CN103926245B - A kind of gold nano colorimetric sensor of DNA modification, and preparation method thereof and purposes - Google Patents
A kind of gold nano colorimetric sensor of DNA modification, and preparation method thereof and purposes Download PDFInfo
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- 239000010931 gold Substances 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 18
- 230000008836 DNA modification Effects 0.000 title claims abstract description 12
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 63
- 235000019152 folic acid Nutrition 0.000 claims abstract description 35
- 239000011724 folic acid Substances 0.000 claims abstract description 35
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229960000304 folic acid Drugs 0.000 claims abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000002245 particle Substances 0.000 claims abstract description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 12
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 6
- 238000006722 reduction reaction Methods 0.000 claims abstract description 6
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 43
- 239000007853 buffer solution Substances 0.000 claims description 23
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 229940064302 folacin Drugs 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 6
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims description 6
- 229960002317 succinimide Drugs 0.000 claims description 6
- 239000006185 dispersion Substances 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 claims description 3
- 230000000007 visual effect Effects 0.000 claims description 3
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 6
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 238000012800 visualization Methods 0.000 abstract description 3
- XEIPQVVAVOUIOP-UHFFFAOYSA-N [Au]=S Chemical compound [Au]=S XEIPQVVAVOUIOP-UHFFFAOYSA-N 0.000 abstract description 2
- 230000000295 complement effect Effects 0.000 abstract description 2
- 239000002773 nucleotide Substances 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 238000011897 real-time detection Methods 0.000 abstract description 2
- 230000009870 specific binding Effects 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 abstract 1
- 238000000862 absorption spectrum Methods 0.000 description 8
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 3
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012455 bioassay technique Methods 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention relates to the gold nano colorimetric sensor of a kind of DNA modification, and preparation method thereof and purposes, use chemical reduction method to synthesize golden nanometer particle, utilize gold sulfide linkage to be modified by the DNA (S1) having modified sulfydryl and prepare S1 Au NPs to particle surface.FR can be with folic acid (FA) specific binding formation FR FA; the DNA (FA DNA) making FA modify is not hydrolyzed by nucleotide excision enzyme I (Exo I); thus reach to protect the purpose of FA DNA; utilize the complementary pairing principle of base between this character and DNA sequence, construct S1 Au NPs colorimetric sensor and achieve the detection FR of directly selecting property.Colorimetric sensor of the present invention preparation is simple, consumes energy low, and selectivity is high, quick and visualization, it is adaptable to the real-time detection of FR, it is expected to be applied to clinical diagnosis.
Description
Technical field
The invention belongs to bioassay technique field, be specifically related to the preparation of gold size solution colorimetric sensor and to folacin receptor
Detection.
Background technology
The vital effect that folacin receptor (FR) has in physiology, pharmacology and cytopathy process, therefore directly selecting property
Detection FR expression in malignant tumor significant.In past 20 years, FRET (fluorescence resonance energy transfer) (FRET), glimmering
Photoanisotropy, capillary electrophoresis (CE), surface plasma body resonant vibration (SPR) etc. are the methods of the most frequently used detection FR.But,
The defect that these methods are intrinsic, as required instrument costliness, complicated, limits it and develops into the important hands detecting FR in real time
Section.
Summary of the invention
It is an object of the invention to provide the gold nano colorimetric sensor of a kind of DNA modification, and preparation method thereof and purposes.This
Invention uses chemical reduction method to synthesize golden nanometer particle, utilizes gold sulfide linkage that the DNA (S1) having modified sulfydryl is modified to particle table
S1-Au NPs is prepared in face.FR can with folic acid (FA) specific binding formation FR-FA, make DNA (FA-DNA) that FA modifies not by
Nucleotide excision enzyme-I (Exo I) is hydrolyzed, thus reaches to protect the purpose of FA-DNA, utilizes between this character and DNA sequence
The complementary pairing principle of base, constructs S1-Au NPs colorimetric sensor and achieves the detection FR of directly selecting property.
Concrete technical scheme is as follows:
The preparation method of the gold nano colorimetric sensor of a kind of DNA modification, comprises the steps:
(1) DNA sequence (sulfhydrylation: S1, FA-DNA:S2) is dissolved in Tris-HCl buffer solution respectively, and saves backup；
(2) FA is attached on the S2 of 3 ' terminal modified amino；
(3) cultivate a few hours in dark after finite concentration S2 and Tris-HCl buffer solution is mixed homogeneously with certain proportion；
(4) this FA-S2 solution is dialysed in PBS buffer solution, separates unnecessary FA；
(5) FR is joined in the above-mentioned solution dialysed, dark is further cultured for a period of time；
(6) addition ExoI is in above-mentioned mixed solution, has hydrolyzed rear S2/FA-FR and has been formed；
(7) being mixed homogeneously with S2/FA-FR solution in step (6) by S1-AuNP, vibration is to obtain more stable solution.
Further, Tris-HCl buffer solution pH7.4 in step (1), and save backup at 4 DEG C.
Further, step (2) use butanimide coupled method EDC-NHS FA is attached to 3 ' terminal modified amino
On S2.
Further, the pH7.4 of Tris-HCl buffer solution in step (3), containing 10mMFA, 1mMEDC, 5mMSulfo-NHS,
And cultivate in 37 DEG C of dark；And/or, in step (3), 0.5mL20 μM of S2 Yu 0.5mL100mMTris-HCl buffering
In 37 DEG C of dark, 3h is cultivated after solution mix homogeneously.
Further, buffer solution pH7.4 in step (4), unnecessary FA molecular weight cutoff: 1000Da.
Further, in step (5), FR is standard series concentration, cultivates in 2h, and/or step (6) in 37 DEG C of dark
Add 400U/mLExoI, and/or hydrolyze 30 minutes.
Further, in step (7), S2/FA-FR solution mix homogeneously in 400 μ LS1-AuNPs and 200 μ L step (6),
Vibration 5 minutes is then molten by obtaining series standard colorimetric after cultivation 2h in this solution and dark to obtain more stable solution gently
Liquid；And/or, in step (7), the preparation of S1-AuNPs uses: utilizes chemical reduction method, first synthesizes the AuNPs of size uniform
Dispersion soln, then the DNA-S1 having sulfydryl modification is modified particle surface.
The gold nano colorimetric sensor of a kind of DNA modification, it is prepared by above-mentioned method.
The purposes of above-mentioned gold nano colorimetric sensor, protects colorimetric detection folacin receptor based on tail end.
The purposes of above-mentioned gold nano colorimetric sensor, it is adaptable to the visual detection in real time of FR, detection range is about 0.5to50 μ g/mL.
Compared with currently available technology, colorimetric sensor of the present invention preparation is simple, consumes energy low, and selectivity is high, quickly
And visualization, it is adaptable to the real-time detection of FR, it is expected to be applied to clinical diagnosis.S1 Yu FR protection on golden nanometer particle
S2/FA between hybridization cause Gold nanoparticle polymerization so that solution colour occur from claret to navy blue substantially
Change, therefore can reach the purpose with the naked eye detecting FR concentration.Prepared Colorimetric System, detection range is about 0.5to50
μg/mL.This Colorimetric System product is stable, and preparation process is simple, inexpensive, it is expected to for deficient remote of medical resource
The detection of FR concentration in the cell of area.This be not only simply by detection FR for treatment of cancer provide one potential
Approach, the openst development prospect of conjoint therapyLian Hezhiliaofa.
Accompanying drawing explanation
Fig. 1 is colorimetric sensor visual detection FR schematic diagram；
The SEM figure that Fig. 2 (a) is the Au NPs of preparation；
Fig. 2 (b) is ultra-violet absorption spectrum, and a is the ultra-violet absorption spectrum of golden nanometer particle, and b is the golden nanometer particle that S1 modifies
Ultra-violet absorption spectrum；
The golden nanometer particle ultra-violet absorption spectrum that the S1 that Fig. 3 (a) is preparation modifies, a is without S2/FA-FR ultra-violet absorption spectrum, b
There is ultra-violet absorption spectrum during S2/FA-FR；
The golden nanometer particle that the S1 that Fig. 3 (b) is preparation modifies is schemed without S2/FA-FR SEM；
The golden nanometer particle that the S1 that Fig. 3 (c) is preparation modifies is schemed containing S2/FA-FR SEM；
Fig. 4 (a) is ultra-violet absorption spectrum corresponding after adding series standard concentration FR；
Fig. 4 (b) is the linear relationship chart of absorptance and concentration；
The uv absorption spectra of Fig. 5 (a) FR, Thrombin, BSA；
Fig. 5 (b) FR, Thrombin, BSA for the colorimetric sensor selectivity to FR.
Detailed description of the invention
Describing the present invention below according to accompanying drawing, it is a kind of preferred embodiment in numerous embodiments of the present invention.
Embodiment one:
The gold nano colorimetric sensor of a kind of DNA modification, protects colorimetric detection folacin receptor based on tail end.A kind of golden nanometer particle ratio
The preparation method of colour sensor, specifically includes:
A, by buy DNA sequence (sulfhydrylation: S1, FA-DNA:S2) be dissolved in 0.05M Tris-HCl (pH7.4) respectively
In buffer solution, and save backup at 4 DEG C.
FA is attached on the S2 of 3 ' terminal modified amino by b, use butanimide coupled method (EDC-NHS).0.5mL20μM S2
With 0.5mL100mM Tris-HCl buffer solution (pH7.4, containing 10mM FA, 1mM EDC, 5mM Sulfo-NHS)
In 37 DEG C of dark, 3h is cultivated after mix homogeneously.This FA-S2 solution (pH7.4) in PBS buffer solution is dialysed, and moves
Walk unnecessary FA (molecular weight cutoff: 1000Da).Again the FR of standard series concentration is joined in the above-mentioned solution dialysed,
37 DEG C of dark are further cultured for 2h.Finally, 400U/mL Exo I is in above-mentioned mixed solution in addition, S2/FA-FR after hydrolyzing 30 minutes
Formed.
S2/FA-FR solution mix homogeneously in c, 400 μ L S1-Au NPs and 200 μ L (b), gently vibration 5 minutes with
Obtain more stable solution, then this solution is obtained series standard colorimetric solution with dark after cultivation 2h.
Embodiment two:
The gold nano colorimetric sensor of a kind of DNA modification, protects colorimetric detection folacin receptor based on tail end.Based on S1-AuNP with
The colorimetric sensor preparation of S2/FA-FR and applying step are as follows:
A, by buy DNA sequence (sulfhydrylation: S1, FA-DNA:S2) be dissolved in respectively in Tris-HCl (pH7.4) buffer solution,
And save backup at 4 DEG C.
FA is attached on the S2 of 3 ' terminal modified amino by b, use butanimide coupled method (EDC-NHS).Finite concentration S2 with
Tris-HCl buffer solution (pH7.4, containing 10mM FA, 1mM EDC, 5mM Sulfo-NHS) is with certain proportion mixing all
Cultivate a few hours in 37 DEG C of dark after even.This FA-S2 solution (pH7.4) in PBS buffer solution is dialysed, and separation is many
Remaining FA (molecular weight cutoff: 1000Da).Again the FR of standard series concentration is joined in the above-mentioned solution dialysed, 37 DEG C
Dark is further cultured for a period of time.Finally, 400U/mL Exo I is in above-mentioned mixed solution in addition, has hydrolyzed rear S2/FA-FR
Formed.
C, being mixed homogeneously with S2/FA-FR solution in (b) by S1-AuNP, vibration several minutes is to obtain more stable solution gently,
Then after this solution and dark cultivate several hours, series standard colorimetric solution is obtained.
Embodiment three:
The preparation of S1-Au NPs: utilize chemical reduction method, first synthesizes the Au NPs dispersion soln of size uniform (14nm),
Again the DNA-S1 having sulfydryl modification is modified particle surface.
Colorimetric sensor based on S1-Au NPs with S2/FA-FR preparation and applying step are as follows:
A, by buy DNA sequence (sulfhydrylation: S1, FA-DNA:S2) be dissolved in 0.05M Tris-HCl (pH7.4) respectively
In buffer solution, and save backup at 4 DEG C.
FA is attached on the S2 of 3 ' terminal modified amino by b, use butanimide coupled method (EDC-NHS).0.5mL20μM S2
With 0.5mL100mM Tris-HCl buffer solution (pH7.4, containing 10mM FA, 1mM EDC, 5mM Sulfo-NHS)
Cultivate 3 hours in 37 DEG C of dark after mix homogeneously.This FA-S2 solution (pH7.4) in PBS buffer solution is dialysed,
Separate unnecessary FA (molecular weight cutoff: 1000Da).Again the FR of standard series concentration is joined in the above-mentioned solution dialysed,
37 DEG C of dark are further cultured for 2 hours.Finally, 400U/mL Exo I is in above-mentioned mixed solution, after hydrolyzing 30 minutes in addition
S2/FA-FR is formed.
S2/FA-FR solution mix homogeneously in c, 400 μ L S1-Au NPs and 200 μ L (b), gently vibration 5 minutes with
Obtain more stable solution, then after this solution and dark are cultivated 2 hours, obtain series standard colorimetric solution.
Embodiment four:
The preparation of S1-AuNP: utilize chemical reduction method, first synthesizes the Au NPs dispersion soln of size uniform (14nm), then
The DNA-S1 having sulfydryl modification is modified particle surface.
Colorimetric sensor based on S1-AuNP with S2/FA-FR preparation and applying step are as follows:
A, by buy DNA sequence (sulfhydrylation: S1, FA-DNA:S2) be dissolved in 0.05M Tris-HCl (pH7.4) respectively
In buffer solution, and save backup at 4 DEG C.
FA is attached on the S2 of 3 ' terminal modified amino by b, use butanimide coupled method (EDC-NHS).Finite concentration S2 with
100mM Tris-HCl buffer solution (pH7.4, containing 10mM FA, 1mM EDC, 5mM Sulfo-NHS) is with certain proportion
Cultivate a few hours in 37 DEG C of dark after mix homogeneously.This FA-S2 solution (pH7.4) in PBS buffer solution is dialysed,
Separate unnecessary FA (molecular weight cutoff: 1000Da).Again the FR of standard series concentration is joined in the above-mentioned solution dialysed,
37 DEG C of dark are further cultured for a period of time.Finally, 400U/mL Exo I is in above-mentioned mixed solution, after having hydrolyzed in addition
S2/FA-FR is formed.
C, being mixed homogeneously with S2/FA-FR solution in (b) by S1-AuNP, vibration several minutes is to obtain more stable solution gently,
Then after this solution and dark cultivate several hours, series standard colorimetric solution is obtained.
Colorimetric sensor of the present invention preparation is simple, consumes energy low, and selectivity is high, quick and visualization, it is adaptable to the reality of FR
Time detection, it is expected to be applied to clinical diagnosis.Hybridization between the S2/FA of S1 and the FR protection on golden nanometer particle causes Jenner
The polymerization of rice ion so that solution colour occurs from claret to navy blue significant change, therefore can reach and with the naked eye detects FR
The purpose of concentration.This Colorimetric System product is stable, and preparation process is simple, inexpensive, it is expected to for deficient inclined of medical resource
The detection of FR concentration in the cell of remote area.This be not only simply by detection FR for treatment of cancer provide one potential
Approach, the openst development prospect of conjoint therapyLian Hezhiliaofa.
Above in conjunction with accompanying drawing, the present invention is exemplarily described, it is clear that the present invention implements and is not subject to the restrictions described above,
As long as have employed method design and the various improvement that carry out of technical scheme of the present invention, or the most improved directly apply to other occasion
, all within protection scope of the present invention.
Claims (6)
1. the preparation method of the gold nano colorimetric sensor of a DNA modification, it is characterised in that comprise the steps:
(1) DNA sequence being dissolved in respectively in the Tris-HCl buffer solution of pH7.4, and save backup at 4 DEG C, described DNA sequence is DNA sequence S1 of sulfhydrylation, DNA sequence S2 of 3 ' terminal modified amino；
(2) butanimide coupled method EDC-NHS is used to be attached to by folic acid FA on the S2 of 3 ' terminal modified amino:
20 μMs of S2 with 0.5mL 100mM Tris-HCl buffer solution of 0.5mL cultivate 3h, the wherein pH7.4 of Tris-HCl buffer solution, buffer solution FA Han 10mM, 1mM EDC, 5 mM after mixing homogeneously in 37 DEG C of dark
N-hydroxy thiosuccinimide；
(3) FA-S2 solution is dialysed in the PBS buffer solution of pH7.4, separates unnecessary FA, the most unnecessary FA molecular weight cutoff: molecular cut off is 1000Da；
(4) folacin receptor FR is joined in the above-mentioned solution dialysed, dark is further cultured for a period of time；
(5) addition ExoI enzyme is in the mixed solution that step (4) obtains, and has hydrolyzed rear S2/FA-FR and has been formed；
(6) first with the AuNPs dispersion soln of chemical reduction method synthesis size uniform, again DNA sequence S1 having sulfydryl modification is modified particle surface, then being mixed homogeneously with S2/FA-FR solution in step (5) by S1-AuNP, vibration is to obtain more stable solution.
2. the preparation method of the gold nano colorimetric sensor of DNA modification as claimed in claim 1, it is characterized in that, in step (4), FR is standard series concentration, cultivates 2h in 37 DEG C of dark, and/or step (5) adds 400U/mL ExoI enzyme, and/or hydrolyze 30 minutes.
3. the preparation method of the gold nano colorimetric sensor of DNA modification as claimed in claim 1 or 2, it is characterized in that, in step (6), S2/FA-FR solution mix homogeneously in 400 μ L S1-AuNPs and 200 μ L step (5), vibration obtains series standard colorimetric solution to obtain more stable solution in 5 minutes after then this solution being cultivated 2h in dark gently.
4. the gold nano colorimetric sensor of a DNA modification, it is characterised in that it is prepared by the method described in claim 1.
5. the purposes of gold nano colorimetric sensor described in a claim 4, it is characterised in that protect colorimetric detection folacin receptor based on tail end.
6. the purposes of gold nano colorimetric sensor described in a claim 4, it is characterised in that being applicable to the visual detection in real time of FR, detection range is 0.5-50 μ g/mL.
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