analysis Under the condition that thing exists, owing to the binding interactions between particle composites and the target analyte produces Aggregation. Separable these aggregations are also analyzed by any suitable mode such as thermal denaturation To detect existing of one or more dissimilar reporter oligonucleotides. When enforcement is of the present invention, excellent Select the nano-particle complex probe.
Therefore, in one aspect of the invention, the particle composites probe comprises a kind of few nucleosides that combines The particle of acid, one or more DNA bar codes, and combine the specific binding of concrete target analyte The oligonucleotides of complement, wherein (i) DNA bar code has at least two-part sequence; (ii) be attached to At least some oligonucleotides on the particle have the sequence with the complementation of DNA bar code first; (iii) Oligonucleotides with specific binding complement and its combination has with DNA bar code second portion mutual The sequence of mending; (iv) the DNA bar code in every type the particle composites probe has different Sequence and as the identifier of concrete target analyte.
In this embodiment on the other hand, the particle composites probe comprises and has at least two types The particle of oligonucleotides and its combination, one or more DNA bar codes have the special of target analyte The property in conjunction with the oligonucleotides of complement and its combination, the widow of first type of wherein being combined with probe nuclear Thuja acid has the sequence with at least a portion complementation of DNA bar code, second kind of being combined with probe The sequence that the oligonucleotides of type has and at least one section with oligonucleotides of specific binding complement The sub-sequence complementation.
In this embodiment on the other hand, the particle composites probe comprise combine oligonucleotides The grain, the specific binding complement of one or more DNA bar codes and target analyte, wherein with At least a portion of the oligonucleotides that burl closes has at least a portion sequence complementation with the DNA bar code Sequence and wherein this DNA bar code as the identifier of specific target analyte.
In another embodiment of the present invention, provide the particle composites probe. Therefore in the present invention An embodiment in, the particle composites probe that provides comprises the particle that combines oligonucleotides, DNA bar code and have the specific binding complement of concrete target analyte and the few nucleosides of its combination Acid, wherein (i) this DNA bar code has at least two parts sequence; (ii) be attached at least one on the particle A little oligonucleotides have the sequence with first's complementation of DNA bar code; (iii) has the specificity knot The oligonucleotides that closes complement and its combination has the sequence with the second portion complementation of DNA bar code; (iv) the DNA bar code in every type particle composites probe has different sequences and usefulness Make the identifier of concrete target analyte.
In another embodiment of the present invention, the particle composites probe that provides comprises and has at least two Type oligonucleotides and the particle of its combination, DNA bar code and have the special of target analyte The property in conjunction with the oligonucleotides of complement and its combination, the widow of first type of wherein being combined with probe nuclear Thuja acid has the sequence with at least a portion complementation of DNA bar code, second kind of being combined with probe The sequence that the oligonucleotides of type has and at least one section with oligonucleotides of specific binding complement The sub-sequence complementation.
In another embodiment of the present invention, the particle composites probe that provides comprises and combines few nuclear The particle of thuja acid, the specific binding complement of DNA bar code and target analyte is wherein with particle In conjunction with at least a portion oligonucleotides have order with at least a portion sequence complementation of DNA bar code Row and wherein the DNA bar code as the identifier of concrete target analyte.
In another embodiment of the present invention, a kind of detector probe is provided, it comprises nano particle; The member that the specific binding of being combined with nano particle is right; What be combined with nano particle is at least a The oligonucleotides of type; The DNA bar code that has respectively at least a type of predetermined sequence, its In at least a portion hybridization of oligonucleotides of every type DNA bar code and at least a type.
Preferred particle comprises above-mentioned nano particle, metal for example, semiconductor, insulator, or magnetic Nano particle. Preferred particle is gold nano grain. The specific binding complement or in conjunction with to the member with Target analyte is the right member of specific binding, and it comprises nucleic acid, oligonucleotides, and peptide nucleic acid, polypeptide, Antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroids, vitamin, medicine, Virus, polysaccharide, lipid, lipopolysaccharides, glycoprotein, lipoprotein, nucleoprotein, oligonucleotides, antibody, Immunoglobulin (Ig), albumin, hemoglobin, clotting factor, the peptides and proteins hormone, non-peptide class swashs Element, interleukins, interferon, cell factor comprises the peptide of tumour-specific epi-position, cell, Cell surface molecule, microorganism, the fragment of microorganism, partly, component or product, organic little branch Son, nucleic acid and oligonucleotides, the metabolite of any above-mentioned substance or antibody.
In another embodiment of the present invention, provide for detection of whether exist a kind of in the sample or The method of multiple target analyte, this target analyte has at least two binding sites. Be somebody's turn to do of the present invention An aspect of embodiment, the method that provides comprises step:
A kind of matrix is provided;
The particle probe of one or more types is provided, and every type probe comprises and has concrete target branch Analyse thing one or more specific binding complements particle and with one or more DNA of its combination Bar code, wherein the specific binding complement of every type particle probe is to a specific target branch Analyse thing and have specificity, and the DNA bar code of every type particle probe is as this concrete target analysis The mark of thing;
This target analyte is fixed on this matrix;
Allowing between the specific binding complement of target analyte and this analyte combination and in the target analysis Form under the condition that thing exists under the condition for validity of compound, with fixing target analyte and a kind of or many Type particle probe contact;
Wash this matrix to remove unconjugated particle probe; With
The optional DNA barcode amplification that makes; With
Detect the DNA bar code whether there is amplification, wherein whether exist this mark be in the sample whether The index that has concrete target analyte.
Aspect of this embodiment of the present invention, this target analyte be protein or haptens and Its specific binding complement is the antibody that comprises monoclonal or polyclonal antibody.
In another aspect of this invention, can use any suitable matrix. This matrix can be arranged with a kind of Or the capturing probe of polytype target analyte.
Of the present invention aspect this, separable bar code. Any suitable by with such as the DNA chip Mode determine that the existence of reporter oligonucleotides or bar code carries out analyte determination indirectly.
The DNA bar code is optional can to increase by any suitable mode that comprises pcr amplification, right Any suitable detector probe of rear use detects by any suitable DNA detection system. Particle Preferably with the DNA bar code of q.s carry out mark with provide enough signals to amplify and eliminate right The demand of DNA barcode amplification. When enforcement is of the present invention, preferably increase by PCR method. The pcr amplification of DNA bar code (this paper is also referred to as BPCR) allows to detect the albumen of attomolar level The matter target. Determination method shown in Figure 6 has been used the oligonucleotides (bio-barcode) with hybridization36Serious function The novel nano particle of changing and polyclone detect antibody and identify target analyte, and namely prostate specific is anti-Former (PSA) (embodiment 5). In addition, the polyamine microparticle (diameter 1 μ m) that has the magnetic oxide core is used PSA monoclonal antibody functionalization (Fig. 6 and embodiment 5). Gold nano grain and polyamine microparticle folder position PSA Target, produce the compound that bar code DNA and protein target have height ratio (for the particle of 13nm, 200 DNA chains of each particle portability; This has represented the upper limit of this size particles). Applying a magnetic field Moment moves magnetic-particle on the reactor wall to, so that people can be with reaction and unreacted microparticle But the nano particle that only responds is separated from reactant mixture. At Nanopure water (18 MOhms) washing aggregation structure in is so that the fixing complement impurity elimination of bar code DNA and nano particle Hand over. Use magnetic separator easily aggregation to be taken out from measure solution, stay bar code DNA can use PCR that it is increased, then with standard DNA detection method (sweep measuring11, Gel electrophoresis, or fluorogen labeling method) identify rapidly. Select PSA as the initial target of these tests Be because its in prostate cancer, i.e. one of modal cancer of U.S. man and be second of cancer mortality Big inducement47，48, earlier detection in importance. Importantly, use (is copied for 10 as low-level Shellfish) PSA of mark identify that palindromia after the surgery for prostate cancer treatment is extremely beneficial and make it possible to into The administration of row therapeutic complementary therapy46，49。
Embodiment 5-6 has confirmed that BPCR uses gel electrophoresis under the condition that background protein exists Or scanning survey microarray detection method detects the protein analyte of low attomolar concentration, i.e. PSA Effective method extremely. This studies confirm that some advantages with respect to present protein detection method. At first, the target binding protein of this determination method is homogeneity. Therefore, can in reactor, add a large amount of magnetic The property particle is to be conducive to detect the binding kinetics between antibody and the target analyte. This causes this mensuration ratio Heterogeneous systems is faster and owing to catch that step is more effective also to make it possible to increase sensitivity. Secondly, use The nano particle bio-barcode provides DNA that PCR can increase and the height ratio of labelled antibody, leads Cause remarkable increase and measure sensitivity. For example, can to detect 3aM dense for the BPCR determination method of this paper report The PSA of degree, and the immunoassay of PCR-based it is reported for identical target analyte detectable limit Be 3fM43 The 3rd, this determination method has avoided connecting the required complex combination of DNA and labelled antibody Process. Bar code DNA when labeled reactant begins, be attached on the nanoparticle probes by hybridization and Use simple washing step to discharge for pcr amplification. Labelled antibody and DNA exist in addition On identical particle, before pcr amplification bar code DNA, do not need to add other antibody or DNA-The protein attachment. In addition, from detect test, take out bar code DNA, to being free on PSA, big Most biological samples, the bar code DNA sample of microparticle and nano particle carries out PCR. This is very big Ground has reduced background signal. At last, this protein detection method has extensive multiple use and one Plant the potentiality that detect simultaneously multiple analytes in the solution. Although the PSA system is used for the confirmation of concept, That this scheme all should generally be suitable for for the almost any target with known binding partners, and by making Use the bio-barcode method based on nano particle36, can prepare distinctive appraisable bar code and be used for several All interested targets.
Embodiment 9 has confirmed that probe of the present invention can be used for detecting and the direct sample target test thing of measuring Amount. Therefore, comprising BPCR is not to realize fabulous sensitivity with the step of amplification bar code DNA With detectable limit necessary (Fig. 6 B (step 4), 9 (illustrations) and 12).
Can use the stromal surface after any suitable wash solution forms from compound to remove unconjugated Probe. Representational example includes, but not limited to PBS (PBS).
Under the condition that target analyte exists, because between nano-particle complex probe and the target analyte Binding interactions and produce nanoparticle aggregate thing compound. Separable these aggregations are also having Process under the condition of effect impurity elimination friendship and release reporter oligonucleotides. Separate then reporter oligonucleotides. If need, reporter oligonucleotides can increase by any suitable mode that comprises pcr amplification. With Any suitable mode depositing by definite reporter oligonucleotides or bio-barcode such as the DNA chip In the detection of indirectly carrying out analyte.
Can detect DNA bar code or reporter oligonucleotides by any suitable mode then. General next Say, before detection, discharge the DNA bar code by handing over the compound impurity elimination. It is any suitable to use Solution or medium carry out the impurity elimination friendship and released dna bar code from compound. Representational medium is water.
Impurity elimination by aggregation hands over the DNA bar code that discharges to use to have the oligonucleotides of seizure and its In conjunction with the matrix direct-detection. This oligonucleotides has at least a portion complementation with reporter oligonucleotides Sequence. Some embodiments that detect the method for DNA bar code use have complementary oligonucleotide with The matrix of its combination catches reporter oligonucleotides. Detect in any suitable manner then these seizure Reporter oligonucleotides. By adopting matrix, the sensitivity that detectable variation (signal) is exaggerated and measures Degree increases.
Can use any suitable method that oligonucleotides is attached on the matrix. For example, can be by for example, Chrisey etc., Nucleic Acids Res., 24,3031-3039 (1996); Chrisey etc., Nucleic Acids Res., 24,3040-3047 (1996); Mucic etc., Chem.Commun., 555 (1996); Zimmermann And Cox, Nucleic Acids Res., 22,492 (1994); Bottomley etc., J.Vac. Sci.Technol.A, 10,591 (1992); With Hegner etc., FEBS Lett., 336,452 (1993) described will Oligonucleotides is attached on the matrix.
Be attached to oligonucleotides on the matrix and have First with reporter oligonucleotides sequence to be detected Divide complementary sequence. The condition for validity of the oligonucleotides on permission matrix and reporter oligonucleotides hybridization Lower, reporter oligonucleotides is contacted with matrix. Reporter oligonucleotides is attached on the matrix like this. Adding Enter such as the preferred any knot of flush away on the matrix before the detector probe of nano particle-oligonucleotides attachment The reporter oligonucleotides of closing.
In one aspect of the invention, the reporter oligonucleotides and of being combined of the oligonucleotides on matrix One type the oligonucleotides that has contacts with the nano particle that it adheres to. This oligonucleotides has and newspaper The sequence of the second portion complementation of road oligonucleotide sequence, and at the oligonucleotides that allows on the nano particle Under the condition for validity of reporter oligonucleotides hybridization, contact. Such first type nanometer Burl is incorporated on the matrix. After nano particle-oligonucleotides attachment was attached on the matrix, washing matrix was gone Fall any unconjugated nano particle-oligonucleotides attachment.
Oligonucleotides on first type nano particle can have all identical sequences or can Have the different sequences of hybridizing from the different piece of reporter oligonucleotides to be measured. When use has different orders During the oligonucleotides of row, each nano particle can have all that different oligonucleotides adhere to it or, Preferred different oligonucleotides is attached on the different nano particles. As selection, in each first kind Oligonucleotides on the nano particle of type can have multiple different sequence, wherein at least a can with treat Observe and predict the part hybridization of oligonucleotides.
Optional is, be attached to first type nano particle on the matrix-oligonucleotides attachment with Having oligonucleotides contacts with the nano particle of its second type of adhering to. These oligonucleotides have Order with at least a portion sequence complementation that is attached to the oligonucleotides on first type the nano particle Row, and in the nanometer that allows oligonucleotides on first type the nano particle and second type Under the condition for validity of the oligonucleotide hybridization on the grain, contact. After the nano particle combination, preferably wash Wash matrix and remove any unconjugated nano particle-oligonucleotides attachment.
Combined hybrid produces detectable change. Except multiple crossing causes detectable change amplification, This detectable change is same as described above. Specifically, because each nano particle tool of first type There is multiple oligonucleotides (having identical or different sequence) to adhere to it, each nanometer of first type Grain-oligonucleotides attachment can with the nano particle of multiple second type-oligonucleotides attachment hybridization. Equally, first type nano particle-oligonucleotides attachment can with the above report to be measured of a part Oligonucleotide hybridization. The amplification that multiple crossing causes can be so that change and can detect for the first time or can improve Detectable change value. This amplification has improved the sensitivity of measuring, and allows to detect a small amount of newspaper The road oligonucleotides.
If need, the nano particle by first and second types of continuous addings-oligonucleotides connects Thing can make up more multi-layered nano particle. Like this, it is fixing further to increase each target nucleic acid molecule The nano particle number, thus the intensity of signal correspondingly increased.
In addition, be designed to first and second types nano particle of mutual direct cross as use Substituting of oligonucleotides attachment can be used and be carried conduct with the result of the oligonucleotide hybridization of combination Nano particle for the oligonucleotides that nano particle is combined.
When using matrix, nano particle that can multiple initial type-oligonucleotides attachment or few examining Thuja acid is attached on the matrix for detection of a plurality of parts of target reporter oligonucleotides with the form of array, uses In detecting multiple different reporter oligonucleotides, perhaps both detect. For example, can have on the matrix Point in a row, every contains be designed to be combined with the part of target reporter oligonucleotides dissimilar Oligonucleotides. Add the sample that contains one or more reporter oligonucleotides to each point, it is suitable to use The mensuration that oligonucleotide nano particle attachment is left in above-mentioned a kind of mode.
On the other hand, can revise with BPCR, and the analyte detection method of direct-detection is to be used for Be included in such as glass gold, silicon, nickel, plastics, the method that the analyte on the matrix that waits detects. Also can revise these methods to detect other biology and Chemical recognition event, for example DNA-protein knot Close event, the combination of physiology protein-protein or dimerization and above-mentioned other known biomolecule Interact.
In an embodiment aspect this, the method comprises one or more of each target analyte The capturing probe of type is attached on the matrix, and matrix is contacted with solution to be measured, chooses wantonly and washes from matrix Remove solution to be measured, subsequently the detector probe of matrix with one or more types of each target analyte contacted, Remove any unconjugated detector probe, and detect observable signal, wherein observable signal Testing result represent to have target analyte in the solution to be measured.
Aspect this, can use any method as herein described to detect observable signal of the present invention. For example, direct-detection bar code DNA detects the bar code DNAs that BPCR-increases, and detects a kind of Or the gathering of polytype detector probe (for example, by visually observing, fluorescence, colorimetric, electrochemistry, Electronics, light densitometry, radioactivity, etc.), perhaps comprise and bar code DNAs by use Report nucleotides and detectable signal half molecule (for example, the fluorescence of sequence of at least a portion complementation Mark).
When using matrix, can produce or further strengthen by the decoration method of dying such as silver or gold dyes can The change that detects. Nano particle for the silver-colored any type of reducing of catalysis all can adopt argentation. Excellent Select nano particle (for example, Jin Heyin) to be made by noble metal. Referring to Bassell, etc., J.Cell Biol, 126,863-876 (1994); Braun-Howland etc., Biotechniques, 13,928-931 (1992). If the nano particle for detection of nucleic acid can not reduce by catalysis silver, so can be with silver ion and nucleic acid network Close with catalytic reduction. Referring to Braun etc., Nature, 391,775 (1998). In addition, known silver-colored dyestuff Can with nucleic acid on phosphate group reaction.
In any mensuration that the matrix that comprises above-mentioned matrix is carried out, can use silver-colored dyestuff to produce or enhancing Detectable change. Specifically, find that silver-colored dyestuff can improve the nanometer that adopts single type in a large number Sensitivity in the mensuration of grain, thus common use to the multi-layer nano particle can be eliminated.
In the mensuration of the detection reporter oligonucleotides that matrix is carried out, can be observed with optical scanner Detectable change. Suitable scanner comprises can be with the usefulness of reflection mode (reflective mode) operation In those scanners that file are scanned into computer (for example, flat-bed scanner), can finish this function Perhaps utilize other device of same type optics, the tonal gradation of any type (greyscale)-sensitivity (for example, improveing is bag according to the standard scan device of matrix of the present invention for scanning for measurement mechanism and improvement The flat-bed scanner that contains the matrix clamper) (so far, not yet finds and to use with transmission mode The scanner of (transmissive mode) operation). The resolution ratio of scanner is essential enough big so that on the matrix Reaction zone greater than the single pixel of scanner. This scanner can be used for any matrix, and prerequisite is at base Can be observed on the matter detectable variation that test produces (for example, gray corrosion, for example silver dyes generation Gray corrosion can be observed in white background, but can not observe at gray background). Scanning Device can be Hei-Bai scanner, perhaps, and preferred colour scanner. Most preferably, scanner is Be used for file is scanned into the standard colour scanner type of computer. This scanner cheaply and easily with Commercial form has been bought. For example, can use Epson Expression 636 (600 * 600dpi), UMAX Astra 1200 (300 * 300dpi), or Microtec 1600 (1600 * 1600dpi). Scanner and loading There is the computer of software to connect the image that obtains by scanning matrix for processing. This software can be to hold The standard software of easily having bought with commercial form, for example Adobe Photoshop 5.2 and Corel Photopaint 8.0. Use this software to calculate the method that the tonal gradation measured value provides the quantitative assay result. This software The number of color of color spot also can be provided and can produce scan image (for example, printout), can carry out it Browse to provide the qualitative determination that has nucleic acid result, nucleic acid quantitatively, perhaps both measure. Meter The calculation machine can be the standard personal computer of having bought with commercial form easily. Therefore, use and be mounted with The standard scan device that the standard computer of standard software links to each other can be provided at when measuring on the matrix and detect With the convenience of quantitative nucleic acid, simple and easy, cheap instrument. Also scanning can be stored in the computer with dimension The record of holding this result is used for further reference or use. Certainly, if need, can use more senior Instrument and software.
In another embodiment of the present invention, the method that whether has one or more target analytes in a kind of test sample is provided, every kind of target analyte has at least two binding sites, and the method comprises:
The capturing probe that is incorporated into one or more types on the matrix is provided, and every type seizure is visited Pin comprises the specific binding complement of first binding site of specific target analyte;
The detector probe of one or more types is provided, and every type detector probe comprises and combines the widow The nano particle of nucleotides, second binding site of this specific target analyte one or more special The property in conjunction with complement with as one or more DNA bar codes of concrete target analyte mark, wherein At least a portion sequence of this DNA bar code and at least some oligonucleotides that are attached on the nano particle Hybridization;
When allowing that the specificity binding interactions takes place between target analyte and the probe and having target analyte, form under the condition for validity of assembling mixture, make sample, capturing probe, detection probes contact;
Washing matrix is to remove unconjugated detection probes;
Detect in the gathering mixture on the matrix whether have this DNA barcode, the detected result that wherein whether has this DNA barcode is the index that whether has target analyte in the sample.
Aspect of this embodiment of the present invention, this detection probes comprises one or more specificitys of second binding site of (i) specific target analyte in conjunction with complement, (ii) with the oligonucleotide of at least a type of nano particle bonded, with the DNA barcode that has with at least a portion complementary predetermined sequence of the oligonucleotide of at least a type, this DNA barcode combines the mark as the specific target analyte with all types of detection probes;
In this embodiment on the other hand, before described detection step, this method also comprises step:
Making mixture impurity elimination friendship and discharging under the condition for validity of this DNA barcode, handle this gathering mixture; With
Choose wantonly and before described detection, make the DNA barcode amplification.
In another aspect of this invention, capturing probe is attached on the magnetic substrate such as magnetic-particle, for example, has the polystyrene MMP of martial ethiops.This allows easily to remove mixture from solution.Use the direct detection of above-mentioned detection technique then or before detection technique, pass through amplification indirect detection DNA barcode based on matrix.Among the embodiment, the nano particle (NPs) based on oligonucleotide-modification has been described below, the method for magnetic corpuscular (MMPs) and detect barcode DNA subsequently as the amplifier of one or more target nucleic acid sequences.Preferably, oligonucleotides-modified nano particle comprises gold nano grain.Whether detect exists target DNA signal (by detector bar font code DNA) preferably to use above-mentioned detection method based on matrix to carry out.
In one aspect, the invention provides the target nucleic acid amplification method that does not rely on PCR method, and it is with oligonucleotides-modified nano particle (NPs), magnetic corpuscular (MMPs) is the basis, and detects the target nucleic acid with the form amplification of barcode DNA.In an embodiment aspect this, oligonucleotides-modified nano particle (NPs) comprises gold nano grain.In another embodiment aspect this, use the detection of amplifying DNA signal (the specific barcode DNA of target sequence) based on the detection method of chip.In another embodiment aspect this, barcode DNA comprises the specific sequence of each interested target nucleic acid molecule, and it allows the multiple target nucleic acid sequence in the specific detection solution to be measured.
In another aspect of this invention, this barcode DNA comprises the specific sequence of each interested concrete target analyte in the test sample, and it allows to detect the multiple specific target in single mensuration/detection solution.As shown in the Examples, (" zepto " order of magnitude is 10 can to reach the limit of detection that is low to moderate about 500 zeptomolar (zM) -21For example in whole 20 μ L samples, 10 copies are arranged).This limit of detection represents that the sensitivity that the no PCR of target nucleic acid molecule detects enlarges markedly.
At this on the one hand, provide two types probe to be used for target DNA detection (DNA-BCA).In certain embodiments, first type probe is the polystyrene MMP with martial ethiops core, uses at least a portion sequence complementary oligonucleotide functionalization with target.The complementary portion of the oligonucleotide of MMP can have all lengths, this depend on concrete condition determination (for example, Laemmli buffer system Laemmli, target nucleic acid sequence, temperature, etc.).
In another embodiment, second kind of probe comprises the oligonucleotides-modified nano particle with two types, a kind of sequence that comprises and be different from least a portion target complement sequence of the target region of being discerned by MMP; Another kind comprises and at least a portion barcode dna sequence dna complementary sequence, and this barcode DNA provides the peculiar identifying mark of particular target sequence.In certain embodiments, this nano particle is a gold nano grain.In another embodiment, gold nano grain comprises 13,20, or the gold nano grain of 30nm.
The barcode DNA on the nano grain surface and the ratio of target binding sequence can change to adapt to each independent assay method.For the no PCR that barcode DNA is provided detects, barcode DNA combines the ratio of DNA with target should be greater than 1: 1, and preferably about at least 25: 1, more preferably about at least 50: 1, most preferably about at least 100: 1.Higher ratio can provide no PCR target amplification, because identify and detector bar font code DNA rather than target sequence in the DNA-BCA method.For example, the 13nm gold nano grain can be supported the DNA chain of at least 100 mercaptanization of each particle 73For 20 and the nano particle of 30nm, suppose have suitable oligonucleotide to load and each particle for spherical, then this particle can be supported 240 and 530 fixed oligonucleotide respectively.
In this embodiment on the other hand, combining complement with nano particle bonded specificity is mono-clonal or polyclonal antibody.
In this embodiment on the other hand, combining complement with capturing probe bonded specificity is monoclonal antibody.
In this embodiment on the other hand, this antibody is anti-PSA antibody.
In this embodiment on the other hand, before described washing step, this method also comprises step:
By applying magnetic field and before washing, separate this gathering mixture to being attached to gathering mixture on the magnetic-particle.
In this embodiment on the other hand, this method also comprises step: hand under the condition for validity of assembling mixture and released dna barcode in impurity elimination, separate this gathering mixture.
In this embodiment on the other hand, make the DNA barcode amplification of release by any suitable technique such as PCR.
In this embodiment on the other hand, target analyte is to have two-part at least nucleic acid.
In this embodiment on the other hand, this target analyte is to have the target nucleic acid of two portions sequence at least, detection probes comprises the nano particle that has with the oligonucleotide of DNA barcode complementary sequence, the specificity of detection probes comprises first target identification oligonucleotide that has with first part's complementary sequence of target nucleic acid in conjunction with complement, and the specificity of capturing probe comprises second target identification oligonucleotide that has with the complementary of the second section at least sequence of target nucleic acid in conjunction with complement.
In this embodiment on the other hand, target analyte is to have the target nucleic acid of two portions sequence at least, detection probes comprises the nano particle that combines oligonucleotide, the DNA barcode has and at least a portion complementary sequence that is attached to the oligonucleotide on the detection probes, specificity comprises the target identification oligonucleotide with at least the first and second partial sequences in conjunction with complement, first part's complementation of first part and target nucleic acid and second section and be attached at least a portion complementation of the oligonucleotide on the nano particle, the specificity of matrix comprise the target identification oligonucleotide that has with second section complementary at least a portion of target nucleic acid in conjunction with complement.
In this embodiment on the other hand, detection probes comprises above-mentioned SD nano particle.
In another embodiment of the present invention, the method that whether has one or more target analytes in a kind of test sample is provided, every kind of target analyte has at least two binding sites, and this method comprises:
The capturing probe of one or more types is provided, and every type capturing probe comprises (i) magnetic-particle; (ii) be attached to first specificity on the magnetic-particle in conjunction with the first right member, wherein first specificity combines with first binding site of specific target analyte in conjunction with the first right member;
Be provided for the detection probes of one or more types of every kind of target analyte, every type detection probes comprises (i) nano particle; (ii) be attached to second specificity on the nano particle in conjunction with the first right member, wherein second specificity combines with second binding site of target analyte in conjunction with the first right member; (iii) with the oligonucleotide of at least a type of nano particle bonded; The (iv) DNA barcode of at least a type, various types of DNA barcodes have with at least a portion complementary predetermined sequence of the oligonucleotide of particular type and as the mark of specific target analyte;
When allowing that the specificity binding interactions takes place between target analyte and the probe and having target analyte, form and assemble under the condition for validity of mixture, make sample, capturing probe, detection probes contact with the magnetic-particle bonded;
The unconjugated detection probes of flush away from the magnetic-particle; With
Detect in the mixture whether have this DNA barcode, wherein the detected result of this DNA barcode is the index that has target analyte.
Aspect of this embodiment, this method also comprised step before described detection step:
Assemble mixture by applying magnetic field separation;
Discharge under the condition for validity of this DNA barcode in the impurity elimination friendship and from assembling mixture, handle this gathering mixture;
Separate the DNA barcode that discharges.
In this embodiment on the other hand, this method also comprises the DNA barcode amplification that makes release.
In this embodiment on the other hand, this method also comprises:
The matrix that combines oligonucleotide is provided, and this oligonucleotide has at least a portion sequence complementary sequence with the DNA barcode;
The nano particle that comprises with its bonded oligonucleotide is provided, and at least a portion that wherein is attached to the oligonucleotide on the nano particle has at least a portion complementary sequence with the DNA barcode; With
Under the condition for validity of between the first part at least that allows the DNA barcode and the complementary oligonucleotide that is attached on the matrix and between the second section of DNA barcode and some oligonucleotide that are attached on the nano particle, hybridizing, make the DNA barcode, be attached to oligonucleotide on the matrix, reach nano particle and contact.
In this embodiment on the other hand, before detection, make the DNA barcode amplification by PCR.
In this embodiment on the other hand, this method also is included in to analyze and assembles preceding this gathering mixture that separates of mixture.
In this embodiment on the other hand, by being applied magnetic field, the gathering mixture separates this gathering mixture.
In this embodiment on the other hand, this nano particle is metal nanoparticle or the semiconductor nanoparticle such as gold nano grain.
In this embodiment on the other hand, the specificity combination is to being antibody and antigen; Acceptor and part; Enzyme and substrate; Medicine and target molecule; Antibody nucleic acid (aptamer) and antibody nucleic acid target; Article two, to small part complementary oligonucleotide chain.
In this embodiment on the other hand, this DNA barcode can be used biotin labeling, radio-labeling, or fluorescent mark.
In arbitrary embodiment, at least two types particle composites probe is provided, and the specificity that first type probe has first binding site on the target analyte has the specificity of second binding site on the probe in conjunction with complement in conjunction with the probe of complement and second type.Multiple particle composites probe can be provided, and every type probe has a specific specificity of different binding sites on the target analyte in conjunction with complement.
Specificity in conjunction with complement and target analyte be specificity in conjunction with right member, it comprises nucleic acid, oligonucleotide, peptide nucleic acid(PNA), polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, VITAMIN, medicine, virus, polysaccharide, lipid, lipopolysaccharides, glycoprotein, lipoprotein, nucleoprotein, oligonucleotide, antibody, immunoglobulin (Ig), albumin, oxyphorase, thrombin, peptide and proteohormone, non-peptide hormone, interleukin-, Interferon, rabbit, cytokine comprises the peptide of tumour-specific epi-position, cell, cell surface molecule, microorganism, the fragment of microorganism, part, component, or product, organic molecule, nucleic acid and oligonucleotide, the meta-bolites of arbitrary above-mentioned substance or antibody.
Nucleic acid and oligonucleotide comprise gene, viral RNA and DNA, DNA of bacteria, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and dna segment, oligonucleotide, synthetic oligonucleotide, the oligonucleotide of modifying, strand and double-strandednucleic acid, natural and nucleic acid and antibody nucleic acid.
Target analyte is that nucleic acid and specificity are oligonucleotide in conjunction with complement.As selection, target analyte is that protein or haptens and specificity are the antibody that comprises mono-clonal or polyclonal antibody in conjunction with complement.As selection, target analyte is to be oligonucleotide from the sequence of genome DNA sample and specificity in conjunction with complement, and this oligonucleotide has and this genomic at least a portion sequence complementary sequence.This genomic dna can be an eucaryon, bacterium, the DNA of fungi or virus.
Specificity is the right member of antibody-part in conjunction with complement and target analyte.
Except its first binding site, target analyte can be modified into and comprise second binding site.
This method can further comprise filtration step to remove the gathering mixture, wherein filters before analyzing the gathering mixture and carries out.Filtration step comprises the film that removes the sample composition that does not comprise the DNA barcode.
In another embodiment of the present invention, the method that whether has one or more target analytes in the test sample is provided, this method comprises:
At least a or polytype particle composites probe is provided, every type probe comprises and its bonded oligonucleotide, one or more specificitys of specific target analyte are in conjunction with complement, be used as one or more DNA barcodes of concrete target analyte mark, wherein at least a portion sequence of this DNA barcode and at least some oligonucleotide hybridizations that are attached on the nano particle;
Under the condition for validity that allows formation gathering mixture under the condition that the specificity binding interactions takes place between target analyte and the particle composites probe and have target analyte, this sample is contacted with the particle composites probe; With
Observe and mixture formation whether occurs assembling.
Aspect this, can use any method as herein described to detect observable signal of the present invention.For example, directly detector bar font code DNA detects the barcode DNAs that BPCR-increases, detect the detection probes of one or more types gathering (for example, by visual inspection, fluorescence, colorimetric, electrochemistry, electronics, light densitometry, radioactivity, or the like), perhaps comprise report Nucleotide and detectable signal half point (for example, fluorescent mark) with at least a portion complementary sequence of barcode DNAs by use.
If there are enough mixtures, but exist or do not having this mixture of visual inspection under the condition of background matrix.Can use any matrix that allows to observe detectable change.Suitable matrix comprises transparent solid surface (for example, glass, quartz, plastics and other polymkeric substance), opaque solid surface (for example, white solid surface, for example TLC silica plate, filter paper, glass fibre filter, nitrocellulose filter, nylon membrane), with conducting solid surface (for example, indium-Xi-oxide compound (ITO)).This matrix can be Any shape or thickness, but generally is flat board and thin slice.Preferably transparent matrix, for example glass (for example, slide glass) or plastics (for example, micro titer plate well).
In one aspect of the invention, provide in a kind of test sample to have target analyte, for example, the method for antibody.The immunoglobulin E (IgE) shown in following embodiment or the antibody of immunoglobulin G (IgG1) can use with suitable haptens (for the IgG1 vitamin H, for IgE dinitrophenyl (DNP), Figure 1A) the oligonucleotides-modified probe of the oligonucleotide chain prehybridization of Xiu Shiing detects 13,14Dna sequence dna during the Proof of Concept that provides among the embodiment is below measured designs by this way, guarantees that promptly probe unwinds Figure 1B with two different aggregations that IgGI and IgE reaction form under differing temps.The probe of IgG1 has longer sequence and the G of Geng Gao, C base contents than IgE.Therefore, the former unwinds under higher temperature than latter sequence at sequence.These sequence differences allow people's preparation to have the unwind probe of mark of difference, this mark as code to identify the target that forms nanoparticle aggregate thing with their reactions.After deliberation three kinds of different systems: (1) provides two kinds of probes and a kind of target antibody (IgG1 or IgE); (2) provide two kinds of probes and two kinds of different target antibodies and (3) that the contrast of no target antibody is provided.
, provide in a kind of test sample to have target analyte aspect this of the present invention, for example, the method for antibody comprises that the nanoparticle probes that will combine oligonucleotide contacts with the sample that contains target analyte.At least some oligonucleotide that are attached on the nano particle combine with the first part of reporter oligonucleotides owing to hybridizing.The second section of reporter oligonucleotides has on the specific conjugated complement of analyte (for example, antigen) and its bonded oligonucleotide owing to hybridization is attached to.Under the condition for validity that allows generation specificity binding interactions between analyte and the nanoparticle probes, contact.When having target analyte, can produce the nanoparticle aggregate thing.Can detect these aggregations by any suitable manner.
In another aspect of this invention, can use the particle composites probe, preferred nano-particle complex probe.These particle composites can produce before carrying out practical measurement or original position generation when measuring.These mixtures comprise a kind of particle that combines oligonucleotide, preferred nano particle, with the specificity of at least a portion bonded reporter oligonucleotides that is attached to the oligonucleotide on the nano particle and target analyte in conjunction with complement.These specificitys can directly or indirectly combine with nano particle in conjunction with complement.For example, specificity can combine with joint or oligonucleotide in conjunction with complement, and the joint of this mark or oligonucleotide are attached on the nano particle then.In one embodiment, DNA barcode or the reporter oligonucleotides nano particle that has two portions sequence at least and combine oligonucleotide by hybridization with have specificity and be connected with its bonded oligonucleotide in conjunction with complement.Being attached to oligonucleotide on the nano particle has with a part of complementary sequence of reporter oligonucleotides and has specificity and combine complement and its bonded oligonucleotide and have second section complementary sequence with reporter oligonucleotides.The nano particle that reporter oligonucleotides has two portions at least and combines oligonucleotide by hybridization with have specificity and be connected with its bonded oligonucleotide in conjunction with complement.When using in the sample that is containing target analyte, nano-particle complex combines with target analyte and assembles.Separable this aggregation also carries out further melting analysis to identify concrete target analyte, wherein can have aforesaid multiple target.In addition, but the aggregation impurity elimination hand over to discharge reporter oligonucleotides.Can choose these reporter oligonucleotides of amplification wantonly, or the DNA barcode, use any suitable detection probes to detect then by any suitable DNA detection system.
Implementing when of the present invention, the nano particle by will combining oligonucleotide combines the oligonucleotide of complement modification and reporter oligonucleotides and hybridizes and can prepare the nano-particle complex probe with the specificity with target analyte.Be attached at least some oligonucleotide on the nano particle and have first part's complementary sequence with reporter oligonucleotides.Have specificity and have second section complementary sequence with reporter oligonucleotides in conjunction with complement and its bonded oligonucleotide.Reporter oligonucleotides be attached to nano particle at least some oligonucleotide and combine complement and its bonded oligonucleotide and hybridize under the condition that allows to hybridize between this composition being enough to having specificity, form the nano-particle complex probe.When the nano-particle complex solution that this composition of preparation permission is fully hybridized, can use any suitable solvent medium and hybridization conditions.Preferably, this composition was at room temperature hybridized about 2-3 hour in the phosphate buffered saline buffer of being made up of 0.3M NaCl and 10mM phosphate buffered saline buffer (pH7) (PBS).The concentration of nano particle in the hybridization mixture-oligonucleotide connector is approximately changing preferably approximately 13nM between 2nM and the about 50nM.The concentration of the oligonucleotide of hapten transformation generally changes between about 50 and about 900, preferably approximately 300nM.The concentration of reporter oligonucleotides generally changes between about 50 and about 900, preferably approximately 300nM.The oligonucleotide of unreacted hapten transformation and reporter oligonucleotides are optional, but preferably can remove by any suitable manner, preferably by centrifugal (12,000rpm, 20 minutes) hybridization mixture and this supernatant of decant subsequently.The mixture of preparation is stored in 0.3M NaCl and 10mM phosphate buffered saline buffer (pH7-7.4) in 4-6 ℃, in 0.01% azide solution.
There is target analyte in the test sample, for example the typical assay method of antibody is as follows: the solution that will contain the nano-particle complex probe mixes with believing the aqueous specimen solution that contains target protein, this nano-particle complex probe comprises the nano particle that combines oligonucleotide, reporter oligonucleotides and have the oligonucleotide of the specificity of target analyte in conjunction with complement.Total protein content in aqueous specimen solution generally changes between about 5 to about 100, is typically about 43ug/ml.The concentration of nano particle generally approximately changing between 2nM and the about 20nM, is typically about～13nM in the reaction mixture.The cumulative volume of gained mixture is generally approximately changing preferably approximately 400uL between 100uL and the about 1000uL.When the aqueous specimen solution that contains target analyte is believed in preparation, can adopt any suitable solvent, preferably contain the PBS of 0.3M NaCl and 10mM phosphate buffered saline buffer (pH7-7.4).
The mensuration mixture of gained changes between about 35 to about 40 ℃ then, preferably change between about 30 to about 60 at incubation under 37 ℃ the temperature, 50 minutes the enough time of preferably approximately is to promote specificity in conjunction with right, for example, protein-haptens, compound action.If there is target protein, particle aggregation then takes place, cause along with precipitating action gold nano grain mass spectrum band (plasmon band) transfer with by red color change to purple.Centrifugal hybridization product (for example, 3000rpm 2 minutes), decant contains the supernatant of unreacted composition before analyzing.
If desired, by mixing the nano particle that all combine oligonucleotide, the oligonucleotide of reporter oligonucleotides and hapten transformation and suspection contain sample in-situ preparing nano-particle complex probe in measuring mixture of target analyte.In order to ensure all compositions, the particularly hybridization fully between the complementary dna chain is measured mixture and can and be stored 24 hours to promote hybridization under 4 ℃ at-15 ℃ of incubations 20 minutes (Boekel Tropicooler Hot/Cold Block Incubator).Yet, when enforcement is of the present invention, preferably before carrying out assaying reaction, prepare the nano-particle complex probe to increase the DNA barcode total amount in the nano-particle complex probe.
To have any protein in order measuring, can carry out the melting analysis of aggregation in solution, the delustring under the monitoring 260nm is as the function of temperature.Referring to, for example, the Fig. 2 among the embodiment 3, it has described to contain one or both known target analytes: i.e. the sample analysis of IgG1 and IgE.As described in embodiment 3, when handling IgG1 by aforesaid method with probe, solution becomes slightly pinkish blueness, shows to have formed the nanoparticle aggregate thing.Do not having target but exist in the proteic control experiment of background, do not having discernible precipitation.The melting analysis of solution shows under 55 ℃ melting temperature(Tm) (Tm) and flipflop occurs.This is the expection transformation of IgG1 target, Fig. 2 A (---).If in new probe solution, add IgE, can be observed identical colour-change but melting analysis provides Tm is 36 ℃ curve, i.e. the expection of this target changes, Fig. 2 A (-).It should be noted that solution becomes mulberry, and melting analysis demonstrates two different conversions (transactions) when adding two kinds of protein targets in probe solution.First derivative (derivative) of this curve demonstrates two peaks under 36 and 55 ℃ respectively, center, Fig. 2 B.This has confirmed to use the two kinds of different assembling forms that produce from the oligonucleotide barcode and the characteristic of unwinding thereof to distinguish two kinds of protein targets.
In another aspect of this invention, can use various above-mentioned method for congregating strategies with the sensitivity that improves said system with increase the target number that can in a kind of solution, detect.Referring to, for example, the Fig. 3 among the embodiment 4.Use this strategy, can be by giving the specified DNA barcode of concrete target analyte or this protein target of peculiar reporter oligonucleotides indirect detection.In general, be used for the appropriate length of target analyte, GC content, and sequence, and being chosen in of reporter oligonucleotides measured preceding being scheduled to.For example, the oligonucleotide of 12-aggressiveness has 4 12Plant different sequences, the wherein multiple barcode that can be used for preparing the interested multivalent protein shown in Figure 1A.In this assay method, the characteristic energy measurement not in solution that unwinds of the aggregation of its formation, and should reporter oligonucleotides in the aggregation or DNA barcode be separated with target molecule with unreacted probe by centrifugal (for example, 3000rpm is 2 minutes).Then by any suitable manner, for example, by in solution, adding water, make this aggregation sex change, to discharge reporter oligonucleotides or bio-barcode.If reporter oligonucleotides can increase by methods known in the art to exist on a small quantity.Referring to, for example, Sambrook etc., Molecular Cloning:A Laboratory Manual (second edition 1989) and B.D.Hames and S.J.Higgins, Eds., Gene Probes l (IRL Press, New York, 1995).Preferred polymeric polymerase chain reaction (PCR) amplification.Can be by any proper tools, for example, centrifugal filter device (Millipore Microcon YM-100,3500rpm 25 minutes) separates with protein particle with reporter oligonucleotides.In case separated reporter oligonucleotides, can on oligonucleotide arrays, catch they also use one of multiple suitable DNA detection test identify they (Fig. 3).For the example that relates to IgG1 and IgE as herein described, on the slide glass of using with half complementary oligonucleotide functionalization (diameter is the point of 250 μ m) of interested barcode, catch reporter oligonucleotides (A3 and B3 among Fig. 1).If barcode is caught by oligonucleotide arrays, the particle of modifying with barcode remainder complementary DNA-can with this hybridization array (seeing experimental section).When passing through the standard scan measuring method [11]When develop (it relate to soup handle), can use quantitatively this result of flat-bed scanner, Fig. 4 11If there is IgG1, then only there is point to demonstrate measurable signal at the IgG1 design.Equally, if IgE is only protein, then only there is point to demonstrate signal at its design.At last, if two kinds of protein all exist, then two kinds of points all show strong signal.
Aspect of this embodiment, the DNA barcode in various types of particle composites probes has different sequences and is used as the identifier of concrete target analyte.
In this embodiment on the other hand, this method also comprises following steps:
Separate and assemble mixture; With
Analyze this gathering mixture and have existing of not homotactic one or more DNA barcodes with mensuration.
In this embodiment on the other hand, this method also comprises the steps:
Separate this gathering mixture;
Assemble under the condition for validity of mixture impurity elimination friendship and released dna barcode at this, handle this gathering mixture;
Separate this DNA barcode; With
Detection has the existence of not homotactic one or more DNA barcodes, and wherein each DNA barcode is the index that has concrete target analyte in the sample.
In this embodiment on the other hand, this method also comprises the steps:
Separate and assemble mixture;
Assemble under the condition for validity of mixture impurity elimination friendship and released dna barcode at this, handle this gathering mixture;
Separate this DNA barcode;
Make the separated DNA barcode amplification; With
Detection has the existence of the DNA barcode of not homotactic one or more amplifications, and wherein each DNA barcode is the index that has concrete target analyte in the sample.
In this embodiment on the other hand, target has plural binding site and at least two types particle composites probe is provided, first type probe has the specificity of first binding site on the target analyte in conjunction with complement, and second type probe has the specificity of second binding site on the probe in conjunction with complement.Multiple particle composites probe can be provided, and every type probe has a specific specificity of different binding sites on the target analyte in conjunction with complement.
In this embodiment on the other hand, specificity in conjunction with complement and target analyte be specificity in conjunction with right member, it comprises nucleic acid, oligonucleotide, peptide nucleic acid(PNA), polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, VITAMIN, medicine, virus, polysaccharide, lipid, lipopolysaccharides, glycoprotein, lipoprotein, nucleoprotein, oligonucleotide, antibody, immunoglobulin (Ig), albumin, oxyphorase, thrombin, peptide and proteohormone, non-peptide hormone, interleukin-, Interferon, rabbit, cytokine comprises the peptide of tumour-specific epi-position, cell, cell surface molecule, microorganism, the fragment of microorganism, part, component, or product, organic molecule, nucleic acid and oligonucleotide, the meta-bolites of arbitrary above-mentioned substance or antibody.
Nucleic acid and oligonucleotide comprise gene, viral RNA and DNA, DNA of bacteria, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and dna segment, oligonucleotide, synthetic oligonucleotide, the oligonucleotide of modifying, strand and double-strandednucleic acid, natural and nucleic acid and antibody nucleic acid.
In one aspect, target analyte can be that nucleic acid and specificity can be oligonucleotide in conjunction with complement.As selection, target analyte is that protein or haptens and specificity are the antibody that comprises mono-clonal or polyclonal antibody in conjunction with complement.
In this embodiment on the other hand, target analyte is to be oligonucleotide from the sequence of genome DNA sample and specificity in conjunction with complement, and this oligonucleotide has and this genomic at least a portion sequence complementary sequence.
In this embodiment on the other hand, this genomic dna can be an eucaryon, bacterium, the DNA of fungi or virus.
On the other hand, specificity is the right member of antibody-part in conjunction with complement and target analyte.
In this embodiment on the other hand, be used to detect and exist the step of one or more DNA barcodes to comprise:
The matrix that combines oligonucleotide is provided, and this oligonucleotide has at least a portion sequence complementary sequence with the DNA barcode;
Provide to comprise oligonucleotide and its bonded nano particle, at least a portion that wherein is attached to the oligonucleotide on the nano particle has at least a portion complementary sequence with the DNA barcode; With
Under the condition for validity of between the first part at least that allows the DNA barcode and the complementary oligonucleotide that is attached on the matrix and between the second section of DNA barcode and some oligonucleotide that are attached on the nano particle, hybridizing, make the DNA barcode, be attached to oligonucleotide on the matrix, reach nano particle and contact; With
Observe detectable variation.
In this embodiment on the other hand, matrix comprises with array way and adheres to the polytype oligonucleotide on it so that allow to detect one or more dissimilar DNA barcodes.
In this embodiment on the other hand, detectable change is to form dark areas on matrix.
In this embodiment on the other hand, detectable variation is observed with optical scanner.
In this embodiment on the other hand, matrix contacts to produce detectable variation with silver-colored stain.
In this embodiment on the other hand, the DNA barcode is under the condition for validity that allows DNA barcode and the complementary oligonucleotide that is attached on the matrix to hybridize, contact with matrix, and will be attached to DNA barcode on the matrix subsequently under the condition for validity of the part hybridization that allows to be attached at least some oligonucleotide on the nano particle and the DNA bar code sequence on the matrix, contact with the nano particle that combines oligonucleotide.
In this embodiment on the other hand, the DNA barcode is allowing the DNA barcode and is being attached under the condition for validity of at least some oligonucleotide hybridizations on the nano particle, contacts with the nano particle that combines oligonucleotide; And will be attached to DNA barcode on the nano particle subsequently under at least a portion sequence that allows to be attached to the DNA barcode on the nano particle and the condition for validity that is attached to the complementary oligonucleotide hybridization on the matrix, contact with matrix.
In this embodiment on the other hand, DNA amplification barcode before contact procedure.
In this embodiment on the other hand, at least two types particle composites probe is provided, first type probe has the specificity of target analyte first binding site in conjunction with complement, and second type probe has the specificity of target analyte second binding site in conjunction with complement.
As mentioned above, nucleic acid and oligonucleotide comprise gene, viral RNA and DNA, DNA of bacteria, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and dna segment, oligonucleotide, synthetic oligonucleotide, the oligonucleotide of modification, strand and double-strandednucleic acid, natural and nucleic acid and antibody nucleic acid.
In another embodiment of the present invention, provide the test kit that comprises above-mentioned particle composites probe.
In this embodiment on the other hand, provide the test kit that whether has one or more target analytes in a kind of test sample, every kind of target analyte has at least two binding sites, and this test kit comprises:
The detection probes that is used at least a type of every kind of target analyte, every type detection probes comprise (i) nano particle; (ii) be attached to specificity on the nano particle in conjunction with a right member; (iii) with nano particle bonded oligonucleotide; (iv) has DNA barcode with at least a portion complementary predetermined sequence of this oligonucleotide.
In this embodiment on the other hand, this test kit comprises:
The capturing probe of at least a type, it comprises (i) matrix; (ii) be attached to first specificity on the matrix in conjunction with first right member, wherein first specificity is attached on first binding site of target analyte in conjunction with the first right member;
The detection probes of at least a type, it comprises (i) nano particle; (ii) be attached to second specificity on the nano particle in conjunction with first right member, wherein this second specificity combines with second binding site of target analyte in conjunction with first right member; (iii) with the oligonucleotide of at least a type of nano particle bonded; The (iv) DNA barcode of at least a type, every type of at least a portion complementary predetermined sequence that has with the oligonucleotide of particular type.
In this test kit, can use any suitable matrix.Preferably, matrix is the magnet such as magnetic corpuscular.
In this embodiment on the other hand, this test kit comprises:
The capturing probe of at least a type, it comprises (i) magnetic-particle; (ii) be attached to first specificity on the magnetic-particle in conjunction with first right member, wherein first specificity is attached on first binding site of target analyte in conjunction with the first right member;
The detection probes of at least a type, it comprises (i) nano particle; (ii) be attached to second specificity on the nano particle in conjunction with first right member, wherein this second specificity combines with second binding site of target analyte in conjunction with first right member; (iii) with the oligonucleotide of at least a type of nano particle bonded; The (iv) DNA barcode of at least a type, every type of at least a portion complementary predetermined sequence that has with the oligonucleotide of particular type.
In this embodiment on the other hand, this test kit comprises:
At least a container that comprises the particle composites probe, this probe comprises the particle that combines oligonucleotide, the DNA barcode, and combine the oligonucleotide of the specificity of target analyte in conjunction with complement, wherein this DNA barcode has the sequence of at least two parts, be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode, combine specificity and have second section complementary sequence with the DNA barcode in conjunction with the oligonucleotide of complement, and wherein this DNA barcode be attached to particle at least some oligonucleotide and with combine that specificity combines the oligonucleotide hybridization of complement and the matrix of choosing any one kind of them is used to observe detectable variation.
In this embodiment on the other hand, this test kit comprises:
At least a or multiple container, container contains one type particle composites probe, this probe comprises the particle that combines oligonucleotide, the DNA barcode, and combine the oligonucleotide of the specificity of concrete target analyte in conjunction with complement, wherein (i) this DNA barcode has the sequence of at least two parts, (ii) be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode, (iii) combine specificity and have second section complementary sequence with the DNA barcode, have different sequences with DNA barcode in (iv) every type the particle composites probe and as the identifier of concrete target analyte in conjunction with the oligonucleotide of complement; Wherein this test kit is optional comprises a kind of matrix and is used to observe detectable variation.
In this embodiment on the other hand, this test kit comprises:
At least one pair of container and the optional matrix that is used to observe detectable variation,
This contains particle probe to first container in the container, this probe comprises the particle that combines oligonucleotide and the DNA barcode with at least two partial sequences, wherein is attached at least some oligonucleotide on the particle and has first part's complementary sequence with the DNA barcode;
This comprises the oligonucleotide that has with the second section complementary sequence of DNA barcode to second container in the container, this oligonucleotide have can be used for covalently bound target analyte specificity in conjunction with half point to complement.
In this embodiment on the other hand, this test kit comprises:
At least two pairs or more to container,
First container in the every pair of container contains the particle composites probe, this probe comprises particle that combines oligonucleotide and the DNA barcode with at least two partial sequences, and at least some oligonucleotide that wherein are attached on the particle have and have first part's complementary sequence of the DNA barcode of at least two parts; With
Second container in the every pair of container comprises the oligonucleotide that has with the second section complementary sequence of DNA barcode, this oligonucleotide have can be used for covalently bound target analyte specificity in conjunction with half point to complement,
Wherein the DNA barcode of every type particle composites probe has different sequences and as the identifier of target analyte and wherein this test kit is optional comprises a kind of matrix and be used to observe detectable variation.
In this embodiment on the other hand, this test kit comprises:
First container and at least two pairs or more to container,
First container contains the particle composites probe, and this probe comprises the particle that combines oligonucleotide;
First container in the every pair of container comprises the DNA barcode with at least two partial sequences, wherein is attached at least some oligonucleotide on the particle and has first part's complementary sequence with the DNA barcode; With
Second container in the every pair of container comprises the oligonucleotide that has with the second section complementary sequence of DNA barcode, this oligonucleotide have can be used for covalently bound target analyte specificity in conjunction with half point to complement,
Wherein the DNA barcode in first container of every pair of container as the identifier of target analyte and have with another to the different sequence of DNA barcode in the container, and wherein this test kit is optional comprises a kind of matrix and be used to observe detectable variation.
In this embodiment on the other hand, this test kit comprises:
Be used as the oligonucleotide sequence of the identifier of concrete target analyte existence.
The mentioned reagent box can comprise the specification sheets that is used to assemble this mensuration and is used to carry out this mensuration.
It should be noted that term " " or " one " entity are meant one or more these entities.For example, " feature " is meant one or more features or at least one feature.Therefore, term " " (or " "), " one or more " and " at least one " are used in this article convertibly.Should also be noted that term " comprises ", " comprising " and " having " used convertibly.The following examples only are used for the purpose of illustration, and should not constitute the restriction to essence of the present invention or scope by any way.
Embodiment
Embodiment 1: Prepare oligonucleotides-modified gold nano grain
A. The preparation gold nano grain
Oligonucleotides-modified 13nm Au particle is pressed the method preparation (～110 oligonucleotide/particles) of document record 18-20Gold colloid (diameter 13nm) is pressed Frens, Nature Phys.Sci., and 241,20 (1973) and Grabar, Anal.Chem., 67,735 (1995) is described by reduce HAuCl with Citrate trianion 4Prepare.Briefly, all glass waress are at chloroazotic acid (3 parts of HCl, 1 part of HNO 3) middle cleaning, use NanopureH 2O rinsing, oven dried before use then.HAuCl 4Buy from Aldrich chemical company with Trisodium Citrate.With HAuCl 4The aqueous solution (1mM 500mL) refluxes while stirring, adds the citric acid three sodium solution of 50mL 38.8mM then rapidly, cause solution colour by pale yellow become dark red.Behind the color change, solution was refluxed 15 minutes again, cool to room temperature, 0.45 micrometer nylon filter by Micron Separations Inc. filters subsequently.The Au colloid uses Hewlett Packard 8452A diode array spectrophotometer to identify by transmission electron microscope inspection (TEM) by UV-vis spectroscopy and use Hitachi 8100 transmission electron microscopes.The typical solution of 13nm diameter gold grain shows the characteristic surface mass spectrum band that concentrates on 518-520nm.The gold grain of 13nm diameter produces the visible colour-change when assembling with the probe oligonucleotides sequence of target and 10-72 Nucleotide scope.
B. Synthesizing of oligonucleotide
Use phosphoramidic acid chemistry (phosphoramidite chemistry) to use Milligene Expedite dna synthesizer synthetic oligonucleotide on 1 micromole (micromole) scale with single post.Eckstein，F.(ed.)Oligonucleotides?and?Analogues?：A?Practical?Approach(IRL?Press，Oxford，1991)。All solution are bought (the synthetic level of DNA) from Milligene.Average coupled efficiency range from 98 to 99.8%, final dimethoxytrityl (DMT) blocking group does not separate so that purifying with oligonucleotide.
For 3 '-sulfydryl-oligonucleotide, sulfydryl modification agent C3 S-S CPG carrier is bought and is used automatic DNA synthesizer DNA from Glen Research.Final dimethoxytrityl (DMT) blocking group is not removed so that purifying.After synthetic, the oligonucleotide on the carrier put into the 1mL strong aqua handle the blocking group that oligonucleotide was separated with solid carrier and remove on the base in 16 hours down at 55 ℃.
Behind the evaporation ammoniacal liquor, by preparation type reversed-phase HPLC use HP ODS Hypersil post (5um, 250 * 4mm) with 0.03 M triethylacetic acid ammonium (TEAA), pH7 and 1%/minute 95%CH 3The gradient of CN/5%0.03M TEAA was with 1mL/ minute flow velocity, and the DNA UV signal of monitoring simultaneously under the 254nm is come this oligonucleotide of purifying.The residence time of the 12-base oligomer that the DMT protection is modified is 30 minutes.Oligonucleotide by soaking purifying in 80% acetic acid solution is 30 minutes subsequently, and then evaporation can be removed DMT; In 500uL water, (3 * 300uL) extract solution with ethyl acetate with the oligonucleotide redispersion.After the solvent evaporation, (10 OD ' s) redispersion spend the night to remove 3 ' disulphide under 50 ℃ in the 0.17M phosphate buffered saline buffer (pH8) in the 0.04 M DTT of 100uL with oligonucleotide.(＜10 OD ' are s) by desalination NAP-5 column purification for the aliquots containig of this solution.Content by the absorbance measurement oligonucleotide under 260nm.By ion-exchange HPLC use DionexNucleopac PA-100 post (250 * 4mm) with 10mM NaOH (pH12) and 2%/minute 10mMNaOH, 1M NaCl gradient is assessed purity with flow velocity while UV signal of monitoring of DNA under 254nm of 1mL/ minute.Observe residence time (T r) be 3 peaks of 18.5,18.9 and 22 minutes.T r=22.0 minutes main unimodal 79% the area that accounts for, it belongs to disulphide.Two peaks with shorter residence time of 18.5 and 18.9 minutes account for respectively～9% and 12% area, and they belong to oxidation impurities and residual sulfydryl oligonucleotide.
5 '-oligonucleotide that alkyl sulfhydryl is modified uses following method preparation: it is synthetic that 1) CPG-bonded, detritylation oligonucleotide use standard method to go up at automatic dna synthesizer (Expedite); 2) take out CPG-cartridge case and disposable syringe linked to each other with end; 3) solution that 200uL is contained 20umole 5-sulfydryl-thinner C6-phosphoramidic acid (Glen Research) in dry acetonitrile (dryacetonitrile) mixes with 200uL standard " tetrazolium activator solution ", and contains in the cartridge case of oligonucleotide-CPG by a syringe importing; 4) this solution was slowly aspirated 10 minutes back and forth by cartridge case, discharge then, then with dry acetonitrile (2 * 1mL) washings; 5) the intermediate phosphite is then used acetonitrile/pyridine (1: 1 with 0.02 M iodine oxidation (30 seconds) in 700uL THF/ pyridine/water; 2 * 1mL) and dry acetonitrile washing.Then by for 3 '-the described separation and purification trityl of alkyl sulfhydryl oligonucleotide oligonucleotide derivative; Then by in dried oligonucleotide sample, adding 15uL (for s) 50mM AgNO of 10 OD ' 3Solution removed trityl-protecting group group in 20 minutes, and this reaction produces milky suspension.Remove excessive Silver Nitrate by the DTT solution (5 minutes reaction times) that adds 20uL 10mg/mL, this reaction forms immediately can be by the centrifugal yellow mercury oxide that removes.(＜10 OD ' aliquots containig s) is transferred on the desalination NAP-5 post and is carried out purifying with oligonucleotide solution then.Use above final content and purity for 5 ' alkyl sulfhydryl oligonucleotide of the described technical evaluation gained of 3 ' alkyl sulfhydryl oligonucleotide.Observe two main peaks by ion-exchange HPLC, its residence time is 19.8 minutes (the mercaptan peak accounts for 16% area) and 23.5 minutes (the disulphide peak accounts for 82% area).
C. Oligonucleotide is connected on the gold nano grain
With the aqueous solution of the aqueous solution of 1mL gold colloid (17nM) and excessive (3.68uM) mercaptan-oligonucleotide (22 bases are long), mixture at room temperature left standstill 12-24 hour.Then, solution is added 0.1M NaCl, in the 10mM phosphate buffered saline buffer (pH7) and it was left standstill 40 hours.Design should " wearing out " step be used to the oligonucleotide base that improves the surface coverage of mercaptan oligonucleotide and replace gold surface.Then with this solution in Eppendorf centrifuge tube 5414 with 14,000rpm contained with generation in centrifugal about 25 minutes most of oligonucleotide (representing) and 7-10% with the absorbancy under the 260nm Radioactive colloidal gold (representing) with the absorbancy under the 520nm extremely shallow pink supernatant and the pipe end fine and close dark gluey residue.Remove supernatant, residue is resuspended in the damping fluid (10mM phosphoric acid salt, 0.1M NaCl) of about 200 μ L and centrifugal again.After removing supernatant solution, residue is sucked 1.0mL damping fluid (10mM phosphoric acid salt, 0.3M NaCl, 0.01%NaN 3) in.The redness of gained is that main solution at room temperature leaves standstill some months, point sample (seeing embodiment 4) and adding 1M NaCl to thin layer of silicon dioxide chromatogram (TLC) flat board, 10mM MgCl 2, or have stability (, keep red and do not assemble) when containing in the solution of high density salmon sperm DNA.
Embodiment 2: The oligonucleotide of preparation hapten transformation
The oligonucleotide of hapten transformation use standard solid-phase DNA synthesis method for A1 with vitamin H-triglycol phosphoramidic acid (triethylene glycol phosphoramidite) and for B1 with 2,4-dinitrophenyl-triglycol phosphoramidic acid (Glen research) prepares 21
The oligonucleotide of biotin modification makes with the following method and prepares: the CPG-bonded, the detritylation oligonucleotide uses standard method 21Go up synthetic at automatic dna synthesizer (Expedite).Take out the CPG-cartridge case then and disposable syringe is linked to each other with end.The solution that 200uL is contained 20umole vitamin H-triglycol phosphoramidic acid in dry acetonitrile mixes with 200uL standard " tetrazolium activator solution ", and contains in the cartridge case of oligonucleotide-CPG by a syringe importing.Then this solution was slowly aspirated 10 minutes back and forth by cartridge case, discharge then, then with dry acetonitrile (2 * 1mL) washings.Subsequently, the intermediate phosphite is then used acetonitrile/pyridine (1: 1 with the 0.02M iodine oxidation (30 seconds) in 700uL THF/ pyridine/water; 2 * 1mL) and dry acetonitrile washing, use nitrogen vapour seasoning pillar subsequently.Do not remove trityl-protecting group group, it helps purifying.Oligonucleotide on the carrier is put into the 1mL strong aqua handle down the blocking group that oligonucleotide was separated with solid carrier and remove on the base in 16 hours at 55 ℃.Behind the evaporation ammoniacal liquor, by preparation type reversed-phase HPLC use HP ODS Hypersil post (5nm, 250 * 4mm) with 0.03M triethylacetic acid ammoniums (TEAA), pH7 and 1%/minute 95%CH 3The gradient of CN/5%0.03M TEAA was with 1mL/ minute flow velocity, and the DNA UV signal of monitoring simultaneously under the 254nm is come this oligonucleotide of purifying.The residence time of the oligonucleotide of DMT protection is～32 minutes.Oligonucleotide by soaking purifying in 80% acetic acid solution is 30 minutes subsequently, and then evaporation can be removed DMT; In 500uL water, (3 * 300uL) extractions are also dry with ethyl acetate for solution with the oligonucleotide redispersion.Can use identical method with 2, the oligonucleotide that the synthetic DNP of 4-dinitrophenyl-triglycol phosphoramidic acid modifies.
Embodiment 3: Use the assay method of nano-particle complex probe
Oligonucleotides-modified 13nm gold grain is pressed embodiment 1 described preparation.The oligonucleotide of hapten transformation press embodiment 2 described for A1 with vitamin H-triglycol phosphoramidic acid and for B1 with 2,4-dinitrophenyl-triglycol phosphoramidic acid (Glen research) use standard solid-phase DNA synthetic method prepares 21The PBS damping fluid that is used for this research is made up of 0.3M NaCl and 10mM phosphate buffered saline buffer (pH7).(Milwaukee, WI) purchase also is dissolved in before use and contains 0.05%Tween 20 (ultimate density: 4.3 * 10 from Sigma Aldrich for IgE and IgGl -8B/ μ 1) and background albumen (10ug/ml N,O-Diacetylmuramidase, anti--digoxin of 1% bovine serum albumin and 5.3ug/ml; Each 10uL) in the 0.3M PBS damping fluid.
In order to prepare this probe, oligonucleotides-modified particle (13nM, 300 μ l) is at room temperature hybridized 2-3h with the complementary oligonucleotide (10 μ l, 10 μ M) and the bio-barcode DNA (10 μ L, 10 μ M) of hapten transformation, sequence provides in Fig. 1.By centrifugal (12,000rpm, 20 minutes) nanoparticle probes and subsequently supernatant decanted remove the oligonucleotide and the bio-barcode of unreacted hapten transformation.
In the typical assay method of IgE and/or IgG1, with target protein (each 40 μ l, 43 μ g/ml) add contain probe (in～13nM) the solution, mixture 37 ℃ of following incubations 50 minutes to help protein-haptens compound.In order to ensure all compositions, the complete reaction between the complementary dna chain particularly, solution is stored 24 hours down to promote hybridization at-15 ℃ of incubations 20 minutes (Boekel Tropicooler Hot/Cold Block Incubator) and at 4 ℃.If there is target protein, particle aggregation then takes place, cause along with the transfer of precipitating action gold nano grain mass spectrum band with by red color change to purple.Centrifugal hybridization product (3000rpm 2 minutes), decant contains the supernatant of unreacted composition before analyzing.To have any protein in order measuring, can carry out melting analysis in solution, the delustring under the monitoring 260nm is as the function of temperature, Fig. 2.When handling IgG1 by aforesaid method with probe, solution becomes slightly pinkish blueness, shows to have formed the nanoparticle aggregate thing.Do not having target but exist in the proteic control experiment of background, do not having discernible precipitation.The melting analysis of solution shows under 55 ℃ melting temperature(Tm) (Tm) and flipflop occurs.This is the expection transformation of IgG1 target, Fig. 2 A (---).If in new probe solution, add IgE, can be observed identical colour-change but melting analysis provides Tm is 36 ℃ curve, i.e. the expection of this target changes, Fig. 2 A (-).It should be noted that solution becomes mulberry, and melting analysis demonstrates two different conversions (transactions) when adding two kinds of protein targets in probe solution.First derivative (derivative) of this curve demonstrates two peaks under 36 and 55 ℃ respectively, center, Fig. 2 B.This has confirmed to use the two kinds of different assembling forms that produce from the oligonucleotide barcode and the characteristic of unwinding thereof to distinguish two kinds of protein targets.
Embodiment 4: Use the assay method of nano-particle complex probe
Can use a kind of should strategy with the sensitivity that improves said system with increase the target number (Fig. 3) that can in a kind of solution, detect.Use this strategy, can pass through this protein target of DNA barcode indirect detection.The oligonucleotide of 12-aggressiveness has 4 12Plant different sequences, the wherein multiple barcode that can be used for preparing the interested multivalent protein shown in Figure 1A.In this assay method, the characteristic energy measurement not in solution that unwinds of the aggregation of its formation, and should the DNA barcode in the aggregation be separated with target molecule with unreacted probe by centrifugal (3000rpm 2 minutes).Make this aggregation sex change by in solution, adding water then, to discharge compound DNA.Available centrifugal filter device (Millipore Microcon YM-100,3500rpm 25 minutes) separates with protein particle with the DNA barcode.In case separated the DNA barcode, can on oligonucleotide arrays, catch they also can use one of multiple DNA detection test identify they (Fig. 3).For the example that relates to IgG1 and IgE as herein described, on the slide glass of using with half complementary oligonucleotide functionalization (diameter is the point of 250 μ m) of interested barcode, catch this barcode (A3 and B3 among Fig. 1).If barcode is caught by oligonucleotide arrays, the particle of modifying with barcode remainder complementary DNA-can with this hybridization array (seeing experimental section).When passing through the standard scan measuring method [11]When develop (it relate to soup handle), can use quantitatively this result of flat-bed scanner, Fig. 4 11If there is IgG1, then only there is point to demonstrate measurable signal at the IgG1 design.Equally, if IgE is only protein, then only there is point to demonstrate signal at its design.At last, if two kinds of protein all exist, then two kinds of points all show strong signal.
For sweep measurement DNA barcode detection,, remove supernatant with DNA/Au nano particle assemblage centrifugal in polystyrene 1.5mL bottle (3000rpm 2 minutes).In aggregation, add PBS damping fluid (700 μ l) and repeat this operation and separate with the mensuration composition with unreacted protein to guarantee aggregation.Then, in the bottle that contains this aggregation, add 500 μ l water and make its sex change.The preparation microarray is also pressed the described method of document and is used the DNA hybridizing method 11,22Nano particle (2nM) pre-mixing that nano particle that separated DNA barcode and A2-are modified or B2-modify contacts with dna microarray, and in hybridization chamber (GRACE BIO-LABS) incubation 3 hours under the room temperature.Use 0.3M NaNO then 3With lOnM NaH 2PO 4/ Na 2HPO 4Damping fluid (pH7) washs this array and is immersed in silver and dyed the middle room temperature of enhancing solution (Sigma) following 3 minutes.Wash slide glass with water, analyze with flat-bed scanner then.
Embodiment 5: Use the assay method of nano-particle complex probe
Implement bio-barcode PCR (BPCR) scheme and detect the protein target with the sensitivity of 3aM, promptly free prostate gland specificity antigen (PSA) (Fig. 6), it is than highly sensitive 6 orders of magnitude of the clinical assays method of the detection PSA of present routine (46).By (the 13nM solution of 10ml is present in and adds polyclone anti-PSA antibody (7 μ g) in the Citrate trianion (～38mM) stable suspension colloid) and prepare the nano particle detection probes to the 13nm of the pH9.0 Au nano particle aqueous solution.After 20 minutes, the nano particle of anti--PSA modification and the barcode DNA that alkyl sulfhydryl adds cap (alkylthiol-capped) are caught chain (0.2OD; 5 ' CAA CTT CAT CCA CGT TCA ACG CTA GTG AAC ACA GTT GTGT-A 10-SH 3 ' SEQ ID NO:9) reaction is 12 hours, and then at 0.1M NaCl/0.01M phosphate buffered saline buffer, salt is stable among the pH7.Then, this solution with the 10%BSA solution-treated of 1ml 20 minutes with passivation and stable gold nano grain.Final solution 4 ℃ centrifugal 1 hour down (20,000g), remove supernatant.Repeating this centrifugally operated is further purified.Then with the specific barcode DNA of PSA-chain (1OD; 5 ' ACA CAA CTG TGT TCA CTA GCG TTG AAC GTG GAT GAA GTT G, 3 ' SEQ ID NO:10) to be connected to nano particle on barcode DNA catch chain hybridization and use similar centrifugal method purifying.
1 μ m diameter magnetic-particle of amino-functionalization is from Polysciences, and Inc obtains.The coupled chemistry of commodity in use glutaraldehyde-amine is connected to them on the protein then.Reach 90% by comparison protein and the coupled front and back of particle in the coupled efficient of the absorbance measurement under the 270nm with the UV-vis spectroscopy.With this particle, the magnetic capturing probe is suspended in the 0.1M PBS damping fluid (pH7.4) of 40ml before use.
In typical PSA test experience (Fig. 6 B), will mix with the aqueous solution (0.1M PBS) of the water dispersion (the magnetic probe solution of 50 μ l 3mg/ml) of the magnetic capturing probe of monoclonal anti-PSA antibody functionization and free PS A (PSA of 10 μ l) and be incorporated in 37 ℃ of 30 minutes (step 1) of Fig. 6 B of stirring down.Can easily PSA bonded magnetic detection probe be separated with unconjugated PSA by applying magnetic field.In order to finish magnetic resolution, will contain the 1.5ml pipe of measuring solution and at room temperature be placed on BioMag The Eppendorf tube separator (Polysciences, Inc.) in.After 15 seconds, magnetic capturing probe-PSA crossbred concentrates on the tube wall.Remove the supernatant solution of PSA molecule (not in conjunction with), the magnetic capturing probe that exists with precipitation forms on tube wall is resuspended among the 0.1M PBS of 50 μ l (repeating 2 times).Add nano particle detection probes (50 μ l 1nM) then with polyclone anti-PSA antibody and crossbred barcode DNA chain functionalization.With detection probes be fixed on the PSA reaction on the capturing probe and be provided for that signal amplifies and the DNA chain (Fig. 6 B step 2) of identification of proteins.Solution was 37 ℃ of following vigorous stirring 30 minutes.Use magnetic separator to wash magnetic-particle to separate magnetic-particle then with 0.3M PBS.This step repeats 4 times, needs 1 minute at every turn, only stays magnetic capturing probe (with PSA bonded nano particle detection probes).Behind the last washing step, the magnetic capturing probe is resuspended in Nanopure (18M Ohm) water (50 μ l), barcode DNA chain and the surface impurity elimination of nano particle detection probes were handed over 2 minutes.Barcode DNA and probe separates and collection (Fig. 6 B step 3) of using magnetic separator easily impurity elimination to be handed over then.
For barcode DNA cloning (step 4, Fig. 6 B), isolating barcode DNA adding is contained in the PCR reaction mixture (20 μ l, final volume) of suitable primer, according to following method this solution is carried out thermal cycling then.The primer that the aliquots containig (8.9 μ l) of free barcode DNA and 0.3 μ l is suitable (is respectively 25 μ M, primer 1:5 ' CAA CTT CAT CCA CGT TCA AC 3 ' SEQ ID NO:11, primer 2: 5 ' ACA CAA CTG TGT TCA CTA GC, 3 ' SEQ ID NO:12), 0.4 μ l DMSO (2% final concentration), with 0.1 μ l Taq archaeal dna polymerase, promptly demonstrate and EasyStart TMThe compatible polysaccharase of system (5U/ μ l, Fisher Scientific) adds EasyStart with the final volume of 20 μ l together TM(Molecular Bio-Products, San Diego is in top wax layer CA) for Micro 20 PCR Mix-in-a-Tube.The final concentration of PCR reaction mixture is as follows: primer 1 and 2,0.37 μ M; The dNTP mixture, 0.2mM; The PCR damping fluid, 1X; And MgCl 2, 2mM.(GeneAmp 9700 then the PCR pipe to be loaded into thermal cycler, Applied Biosystems) carry out in and under 94 ℃ 7 minutes " hot start ", with 94 ℃ 30 seconds, 58 ℃ of 30 seconds and 72 ℃ circulation in 1 minute 25 times, 72 ℃ of last extensions 7 minutes, then carry out 4 ℃ of immersions.
At first carry out the primer dimer formation after pcr amplification is assessed in control experiment.Add methyl-sulphoxide (DMSO) with the melting temperature(Tm) that reduces pseudo-hybridized primer and minimize that primer dimer forms and the possibility of amplification (Fig. 7, in swimming lane 1-5 and swimming lane 6-10 with 0.5% increment from 0 to 2%).In addition, the number of times of thermal cycling is set at 25 times.As shown in Figure 7, the clear band (swimming lane 1-5) of barcode DNA cloning is arranged, and when only having primer to carry out thermal cycling, do not having observable band (swimming lane 6-10) in the presence of the Taq polysaccharase.Therefore, in all BPCR reactions, add 2%DMSO, keep amplification because under this concentration, there not being observable trace band (Fig. 7, swimming lane 10) barcode DNA.
Mix with ethidium bromide staining barcode DNA cloning and with sample dyestuff on the gel.Carry out gel electrophoresis whether increase to measure (Fig. 8, A and C group) then.For all electrophoresis experiments, the aliquots containig of PCR mixture (15 μ l) dyes with ethidium bromide (1mg), with (the 3 μ l of sample dyestuff in the gel electrophoresis, 6X, Promega, Madison, WI) mix, in flowing damping fluid, 1X TAE carries out gel electrophoresis (2% sepharose, 110V, 35 minutes).Bio-barcode standard (1 μ l, 6 μ M bio-barcode duplexs) is added in the gel with comparing.By bio-barcode DNA being added preparation bio-barcode standard (40-aggressiveness) in its complementary strand among the 0.3M PBS.Use Kodak DC-120 digital camera and Kodak ID 2.0.2 imaging software to carry out all gels photograph and band strength mensuration.Behind the electrophoresis by gel is immersed in the ethidium bromide 35 minutes (0.5 μ g/ml is in 1X TAE flows damping fluid) also with the ethidium bromide staining gel band and obtained to electrophoresis before ethidium bromide added qualitative results similar in the PCR reactant.
The relative intensity of ethidium bromide staining band allows to be used to assess the relative concentration (Fig. 8, B and D group) of PSA.The staining power of pcr amplification has been represented PSA concentration, is respectively 300aM in swimming lane 3-8,3fM, 30fM, 300fM, 3pM, and 30pM.The non-specific binding of nanoparticle probes and magnetic probe can be ignored, because BPCR does not produce very little signal (Fig. 8, A group, swimming lane 1 and 2, C group, swimming lane 1) when having PSA.For the B group, the strength ratio of contrast band exists the band of PSA to hang down at least 8 times.In the figure that the PSA of expression lower concentration (swimming lane 2-7 is respectively 3aM to 300fM for Fig. 8, B group) detects, higher 2.5 times than the relative intensity of negative control (swimming lane 1) corresponding to the gel band (swimming lane 2) of 3aM.
Embodiment 6: Use the assay method of nano-particle complex probe
Although gel electrophoresis is generally used for the assay determination result, in general, the sweep measuring method provides higher sensitivity and has compared based on the easier enforcement of the method for gel.Therefore, this paper has reported the result of sweep measuring method.Half complementation of chip fixed DNA 20-aggressiveness and target bar code sequence (40-aggressiveness), it is used to catch the barcode dna sequence dna of amplification, and gold nano grain is used for second half of this sequence of sandwich assay formal notation.The amplification duplex barcode DNA at first sex change to be implemented in barcode DNA, chip surface, and the hybridization between the gold nano grain probe.Therefore, barcode DNA cloning is taken out from original PCR pipe and add in (5 μ l) gold nano grain probe solution (5 μ l, 10nM is present among the 0.3M PBS).This solution is diluted to final volume 100 μ l with 0.3MPBS (90 μ l) in the 0.2ml of cleaning PCR pipe.For barcode dna single chain (40-aggressiveness) and nano particle bonded complement (20-aggressiveness) are hybridized, PCR pipe is added in the thermal cycler 95 ℃ of heating 3 minutes with sex change barcode dna double spiral, and the hybridization temperature 2 minutes that is cooled to 45 ℃ then is so that nanoparticle probes combines with its complementary barcode dna sequence dna.This mixture taken out from the PCR pipe and add and be fixed with the microarray of catching chain (20-aggressiveness) (GMC417 Arrayer is GeneticMicroSystems) on the chip.The detection solution that will be used for each experiment is limited in the array with 100 μ l hybridization hole, and (OR) hybridized 45 minutes in the humidity watch-keeping cubicle active zone for Grace BioLabs, Bend.After the hybridization, chip 0.1M NaNO 3/ 0.01 M phosphate buffered saline buffer, pH7.0 45 ℃ of following rinsings to remove excessive gold grain (repeating 2 times).(Redding CA), with Nanopure (18MOhm) water rinse, and uses the desk centrifuge drying for 6 minute reaction times, Ted Pella Inc. with silver enhancement solution the chip with hybridization nanometer particle probe to be carried out the silver amplification then.Silver after the gold grain combination amplifies the available Verigene ID of generation system (Nanosphere, the gray corrosion that Inc.) reads, the spot dispersive light of this systematic survey from forming 11,51Can easily detect target PSA concentration (Fig. 9) from 300fM to 3aM by this method.The sample of 3aM is corresponding to all in the sample 18 protein molecules being arranged.Contain incomplementarity catch DNA (5 ' SH-C6-A10-GGCAGCTCGTGGTGA-3 ', SEQ ID NO:13) control point does not have signal (Fig. 9, spotting template) and seldom have when not having PSA the observations (Fig. 9, contrast) of discernible signal to confirm that the selectivity of barcode dna sequence dna is splendid.
Embodiment 7: Use BPCR to detect proteinic theoretical limit
To use BPCR to detect the lower limit of proteinic theory in order checking, the barcode DNA concentration of the serial dilution that is used for pcr amplification to be carried out PCR/ gel electrophoresis (Figure 10).Exist the signal of the barcode DNA cloning period of the day from 11 p.m. to 1 a.m from the contrast band, to identify (swimming lane 10) fully, even in the PCR reactant, only add the barcode DNA (swimming lane 9) of 30 copies.Suppose that each nanoparticle probes has about 50 barcode DNA chains, then BPCR can produce detection signal with single bonded nanoparticle probes in theory.
Embodiment 8: PSA in the detection of complex medium
In order to confirm the suitability of BPCR amplification method in the sample solution that more is equivalent to clinical settings,, the PSA target carries out embodiment 5 and 6 described assay methods, detector bar font code after the BPCR amplification step in the lowlenthal serum by being dissolved in.With PSA serial dilution in 0.1M PBS, add then undiluted lowlenthal serum (ICN Biomedicals, Inc., Aurora, Ohio) in.Lowlenthal serum can be simulated 1X physiological saline effectively, (for example, any biology system aspect ionic strength and pH).
But this data acknowledgement in complex dielectrics the low target analyte (being PSA here) of method detectable level of the present invention to 30aM.The signal that produces under this concentration can clearly be discerned (Figure 11) with control experiment.Can reduce background signal by the optional step of introducing with the membrane filtration barcode DNA sample that can remove great majority pollution composition.Filtration step that should be optional can be with any known method lightning strip font code DNA and impurity, for example, and by big or small (molecular weight), shape, electric charge, or hydrophobicity/hydrophilic separation method.
Embodiment 9: The direct detection of barcode DNA and measurement (amplification of nothing-BPCR)
Basically press above embodiment 5 described preparation gold nano grain (NP) probes.Briefly, the polyclonal antibody (Abs) (3.5 μ g) with anti-PSA adds in the 30 nm gold NPs aqueous solution (1ml 40pM NP solution) of pH 9.0.After 20 minutes, (gold grain of 30nm is 1OD to the NPs that Ab is modified with barcode DNA seizure chain that alkyl sulfhydryl adds cap; 5 '-CAA CTT CAT CCA CGT TCAACGCTA GTG AAC ACA GTT GTGT-A 10-(CH 2) 3-SH 3 ') reaction is 16 hours, carries out salt with 0.1MNaCl then and stablizes.This solution with 0.3ml 10%BSA solution-treated 30 minutes with passivation with stablize golden NPs.Final solution 4 ℃ centrifugal 1 hour down (20,000g), remove supernatant.Repeating this centrifugation step is further purified.With final NP probe redispersion in the 0.1M of pH7.4 NaCl/0.01M phosphate buffered saline buffer.Then with PSA-specificity barcode DNA chain (1OD; 5 '-ACA CAACTG TGT TCA CTA GCG TTG AAC GTG GAT GAA GTT G-3 ') catch chain hybridization and use similar centrifugally operated purifying to bio-barcode DNA on being connected to NPs.The oligonucleotide charging ratio is measured by the method for Demers etc., referring to: L.M.Demers etc., Anal.Chem.72,5535 (2000).The MMPs of 1 μ m diameter of amino functional is from Polysciences, and Inc. obtains.The coupled chemistry of commodity in use glutaraldehyde-amine is connected to MMPs on the mAbs of anti-PSA.Use the UV-vis spectroscopy to reach 90% in the coupled efficient of the absorbance measurement under the 270nm by comparison protein and the coupled front and back of MMPs.Before using MMPs is suspended in the 40ml 0.1M PBS damping fluid (pH7.4).
Generally directly measure the amount of barcode DNA in the solution based on the assay method of chip, but do not use amplification step by BPCR by embodiment 6 described uses.Owing to do not use PCR, this method has been got rid of the needs that the duplex DNA that forms during the pcr amplification carried out sex change.Therefore, in protein detection with after barcode DNA separates, with the aliquots containig (10 μ l) of isolating barcode sample and golden detection probes (sequence is the same for 5 μ l, the 500pM final concentration) mixing of 0.6M PBS (85 μ l) and 13nm.This mixture is added by in the Verigene hole on the above-mentioned chip hybridization chamber that is arranged with suitable seizure chain.With this sample 42 ℃ of following incubations 2 hours.Behind the incubation, remove reaction mixture, chip 0.5M NaNO 3The washing of/0.01M phosphate buffered saline buffer is to remove excessive gold nano grain.The gold grain that is fixed in the surface is used the Nanopure water washing with silver enhancement solution (Ted Pella) dyeing 6 minutes, and with Verigene ID system imaging, all by mentioned above.
Although used 30nm gold NPs in this case, expect that 13nm gold NPs also can be used for this method with direct detector bar font code DNA.Yet, by increasing the size of NP probe in the protein detection step, can significantly increase the DNA chain number on each NP, help direct detection (in theory, suppose that 100 DNA chains are attached on the 13nm gold NP, then have nearly 532 DNA chains on each 30nm gold NP).The size that can regulate specific nanoparticle probes is to optimize some aspect.
This result (Figure 12) has confirmed to use the directly low PSA target to 30attomolar of detectable level of 30nm gold NP (20pM) under the condition of the amplification of no BPCR.Although this expression with respect to its loss of sensitivity of BPCR amplification method an order of magnitude, this method is still very sensitive and significantly reduced cost, work, and time by cancellation PCR step.
Embodiment 10: Directly detect the target nucleic acid sequence of ZeptoMolar concentration
For this experiment, select the dna sequence dna relevant with anthrax lethal (5 '-GGA TTA TTGTTAAATATT GATAAGGAT-3 '; SEQ ID NO:14) as initial target, because this sequence is used significant for biology-terrorism and biology-war and sufficient research is arranged in the literature 63,67-692 contrasts of adding dna sequence dnas in each 20 μ L test sample [1 μ L 10pM (5 '-CTATTA TAA TAA AAT ATT TAT ATA GCA-3 '; SEQ ID NO:15) and the contrast 2 of 1 μ L 10pM (5 '-GAATTATAG TTAACTATA GCTAAGGAT-3 '; SEQ ID NO:16)].Before the use, barcode DNA is loaded into (the 20nm probe of 400pM on the nanoparticle probes by hybridization; The 30nm probe of 200pM).Barcode DNA imports so that finish hybridization in suitable hybridization buffer with the concentration of 10 μ M.Centrifugal subsequently this particle is also used the washing of PBS damping fluid.Then this probe is suspended in the suitable storage buffer, stores standby.
For MMPs, the DNA that the granules of polystyrene of amine functional polysiloxaneization and alkyl sulfhydryl add cap is by with them and sulfosuccinimide base 4-[p-dimaleoyl imino phenyl] reaction of butyric ester (sulfo-SMPB) bifunctional linker is connected, and primary amine on this joint and the MMPs and the sulfydryl that forms on the oligonucleotide of specific recognition binding site of target sequence react.MMPs is before use by adding 10%BSA bovine serum albumin passivation in the solution that contains them.Centrifugal then this probe, with the washing of PBS damping fluid, and with the 2mg/mL resuspension to produce active probe (Figure 13 A).
Carry out this mensuration by in the solution of the target DNA that contains single stranded form, adding 50 μ LMMP probes (2mg/mL).This system at room temperature left standstill 10 minutes.After leaving standstill in 10 minutes, in solution, add 50 μ l NP probes [50 μ L 400pM (20nm NP probe solution) or 50 μ l 200pM (30nm NP probe solution)] and make its hybridization 50 minutes.After the hybridization, to reaction tubes apply magnetic field (BioMag multi-6 Eppendorf tube separator, Polysciences, Incorporated, Warrington, PA), it is the target dna strand of MMPs and NPs clamping, and unreacted MMPs moves on the reaction tube wall.Wash any remaining unreacted solution composition of flush away several times with the PBS damping fluid, particularly not with the NPs of MMPs specific hybrid.Remove magnetic field then and in reaction tubes, add 50 μ l NANOpure water (Bamstead International, Dubuque, IA), with this system be heated to 55 ℃ 3 minutes with released strip font code DNA.Introduce magnetic field again and from solution, remove all MMPs, stay barcode DNA and be used for detecting.
In order to analyze amount and the identity of the barcode DNA in the end reaction solution, use the sweep measurement method 67The sweep measurement method is based on the DNA detection method of chip, it depend on oligonucleotides-modified golden NP probe (5 '-TCT CAA CTC GTA GCT-A 10-SH-3 '-Au; SEQ ID NO:17) and the silver reduction (I) that starts of NP-carry out signal and amplify.For this concrete assay method, use the dna microarray instrument with 5 ' seizure DNA chain (5 '-SH-A 10-CGT CGC ATT CAG GAT-3 '; SEQ ID NO:18) on the amine-modified glass-chip of maleimide point sample (the sampling point diameter is 175 μ m, and the distance between two sampling points is 375 μ m; GMS 417 Arrayer, Genetic MicroSystems, Wobum, Massachusetts).The no spot area A on surface 10Sequence (10 μ M 5 '-SH-AAA AAA AAA A-3 '; SEQ IDNO:19) passivation is spent the night.In case chip surface is contacted with the barcode dna solution, on the chip of barcode/seizure dna modification, adds and target sequence solution blended NP probe.Sampling point on the chip NP probe and target dna strand mark.(Ted Pella, Redding CA) contact further enhancing signal with the chip of point sample and silver enhancement solution then.(Northbrook IL) reads the development spot for Nanosphere, Incorporated to use Verigene ID (identification) system then.The dispersed light of Verigene ID systematic survey development spot and the permanent recording that is provided for analyzing.Figure 14 A has shown the 20-nm NP probe that uses in the barcode DNA detection of " amplification ".The spot intensity of the target DNA concentration from 5fM to 50aM is obviously compared according to spot stronger.In order to measure the spot intensity of each concentration, three spots are distributed on the chip also with Verigene ID system imaging.The grey level and the spot intensity photo that calculate spot with image software (AdobePhotoshop) show in Figure 15.This photo shows that 20-nm NP probe can clearly detect the target DNA of about 50aM, but can not make a distinction with background signal under 5aM concentration.
When using 30nm NP probe to carry out the DNA-BCA detection, can detect low target DNA concentration (Figure 14 B) to 500zM.20 with 30nm NP probe this difference on the limit of detection may be since on the NP probe of different table area the absolute number of barcode DNA chain have any different.The intensity photo show 30-nm NP probe system under all samples concentration than 20-nm NP probe system speckle signal stronger (Figure 15).The 20 μ L sample volumes of 500zM have been represented the gross sample number of about 10 target dna strands, and its sensitivity is comparable to the assay method that adopts with the coupled PCR-based method of molecular fluorescence probe 58-60
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Claims (130)

1. one kind is used for the method whether test sample exists one or more target analytes, and this target analyte has at least two binding sites, and the method comprising the steps of:
A kind of matrix is provided;
The particle probe of one or more types is provided, every type probe comprises one or more specificitys with specific target analyte in conjunction with the particle of complement and one or more and this particle bonded DNA barcode, wherein the specificity of every type particle probe has specificity in conjunction with complement to concrete target analyte, and the DNA barcode of every type particle probe is as the mark of this concrete target analyte;
This target analyte is fixed on this matrix;
Allow target analyte combine with the specificity of this analyte between the complement in conjunction with and under the condition for validity of formation mixture under the condition that target analyte exists, this fixed target analyte is contacted with the particle probe of one or more types;
Wash this matrix to remove unconjugated particle probe; With
The optional DNA barcode amplification that makes; With
Detect whether there is this DNA barcode, wherein whether having this mark is the index that whether has concrete target analyte in the sample.
2. the process of claim 1 wherein that this target analyte is that protein or haptens and its specificity are the antibody that comprises mono-clonal or polyclonal antibody in conjunction with complement.
3. the process of claim 1 wherein that the DNA barcode increases by PCR.
4. the process of claim 1 wherein that this particle carries out mark with at least two DNA barcodes.
5. the process of claim 1 wherein the capturing probe of having arranged one or more types of target analyte on this matrix.
6. whether have the method for one or more target analytes in the test sample, every kind of target analyte has at least two binding sites, and this method comprises:
The capturing probe that is incorporated into one or more types on the matrix is provided, and every type capturing probe comprises the specificity of first binding site of concrete target analyte in conjunction with complement;
The detection probes of one or more types is provided, every type detection probes comprises the nano particle that combines oligonucleotide, one or more specificitys of second binding site of this concrete target analyte are in conjunction with complement, with as one or more DNA barcodes of concrete target analyte mark, wherein at least a portion sequence of DNA barcode and at least some oligonucleotide hybridizations that are attached on the nano particle;
When allowing that the specificity binding interactions takes place between target analyte and the probe and having target analyte, form under the condition for validity of assembling mixture, make sample, capturing probe, detection probes contact;
Washing matrix is to remove unconjugated detection probes;
Detect in any gathering mixture on the matrix whether have this DNA barcode, the detected result that wherein whether has this DNA barcode is the index that whether has target analyte in the sample.
7. the method for claim 6, wherein this detection probes comprises one or more specificitys of second binding site of (i) concrete target analyte in conjunction with complement, (ii) with the oligonucleotide of at least a type of nano particle bonded, with the DNA barcode that has with at least a portion complementary predetermined sequence of the oligonucleotide of at least a type, with the mark of every type of detection probes bonded DNA barcode as concrete target analyte.
8. the method for claim 6 wherein before described detection step, also comprises step:
Under the condition for validity that makes mixture impurity elimination friendship and released dna barcode, handle this gathering mixture; With
Before described detection, make the DNA barcode amplification.
9. the method for claim 8, wherein this DNA barcode passes through pcr amplification.
10. the method for claim 6, wherein this capturing probe is attached on the magnetic substrate.
11. the method for claim 10, wherein this matrix is magnetic-particle.
12. the method for claim 6, the specificity that wherein is attached on the nano particle is mono-clonal or polyclonal antibody in conjunction with complement.
13. the method for claim 12, wherein this antibody is anti-PSA antibody.
14. the method for claim 11 wherein, before described washing step, also comprises step:
Before washing by applying magnetic field and separate this gathering mixture to being attached to gathering mixture on the magnetic-particle.
15., also comprise step according to the method for claim 14:
Hand over and discharge under the condition for validity of this DNA barcode in this gathering mixture impurity elimination, handle isolating gathering mixture.
16. according to the method for claim 15, the DNA barcode of the described release of wherein increasing.
17., wherein pass through the DNA barcode of the described release of pcr amplification according to the method for claim 16.
18. according to the method for claim 6, wherein this target analyte is to have two-part at least nucleic acid.
19. method according to claim 6, wherein target analyte is to have the target nucleic acid of two portions sequence at least, this detection probes comprises the nano particle that combines oligonucleotide, the at least a portion that is attached to this oligonucleotide on the nano particle has and this DNA barcode complementary sequence, the specificity of this detection probes comprises first target identification oligonucleotide that has with first part's complementary sequence of target nucleic acid in conjunction with complement, and the specificity of capturing probe comprises second target identification oligonucleotide that has with the complementary of the second section at least sequence of target nucleic acid in conjunction with complement.
20. method according to claim 6, wherein this target analyte is the target nucleic acid with sequence of at least two parts, this detection probes comprises the nano particle that combines oligonucleotide, this DNA barcode has and at least a portion complementary sequence that is attached to the oligonucleotide on this detection probes, this specificity comprises the target identification oligonucleotide of the sequence with at least the first and second parts in conjunction with complement, the first part of this first part and target nucleic acid complementary and this second section and at least a portion complementation that is attached to the oligonucleotide on the nano particle, the specificity of this matrix comprises target identification oligonucleotide in conjunction with complement, at least a portion of this target identification oligonucleotide and the second section complementation of target nucleic acid.
21. the method for claim 6, wherein this detection probes comprises SD.
22. whether have the method for one or more target analytes in the test sample, every kind of target analyte has at least two binding sites, this method comprises:
The capturing probe of one or more types is provided, and every type capturing probe comprises (i) magnetic-particle; (ii) be attached to first specificity on the magnetic-particle in conjunction with first right member, wherein first specificity is in conjunction with first binding site of the first right member in conjunction with concrete target analyte;
The detection probes of one or more types of every kind of target analyte is provided, and every type detection probes comprises (i) nano particle; (ii) be attached to second specificity on the nano particle in conjunction with first right member, wherein this second specificity combines with second binding site of target analyte in conjunction with first right member; (iii) with the oligonucleotide of at least a type of nano particle bonded; The (iv) DNA barcode of at least a type, every type DNA barcode have with at least a portion complementary predetermined sequence of the oligonucleotide of particular type and as the mark of concrete target analyte;
Assemble under the condition for validity of mixture allowing to carry out the specificity binding interactions between target analyte and the probe and exist to form with the magnetic-particle bonded under the condition of target analyte, this sample is contacted with detection probes with capturing probe;
Unconjugated detection probes on the flush away magnetic-particle; With
Detect in the mixture whether have the DNA barcode, wherein the detected result of this DNA barcode is the index that target analyte exists.
23. the method for claim 22 also comprises step before described detection step:
By applying this gathering mixture of magnetic field separation;
Discharge under the condition for validity of this DNA barcode in impurity elimination friendship and gathering mixture, handle this gathering mixture;
The DNA barcode that separates this release.
24. the method for claim 23 also comprises the DNA barcode amplification that makes release.
25. the method for claim 22 or 23 also comprises:
The matrix that combines oligonucleotide is provided, and this oligonucleotide has at least a portion sequence complementary sequence with the DNA barcode;
Provide to comprise oligonucleotide and its bonded nano particle, at least a portion that wherein is attached to the oligonucleotide on the nano particle has at least a portion complementary sequence with the DNA barcode; With
Under the condition for validity of between the first part at least that allows the DNA barcode and the complementary oligonucleotide that is attached on the matrix and between the second section of DNA barcode and some oligonucleotide that are attached on the nano particle, hybridizing, make the DNA barcode, be attached to oligonucleotide on the matrix, reach nano particle and contact.
26. the method for claim 25 wherein makes the DNA barcode amplification by PCR before detection.
27. the method for claim 22 also is included in to analyze and assembles preceding this gathering mixture that separates of mixture.
28. the method for claim 27 is wherein separated this gathering mixture by the gathering mixture is applied magnetic field.
29. the method for claim 22, wherein this nano particle is metal nanoparticle or semiconductor nanoparticle.
30. the method for claim 29, wherein this nano particle is a gold nano grain.
31. the method for claim 22, wherein this specificity combination is to being antibody and antigen.
32. the method for claim 22, wherein this specificity combination is to being acceptor and part.
33. the method for claim 22, wherein this specificity combination is to being enzyme and substrate.
34. the method for claim 22, wherein this specificity combination is to being medicine and target molecule.
35. the method for claim 22, wherein this specificity in conjunction with to be two to small part complementary oligonucleotide chain.
36. the method for claim 22, wherein this DNA barcode is biotin labeled.
37. the method for claim 22, wherein this DNA barcode is radiolabeled.
38. the method for claim 22, wherein this DNA barcode is fluorescently-labeled.
39. each method in the claim 1,6 and 22, wherein this target has the binding site above 2.
40. the method for claim 39, at least two types particle composites probe wherein is provided, first type probe has the specificity of first binding site on the target analyte in conjunction with complement, and second type probe has the specificity of second binding site on the probe in conjunction with complement.
41. the method for claim 39 wherein provides multiple particle composites probe, every type probe has the specificity of different binding sites on the target analyte in conjunction with complement.
42. each method in the claim 1,6 or 22, wherein specificity is that specificity is in conjunction with right member in conjunction with complement and target analyte.
43. the method for claim 42, wherein specificity comprises nucleic acid in conjunction with right member, oligonucleotide, peptide nucleic acid(PNA), polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, VITAMIN, medicine, virus, polysaccharide, lipid, lipopolysaccharides, glycoprotein, lipoprotein, nucleoprotein, oligonucleotide, antibody, immunoglobulin (Ig), albumin, oxyphorase, thrombin, peptide and proteohormone, non-peptide hormone, interleukin-, Interferon, rabbit, cytokine comprises the peptide of tumour-specific epi-position, cell, cell surface molecule, microorganism, the fragment of microorganism, part, component, or product, organic molecule, nucleic acid and oligonucleotide, the meta-bolites of any above-mentioned substance or antibody.
44. the method for claim 43, its amplifying nucleic acid and oligonucleotide comprise gene, viral RNA and DNA, DNA of bacteria, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and dna segment, oligonucleotide, synthetic oligonucleotide, the oligonucleotide of modifying, strand and double-strandednucleic acid, and natural and nucleic acid.
45. according to each method in the claim 1,6 or 22, wherein target analyte is that nucleic acid and specificity are oligonucleotide in conjunction with complement.
46. according to each method in the claim 1,6 or 22, wherein target analyte is that protein or haptens and specificity are the antibody that comprises mono-clonal or polyclonal antibody in conjunction with complement.
47. according to each method in the claim 1,6 or 22, wherein target analyte is that sequence and specificity from genome DNA sample are oligonucleotide in conjunction with complement, this oligonucleotide has and this genomic at least a portion sequence complementary sequence.
48. the method for claim 47, wherein this genomic dna is an eucaryon, bacterium, the DNA of fungi or virus.
49. according to each method in the claim 1,6 or 22, wherein specificity is the right member of antibody-part in conjunction with complement and target analyte.
50. according to each method in the claim 1,6 or 22, wherein except its first binding site, target analyte is modified into and comprises second binding site.
51. the method for claim 22 also comprises filtration step, wherein filters before analyzing the gathering mixture and carries out.
52. the method for claim 51, wherein filtration step comprises film, and this film removes the sample composition that does not comprise the DNA barcode.
53. whether have the method for one or more target analytes in the test sample, comprising:
At least a or polytype particle composites probe is provided, every type probe comprises and its bonded oligonucleotide, one or more specificitys of concrete target analyte are in conjunction with complement, be used as one or more DNA barcodes of concrete target analyte mark, wherein at least a portion sequence of this DNA barcode and at least some oligonucleotide hybridizations that are attached on the nano particle;
Under the condition for validity that allows formation gathering mixture under the condition that the specificity binding interactions takes place between target analyte and the particle composites probe and have target analyte, this sample is contacted with the particle composites probe; With
Observe and mixture formation whether occurs assembling.
54. according to the method for claim 53, wherein the DNA barcode in every type the particle composites probe has difference and as the sequence of concrete target analyte identifier.
55., also comprise following steps according to the method for claim 53:
Separate and assemble mixture; With
Analyze this gathering mixture and have existing of not homotactic one or more DNA barcodes with mensuration.
56. the method according to claim 53 also comprises the steps:
Separate this gathering mixture;
Assemble under the condition for validity of mixture impurity elimination friendship and released dna barcode at this, handle this gathering mixture;
Separate this DNA barcode; With
Detection has the existence of not homotactic one or more DNA barcodes, and wherein each DNA barcode is the index that has concrete target analyte in the sample.
57. the method according to claim 53 also comprises the steps:
Separate and assemble mixture;
Assemble under the condition for validity of mixture impurity elimination friendship and released dna barcode at this, handle this gathering mixture;
Separate this DNA barcode;
Make the separated DNA barcode amplification; With
Detection has the existence of the DNA barcode of not homotactic one or more amplifications, and wherein each DNA barcode is the index that has concrete target analyte in the sample.
58. the method for claim 53, wherein this target has at least two binding sites.
59. the method for claim 58, at least two types particle composites probe wherein is provided, first type probe has the specificity of first binding site on the target analyte in conjunction with complement, and second type probe has the specificity of second binding site on the probe in conjunction with complement.
60. the method for claim 58 wherein provides multiple particle composites probe, every type probe has the specificity of different binding sites on the target analyte in conjunction with complement.
61. the method for claim 53, wherein specificity is that specificity is in conjunction with right member in conjunction with complement and target analyte.
62. the method for claim 61, wherein specificity comprises nucleic acid in conjunction with right member, oligonucleotide, peptide nucleic acid(PNA), polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, VITAMIN, medicine, virus, polysaccharide, lipid, lipopolysaccharides, glycoprotein, lipoprotein, nucleoprotein, oligonucleotide, antibody, immunoglobulin (Ig), albumin, oxyphorase, thrombin, peptide and proteohormone, non-peptide hormone, interleukin-, Interferon, rabbit, cytokine comprises the peptide of tumour-specific epi-position, cell, cell surface molecule, microorganism, the fragment of microorganism, part, component, or product, organic molecule, nucleic acid and oligonucleotide, the meta-bolites of any above-mentioned substance or antibody.
63. the method for claim 62, its amplifying nucleic acid and oligonucleotide comprise gene, viral RNA and DNA, DNA of bacteria, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and dna segment, oligonucleotide, synthetic oligonucleotide, the oligonucleotide of modifying, strand and double-strandednucleic acid, and natural and nucleic acid.
64. the method for claim 53, wherein target analyte is that nucleic acid and specificity are oligonucleotide in conjunction with complement.
65. the method for claim 53, wherein target analyte is that protein or haptens and specificity are the antibody that comprises mono-clonal or polyclonal antibody in conjunction with complement.
66. the method for claim 53, wherein target analyte is that sequence and specificity from genome DNA sample are oligonucleotide in conjunction with complement, and this oligonucleotide has and this genomic at least a portion sequence complementary sequence.
67. the method for claim 65, wherein this genomic dna is an eucaryon, bacterium, the DNA of fungi or virus.
68. the method for claim 53, wherein specificity is the right member of antibody-part in conjunction with complement and target analyte.
69. the method for claim 53, wherein except its first binding site, target analyte is modified into and comprises second binding site.
70. the method for claim 51, wherein this particle comprises nano particle.
71. the method for claim 70, wherein this particle comprises metal, semi-conductor, isolator, or magnetic nanoparticle.
72. the method for claim 71, wherein this particle comprises gold nano grain.
73. according to claim 58 or 59 each methods, the existence that wherein detects one or more DNA barcodes comprises:
The matrix that combines oligonucleotide is provided, and this oligonucleotide has at least a portion sequence complementary sequence with the DNA barcode;
Provide to comprise oligonucleotide and its bonded nano particle, at least a portion that wherein is attached to the oligonucleotide on the nano particle has at least a portion complementary sequence with the DNA barcode; With
Under the condition for validity of between the first part at least that allows the DNA barcode and the complementary oligonucleotide that is attached on the matrix and between the second section of DNA barcode and some oligonucleotide that are attached on the nano particle, hybridizing, make the DNA barcode, be attached to oligonucleotide on the matrix, reach nano particle and contact; With
Observe detectable variation.
74. according to the method for claim 73, its mesostroma comprises with array way and adheres to the polytype oligonucleotide on it so that allow to detect one or more dissimilar DNA barcodes.
75. according to the method for claim 73, wherein detectable variation is to form dark areas on matrix.
76. according to the method for claim 73, wherein detectable variation is observed with optical scanner.
77. according to the method for claim 73, its mesostroma contacts to produce detectable variation with silver-colored stain.
78. method according to claim 73, wherein under the condition for validity that allows DNA barcode and the complementary oligonucleotide that is attached on the matrix to hybridize, this DNA barcode contacts with matrix, and under the condition for validity of the part hybridization that allows to be attached at least some oligonucleotide on the nano particle and the DNA bar code sequence on the matrix, the DNA barcode that is attached on the matrix is contacted with the nano particle that combines oligonucleotide subsequently.
79. according to the method for claim 73, wherein allowing the DNA barcode and be attached under the condition for validity of at least some oligonucleotide hybridizations on the nano particle, this DNA barcode contacts with the nano particle that combines oligonucleotide; And under the condition for validity that at least a portion sequence that allows to be attached to the DNA barcode on the nano particle and the complementary oligonucleotide that is attached on the matrix are hybridized, the DNA barcode that is attached on the nano particle is contacted with matrix subsequently.
80. according to the method for claim 73, DNA amplification barcode before contact procedure wherein.
81. according to the method for claim 53, wherein this target analyte has at least two binding sites.
82. 1 method according to Claim 8, at least two types particle composites probe wherein is provided, first type probe has the specificity of target analyte first binding site in conjunction with complement, and second type probe has the specificity of target analyte second binding site in conjunction with complement.
83. method according to claim 53, wherein the particle composites probe comprises the particle that combines oligonucleotide, one or more DNA barcodes, and combine the oligonucleotide of the specificity of concrete target analyte in conjunction with complement, wherein (i) DNA barcode has the sequence of at least two parts; (ii) be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode; (iii) oligonucleotide, it combines to have with the specificity of the second section complementary sequence of DNA barcode and combines complement; The (iv) DNA barcode in every type the particle composites probe, they have different sequences and as the identifier of concrete target analyte.
84. method according to claim 53, wherein the particle composites probe comprises oligonucleotide and its bonded particle with at least two types, one or more DNA barcodes, and combine the oligonucleotide of the specificity of target analyte in conjunction with complement, wherein the oligonucleotide with the probe bonded first kind has and DNA barcode at least a portion complementary sequence, has with the oligonucleotide of second type of probe bonded and has at least a portion sequence complementary sequence that specificity combines the oligonucleotide of complement.
85. method according to claim 53, wherein the particle composites probe comprises the particle that combines oligonucleotide, one or more DNA barcodes, with the specificity of target analyte in conjunction with complement, wherein with at least a portion of particle bonded oligonucleotide have with at least a portion sequence complementary sequence of DNA barcode and wherein this DNA barcode as the identifier of concrete target analyte.
86. a particle composites probe, it comprises the particle that combines oligonucleotide, the DNA barcode, and combine the oligonucleotide of the specificity of concrete target analyte in conjunction with complement, wherein (i) DNA barcode has the sequence of at least two parts; (ii) be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode; (iii) combine specificity and have second section complementary sequence with the DNA barcode in conjunction with the oligonucleotide of complement; (iv) the DNA barcode in every type the particle composites probe has difference and as the sequence of concrete target analyte identifier.
87. particle composites probe, it comprises oligonucleotide and its bonded particle with at least two types, the DNA barcode, and combine the oligonucleotide of the specificity of target analyte in conjunction with complement, wherein the oligonucleotide with the probe bonded first kind has and DNA barcode at least a portion complementary sequence, has with the oligonucleotide of second type of probe bonded and has at least a portion sequence complementary sequence that specificity combines the oligonucleotide of complement.
88. particle composites probe, it comprises the particle that combines oligonucleotide, the DNA barcode, with the specificity of target analyte in conjunction with complement, wherein with at least a portion of particle bonded oligonucleotide have with at least a portion sequence complementary sequence of DNA barcode and wherein this DNA barcode as the identifier of concrete target analyte.
89. a detection probes, it comprises
(a) nano particle;
(b) combine a right member with nano particle bonded specificity;
(c) with the oligonucleotide of at least one type of nano particle bonded; With
(d) have the DNA barcode of at least one type of predetermined sequence respectively,
Wherein at least a portion of every type DNA barcode and at least a type oligonucleotide hybridization.
90. each probe among the 6-88 according to Claim 8, wherein this particle comprises nano particle.
91. each probe among the 6-89 according to Claim 8, wherein this nano particle is a metal, semi-conductor, isolator, or magnetic nanoparticle.
92. each probe among the 6-89 according to Claim 8, wherein this particle is a gold nano grain.
93. each probe among the 6-89 according to Claim 8, wherein this target has at least two binding sites.
94. the probe of claim 93, at least two types particle composites probe wherein is provided, first type probe has the specificity of first binding site on the target analyte in conjunction with complement, and second type probe has the specificity of second binding site on the probe in conjunction with complement.
95. the probe of claim 93 wherein provides multiple particle composites probe, every type probe has the specificity of different binding sites on the target analyte in conjunction with complement.
96. each probe among the 6-89 according to Claim 8, wherein specificity is that specificity is in conjunction with right member in conjunction with complement and target analyte.
97. the probe of claim 96, wherein specificity comprises nucleic acid in conjunction with right member, oligonucleotide, peptide nucleic acid(PNA), polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, VITAMIN, medicine, virus, polysaccharide, lipid, lipopolysaccharides, glycoprotein, lipoprotein, nucleoprotein, oligonucleotide, antibody, immunoglobulin (Ig), albumin, oxyphorase, thrombin, peptide and proteohormone, non-peptide hormone, interleukin-, Interferon, rabbit, cytokine comprises the peptide of tumour-specific epi-position, cell, cell surface molecule, microorganism, the fragment of microorganism, part, component, or product, organic molecule, nucleic acid and oligonucleotide, the meta-bolites of any above-mentioned substance or antibody.
98. the probe of claim 97, its amplifying nucleic acid and oligonucleotide comprise gene, viral RNA and DNA, DNA of bacteria, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and dna segment, oligonucleotide, synthetic oligonucleotide, the oligonucleotide of modifying, strand and double-strandednucleic acid, and natural and nucleic acid.
99. each probe among the 6-89 according to Claim 8, wherein target analyte is that nucleic acid and specificity are oligonucleotide in conjunction with complement.
100. each probe among the 6-89 according to Claim 8, wherein target analyte is that protein or haptens and specificity are the antibody that comprises mono-clonal or polyclonal antibody in conjunction with complement.
101. each probe among the 6-89 according to Claim 8, wherein target analyte is that sequence and specificity from genome DNA sample are oligonucleotide in conjunction with complement, and this oligonucleotide has and this genomic at least a portion sequence complementary sequence.
102. the probe of claim 100, wherein this genomic dna is an eucaryon, bacterium, the DNA of fungi or virus.
103. each probe among the 6-89 according to Claim 8, wherein specificity is the right member of antibody-part in conjunction with complement and target analyte.
104. each probe among the 6-89 according to Claim 8, wherein except its first binding site, target analyte is modified into and comprises second binding site.
105. test kit comprises among the claim 86-89 each probe.
106. whether have the test kit of one or more target analytes in the test sample, every kind of target analyte has at least two binding sites, this test kit comprises:
The detection probes that is used at least a type of every kind of target analyte, every type detection probes comprise (i) nano particle; (ii) be attached to specificity on the nano particle in conjunction with a right member; (iii) with nano particle bonded oligonucleotide; (iv) has DNA barcode with at least a portion complementary predetermined sequence of this oligonucleotide.
107. whether have the test kit of one or more target analytes in the test sample, every kind of target analyte has at least two binding sites, this test kit comprises:
The capturing probe of at least a type, it comprises (i) matrix; (ii) be attached to first specificity on the matrix in conjunction with first right member, wherein first specificity is in conjunction with first binding site of the first right member in conjunction with target analyte;
The detection probes of at least a type, it comprises (i) nano particle; (ii) be attached to second specificity on the nano particle in conjunction with first right member, wherein this second specificity combines with second binding site of target analyte in conjunction with first right member; (iii) with the oligonucleotide of at least a type of nano particle bonded; The (iv) DNA barcode of at least a type, every type of at least a portion complementary predetermined sequence that has with the oligonucleotide of particular type.
108. the test kit of claim 107, wherein this matrix is magnetic-particle.
109. whether have the test kit of one or more target analytes in the test sample, every kind of target analyte has at least two binding sites, this test kit comprises:
The capturing probe of at least a type, it comprises (i) magnetic-particle; (ii) be attached to first specificity on the magnetic-particle in conjunction with first right member, wherein first specificity is attached on first binding site of target analyte in conjunction with the first right member;
The detection probes of at least a type, it comprises (i) nano particle; (ii) be attached to second specificity on the nano particle in conjunction with first right member, wherein this second specificity combines with second binding site of target analyte in conjunction with first right member; (iii) with the oligonucleotide of at least a type of nano particle bonded; The (iv) DNA barcode of at least a type, every type of at least a portion complementary predetermined sequence that has with the oligonucleotide of particular type.
110. the test kit of the target analyte in the test sample, this test kit comprises: at least a container that the particle composites probe is housed, this probe comprises the particle that combines oligonucleotide, the DNA barcode, and combine the oligonucleotide of the specificity of target analyte in conjunction with complement, wherein this DNA barcode has the sequence of at least two parts, be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode, combine specificity and have second section complementary sequence with the DNA barcode in conjunction with the oligonucleotide of complement, and wherein this DNA barcode be attached to particle at least some oligonucleotide and combine the oligonucleotide hybridization of complement with combining specificity, choose wantonly and comprise that matrix is used to observe detectable variation.
111. the test kit of one or more target analytes in the test sample, this test kit comprises at least a or multiple container, the particle composites probe is housed in the container, this probe comprises the particle that combines oligonucleotide, the DNA barcode, and combine the oligonucleotide of the specificity of concrete target analyte in conjunction with complement, wherein (i) this DNA barcode has the sequence of at least two parts, (ii) be attached at least some oligonucleotide on the particle and have first part's complementary sequence with the DNA barcode, (iii) combine specificity and have second section complementary sequence with the DNA barcode, have different sequences with DNA barcode in (iv) every type the particle composites probe and as the identifier of concrete target analyte in conjunction with the oligonucleotide of complement; Wherein this test kit is optional comprises a kind of matrix and is used to observe detectable variation.
112. a test kit that detects target analyte, this test kit comprise at least one pair of container and the optional matrix that is used to observe detectable variation that comprises,
This contains particle probe to first container in the container, this probe comprises the particle that combines oligonucleotide and the DNA barcode with at least two partial sequences, wherein is attached at least some oligonucleotide on the particle and has first part's complementary sequence with the DNA barcode;
This comprises the oligonucleotide that has with the second section complementary sequence of DNA barcode to second container in the container, this oligonucleotide have can be used for covalently bound target analyte specificity in conjunction with half point to complement.
113. the test kit of the multiple target analyte in the test sample, this test kit comprises at least two pairs or more to container,
First container in the every pair of container contains the particle composites probe, this probe comprises particle that combines oligonucleotide and the DNA barcode with at least two partial sequences, and at least some oligonucleotide that wherein are attached on the particle have and have first part's complementary sequence of the DNA barcode of at least two parts; With
Second container in the every pair of container comprises the oligonucleotide that has with the second section complementary sequence of DNA barcode, this oligonucleotide have can be used for covalently bound target analyte specificity in conjunction with half point to complement,
Wherein the DNA barcode of every type particle composites probe has different sequences and as the identifier of target analyte, and wherein this test kit is optional comprises a kind of matrix and be used to observe detectable variation.
114. the test kit of the multiple target analyte in the test sample, this test kit comprise first container and at least two pairs or more to container,
First container contains the particle composites probe, and this probe comprises the particle that combines oligonucleotide;
First container in the every pair of container comprises the DNA barcode with at least two partial sequences, wherein is attached at least some oligonucleotide on the particle and has first part's complementary sequence with the DNA barcode; With
Second container in the every pair of container comprises the oligonucleotide that has with the second section complementary sequence of DNA barcode, this oligonucleotide have can be used for covalently bound target analyte specificity in conjunction with half point to complement,
Wherein the DNA barcode in first container of every pair of container as the identifier of target analyte and have with another to the different sequence of DNA barcode in the container, and wherein this test kit is optional comprises a kind of matrix and be used to observe detectable variation.
115. the test kit of claim 106, wherein this DNA barcode comprises the oligonucleotide sequence of the identifier that exists as concrete target analyte.
116. the test kit of claim 106, wherein this specific target analyte is an antibody.
117. the test kit of claim 115, wherein this specificity comprises antibody or antigen in conjunction with right member.
118. the test kit of claim 115, wherein this specificity comprises acceptor or part in conjunction with right member.
119. the test kit of claim 115, wherein this specificity comprises enzyme or substrate in conjunction with right member.
120. the test kit of claim 115, wherein this specificity comprises medicine or target molecule in conjunction with right member.
121. the test kit of claim 115, wherein this specificity comprises two to small part complementary oligonucleotide chain in conjunction with right member.
122. the test kit of claim 115, wherein this DNA barcode is biotin labeled.
123. the test kit of claim 115, wherein this DNA barcode is radiolabeled.
124. the method for claim 115, wherein this DNA barcode is fluorescently-labeled.
125. the test kit of claim 115 wherein is attached to oligonucleotide on the particle with 10picomoles/cm at least 2Density be present in particle surface.
126. the test kit of claim 115 wherein is attached to oligonucleotide on the particle with 15picomoles/cm at least 2Density be present in particle surface.
127. the test kit of claim 115 wherein is attached to oligonucleotide on the particle with from about 15picomoles/cm 2To about 40picomoles/cm 2Density be present in particle surface.
128. the test kit of claim 115, wherein this nano particle is a metal nanoparticle, or semiconductor nanoparticle.
129. the test kit of claim 115, wherein this nano particle is a gold nano grain.
130. the test kit of claim 115, wherein this semiconductor nanoparticle is made by CdSe/ZnS (core/shell).
CN 200480024522 2003-06-27 2004-06-25 Bio-barcode based detection of target analytes Pending CN101001960A (en)

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