CN101561398A - Target molecule detection method based on nano-Au and nucleic acid structure - Google Patents
Target molecule detection method based on nano-Au and nucleic acid structure Download PDFInfo
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- CN101561398A CN101561398A CNA2008100362191A CN200810036219A CN101561398A CN 101561398 A CN101561398 A CN 101561398A CN A2008100362191 A CNA2008100362191 A CN A2008100362191A CN 200810036219 A CN200810036219 A CN 200810036219A CN 101561398 A CN101561398 A CN 101561398A
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Abstract
The invention discloses a target molecule detection method based on nano-Au and nucleic acid structure. The detection method sequentially comprises the following steps: (1) specific DNA which can specifically react with target molecules is sufficiently hybridized with the cDNA of the DNA to form a double-chain capture probe, wherein the target molecules are proteins or ions; (2) target molecule solution is added for full reaction; (3) nano-Au solution with the mol number of 0.01-1 time of that of the specific DNA is added, and the solution is red after reaction; (4) high salting solution with the final concentration of 1-100 mM is added, and the color change of the solution is observed. The detection method has commonality, wide application range, good specificity and high sensitivity, can quickly detect any proteins or any ions with low cost, and does not need DNA mark or dear instruments.
Description
Technical field
The present invention be more particularly directed to a kind of target molecule detecting method 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-molecule 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.
The application of nanogold particle in biology is very extensive, and at present, the application of nucleic acid structure-nano-complex in analyzing and testing also received concern.1996, Mirkin and Alivisatos place working group reported the important application of nm of gold-DNA compound respectively, and it is developed into novel detection platform based on nanostructured.By the optics and the electrical properties of understanding the gold size uniqueness in depth, people such as Mirkin have been developed serial of methods subsequently DNA and protein have been carried out the ultramicron detection.Their method depends on the modification of sulfydryl dna probe on the gold size surface, when adding target, can cause the reunion or the depolymerization of gold size, thereby causes that macroscopic view goes up the red-blue stainization of color.
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.Normally, the nucleic acid structure of individualism is the random coil state in the solution, can be adsorbed on the nm of gold surface and improve its stability.But when target molecule existed, nucleic acid molecules can form the secondary structure of rigidity, can not be adsorbed on the nm of gold surface.Owing to the dna molecular difference of target molecule being carried out specific recognition, the secondary structure of formation is also variant, for example can form structures such as tetrad, stem ring, clover, projection, false knot.But the effect of these structures and nm of gold is similar certain difference is arranged again, so in specific operation process, need man-to-man the design, be unfavorable for its application.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly to overcome in the colourimetry that existing nm of gold detects target molecule different target molecules, need carry out the defective of different designs to detection scheme, provide a kind of general nm of gold to detect the colourimetry of target molecule, can realize detection easily any protein or ion.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of target molecule detecting method based on nm of gold and nucleic acid structure can may further comprise the steps:
1) can fully hybridize the double-stranded capture probe of formation with specific DNA and its cDNA of target molecule generation specific reaction, described target molecule is protein substance or ion;
2) add target molecule solution, fully reaction;
3) adding molal quantity is 0.01～1 times nano-Au solution of specific DNA, and reaction back solution colour takes on a red color;
4) add the high level salt solution of final concentration between 1～100mM, observe solution colour and change.
" protein substance " of the present invention is meant the compound that comprises amino acid or amino acid residue, as protein, polypeptide, amino acid, glycoprotein, lipoprotein, enzyme even whole virion etc.The present invention can realize arbitrary protein matter class material and the detection of ion arbitrarily.Wherein, described protein substance is preferable can be fibrin ferment (thrombin) or lysozyme enzymes such as (lysozyme).Ion of the present invention is meant the atom or the atomic group of positively charged or negative charge, and preferable ion is metallic ion or hydrogen ion.Wherein, metallic ion is preferable can be mercury ion.When target molecule is hydrogen ion, detection be hydrionic concentration, i.e. pH value.
Preferable, the described DNA that can combine with the target molecule specificity of step 1) can be the oligonucleotides of aptamer, specificity coupled ion or the oligonucleotide with i-motif structure.Wherein, the oligonucleotides of described specificity coupled ion is preferable can be specificity in conjunction with the oligonucleotides of mercury ion (mercury-specific Oligonuclide, MSO).
Preferable, the concentration of the described DNA that can combine with the target molecule specificity of step 1) and its cDNA solution respectively can be 10～500 μ M.Better, described can equating with the molar weight of its cDNA solution with the DNA of target molecule specificity combination.
Better, can carry out suitable modification to complementary series in the double-chain probe, improve the consistance of cDNA structure in the different double-chain probes, when even cDNA dissociates in solution, also be consistent with the adhesion of nm of gold, thereby make the accuracy, repeatability and the general degree that detect all improve a lot.
With conventional, can add hybridization buffer solution in the reaction system when the described hybridization of step 1) forms double-stranded capture probe.
Preferable, the final concentration of the described nano-Au solution of step 3) can be 1～100nM, nm of gold particle diameter 10～20nm.
Preferable, the described high level salt solution of step 4) is the buffer solution that contains salt; Wherein, described salt can be: MX and/or M ' X
2, wherein M is Na or K ion, and M ' is Mg or Ca ion, and X is Cl
-, Br
-, I
-, NO
3 -Or ClO
4 -Described buffer system can be conventional Tris, PBS or arsonate system.
With conventional, the present invention can adopt water or target molecule analog replacement step 2) in added target molecule solution organize in contrast.Analogue that described target molecule analog can be a target molecule or character analog.
Preferable, the method that the described observation solution colour of step 4) changes can be visual colorimetry or spectrophotometric method.Visual colorimetry can be visual inspection: solution colour keeps red, and target molecule is positive; Solution colour is by red stain indigo plant, and target molecule is negative.Spectrophotometric method can be the variation of the uv-vis spectra of instrument detecting solution: spectrum remains unchanged, and target molecule is positive; Spectrum 520nm place absorbs reduction, the absorption of long wave place raises, and target molecule is negative.
The detectable concentration of the target molecule that the present invention is preferable is 100nM～10mM, and minimal detectable concentration can reach 1pmol.
The system of entire reaction of the present invention can be controlled in the microlitre rank, and the volume of preferable reaction system is 5～500 microlitres.
The optimization of technical solution of the present invention is implemented and can be expressed as follows, get described specific DNA solution of 2 μ L and 2 μ L cDNA solution respectively with isoconcentration, and the final concentration of control DNA and cDNA is 1-50 μ M, the hybridization buffer solution that adds 18 μ L makes it fully hybridize the formation double-chain probe under the room temperature condition.In hybridization solution, add 2 μ L target molecule solution then, (appropriate condition has comprised the ion concentration of proper strength usually to select appropriate condition, suitable pH etc., the condition of every kind of target material is all not quite identical, it is fixed to come according to the condition of nucleic acid structure and target molecule) fully react.Simultaneously not add one 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 the described nano-Au solution of 5～500 μ L, behind the room temperature reaction 5min, adds described high salt buffer solution.The nm of gold change color of observation experiment group and control group also writes down uv-vis spectra.
Ultimate principle of the present invention can be summarized as: utilize nm of gold that the suction-operated difference of different structure DNA is realized differentiation to dna structure.Specific DNA-cDNA the two strands that designs any target adds the cDNA that discharges behind the target and exists with single stranded form as capture probe, and target and specific DNA form secondary structure.Because single stranded DNA can be adsorbed onto the nanogold particle surface by surface amino groups; thereby protect nm of gold not to be subjected to the influence of extraneous polyelectrolyte effectively; when adding high salt concentration; nm of gold still is disperse state; keep red on the macroscopic view, and the secondary structure of double-stranded DNA and other rigidity all can not be adsorbed onto the nm of gold surface, therefore when running into high salt; nm of gold is assembled, and presents on the macroscopic view by red to blue change color.Detection schematic diagram of the present invention can be referring to Fig. 1.
Than prior art, advantage of the present invention is as follows:
Detection method of the present invention has versatility, applied range.Detected object of the present invention can be arbitrary protein matter class material or any ion, as protein, polypeptide, amino acid, glycoprotein, lipoprotein, enzyme, virion, Hg
2+And H
+Deng.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.
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.
Detection method cheapness of the present invention, quick, highly sensitive.The present invention utilizes nm of gold to carry out colorimetric detection, and DNA need not mark, need not expensive instrument, even only need just can judge the existence of target molecule by the change color of visual inspection solution.The system of entire reaction is controlled at the microlitre rank, detection highly sensitive.
Description of drawings
Below in conjunction with the description of drawings the features and advantages of the present invention.
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 ultraviolet-visible spectrogram of nm of gold reactant liquor of the present invention.1, the fibrin ferment group; 2, the BSA group; 3, blank group.
Embodiment
With the present invention the embodiment that fibrin ferment, lysozyme, mercury ion and 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.
Uv-vis spectra detects: Hitachi UV-3010 spectrophotometer, wavelength coverage 300～800nm with 50 μ L quartz colorimetric utensils, gets 50 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, and are all synthetic by Shanghai biotechnology company limited according to the sequence of having announced.Wherein, detecting fibrin ferment and the used specific DNA of lysozyme is aptamer, the DNA that detection mercury ion, the used specific DNA of pH value are respectively MSO and have the i-motif structure.Wherein, the cDNA of fibrin ferment, lysozyme and pH value modifies, and the sequence before modifying is seen sequence in the bracket, and different bases mark with underscore.
Table 1 oligonucleotide sequence
| Target molecule | DNA character | Base composition (5 '-3 ') | Length (mer) |
| Fibrin ferment | Aptamer | GG TT GG TGT GG TT GG | 15 |
| cDNA | C AA CC ACA CC AAC (before the modification: CC AA CC ACA CC AA C C) | 13 | |
| Lysozyme | Aptamer | ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG | 42 |
| cDNA | AAC TCT GCA CTC TTT AGC CCT GAT GAA TTC (before the modification: CTA AGT AAC TCT GCA CTC TTT AGC CCT GAT GAA TTC GTA GAT) | 30 | |
| Hg 2+ | MSO | TTC TTT CTT CCCC TTG TTT GTT | 22 |
| cDNA | AAC AAA CAA GGGG AAG AAA GAA | 22 | |
| The pH value | i-motif | CCC TAA CCC TAA CCC TAA CCC | 21 |
| cDNA | G TTA GTG TTA GGT TTA G (before the modification: GGG TTA G GG TTA GG G TTA G GG) | 17 |
The detection of embodiment 1 fibrin ferment
Step: the aptamer solution and the 2 μ L concentration of getting 2 μ L concentration respectively and be the fibrin ferment of 100 μ M are the cDNA solution of 100 μ M, add buffer solution (20mM Tris-acetate, pH7.4,140mM NaCl, the 1mM CaCl of 18 μ L
2, 1mM MgCl
2) after, room temperature condition reaction 30min down makes it fully hybridize the formation double-chain probe.Adding 2 μ L concentration then in hybridization solution is the aqueous solution of the fibrin ferment of 0.1mM, and room temperature condition reaction 30min down fully reacts it.Not add fibrin ferment, 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, 3.5nM) and the molal quantity of controlling DNA be about 50 times of nano Au particle.Behind the room temperature reaction 5min, (10mM PB, pH7.0 0.2MNaCl), make ion concentration in the system about 50mM to add the 0.2M PBS of 30 μ L.The nm of gold change color of observation experiment group and control group also writes down uv-vis spectra.
The result: in containing one group of solution of fibrin ferment, fibrin ferment combines with aptamer in the double-chain probe, discharges the cDNA strand, thereby is adsorbed onto the brine tolerance that the nm of gold surface effectively raises nm of gold, still keeps red.And do not add a group of fibrin ferment, and and BSA group, the structure of double-chain probe does not change, and therefore adds after the salt nm of gold and takes place to reunite and show as blueness.The ultraviolet-visible spectrogram is seen Fig. 2, and Fig. 2 shows that the fibrin ferment group does not have significant change at the 520nm place, and BSA group and blank group obviously reduce at the 520nm place, and the long wave place absorbs obviously and raises, and proves that major part has formed aggregation.Utilize this method can the detectable concentration scope at the fibrin ferment of 100nM～10mM, the volume of solution can be controlled at 10～500 microlitres.By optimal conditions, for example control liquor capacity at 10 microlitres, detectable concentration when 100nM, the minimum fibrin ferment that can detect 1pmol.
The detection of embodiment 2 lysozymes
Step: the aptamer solution and the 2 μ L concentration of getting 2 μ L concentration respectively and be the lysozyme of 100 μ M are the cDNA solution of 100 μ M, buffer solution (the 10mM PB that adds 18 μ L, pH7.0,0.2MNaCl) after, room temperature condition down reaction 30min makes it fully hybridize the formation double-chain probe.Adding 2 μ L concentration then in hybridization solution is the aqueous solution of the lysozyme of 0.01～1mM, and room temperature condition reaction 30min down fully reacts it.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, 3.5nM) and the molal quantity of controlling DNA be about 50 times of nano Au particle.Behind the room temperature reaction 5min, add 0.2M buffer solution (10mM PB, pH7.0, the 0.2M MgCl of 15 μ L
2), make ion concentration in the system about 30mM.The nm of gold change color of observation experiment group and control group also writes down uv-vis spectra.
The result: in containing one group of solution of lysozyme, lysozyme combines with aptamer in the double-chain probe, discharges the cDNA strand, thereby is adsorbed onto the brine tolerance that the nm of gold surface effectively raises nm of gold, still keeps red.And do not add a group of lysozyme, and and BSA group, the structure of double-chain probe does not change, and therefore adds salt (PBS) back nm of gold and takes place to reunite and show as blueness.Uv-vis spectra shows that the lysozyme group does not have significant change at the 520nm place, and BSA group and blank group obviously reduce at the 520nm place, and the long wave place absorbs obviously and raises, and proves that major part has formed aggregation.Utilize this method can detect the lysozyme of 100nM～10mM, the volume of solution can be controlled at 10～500 microlitres.By optimal conditions, for example control liquor capacity at 10 microlitres, detectable concentration when 100nM, the minimum lysozyme that can detect 1pmol.
The detection (one) of embodiment 3 dimercurions
Step: MSO (specificity is in conjunction with the oligonucleotides of the mercury ion) solution and the 2 μ L concentration of getting 2 μ L concentration respectively and be 100 μ M are the cDNA solution of 100 μ M, buffer solution (the 10mM arsenic acid-arsenic acid sodium that adds 18 μ L, pH6.8,0.3M NaCl), room temperature condition reaction 30min down makes it fully hybridize the formation double-chain probe.Adding 2 μ L concentration then in hybridization solution is the Hg of 0.01mM
2+Aqueous solution, room temperature condition down reaction 5min fully reacts it.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+).Behind 60 times of the above-mentioned reaction solution dilute with waters, get respectively then that 2 μ L add that 100 μ L as above prepare and nano-Au solution (13nm, 3.5nM) and the molal quantity of controlling DNA be about 1 times of nano Au particle.Behind the room temperature reaction 5min, add 0.2M arsenic acid-arsenic acid sodium/NaClO of 15 μ L
4(10mM arsenic acid-arsenic acid sodium, pH6.8,0.2M NaClO
4), make ion concentration in the system about 15mM.The nm of gold change color of observation experiment group and control group also writes down uv-vis spectra.
Result: containing Hg
2+One group of solution in, Hg
2+Combine with the MSO in the double-chain probe, discharge the cDNA strand, thereby be adsorbed onto the brine tolerance that the nm of gold surface effectively raises nm of gold, still keep red.And do not add Hg
2+One group, and two groups of control groups, the structure of double-chain probe does not change, and therefore adds salt (PBS) back nm of gold and takes place to reunite and show as blueness.Uv-vis spectra shows Hg
2+Group does not have significant change at the 520nm place, and control group and blank group obviously reduce at the 520nm place, and the long wave place absorbs obviously and raises, and proves that major part has formed aggregation.Utilize this method can detect the Hg of 100nM～10mM
2+, the volume of solution can be controlled at 10～500 microlitres.By optimal conditions, for example control liquor capacity at 10 microlitres, detectable concentration when 100nM, the minimum Hg that can detect 1pmol
2+
The detection (two) of embodiment 4 dimercurions
Step: MSO (specificity is in conjunction with the oligonucleotides of the mercury ion) solution and the 2 μ L concentration of getting 2 μ L concentration respectively and be 10 μ M are the cDNA solution of 10 μ M, buffer solution (the 10mM arsenic acid-arsenic acid sodium that adds 18 μ L, pH6.8,0.3M NaCl), room temperature condition reaction 30min down makes it fully hybridize the formation double-chain probe.Adding 2 μ L concentration then in hybridization solution is the Hg of 0.01mM
2+Aqueous solution, room temperature condition down reaction 5min fully reacts it.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+).Behind 60 times of the above-mentioned reaction solution dilute with waters, get respectively then that 2 μ L add that 3500 μ L as above prepare and nano-Au solution (13nm, 0.01nM) and the molal quantity of controlling DNA be about 1 times of nano Au particle.Behind the room temperature reaction 30min, add 0.2M arsenic acid-arsenic acid sodium/NaClO of 15 μ L
4(10mM arsenic acid-arsenic acid sodium, pH6.8,0.2M NaClO
4), make ion concentration in the system about 15mM.The nm of gold change color of observation experiment group and control group also writes down uv-vis spectra.
Result:, contain Hg with embodiment 3
2+One group of solution keep red, and do not add Hg
2+One group, and the color of two groups of control groups becomes blueness.Uv-vis spectra has proved the whether formation of aggregation.
The detection (three) of embodiment 5 dimercurions
Step: MSO (specificity is in conjunction with the oligonucleotides of the mercury ion) solution and the 2 μ L concentration of getting 2 μ L concentration respectively and be 500 μ M are the cDNA solution of 500 μ M, buffer solution (the 10mM arsenic acid-arsenic acid sodium that adds 18 μ L, pH6.8,0.3M NaCl), room temperature condition reaction 30min down makes it fully hybridize the formation double-chain probe.Adding 2 μ L concentration then in hybridization solution is the Hg of 10mM
2+Aqueous solution, room temperature condition down reaction 5min fully reacts it.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 250mM of μ L in addition
2+, Mg
2+, other one group of hybrid ionic (Fe that adds each 5mM
2+, Cu
2+, Co
2+, Mn
2+, Ni
2+, Zn
2+, Cd
2+).Get respectively then that the above-mentioned reaction solution of 2 μ L adds that 50 μ L as above prepare and nano-Au solution (10nm, 100nM) and the molal quantity of controlling DNA be about 20 times of nano Au particle.Behind the room temperature reaction 3min, add 0.2M arsenic acid-arsenic acid sodium/NaClO of 50 μ L
4(10mM arsenic acid-arsenic acid sodium, pH6.8,0.2M NaClO
4), make ion concentration in the system about 100mM.The nm of gold change color of observation experiment group and control group also writes down uv-vis spectra.
Result:, contain Hg with embodiment 3
2+One group of solution keep red, and do not add Hg
2+One group, and the color of two groups of control groups becomes blueness.Uv-vis spectra has proved the whether formation of aggregation.
The detection (one) of embodiment 6pH value
Step: pH-DNA (DNA that promptly the has the i-motif structure) solution and the 2 μ L concentration of getting 2 μ L concentration respectively and be 10 μ M are the cDNA solution of 10 μ M, buffer solution (the 10mM PB that adds 18 μ L, pH7.0,0.3M NaCl), room temperature condition reaction 30min down makes it fully hybridize the formation double-chain probe.(20nm, pH 1nM) are 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 hybridization reaction solution of getting 2 μ L then respectively adds in the nano-Au solution of this 100 μ L with different pH values and the molal quantity of controlling DNA is 20 times of nano Au particle.Room temperature reaction 5min in the nano-Au solution of double-chain probe under above-mentioned condition of different pH adds the 0.1M buffer solution (10mM PB, pH7.0,0.1M NaI) of 10 μ L then, makes ion concentration in the system about 1mM.The nm of gold change color of observation experiment group also writes down uv-vis spectra.
The result: in pH was one group of solution of 5.5, the rich C sequence pH-DNA in the double-chain probe formed the i-motif structure, discharges the cDNA strand, thereby was adsorbed onto the brine tolerance that the nm of gold surface effectively raises nm of gold, still kept red.And pH is one group of 8.5, and the structure of double-chain probe does not change, and therefore adds the nm of gold generation reunion of salt (PBS) back and shows as blueness.Uv-vis spectra shows that pH is 5.5 groups and does not have significant change at 520nm place, and pH is 8.5 groups and obviously reduces at 520nm place, and the long wave place absorbs obvious rising, proves that major part has formed aggregation.Utilize that this method can detect scope be pH5.5～7.0.In addition, utilize half transfer point of i-motif structure that the present invention also obtains DNA near pH6.3, this is very identical with structure half transfer point that detects the pH6.2 that obtains by the CD spectrum, further specifies the accuracy of the inventive method.
The detection (two) of embodiment 7pH value
Step:
1, obtains standard working curve
Getting pH-DNA solution and the 2 μ L concentration that 2 μ L concentration are 15 μ M respectively is the cDNA solution of 15 μ M, add the buffer solution (10mM PB, pH7.0,0.3M NaCl) of 18 μ L after, room temperature condition down reaction 30min makes it fully hybridize the formation double-chain probe.(13nm, pH 3.5nM) are 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 molal quantity that the above-mentioned hybridization reaction solution of getting 2 μ L then respectively adds the nano-Au solution of these 100 μ L with different pH values and controls DNA is 10 times of nano Au particle.Room temperature reaction 5min in the nano-Au solution of double-chain probe under above-mentioned condition of different pH, (10mM PB, pH7.0 0.2MNaCl), make ion concentration in the system about 20mM to add the 0.2M PBS of 20 μ L then.Observe the nm of gold change color of each group and write down uv-vis spectra, 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 (pure water solution of nm of gold) in (13nm, 3.5nM).The hybridization reaction solution that adds step 1 gained of 2 μ L again, room temperature reaction 5min.The 0.2M PBS (10mM PB, pH7.0,0.2M NaCl) that adds 20 μ L then observes the change color of nano-Au solution and writes down uv-vis spectra, according to typical curve, determines the pH value of solution to be measured.
The result: the pH scope of the sample of detectable pH value is pH5～10.
Claims (10)
1, a kind of target molecule detecting method based on nm of gold and nucleic acid structure is characterized in that, may further comprise the steps:
1) can fully hybridize the double-stranded capture probe of formation with specific DNA and its cDNA of target molecule generation specific reaction, described target molecule is protein substance or ion;
2) add target molecule solution, fully reaction;
3) adding molal quantity is 0.01～1 times nano-Au solution of specific DNA, and reaction back solution colour takes on a red color;
4) add the high level salt solution of final concentration between 1～100mM, observe solution colour and change.
2, target molecule detecting method according to claim 1 is characterized in that, the described protein substance of step 1) is an enzyme: fibrin ferment or lysozyme.
3, target molecule detecting method according to claim 1 is characterized in that, the described ion of step 1) is mercury ion or hydrogen ion.
4, target molecule detecting method according to claim 1 is characterized in that, the described specific DNA that can combine with the target molecule specificity of step 1) is the oligonucleotides of aptamer, specificity coupled ion or the oligonucleotide with i-motif structure.
5, target molecule detecting method according to claim 4 is characterized in that, the oligonucleotides of described specificity coupled ion is the oligonucleotides of specificity in conjunction with mercury ion.
6, target molecule detecting method according to claim 1 is characterized in that, the concentration of the described DNA that can combine with the target molecule specificity of step 1) and its cDNA solution respectively is 1～50 μ M.
7, target molecule detecting method according to claim 6 is characterized in that, described can equating with the molar weight of its cDNA solution with the DNA of target molecule specificity combination.
8, target molecule detecting method according to claim 1 is characterized in that, the final concentration of the described nano-Au solution of step 3) is 1～100nM, nm of gold particle diameter 10～20nm.
9, target molecule detecting method according to claim 1 is characterized in that, the described high level salt solution of step 4) is the buffer solution that contains salt; Wherein, described salt is: MX/M ' X
2, M=Na
+/ K
+, M '=Mg
2+/ Ca
2+, X=Cl
-/ Br
-/ I
-/ NO
3 -/ ClO
4 -Described buffer system is conventional Tris, PBS or arsonate system.
10, target molecule detecting method according to claim 1 is characterized in that, the method that the described observation solution colour of step 4) changes is visual colorimetry or spectrophotometric method.
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