CN103320515B - Method for detecting DNA (Deoxyribonucleic Acid) based on chiral tetrahedron conformation change - Google Patents
Method for detecting DNA (Deoxyribonucleic Acid) based on chiral tetrahedron conformation change Download PDFInfo
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Abstract
The invention discloses a method for detecting DNA (Deoxyribonucleic Acid) based on chiral tetrahedron conformation change and belongs to the technical field of nanobiology. The invention relates to the preparation of a chiral nanometer material applied to DNA ultra-sensitive detection and a detection method of the DNA. The method comprises the synthesis of a gold nanoparticle, the synthesis of a DNA-gold nanoparticle composite, the assembly of a gold nanoparticle tetrahedron, the construction of a chiral tetrahedron and the establishment of a target DNA chiral sensor. The invention provides a preparation method of a novel chiral material; according to the method, an achiral nanometer material is converted to a chiral nanometer material based on the original achiral nanometer assembly material by introducing foreign DNA reconstructing the geometrical conformation; and the fast, effective, and ultrasensitive detection of the target DNA at an Ahmed Ammor (10-18) level can be achieved.
Description
Technical field
Change the method that DNA is detected based on chirality tetrahedral conformation, belong to field of nano biotechnology.Relate to a kind of preparation of the chiral nanomaterial for DNA super sensitivity detection and the detection method of DNA.
Background technology
Thymus nucleic acid (DNA) is the undertaker of genetic information, can form genetic command, guides biological development and vital functions to operate.Because the variation of base sequence in DNA molecular is relevant with the many genetic diseasess of the mankind, therefore, to the analysis of the DNA of particular sequence and to the detection of base mutation in DNA chain, there is in the early diagnosis and therapy of genescreen, genetic diseases very profound significance.The detection method of tradition DNA is mainly based on polymerase chain reaction (polymerase chain reaction, PCR).PCR is a kind of important technology of amplification of DNA fragments in vitro.When there is template DNA, substrate, upstream and downstream primer and heat-resisting archaeal dna polymerase, through the working cycle of repeatedly " denaturation renaturation-extension ", trace template DNA can be expanded to millions of times.Partial dna sequence can copy by round pcr, and signal amplifies, and represents the limit of detection in sensitivity.However, still there are some intrinsic shortcomings in round pcr, as high in cost, need professional skill and equipment and rely on polysaccharase operation, easily there is false positive etc., these features all seriously hinder the widespread use of PCR.
Along with the rise of nanoscale science and technology, nano material has become the mainstay preparing biosensor due to the physics and chemistry attribute of its uniqueness.Nano material is the material of size between 1-100nm scope of a class formation unit, and because its size is close to the coherence length of electronics, great changes will take place because the strong relevant self-organization brought makes character for its character.Further, its yardstick is close to the wavelength of light, and add that it has the special effects on large surface, therefore its characteristic showed, such as fusing point, magnetic, optics, heat conduction, conductive characteristic etc., be often different from the character that this material shows when integrality.Nano-sensor is because instrument is simple, cheap, mensuration has quick and precisely been widely used in the detection of environment, medicine, food etc. with method sensitivity high.Chiral nanomaterial combines mirror symmetry in the special physical properties of nano material and chiral molecules chemical structure, and chiral signal can be transferred to visible region from ultraviolet region by this advanced composite material, thus has greatly expanded its range of application.
Summary of the invention
The object of this invention is to provide and a kind ofly change the method that DNA is detected based on chirality tetrahedral conformation, relate to a kind of preparation of the chiral nanomaterial for DNA super sensitivity detection and the detection method of DNA.
Technical scheme of the present invention: a kind ofly change the method that DNA is detected based on chirality tetrahedral conformation,
Comprise the foundation of the synthesis of golden nanometer particle, the finishing of golden nanometer particle, the synthesis of DNA-golden nanometer particle mixture, the tetrahedral assembling of golden nanometer particle, the tetrahedral structure of chirality and target dna chiral sensor.Processing step is:
(1) synthesis of golden nanometer particle
Citric acid and Weibull two step synthesis 10nm golden nanometer particle;
(2) finishing of golden nanometer particle
Two (p-sulfonyl-phenyl) the Phenylphosphine di-potassium solution of golden nanometer particle two hydrations that step (1) is synthesized wraps up, and makes nanoparticle surface negative charge more, to improve the stability of Radioactive colloidal gold in high level salt solution;
(3) synthesis of DNA-golden nanometer particle mixture
The golden nanometer particle of finishing that step (2) is obtained and the DNA hybrid reaction according to a certain percentage of end strips sulfydryl; DNA used has four kinds (Y1, Y2, Y3, Y4), forms four kinds of different Au-DNA mixtures (Au-Y1, Au-Y2, Au-Y3, Au-Y4);
(4) the tetrahedral assembling of golden nanometer particle
Four kinds of Au-DNA mixtures that step (3) is synthesized, equal proportion Hybrid assembling forms golden nanometer particle tetrahedron;
(5) the tetrahedral structure of chirality
In four kinds of DNA that step (3) is used, wherein embedded in section of DNA ring (containing 13 bases) in the middle of Y3, so in the golden nanometer particle tetrahedron assembled in step (4), a DNA ring is contained on one of them limit.When adding the sequence Y5 with this segment DNA ring complete complementary, DNA circle is opened, distance in tetrahedron between two particles becomes large, and originally symmetrical golden nanometer particle tetrahedron becomes unsymmetrical structure, thus makes originally do not have the structure of chiral signal to occur chiral signal;
(6) foundation of target dna chiral sensor
Method according to step (5) builds the DNA sensor detected based on chirality.
Described changes based on chirality tetrahedral conformation the method detected DNA, and step is:
(1) synthesis of golden nanometer particle
The method of citric acid and Weibull two step synthesis golden nanometer particle is: get two clean triangular flasks respectively, add 158mL ultrapure water and 2mL 1% hydrochloro-auric acid in A bottle; 8mL 1% trisodium citrate is added, 0.2mL 1% Weibull, 0.2mL 25mM salt of wormwood, 31.6mL ultrapure water in B bottle.A, B liquid is all heated to 60 DEG C, then under high velocity agitation B liquid is added rapidly in A liquid, and mixed solution continues to stir 40min to formation dark red solution at 60 DEG C.Last cool to room temperature forms the stable golden nanometer particle of citric acid, and transmission electron microscope display median size is 10 ± 2nm.
(2) finishing of golden nanometer particle
The stable golden nanometer particle of the 10nm citric acid that synthesizes of step (1) gets 100mL in centrifuge tube, it is 50nM that 13000r/min is concentrated into final concentration, add two (p-sulfonyl-phenyl) the Phenylphosphine di-potassium solution of 10 μ L 20mg/mL bis-hydrations, room temperature concussion reaction 10h; Remove supernatant liquor with after the centrifugal 10nm of 13000r/min, add ultrapure water and return to original volume;
(3) synthesis of DNA-golden nanometer particle mixture
The golden nanometer particle of the finishing that step (2) is obtained respectively gets 50 μ L in four PCR pipe, four kinds of DNA:Y1 of 1 μ L 10 μMs are sequentially added separately in 4 pipes, Y2, Y3 and Y4, after mixing, 5 μ L 5 × tris-borate buffers and 1.25 μ L 1M NaCl solution are added, rocked at room temperature reaction 2h in each system; Synthetic centrifugal 10 min of Au-DNA mixture 13000r/min, remove supernatant liquor, and precipitation adds 1 × tris-borate buffer to original volume, form four kinds of different Au-DNA mixtures: Au-Y1, Au-Y2, Au-Y3, Au-Y4;
(4) the tetrahedral assembling of golden nanometer particle
By mixture (Au-Y1, Au-Y2, the Au-Y3 of four kinds of Au-DNA that step (3) is synthesized, Au-Y4) respectively get 50 μ L to be mixed in 1.5mL centrifuge tube, add 5 μ L 1M NaCl solution, room temperature reaction spends the night, form symmetrical golden nanometer particle tetrahedron, this structure does not have chiral signal.
(5) the tetrahedral structure of chirality
In the preparation process of step (4), add the sequence Y5 of the DNA circle complete complementary embedded with Y3, now Y5 can, with the DNA circle Complementary hybridization in Au-Y3, make DNA circle open completely, and the distance in tetrahedron between two nanoparticles widens.Originally the symmetrical tetrahedral conformation of golden nanometer particle is changed, and become asymmetric space tetrahedral structure, this structure shows obvious characteristic peak at circular dichroism spectrum 520nm place.
(6) foundation of target dna chiral sensor
Based on the method establishment target dna chiral sensor of step (5), concrete operations are: the target dna (Y5) adding different concns in the preparation process of step (4), according to the ratio of 520nm place circular dichroism signal and the concentration Criterion curve of target dna.In this experiment, the detection of DNA is limited to 43aM, and linearity range is at 0.05fM-50fM.
The sequence of DNA used in experiment:
Y1：5’-TTT GCC TGG AGA TAC ATG CAC ATT ACG GCT TTC CCT ATT AGA AGG TCT CAG GTG CGC GTT TCG GTA AGT AGA CG-3’
Y2：5’- TTT CGC GCA CCT GAG ACC TTC TAA TAG GGT TTG CGA CAG TCG TTC AAC TAG AAT GCC CTT TGG GCT GTT CCG GGT GTG GCT CGT CGG-3’
Y3：5’-TTT GGC CGA GGA CTC CTG CTC CGC TGC GGT TTG GCG AAC TGG TCC TC ATG TCT CAT CT CGT CTA CTT ACC GTT TCC GAC GAG CCA CAC CCG GAA CAG CCC-3’
Y4：5’-TTT GCC GTA ATG TGC ATG TAT CTC CAG GCT TTC CGC AGC GGA GCA GGA GTC CTC GGC CTT TGG GCA TTC TAG TTG AAC GAC TGT CGC-3’
Y5：5’-AG ATG AGA CAT GA GGA CCA GTT CGC C-3’。
Note: DNA used in the present invention all purchased from Chinese Shanghai Sheng Gong biotechnology company limited, and carries out purifying by polyacrylamide gel electrophoresis.
Beneficial effect of the present invention: the invention discloses a kind of induction by target dna is the method for chiral structure achirality structural transformation.Based on this method, we establish the novel method of a kind of signal intensity rapid detection DNA according to circular dichroism spectrum.The method has started a kind of construction process of novel hand-type structure, achieves quick, efficient, the overdelicate chemical detection of DNA simultaneously.
Accompanying drawing explanation
Fig. 1: the tetrahedral transmission electron microscope picture of golden nanometer particle;
Fig. 2: the tetrahedral transmission electron microscope picture after the conformational change of target induction;
Fig. 3: add the circular dichroism change curve after different concns target dna (Y5) in golden nanometer particle tetrahedron; Along the direction of arrow, DNA concentration is followed successively by 0fM, 0.05fM, 0.5fM, 1fM, 5fM, 10fM, 50fM;
Fig. 4: the canonical plotting of DNA detection.
Embodiment
Embodiment 1:
(1) synthesis of golden nanometer particle
The method of citric acid and Weibull two step synthesis golden nanometer particle is: get two clean triangular flasks respectively, add 158mL ultrapure water and 2mL 1% hydrochloro-auric acid in A bottle; 8mL 1% trisodium citrate is added, 0.2mL 1% Weibull, 0.2mL 25mM salt of wormwood, 31.6mL ultrapure water in B bottle.A, B liquid is all heated to 60 DEG C, then under high velocity agitation B liquid is added rapidly in A liquid, and mixed solution continues stirring and within 40 minutes, arrives formation dark red solution at 60 DEG C.Last cool to room temperature forms the stable golden nanometer particle of citric acid, and transmission electron microscope display median size is 10 ± 2nm.
(2) finishing of golden nanometer particle
The golden nanometer particle that 10 citrates that step (1) is synthesized are modified gets 100mL in centrifuge tube, it is 50nM that 13000r/min is concentrated into final concentration, add two (p-sulfonyl-phenyl) the Phenylphosphine di-potassium solution of 10 μ L 20mg/mL bis-hydrations, room temperature concussion reaction 10h; With 13000r/min, remove supernatant liquor after centrifugal 10 min, add water and return to original volume;
(3) synthesis of DNA-golden nanometer particle mixture
The golden nanometer particle that step (2) has been modified respectively gets 50 μ L in four PCR pipe, sequentially adds the Y1 of 1 μ L 10 μMs separately, Y2 in 4 pipes, Y3 and Y4, after mixing, in each system, add 5 μ L 5 × tris-borate buffers and 1.25 μ L 1M NaCl solution, rocked at room temperature reaction 2h; Synthetic centrifugal 10 min of Au-DNA mixture 13000r/min, remove supernatant liquor, and precipitation adds 1 × tris-borate buffer to original volume;
(4) the tetrahedral assembling of golden nanometer particle
Mixture (Au-Y1, Au-Y2, the Au-Y3 of four kinds of Au-DNA prepared by step (3), Au-Y4) respectively get 50 μ L to be mixed in 1.5mL centrifuge tube, add 5 μ L 1M NaCl solution, room temperature reaction spends the night, form symmetrical golden nanometer particle tetrahedron, this structure does not have chiral signal.
(5) the tetrahedral structure of chirality
In the preparation process of step (4), add the sequence Y5 of the DNA circle complete complementary embedded with Y3, now Y5 can, with the DNA circle Complementary hybridization in Au-Y3, make DNA circle open completely, and the distance in tetrahedron between two nanoparticles widens.Originally the symmetrical tetrahedral conformation of golden nanometer particle is changed, and become asymmetric space tetrahedral structure, this structure shows obvious characteristic peak at circular dichroism spectrum 520nm place.
(6) foundation of target dna chiral sensor
Based on the method establishment target dna chiral sensor of step (5), concrete operations are: the target dna (Y5) adding different concns in the preparation process of step (4), according to the ratio of 520nm place circular dichroism signal and the concentration Criterion curve of target dna.In this experiment, the detection of DNA is limited to 43aM, and linearity range is at 0.05fM-50fM.
The sign of assembling tetrahedral structure:
The above-mentioned product assembled is carried out the centrifugal 10min of 13000r/min, abandons supernatant, precipitation is heavily distributed in the ultrapure water of 120 μ L, and ultrapure washing once.Electronic Speculum characterizes: the sample drop of the above-mentioned process of 7 μ L is added on the copper mesh of carbon film support, under infrared lamp, carry out drying.Projection Electronic Speculum adopts the Electronic Speculum of JEOL JEM-2100 model, and its acceleration voltage is 200 kV, as illustrated in fig. 1 and 2.Circular dichroism spectrum characterizes: get the system 100 μ L that assembles in cuvette, do blank with ultrapure water.Circular dichroism spectrometer adopts French Bio-Logic MOS-450+SMF-300, as shown in Figure 3.
Y1：5’-TTT GCC TGG AGA TAC ATG CAC ATT ACG GCT TTC CCT ATT AGA AGG TCT CAG GTG CGC GTT TCG GTA AGT AGA CG-3’
Y2：5’- TTT CGC GCA CCT GAG ACC TTC TAA TAG GGT TTG CGA CAG TCG TTC AAC TAG AAT GCC CTT TGG GCT GTT CCG GGT GTG GCT CGT CGG-3’
Y3：5’-TTT GGC CGA GGA CTC CTG CTC CGC TGC GGT TTG GCG AAC TGG TCC TC ATG TCT CAT CT CGT CTA CTT ACC GTT TCC GAC GAG CCA CAC CCG GAA CAG CCC-3’
Y4：5’-TTT GCC GTA ATG TGC ATG TAT CTC CAG GCT TTC CGC AGC GGA GCA GGA GTC CTC GGC CTT TGG GCA TTC TAG TTG AAC GAC TGT CGC-3’
Y5：5’-AG ATG AGA CAT GA GGA CCA GTT CGC C-3’
Claims (2)
1. change based on chirality tetrahedral conformation and be used for the method that DNA is detected of non-diagnostic object, it is characterized in that: the foundation of the synthesis of the synthesis of golden nanometer particle, the finishing of golden nanometer particle, DNA-golden nanometer particle mixture, the tetrahedral assembling of golden nanometer particle, the tetrahedral structure of chirality and target dna chiral sensor; Processing step is:
(1) synthesis of golden nanometer particle
Citric acid and Weibull two step synthesis 10nm golden nanometer particle;
(2) finishing of golden nanometer particle
Two (p-sulfonyl-phenyl) the Phenylphosphine di-potassium solution of golden nanometer particle two hydrations that step (1) is synthesized wraps up, and makes nanoparticle surface negative charge more, to improve the stability of Radioactive colloidal gold in high level salt solution;
(3) synthesis of DNA-golden nanometer particle mixture
The golden nanometer particle of finishing that step (2) is obtained and the DNA hybrid reaction according to a certain percentage of end strips sulfydryl; DNA used has four kinds: Y1, Y2, Y3, Y4, forms four kinds of different Au-DNA mixtures: Au-Y1, Au-Y2, Au-Y3, Au-Y4;
(4) the tetrahedral assembling of golden nanometer particle
Four kinds of Au-DNA mixtures that step (3) is synthesized, equal proportion Hybrid assembling forms golden nanometer particle tetrahedron;
(5) the tetrahedral structure of chirality
In four kinds of DNA that step (3) is used, wherein embedded in one section of DNA circle containing 13 bases in the middle of Y3, so in the golden nanometer particle tetrahedron assembled in step (4), a DNA ring is contained on one of them limit, when adding the sequence Y5 with this segment DNA ring complete complementary, DNA circle is opened, and the distance in tetrahedron between two particles becomes large, originally symmetrical golden nanometer particle tetrahedron becomes unsymmetrical structure, thus makes originally do not have the structure of chiral signal to occur chiral signal;
(6) foundation of target dna chiral sensor
Based on the method establishment target dna chiral sensor of step (5);
Y1：5’-TTT GCC TGG AGA TAC ATG CAC ATT ACG GCT TTC CCT ATT AGA AGG TCT CAG GTG CGC GTT TCG GTA AGT AGA CG-3’
Y2：5’- TTT CGC GCA CCT GAG ACC TTC TAA TAG GGT TTG CGA CAG TCG TTC AAC TAG AAT GCC CTT TGG GCT GTT CCG GGT GTG GCT CGT CGG-3’
Y3：5’-TTT GGC CGA GGA CTC CTG CTC CGC TGC GGT TTG GCG AAC TGG TCC TC ATG TCT CAT CT CGT CTA CTT ACC GTT TCC GAC GAG CCA CAC CCG GAA CAG CCC-3’
Y4：5’-TTT GCC GTA ATG TGC ATG TAT CTC CAG GCT TTC CGC AGC GGA GCA GGA GTC CTC GGC CTT TGG GCA TTC TAG TTG AAC GAC TGT CGC-3’
Y5：5’-AG ATG AGA CAT GA GGA CCA GTT CGC C-3’。
2. the method detected DNA being used for non-diagnostic object based on the change of chirality tetrahedral conformation according to claim 1, is characterized in that:
(1) synthesis of golden nanometer particle
The method of citric acid and Weibull two step synthesis golden nanometer particle is: get two clean triangular flasks respectively, add 158mL ultrapure water and 2mL 1% hydrochloro-auric acid in A bottle; 8mL 1% trisodium citrate is added, 0.2mL 1% Weibull, 0.2mL 25mM salt of wormwood, 31.6mL ultrapure water in B bottle; A, B liquid is all heated to 60 DEG C, then under high velocity agitation B liquid is added rapidly in A liquid, and mixed solution continues to stir 40min to formation dark red solution at 60 DEG C; Last cool to room temperature forms the stable golden nanometer particle of citric acid, and transmission electron microscope display median size is 10 ± 2nm;
(2) finishing of golden nanometer particle
The stable golden nanometer particle of the 10 nm citric acids that synthesize of step (1) gets 100mL in centrifuge tube, it is 50nM that 13000r/min is concentrated into final concentration, add two (p-sulfonyl-phenyl) the Phenylphosphine di-potassium solution of 10 μ L 20mg/mL bis-hydrations, room temperature concussion reaction 10h; Remove supernatant liquor with after the centrifugal 10min of 13000r/min, add ultrapure water and return to original volume;
(3) synthesis of DNA-golden nanometer particle mixture
The golden nanometer particle of the finishing that step (2) is obtained respectively gets 50 μ L in four PCR pipe, four kinds of DNA:Y1 of 1 μ L 10 μMs are sequentially added separately in 4 pipes, Y2, Y3 and Y4, after mixing, 5 μ L 5 × tris-borate buffers and 1.25 μ L 1M NaCl solution are added, rocked at room temperature reaction 2h in each system; Synthetic centrifugal 10 min of Au-DNA mixture 13000r/min, remove supernatant liquor, and precipitation adds 1 × tris-borate buffer to original volume, form four kinds of different Au-DNA mixtures: Au-Y1, Au-Y2, Au-Y3, Au-Y4;
(4) the tetrahedral assembling of golden nanometer particle
Mixture by four kinds of Au-DNA that step (3) is synthesized: Au-Y1, Au-Y2, Au-Y3, Au-Y4, respectively gets 50 μ L and is mixed in 1.5mL centrifuge tube, adds 5 μ L 1M NaCl solution, room temperature reaction spends the night, and form symmetrical golden nanometer particle tetrahedron, this structure does not have chiral signal;
(5) the tetrahedral structure of chirality
The sequence Y5 of the DNA circle complete complementary embedded with Y3 is added in the preparation process of step (4), now Y5 can with the DNA circle Complementary hybridization in Au-Y3, DNA circle is opened completely, distance in tetrahedron between two nanoparticles widens, originally the symmetrical tetrahedral conformation of golden nanometer particle is changed, become asymmetric space tetrahedral structure, this structure shows obvious characteristic peak at circular dichroism spectrum 520nm place;
(6) foundation of target dna chiral sensor
Based on the method establishment target dna chiral sensor of step (5), concrete operations are: the target dna adding different concns in the preparation process of step (4): Y5, according to the ratio of 520nm place circular dichroism signal and the concentration Criterion curve of target dna, the detection of DNA is limited to 43aM, and linearity range is at 0.05fM-50fM.
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