CN101430334A - Detection method for target substance based on aptamer and its solid phase biological inductor - Google Patents
Detection method for target substance based on aptamer and its solid phase biological inductor Download PDFInfo
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- CN101430334A CN101430334A CNA2008102022777A CN200810202277A CN101430334A CN 101430334 A CN101430334 A CN 101430334A CN A2008102022777 A CNA2008102022777 A CN A2008102022777A CN 200810202277 A CN200810202277 A CN 200810202277A CN 101430334 A CN101430334 A CN 101430334A
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Abstract
The invention discloses a method which is used for detecting and quantifying target material and based on nucleic acid aptamer and a solid-phase biosensor used for detecting, wherein, the method comprises the following steps: (1) capture probes are connected to the surface of magnetic particle, and the sequence of the capture probes comprises the nucleic acid aptamer sequence of the target material; (2) a sample to be tested is added for reaction; (3) signal probes and bridge molecules are hybridized to obtain double-stranded nucleic acids, and the signal probes are added into the reaction system obtained by step (2) through a double-stranded mode for reaction, the magnetic separation is then carried out to remove free signal probes; (4) the signal probes are separated from the surface of the magnetic particle by a nucleic acid denaturation way, and the fluorescent signals on the signal probes are detected. The method is suitable for detecting wide target materials comprising protein, organic micromolecule, metallic ions, and the like. The method not only has higher sensitivity, but also solves the nonspecific problem and has important meanings in bioinstrumentation, early diagnosis and treatment of disease.
Description
Technical field
The invention belongs to the biological inductor field, particularly a kind of based on aptamer be used to detect and the method for quantifying target material and the solid phase biological inductor that detects usefulness.
Background technology
Protein is one of important component part that constitutes biosome, detects with quantitatively some specified protein is significant at aspects such as medical diagnosis on disease, treatment and new drug developments.At present, the method of protein detection that is based on antibody commonly used in research and the medical diagnosis is (as ELISA, Western Blotting), these classic methods have good, the highly sensitive advantage of specificity, but often need miscellaneous operations such as radioactive label, gel electrophoresis and radioautograph, also be not suitable for quick, high-throughout multivariate detection.And the aptamer that latest developments are got up (Aptamer), the high degree of specificity that not only has similar antibody, and can screen, directly mechanochemical method is synthetic, cost is lower and chemical stability good, for the design that detects the protein sensing device provides new approach.
Aptamer (Aptamer) is the section of DNA or the RNA molecule that can combine with specific ligand molecular (ligand) that a class is come out by SELEX (Systematic Evolution of Ligandsby Exponential Enrichment) technology in-vitro screening.Because for a certain specific target molecule, can find one or more aptamers of specific bond with it, and they have affinity and specificity highly with the target molecule.Aptamer is considered to one of optimal recognition component of field of biosensors.At present, aptamer has been used to a lot of sensing detection field.The in-vitro screening aptamer has not only strengthened the understanding of people to nucleic acid, protein interaction, and also the design for protein biosensor provides a new approach.Up to now, screened the aptamer of multiple proteins molecules such as HIV, HCV, tumour correlation factor, fibrin ferment, elastoser.Antibiotic aptamer has been showed good prospects for application in treatment.Can predict that aptamer will be well worth doing in the protein biosensor field.
As the fibrin ferment of one of protein, and the existing big quantity research of the specific reaction between its aptamer.(L.C.Bock such as Bock in 1992, L.C.Griffin, J.A.Latham, E.H.Vermaas, J.J.Toole, Nature 1992,355,564) first in in-vitro separation and screened single stranded DNA the aptamer 5 '-GGTTGGTGTGGTTGG-3 ' of the 15mer with antithrombase agglutination activity.The dissociation constant Kd of this aptamer can reach 25～200nM.(D.M.Tasset such as Tasset in 1997, M.F.Kublik, W.Steiner, J.Mol.Biol.1997,272,688) screen the fibrin ferment single stranded DNA aptamer of another 29mer, external fibrin agglutinating reaction that also can Trombin inhibiting catalysis, its dissociation constant Kd can reach 0.5nM, knows body in conjunction with more tight than the nucleic acid of 15mer.In these two kinds of aptamers, particularly concrete to the research of the aptamer of 15mer, this aptamer be G-tetramer structure after fibrin ferment combines, and this also is the basis of most of experimental designs.
Developed at present the new method that a lot of fibrin ferments detect, as optical detection, Electrochemical Detection, QCM (Quartz Crystal Microbalance) and surface plasma resonance.In these methods, (Y.Xiao such as Xiao, A.A.Lubin, A.J.Heeger, K.W.Plaxco, Label-free electronic detection of thrombin inblood serum by using an aptamer-based sensor.Angew.Chem.Int.Ed.2005,44,5456) Bao Dao the electrochemical method of exempting from mark need not other reagent to the detection of fibrin ferment, the selectivity height.But the sensitivity that detects in blood is limited, and it is the mode of operation of a kind of " singal-off ".When detecting,, thereby make the signal that specific bond produced of target be difficult to distinguish because the impurity that the degraded of aptamer or reduction label produces usually can produce the false positive signal with this pattern.Other as with QCM (Quartz Crystal Microbalance) (S.Fukusho, H.Furusawa, Y.Okahata, Chem.Commun.2002,88) or surface plasma body resonant vibration (S.H.L.Verhelst, P.J.A.Michiels, G.A.v.d.Marel, C.A.A.v.Boeckel, J.H.v.Boom, Chembiochem 2004,5, and 937; S.Tombelli, M.Minunni, M.Mascini, Anal.Lett.2002,35,599) though for the aptamer biology sensor of detection mode has higher sensitivity and short detection time, thereby but cause easily that all non-special absorption produces false positive results; And other report (T.Hianik, V.Ostatna, Z.Zajacova, E.Stoikova, G.Evtugyn, Bioorg.Med.Chem.Lett.2005,15,291) a kind of electrochemical method based on aptamer detection fibrin ferment needs the multistep operating process, and need add the reduction indicator, also have the shortcoming that is difficult to overcome.As seen, development high specific, highly sensitive biological detecting method are to realize one of protein detection and quantitative significant challenge that faces.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly at the prior art deficiency that material detect to exist that hits, and provides a kind of and is used to detect with the method for quantifying target material and detects the solid phase biological sensor of usefulness, and it has more high specific and sensitivity.
The present invention solves the technical scheme that above-mentioned first technical matters adopts: a kind of based on aptamer be used to detect method with quantifying target material, may further comprise the steps:
1) with the magnetic-particle is solid phase carrier, capture probe is connected the magnetic-particle surface, get magnetic-particle-capture probe compound; This capture probe is a single-chain nucleic acid, and its sequence comprises the aptamer sequence of target material;
2) add testing sample in magnetic-particle-capture probe compound and react, magnetic resolution is removed free composition then;
3) signal probe and bridge molecule hybridization gained double-strandednucleic acid is joined step 2) react in the reaction system of gained, magnetic resolution is removed free composition then, comprises free signal probe; This signal probe is the single-chain nucleic acid of a fluorescently-labeled arbitrary sequence, and bridge molecule also is a single-chain nucleic acid, and its sequence comprises: an end is and the sequence of signal probe complementation that the other end is the aptamer sequence of target material;
4) by the method for nucleic acid denaturation signal probe is got off from the magnetic-particle surface isolation, the fluorescence signal on this signal probe is detected.
Target material of the present invention can be any material, as long as it has an aptamer at least, and when having only an aptamer, this target material and this aptamer have two binding sites at least.
According to the present invention, step 1) is: be solid phase carrier with the magnetic-particle, capture probe is connected the magnetic-particle surface, get magnetic-particle-capture probe compound; This capture probe is a single-chain nucleic acid, and its sequence comprises the aptamer sequence of target material.Wherein, (magneticmicro-particles is conventional to react nanometer or the micron order colloidal particles that forms by organic or inorganic matrix and magnetic superfine powder (comprising magnetic metal, metal oxide etc.) MMP) to magnetic-particle of the present invention.This material has superparamagnetism makes it remain on stability in the colloidal solution by motion, and there is magnetic responsiveness can guarantee separating of in externally-applied magnetic field particle and suspending medium, after removing its external magnetic field, particulate itself suspended dispersed is not again assembled, and does not have the magnetic Memorability.Compound by macromolecule or organic reagent and Magnaglo, can introduce functional groups such as amino, carboxyl, sulfydryl or hydroxyl at particle surface, by covalent effect biomolecule such as enzyme, antibody, cell and nucleic acid are fixed on the surface, so the immobilization, immunologic surveillance, cell sorting, pharmaceutical carrier and the purification of nucleic acids that are applied to enzyme with biological, medical domain such as separate.The preparation method of magnetic-particle has many, existing at present numerous commercial magnetic-particle listings.The kind of magnetic-particle of the present invention comprises conventional the metal oxide magnetic particle that superparamagnetism is arranged, compound substance magnetic-particle, preferable alloy oxide magnetic particle.The diameter of magnetic-particle of the present invention is 800nm～1200nm, and preferably 800nm～1000nm is more preferably 1000nm.Described capture probe and being connected of magnetic-particle are preferable passes through the part (or acceptor) that the magnetic-particle surface has and is connected with specificity affinity interaction between the acceptor (or part) that capture probe has.The better conventional biotin-avidin effect of passing through connects.Capture probe of the present invention is a single-chain nucleic acid, comprises the aptamer sequence of a target material in its sequence.Preferable, the sequence of capture probe also comprises an intervening sequence (spacer) between aptamer and magnetic-particle junction.Described intervening sequence is conventional adenine base or thymine alkali bases sequence, preferable attach most importance to compound gland purine bases or the thymine alkali bases of repeating in this area.That the length of intervening sequence is preferable is 5bp～20bp, and that better is 12bp～15bp.That the reaction ratio of described magnetic-particle and capture probe is preferable is 1mg:1.25nmol～1mg:1nmol, and that better is 1mg:1.25nmol.
According to the present invention, in step 2) before described magnetic-particle-capture probe compound and testing sample react, preferable is further comprising the steps of: add confining liquid and react in magnetic-particle-capture probe compound, with the vacant binding site on sealing magnetic-particle surface.Described confining liquid can be a closed reagent commonly used in this area, preferable bovine serum albumin(BSA) (BSA), PEG (polyglycol), casein, skimmed milk power and the calf serum etc. of being selected from, and that better is BSA.Enclosure method is the ordinary skill in the art.That the reaction density of confining liquid is preferable is 0.5～10wt%, and that better is 1～2wt%, and that best is 2wt%.What the temperature of capping was preferable is 20 ℃～30 ℃, and better is 23 ℃～27 ℃.What the time of capping was preferable is 20～60 minutes, and better is 20～30 minutes.
According to the present invention, step 2) be: add testing sample and react in magnetic-particle-capture probe compound, magnetic resolution is removed free composition then.Wherein, in the reaction of described magnetic-particle-capture probe compound and testing sample, that the reaction density of testing sample is preferable is 1nM～500nM, and that better is 10nM～250nM.What the reaction density of magnetic-particle-capture probe compound was preferable is 0.5
μ M～1.25 μ M, that better is 1 μ M～1.25 μ M.Preferable 35 ℃～42 ℃ of temperature of reaction, better is 35 ℃～37 ℃.60min～90min that reaction time is preferable, that better is 50min～70min.The method of magnetic resolution is the method for the magnetic-particle magnetic resolution of this area routine.
According to the present invention, step 3) is: signal probe and bridge molecule hybridization gained double-strandednucleic acid is joined step 2) react in the reaction system of gained, magnetic resolution is removed free composition then, comprises free signal probe; This signal probe is the single-chain nucleic acid with a fluorescently-labeled arbitrary sequence, and bridge molecule also is a single-chain nucleic acid, and its sequence comprises: an end is and the sequence of signal probe complementation that the other end is the aptamer sequence of target material.
Wherein, the sequence of described signal probe is an arbitrary sequence, and that length is preferable is 10bp～20bp, and that better is 12bp～15bp.Described fluorescence labeling is the fluorescence labeling of this area routine, preferable FAM (fluorescein), FITC (fluorescein isothiocynate), Alexa 488, rhodamine 110 and the Cy2 of being selected from, better FAM and the FITC of being selected from.The sequence of described bridge molecule comprises: with the sequence of signal probe complementation and the aptamer sequence of target material.And the aptamer sequence of the sequence of signal probe complementation and target material between the preferable intervening sequence that also comprises.Described intervening sequence is conventional adenine base or thymine alkali bases sequence, preferable attach most importance to compound gland purine bases or the thymine alkali bases of repeating in this area.That the length of intervening sequence is preferable is 4bp～12bp, and that better is 5bp～10bp.The method of described signal probe and bridge molecule hybridization is with conventional, and preferable, the hybridization reaction temperature is 25 ℃～37 ℃, and the reaction time is 10min～30min.The double-strandednucleic acid of signal probe and bridge molecule hybridization gained is joined step 2) react in the reaction system of gained, the reaction density of double-strandednucleic acid is 0.5 μ M～2.5 μ M, that preferable is 0.5 μ M～1.25 μ M.Step 2) reaction density of the magnetic-particle compound of gained is 0.5 μ M～1.25 μ M, and that preferable is 0.5 μ M～1 μ M.The concentration of double-strandednucleic acid is preferable is in same system in the magnetic-particle compound 1～2 of capture probe concentration times.What temperature of reaction was preferable is 35 ℃～42 ℃, and better is 35 ℃～37 ℃, and that the reaction time is preferable is 60min～90min, and that better is 50min～70min.
Among the present invention, the aptamer sequence that all one section sequence must to be arranged in capture probe and the bridge molecule be target material to be detected.Whether these two capture probes are identical with sequence in the bridge molecule, depend on that the target material has several aptamers, depend on that also target material and aptamer have several binding sites.These two sequences meet the following conditions: when the target material has only an aptamer, and target material and this aptamer be when having two binding sites, and these two sequences are identical, promptly are all this aptamer sequence; When the target material exist two and more than not homotactic aptamer, and target material and each aptamer be when having only a binding site, these two sequences are inequality, promptly are respectively to be selected from two different in this different aptamer sequence; When the target material has two and above aptamer, and during more than one of the binding site of target material and each aptamer, these two sequences can be identical also can be inequality, promptly be respectively any one that is selected from this not homotactic aptamer sequence.
According to the present invention, step 4) is: the method for nucleic acid denaturation is got off signal probe from the magnetic-particle surface isolation, and the fluorescence signal on this signal probe is detected.
Wherein, the method for described nucleic acid denaturation is the denaturation method of this area routine, the preferred bases denaturation method.Denaturant can be this area various aqueous slkalis commonly used, and that preferable is the NaOH of 20～100mM, and that best is 50mM NaOH.The detection method of described fluorescence signal can be this area fluorescence signal detection method commonly used, the preferred spectrophotometric method of the present invention.
Wherein, when detecting fluorescence signal, preferable elder generation adds the water miscible conjugated polymer with fluorescence signal multiplication function in fluorescently-labeled signal probe, carries out fluorescence signal again and detects.Utilize the FRET (fluorescence resonance energy transfer) (FRET) between conjugated polymer and the fluorescein to realize signal multiplication, thereby improve detection sensitivity.Described conjugated polymer (Conjugated Polymers): be meant and contain the polymkeric substance that single, double key replaces conjugated system on the carbochain skeleton.Conjugated polymer exists the pi-electron conjugated system, has very strong light capture ability, has fluorescence signal multiplication function.Described conjugated polymer preferable for being selected from poly-fluorene derivative, polythiofuran derivative, polyphenylene ethylene, polyhenylene acetylene and the polypyrrole one or more.Specifically select which kind of polymkeric substance for use, relevant with the fluorophor of employed marking signal probe, as long as between polymkeric substance and the fluorophor FRET (fluorescence resonance energy transfer) efficiently can take place.Preferred FAM of fluorescence labeling of the present invention and FITC, so the preferred water-soluble poly fluorenes of conjugated polymer, its structural formula is as follows:
In the said structure, R=(CH
2)
3N
+(CH
3)
2CH
2CH
3Br, n 〉=10.When the conjugated polymer of adding fluorescence signal multiplication carried out the fluorescence signal detection, that the concentration of described fluorescently-labeled signal probe is preferable was 1nM～500nM, and that better is 10nM～250nM; That the concentration of conjugated polymer is preferable is 200nM～1000nM, and that better is 10nM～250nM; The concentration of conjugated polymer is 1～2 times of signal probe concentration, and better is 1～1.5 times, preferred 1 times.
The present invention solves above-mentioned second technical scheme that technical matters adopted: a kind ofly adopt described method to detect solid phase biological sensor with quantifying target material, comprise solid phase carrier, capture probe, signal probe and bridge molecule, wherein said solid phase carrier is a magnetic-particle, and surface combination part or the acceptor that the specificity affinity interaction can take place; Described capture probe be one in conjunction with can with part or the acceptor of acceptor generation specificity affinity interaction or the single-chain nucleic acid of part on described magnetic-particle surface, its sequence comprises the aptamer sequence of target material; Described signal probe is the single-chain nucleic acid of a fluorescently-labeled arbitrary sequence; Described bridge molecule also is a single-chain nucleic acid, and its sequence comprises: an end is and the sequence of signal probe complementation that the other end is the aptamer sequence of target material.
Wherein, described magnetic-particle as mentioned above, diameter is that the kind of 800nm～1200nm is Fe in the magnetic-particle of the preferred this area of the present invention routine
3O
4, γ-Fe
2O
3Magnetic-particle.Described part or the acceptor that the specificity affinity interaction can take place, preferred Avidin or biotin.The sequence of described capture probe comprises the aptamer sequence of target material, wherein the preferable intervening sequence that also comprises between aptamer sequence and the junction in conjunction with acceptor or part; Intervening sequence is preferably repetition adenine base or the thymine alkali bases of 12bp～15bp.Described signal probe is the single-chain nucleic acid of a fluorescently-labeled arbitrary sequence, and preferred sequence length is 12bp～15bp, and described fluorescence labeling is FAM or FITC preferably.The sequence of described bridge molecule, preferable and the aptamer sequence of the sequence of signal probe complementation and target material between also comprise an intervening sequence, intervening sequence is preferably repetition adenine base or the thymine alkali bases of 5bp～10bp.
Preferable, solid phase biological sensor of the present invention also comprises confining liquid, denaturant and/or conjugated polymer.BSA that is selected from mass percent 0.5～10% that described confining liquid is preferable and 0.5～10% PEG, mass percent 2%BSA most preferably, NaOH that is selected from 20～100mM and KOH that denaturant is preferable, 50mM NaOH most preferably, the preferred aforesaid water-soluble poly fluorenes of conjugated polymer.
Solid phase biological inductor of the present invention adopts the detection method of the invention described above.In theory, arbitrary substance can both find one or more aptamers of specific bond with it.In fact, there have been numerous materials to find its aptamer, as protein (fibrin ferment, growth factor, HIV related polypeptide etc.), organic molecule (cAMP, ATP, cocaine etc.) and metallic ion (K
+, Hg
2+, Pb
2+Deng).These materials can be as target material to be checked, as long as this testing sample has an aptamer at least, and when it has only an aptamer, this testing sample and aptamer have two binding sites at least, all can adopt solid phase biological inductor of the present invention and detection method, carry out quantitatively or detection with corresponding nucleic acids is fit.Preferably, described target material is a protein, and is better for fibrin ferment.The present invention is that example illustrates the specific embodiment of the present invention with fibrin ferment (thrombin), wherein, capture probe is 5 '-biotin-TTTTTTTTTTTTTTTGGTTGGTGTGGTTG G-3 ', or 5 '-biotin-TTTTTTTT TTTTTTTAGTCCGTGGTAGGGCAGG TTGG GGT GACT-3 ', signal probe is 5 '-fluorescein-GTATGCTGATCTGAT-3 ', bridge molecule is 5 '-GGTTGGTGTGGTTGGTT TTTATCAGATCAGCATAC-3 '.
The detection principle of fibrin ferment scheme of the present invention can be sketched to shown in Figure 1.
The present invention is except that specifying, used number percent all is mass percent.
Than prior art, beneficial effect of the present invention is as follows: of the present invention applicable to detecting various different types of target materials, as protein, organic molecule and metallic ion etc.As long as this target material has an aptamer at least, and when it has only an aptamer, this testing sample and aptamer have two binding sites at least, all can adopt solid phase biological inductor of the present invention and detection method, carry out quantitatively or detection with corresponding nucleic acids is fit.The present invention is very high to the detection sensitivity of target material, is 500pM as the detection sensitivity to fibrin ferment.The present invention not only has higher sensitivity, but also has solved non-specific problem, has great significance in the early diagnosis and therapy of biological detection, disease.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 for the present invention preferably based on the fundamental diagram of the target material detection method of magnetic-particle and soluble conjugated polymer.
Fig. 2 is the testing result contrast when not carrying out the signal amplification with soluble conjugated polymer.A is blank; B is that concentration is the fibrin ferment of 100nM.
Fig. 3 is result's contrast that two kinds of systems when whether adopting the sealing measure detect fibrin ferments.A figure is the FRET collection of illustrative plates, and B figure is corresponding FRET efficient.A, b are respectively untight blank and target; C, d are respectively blank and the target after the sealing.Target level is 25nM.
Fig. 4 detects the sensitivity analysis of fibrin ferment for the magnetic-particle that uses sealing.A figure is the FRET collection of illustrative plates, and B figure is corresponding FRET efficient.A is a blank, and b is the fibrin ferment of 500pM.
Embodiment
Below in conjunction with accompanying drawing, further specify the present invention with the detection embodiment of fibrin ferment, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Described " room temperature " is meant 20 ℃～25 ℃.
Used instrument is fluorospectrophotometer (Hitachi F-4500, a Japan).The fluorescence spectral measuring condition: xenon lamp excites, and excites and launch narrow peak width to be 2.5nm, voltage PMT 950V, response time 2S.Excitation wavelength is 380nm, emission wavelength sweep limit 390-700nm.Measure sample volume 1mL, room temperature with the 3mL quartz colorimetric utensil.
The supperparamagnetic particles that used streptavidin is modified in the embodiment of the invention is purchased the company in Promega, and particle diameter is 1.0 μ m, and solid content is 1mg/mL, and binding ability is 1.25nmol probe/mg MMPs.
Used soluble conjugated polymer is a cationic poly-fluorene derivative (PF) in the embodiment of the invention, and synthetic according to document (F.Huang, H.Wu, D.Wang, W.Yang, Y.Cao, Chem.Mater.2004,16,708), its structural formula is as follows:
In the said structure, R=(CH
2)
3N
+(CH
3)
2CH
2CH
3Br, n 〉=10.
Various dna sequence dnas (available from Shanghai biotechnology company limited) used in the embodiment of the invention are as shown in table 1, and used various solution compositions are as shown in table 2.
Table 1. oligonucleotide sequence
| Title | Base composition | Length |
| Capture probe 1 | 5 '-biotin-TTTTT TTTTT TTTTT GGTTGGTGTGGTTGG-3 ' | 30bp |
| Capture probe 2 | 5 '-biotin-TTT TTT TTT TTT TTT AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 ' | 44bp |
| Bridge molecule | 5′-GGTTGGTGTGGTTGG TTTTT ATCAGATCAGCATAC-3’ | 35bp |
| Signal probe | 5 '-fluorescein-GTATGCTGATCTGAT-3 ' | 15bp |
The various solution composition component lists of table 2.
| The solution title | Solution composition |
| 0.5×SSC | 75mM NaCl, the 7.5mM sodium citrate, pH 7.4 |
| TTL | 100mM Tris-HCl，0.1％ Tween 20，1M LiCl，pH 8.0 |
| TTA | 250mM Tris-HCl，0.1％ Tween20，5％BSA，pH 7.4 |
| In conjunction with liquid | 34mM Tris-HCl，233mM NaCl，10mM KCl，1.7mM CaCl 2，1.7mM MgCl 2, 10% glycerine, pH 7.4 |
Embodiment 1 preparation magnetic-particle-capture probe compound
By the product description requirement, MMPs at first uses 0.5 * SSC damping fluid washing 3 times before use, with 5 μ l concentration be then the biotin labeled capture probe 2 of 25 μ M join 100 μ l contain 10 μ g MMPs the TTL damping fluid in, at room temperature mixed gently 10 minutes.The superficial density of capture probe is about 4～6 * 10
11Chain/cm
2Then the compound (MMPs-capture probe is designated as MMPs-cp1) of MMPs and capture probe is washed twice with the TTA damping fluid, be suspended in conjunction with in the liquid, suspension concentration is 1.25 μ M, and 4 ℃ of refrigerations are standby.
Embodiment Bridge 2 beam molecule and signal probe hybridization
2.17 μ l concentration is the bridge molecule of 46 μ M and signal probe that 1.62 μ l concentration are 62 μ M to join the hybridization buffer of 50 μ l (composition of hybridization buffer is 750mM NaCl, the 75mM sodium citrate, pH 7.4) in, room temperature hybridization 20 minutes gets double-stranded signal probe.
The sealing in embodiment 3 magnetic-particles-vacant site of capture probe compound
Get above-mentioned preparation gained in the suspending liquid 0.1ml of 4 ℃ of standby magnetic-particle-capture probe compounds (MMPs-cp1) of refrigeration, then with TTA damping fluid washing three times, after supernatant is removed in magnetic resolution and suction, adding 0.1ml concentration is bovine serum albumin (BSA) solution of 2wt%, room temperature sealing 30 minutes.Wash twice with the TTA damping fluid then, be suspended in conjunction with in the liquid, the suspension concentration of magnetic-particle in the suspending liquid-capture probe compound is preceding identical with sealing, 4 ℃ of refrigerations standby (being designated as MMPs-cp2).
Embodiment 4 detects
Detect 1:
With 2.5 μ l concentration is in the suspending liquid of the fibrin ferment of the 500nM MMPs-cp 1 that joins 50 μ l embodiment, 1 preparation gained, 37 ℃ of reactions 1 hour, and magnetic resolution is removed free components.Add the double-stranded signal probe of the hybridization of 50 μ l embodiment, 2 preparation gained then, 37 ℃ were reacted 1 hour.Use then in conjunction with liquid washing 3～5 times, in the separation of supernatant that washing obtains till the no fluorescence signal.Then to through magnetic resolution, siphon away that to add 100 μ L concentration in the magnetic-particle compound after the supernatant be the NaOH of 50mM, room temperature sex change 5 minutes, magnetic resolution, collect the sex change supernatant, neutralize, add 10mMTris-HCl (pH 8.0) buffer solution (10mMTris-HCl of 0.8ml again to wherein adding equimolar amounts HCl, 0.5M NaCl, 2mM MgCl
2) carry out fluoroscopic examination (Ex:480nm, Em:490～700nm), and then to add 5 μ l concentration be the PF solution of the 27.08 μ M detection carrying out the conjugated polymer signal and amplify (Ex:380nm, Em:390～700nm).
Simultaneously, do following test as a comparison.
Detect 2: (carcinoembryonicantigen, CEA) (Sigma company of production firm) antibody replaces fibrin ferment to test, and is as negative control, identical in other condition and the detection 1 with the carcinomebryonic antigen of isopyknic same concentrations.
Detect 3: replace fibrin ferment to test with equal-volume water, as blank, identical in other condition and the detection 1.
Detect 4: test with any DNA sequence of a biotin modification (5 '-biotin-TTTTT TTTTTTTTTT AGT CTG GTT AGT CGT GGT TCG AAT GCT TA-3 ') replacement capture probe 2, as the specificity contrast, other condition is with last identical.
Detect 5: replace capture probe 2 to test with capture probe 1, other condition is with last identical.
Detect 6:
With 2.5 μ l concentration is that the fibrin ferment of 500nM joins in the suspending liquid of the MMPs-cp2 that 50 μ l of embodiment 3 preparation gained seals and tests the condition in other steps and the condition same " detection 1 ".
Simultaneously, do following test as a comparison.
Detect 7: test with equal-volume water replacement fibrin ferment, be blank, identical in other step and condition and the detection 6.
Detect 8: with concentration is that the fibrin ferment replacement concentration of 500pM is that the fibrin ferment of 25nM is tested, identical in other step and condition and the detection 6.
The result:
Negative control CEA antibody (promptly detecting 2) is suitable with the fluorescence signal of two kinds of fluoroscopic examinations of blank (promptly detecting 3).As seen this sensor of the present invention has the specificity of height to the detection of fibrin ferment.
Any DNA sequence with a biotin modification is that capture probe (promptly the detecting 4) signal that detects is suitable with the signal of blank.As seen this sensor of the present invention has sequence-specific preferably.
When being probe with long capture probe 2, the testing result to the testing result of fibrin ferment during than capture probe 1 is better.Aptamer sequence in the capture probe 1 is the aptamer sequence (ap1) of fibrin ferment, (list of references is L.C.Bock with fibrin ferment two affine sites of specificity, L.C.Griffin, J.A.Latham, E.H.Vermaas, J.J.Toole, Selection of single-strandedDNA molecules that bind and inhibit human thrombin.Nature 1992,355,564-566.).Aptamer sequence in the capture probe 2 is another aptamer sequence (ap2) of fibrin ferment, (list of references is D.M.Tasset with fibrin ferment an affine site of specificity, M.F.Kublik, W.Steiner, Oligonucleotide inhibitor of human thrombin that binddistinct epitopes.J.Mol.Biol.1997,272,688-698.).Aptamer sequence in the bridge molecule all is ap1.The aptamer of capture probe 1 has two different binding sites with fibrin ferment, and the aptamer of capture probe 2 and fibrin ferment have only a binding site.When being probe with capture probe 1, connected after the fibrin ferment, bridge molecule can only carry out combination with the another one site of fibrin ferment, in conjunction with probability less; When being probe with capture probe 2, connected after the fibrin ferment, bridge molecule can with the arbitrary site combination in two of the fibrin ferment different binding sites, higher in conjunction with probability.Therefore, contained sequence is an aptamer sequence inequality in preferred capture probe of the present invention and the bridge molecule.As seen, the sensing strategy based on magnetic-particle and aptamer-target material binding mechanism of the present invention has higher specificity.When being capture probe, all can realize qualitative detection to fibrin ferment with any one aptamer.
Fig. 2 is fluoroscopic examination (Ex:480nm, the result of Em:490～700nm).As seen, add water miscible conjugated polymer, when carrying out the signal amplification, can't distinguish the signal of blank and target with fluorescence signal multiplication function.Wherein the faint peak at about 580nm place is not desired characteristic peak among the figure, may be that damping fluid causes, this peak had just been covered naturally when signal was strong, so can not consider.
Add PF solution and carry out detection (Ex:380nm, the Em:390-700nm) the results are shown in Figure 3 that the conjugated polymer signal amplifies.Among Fig. 2 and 3, curve a is the preceding blank test findings (promptly detecting 3) of sealing; Curve b is the preceding 25nM crosby test result (promptly detecting 1) of sealing; Curve c is the blank test findings (promptly detecting 7) after sealing; Curve d is the 25nM crosby test result (promptly detecting 6) after sealing.As seen from the figure, adopt after the sealing measure, (curve signal a) significantly reduces blank, shows that the non-specific adsorption in the system obviously reduces; The signal of same concentrations target (curve d) then slightly raises.This is because the cause that the minimizing of non-specific adsorption increases the joint efficiency of fibrin ferment.As seen, use BSA has carried out the magnetic-particle surface after the sealing, the background signal when having reduced this sensor fibrin ferment, and the signal to noise ratio (S/N ratio) of detection improves greatly, has further improved the sensitivity that fibrin ferment detects.
Fig. 4 carries out the detection that the conjugated polymer signal amplifies (wherein curve a is sealing back blank test findings (promptly detecting 7) for Ex:380nm, the result of Em:390-700nm) for adding PF solution; Curve b is the test findings (promptly detecting 8) of sealing back 500pM fibrin ferment.As shown in the figure, use BSA that sealing has been carried out on the magnetic-particle surface after, can detect the 500pM fibrin ferment, improved at least two orders of magnitude when not sealing.The sensitivity based on the fluorescence detection method of molecular beacon of reports such as this and Heyduk (E.Heyduk, T.Heyduk, Anal.Chem.2005,77,1147) is suitable.Compare with the electrochemical sensor of Xiao etc. (Y.Xiao, A.A.Lubin, A.J.Heeger, K.W.Plaxco, Angew.Chem.Int.Ed.2005,44,5456) report, sensitivity also improves 1～2 order of magnitude.
Claims (16)
1, a kind of based on aptamer be used to detect and the method for quantifying target material, may further comprise the steps:
1) with the magnetic-particle be solid phase carrier, capture probe is connected the magnetic-particle surface, get magnetic-particle-capture probe compound, this capture probe is a single-chain nucleic acid, and its sequence comprises the aptamer sequence of target material;
2) add testing sample in magnetic-particle-capture probe compound and react, magnetic resolution is removed free composition then;
3) signal probe and bridge molecule are hybridized double-strandednucleic acid, with this double-stranded form signal probe is added step 2) react in the reaction system of gained, magnetic resolution is removed free composition then; This signal probe is the single-chain nucleic acid of a fluorescently-labeled arbitrary sequence, and bridge molecule also is a single-chain nucleic acid, and its sequence comprises: an end is and the sequence of signal probe complementation that the other end is the aptamer sequence of target material;
4) by the method for nucleic acid denaturation signal probe is got off from the magnetic-particle surface isolation, the fluorescence signal on this signal probe is detected.
2, method according to claim 1 is characterized in that, be connected part by the surface or the acceptor of the described aptamer of step 1) and magnetic-particle are connected with specificity affinity interaction between acceptor or the part.
3, method according to claim 1, it is characterized in that, in step 2) before described magnetic-particle-capture probe compound and testing sample react, also comprise step: in magnetic-particle-capture probe compound, add confining liquid and react, with the vacant binding site on sealing magnetic-particle surface.
4, method according to claim 1 is characterized in that, the method for the described nucleic acid denaturation of step 4) is alkaline denaturation method commonly used.
5, method according to claim 1 is characterized in that, when step 4) detects fluorescence signal, adds the water miscible conjugated polymer with fluorescence signal multiplication function earlier in fluorescently-labeled signal probe, carries out fluorescence signal again and detects.
6, method according to claim 1 or 5 is characterized in that the detection method of described fluorescence signal is the spectrophotometric method of using always.
7, a kind ofly adopt the described method of claim 1 to detect and the solid phase biological sensor of quantifying target material, comprise solid phase carrier, capture probe, signal probe and bridge molecule, it is characterized in that, wherein said solid phase carrier is a magnetic-particle, and surface combination part or the acceptor that the specificity affinity interaction can take place; Described capture probe be one in conjunction with can with part or the acceptor of acceptor generation specificity affinity interaction or the single-chain nucleic acid of part on described magnetic-particle surface, its sequence comprises the aptamer sequence of target material; Described signal probe is the single-chain nucleic acid of a fluorescently-labeled arbitrary sequence; Described bridge molecule also is a single-chain nucleic acid, and its sequence comprises: an end is and the sequence of signal probe complementation that the other end is the aptamer sequence of target material; Described target material has an aptamer at least, and when the target material had only an aptamer, target material and this aptamer had two binding sites at least.
8, solid phase biological sensor according to claim 7 is characterized in that, described part or the acceptor that the specificity affinity interaction can take place is Avidin or biotin.
9, solid phase biological sensor according to claim 7, it is characterized in that, the sequence of described capture probe also comprises an intervening sequence between aptamer and magnetic-particle junction, in the described bridge molecule and the aptamer sequence of the sequence of signal probe complementation and target material between also comprise an intervening sequence.
10, solid phase biological sensor according to claim 9 is characterized in that, described intervening sequence repeats adenine base or thymine alkali bases.
11, solid phase biological sensor according to claim 7 is characterized in that, described fluorescence labeling is selected from FAM and FITC.
12, solid phase biological sensor according to claim 7, it is characterized in that, the aptamer sequence of the target material in the described capture probe and the aptamer sequence of the target material in the bridge molecule, meet the following conditions: when the target material has only an aptamer, and when target material and this aptamer have two binding sites, these two sequences are identical, promptly are all this aptamer sequence; When the target material exist two and more than not homotactic aptamer, and target material and each aptamer be when having only a binding site, these two sequences are inequality, promptly are respectively to be selected from two different in this different aptamer sequence; When the target material has two and above aptamer, and during more than one of the binding site of target material and each aptamer, these two sequences are identical or inequality, promptly are respectively any one that is selected from this not homotactic aptamer sequence.
13, solid phase biological sensor according to claim 7 is characterized in that, also comprises being selected from confining liquid, denaturant and the conjugated polymer one or more.
14, solid phase biological sensor according to claim 13 is characterized in that, described confining liquid is a bovine serum albumin(BSA), and described denaturant is selected from NaOH solution and KOH solution, and described conjugated polymer is the water-soluble poly fluorenes, and its structural formula is as follows:
In the said structure, R=(CH
2)
3N
+(CH
3)
2CH
2CH
3Br, n 〉=10.
15, solid phase biological sensor according to claim 7 is characterized in that, described target material is a protein.
16, solid phase biological sensor according to claim 15 is characterized in that, described target material is a fibrin ferment.
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