CN104059976A - Preparation method and application of non-sulfydryl nucleic acid-nanogold conjugate - Google Patents
Preparation method and application of non-sulfydryl nucleic acid-nanogold conjugate Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a non-sulfydryl nucleic acid-nanogold conjugate. The preparation method comprises the following steps of: (1) adding oligonucleotide with a terminal containing poly adenine PolyAx to a gold nanoparticle dispersion solution, adding a sodium citrate buffer solution, uniformly mixing, hatching, and then centrifuging to remove free oligonucleotide and retain a compound of the oligonucleotide and gold nanoparticles; (2) resuspending the compound finally obtained in the step (1) through a neutral phosphate buffer solution, adding the oligonucleotide, a sealing reagent and a sodium chloride solution, hatching again, centrifuging to remove the free oligonucleotide and the seal reagent to obtain the non-sulfydryl nucleic acid-nanogold conjugate. The preparation method disclosed by the invention can be applied to the visualized detection of DNA (Deoxyribose Nucleic Acid) and the preparation of a DNA detection kit. The nucleic acid-nanogold prepared through the method disclosed by the invention is high in stability, has the advantages of fastness, sensitiveness, wide dynamic range, low cost, easy and convenient operation and the like when applied to nucleic acid detection and is suitable for popularization and application.
Description
Technical field
The present invention relates to preparation method and application thereof that a kind of oligonucleotide that is connected poly VITAMIN B4 based on nano Au particle with end forms mixture, particularly, in the application detecting in DNA, belong to biological technical field.
Background technology
DNA is the undertaker of genetic information, is the essential substance basis of biological heredity.Due to concerning each organism, the sequence of nucleic acid is all unique.Therefore,, in the time of diagnosis and identification various diseases, the nucleotide sequence of these bacteriums, virus, pathogenic agent just becomes research object.At present, the sequence information of a lot of diseases is grasped by people, in order effectively to resist these diseases, surveys early and exactly DNA sequence dna and seems particularly important.DNA detection means based on nano material have great advantage at aspect tools such as location, visual, Multiple detections, and its amplifying nucleic acid-nanometer gold conjugate obtains a wide range of applications in detection of nucleic acids.Therefore, be necessary very much to develop the method for detection DNA easy, sensitive, with low cost a kind of.
Traditional nucleic acid-nanometer conjugate preparation realizes the self-assembly of sulfhydrylation oligonucleotide on golden nanometer particle surface based on Au-S covalent linkage.Although this method is used widely, need to overcome but also there are the following problems: in (one) the method preparation process, need to utilize the NaCl of high density to order about the end modified nucleic acid of sulfydryl near nanometer gold surface, and this process operation is very loaded down with trivial details, nanometer gold is easily reunited under the effect of high salt concentration, preparation process poor repeatability; (2) adopt nucleic acid-nanometer gold conjugate of sulfhydrylation as probe and target nucleic acid hybridization, because products therefrom frequent degree is large, easily precipitation, the responding range narrow (only 1-2 order of magnitude concentration) of detection; (3) the nucleotide sequence cost of sulfhydrylation is higher; .Recently, studies have reported that the method for employing poly VITAMIN B4 end nucleic acid (PolyA-DNA) coupled to Nano gold, can improve the sensitivity of the detection of nucleic acids that nucleic acid-Nano-Au probe is applied to, shorten detection time, (Langmuir 2012,28,17,053 17060).But, the preparation method of existing PolyA-DNA coupled to Nano gold, stability and repeatability are poor, and in the time being applied to detection of nucleic acids, also have the narrower shortcoming of dynamic detection range.
Summary of the invention
One of object of the present invention is to provide a kind of preparation method of non-sulfydryl nucleic acid-nanometer gold conjugate, to overcome the deficiencies in the prior art.
Two of object of the present invention is to provide the preparation method of described non-sulfydryl nucleic acid-nanometer gold conjugate in the application detecting in DNA.
Three of object of the present invention is to provide a kind of visual DNA detection method.
Four of object of the present invention is to provide a kind of DNA detection test kit.
For realizing aforementioned goal of the invention, the technical solution used in the present invention comprises:
A preparation method for non-sulfydryl nucleic acid-nanometer gold conjugate, comprising:
(1) oligonucleotide that end is contained to poly VITAMIN B4 PolyAx adds the dispersion liquid of golden nanometer particle, and add sodium citrate buffer solution to mix, hatch, the described oligonucleotide that then centrifugal removal is free, and retain the mixture of described oligonucleotide and golden nanometer particle;
(2) by resuspended with neutral phosphonic phthalate buffer the mixture of final step (1) the oligonucleotide that obtains and golden nanometer particle, again add described oligonucleotide, closed reagent and sodium chloride solution, again hatch, and centrifugally remove free described oligonucleotide and closed reagent, obtain described non-sulfydryl nucleic acid-nanometer gold conjugate.
Further, described closed reagent can be selected from dATP or sulfydryl undecanoic acid, and is not limited to this.
Further, the particle diameter of described golden nanometer particle is preferably 15-50nm.
Aforementioned any method is in the application detecting in DNA or preparation DNA detection test kit.
A kind of visual DNA detection method, comprising:
Provide 5 ' to hold the first oligonucleotide and the golden nanometer particle that contain poly VITAMIN B4 PolyAx, and adopt in claim 1-2 method described in any one to be made as non-sulfydryl nucleic acid-nanometer gold conjugate of the first probe;
Provide 3 ' to hold the second oligonucleotide and the golden nanometer particle that contain poly VITAMIN B4 PolyAx, and adopt in claim 1-2 method described in any one to be made as non-sulfydryl nucleic acid-nanometer gold conjugate of the second probe, x >=10;
And, described the first probe and the second probe are mixed, and add may contain can with the sample of the target dna of the nucleotide sequence of the complementary hybridization of described the first oligonucleotide and the second oligonucleotide, in the environment that is suitable for carrying out nucleic acid hybridization reaction, carry out complementary hybridization, by measuring the visible absorption Strength Changes of hybridization system, realize the detection to sample.
Further, described visual DNA detection method specifically can comprise:
Getting described the first probe and the second probe evenly mixes according to the mol ratio of 1:1, and add hybridization buffer and a series of target dna standard model with different concns, form a series of different mixed systems and carry out nucleic acid hybridization reaction, after reaction finishes, detect the fit series visible-light absorbance value of a series of different blended, determine thus the typical curve of optical absorption intensity with target dna change in concentration;
And, getting described the first probe and the second probe evenly mixes according to the mol ratio of 1:1, and add hybridization buffer and detected sample to carry out nucleic acid hybridization reaction, reaction finishes the visible ray absorbance value of mixed reactant that rear detection obtains, and calculates the concentration of the contained target dna of detected sample according to described typical curve.
Further, described visible light wavelength is preferably 520 nm, 650 nm.
Further, described hybridization buffer preferably adopts pH=7.4, and contains phosphate buffered saline buffer 150-300mM NaCl, that concentration is 0.01M.
A kind of DNA detection test kit, comprising:
The first probe, mainly holds the first oligonucleotide and the golden nanometer particle that contain poly VITAMIN B4 PolyAx to form through the made non-sulfydryl nucleic acid-nanometer gold conjugate of aforementioned any method by 5 ';
The second probe, mainly holds the second oligonucleotide and the golden nanometer particle that contain poly VITAMIN B4 PolyAx to form through the made non-sulfydryl nucleic acid-nanometer gold conjugate of aforementioned any method by 3 ';
And, in order to form the auxiliary reagent that is suitable for the liquid phase environment that carries out nucleic acid hybridization reaction,
Wherein, described the first oligonucleotide and the second oligonucleotide can be hybridized from the different nucleic acid array complementations in target dna respectively, x >=10.
Further, described auxiliary reagent comprises and contains the phosphate buffered saline buffer that concentration is the NaCl of 150-300mM.
Compared with prior art, advantage of the present invention comprises:
A) by adopting the nucleic acid probe of non-sulfydryl end to prepare nucleic acid-gold nano conjugate, can significantly reduce nucleic acid reagent cost, thereby significantly reduce testing cost, and, adopt nucleic acid-nanometer gold conjugate of this PolyA end probe to detect nucleic acid, owing to controlling the aggregation extent of AuNPs after nucleic acid hybridization, thereby improve the sensitivity and linear measurement range of this DNA-AuNPs probe in detecting, particularly, by adopting two-step approach absorption nucleic acid, can effectively solve under high salt concentration, AuNPs absorption PolyA-DNA easily produces the problem of precipitation, preparation poor repeatability;
B) further, by adopting dATP, sulfydryl undecanoic acid etc. are further modified AuNPs, can improve the stability of PolyA-DNA--AuNPs probe hybridization;
C), compared with the preparation method of existing PolyA-DNA-AuNPs probe, the present invention has that repeatability, stability prepared by probe are higher, dynamic detection range is wide while detecting nucleic acid for light-intensity method, the advantages such as sensitivity height.
Brief description of the drawings
Fig. 1 is preparation technology's schema of a kind of PolyA-DNA-AuNP probe among the present invention's one typical embodiments;
Fig. 2 is the schematic diagram of a kind of DNA detection method among the present invention's one typical embodiments;
Fig. 3 a-3c be in the present invention's one exemplary embodiments before the hybridization of DNA-AuNP probe and target dna (a) and with concentration be the transmission electron microscope photo after the target dna hybridization of 1.0nM (b), 100nM (c);
Fig. 4 is to AuNP-DNA in the typical case study on implementation of the present invention one
1and AuNP-DNA
2mixing solutions in add the target dna of different concns and the uv absorption spectra of the varying strength that causes;
Fig. 5 is target dna concentration in the present invention's one exemplary embodiments and the canonical plotting of uv-absorbing intensity.
Embodiment
Prepare and applied defect based on non-sulfhydrylation oligonucleotide and nano-Au composite in view of existing, this case contriver has proposed a kind of improved technical scheme, and it is low and be applied to the shortcoming of detection of nucleic acids narrow dynamic range that this technical scheme has overcome in existing non-sulfhydrylation oligonucleotide-nano-Au composite preparation process easy precipitation, repeatability.
Technical scheme of the present invention is the technological method that based on a kind of two-step approach, nucleic acid probe is adsorbed onto to nanometer gold surface, nucleic acid probe connects oligonucleotide multiple, particularly 10 above continuous base A by end and forms, the complementation hybridization of prepared mixture and target dna and the detection of realize target DNA.
More specifically say, what one aspect of the present invention was related is a kind of DNA photometric detection method based on nucleic acid-nano-Au composite and target dna hybridization, and the method comprises:
The first oligonucleotide that end is contained to poly VITAMIN B4 (can be labeled as DNA
1), end the second oligonucleotide of containing poly VITAMIN B4 (can be labeled as DNA
2) and golden nanometer particle (AuNP) coupling respectively, form respectively the first probe (AuNP-DNA
1) and the second probe (AuNP-DNA
2),
The first probe and the second probe are mixed, and add may contain can with the sample of the target dna of the nucleotide sequence of the complementary hybridization of the first oligonucleotide and the second oligonucleotide, in the environment that is suitable for carrying out nucleic acid hybridization reaction, carry out complementary hybridization (its principle can be consulted Fig. 2), by measuring the visible absorption Strength Changes of hybridization system, realize the detection to sample.
As one of comparatively preferred embodiment, consult Fig. 1, the preparation method of described the first probe or the second probe can comprise the steps:
(1) hold the first oligonucleotide or 3 ' that contains poly VITAMIN B4 PolyAx to hold the second oligonucleotide that contains poly VITAMIN B4 PolyAx to add in the dispersion liquid of golden nanometer particle by 5 ', and add sodium citrate buffer solution to mix, hatch, the first oligonucleotide or the second oligonucleotide that then centrifugal removal is free, and the mixture of reservation the first oligonucleotide or the second oligonucleotide and golden nanometer particle;
(2) by resuspended with neutral phosphonic acid buffer the mixture of final step (1) the oligonucleotide of winning the first place or the second oligonucleotide and golden nanometer particle, again add the first oligonucleotide or the second oligonucleotide, closed reagent and NaCl solution, again hatch, and centrifugal the first free oligonucleotide or the second oligonucleotide and the closed reagent removed, obtain described the first probe and the second probe
Wherein, described closed reagent comprises dATP or sulfydryl undecanoic acid.
What another aspect of the present invention was related is a kind of DNA detection test kit, and this test kit comprises:
The first probe, the conjugate that comprises golden nanometer particle and the first oligonucleotide, wherein, the poly VITAMIN B4 PolyAx that described the first oligonucleotide comprises with end and described golden nanometer particle coupling,
The second probe, the conjugate that comprises golden nanometer particle and the second oligonucleotide, wherein, the poly VITAMIN B4 PolyAx that described the second oligonucleotide comprises with end and described golden nanometer particle coupling,
And, in order to form the auxiliary reagent that is suitable for the liquid phase environment that carries out nucleic acid hybridization reaction,
Wherein, described the first oligonucleotide and the second oligonucleotide respectively can from the different nucleic acid array complementations in target dna, x >=10.
For example, in a typical case study on implementation of the present invention, the visual DNA detection method that should form based on nanometer gold can comprise the steps:
(1) AuNP and DNA coupling
The DNA that golden nanometer particle (AuNP, diameter 15-50nm) prepared by Trisodium Citrate reduction method and 3 ' end contain poly VITAMIN B4 (PolyAx, x>=10)
1, 5 ' end contains PolyAx(x>=10) DNA
2carry out respectively coupling and form AuNP-DNA
1, AuNP-DNA
2mixture.For example, the dispersion liquid of getting AuNP adds respectively the DNA of 100uM
1, DNA
2, then add the sodium citrate buffer solution that 500mM pH value is 3, concussion mixes, hatch 3 minutes above centrifugal, remove free DNA
1, DNA
2molecule, uses neutral phosphonic acid buffer (0.01M) resuspended to original volume, then adds the DNA of concentration 100uM
1, DNA
2solution, the sulfydryl undecanoic acid of the dATP(of concentration 100uM or concentration 10 uM), then in batches, for example every two hours add the NaCl solution of a concentration 0.1M, until contain the PB(phosphoric acid salt that concentration is 0.01M in the mixed system forming), the concentration NaCl that is 100mM, continue to hatch after 30h, add closed reagent sealing AuNP-DNA
1, AuNP-DNA
2conjugate, hatch 10h, centrifugal, remove free DNA
1, DNA
2, dATP molecule, obtain AuNP-DNA
1and AuNP-DNA
2.
(2) mixing solutions of AuNP-DNA1 and AuNP-DNA2 and target dna hybridization, get respectively the AuNP-DNA that step (1) is prepared
1and AuNP-DNA
2ratio with 1:1 mixes, and then in this mixing solutions, adds the target dna standard model of different concns to carry out complementary hybridization (hybridization buffer can be selected and contain the phosphate buffered saline buffer that concentration is 150-300mM NaCl solution).Gained mixing solutions reaction 15 minutes and above after, adopt spectrophotometer to detect the absorbance value of 520 nm and 650 nm wavelength.
The present invention has realized preparation PolyA end DNA by two-step approach and nano-Au composite is stable and repeatability is high, by this nucleic acid nano Au probe and target nucleic acid are hybridized, nanometer gold is assembled to a certain extent, absorption spectrum changes, compared with detecting nucleic acid with the light-intensity method of nucleic acid-nanometer gold conjugate of reporting, the light-intensity method based on nucleic acid-nanometer gold conjugate of the present invention has fast (time is in 10 minutes), sensitive (detection sensitivity can reach 0.1 nM), detection dynamicrange can reach the advantages such as 3 orders of magnitude.
Below in conjunction with some embodiment and accompanying drawing, technical scheme of the present invention is done to more specific detail.
Related DNA in following examples
1, DNA
2, target dna sequence as shown in table 1 below, it all can be by mode well known to those skilled in the art, the modes such as such as synthetic are obtained.Postscript, it should be noted that, this case contriver utilizes the DNA with other sequence
1, DNA
2, target dna tests according to previously described mode, also can obtain the detection effect close with following embodiment.
the design example of table 1 embodiment 1-3 amplifying nucleic acid-Nano-Au probe (AuNP-DNA)
| Sequence (from 5 '-3 ' end) | |
| DNA 1 | TA CTA CCG AGG TTT TTT AAA AAA AAA A |
| DNA 2 | A AAA AAA AAA TTT TTT C TGT CAT TAG G |
| Target dna | CCT CGG TAG TAC CTA ATG ACA G |
embodiment 1
(1) AuNP and DNA coupling
The DNA that golden nanometer particle dispersion liquid (concentration 4nM) and the 5 ' end of median size 15nm standby Trisodium Citrate reduction method legal system are contained to poly VITAMIN B4 (PolyA10)
1, 3 ' hold the DNA that contains PolyA10
2carry out respectively coupling and form AuNP-DNA
1, AuNP-DNA
2complex body: by concentrated golden nanometer particle twice, it is the DNA of 100uM that the concentrated solution 1mL that gets AuNP adds respectively 30uL concentration
1, DNA
2, then adding 20uL concentration is the sodium citrate buffer solution that 500mM pH value is 3, concussion mixes, hatch 3 minutes centrifugal, remove free DNA
1, DNA
2molecule, resuspended to original volume with the PB of 0.01M, then to add 30uL concentration be the DNA of 100uM
1, DNA
2with the dATP that 10uL concentration is 100uM, then every two hours add the NaCl solution of a 20uL concentration 0.1M, make to contain the PB that concentration is 0.01M, the NaCl solution that concentration is 100mM in system, continue to hatch after 40h, centrifugal three times, remove free DNA
1, DNA
2, dATP molecule, taking containing concentration resuspended to original volume as the Nacl damping fluid of 100mM as PB, the concentration of 0.1M, obtain AuNP-DNA
1and AuNP-DNA
2complex body.
(2) AuNP-DNA
1and AuNP-DNA
2mixing solutions and target dna hybridization
Get respectively the AuNP-DNA that 50 uL steps (1) are prepared
1and AuNP-DNA
2concentration ratio with 1:1 mixes, adding NaCl solution to make NaCl final concentration in mixed system is 200mM, then in this mixing solutions, add the target dna of 100 uL different concns (0-300nM) to carry out complementary hybridization (hybridization buffer: containing concentration is the phosphate buffered saline buffer of the NaCl solution of 200mM), react after 10 minutes, adopt spectrophotometer to detect the absorbance value of 520 nm and 650 nm wavelength, its result as shown in Figure 4, obtains typical curve as shown in Figure 5 thus.Can reach 0.1 nM to the lowest detectable limit of single stranded DNA, detect dynamicrange 0.3-300 nM, can reach 3 orders of magnitude.
In the present embodiment, AuNP-DNA
1, AuNP-DNA
2before hybridizing with target dna, in reaction process, reacted after, also characterize for reaction mixture, wherein typical characterization result is respectively as shown in Fig. 3 a-Fig. 3 c.
embodiment 2
(1) AuNP and DNA coupling
The DNA that the golden nanometer particle of average out to 30nm standby Trisodium Citrate reduction method legal system (concentration is 2nM) and 5 ' end are contained to poly VITAMIN B4 (PolyAx, x=15)
1, 3 ' end contains PolyAx(x=15) DNA
2carry out respectively coupling and form AuNP-DNA
1, AuNP-DNA
2complex body: by concentrated golden nanometer particle twice, it is the DNA of 100uM that the concentrated solution 1mL that gets AuNP adds respectively 30uL concentration
1, DNA
2solution, then adding 20uL concentration is the sodium citrate buffer solution that the pH value of 500mM is 3, concussion mixes, hatch 3 minutes centrifugal, remove free DNA
1, DNA
2molecule, water is resuspended to original volume, then to add 30uL concentration be the DNA of 100uM
1, DNA
2with the dATP solution that 10uL concentration is 100uM, the NaCl solution that then every two hours to add 20uL concentration be 0.1M, making to contain concentration in system is the PB of 0.01M, the NaCl of concentration 100mM, continues to hatch after 40h, centrifugal three times, removes free DNA
1, DNA
2, dATP molecule, and resuspended to original volume as the Nacl damping fluid of PB, the concentration 100mM of 0.1M to contain concentration, obtain AuNP-DNA
1and AuNP-DNA
2complex body.
(2) AuNP-DNA
1and AuNP-DNA
2mixing solutions and target dna hybridization
Get respectively the AuNP-DNA that 50 uL steps (1) are prepared
1and AuNP-DNA
2concentration ratio with 1:1 mixes, adding NaCl to make NaCl final concentration in mixed system is 200mM, then in this mixing solutions, adds the target dna of 100uL different concns to carry out complementary hybridization (hybridization buffer: contain the phosphate buffered saline buffer that concentration is the NaCl of 200mM).React after 15 minutes, adopt spectrophotometer to detect the absorbance value of 520 nm and 650 nm wavelength.And according to detected result that the present embodiment obtains, also can draw the typical curve close with embodiment 1.
embodiment 3
(1) AuNP and DNA coupling
The DNA that the golden nanometer particle that is 50nm by median size standby Trisodium Citrate reduction method legal system (concentration 1 nM) and 5 ' end contain poly VITAMIN B4 (PolyAx, x=20)
1, 3 ' end contains PolyAx(x=20) DNA
2carry out respectively coupling and form AuNP-DNA
1, AuNP-DNA
2complex body: by concentrated golden nanometer particle twice, the concentrated solution 1mL that gets AuNP adds respectively the DNA of 30uL concentration 100uM
1, DNA
2, then add the sodium citrate buffer solution that 20uL concentration 500mM, pH value are 3, concussion mixes, hatch 3 minutes centrifugal, remove free DNA
1, DNA
2molecule, uses the PB of 0.01M resuspended to original volume, then adds the DNA of 30uL100uM
1, DNA
2, and the sulfydryl undecanoic acid of 10uL10 uM, the NaCl solution that then every two hours to add 20uL concentration be 0.1M, makes to contain concentration 0.01M PB, 100mM NaCl in system, continues to hatch after 40h, centrifugal three times, removes free DNA
1, DNA
2, dATP molecule, and containing concentration 0.1M PB, 100mM Nacl damping fluid is resuspended to original volume, obtains AuNP-DNA
1and AuNP-DNA
2complex body.
(2) AuNP-DNA
1and AuNP-DNA
2mixing solutions and target dna hybridization
Get respectively the AuNP-DNA that 50 uL steps (1) are prepared
1and AuNP-DNA
2concentration ratio with 1:1 mixes, adding NaCl to make NaCl final concentration in mixed system is 200mM, then in this mixing solutions, adds the target dna of 100uL different concns to carry out complementary hybridization (hybridization buffer: the phosphate buffered saline buffer that contains concentration 200mM NaCl).React after 10 minutes, adopt spectrophotometer to detect the absorbance value of 520 nm and 650 nm wavelength, and according to detected result that the present embodiment obtains, also can draw the typical curve close with embodiment 1.
It will be appreciated by those skilled in the art that only illustrative principle of the present invention and effect thereof of above-described embodiment, but not for limiting the present invention.Any person skilled in the art scholar all can, under spirit of the present invention and category, modify or change above-described embodiment.Therefore, such as in affiliated technical field, have and conventionally know that the knowledgeable, not departing from all equivalence modifications that complete under disclosed spirit and technological thought or changing, must be contained by claim of the present invention.
Claims (10)
1. a preparation method for non-sulfydryl nucleic acid-nanometer gold conjugate, is characterized in that comprising:
(1) oligonucleotide that end is contained to poly VITAMIN B4 PolyAx adds the dispersion liquid of golden nanometer particle, and add sodium citrate buffer solution to mix, hatch, the described oligonucleotide that then centrifugal removal is free, and retain the mixture of described oligonucleotide and golden nanometer particle;
(2) by resuspended with neutral phosphonic phthalate buffer the mixture of final step (1) the oligonucleotide that obtains and golden nanometer particle, again add described oligonucleotide, closed reagent and sodium chloride solution, again hatch, and centrifugally remove free described oligonucleotide and closed reagent, obtain described non-sulfydryl nucleic acid-nanometer gold conjugate;
Wherein, described closed reagent comprises dATP or sulfydryl undecanoic acid.
2. the preparation method of non-sulfydryl nucleic acid-nanometer gold conjugate according to claim 1, the particle diameter that it is characterized in that described golden nanometer particle is 15-50nm.
3. the application of method in detection DNA or preparation DNA detection test kit described in any one in claim 1-2.
4. a visual DNA detection method, is characterized in that comprising:
Provide 5 ' to hold the first oligonucleotide and the golden nanometer particle that contain poly VITAMIN B4 PolyAx, and adopt in claim 1-2 method described in any one to be made as non-sulfydryl nucleic acid-nanometer gold conjugate of the first probe;
Provide 3 ' to hold the second oligonucleotide and the golden nanometer particle that contain poly VITAMIN B4 PolyAx, and adopt in claim 1-2 method described in any one to be made as non-sulfydryl nucleic acid-nanometer gold conjugate of the second probe, x >=10;
And, described the first probe and the second probe are mixed, and add may contain can with the sample of the target dna of the nucleotide sequence of the complementary hybridization of described the first oligonucleotide and the second oligonucleotide, in the environment that is suitable for carrying out nucleic acid hybridization reaction, carry out complementary hybridization, by measuring the visible absorption Strength Changes of hybridization system, realize the detection to sample.
5. visual DNA detection method according to claim 4, is characterized in that specifically comprising:
Getting described the first probe and the second probe evenly mixes according to the mol ratio of 1:1, and add hybridization buffer and a series of target dna standard model with different concns, form a series of different mixed systems and carry out nucleic acid hybridization reaction, after reaction finishes, detect the fit series visible-light absorbance value of a series of different blended, determine thus the typical curve of optical absorption intensity with target dna change in concentration;
And, getting described the first probe and the second probe evenly mixes according to the mol ratio of 1:1, and add hybridization buffer and detected sample to carry out nucleic acid hybridization reaction, reaction finishes the visible ray absorbance value of mixed reactant that rear detection obtains, and calculates the concentration of the contained target dna of detected sample according to described typical curve.
6. according to the visual DNA detection method described in claim 4 or 5, it is characterized in that described visible light wavelength is 520 nm, 650 nm.
7. according to the visual DNA detection method described in claim 4 or 5, it is characterized in that described hybridization buffer adopts pH=7.4, and contain phosphate buffered saline buffer 150-300mM NaCl, that concentration is 0.01M.
8. a DNA detection test kit, is characterized in that comprising:
The first probe, mainly by 5 ' hold the first oligonucleotide of containing poly VITAMIN B4 PolyAx and golden nanometer particle in claim 1-2 described in any one the made non-sulfydryl nucleic acid-nanometer gold conjugate of method form;
The second probe, mainly by 3 ' hold the second oligonucleotide of containing poly VITAMIN B4 PolyAx and golden nanometer particle in claim 1-2 described in any one the made non-sulfydryl nucleic acid-nanometer gold conjugate of method form;
And, in order to form the auxiliary reagent that is suitable for the liquid phase environment that carries out nucleic acid hybridization reaction,
Wherein, described the first oligonucleotide and the second oligonucleotide can be hybridized from the different nucleic acid array complementations in target dna respectively, x >=10.
9. DNA detection test kit according to claim 8, the particle diameter that it is characterized in that described golden nanometer particle is 15-50nm.
10. DNA detection test kit according to claim 8, it is characterized in that described auxiliary reagent comprises contains the phosphate buffered saline buffer that concentration is the NaCl of 150-300mM.
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| CN105695452A (en) * | 2014-11-26 | 2016-06-22 | 中国科学院上海应用物理研究所 | New nano-gold compound and preparation method thereof |
| CN106338539A (en) * | 2016-11-03 | 2017-01-18 | 上海市计量测试技术研究院 | Multi-adenine based DNA capture probe, biosensor and detection method thereof |
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| CN106338539B (en) * | 2016-11-03 | 2019-07-12 | 上海市计量测试技术研究院 | DNA capture probe, biosensor and its detection method based on polyadenous purine |
| CN107402297A (en) * | 2017-06-04 | 2017-11-28 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Enterotoxin B detection kit and detection method based on immune bio-barcode |
| CN111299568A (en) * | 2020-02-09 | 2020-06-19 | 浙江大学 | Method for quickly modifying nanogold through polyA-mediated nucleic acid ligand |
| CN111299568B (en) * | 2020-02-09 | 2021-11-30 | 浙江大学 | Method for quickly modifying nanogold through polyA-mediated nucleic acid ligand |
| CN112301106A (en) * | 2020-10-30 | 2021-02-02 | 南京师范大学 | DNA-AuNP probe of DNA modified gold nanoparticles with photo-thermal response and preparation method and application thereof |
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