CN104059976B - The preparation method and applications of non-sulfydryl nucleic acid-nanometer gold conjugate - Google Patents

The preparation method and applications of non-sulfydryl nucleic acid-nanometer gold conjugate Download PDF

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CN104059976B
CN104059976B CN201410288696.2A CN201410288696A CN104059976B CN 104059976 B CN104059976 B CN 104059976B CN 201410288696 A CN201410288696 A CN 201410288696A CN 104059976 B CN104059976 B CN 104059976B
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oligonucleotide
nucleic acid
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sulfydryl
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CN104059976A (en
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彭池方
泮秋立
刘春丽
刘丽强
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Jiangnan University
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Abstract

The invention discloses the preparation method and applications of a kind of non-sulfydryl nucleic acid nano-gold conjugate.The method includes: end is contained the dispersion liquid of the oligonucleotide addition golden nanometer particle of poly adenine PolyAx by (1), and add sodium citrate buffer solution mix homogeneously, hatch, the described oligonucleotide that then centrifugal segregation is free, and retain the complex of described oligonucleotide and golden nanometer particle；(2) step (1) obtained complex is resuspended with neutral phosphate buffer liquid, again add described oligonucleotide, closed reagent and sodium chloride solution, again hatch, and be centrifuged off described oligonucleotide and the closed reagent dissociated, it is thus achieved that described non-sulfydryl nucleic acid nano-gold conjugate.The method can be applicable to Visual retrieval and the preparation DNA detection test kit of DNA.Nucleic acid nano-gold stability prepared by the present invention is high, is applied to during detection of nucleic acids to have quick, sensitive, wide dynamic range, low cost, the advantage such as easy and simple to handle, is suitable to popularization and application.

Description

The preparation method and applications of non-sulfydryl nucleic acid-nanometer gold conjugate
Technical field
The present invention relates to a kind of oligonucleotide being connected poly adenine based on nano Au particle with end and form complex Preparation method and applications, particularly detection DNA in application, belong to biological technical field.
Background technology
DNA is the undertaker of hereditary information, is the main matter basis of biological heredity.Owing to each organism is come Saying, the sequence of nucleic acid is all unique.Therefore, when diagnosing and identifying various disease, these antibacterials, virus, pathogen Nucleotide sequence just become object of study.At present, the sequence information of a lot of diseases is grasped by people, in order to the most anti- Hit these diseases, detect DNA sequence early and exactly and be particularly important.DNA detection means based on nano material then exist The aspect tools such as location, visualization, Multiple detection have great advantage, and its amplifying nucleic acid-nanometer gold conjugate obtains in detection of nucleic acids It is widely applied.Therefore, it is highly desirable to develop a kind of simplicity, the method for detection DNA sensitive, with low cost.
Traditional nucleic acid-nanoconjugate preparation realizes sulfhydrylation oligonucleotide at golden nanometer particle based on Au-S covalent bond The self assembly on surface.Although this method has been used widely, but there is also following problem and need to overcome: (one) the method Preparation process need utilize the NaCl of high concentration to order about the end modified nucleic acid of sulfydryl near nanometer gold surface, and this process Operate the most loaded down with trivial details, nanometer gold easily to reunite under the effect of high salt concentration, preparation process poor repeatability；(2) sulfhydrylation is used Nucleic acid-nanometer gold conjugate hybridize as probe and target nucleic acid, owing to products therefrom frequent degree is big, easily sink Forming sediment, the responding range of detection is narrow (only 1-2 order of magnitude concentration)；(3) nucleotide sequence of sulfhydrylation is relatively costly；.? Closely, the method that studies have reported that employing poly adenine end nucleic acid (PolyA-DNA) coupled to Nano gold, it is possible to increase core The sensitivity of the detection of nucleic acids that acid-Nano-Au probe is applied to, shortens the detection time, (Langmuir 2012,28,17053 17060).But, the preparation method of existing PolyA-DNA coupled to Nano gold, stability and repeatability are poor, and are being applied to Also there is during detection of nucleic acids the shortcoming that dynamic detection range is narrower.
Summary of the invention
An object of the present invention is to provide the preparation method of a kind of non-sulfydryl nucleic acid-nanometer gold conjugate, to overcome The deficiencies in the prior art.
The two of the purpose of the present invention are that the preparation method providing described non-sulfydryl nucleic acid-nanometer gold conjugate is in detection Application in DNA.
The three of the purpose of the present invention are to provide a kind of visualization DNA detection method.
The four of the purpose of the present invention are to provide a kind of DNA detection test kit.
For realizing aforementioned invention purpose, the technical solution used in the present invention includes:
A kind of preparation method of non-sulfydryl nucleic acid-nanometer gold conjugate, including:
(1) oligonucleotide that end contains poly adenine PolyAx adds the dispersion liquid of golden nanometer particle, and adds Sodium citrate buffer solution mix homogeneously, hatch, the described oligonucleotide that then centrifugal segregation is free, and retain described oligonucleotide Complex with golden nanometer particle；
(2) by the complex of step (1) obtained oligonucleotide and golden nanometer particle with neutral phosphate buffer liquid weight Outstanding, again add described oligonucleotide, closed reagent and sodium chloride solution, again hatch, and be centrifuged off dissociating described Oligonucleotide and closed reagent, it is thus achieved that described non-sulfydryl nucleic acid-nanometer gold conjugate.
Further, described closed reagent is selected from dATP or Mercaptoundecanoic acid, and is not limited to this.
Further, the particle diameter of described golden nanometer particle is preferably 15-50nm.
Any one method aforementioned application in detection DNA or preparation DNA detection test kit.
A kind of visualization DNA detection method, including:
5 ' the ends the first oligonucleotide containing poly adenine PolyAx and golden nanometer particle are provided, and use claim Method according to any one of 1-2 is made as the non-sulfydryl nucleic acid-nanometer gold conjugate of the first probe；
3 ' the ends the second oligonucleotide containing poly adenine PolyAx and golden nanometer particle are provided, and use claim Method according to any one of 1-2 is made as the non-sulfydryl nucleic acid-nanometer gold conjugate of the second probe, x >=10；
And, described first probe and the second probe are mixed, and add may be containing can be with described first oligonucleoside The sample of the target dna of the nucleotide sequence of acid and the second oligonucleotide Complementary hybridization, is being adapted for the ring of nucleic acid hybridization reaction Border carries out Complementary hybridization reaction, by measuring the visible absorption Strength Changes of hybridization system, it is achieved the inspection to sample Survey.
Further, described visualization DNA detection method specifically may include that
Take described first probe and the second probe uniformly mixes according to the mol ratio of 1:1, and add hybridization buffer and Series has the target dna standard sample of variable concentrations, forms a series of different mixed system and carries out nucleic acid hybridization reaction, reaction After end, detect a series of different blended zoarium series visible-light absorbance value, thereby determine that optical absorption intensity becomes with target DNA concentration The standard curve changed；
And, take described first probe and the second probe uniformly mixes according to the mol ratio of 1:1, and add hybridization buffer Carrying out nucleic acid hybridization reaction with detected sample, reaction detects the visible ray absorbance value of obtained mixed reactant after terminating, and The concentration of target dna contained by detected sample is calculated according to described standard curve.
Further, the wavelength of described visible ray is preferably 520 nm, 650 nm.
Further, described hybridization buffer preferably employs pH=7.4, and containing 150-300mM NaCl, concentration be The phosphate buffer of 0.01M.
A kind of DNA detection test kit, including:
First probe, is mainly held the first oligonucleotide containing poly adenine PolyAx premenstrual with golden nanometer particle by 5 ' State the non-sulfydryl nucleic acid made by any one method-nanometer gold conjugate composition；
Second probe, is mainly held the second oligonucleotide containing poly adenine PolyAx premenstrual with golden nanometer particle by 3 ' State the non-sulfydryl nucleic acid made by any one method-nanometer gold conjugate composition；
And, in order to constitute the auxiliary reagent of the liquid phase environment being adapted for nucleic acid hybridization reaction,
Wherein, described first oligonucleotide and the second oligonucleotide are able to and the different IPs acid sequence in target dna Complementary hybridization, x >=10.
Further, described auxiliary reagent includes the phosphate buffer containing the NaCl that concentration is 150-300mM.
Compared with prior art, the invention have the advantages that
A) by using the nucleic probe of non-sulfydryl end to prepare nucleic acid-gold nano conjugate, nucleic acid examination can be significantly reduced Agent cost, thus significantly reduce testing cost, and, use the nucleic acid-nanometer gold conjugate detection of this PolyA terminal probe Nucleic acid, owing to can control the aggregation extent of AuNPs after nucleic acid hybridization, thus improves the sensitive of this DNA-AuNPs probe in detecting Degree and the range of linearity, particularly, by using two-step method absorption nucleic acid, can effectively solve under high salt concentration, and AuNPs adsorbs PolyA-DNA easily produces precipitation, the problem of preparation poor repeatability；
B) further, by using dATP, Mercaptoundecanoic acid etc. is modified AuNPs further, can be improved PolyA- The stability of DNA--AuNPs probe hybridization；
C) compared with the preparation method of existing PolyA-DNA-AuNPs probe, the present invention have repeatability prepared by probe, Stability is higher, dynamic detection range width when photometry detection nucleic acid, sensitivity advantages of higher.
Accompanying drawing explanation
Fig. 1 is the preparation technology flow chart of a kind of PolyA-DNA-AuNP probe among the present invention one typical embodiments；
Fig. 2 is the schematic diagram of a kind of DNA detection method among the present invention one typical embodiments；
Fig. 3 a-3c is (a) and and concentration before DNA-AuNP probe and target dna hybridize in the present invention one exemplary embodiments For the transmission electron microscope photo after the target dna hybridization of 1.0nM (b), 100nM (c)；
Fig. 4 is to AuNP-DNA in the present invention one exemplary embodiments1And AuNP-DNA2Mixed solution in add difference The target dna of concentration and the uv absorption spectra of varying strength that causes；
Fig. 5 is the canonical plotting of the target DNA concentration in the present invention one exemplary embodiments and uv absorption intensity.
Detailed description of the invention
Preparing and applied defect with nano-Au composite based on non-sulfhydrylation oligonucleotide in view of existing, inventor carries Having gone out the technical scheme of a kind of improvement, this technical scheme overcomes existing non-sulfhydrylation oligonucleotide-nano-Au composite and prepares During easily precipitation, repeatability is low and the shortcoming that is applied to detection of nucleic acids narrow dynamic range.
The technical scheme is that the technical method that based on a kind of two-step method, nucleic probe is adsorbed onto nanometer gold surface, Nucleic probe is connected oligonucleotide multiple, continuous base A of particularly more than 10 and constitutes by end, prepared complex The detection of target dna is realized with the Complementary hybridization of target dna.
More specifically saying, involved by one aspect of the present invention is a kind of based on nucleic acid-nano-Au composite and mesh The DNA photometric detection method of mark DNA hybridization, the method includes:
The first oligonucleotide that end contains poly adenine (can be labeled as DNA1), end contain poly adenine Second oligonucleotide (can be labeled as DNA2) and golden nanometer particle (AuNP) coupling respectively, form the first probe (AuNP-respectively DNA1) and the second probe (AuNP-DNA2),
First probe and the second probe are mixed, and add may be containing can be with the first oligonucleotide and the second oligonucleoside The sample of the target dna of the nucleotide sequence of acid Complementary hybridization, carries out complementary miscellaneous in the environment be adapted for nucleic acid hybridization reaction Hand over reaction (its principle see Fig. 2), by measuring the visible absorption Strength Changes of hybridization system, it is achieved to sample Detection.
As more one of preferred embodiment, refering to Fig. 1, the preparation method of described first probe or the second probe can To comprise the steps:
(1) the first oligonucleotide or the 3 ' ends that 5 ' ends are contained poly adenine PolyAx contain poly adenine PolyAx The second oligonucleotide add in the dispersion liquid of golden nanometer particle, and add sodium citrate buffer solution mix homogeneously, hatch, then The first oligonucleotide that centrifugal segregation is free or the second oligonucleotide, and retain the first oligonucleotide or the second oligonucleotide and gold The complex of nanoparticle；
(2) by the complex of step (1) won the first place oligonucleotide or the second oligonucleotide and golden nanometer particle with Neutral phosphor acid buffer is resuspended, again adds the first oligonucleotide or the second oligonucleotide, closed reagent and NaCl solution, then Secondary hatch, and be centrifuged off the first oligonucleotide of dissociating or the second oligonucleotide and closed reagent, it is thus achieved that described first probe With the second probe,
Wherein, described closed reagent includes dATP or Mercaptoundecanoic acid.
Involved by another aspect of the present invention is a kind of DNA detection test kit, and this test kit includes:
First probe, comprises golden nanometer particle and the conjugate of the first oligonucleotide, wherein, described first oligonucleotide with The poly adenine PolyAx that end comprises and described golden nanometer particle coupling,
Second probe, comprises golden nanometer particle and the conjugate of the second oligonucleotide, wherein, described second oligonucleotide with The poly adenine PolyAx that end comprises and described golden nanometer particle coupling,
And, in order to constitute the auxiliary reagent of the liquid phase environment being adapted for nucleic acid hybridization reaction,
Wherein, described first oligonucleotide and the second oligonucleotide are able to and the different IPs acid sequence in target dna Complementation, x >=10.
Such as, in an exemplary embodiments of the present invention, being somebody's turn to do visualization DNA detection method based on nanometer gold formation can To comprise the steps:
(1) AuNP and DNA coupling
The golden nanometer particle (AuNP, diameter 15-50nm) reduction of sodium citrate method prepared and 3 ' ends contain poly gland The DNA of purine (PolyAx, x >=10)1, 5 ' ends are containing PolyAx(x >=10) DNA2Carry out coupling respectively and form AuNP-DNA1、 AuNP-DNA2Complex.Such as, the dispersion liquid taking AuNP is separately added into the DNA of 100uM1、DNA2, it is subsequently adding 500mM pH value It is the sodium citrate buffer solution of 3, concussion mixing, hatch more than 3 minutes and be centrifuged, remove free DNA1、DNA2Molecule, by neutrality Phosphate buffer (0.01M) is resuspended to original volume, adds the DNA of concentration 100uM1、DNA2Solution, the dATP of concentration 100uM (or Mercaptoundecanoic acid of concentration 10 uM), the most in batches, the most every two hours adds the NaCl solution of concentration 0.1M, directly Be the PB(phosphate of 0.01M containing concentration to the mixed system formed), concentration be the NaCl of 100mM, continue to hatch 30h After, add closed reagent and close AuNP-DNA1 、AuNP-DNA2Conjugate, hatch 10h, centrifugal, remove free DNA1、 DNA2, dATP molecule, obtain AuNP-DNA1And AuNP-DNA2。
(2) mixed solution of AuNP-DNA1 and AuNP-DNA2 hybridizes with target dna, takes what step (1) was prepared respectively AuNP-DNA1And AuNP-DNA2Mix with the ratio of 1:1, in this mixed solution, then add the target dna mark of variable concentrations Quasi-sample carries out Complementary hybridization, and (hybridization buffer can be selected for containing the phosphate-buffered that concentration is 150-300mM NaCl solution Liquid).After gained mixed solution reacts 15 minutes and be above, spectrophotometer is used to detect 520 nm and the light of 650 nm wavelength Absorption value.
The present invention achieves preparation PolyA end DNA by two-step method and stablizes with nano-Au composite and repeatability height, logical Cross and this nucleic acid nano Au probe is hybridized with target nucleic acid so that nanometer gold is assembled to a certain extent, and absorption spectrum becomes Changing, compared with the photometry of the nucleic acid reported-nanometer gold conjugate detection nucleic acid, the present invention's is even based on nucleic acid-nanometer gold The photometry of connection thing has quickly (time is within 10 minutes), sensitive (detection sensitivity is up to 0.1 nM), detects dynamic model Enclose advantages such as can reach 3 orders of magnitude.
Below in conjunction with some embodiments and accompanying drawing, technical scheme is made more specific detail.
DNA involved in following example1、DNA2, target dna sequence as shown in table 1 below, it all can pass through ability The modes such as the mode known to field technique personnel, such as synthetic obtain.Postscript, it should be noted that inventor utilizes There is the DNA of other sequence1、DNA2, target dna test according to manner described above, also can obtain and below embodiment Close Detection results.
The design example of table 1 embodiment 1-3 amplifying nucleic acid-Nano-Au probe (AuNP-DNA)
Sequence (from 5 '-3 ' end)
DNA1 TA CTA CCG AGG TTT TTT AAA AAA AAA A
DNA2 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 golden nanometer particle dispersion liquid (concentration 4nM) of mean diameter 15nm prepared by reduction of sodium citrate method method and 5 ' ends The end DNA containing poly adenine (PolyA10)1, 3 ' ends are containing the DNA of PolyA102Carry out coupling respectively and form AuNP- DNA1、AuNP-DNA2Complex: golden nanometer particle is concentrated twice, takes the concentrated solution 1mL of AuNP and is separately added into 30uL concentration and be The DNA of 100uM1、DNA2, be subsequently adding 20uL concentration be 500mM pH value be the sodium citrate buffer solution of 3, concussion mixing, incubate Educate 3 minutes and be centrifuged, remove free DNA1、DNA2Molecule, resuspended to original volume with the PB of 0.01M, adding 30uL concentration is The DNA of 100uM1、DNA2It is the dATP of 100uM with 10uL concentration, the most every two hours adds the NaCl of 20uL concentration 0.1M Solution, make in system containing concentration be the PB of 0.01M, concentration be the NaCl solution of 100mM, after continuing to hatch 40h, centrifugal three Secondary, remove free DNA1、DNA2, dATP molecule, with resuspended containing the concentration PB as 0.1M, the concentration Nacl buffer as 100mM To original volume, obtain AuNP-DNA1And AuNP-DNA2Complex.
(2) AuNP-DNA1And AuNP-DNA2Mixed solution and target dna hybridize
Take the AuNP-DNA that 50 uL steps (1) are prepared respectively1And AuNP-DNA2With the concentration ratio mixing of 1:1, add NaCl solution makes the final concentration of 200mM of NaCl in mixed system, then adds 100 uL variable concentrations (0-in this mixed solution Target dna 300nM) carries out Complementary hybridization (hybridization buffer: containing the phosphate-buffered of the NaCl solution that concentration is 200mM Liquid), after reacting 10 minutes, use spectrophotometer to detect 520 nm and the absorbance value of 650 nm wavelength, its result such as Fig. 4 Shown in, it is derived from standard curve as shown in Figure 5.The lowest detectable limit of single stranded DNA be can reach 0.1 nM, detect dynamic range 0.3-300 nM, can reach 3 orders of magnitude.
In the present embodiment, AuNP-DNA1、AuNP-DNA2Before hybridizing with target dna, in course of reaction, react After completing, being characterized also for reactant mixture, wherein typical characterization result is respectively as shown in Fig. 3 a-Fig. 3 c.
Embodiment 2
(1) AuNP and DNA coupling
The golden nanometer particle (concentration is 2nM) of average out to 30nm reduction of sodium citrate method method prepared and 5 ' ends contain The DNA of poly adenine (PolyAx, x=15)1, 3 ' ends are containing PolyAx(x=15) DNA2Carry out coupling respectively and form AuNP- DNA1、AuNP-DNA2Complex: golden nanometer particle is concentrated twice, takes the concentrated solution 1mL of AuNP and is separately added into 30uL concentration and be The DNA of 100uM1、DNA2Solution, is subsequently adding the sodium citrate buffer solution that pH value is 3 that 20uL concentration is 500mM, and concussion is mixed Even, hatch 3 minutes and be centrifuged, remove free DNA1、DNA2Molecule, resuspended to original volume with water, adding 30uL concentration is The DNA of 100uM1、DNA2With the dATP solution that 10uL concentration is 100uM, the most every two hours adding a 20uL concentration is 0.1M NaCl solution, making in system containing concentration is the NaCl of the PB of 0.01M, concentration 100mM, after continuing to hatch 40h, centrifugal three Secondary, remove free DNA1、DNA2, dATP molecule, and with containing the concentration PB as 0.1M, the Nacl buffer weight of concentration 100mM Hang to original volume, obtain AuNP-DNA1And AuNP-DNA2Complex.
(2) AuNP-DNA1And AuNP-DNA2Mixed solution and target dna hybridize
Take the AuNP-DNA that 50 uL steps (1) are prepared respectively1And AuNP-DNA2With the concentration ratio mixing of 1:1, add NaCl makes the final concentration of 200mM of NaCl in mixed system, then adds the target of 100uL variable concentrations in this mixed solution DNA carries out Complementary hybridization (hybridization buffer: containing the phosphate buffer of the NaCl that concentration is 200mM).After reacting 15 minutes, Spectrophotometer is used to detect 520 nm and the absorbance value of 650 nm wavelength.And according to the obtained testing result of the present embodiment, also The standard curve close with embodiment 1 can be drawn.
Embodiment 3
(1) AuNP and DNA coupling
The golden nanometer particle that mean diameter is 50nm (concentration 1 nM) prepared by reduction of sodium citrate method method and 5 ' ends DNA containing poly adenine (PolyAx, x=20)1, 3 ' ends are containing PolyAx(x=20) DNA2Carry out coupling formation respectively AuNP-DNA1、AuNP-DNA2Complex: golden nanometer particle is concentrated twice, taking the concentrated solution 1mL of AuNP, to be separately added into 30uL dense The DNA of degree 100uM1、DNA2, it is subsequently adding 20uL concentration 500mM, pH value is the sodium citrate buffer solution of 3, concussion mixing, incubate Educate 3 minutes and be centrifuged, remove free DNA1、DNA2Molecule, resuspended to original volume with the PB of 0.01M, add 30uL100uM's DNA1、DNA2, and the Mercaptoundecanoic acid of 10uL10 uM, the most every two hours add the NaCl that 20uL concentration is 0.1M molten Liquid, makes containing concentration 0.01M PB, 100mM NaCl in system, after continuing to hatch 40h, centrifugal three times, removes free DNA1、 DNA2, dATP molecule, and with resuspended to original volume containing concentration 0.1M PB, 100mM Nacl buffer, obtain AuNP-DNA1With AuNP-DNA2Complex.
(2) AuNP-DNA1And AuNP-DNA2Mixed solution and target dna hybridize
Take the AuNP-DNA that 50 uL steps (1) are prepared respectively1And AuNP-DNA2With the concentration ratio mixing of 1:1, add NaCl makes the final concentration of 200mM of NaCl in mixed system, then adds the target of 100uL variable concentrations in this mixed solution DNA carries out Complementary hybridization (hybridization buffer: containing the phosphate buffer of concentration 200mM NaCl).After reacting 10 minutes, adopt Detect 520 nm and the absorbance value of 650 nm wavelength with spectrophotometer, and according to the obtained testing result of the present embodiment, also may be used Draw the standard curve close with embodiment 1.
It will be appreciated by those skilled in the art that principle and effect thereof of above-described embodiment only illustrative present invention, and Non-for limiting the present invention.Any person skilled in the art all can be under the spirit and the scope of the present invention, to above-mentioned Embodiment is modified or changes.Therefore, art has usually intellectual such as taken off without departing from the present invention All equivalences completed under the spirit shown and technological thought are modified or change, and must be contained by the claim of the present invention.

Claims (4)

1. the preparation method of non-sulfydryl nucleic acid-nanometer gold conjugate, it is characterised in that including:
(1) end is contained poly adenine PolyAx oligonucleotide add particle diameter be 15-50nm golden nanometer particle point Dissipate liquid, and add sodium citrate buffer solution mix homogeneously, hatch, the described oligonucleotide that then centrifugal segregation is free, and retain Described oligonucleotide and the complex of golden nanometer particle, x >=10；
(2) step (1) obtained oligonucleotide is resuspended with neutral phosphate buffer liquid with the complex of golden nanometer particle, then The described oligonucleotide of secondary addition, closed reagent and sodium chloride solution, hatch again, and is centrifuged off the described oligonucleoside dissociated Acid and closed reagent, it is thus achieved that described non-sulfydryl nucleic acid-nanometer gold conjugate；
Wherein, described closed reagent is selected from dATP or Mercaptoundecanoic acid.
2. the application in preparation DNA detection test kit of the method described in claim 1.
3. a DNA detection test kit, it is characterised in that including:
First probe, the first oligonucleotide mainly being contained poly adenine PolyAx by 5 ' ends is wanted through right with golden nanometer particle Seek the non-sulfydryl nucleic acid made by method described in 1-nanometer gold conjugate composition；
Second probe, the second oligonucleotide mainly being contained poly adenine PolyAx by 3 ' ends is wanted through right with golden nanometer particle Seek the non-sulfydryl nucleic acid made by method described in 1-nanometer gold conjugate composition；
And, in order to constitute the auxiliary reagent of the liquid phase environment being adapted for nucleic acid hybridization reaction, described auxiliary reagent pH value= 7.4, and containing 150-300mM NaCl, concentration be the phosphate buffer of 0.01M；
Wherein, described first oligonucleotide and the second oligonucleotide are able to and the different IPs sequence complementary in target dna Hybridization, x >=10, the particle diameter of described golden nanometer particle is 15-50nm；
The using method of described test kit includes: described first probe and the second probe mixed, and add may be containing can With described first oligonucleotide and the sample of the target dna of the nucleotide sequence of the second oligonucleotide Complementary hybridization, it is being adapted for The liquid phase environment of nucleic acid hybridization reaction carries out Complementary hybridization reaction, by measuring the visible absorption intensity of hybridization system Change, it is achieved the detection to sample, the wavelength of described visible ray is 520nm, 650nm.
DNA detection test kit the most according to claim 3, it is characterised in that the using method of described test kit is specifically wrapped Include:
Take described first probe and the second probe uniformly mixes according to the mol ratio of 1:1, and add hybridization buffer and a series of Having the target dna standard sample of variable concentrations, form a series of different mixed system and carry out nucleic acid hybridization reaction, reaction terminates After, detect a series of different blended zoarium series visible-light absorbance value, thereby determine that what optical absorption intensity changed with target DNA concentration Standard curve；
And, take described first probe and the second probe uniformly mixes according to the mol ratio of 1:1, and add hybridization buffer and treat Detection sample carries out nucleic acid hybridization reaction, and reaction detects the visible ray absorbance value of obtained mixed reactant, and foundation after terminating Described standard curve and calculate the concentration of target dna contained by detected sample.
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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
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US20110171749A1 (en) * 2009-03-02 2011-07-14 Board Of Trustees Of Michigan State University Nanoparticle tracer-based electrochemical dna sensor for detection of pathogens-amplification by a universal nano-tracer (aunt)
CN103540651B (en) * 2012-07-12 2017-05-10 中国科学院上海应用物理研究所 Nanometer gold complex as well as preparation and application thereof
CN103007294A (en) * 2012-12-28 2013-04-03 中国科学院上海应用物理研究所 Multivalent immunostimulation nanoformula, preparation method and application thereof

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