CN110296961A - The building and application of controllable Nano-Au probe based on double block D NA - Google Patents
The building and application of controllable Nano-Au probe based on double block D NA Download PDFInfo
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- CN110296961A CN110296961A CN201810242069.3A CN201810242069A CN110296961A CN 110296961 A CN110296961 A CN 110296961A CN 201810242069 A CN201810242069 A CN 201810242069A CN 110296961 A CN110296961 A CN 110296961A
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The present invention relates to field of biotechnology, more particularly to a kind of Nano-Au probe, it include first chain and Article 2 chain including nanogold particle and the double-chain probe being connected on the nanogold particle, the double-chain probe, first chain includes 5 ' -3 ' the first sections and the second section being arranged successively；First section and the Article 2 chain are complementary；Second section is as cohesive end, target sequence for identification；The Article 2 chain is modified by fluorophor.Nano-Au probe provided by the invention realizes the hybridization efficiency of regulation identification sequence and target sequence by adjusting the nucleotide number of the second section, and then regulates and controls Nano-Au probe to the recognition capability of target sequence.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of controllable Nano-Au probe based on double block D NA and
Its preparation and application.
Background technique
The detection of DNA/RNA has wide application space in biomedical research and clinical diagnosis.DNA detects point in disease
Sub diagnosis, gene therapy, biomedical research, biological warfare agent quickly detect and forensic application etc. is all with of crucial importance
Meaning.RNA plays an important role in bioprocess, influences cell, the development of tissue and individual, and lysis causes specifically
Property RNA is increased in body fluid.Such as the mRNA/miRNA of tumour-specific can be used for cancer diagnosis and parting and by extensive
Concern.
Traditional method of detection of nucleotides is divided into two classes, and one kind is the detection method based on probe hybridization technique, including
Northern blotting, micro-array chip and Microspheres Technique, hybridization in situ technique etc.；Another kind of is based on amplified reaction
Detection method, including rolling circle amplification, quantitative PCR etc..That often there is sensitivity is low for these conventional methods, specificity it is low or
The disadvantages of high, is required to sample, laboratory apparatus.
Compared to conventional method, biosensor possesses the characteristics such as efficient, quick, sensitive.Nanogold (AuNPs) is excellent due to its
Good biocompatibility is received to be widely applied in biosensor with unique optical property.Nanogold can make nearly all
Fluorescent quenching is on its surface.Nanogold has very high specific surface area, can assemble a large amount of biomolecule, such as DNA, protein and anti-
Body is simultaneously applied to drug delivery, biological detection and field of biomedical research.With the development of DNA chemical synthesising technology, DNA at
For a kind of biomaterial simple and easy to get.Single stranded DNA may be used as probe, metal ion, small molecule and the albumen of complementary series
Matter aptamers.
The conventional method that DNA is assembled in nanometer gold surface is usually that the DNA of use-SH modification and nanometer gold surface utilize Au-S
Key absorption.However, there are drawbacks for this method: the DNA of-SH modification is at high cost, and single stranded DNA is easy to happen non-spy in nanometer gold surface
Opposite sex absorption, sulfydryl are adsorbed on a nanometer gold surface and make probe steric hindrance big.It is low that these reasons result in hybridization efficiency, thus
Reduce molecule distinguishability.
Most of DNA/RNA biosensors are all based on the base pairing of complementary base sequence.It common are three kinds of inspections
Survey strategy: the first strategy use antisense oligonucleotides modifies AuNPs and detects target sequence.Target sequence and antisense oligonucleotides
Complementation, hybridization cause nanogold to be assembled.Second of strategy has very strong affinity to gold nano gold surface using single-stranded nucleotide,
And double chain nucleotide does not have suction-operated.By addition target sequence and with antisense oligonucleotides acid hybridization, DNA to nanogold inhale
Attached ability reduction causes to reduce with gold nanoparticle stability.The third strategy has the hair of fluorescent molecule and quencher with both ends
Clamping structure is added target sequence and opens target sequence, and fluorescence intensity increases.However, although each have efficiency, sensitivity and
Convenience, but none of these methods has regulatable sensitivity and signal.Improve the dynamics and thermodynamic property pair of probe
Recognition capability is improved to be of great significance.Currently, most of available methods are optimized based on empirical parameter.Therefore, having very much must
Develop a kind of regulatable Nano-Au probe technology for DNA/RNA detection.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide based on the controllable of double block D NA
Nano-Au probe and its preparation and application.
The present invention is achieved by the following technical solutions:
The first aspect of the present invention provides a kind of Nano-Au probe, including nanogold particle and is connected to the nanometer
Double-chain probe, the double-chain probe on gold particle include first chain and Article 2 chain, first chain include 5 ' -3 ' according to
The first section and the second section of secondary arrangement；First section and the Article 2 chain are complementary；Second section is as viscous
Property end, for identification target sequence；The Article 2 chain is modified by fluorophor.
First chain in Nano-Au probe provided by the invention also referred to as identifies sequence, identifies that the length of sequence subtracts
The length of first section is the length of the second section, therefore the length that the length of the first section is equal to Article 2 chain can pass through
The length of Article 2 chain is adjusted to adjust the length of the second section, so as to regulate and control the hybridization effect of identification sequence and target sequence
Rate, and then regulate and control Nano-Au probe to the recognition capability of target sequence.
In one possible implementation, the length of second section is 1-11nt.It is preferred that 1nt or 3nt is used,
Or 5nt or 7nt or 9nt or 11nt.
In one possible implementation, the double-chain probe is connected on the nanogold particle by Poly A.
In one possible implementation, first chain of the double-chain probe includes the Poly that 5'-3' is arranged successively
A, the first section and the second section, the Poly A are incorporated in the nanogold particle surface.
PolyA, also that is, polyadenylic acid, the nucleotide fragments formed for multiple continuous adenylate A.
In Nano-Au probe provided by the invention, the steric hindrance of the effect length chain substitution reaction of Poly A, thus
Chain substitution reaction macroscopic property is adjusted, it therefore, can be miscellaneous by the length-adjusting target sequence and identification sequence that adjust Poly A
Efficiency is handed over, to regulate and control Nano-Au probe to the recognition capability of target sequence.
In one possible implementation, the length of the Poly A is 30-50nt.It is preferred that using PolyA30 or
PolyA40 or PolyA50.
In one possible implementation, the fluorophor modification is in 3 ' ends of the Article 2 chain.
In one possible implementation, the transmitted wave of the excitation wavelength of the fluorophor and the nanogold particle
It is long to overlap or be all overlapped.Preferably, the fluorophor be selected from fluorescein isothiocynate, FAM, ROX, Cy3 or
Cy5。
Preferably, the partial size of the nanogold is 25~35nm.The nanogold is the prior art, can both have been referred to existing
Document preparation, can also be bought by commercial sources.
The second aspect of the present invention additionally provides a kind of preparation method of Nano-Au probe described in first aspect, the side
Method includes the following steps:
(1) first chain and the Article 2 chain are hybridized, obtains double-chain probe；
(2) double-chain probe and the nanogold particle mixing obtained step (1), obtains mixed liquor；
(3) pH for the mixed liquor that step (2) obtains is adjusted to acidity, after standing the first preset time, then is adjusted to neutrality, it is quiet
After setting the second preset time, centrifugation removes supernatant, obtains the Nano-Au probe.
In one possible implementation, the double-chain probe connects Poly A.Preferably, the 5 ' of first section
End connects the Poly A.
In one possible implementation, in the step (3), the pH tune of the mixed liquor that step (2) is obtained
To acidity, the first preset time is stood specifically, the trisodium citrate of 2ul pH=3 500mM is added in every 100ul mixed solution
PH is adjusted to acidity, stands 5-10 minutes after mixing.Preferably, stand 5 minutes or 6 minutes after mixing or 7 minutes or 8 minutes or
9 minutes or 10 minutes.
It is described to be adjusted to neutrality in a kind of possible implementation kind, the step (3), stand the second preset time tool
Body is, every 100ul mixed solution is added during pH is adjusted to by the phosphate buffer (final concentration 30mM) of 18ul PH=7.2 200mM
Property, 3-10 minutes are stood after mixing.Preferably, 3 minutes or 4 minutes are stood after mixing or 5 minutes or 6 minutes or 7 minutes or 8 points
Clock or 9 minutes or 10 minutes.
In one possible implementation, the centrifugation is specially 7500g-8500g 10min.Preferably, centrifugation speed
Degree or 7500g or 7600g or 7700g or 7800g or 7900g or 8000g.
In one possible implementation, in the step (2), the double-chain probe that step (1) is obtained and
Nanogold particle is mixed into, and every 500-2000 moles of double-chain probe and 1 mole of nanogold particle are mixed.It is preferred every
500 moles or every 800 moles or every 1000 moles or every 1500 moles or every 2000 moles of double-chain probe and 1 mole of nanometer
Gold particle mixing.
The third aspect of the present invention discloses a kind of method of the detection target molecule of non-disease diagnostic purpose, including as follows
Step: Nano-Au probe described in target molecule and first aspect is mixed, then carries out fluorescent value detection.
Preferably, the target molecule includes DNA, miRNA, mRNA.
Fourth aspect present invention additionally provides Nano-Au probe described in first aspect in preparation DNA, miRNA or mRNA inspection
Application in test agent.
Fifth aspect present invention provides a kind of kit for detecting nucleic acid, including Nano-Au probe described in first aspect.
Compared with prior art, the invention has the benefit that
(1) by adjusting cohesive end, the i.e. nucleotide number of the second section, regulation identification sequence and target sequence are realized
Hybridization efficiency, and then regulate and control Nano-Au probe to the recognition capability of target sequence.Further, by adjusting cohesive end and
The nucleotide number of Poly A, realizes the hybridization efficiency of regulation identification sequence and target sequence, and then regulates and controls Nano-Au probe pair
The recognition capability of target sequence；To solve the absorption of identification sequence-nonspecific, identification sequence and mesh existing in the prior art
The problems such as sequence hybridization steric hindrance is big is marked, photochemistry detection small molecule nucleic acid (DNA, miRNA or mRNA) is substantially increased
Accuracy and sensitivity have the advantages such as convenient, cheap.
(2) nanogold provided by the invention can be used in simulating serum sample detection, have detection of biological samples potential.
(3) for the present invention using nanogold as fluorescent quenching group, report sequence can connect different fluorophors, for simultaneously
Detect plurality of target molecule.It is easy to operate, quick, it is at low cost.
Detailed description of the invention
Fig. 1: Nano-Au probe structural schematic diagram provided in an embodiment of the present invention.
Fig. 2: Nano-Au probe detection schematic diagram provided in an embodiment of the present invention.
Fig. 3 A: the phenogram for the Nano-Au probe that the embodiment of the present invention 1 assembles shows under transmission electron microscope (AFM)
Shown nanogold particle size all be in monodisperse.
Fig. 3 B: the phenogram for the Nano-Au probe that the embodiment of the present invention 1 assembles shows nanogold particle and diblock
Nano-Au probe ultra-violet absorption spectrum after DNA assembling.
Fig. 3 C: the phenogram for the Nano-Au probe that the embodiment of the present invention 1 assembles shows nanogold particle and diblock
Nano-Au probe after DNA assembling is hydrated radius.
Fig. 4: poly A length shows nanogold the result displaying of the regulating and controlling effect of number of probes in the embodiment of the present invention 2
Figure.In Fig. 4, (A), which is shown, adjusts Poly A nucleotide number, and nanometer gold surface diblock DNA assembles the variation of number；
(B) the corresponding Standardization curve for fluorescence intensity of various concentration fluorescent molecule is shown.
Fig. 5: result display diagram of the poly A length to the regulation of target molecule recognition capability in the embodiment of the present invention 3.?
In Fig. 5, (A) shows AuNPs-PolyA30 probe to the recognition capability of target molecule, and detection is limited to 100pM；(B) it shows
AuNPs-PolyA40 probe detects to the recognition capability of target molecule and is limited to 50pM；(C) AuNPs-PolyA50 probe is shown
To the recognition capability of target molecule, detection is limited to 10pM.It is found that nanogold surface molecular is known as PolyA number gradually increases
Other ability gradually increases.
Fig. 6: cohesive end nucleotide number connects the tune of probe to nanometer under difference poly A length in the embodiment of the present invention 4
The result display diagram of control effect；Wherein, the result of regulating and controlling effect shows as probe signal-to-noise ratio (SNR) under same concentrations.In Fig. 6
In, (A) shows the signal-to-noise ratio of Poly A30 diblock Nano-Au probe；(B) spy of Poly A40 diblock nanogold is shown
The signal-to-noise ratio of needle；(C) signal-to-noise ratio of PolyA50 diblock Nano-Au probe is shown.
Fig. 7: the specific display diagram of Nano-Au probe in the embodiment of the present invention 5.(A) shows (A) AuNPs- in Fig. 7
PolyA30 is to final concentration 5nM target compound, final concentration 50nM non-complementary sequence 1 (seq1) and final concentration 50nM incomplementarity sequence
The fluorescence response result of column 2 (seq2)；(B) AuNPs-PolyA40 is shown to final concentration 5nM target compound, final concentration
The fluorescence response result of 50nM non-complementary sequence 1 (seq1) and final concentration 50nM non-complementary sequence 2 (seq2)；(C) it shows
For AuNPs-PolyA40 to final concentration 5nM target compound, final concentration 50nM non-complementary sequence 1 (seq1) and final concentration 50nM are non-
The fluorescence response result of complementary series 2 (seq2).
Fig. 8: what the embodiment of the present invention 6 assembled carries the testing result displaying of the Nano-Au probe of different identification sequences
Figure.Difference identification sequence hybridizes from the report molecule of different fluorophors modification respectively.
Fig. 9: in the embodiment of the present invention 7, the result display diagram of DNA in Nano-Au probe detection simulation serum sample.In Fig. 9
In, (A) shows the result of DNA in Poly A30 diblock Nano-Au probe detection simulation serum sample；(B) Poly is shown
The result of DNA in A40 diblock Nano-Au probe detection simulation serum sample；(C) Poly A50 diblock nanogold is shown
Probe in detecting simulates the result of DNA in serum sample.
Specific embodiment
Embodiment 1
(1) material and equipment
The reagent that the present embodiment uses: trisodium citrate (C6H5Na3O72H2O), phosphate, NaCl, MgCl2 and KCl
Equal reagents are bought from Chinese Chinese medicines group company.
Mentioned reagent is that analysis is pure, and is not further purified.
The water that the present embodiment uses is MilliQ water；MilliQ water: 18.2M Ω .cm (Millipore).The present embodiment is adopted
Equipment includes transmission electron microscope (TEM), ultraviolet specrophotometer (Hitachi U-3010), sepectrophotofluorometer
(F-900, Edinburg), PH meter, high-speed refrigerated centrifuge (Hitachi).
Diblock DNA used in the embodiment of the present invention, report molecule and target molecules be purchased from raw work bioengineering (on
Sea) limited liability company and use high-efficient liquid phase chromatogram purification.
The embodiment of the present invention use 3 diblock DNA, respectively 2blockPolyA30 (SEQ IDNO.1),
2blockPolyA40(SEQ ID NO.2),2blockPolyA50(SEQ ID NO.3)；Its sequence is as shown in table 1.
Report molecule used in the embodiment of the present invention is that reporter7 (SEQ ID NO.4) its sequence is as shown in table 1,
3 ' ends are modified by fluorescein isothiocynate (fluorescein isothiocyanate, FITC).
Table 1
(2) preparation and characterization of Nano-Au probe
Nanogold can be bought by commercial sources, the method that can also be recorded by the prior art prepares nanogold.
By diblock DNA and report molecule hybridization, diblock probe is obtained.
The preparation of Nano-Au probe: it is broadly divided into following four step: first, it is 0.5nM that diblock probe, which is added to concentration,
Nano-Au solution in, the final concentration ratio 1000:1 both made, incubation 10 minutes；Second, by trisodium citrate (pH=3,
It 500mM) is added into above-mentioned solution, makes its final concentration of 10mM, be quickly vortexed, stand 3-5 minutes；Third, by above-mentioned solution
PH is adjusted to neutrality, with 200mM PB solution (PH=7.6), makes its final concentration of 30mM, is quickly vortexed, and stands 10-15 minutes；Most
Afterwards, three times by above-mentioned nano-Au solution 8000rpm centrifuge washing, washing solution is 10mM PB (pH=7.6).By above-mentioned preparation
Good solution is dissolved in 1xPBS hybridization solution, in case subsequent use.(ultraviolet specrophotometer surveys emission peak in 530nm, and ε=
3.585*109Lmol-1cm-1)。
By the Nano-Au probe solution drop after nano-Au solution and assembling in copper mesh, natural air drying prepares TEM sample.Make
Front and back nanogold and Nano-Au probe are assembled with tem observation.
Nano-Au probe solution after nano-Au solution and assembling is added in cuvette, ultraviolet specrophotometer is used
Scan emission peak positions.
Nano-Au probe solution after nano-Au solution and assembling is added in cuvette, with DelsaTM nanometers of sub-micros
Rice particle size instrument.Under the theory of DLS (dynamic light scattering), with the included software statistics of instrument and each group nanogold is fitted
Hydrated diameter Fig. 3 A of probe, Fig. 3 B, the phenogram that Fig. 3 C is the Nano-Au probe that embodiment 1 synthesizes, wherein Au indicates nanometer
Gold particle, Au-PolyA30, Au-PolyA40, Au-PolyA50 respectively indicate by 2blockPolyA30,2blockPolyA40,
The Nano-Au probe of 2blockPolyA50 preparation.By Fig. 3 A it is found that shown nanogold particle size is equal under projection electron microscope
One, it is in monodisperse.By Fig. 3 B it is found that assembling front and back nanogold ultra-violet absorption spectrum (left side), nanogold ultraviolet absorption peak after assembling
5nm is moved to right to 530nm.
By Fig. 3 C it is found that the hydration radius of assembling front and back nano Au particle.Assemble the diblock of different Poly A length
DNA, nanogold are hydrated radius and increase different numerical value.
Embodiment 2
In the present embodiment, to the diblock DNA of different Poly A length to the regulation of nanogold surface-assembled number into
Row is inquired into.
Diblock DNA Nano-Au probe Solutions Solution with different length Poly A prepared by embodiment 1, first with purple
Outer spectrophotometer quantitative concentrations.Calculation formula is CAuNPs-PolyA=A520nm*2.79*10-10M.By diblock nanometer
Mercaptoethanol (final concentration of 20mM) is added in Au probe (final concentration of 0.15nM) probe, at room temperature shaken over night.Then it is centrifuged
Method collects the DNA (fluorophor FAM, excitation wavelength 494nm, launch wavelength 520nm) of supernatant fluorescent marker, and detection fluorescence is strong
Degree.Standard curve before finally fluorescent value is substituted into calculates fluorescent label DNA concentration.As shown in figure 4, (A) is with PolyA
The diblock DNA number of the increase of length, nanogold surface-assembled gradually decreases.It is consistent with theory group loading amount (with PolyA long
The increase of degree, theory group loading amount gradually decrease)；(B) different fluorescent molecule concentration and corresponding Standardization curve for fluorescence intensity.Note: mark
Directrix curve is drawn under same ionic strength, pH, concentration buffer solution.
Embodiment 3
In the present embodiment, to different Poly A few nucleotide purpose diblock DNA to the tune of target sequence recognition capability
Control is inquired into.
The Nano-Au probe with different length Poly A provided using embodiment 1.In different groups of Nano-Au probes
Various concentration target molecule is added, is respectively as follows: 0,0.01,0.05,0.1,0.5,1,10,50,100,150,200nM, after incubation
Detect each group fluorescence intensity.Every group of experiment is repeated three times.In the present embodiment, the sequence of target molecule such as SEQ ID NO.11 institute
Show, specially 5 ' AGCCC CTGCC CACCG CACAC TG3 '.
As a result as shown in figure 5, the intensity of fluorescence signal enhances with the addition of target molecule, plateau is finally reached.
Different Poly A numbers have regulating and controlling effect to the minimum detection limit of probe.As adenylate number increases in PolyA, probe
Detection limit is respectively 100pM, 50pM, 10pM.Meanwhile with the increase of Poly A number, nanogold surface-assembled amount is reduced, because
The range of linearity of this probe in detecting decreases.
Embodiment 4
Cohesive end nucleotide number inquires into the regulation of identification recognition sequence ability in the present embodiment.
For same target molecule, the report molecule for designing different length hybridizes with identification sequence, regulates and controls cohesive end core
Thuja acid number.Target molecule in the present embodiment is using target molecule shown in SEQ ID NO.11 in embodiment 3；Diblock
DNA be embodiment 1 in 2blockPolyA30 (SEQ ID NO.1), 2blockPolyA40 (SEQ ID NO.2),
2blockPolyA50(SEQ ID NO.3).The report sequence molecule of design be Reporter-1 (SEQ ID NO.5),
Reporter-3(SEQ ID NO.6)、Reporter-5(SEQ ID NO.8)、Reporter-7(SEQ ID NO.4)、
Reporter-9 (SEQ ID NO.9), Reporter-11 (SEQ ID NO.10), their sequence is as shown in table 2.
Table 2
Corresponding identification sequence cohesive end nucleotide number is respectively 1,3,5,7,9,11, is denoted as T-1, T-3, T- respectively
5, T-7, T-9, T-11.The above-mentioned target molecule of 5nM, incubation at room temperature is added；By probe solution centrifuging and taking supernatant, with F-900 fluorescence
Detector test each group background fluorescence and signal fluorescence intensity (fluorophor FAM, excitation wavelength 494nm, launch wavelength
520nm), signal-to-noise ratio is calculated.Every group of experiment is in triplicate.As a result as shown in Figure 6.It will be appreciated from fig. 6 that with cohesive end nucleotide
There is ascendant trend in the signal-to-noise ratio presentation of the increase of number, probe.When cohesive end nucleotide number increases to 7, probe is gathered around
There is optimum signal-noise ratio.
Embodiment 5
In the present embodiment, the specificity of the Nano-Au probe provided invention is inquired into.
Complementary DNA target mark and other incomplementarity targets (non-target DNA) are installed with example 1 group respectively
Diblock Nano-Au probe (0.15nM) mixing, be incubated for.Centrifuging and taking supernatant surveys fluorescence intensity (in the present embodiment, using fluorescence
Group FAM, excitation wavelength 494nm, launch wavelength 520nm).Hybridization system is 200ul, and target molecule target hybridization is dense
5nM is spent, incomplementarity target molecules seq-1 hybridizes concentration 50nM, and incomplementarity target molecules seq-2 hybridizes concentration 50nM.Every group real
It tests and is repeated three times.
As shown in fig. 7,50nM is added in incomplementarity target, 5nM is added in target molecule.Target molecule is added and makes fluorescence signal
Strongly, incomplementarity target fluorescence signal intensity and control group no significant difference is added.
Embodiment 6
In the present embodiment, the miRNA polychrome detection of the Nano-Au probe provided invention is inquired into.Design diblock
DNA is used for miRNA210 (SEQ ID NO.11), miRNA155 (SEQ ID NO.12), miRNA196a (SEQ ID NO.13)
Detection.Corresponding diblock DNA is respectively 2blockA50 (for miRNA210) (SEQ ID NO.3), 2blockA50 (for
miRNA155)(SEQ ID NO.14),2blockA50(for miRNA196a)(SEQ ID NO.15).Corresponding sequence such as table
3.Diblock DNA hybridizes in advance from three kinds of report molecules that different fluorophors (respectively FAM, ROX, Cy5) are modified, according to 1:
1:1 mixing.Nano-Au solution is added according to the ratio of 1000:1 in the DNA sequence dna mixed to be incubated for 10 minutes.Then, according to reality
It applies method described in example 1 and continues assemble nanometer Au probe.
MiRNA210, miRNA155, the miRNA196a that concentration is 10nM is added simultaneously in resulting probe, is incubated for.From
The heart takes supernatant to survey fluorescence intensity (fluorophor FAM, excitation wavelength 494nm, launch wavelength 520nm；Fluorophor ROX, excitation wave
Long 588nm, launch wavelength 608nm；Fluorophor Cy5, excitation wavelength 647nm, launch wavelength 670nm).
As shown in figure 8, probe fluorescence signal and control significant difference in the case where three kinds of miRNA are added are assembled altogether,
Assembling probe has detectability simultaneously altogether.
Table 3
Embodiment 7
In the present embodiment, the Nano-Au probe that invention provides is inquired into for simulating detection of biological samples.
In order to detect miRNA in diluted fetal calf serum, 1xPBS (pH=7.6,100mM) dilutes fetal calf serum extremely
10%.Target molecule is diluted with 10%FBS, i.e. simulation serum sample；Target molecule is using target shown in SEQ ID NO.11
Molecule.The diblock DNA that Nano-Au probe uses embodiment 1 to prepare is the Nano-Au probe of 2blockPolyA50.Nanometer used
The concentration of Au probe is 0.15nM.Simulation serum sample is added in gained probe to the final concentration of 5nM of target molecule, is incubated for.
Centrifuging and taking supernatant surveys fluorescence intensity (fluorophor FAM, excitation wavelength 494nm, launch wavelength 520nm；).Every group of experiment counterpoise
Again three times.
As can be seen from Figure 8, DNA detection is carried out in the serum environment of simulation, sensitivity and specificity are very high,
Target is added in AuNPs-PolyA30 probe, and fluorescence signal increases 10 times；Target fluorescence is added in AuNPs-PolyA40 probe
Signal increases 8 times；Target is added in AuNPs-PolyA50 Nano-Au probe, and fluorescence signal increases 10 times.
Since then, it can prove that Nano-Au probe provided in an embodiment of the present invention can be applied in simulation serum, be ground to be subsequent
Study carefully actual sample to lay a good foundation.
It can be seen that Nano-Au probe provided in an embodiment of the present invention overcomes many disadvantages in the prior art, have
Good application prospect.
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that under the premise of not departing from the method for the present invention, can also be made for those skilled in the art
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are equivalent embodiment of the invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical solution of the present invention
It is interior.
Claims (11)
1. a kind of Nano-Au probe, which is characterized in that including nanogold particle and be connected to double on the nanogold particle
Chain probe, the double-chain probe include first chain and Article 2 chain, and first chain includes first that 5'-3' is arranged successively
Section and the second section；First section and the Article 2 chain are complementary；Second section is as cohesive end, for knowing
Other target sequence；The Article 2 chain is modified by fluorophor.
2. Nano-Au probe according to claim 1, which is characterized in that the double-chain probe is connected to institute by Poly A
It states on nanogold particle.
3. Nano-Au probe according to claim 2, which is characterized in that first chain of the double-chain probe includes 5'-
Poly A, the first section and the second section that 3' is arranged successively, the Poly A are incorporated in the nanogold particle surface.
4. Nano-Au probe according to claim 2 or 3, which is characterized in that the length of the Poly A is 30-50nt.
5. Nano-Au probe according to claim 1, which is characterized in that the length of second section is 1-11nt.
6. Nano-Au probe according to claim 1, which is characterized in that the fluorophor modification is in the Article 2 chain
3 ' end.
7. a kind of preparation method of Nano-Au probe described in any one of claims 1-6, which is characterized in that the preparation method
Include the following steps:
(1) first chain and the Article 2 chain are hybridized, obtains double-chain probe；
(2) double-chain probe and the nanogold particle mixing obtained step (1), obtains mixed liquor；
(3) pH for the mixed liquor that step (2) obtains is adjusted to acidity, after standing the first preset time, then is adjusted to neutrality, stand the
After two preset times, centrifugation removes supernatant, obtains the Nano-Au probe.
8. preparation method according to claim 7, which is characterized in that described to obtain step (1) in the step (2)
To double-chain probe and nanogold particle be mixed into, by every 500-2000 moles of double-chain probe and 1 mole of nanogold particle
Mixing.
9. a kind of method of the detection target molecule of non-disease diagnostic purpose, includes the following steps: by target molecule and such as right
It is required that the described in any item Nano-Au probe mixing of 1-6, then carry out fluorescent value detection.
10. Nano-Au probe as claimed in any one of claims 1 to 6 is preparing answering in DNA, miRNA or mRNA detection reagent
With.
11. a kind of kit for detecting nucleic acid, including Nano-Au probe as claimed in any one of claims 1 to 6.
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