CN101832935A - Target molecule detection method based on nanometer-gold and nucleic acid structure and kit thereof - Google Patents
Target molecule detection method based on nanometer-gold and nucleic acid structure and kit thereof Download PDFInfo
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Abstract
The invention relates to a target molecule detection method based on nanometer-gold and nucleic acid structure and a kit thereof. The target molecule detection method comprises the following steps of: (1) reacting specific DNA which can be specifically combined with the target molecules with a solution to be detected; (2) mixing the reaction solution obtained in the step (1) with a nanometer-gold solution for reaction; and (3) observing the fluorescence spectrum of the reaction solution finished in the step (2). The detection method has generality, the detected object can be an arbitrary target molecule, and the application range is wide. By utilizing the invention, the target molecules can be fast, sensitively and selectively detected.
Description
Technical field
The present invention be more particularly directed to a kind of target molecule detecting method and kit thereof based on nm of gold and nucleic acid structure.
Background technology
Aptamer (aptamer) is meant that utilization is set up at the end of last century and SELEX (systematic evolution of ligands by exponential enrichment) the in-vitro screening technology that grows up, the short single stranded oligonucleotide aglucon that screening obtains from the random oligonucleotide library, it can with the target molecule specific bond, thereby occurred conformation own changes.
By the in-vitro screening technology, can screen the aptamer of arbitrary substance in theory, the technical characterstic and the aptamer of adding high flux screening accurately discern, easy characteristics such as external synthetic and modification, makes aptamer have broad application prospects aspect analytical chemistry and the biological medicine research.For example, aptamer has vital role in the design of biology sensor, be applied to the detection in fields such as biology, environment, safety, comprise protein (fibrin ferment, growth factor, HIV related polypeptide etc.), organic molecule (cAMP, ATP, cocaine etc.) and metallic ion (K
+, Hg
2+, Pb
2+Deng), other new infusive application comprise based on the protein-chip of aptamer and in proteomics, be used for the high flux imaging, based on logic dna molecular computing machine of the molecular scale of DNA enzyme and RNA enzyme etc.
In addition, also have some other dna sequence dna also can under a certain specific environment, conformation change take place.Aptamer and these DNA have its specific nucleic acid structure concerning target material separately, can with target material generation specific reaction, thereby the variation of occurred conformation own.
This nucleic acid structure run into corresponding target molecule carry out in conjunction with the time usually can be with tangible conformation change.The difference of this structure has formed the theoretical foundation based on the nucleic acid structure sensor.An oligonucleotide sequence (acceptor of electron transport or NE BY ENERGY TRANSFER and donor) that typically comprises a double-tagging based on the sensor of nucleic acid structure, therefore, the structural change that the target molecule combination causes can directly have influence on the distance between acceptor and the donor, thereby can highly sensitively detect the generation of electric signal or light signal.On this basis, the application of nucleic acid structure aspect analytical chemistry becomes the focus that people pay close attention to gradually, for example, Tan etc. are applied to molecular beacon research with aptamer, develop a kind of method a part aptamer beacon (MAB) of efficient, detecting biomolecule with high sensitivity, and be further used for the detection of fibrin ferment and platelet derived growth factor (PDGF).In addition, because aptamer and antibody have similar specific bond character, therefore in many application such as ELISA, immune analysis, Western blotting and biology sensor, have bigger development potentiality, and obtain PRELIMINARY RESULTS.
, mainly be to use the double-tagging nucleotide sequence at present based on the method for the molecular beacon of DNA conformation change, an end mark fluorescent group, other end mark quencher group, and the quencher group is mainly based on Dabcyl.In order further to improve the sensitivity of fluorescent quenching efficient and detection, scientists is is constantly researched and developed novel quencher.In recent years, scientists is compared by discovering with conventional quencher group DABCYL, and nm of gold can be in can seeing near infrared wavelength coverage, various fluorescent dyes of quencher effectively, and the quencher rate reaches more than 90%.
Analyzing detecting method based on nm of gold and nucleic acid structure also receives publicity.Usefulness nanogold particle (GNPs) such as Huang and aptamer 5 ' end-bond (being Apt-AuNPs) that SH directly acts on formation detects PDGF and acceptor (PDGFR) thereof, find because Apt-AuNPs has simplification and selectivity, so be very suitable for protein analysis and cancer diagnosis.Parlor etc. also are used for Apt-AuNPs the optical detection fibrin ferment.It is fit that Dwarakanath etc. are used for labeling nucleic acid with the semiconductor nano crystal grain-quantum dot of fluorescence property excellence, opened up the beginning that technology of quantum dots and SELEX technical tie-up are used.Effect by biotin and antibiotin such as Korgd is connected to the aptamer of prostate selectivity membranous antigen (PSMA) on the CdTe quantum dot with near-infrared luminous performance and detects prostate gland cancer cell, obtained result preferably, this provides new approaches for the quantum dot-labeled analyzing and testing in living cells and biosome of aptamer.But these methods are based on mainly that the assembling of nm of gold and HS-DNA carries out, because DNA needs mark, and the process of assembling is comparatively loaded down with trivial details, and length consuming time, cost height are unfavorable for applying.
Recently, people such as Rothberg have reported that a kind of utilization does not detect the colourimetry (PNAS 101,14036 for H.Li, L.Rothberg, and September 28,2004) of target DNA through the gold size of modifying.This method ultimate principle be nanogold particle can and single stranded DNA, instantaneous absorption combination takes place by electrostatic interaction, thus can be under the high ionic strength condition stabilized nanoscale gold effectively, double-stranded DNA does not then have this effect.But this method only detects specific DNA, and is not suitable for the detection of any target material, and the sensitivity of colourimetry is limited.
Summary of the invention
The technical problem to be solved in the present invention is exactly to overcome in the colourimetry that existing nm of gold detects target DNA different target molecules, need carry out different designs, complicated operation, the limited defective of sensitivity to detection scheme, a kind of colourimetry and kit thereof easy, quick, universal, highly sensitive nm of gold detection target material are provided, can realize detection easily any target material.
The inventor is through discovering, nucleic acid structure is the random coil state in solution, can be adsorbed on the nm of gold surface, therefore the end with dna sequence dna carries out mark with fluorescein, when it is adsorbed on the nm of gold surface, its fluorophor is very near (less than 10nm) from the nm of gold surface, and fluorescence is by quencher; And when target molecule existed, nucleic acid molecules can form the secondary structure of rigidity, can not be adsorbed on the nm of gold surface, and the distance of fluorescein and nm of gold is enough far away, the fluorescence of nm of gold on can not quencher DNA.Therefore, the inventor has proposed new detection strategy: utilize the character of the efficient quench fluorescence of nm of gold that it is developed into the probe that detects nucleic acid structure, can realize the highly sensitive detection to any target material easily, thereby finish the present invention.
It is that a kind of target molecule detecting method based on nm of gold and nucleic acid structure may further comprise the steps that the present invention solves the problems of the technologies described above one of technical scheme of being adopted:
1) fluorescently-labeled specific DNA that can combine with the target molecule specificity and solution to be checked are reacted;
2) reaction solution with step 1) mixes with nano-Au solution, reacts;
The fluorescence spectrum of reaction solution 3) observation step 2).
Step 1) of the present invention is for to react fluorescently-labeled specific DNA that can combine with the target molecule specificity and solution to be checked.Wherein, described target molecule can be the detection of non-DNA target molecules such as protein, organic molecule, metallic ion even whole virion.Preferable target molecule can be atriphos (ATP), cocaine (Cocaine), platelet-derived property growth factor (PDGF), fibrin ferment (thrombin), lysozyme (lysozyme), potassium ion, mercury ion or hydrogen ion (pH value).The fluorescently-labeled specific DNA that can combine with the target molecule specificity of the present invention is other a dna sequence dna of aptamer and some, conformation change also can take place in these dna sequence dnas under a certain specific environment, aptamer and these DNA have its specific nucleic acid structure concerning target material separately, can with target material generation specific reaction, thereby the variation of occurred conformation own.Preferable specific DNA be aptamer, specificity in conjunction with the oligonucleotides of mercury ion (MSO, mercury-specificOligonuclide), specificity is in conjunction with the oligonucleotides (PSO) of potassium ion or the oligonucleotide with i-motif structure.The used fluorophor of described fluorescence labeling is preferable is selected from fluorescein (FAM), cyanine dye Cy3 and Cy5, rhodamine B (Rodamine B), fluorescein isothiocynate (FITC) and 6-carbonyl-rhodamine (ROX).Preferable, the reaction density of described specific DNA is 1～100 μ M, and the reaction density of solution to be checked is 10nM～10mM, and the mol ratio of solution to be checked and specific DNA is 100: 1～1: 100.With conventional, described reaction preferably can be carried out in buffer solution, buffer solution is preferable can be Tris, PBS, arsonate buffer solution or citrate buffer solution, the reaction density of buffer solution is preferable can be 0～25mM, and the preferable in addition final concentration that also adds is the NaCl of 0～600mM or the MgCl of 0～50mM
2Regulate ionic strength, to satisfy combining of target molecule and DNA.Preferable temperature of reaction is 4～37 ℃, and the reaction time is 1～30 minute.
Step 2 of the present invention) is that reaction solution with step 1) mixes with nano-Au solution, reacts.What wherein, described nm of gold was preferable is particle diameter 10～20nm nano-Au solution.That the reaction density of nm of gold is preferable is 1～200nM, and what the mol ratio of nm of gold and specific DNA was preferable is 4: 1～1: 200.What temperature of reaction was preferable is 4～37 ℃, and what the reaction time was preferable is 1～5 minute.
Step 3) of the present invention is for observing step 2) the fluorescence spectrum of reaction solution.Wherein, reaction solution described step 2) preferably can be observed fluorescence spectrum again after adding buffer solution.With conventional, described buffer solution is preferable can be Tris, PBS or arsonate buffer solution, and reaction density is preferable can be 0～25Mm, and the preferable in addition final concentration that also adds is the NaCl of 0～600mM or the MgCl of 0～50mM
2Regulate ionic strength.The method of described observation fluorescence spectrum is preferable can be spectrophotometric method.Spectrophotometric method can be the variation of the fluorescence spectrum of instrument detecting solution: spectrum remains unchanged, and solution to be checked contains target molecule and is positive; For FAM, observe that fluorescence spectrum 530nm place absorb to reduce, solution to be checked does not contain target molecule and is negative.
With conventional, the present invention can adopt water or target molecule analog replacement step 1) in added target molecule solution organize in contrast.Analogue that described target molecule analog can be a target molecule or character analog.
It is that a kind of aforesaid kit that detects based on the target molecule detecting method of nm of gold and nucleic acid structure comprises that the present invention solves the problems of the technologies described above two of the technical scheme that adopted
1) the fluorescently-labeled specific DNA that can combine with the target molecule specificity;
2) nano-Au solution;
3) damping fluid.
According to the present invention, in this kit, described specific DNA preferable for specific DNA be the aptamer of atriphos, cocaine, platelet-derived property growth factor, fibrin ferment or lysozyme, specificity is in conjunction with the oligonucleotides of potassium ion, and specificity is in conjunction with the oligonucleotides of mercury ion or the oligonucleotide with i-motif structure.The used fluorophor of described fluorescence labeling is preferable is selected from fluorescein, Cy3, Cy5, rhodamine B, fluorescein isothiocynate and 6-carbonyl-rhodamine.That the particle diameter of the nm of gold in the described nano-Au solution is preferable is 10～20nm.That the concentration of described nano-Au solution is preferable is 1～200nM.What described damping fluid was preferable is conventional Tris, PBS, citrate buffer solution.That the concentration of described damping fluid is preferable is 5～50mM.
The system of entire reaction of the present invention can be controlled in the microlitre rank, the volume of preferable reaction system is 5～500 microlitres, optimizing embodiment can be expressed as follows: (operable fluorophor comprises: organic dyestuff such as FAM, Cy3, Cy5, Rodamine B, FITC or ROX) solution and 2 μ L reaction densities are the target molecule solution of 10nM～10mM, and other solution such as the damping fluid that add 18 μ L fully react to get the fluorescently-labeled specific DNA that 2 μ L reaction densities are 1～100 μ M respectively.Simultaneously not add a group of target molecule solution and to add one group of the target molecule analog in contrast.The above-mentioned reaction solution of getting 2 μ L then respectively adds nm of gold (particle diameter 10～20nm of 10～1000 μ L, reaction density is in 1～100nM) solution (the molar ratio scope of Au: DNA was at 1: 1～1: 100), reacted 1～5 minute under 4～37 ℃ of conditions, that preferable is 5min.(routine biochemistry systems such as Tris, PBS, arsonate, reaction density is generally 0.1～10mM) and supplies cumulative volume 0.2～1mL, the fluorescence spectrum of record experimental group and control group to add buffer solution then.
Ultimate principle of the present invention can be summarized as: utilize the suction-operated difference of nm of gold to different structure DNA, and the character of the efficient quencher dna marker of nm of gold fluorescence realizes the differentiation to dna structure.In solution, nucleic acid structures such as aptamer mainly exist with the single stranded form of random coil, mainly show as flexibility.Single stranded DNA can be adsorbed onto the nanogold particle surface by the free amine group on the base on the chain, the distance of furthered fluorescein and nm of gold, and nm of gold quench fluorescence very efficiently, so fluorescence is by quencher.And after nucleic acid structures such as aptamer combine target molecule, show stronger rigidity, for example mainly exist with forms such as two strands, tetrad, stem ring, false knot, bulge loops.Because base is comprised in rigid structure inside, can not be adsorbed onto the nm of gold surface, so the effective quench fluorescence of nm of gold.Detection schematic diagram of the present invention can be referring to Fig. 1.
Than prior art, beneficial effect of the present invention is as follows:
1) the present invention has changed present employed single-stranded probe and has depended on the detecting pattern that the structural change of probe own provides signal, has set up a kind of general mode of operation.That is to say that detection method of the present invention has versatility, detected object can be any target molecule, applied range.Because any in theory target material can search out specific aptamer by the SELEX technology, and occurring in nature also exists in a large number the dna fragmentation that can occurred conformation changes to be based on a certain specific environment and predetermined substance.
2) detection method of the present invention has high degree of specificity.Because nucleic acid structure all has very high affinity usually, and the feasible target molecule that can detect denier easily of high-affinity, and reaction signal directly is transferred to detecting element reads, be not subjected to the interference of other non-target materials simultaneously again, realize specific detection to target molecule.
3) detection method of the present invention is highly sensitive, utilizes fluorescein and nm of gold, can improve the sensitivity to 10 of detection
-14The mol order of magnitude.
4) the present invention detects fast.The present invention has set up a kind of general mode of operation, utilizes nm of gold to carry out fluoroscopic examination, can realize the detection fast to target molecule easily.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 is the detection principle schematic of nm of gold of the present invention-nucleic acid structure probe to the target material.
Fig. 2 is the fluorescence spectrum figure of the present invention's nano-Au solution of detecting cocaine.
Embodiment
With the present invention the embodiment that atriphos (ATP), cocaine, fibrin ferment, platelet-derived property growth factor (PDGF), lysozyme, mercury ion, potassium ion, pH value (hydrogen ion) detect is further specified the present invention below, 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." room temperature " wherein is meant the laboratory temperature that carries out the embodiment operation, is 22～25 ℃.
Fluorescence spectrum carries out on Hitachi F-4500Fluorescence spectrophotometer, the excitation wavelength 480nm of fluorescein FAM, and emission wavelength ranges 490～900nm with 200 μ L quartz colorimetric utensils, gets 200 μ L samples and measures.
Used nanogold particle list of references in the embodiment of the invention (S.Dwarakanath et al., Biochemical and Biophysical Research Communications 325,739,2004) preparation.Concrete steps are as follows, the chlorauric acid solution of 100mL 0.01% (w/w) is heated to boiling after, under vigorous stirring, add the citric acid three sodium solution (10mg/mL) of 2～5mL fast and continue heating 15～30min, solution becomes claret.After stopping heating continuation stirring 30min, standing over night.Filter with 0.22 μ m filter membrane then, and be placed on 4 ℃ of preservations of refrigerator.Obtaining particle diameter according to this method is 10～20nm, and concentration is the nano-Au solution of 8～1nM.In preparation during nm of gold, use be the chlorauric acid solution of same concentration, the particle of generation is big more, corresponding concentration is just low more.Herein, the concentration of 10nm gold is about 8nM, and the concentration of the gold of 20nm is about 1nM.
Various dna sequence dnas used in the embodiment of the invention are as shown in table 1.5 ' end of these sequences all has fluorescence labeling FAM.In this area, other fluorescence labeling also obviously is applicable to the reaction among the embodiment, therefore repeats no more.These sequences are all synthetic by Shanghai biotechnology company limited according to the sequence of having announced.Wherein, detecting ATP, PDGF, cocaine, fibrin ferment and the used specific DNA of lysozyme is aptamer, the DNA that detection mercury ion, potassium ion, the used specific DNA of pH value are respectively MSO, PSO and have the i-motif structure.
The oligonucleotide sequence of the fluorescently-labeled specific DNA of table 1.
The detection of embodiment 1 cocaine
Step: getting cocaine-aptamer solution and the 2 μ L concentration that 2 μ L concentration are 100 μ M respectively is the aqueous solution of the cocaine hydrochloride of 1～100 μ M, add 16 μ L buffer solution (25mM Tris, pH8.2,0.3M NaCl) the back mixing, room temperature condition is reaction 10min down.Simultaneously, add 2 μ L water and organize, and add benzoylecgonine (N1), ecgonine methyl esters (N2) two groups of 2 μ L 1mM in contrast as blank not add cocaine.The above-mentioned reaction solution of getting 2 μ L then respectively adds the nano-Au solution (13nm that 100 μ L as above prepare and get, 3.5nM), behind the room temperature reaction 5min, add as above buffer solution (25mM Tris, pH8.2,0.3M NaCl) supplies the fluorescence spectrum of record experimental group and control group behind the 0.2mL.
The result: fluorescence spectrum figure sees Fig. 2, and visible in containing one group of solution of cocaine, cocaine-aptamer and cocaine effect form the duplex structure of rigidity, can not be adsorbed onto the nm of gold surface, so fluorescence does not obviously reduce.And cocaine-aptamer itself, and after running into N1, N2, DNA all keeps original strand state, fluorescent quenching is more than 80%.。Pass through optimal conditions, the minimum concentration of cocaine in the nano-Au solution that can detect is at 10nM, volume is that 0.2mL calculates during according to detection, can detect the cocaine of 2pmol, if with nano-drop fluorescence spectrum detection system, the sample volume minimum is 1 μ L during detection, this moment can lowest detection to the cocaine of 10fmol.
The detection of embodiment 2ATP
Step: getting ATP-aptamer solution and the 2 μ L concentration that 2 μ L concentration are 100 μ M respectively is the aqueous solution of the ATP of 0.1 μ M～100mM, adds 16 μ L buffer solution (25mM Tris, pH8.2,0.15M NaCl) back mixing, and room temperature condition is reaction 30min down.Simultaneously not add ATP, add one group of 2 μ L water as blank and add CTP, UTP, GTP three groups of 2 μ L 1mM in contrast.The above-mentioned reaction solution of getting 2 μ L then respectively add 100 μ L as above preparation and nano-Au solution (20nm 1nM), behind the room temperature reaction 5min, adds the fluorescence spectrum that above-mentioned buffer solution is supplied record experimental group and control group behind the 0.2mL.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows that the one group of fluorescence that adds ATP does not have significant change, and blank group and control group fluorescence obviously descend.Sensitivity and the detectability reference example 1 of utilizing this method to detect.
The detection of 3 pairs of dimercurions of embodiment
Step: getting 1 μ L concentration respectively is the Hg of 1 μ M
2+-MSO solution and 1 μ L concentration are the Hg of 10nM～0.1mM
2+Aqueous solution, add mixing behind the 18 μ L water, room temperature condition is reaction 1min down.Simultaneously not add Hg
2+, one group that adds 1 μ L water as blank assay.The selectivity analysis is tested by two groups and is carried out one group of Ca that adds 2 each 25mM of μ L in addition
2+, Mg
2+, other one group of hybrid ionic (Fe that adds each 0.5mM
2+, Cu
2+, Co
2+, Mn
2+, Ni
2+, Zn
2+, Cd
2+).The above-mentioned reaction solution of getting 5 μ L then respectively adds the nano-Au solution (10nm that 5 μ L as above prepare and get, 200nM), behind the room temperature reaction 5min, add 0.034% citric acid solution (prevent the nm of gold instability, cause and assemble and variable color) and supply the fluorescence spectrum of record experimental group and control group behind the 0.2mL.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows, adds Hg
2+One group of fluorescence do not have significant change, and blank group and control group fluorescence obviously descend.Sensitivity and the detectability reference example 1 of utilizing this method to detect.
The detection of 4 pairs of dimercurions of embodiment
Step: getting 2 μ L concentration respectively is the Hg of 100 μ M
2+-MSO solution and 2 μ L concentration are the Hg of 0.01～10mM
2+Arsonate buffering (10mM, pH8.0) solution add mixing behind the 16 μ L water, and room temperature condition is reaction 1min down.Simultaneously not add Hg
2+, one group that adds 2 μ L water as blank assay.The selectivity analysis is tested by two groups and is carried out one group of Ca that adds 2 each 25mM of μ L in addition
2+, Mg
2+, other one group of hybrid ionic (Fe that adds each 0.5mM
2+, Cu
2+, Co
2+, Mn
2+, Ni
2+, Zn
2+, Cd
2+).The above-mentioned reaction solution of getting 2 μ L then respectively adds the nano-Au solution that 1000 μ L as above prepare and get, and (10nm 1nM), behind 37 ℃ of reaction 1min, writes down the fluorescence spectrum of experimental group and control group.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows, adds Hg
2+One group of fluorescence do not have significant change, and blank group and control group fluorescence obviously descend.Sensitivity and the detectability reference example 1 of utilizing this method to detect.
The detection of 5 pairs of potassium ions of embodiment
Step: getting PSO solution and the 2 μ L concentration that 2 μ L concentration are 100 μ M respectively is the K of 100nM～10mM
+Aqueous solution, add mixing behind the 16 μ L water, react 30min under 4 ℃ of conditions.Simultaneously not add K
+, one group that adds 2 μ L water as blank assay, and 0.1M NaCl organizes in contrast.The above-mentioned reaction solution of getting 5 μ L then respectively adds the nano-Au solution (13nm that 100 μ L as above prepare and get, 10nM), behind 4 ℃ of reaction 5min, add 0.034% citric acid solution (prevent the nm of gold instability, cause and assemble and variable color) and supply the fluorescence spectrum of record experimental group and control group behind the 0.2mL.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows, adds K
+One group of fluorescence do not have significant change, and blank group and control group fluorescence obviously descend.Sensitivity and the detectability reference example 1 of utilizing this method to detect.
The detection of 6 pairs of pH-DNA structures of embodiment
Step: getting 2 μ L concentration respectively is the pH-DNA solution of 100 μ M, join 18 μ L citric acid solutions (0.05M, pH5.5) and 18 μ L Na
2CO
3/ NaHCO
3In the buffer solution (50mM, pH8.5), room temperature condition is reaction 5min down, and the pH-DNA final concentration is 10 μ M.Simultaneously in contrast, as above operation with comparing dna 1.(13nm, pH value 10nM) is respectively 4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,10.0 to regulate the nano-Au solution that as above prepares and get with HCl or NaOH.The above-mentioned reaction solution of getting 2 μ L then respectively adds the nano-Au solution of 100 μ L of identical pH value, behind the room temperature reaction 5min, adds the fluorescence spectrum that above-mentioned buffer solution is supplied record experimental group and control group behind the 0.2mL.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows, for the pH-DNA experiment, pH is that one group of fluorescence of 5.5 does not have significant change, and pH to be one group of fluorescence of 8.5 obviously descend.Utilize this method to obtain half transfer point of pH-DNA near 6.3.1 experiment of other one group of comparing dna, pH5.5 and 8.5 o'clock, fluorescence all obviously descended, and proves that pH causes the structural change of specificity pH-DNA.
The detection of 7 couples of pH of embodiment
Step:
1, obtains standard working curve
(1) getting 2 μ L concentration respectively is the pH-DNA solution of 100 μ M, join 18 μ L citric acid solutions (0.05M, pH5.5) and 18 μ L Na
2CO
3/ NaHCO
3(0.05M, pH8.5), room temperature condition is reaction 5min down in the buffer solution.
(2) prepare pH with HCl or NaOH and be respectively 4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,10.0 aqueous solution, get respectively then that aqueous solution that 50 μ L should difference pH value joins that 50 μ L as above prepare and nano-Au solution (13nm, 3.5nM) in, mix.
(3) reaction solution of getting the step (1) of 2 μ L respectively joins in the nano-Au solution of 100 μ L of different pH values of step (2), and room temperature reaction 5min adds the fluorescence spectrum that above-mentioned buffer solution is supplied record experimental group and control group behind the 0.2mL.Obtain standard working curve.
2, the detection of sample
The sample of getting 50 μ L pH value to be measured join that 50 μ L as above prepare and nano-Au solution in (13nm, 3.5nM).The reaction solution that adds the step (1) of 2 μ L again, room temperature reaction 5min adds after above-mentioned buffer solution supplies 0.2mL then, and the fluorescence spectrum of record experimental group and control group according to typical curve, is determined the pH value of solution to be measured.
The result: the pH scope of the sample of detectable pH value is pH5～10.
The detection of 8 pairs of fibrin ferments of embodiment
Step: getting fibrin ferment-aptamer solution and the 2 μ L concentration that 2 μ L concentration are 1 μ M respectively is the aqueous solution of the fibrin ferment of 10nM～0.1mM, adds 16 μ L buffer solution (20mM Tris-acetate, pH7.4,140mM NaCl, 1mM CaCl
2, 1mM MgCl
2) the back mixing, room temperature condition is reaction 30min down.Not add fibrin ferment, one group that adds 2 μ L water as blank assay simultaneously.And add one group of BSA (bovine serum albumin(BSA)) of 2 μ L1mM in contrast.The above-mentioned reaction solution of getting 2 μ L then respectively add that 100 μ L as above prepare and nano-Au solution (10nm 5nM), behind the room temperature reaction 5min, adds the fluorescence spectrum that above-mentioned buffer solution is supplied record experimental group and control group behind the 0.2mL.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows that the one group of fluorescence that adds fibrin ferment does not have significant change, and blank group and control group fluorescence obviously descend.Sensitivity and the detectability reference example 1 of utilizing this method to detect.
The detection of 9 pairs of lysozymes of embodiment
Step: getting lysozyme-aptamer solution and the 2 μ L concentration that 2 μ L concentration are 100 μ M respectively is the aqueous solution of the lysozyme of 10 μ M～1mM, adds 16 μ L buffer solution (10mM PB, pH7.0,0.1M NaCl) back mixing, and room temperature condition is reaction 30min down.Not add lysozyme, one group that adds 2 μ L water as blank assay simultaneously.And add one group of BSA of 2 μ L 1mM in contrast.The above-mentioned reaction solution of getting 2 μ L then respectively add that 100 μ L as above prepare and nano-Au solution (13nm 10nM), behind the room temperature reaction 5min, adds after above-mentioned buffer solution supplies 0.2mL, the fluorescence spectrum of record experimental group and control group.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows that the one group of fluorescence that adds lysozyme does not have significant change, and blank group and control group fluorescence obviously descend.Sensitivity and the detectability reference example 1 of utilizing this method to detect.
The detection of 10 couples of PDGF of embodiment
Step: getting PDGF-aptamer solution and the 2 μ L concentration that 2 μ L concentration are 100 μ M respectively is the aqueous solution of the PDGF of 10 μ M～1mM, and (0.2MNaCl) the back mixing reacts 30min under 37 ℃ of conditions for 5mM PB, pH7.0 to add 16 μ L buffer solution.Not add PDGF, one group that adds 2 μ L water as blank assay simultaneously.And add one group of BSA of 2 μ L 1mM in contrast.The above-mentioned reaction solution of getting 2 μ L then respectively add that 100 μ L as above prepare and nano-Au solution (13nm 10nM), behind 37 ℃ of reaction 5min, adds after above-mentioned buffer solution supplies 0.2mL, the fluorescence spectrum of record experimental group and control group.
The result: according to principle and operation steps as shown in Figure 1, fluorescence spectrum shows that the one group of fluorescence that adds PDGF does not have significant change, and blank group and control group fluorescence obviously descend.Sensitivity and the detectability reference example 1 of utilizing this method to detect.
Sequence table
＜110〉Suzhou City Yangtze River Delta Institute of Science and Technology of Biology Co., Ltd
＜120〉a kind of target molecule detecting method and kit thereof based on nm of gold and nucleic acid structure
<130>P4-081493C
<140>200910047299.5
<141>2009-03-10
<160>9
<170>PatentIn?version?3.4
<210>1
<211>30
<212>DNA
<213>Artificial
<220>
＜223〉cocaine aptamer
<400>1
gacaaggaaa?atccttcaat?gaagtgggtc 30
<210>2
<211>27
<212>DNA
<213>Artificial
<220>
＜223〉ATP aptamer
<400>2
acctggggga?gtattgcgga?ggaaggt 27
<210>3
<211>15
<212>DNA
<213>Artificial
<220>
＜223〉fibrin ferment aptamer
<400>3
ggttggtgtg?gttgg 15
<210>4
<211>42
<212>DNA
<213>Artificial
<220>
＜223〉lysozyme aptamer
<400>4
atctacgaat?tcatcagggc?taaagagtgc?agagttactt?ag 42
<210>5
<211>22
<212>DNA
<213>Artificial
<220>
<223>Hg2+-DNA(MSO)
<400>5
ttctttcttc?cccttgtttg?tt 22
<210>6
<211>21
<212>DNA
<213>Artificial
<220>
<223>K+-DNA(PSO)
<400>6
gggttagggt?tagggttagg?g 21
<210>7
<211>21
<212>DNA
<213>Artificial
<220>
<223>pH-DNA
<400>7
ccctaaccct?aaccctaacc?c 21
<210>8
<211>35
<212>DNA
<213>Artificial
<220>
＜223〉PDGF aptamer
<400>8
caggctacgg?cacgtagagc?atcaccatga?tcctg 35
<210>9
<211>24
<212>DNA
<213>Artificial
<220>
＜223〉comparing dna 1
<400>9
agcaacctca?aacagacacc?atgg 24
Claims (17)
1. the target molecule detecting method based on nm of gold and nucleic acid structure is characterized in that, may further comprise the steps:
1) fluorescently-labeled specific DNA that can combine with the target molecule specificity and solution to be checked are reacted;
2) reaction solution with step 1) mixes with nano-Au solution, reacts;
The fluorescence spectrum of reaction solution 3) observation step 2).
2. target molecule detecting method according to claim 1 is characterized in that, the described target molecule of step 1) is atriphos, cocaine, platelet-derived property growth factor, fibrin ferment, lysozyme, potassium ion, mercury ion or hydrogen ion; Described specific DNA is that specific DNA is the aptamer of atriphos, cocaine, platelet-derived property growth factor, fibrin ferment or lysozyme, specificity is in conjunction with the oligonucleotides of potassium ion, and specificity is in conjunction with the oligonucleotides of mercury ion or the oligonucleotide with i-motif structure.
3. target molecule detecting method according to claim 1, it is characterized in that, the reaction density of the described specific DNA of step 1) is 1～100 μ M, and the reaction density of solution to be checked is 10nM～10mM, and the mol ratio of solution to be checked and specific DNA is 100: 1～1: 100.
4. target molecule detecting method according to claim 1 is characterized in that, described being reflected in the buffer solution of step 1) carried out.
5. target molecule detecting method according to claim 1 is characterized in that, the temperature of reaction of the described reaction of step 1) is 4～37 ℃, and the reaction time is 1～30 minute.
6. target molecule detecting method according to claim 1 is characterized in that step 2) reaction density of described nm of gold is 1～200nM.
7. target molecule detecting method according to claim 1 is characterized in that step 2) mol ratio of described nm of gold and specific DNA is 1: 1～1: 100.
8. target molecule detecting method according to claim 1 is characterized in that step 2) temperature of reaction of described reaction is 4～37 ℃, the reaction time is 1～5 minute.
9. target molecule detecting method according to claim 1 is characterized in that, the described reaction solution of step 3) is observed fluorescence spectrum again after adding buffer solution.
10. target molecule detecting method according to claim 9 is characterized in that, described buffer solution is conventional Tris, PBS, arsonate buffer solution or citric acid solution.
11. target molecule detecting method according to claim 1 is characterized in that, the method for the described observation fluorescence spectrum of step 3) is a spectrophotometric method.
12. one kind as each described kit that detects based on the target molecule detecting method of nm of gold and nucleic acid structure of claim 1～11, it is characterized in that, comprises
1) the fluorescently-labeled specific DNA that can combine with the target molecule specificity;
2) nano-Au solution;
3) damping fluid.
13. kit according to claim 12 is characterized in that, the used fluorophor of described fluorescence labeling is selected from fluorescein, Cy3, Cy5, rhodamine B, fluorescein isothiocynate and 6-carbonyl-rhodamine.
14. kit according to claim 12 is characterized in that, the particle diameter of the nm of gold in the described nano-Au solution is 10～20nm.
15. kit according to claim 12 is characterized in that, the concentration of described nano-Au solution is 1～200nM.
16. kit according to claim 12 is characterized in that, described damping fluid is conventional Tris, PBS, arsonate buffer solution or citric acid solution.
17. kit according to claim 12 is characterized in that, the concentration of described damping fluid is 5～50mM.
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| CN102253017A (en) * | 2011-04-07 | 2011-11-23 | 鲁东大学 | Fluorescence detection method for potassium ions |
| CN102507689A (en) * | 2011-10-19 | 2012-06-20 | 青岛科技大学 | Manufacturing method and application of electrochemiluminescence sensor for detecting thrombin |
| CN102590170A (en) * | 2012-02-28 | 2012-07-18 | 江南大学 | Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer |
| CN102676508A (en) * | 2012-04-20 | 2012-09-19 | 华森新科（苏州）纳米技术有限公司 | Small molecule probe based on nano-gold and aptamer and preparation method of small molecule probe |
| CN102735669A (en) * | 2012-07-02 | 2012-10-17 | 湖南大学 | Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor |
| CN102759526A (en) * | 2012-06-28 | 2012-10-31 | 宁波大学 | Method for quantitative detection of mercury ions through gold label silver stain and kit thereof |
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| CN102253017A (en) * | 2011-04-07 | 2011-11-23 | 鲁东大学 | Fluorescence detection method for potassium ions |
| CN102507689B (en) * | 2011-10-19 | 2013-07-10 | 青岛科技大学 | Manufacturing method and application of electrochemiluminescence sensor for detecting thrombin |
| CN102507689A (en) * | 2011-10-19 | 2012-06-20 | 青岛科技大学 | Manufacturing method and application of electrochemiluminescence sensor for detecting thrombin |
| CN102590170A (en) * | 2012-02-28 | 2012-07-18 | 江南大学 | Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer |
| CN102676508A (en) * | 2012-04-20 | 2012-09-19 | 华森新科（苏州）纳米技术有限公司 | Small molecule probe based on nano-gold and aptamer and preparation method of small molecule probe |
| CN102759526A (en) * | 2012-06-28 | 2012-10-31 | 宁波大学 | Method for quantitative detection of mercury ions through gold label silver stain and kit thereof |
| CN102735669A (en) * | 2012-07-02 | 2012-10-17 | 湖南大学 | Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor |
| CN104745586A (en) * | 2013-12-27 | 2015-07-01 | 上海市刑事科学技术研究院 | Cocaine aptamer, detection kit and application thereof |
| CN104745586B (en) * | 2013-12-27 | 2020-02-04 | 上海市刑事科学技术研究院 | Cocaine aptamer, detection kit and application thereof |
| CN103776827A (en) * | 2014-01-16 | 2014-05-07 | 无限极（中国）有限公司 | Nanogold kit for detecting content of mercury in Chinese herbal medicine rapidly as well as using method of kit |
| CN106908595A (en) * | 2015-12-23 | 2017-06-30 | 周勇 | A kind of test paper of quick detection mercury ion and preparation method thereof |
| CN106645050A (en) * | 2016-10-13 | 2017-05-10 | 南京大学 | Aptamer used for detecting lactoferrin content, and application thereof |
| CN110082325A (en) * | 2019-04-28 | 2019-08-02 | 山西中医药大学 | Fluorescence sense system and its construction method and application |
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