CN105784666A - Nano fluorescence biosensor as well as preparation method and application thereof - Google Patents
Nano fluorescence biosensor as well as preparation method and application thereof Download PDFInfo
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- 229910052737 gold Inorganic materials 0.000 claims abstract description 38
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- 238000001514 detection method Methods 0.000 claims abstract description 16
- 238000009396 hybridization Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000005516 engineering process Methods 0.000 claims abstract description 10
- 239000002105 nanoparticle Substances 0.000 claims abstract description 4
- 239000002091 nanocage Substances 0.000 claims abstract 4
- 239000000243 solution Substances 0.000 claims description 31
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 7
- 229940043267 rhodamine b Drugs 0.000 claims description 7
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
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- 239000002539 nanocarrier Substances 0.000 abstract description 4
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- 229940079593 drug Drugs 0.000 abstract description 3
- 238000007626 photothermal therapy Methods 0.000 abstract description 3
- 230000000903 blocking effect Effects 0.000 abstract 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 48
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 47
- 238000011160 research Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 108091023037 Aptamer Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 101000926206 Homo sapiens Putative glutathione hydrolase 3 proenzyme Proteins 0.000 description 2
- 102100034060 Putative glutathione hydrolase 3 proenzyme Human genes 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
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- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 description 1
- -1 amido modified ATP Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
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- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 229910000664 lithium aluminum titanium phosphates (LATP) Inorganic materials 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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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"
-
- 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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- General Health & Medical Sciences (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a new nano fluorescence biosensor with a biological release control function as well as a preparation method and application thereof, wherein gold nano particles are used as an orifice cap of a gold nano cage. The sensor has the following characteristics: a DNA hybridization technology is adopted, and the gold nano particles are assembled on the surface of the gold nano cage to form an orifice cap switch to realize blocking of matters in the gold nano cage. The nano biosensor provided by the invention has the characteristics of simple structure, sensitive signal, strong controllability and the like. By adopting the nano fluorescence biosensor provided by the invention, high-sensitivity detection of ATP can be realized in a range of 1.0*10<-9>-1.0*10<-7>M; and the nano fluorescence biosensor has the advantages of sensitivity, economy, easiness in operation and the like, gains relatively great application potential and broad application prospects in the fields such as biomedicine and life science, and provides a new way and method for early diagnosis and treatment of serious diseases and particularly for the study and application of tumor-targeted drugs and photo-thermal therapy based on the nano carrier release control technology.
Description
Technical field
The invention belongs to biochemistry and life sciences analytical field, be specifically related to a kind of nano fluorescent biosensor and
Its preparation method and application.
Background technology
In recent years, nanotechnology is studied widely at biologic medical and life science and is applied, wherein, and Jenner
Rice cage receives much concern as a kind of emerging gold nano-material.This nano material is the caged grain structure of a kind of hollow, porous,
It is dispersed with aperture on smooth cage wall surface.Compared with traditional ball-type gold nano grain, local surface of gold nanometer cage etc. from
Sub-resonance peak is positioned near infrared region 700～900nm, and this wave band is significant, especially for biological tissue for biomedicine
And the research of cell.By the inspection of the bioactive molecule that gold nanometer cage combines with golden nanometer particle hole cap and enzyme action amplifying technique
Survey technology yet there are no document report.
ATP (adenosine triphosphate) is a kind of coenzyme being widely present in biological cell, by adenosine and three phosphate institutes
Composition, chemical formula C10H16N5O13P3, molecular weight 507.184, the content in cell is 1-10mM.ATP is in the new old generation of cell
Thank and the energy supply of various biochemical reaction plays extremely important role, being the main of energy needed for in-vivo tissue cell
Source.Storing as intracellular energy and " the molecule currency " of transmission, it participates in protein, fat, sugar and the synthesis of nucleotide,
Cell many metabolic processes all there is important regulation effect.
The variation of the change of ATP content directly reflection cell and damage.When after cell damage or death, intracellular ATP
Content substantially reduces.It addition, as the most basic energy source of living cells, the line that ATP level is proportionate with living cells numerical value
Sexual relationship.Therefore, ATP has obtained research widely and report as one of the mark of major disease.Set up quick, sensitive
And ATP detection method not only facilitates early diagnosis and the treatment of the major diseases such as cancer accurately, cure simultaneously for clinic
The numerous areas such as, life sciences, food hygiene, environmental monitoring, medicine and cosmetics also have great importance.
The detection method of conventional ATP in addition to high performance liquid chromatography, other also have electrophoresis method, spectrophotography and
Biloluminescence method etc..These methods need to improve at aspects such as sensitivity, accuracy, selectivitys.To this end, in the urgent need to exploitation
A kind of selective detection technique highly sensitive, high is for the detection of ATP.
Summary of the invention
For the deficiency overcoming prior art to exist, for the nano fluorescent based on golden nanometer particle hole cap of detection ATP
Biosensor has no report, therefore, and the first object of the present invention: build and prepare a kind of novel can be used for and detect ATP's
Nano fluorescent biosensor, amplifies the gold nanometer cage material with golden nanometer particle hole cap switch with the circulation of DNA excision enzyme
Technology combines, and makes this nano fluorescent biosensor not only have controlled nano-pore cap switch, and through DNA excision enzyme
Circulation amplification, discharge a large amount of fluorescence molecule, by the detection of the fluorescence signal being obviously enhanced is realized height to ATP
Sensitive Detection；The second object of the present invention: the preparation method of this nano fluorescent biosensor a kind of is provided；The 3rd of the present invention
Purpose: the method that this nano fluorescent biosensor of a kind of application detection ATP is provided.The nano fluorescent present invention proposed is biological
Sensor is for the fluoroscopic examination of ATP, it is possible to significantly improve the sensitivity of ATP detection.
The present invention is achieved through the following technical solutions goal of the invention.The nano fluorescent biosensor of the present invention be with
Gold nanometer cage, as carrier, utilizes the architectural characteristic of its hollow, porous, loads fluorescence molecule therein, in order to prevent fluorescence
Leaking of molecule, by its surface-assembled golden nanometer particle, forms hole cap switch, is blocked in the hole on gold nanometer cage surface；Institute
The formation of the golden nanometer particle hole cap switch stated first passes through Au-S key effect and single stranded DNA S1 is modified gold nanometer cage surface,
It is subsequently adding the golden nanometer particle modified by single stranded DNA S2, complementary by the hybridization of DNAS2 Yu DNAS1, by golden nanometer particle group
Install to gold nanometer cage surface, form hole cap switch.
Preferably, above-mentioned nano fluorescent biosensor,
The sequence of described single stranded DNA S1 is: 5'-CAC TGG GTT GGG CGG GATTTT TTT TTT-SH-3'；
The sequence of described single stranded DNA S2 is: 5'-SH-TTT TTTATC CCG CCCAAC CCA GTGATT GCG
GAG G-3'；
Described golden nanometer particle is combined with the 5' end of DNAS2 by the effect of Au-S key；
Wherein, in DNAS2 in addition to containing the sequence complementary with DNAS1, possibly together with the sequence complementary with primer strand DNAS3
Row；
The sequence of described primer strand DNAS3 is: 5'-CCT CCG CAAT-3'；
Preferably, above-mentioned nano fluorescent biosensor, described fluorescence molecule is rhodamine B.
A kind of preparation method preparing above-mentioned nano fluorescent biosensor, comprises the steps:
(1) in the complex of magnetic bead-gold nanometer cage, add DNAS1 and DTT solution, be shaken at room temperature overnight, DNAS1 is repaiied
Decorations are to gold nanometer cage surface；
(2) the above-mentioned solution of Magneto separate, adds rhodamine B solution, is shaken at room temperature overnight after cleaning with PBS buffer solution；
(3) mixed solution of golden nanometer particle with DNA S2 and DTT is placed in it is shaken at room temperature overnight；
(4) (3) step gained solution is mixed with (2) step gained solution, the hybridization of DNAS2 Yu DNAS1 occurs, makes gold
Nanoparticle is assembled into gold nanometer cage surface, forms the hole cap that can be blocked in gold nanometer cage hole, prevents leaking of rhodamine B,
Magneto separate, removes supernatant, again disperses with after PBS, i.e. prepares nano fluorescent biosensor；
Wherein, the complex of described magnetic bead-gold nanometer cage is prepared as follows forming: by magnetic bead and gold nanometer cage
Mixed solution is placed in shaken at room temperature reaction 10h, Magneto separate, cleans with PBS buffer solution, removes supernatant, to obtain final product.
A kind of utilize the nano fluorescent biosensor of the present invention for the detection method of ATP, comprise the steps:
(1) in the fit complex of ATP, ATP sample solution is added, 37 DEG C of constant temperature oscillation reaction fit spies of 1h, ATP and ATP
Anisogamy, by competitive displacement reaction, primed DNA S3 is competed；
(2) the above-mentioned solution of Magneto separate, takes the PBS suspension that supernatant joins the nano fluorescent biosensor of the present invention
In, make to be competed primer S3 Yu S2 got off and hybridization occurs, add excision enzyme Exo III, endonuclease reaction occurs, makes double-strand
In S2 be sheared, hole cap is constantly opened, and discharges fluorescence molecule；
(4) the above-mentioned solution of Magneto separate, collects supernatant, detects its fluorescence signal；
Wherein, described ATP is fit, and complex is prepared as follows forming: by carboxyl magnetic bead and amido modified ATP
Aptamer solutions mixes, and is shaken at room temperature overnight, Magneto separate, removes supernatant, adds primed DNA S3 solution, and 37 DEG C of constant temperature oscillations are anti-
Answer 1h, generate by the duplex structure of the fit hybridization of primer S3 and ATP, Magneto separate, to obtain final product；Wherein, fit for ATP sequence
For: 5'-NH2-TTT TAAACC TGG GGGAGTATT GCG GAG GAA GGT-3'。
Beneficial effects of the present invention
Nano fluorescent biosensor that the present invention proposes and its preparation method and application is by nano-pore cap and DNA hybridization skill
Art combines, and is reacted and enzyme action amplifying technique by target molecule and fit specific molecular recognition thereof, can realize ATP high
Selective detection sensitive, high.Compared with traditional aptamer biosensors technology, it is not necessary to carry out in structure ATP is fit
Modify, it is to avoid modification in any structure and the fit specificity that causes and the reduction of associativity and consequent sensitive
The reduction of degree.Utilize this nano fluorescent biosensor to detect ATP, cause DNA hybridization and shearing by primer strand and excision enzyme
Circular response, make cap switch in golden nanometer particle hole constantly be opened, discharge fluorescence molecule, it is achieved that putting of fluorescence signal
Greatly, the detection sensitivity making ATP is significantly improved.
The nano fluorescent biosensor structure of the present invention is simple, and fluorescence signal is sensitive, and controllability is strong, has the choosing of excellence
Selecting property, has bigger application potential and wide application prospect in fields such as biomedicine, life sciences, can be used for tumor mark
Highly sensitive, the specific detection of will thing such as ATP, for early diagnosis and the treatment of major disease, particularly tumor targeted drug,
The research of the controlled-release technology based on nano-carrier such as photo-thermal therapy and application provide new approaches and methods.
Accompanying drawing explanation
The Cleaning Principle figure preparing and being used for ATP of the nano fluorescent biosensor of Fig. 1 present invention.
The fluorescence signal intensity of Fig. 2 difference ATP concentration.
Fig. 3 ATP concentration and the linear relationship of fluorescence signal intensity.
Detailed description of the invention
The following is the specific embodiment that the present invention relates to, technical scheme is described further, but this
Bright protection domain is not limited to these embodiments.Every change without departing substantially from the present invention or equivalent replacement are included in the present invention
Protection domain within.
Specifically describe the present invention below by embodiment, but the present invention is not limited by following embodiment.
Experimental apparatus: F-4600 spectrofluorophotometer (Hitachi, Japan)；(happy chromatograph skill is thought in Tianjin to magnetic separation rack again
Art development centre)；THZ-82A gas bath constant temperature oscillator (medical apparatus and instruments factory of Jintan City).
Experiment reagent: DNA artificial sequence synthetic used is buied by Beijing SBS Genetech biological engineering company limited；3-4μm
Carboxyl modified magnetic bead, sulfydryl modification magnetic bead (Tianjin thinks happy chromatographic technique development centre again)；Rhodamine B (RhB, Aladdin)；
Adenosine triphosphate (ATP, Aladdin)；Dithiothreitol, DTT (DTT, Aladdin)；Exo III exonuclease and buffer thereof
(Thermo Scientific, the U.S.)；PBS solution is 0.01M (pH 7.4, Na2HPO4-NaH2PO4)。
Embodiment 1:
A kind of preparation method preparing nano fluorescent biosensor of the present invention, comprises the steps:
(1) in the complex of magnetic bead-gold nanometer cage, 10 μ L 1.0 × 10 are added-5M DNAS1 and 10 μ L 1.0 × 10-3M
DTT solution, is diluted to 100 μ L with PBS, is shaken at room temperature overnight, DNAS1 modifies gold nanometer cage surface；
(2) the above-mentioned solution of Magneto separate, adds 100 μ L1.0 × 10 after cleaning with PBS buffer solution-5M rhodamine B solution, room
Temperature shaken overnight；
(3) by 200 μ L golden nanometer particles and 10 μ L 1.0 × 10-5M DNA S2 and 10 μ L 1.0 × 10-3M DTT's is mixed
Conjunction solution is placed in and is shaken at room temperature overnight；
(4) (3) step gained solution is mixed with (2) step gained solution, react 2h under room temperature, occur DNAS2's Yu DNAS1
Hybridization, makes golden nanometer particle be assembled into gold nanometer cage surface, forms the hole cap that can be blocked in gold nanometer cage hole, prevents sieve
Red bright B's leaks, and Magneto separate removes supernatant, again disperses with after PBS, i.e. prepares nano fluorescent biosensor；
Wherein, the complex of described magnetic bead-gold nanometer cage is prepared as follows forming: by 20 μ L sulfydryl magnetic beads with
400 μ L gold nanometer cages uniformly mix, shaken at room temperature reaction 10h, and Magneto separate cleans by PBS solution, removes supernatant, to obtain final product；Institute
The sulfydryl magnetic bead stated is the commodity (Tianjin thinks happy chromatographic technique development centre again) bought；Described golden nanometer particle (Au
NPs) press literature method and obtain (L.Tang, I.S.Chun, Z.Wang, J.Li, X.Li, Y.Lu.Analytical Chemistry
2013,85(20):9522-9527)；Described gold nanometer cage press literature method obtain (G.D.Moon, S.W.Choi, X.Cai,
W.Y.Li,E.C.Cho,U.Jeong,L.V.Wang and Y.N.Xia.J.Am.Chem.Soc.2011,133,4762-
4765)。
Embodiment 2:
A kind of nano fluorescent biosensor detection for ATP utilizing the present invention, method is as follows:
(1) in the fit complex of ATP, add 10 μ LATP sample solutions, be diluted to 100 μ L, 37 DEG C of constant temperature oscillations with PBS
1h, ATP are fit specific binding with it, by competitive displacement reaction, are competed by primed DNA S3；
(2) the above-mentioned solution of Magneto separate, takes the PBS suspension that supernatant joins the nano fluorescent biosensor of the present invention
In, there is hybridization in 37 DEG C of reactions 2h, primer S3 Yu S2, adds excision enzyme Exo III, 37 DEG C of reaction 2h, and endonuclease reaction will
S2 in double-strand shears, and primer S3 enters circulation, and cap switch in hole is constantly opened, and discharges fluorescence molecule；
(3) the above-mentioned solution of Magneto separate, collect supernatant, detect its fluorescence signal, fluoroscopic examination condition: excitation wavelength and send out
Ejected wave length is respectively 530,573nm；
Wherein, described ATP is fit, and complex is prepared as follows forming: 10 μ L carboxyl magnetic beads and 10 μ L 1.0 ×
10-5Chain mixing that ATP amido modified for M is fit, is diluted to 100 μ L with PBS, is shaken at room temperature overnight, Magneto separate, removes supernatant,
Add 100 μ L 1.0 × 10-6M primed DNA S3 solution, 37 DEG C of constant temperature oscillation reaction 1h, generate and formed by primer S3 and ATP is fit
Duplex structure, Magneto separate, remove supernatant, to obtain final product；Wherein, fit for ATP sequence is: 5'-NH2-TTT TAAACC TGG
GGGAGTATT GCG GAG GAA GGT-3'。
Fig. 1 is the preparation of the nano fluorescent biosensor of the present invention and for the Cleaning Principle figure of ATP.Fig. 2 is not
With the fluorescence signal intensity of ATP concentration, the concentration of ATP is respectively (0；1.0×10-9；5.0×10-9；1.0×10-8；2.0×
10-8；5.0×10-8；8.0×10-8；1.0×10-7；2.5×10-7；5.0×10-7M).Fig. 3 is that ATP concentration is strong with fluorescence signal
The linear relationship of degree.Result shows, ATP concentration is 1.0 × 10-9～1.0 × 10-7During M, the concentration of fluorescence signal △ F Yu ATP
Presenting good linear relationship, its linear equation is: △ F=64.1209+54.0283CATP(10-8M), linearly dependent coefficient R=
0.9933。
Golden nanometer particle, DNA hybridization technology, biological enzyme action cycle signal amplifying technique are tied mutually by the present invention with nano-carrier
Close, propose a kind of new based on the nano fluorescent biosensor that golden nanometer particle is hole cap switch, it is achieved that the Gao Ling of ATP
Selective detection quick, high.The present invention reacts particular by the specific recognition of biomolecule, utilizes primer strand and biology to cut
Cut the circular response that the effect of enzyme causes chain hybridization and chain to shear, make golden nanometer particle hole cap switch constantly be opened, discharge
Fluorescence molecule, it is achieved that the multiplication of fluorescence signal is amplified, and significantly improves the detection sensitivity of ATP.
The nano fluorescent biosensor structure of the present invention is simple, and fluorescence signal is sensitive, and controllability is strong, has the choosing of excellence
Selecting property, can be applicable to the fluoroscopic examination of ATP in living things system.Test result indicate that, the nano fluorescent using the present invention to propose is raw
Thing sensor can be 1.0 × 10-9～1.0 × 10-7Realize the highly sensitive detection to ATP in M concentration range, there is sensitive, warp
Ji, simple operation and other advantages, before the fields such as biomedicine, life sciences have bigger application potential and wide application
Scape, for controls based on nano-carrier such as early diagnosis and the treatments of major disease, the particularly targeted drug of tumor, photo-thermal therapy
Release the research of technology and the approaches and methods that application offer is new.
Claims (7)
1. a nano fluorescent biosensor, it is characterised in that: using golden nanometer particle as the hole cap of gold nanometer cage, it is used for sealing
Material in stifled gold nanometer cage, prevents it from leaking.
2. a nano fluorescent biosensor as claimed in claim 1, it is characterised in that: described golden nanometer particle is logical
Cross DNA hybridization technology and be assembled into gold nanometer cage surface.
3. a nano fluorescent biosensor as claimed in claim 1, it is characterised in that: the described thing in gold nanometer cage
Matter can be fluorescence molecule.
4. a nano fluorescent biosensor as claimed in claim 1, it is characterised in that:
Described golden nanometer particle is to be assembled into gold nanometer cage by the hybridization complementary action of single stranded DNA S2 Yu single stranded DNA S1
Surface, wherein, single stranded DNA S1 can be modified gold nanometer cage surface by Au-S key effect.
5. a nano fluorescent biosensor as claimed in claim 4, it is characterised in that:
The sequence of single stranded DNA S1 is: 5'-CAC TGG GTT GGG CGG GAT TTT TTT TTT-SH-3'；
The sequence of single stranded DNA S2 is: 5'-SH-TTT TTT ATC CCG CCC AAC CCA GTG ATT GCG GAG G-3'；
Described golden nanometer particle is combined with the 5' end of DNAS2 by the effect of Au-S key.
6. the preparation method of a nano fluorescent biosensor as claimed in claim 1, it is characterised in that step is as follows:
(1) in the complex of magnetic bead-gold nanometer cage, add DNAS1 and DTT solution, be shaken at room temperature overnight, S1 is modified gold
Nanocages surface；
(2) the above-mentioned solution of Magneto separate, adds rhodamine B solution, is shaken at room temperature overnight after cleaning with PBS buffer solution；
(3) mixed solution of golden nanometer particle with DNA S2 and DTT is placed in it is shaken at room temperature overnight；
(4) (3) step gained solution is mixed with (2) step gained solution, the hybridization of DNAS2 Yu DNAS1 occurs, makes gold nano
Particle is assembled into gold nanometer cage surface, forms the hole cap that can be blocked in gold nanometer cage hole, prevents leaking of rhodamine B, and magnetic divides
From, remove supernatant, again disperse with after PBS, i.e. prepare nano fluorescent biosensor；
Wherein, the complex of described magnetic bead-gold nanometer cage is prepared as follows forming: by the mixing of magnetic bead Yu gold nanometer cage
Solution is placed in shaken at room temperature reaction 10h, Magneto separate, cleans with PBS buffer solution, removes supernatant, to obtain final product.
7. the application of the nano fluorescent biosensor as according to any one of claim 1-5, it is characterised in that be used for
The detection of ATP, method is as follows:
(1) in the fit complex of ATP, ATP sample solution is added, 37 DEG C of constant temperature oscillation reaction fit specificitys of 1h, ATP and ATP
In conjunction with, primed DNA S3 is competed；
(2) the above-mentioned solution of Magneto separate, takes supernatant and joins the nano fluorescent biology biography as according to any one of claim 1-5
In the PBS suspension of sensor, make to be competed the primer S3 got off and hybridization occurs, add excision enzyme Exo III, enzyme action occurs
Reaction, makes hole cap constantly be opened, discharges fluorescence molecule；
(3) the above-mentioned solution of Magneto separate, collects supernatant, detects its fluorescence signal；
Wherein, described ATP is fit, and complex is prepared as follows forming: by fit with amido modified ATP for carboxyl magnetic bead
Solution mixes, and is shaken at room temperature overnight, Magneto separate, removes supernatant, adds primed DNA S3 solution, and 37 DEG C of constant temperature oscillations react 1h,
Generate by the duplex structure of the fit hybridization of primer S3 and ATP, Magneto separate, to obtain final product；Wherein, the sequence of primed DNA S3 is:
5'-CCT CCG CAA T-3'。
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