CN103320515A - 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 PDF

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CN103320515A
CN103320515A CN2013102764034A CN201310276403A CN103320515A CN 103320515 A CN103320515 A CN 103320515A CN 2013102764034 A CN2013102764034 A CN 2013102764034A CN 201310276403 A CN201310276403 A CN 201310276403A CN 103320515 A CN103320515 A CN 103320515A
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dna
nanometer particle
golden nanometer
tetrahedron
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CN103320515B (en
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徐丽广
胥传来
严文静
马伟
刘丽强
匡华
王利兵
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Jiangnan University
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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

A kind of method that DNA is detected based on chirality tetrahedron conformational change
Technical field
A kind of based on chirality tetrahedron conformational change to the method that DNA detects, belong to the nanometer biotechnology field.Relate to a kind of chirality preparations of nanomaterials for the 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, guiding biological development and vital functions running.Because the variation of base sequence is relevant with human many genetic diseasess in the dna molecular, therefore, has very profound significance aspect the early diagnosis and therapy of genescreen, genetic diseases to the analysis of the DNA of particular sequence and to the detection of base mutation in the DNA chain.The detection method of tradition DNA mainly is based on polymerase chain reaction (polymerase chain reaction, PCR).PCR is a kind of important technology at the amplification in vitro dna fragmentation.When having template DNA, substrate, upstream and downstream primer and heat-resisting archaeal dna polymerase, through the repeatedly working cycle of " denaturation renaturation-extension ", the trace template DNA can be expanded to millions of times.Round pcr can copy partial dna sequence, and signal amplifies, and has represented the limit that detects aspect sensitivity.However, still there are some intrinsic shortcomings in round pcr, high such as cost, need professional skill and equipment and rely on polysaccharase operation, easily false positive etc. appears, these characteristics have all seriously hindered the widespread use of PCR.
Along with the rise of nanoscale science and technology, nano material is because its unique physics and chemistry attribute has become the mainstay of preparation biosensor.Nano material is the material of size between the 1-100nm scope of a class formation unit because its size is near the coherence length of electronics, its character because the strong relevant self-organization that brings so that character great changes will take place.And its yardstick adds that near light wavelength it has the special effects on large surface, so its characteristic that shows, and for example fusing point, magnetic, optics, heat conduction, conductive characteristic etc. often are different from the character that this material shows when integrality.Nano-sensor is simple, cheap because of instrument, measure quick and precisely with the characteristics such as method sensitivity height and be widely used in the detection of environment, medicine, food etc.The chirality nano material combines mirror image symmetry on the special physical properties of nano material and the chiral molecules chemical structure, and this advanced composite material can be transferred to visible region from ultraviolet region with chiral signal, thereby has greatly expanded its range of application.
Summary of the invention
The purpose of this invention is to provide a kind of based on chirality tetrahedron conformational change to the method that DNA detects, relate to a kind of chirality preparations of nanomaterials for the DNA super sensitivity detection and the detection method of DNA.
Technical scheme of the present invention: a kind of method that DNA is detected based on chirality tetrahedron conformational change,
Comprise the foundation of synthetic, the tetrahedral assembling of golden nanometer particle, the tetrahedral structure of chirality and the target dna chiral sensor of finishing, the DNA-golden nanometer particle mixture of synthetic, the golden nanometer particle of golden nanometer particle.Processing step is:
(1) golden nanometer particle is synthetic
Citric acid and Weibull two step synthesis 10nm golden nanometer particle;
(2) finishing of golden nanometer particle
The synthetic golden nanometer particle of step (1) wraps up with two (p-sulfonyl-phenyl) the Phenylphosphine di-potassium solution of two hydrations, so that the nanoparticle surface negative charge is more, to improve the stability of Radioactive colloidal gold in high level salt solution;
(3) DNA-golden nanometer particle mixture is synthetic
The golden nanometer particle of the finishing that step (2) makes and terminal DNA with sulfydryl be hybrid reaction according to a certain percentage; Used DNA have four kinds (Y1, Y2, Y3, Y4), form 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 synthetic, the equal proportion Hybrid assembling forms the golden nanometer particle tetrahedron;
(5) the tetrahedral structure of chirality
Among the used four kinds of DNA of step (3), wherein embedded section of DNA ring (containing 13 bases) in the middle of the Y3, so in the golden nanometer particle tetrahedron of step (4) assembling, a DNA ring is contained on one of them limit.When the sequence Y5 that adds with this segment DNA ring complete complementary, dna circle is opened, distance in the tetrahedron between two particles becomes large, and symmetrical golden nanometer particle tetrahedron becomes unsymmetrical structure originally, thereby so that originally do not have the structure of chiral signal chiral signal to occur;
(6) foundation of target dna chiral sensor
Method according to step (5) makes up the DNA sensor that detects based on chirality.
The described method that DNA is detected based on chirality tetrahedron conformational change, step is:
(1) golden nanometer particle is synthetic
The method of citric acid and Weibull two step synthesis golden nanometer particle is: get respectively the triangular flask of two cleanings, add 158mL ultrapure water and 2mL 1% hydrochloro-auric acid in the A bottle; Add 8mL 1% trisodium citrate, 0.2mL 1% Weibull, 0.2mL 25mM salt of wormwood, 31.6mL ultrapure water in the B bottle.A, B liquid all are heated to 60 ℃, then add rapidly in the A liquid at lower B liquid of high-speed stirring, and mixed solution arrives the formation dark red solution at 60 ℃ of lower stirring 40min that continue.Last cool to room temperature forms the stable golden nanometer particle of citric acid, and transmission electron microscope shows that median size is 10 ± 2nm.
(2) finishing of golden nanometer particle
The synthetic stable golden nanometer particle of 10nm citric acid of step (1) is got 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, two hydrations, room temperature concussion reaction 10h; With removing supernatant liquor behind the centrifugal 10nm of 13000r/min, add ultrapure water and return to original volume;
(3) DNA-golden nanometer particle mixture is synthetic
The golden nanometer particle of the finishing that step (2) makes is respectively got 50 μ L in four PCR pipes, four kinds of DNA:Y1 that sequentially add separately 1 μ L, 10 μ M in 4 pipes, Y2, Y3 and Y4, behind the mixing, in each system, add 5 μ L 5 * tris-borate buffers and 1.25 μ L 1M NaCl solution, room temperature jolting reaction 2h; Synthetic Au-DNA mixture is removed supernatant liquor with centrifugal 10 min of 13000r/min, and precipitation adds 1 * tris-borate buffer to original volume, forms four kinds of different Au-DNA mixtures: Au-Y1, Au-Y2, Au-Y3, Au-Y4;
(4) the tetrahedral assembling of golden nanometer particle
Mixture (Au-Y1, Au-Y2, the Au-Y3 of four kinds of Au-DNA that step (3) is synthetic, Au-Y4) respectively get 50 μ L and be mixed in the 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 with the embedded dna circle complete complementary of Y3, this moment Y5 can with Au-Y3 in the complementary hybridization of dna circle, dna circle is opened fully, the distance in the tetrahedron between two nanoparticles widens.Originally the symmetrical tetrahedral conformation of golden nanometer particle is changed, and becomes asymmetric space tetrahedral structure, and this structure shows obvious characteristic peak at circular dichroism spectrum 520nm place.
(6) foundation of target dna chiral sensor
Method based on step (5) is set up the target dna chiral sensor, concrete operations are: add the target dna (Y5) of 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 used DNA in the 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’。
Annotate: DNA used in the present invention all gives birth to worker's biotechnology company limited available from Chinese Shanghai, and carries out purifying by polyacrylamide gel electrophoresis.
Beneficial effect of the present invention: the invention discloses a kind ofly by the inducing of target dna, is the achirality structural transformation method of chiral structure.We have set up the novel method of a kind of signal intensity rapid detection DNA according to circular dichroism spectrum based on this method.The method has been started a kind of construction process of novel hand-type structure, has realized simultaneously quick, efficient, the overdelicate chemical detection of DNA.
Description of drawings
Fig. 1: the tetrahedral transmission electron microscope picture of golden nanometer particle;
Fig. 2: the tetrahedral transmission electron microscope picture behind the conformational change that target is induced;
Fig. 3: the circular dichroism change curve in the golden nanometer particle tetrahedron behind the adding different concns target dna (Y5); 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) golden nanometer particle is synthetic
The method of citric acid and Weibull two step synthesis golden nanometer particle is: get respectively the triangular flask of two cleanings, add 158mL ultrapure water and 2mL 1% hydrochloro-auric acid in the A bottle; Add 8mL 1% trisodium citrate, 0.2mL 1% Weibull, 0.2mL 25mM salt of wormwood, 31.6mL ultrapure water in the B bottle.A, B liquid all are heated to 60 ℃, then add rapidly in the A liquid at lower B liquid of high-speed stirring, and mixed solution stirs 40 minutes to forming dark red solution 60 ℃ of lower continuation.Last cool to room temperature forms the stable golden nanometer particle of citric acid, and transmission electron microscope shows that median size is 10 ± 2nm.
(2) finishing of golden nanometer particle
The golden nanometer particle that 10 synthetic citrates of step (1) are modified is got 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, two hydrations, room temperature concussion reaction 10h; Use 13000r/min, remove supernatant liquor behind centrifugal 10 min, add water and return to original volume;
(3) DNA-golden nanometer particle mixture is synthetic
Step (2) is modified good golden nanometer particle and is respectively got 50 μ L in four PCR pipes, sequentially adds separately the Y1 of 1 μ L, 10 μ M in 4 pipes, Y2, Y3 and Y4, behind the mixing, in each system, add 5 μ L 5 * tris-borate buffers and 1.25 μ L 1M NaCl solution, room temperature jolting reaction 2h; Synthetic Au-DNA mixture is removed supernatant liquor with centrifugal 10 min of 13000r/min, 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 of step (3) preparation, Au-Y4) respectively get 50 μ L and be mixed in the 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 with the embedded dna circle complete complementary of Y3, this moment Y5 can with Au-Y3 in the complementary hybridization of dna circle, dna circle is opened fully, the distance in the tetrahedron between two nanoparticles widens.Originally the symmetrical tetrahedral conformation of golden nanometer particle is changed, and becomes asymmetric space tetrahedral structure, and this structure shows obvious characteristic peak at circular dichroism spectrum 520nm place.
(6) foundation of target dna chiral sensor
Method based on step (5) is set up the target dna chiral sensor, concrete operations are: add the target dna (Y5) of 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 that assembles is carried out the centrifugal 10min of 13000r/min, abandon supernatant, precipitation heavily is distributed in the ultrapure water of 120 μ L, and ultrapure washing once.Electronic Speculum characterizes: the sample drop of the above-mentioned processing of 7 μ L is added on the copper mesh of carbon film support, carries out drying under infrared lamp.The 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 assemble in cuvette, do blank with ultrapure water.The 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. One kind based on chirality tetrahedron conformational change to the method that DNA detects, it is characterized in that: the foundation of synthetic, the tetrahedral assembling of golden nanometer particle of the finishing of synthetic, the golden nanometer particle of golden nanometer particle, DNA-golden nanometer particle mixture, the tetrahedral structure of chirality and target dna chiral sensor; Processing step is:
    (1) golden nanometer particle is synthetic
    Citric acid and Weibull two step synthesis 10nm golden nanometer particle;
    (2) finishing of golden nanometer particle
    The synthetic golden nanometer particle of step (1) wraps up with two (p-sulfonyl-phenyl) the Phenylphosphine di-potassium solution of two hydrations, so that the nanoparticle surface negative charge is more, to improve the stability of Radioactive colloidal gold in high level salt solution;
    (3) DNA-golden nanometer particle mixture is synthetic
    The golden nanometer particle of the finishing that step (2) makes and terminal DNA with sulfydryl be hybrid reaction according to a certain percentage; Used DNA has four kinds: Y1, and 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 synthetic, the equal proportion Hybrid assembling forms the golden nanometer particle tetrahedron;
    (5) the tetrahedral structure of chirality
    Among the used four kinds of DNA of step (3), wherein embedded one section dna circle that contains 13 bases in the middle of the Y3, so in the golden nanometer particle tetrahedron of step (4) assembling, a DNA ring is contained on one of them limit, when the sequence Y5 that adds with this segment DNA ring complete complementary, dna circle is opened, and the distance in the tetrahedron between two particles becomes large, originally symmetrical golden nanometer particle tetrahedron becomes unsymmetrical structure, thereby so that originally do not have the structure of chiral signal chiral signal to occur;
    (6) foundation of target dna chiral sensor
    Method based on step (5) is set up the target dna chiral sensor;
    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. According to claim 1 based on chirality tetrahedron conformational change to the method that DNA detects, it is characterized in that:
    (1) golden nanometer particle is synthetic
    The method of citric acid and Weibull two step synthesis golden nanometer particle is: get respectively the triangular flask of two cleanings, add 158mL ultrapure water and 2mL 1% hydrochloro-auric acid in the A bottle; Add 8mL 1% trisodium citrate, 0.2mL 1% Weibull, 0.2mL 25mM salt of wormwood, 31.6mL ultrapure water in the B bottle; A, B liquid all are heated to 60 ℃, then add rapidly in the A liquid at lower B liquid of high-speed stirring, and mixed solution arrives the formation dark red solution at 60 ℃ of lower stirring 40min that continue; Last cool to room temperature forms the stable golden nanometer particle of citric acid, and transmission electron microscope shows that median size is 10 ± 2nm;
    (2) finishing of golden nanometer particle
    The synthetic stable golden nanometer particle of 10 nm citric acids of step (1) is got 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, two hydrations, room temperature concussion reaction 10h; With removing supernatant liquor behind the centrifugal 10min of 13000r/min, add ultrapure water and return to original volume;
    (3) DNA-golden nanometer particle mixture is synthetic
    The golden nanometer particle of the finishing that step (2) makes is respectively got 50 μ L in four PCR pipes, four kinds of DNA:Y1 that sequentially add separately 1 μ L, 10 μ M in 4 pipes, Y2, Y3 and Y4, behind the mixing, in each system, add 5 μ L 5 * tris-borate buffers and 1.25 μ L 1M NaCl solution, room temperature jolting reaction 2h; Synthetic Au-DNA mixture is removed supernatant liquor with centrifugal 10 min of 13000r/min, and precipitation adds 1 * tris-borate buffer to original volume, forms four kinds of different Au-DNA mixtures: Au-Y1, Au-Y2, Au-Y3, Au-Y4;
    (4) the tetrahedral assembling of golden nanometer particle
    The mixture of four kinds of Au-DNA that step (3) is synthetic: Au-Y1, Au-Y2, Au-Y3, Au-Y4 respectively gets 50 μ L and is mixed in the 1.5mL centrifuge tube, adds 5 μ L 1M NaCl solution, room temperature reaction spends the night, and forms symmetrical golden nanometer particle tetrahedron, and this structure does not have chiral signal;
    (5) the tetrahedral structure of chirality
    In the preparation process of step (4), add the sequence Y5 with the embedded dna circle complete complementary of Y3, this moment Y5 can with Au-Y3 in the complementary hybridization of dna circle, dna circle is opened fully, distance in the 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
    Method based on step (5) is set up the target dna chiral sensor, concrete operations are: the target dna that adds 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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CN112432906A (en) * 2020-09-17 2021-03-02 宁波大学 Chiral substance qualitative and quantitative analysis method based on circular dichroism spectrum technology

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Publication number Priority date Publication date Assignee Title
CN108680554A (en) * 2018-03-28 2018-10-19 国家纳米科学中心 A method of enhancing induction phasmon circular dichroism
CN112432906A (en) * 2020-09-17 2021-03-02 宁波大学 Chiral substance qualitative and quantitative analysis method based on circular dichroism spectrum technology
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