CN105784666B - A kind of nano fluorescent biosensor and its preparation method and application - Google Patents
A kind of nano fluorescent biosensor and its preparation method and application Download PDFInfo
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- CN105784666B CN105784666B CN201610344647.5A CN201610344647A CN105784666B CN 105784666 B CN105784666 B CN 105784666B CN 201610344647 A CN201610344647 A CN 201610344647A CN 105784666 B CN105784666 B CN 105784666B
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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"
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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
Abstract
The present invention proposes a kind of new nano fluorescent biosensor and its preparation method and application with biological controlled-released effect using gold nanoparticle as the hole cap of gold nanometer cage.The characteristics of sensor is：Using DNA hybridization technology, gold nanoparticle is assembled into gold nanometer cage surface, hole cap switch is formed, realizes the closure to substance in gold nanometer cage.The nano biological sensor of the present invention has simple in structure, the characteristics such as signal is sensitive, and controllability is strong.It can be 1.0 × 10 using nano fluorescent biosensor proposed by the present invention‑9~1.0 × 10‑7The highly sensitive detection to ATP is realized within the scope of M, with sensitive, economy, simple operation and other advantages, there is larger application potential and wide application prospect in fields such as biomedicine, life sciences, new approaches and methods are provided for the early diagnosis and treatment of major disease, the especially research to tumor-targeting drug, photo-thermal therapy based on nano-carrier controlled-release technology and application.
Description
Technical field
The invention belongs to biochemistry and life sciences analytical fields, and in particular to a kind of nano fluorescent biosensor and
Preparation method and application.
Background technology
In recent years, nanotechnology is widely studied and is applied in biologic medical and life science, wherein Jenner
Rice cage is concerned as a kind of emerging gold nano-material.The nano material is a kind of hollow, porous caged grain structure,
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 located at 700~900nm of near infrared region, and the wave band is for biomedical significant, especially for living tissue
And the research of cell.The inspection for the bioactive molecule that gold nanometer cage is combined with gold nanoparticle hole cap and digestion amplifying technique
Survey technology yet there are no document report.
ATP (atriphos) 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.New old generations of the ATP in cell
Thank and the energy supply of various biochemical reactions in play extremely important role, be the main of energy needed for in-vivo tissue cell
Source." the molecule currency " for storing and transmitting as intracellular energy, it participates in the synthesis of protein, fat, sugar and nucleotide,
There is important adjustment effect to many metabolic processes of cell.
The variation of ATP contents directly reflects the variation and damage of cell.After cell damage or death, intracellular ATP
Content is substantially reduced.In addition, the energy source most basic as living cells, the line that ATP levels are proportionate with living cells numerical value
Sexual intercourse.Therefore, markers one of of the ATP as major disease has obtained extensive research and report.It establishes quick, sensitive
And accurately ATP detection methods not only facilitate the early diagnosis and treatment of the major diseases such as cancer, meanwhile, clinic is cured
The numerous areas such as, life science, food hygiene, environmental monitoring, medicine and cosmetics also have great importance.
The detection method of conventional ATP other than high performance liquid chromatography, it is other also have electrophoresis, spectrophotometry and
Biloluminescence method etc..These methods need to be improved in sensitivity, accuracy, selectivity etc..For this purpose, there is an urgent need to develop
A kind of highly sensitive, highly selective detection technique is used for the detection of ATP.
Invention content
In order to overcome the shortcomings of the prior art, for the nano fluorescent based on gold nanoparticle hole cap of detection ATP
Biosensor has not been reported, therefore, the first object of the present invention：It builds and prepares and a kind of novel can be used for detecting ATP's
Nano fluorescent biosensor amplifies the gold nanometer cage material switched with gold nanoparticle hole cap and DNA excision enzymes cycle
Technology is combined, and the nano fluorescent biosensor is made not only to there is controllable nano-pore cap to switch, and passes through DNA excision enzymes
Cycle amplification, release a large amount of fluorescent moleculars, realized to the height of ATP by the detection of the fluorescence signal to significantly increasing
Sensitive Detection；The second object of the present invention：A kind of preparation method of the nano fluorescent biosensor is provided；The third of the present invention
Purpose：A kind of method of application nano fluorescent biosensor detection ATP is provided.By nano fluorescent proposed by the present invention biology
Sensor is used for the fluoroscopic examination of ATP, can significantly improve the sensitivity of ATP detections.
The present invention is achieved through the following technical solutions goal of the invention.The present invention nano fluorescent biosensor be with
Gold nanometer cage loads fluorescent molecular, in order to prevent fluorescence inside it as carrier, architectural characteristic hollow using its, porous
Molecule leaks, by its surface-assembled gold nanoparticle, forming hole cap switch, the hole on gold nanometer cage surface being blocked；Institute
The formation for the gold nanoparticle hole cap switch stated first is modified single stranded DNA S1 to gold nanometer cage surface by the effect of Au-S keys,
Then the gold nanoparticle modified by single stranded DNA S2 is added, complementation is hybridized by DNAS2 and DNAS1, by gold nanoparticle group
It is attached to gold nanometer cage surface, forms hole cap switch.
Preferably, above-mentioned nano fluorescent biosensor,
The sequence of the single stranded DNA S1 is：5'-CAC TGG GTT GGG CGG GATTTT TTT TTT-SH-3'；
The sequence of the single stranded DNA S2 is：5'-SH-TTT TTTATC CCG CCCAAC CCA GTGATT GCG
GAG G-3'；
The gold nanoparticle is combined by the effect of Au-S keys with the ends 5' of DNAS2；
Wherein, in DNAS2 other than containing with the sequence of DNAS1 complementations, also contain the sequence with primer strand DNAS3 complementations
Row；
The sequence of the primer strand DNAS3 is：5'-CCT CCG CAAT-3'；
Preferably, above-mentioned nano fluorescent biosensor, the fluorescent molecular are rhodamine Bs.
A kind of preparation method preparing above-mentioned nano fluorescent biosensor, includes the following steps：
(1) DNAS1 and DTT solution is added in the compound of magnetic bead-gold nanometer cage, is shaken at room temperature overnight, DNAS1 is repaiied
Adorn gold nanometer cage surface；
(2) the above-mentioned solution of Magneto separate is added rhodamine B solution after being cleaned with PBS buffer solutions, is shaken at room temperature overnight；
(3) mixed solution of gold nanoparticle and DNA S2 and DTT is placed in and is shaken at room temperature overnight；
(4) (3) step acquired solution is mixed with (2) step acquired solution, the hybridization reaction of DNAS2 and DNAS1 occurs, makes gold
Nano-particle is assembled into gold nanometer cage surface, and the hole cap that gold nanometer cage hole blocks can be prevented leaking for rhodamine B by formation,
Magneto separate removes supernatant, disperses again after being cleaned with PBS, obtain nano fluorescent biosensor；
Wherein, the compound of the magnetic bead-gold nanometer cage is prepared as follows：By magnetic bead and gold nanometer cage
Mixed solution is placed in shaken at room temperature reaction 10h, and Magneto separate clean with PBS buffer solutions, removal supernatant to get.
A kind of detection method for utilizing the nano fluorescent biosensor of the present invention to be used for ATP, includes the following steps：
(1) ATP sample solutions are added in ATP aptamer compounds, 37 DEG C of constant temperature oscillation reaction 1h, ATP and ATP aptamers are special
The opposite sex combines, and is reacted by competitiveness displacement, primed DNA S3 is competed；
(2) the above-mentioned solution of Magneto separate takes supernatant to be added to the PBS suspension of the nano fluorescent biosensor of the present invention
In, make to be competed the primer S3 and S2 generation hybridization reactions to get off, adds excision enzyme Exo III, endonuclease reaction occurs, makes double-strand
In S2 be sheared, hole cap is constantly opened, and releases fluorescent molecular；
(4) the above-mentioned solution of Magneto separate collects supernatant, detects its fluorescence signal；
Wherein, the ATP aptamer compounds are prepared as follows：By carboxyl magnetic bead and amido modified ATP
Aptamer solutions mix, and are shaken at room temperature overnight, Magneto separate, remove supernatant, and primed DNA S3 solution is added, and 37 DEG C of constant temperature oscillations are anti-
Answer 1h, generate duplex structure made of being hybridized by primer S3 and ATP aptamer, Magneto separate to get；Wherein, the sequence of ATP aptamers
For：5'-NH2-TTT TAAACC TGG GGGAGTATT GCG GAG GAA GGT-3'。
Beneficial effects of the present invention
Nano fluorescent biosensor proposed by the present invention and its preparation method and application is by nano-pore cap and DNA hybridization skill
Art is combined, by molecular recognition reaction specific between target molecule and its aptamer and digestion amplifying technique, it can be achieved that ATP high
Sensitive, highly selective detection.Compared with traditional aptamer biosensors technology, without being carried out in structure to ATP aptamers
Modification avoids caused by the modification in any structure the reduction of aptamer specificity and associativity and resulting sensitive
The reduction of degree.ATP is detected using the nano fluorescent biosensor, DNA hybridization and shearing are caused by primer strand and excision enzyme
Circular response, make gold nanoparticle hole cap switch be constantly opened, release fluorescent molecular, realize putting for fluorescence signal
Greatly, the detection sensitivity of ATP is made to be significantly improved.
The nano fluorescent biosensor structure of the present invention is simple, and fluorescence signal is sensitive, and controllability is strong, has excellent choosing
Selecting property has larger application potential and wide application prospect in fields such as biomedicine, life sciences, can be used for tumour mark
Highly sensitive, the specific detection of will object such as ATP, be major disease early diagnosis and treatment, especially the targeted drug of tumour,
The research and application of the controlled-release technology based on nano-carrier such as photo-thermal therapy provide new approaches and methods.
Description of the drawings
The preparation of the nano fluorescent biosensor of Fig. 1 present invention and its detection principle diagram for ATP.
The fluorescence signal intensity of Fig. 2 difference ATP concentration.
The linear relationship of Fig. 3 ATP concentration and fluorescence signal intensity.
Specific implementation mode
It is specific embodiment of the present invention below, technical scheme of the present invention is described further, but this hair
Bright protection domain is not limited to these examples.It is every to be included in the present invention without departing substantially from the change of the present invention or equivalent substitute
Protection domain within.
The present invention is specifically described below by embodiment, but the present invention is not limited by following embodiments.
Laboratory apparatus：F-4600 sepectrophotofluorometers (Hitachi, Japan)；Magnetic separation rack (thinks happy chromatography skill in Tianjin again
Art development centre)；THZ-82A gas baths constant temperature oscillator (medical apparatus and instruments factory of Jintan City).
Experiment reagent：DNA artificial sequence synthetic used is bought by Beijing SBS Genetech bioengineering Co., Ltd；3-4μm
Carboxyl modified magnetic bead, sulfydryl modification magnetic bead (think happy chromatographic technique development centre in Tianjin) again；Rhodamine B (RhB, Aladdin)；
Atriphos (ATP, Aladdin)；Dithiothreitol (DTT) (DTT, Aladdin)；III exonucleases of Exo and its buffer
(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, includes the following steps：
(1) 10 μ L 1.0 × 10 are added in the compound of magnetic bead-gold nanometer cage-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, by DNAS1 modifications to gold nanometer cage surface；
(2) 100 L1.0 × 10 μ are added in the above-mentioned solution of Magneto separate after being cleaned with PBS buffer solutions-5M rhodamine B solutions, room
Warm shaken overnight；
(3) by 200 μ L gold nanoparticles and 10 μ L 1.0 × 10-5M DNA S2 and 10 μ L 1.0 × 10-3M DTT's is mixed
Conjunction solution, which is placed in, to be shaken at room temperature overnight；
(4) (3) step acquired solution is mixed with (2) step acquired solution, reacts 2h at room temperature, occur DNAS2's and DNAS1
Hybridization reaction, makes gold nanoparticle be assembled into gold nanometer cage surface, and the hole cap that gold nanometer cage hole blocks can be prevented sieve by formation
The bright B's of pellet leaks, Magneto separate, removes supernatant, disperses again after being cleaned with PBS, obtain nano fluorescent biosensor；
Wherein, the compound of the magnetic bead-gold nanometer cage is prepared as follows：By 20 μ L sulfydryls magnetic beads with
400 μ L gold nanometer cages uniformly mix, and shaken at room temperature reacts 10h, and Magneto separate cleans with PBS solution, remove supernatant to get；Institute
The sulfydryl magnetic bead stated is the commodity (thinking happy chromatographic technique development centre again in Tianjin) of purchase；Gold nanoparticle (the Au
NPs it) presses literature method and obtains (L.Tang, I.S.Chun, Z.Wang, J.Li, X.Li, Y.Lu.Analytical Chemistry
2013,85(20):9522-9527)；The gold nanometer cage by 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 detection for utilizing the nano fluorescent biosensor of the present invention to be used for ATP, method are as follows：
(1) 10 μ LATP sample solutions are added in ATP aptamer compounds, 100 μ L, 37 DEG C of constant temperature oscillations are diluted to PBS
1h, ATP are specifically bound with its aptamer, are reacted by competitiveness displacement, primed DNA S3 is competed；
(2) the above-mentioned solution of Magneto separate takes supernatant to be added to the PBS suspension of the nano fluorescent biosensor of the present invention
In, hybridization reaction occurs for 37 DEG C of reactions 2h, primer S3 and S2, adds excision enzyme Exo III, 37 DEG C of reaction 2h, endonuclease reaction will
S2 shearings in double-strand, primer S3 enter cycle, and cap switch in hole is constantly opened, and releases fluorescent molecular；
(3) the above-mentioned solution of Magneto separate collects supernatant, detects its fluorescence signal, fluoroscopic examination condition：Excitation wavelength and hair
Ejected wave length is respectively 530,573nm；
Wherein, the ATP aptamer compounds are prepared as follows：10 μ L carboxyls magnetic beads and 10 μ L 1.0 ×
10-5ATP aptamer chains mixing amido modified M, is diluted to 100 μ L with PBS, is shaken at room temperature overnight, Magneto separate, remove supernatant,
100 μ L 1.0 × 10 are added-6M primed DNA S3 solution, 37 DEG C of constant temperature oscillations react 1h, and generation is made of primer S3 and ATP aptamer
Duplex structure, Magneto separate, remove supernatant to get；Wherein, the sequence of ATP aptamers 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 its detection principle diagram for 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 and fluorescence signal are strong
The linear relationship of degree.The result shows that ATP concentration is 1.0 × 10-9~1.0 × 10-7When M, the concentration of fluorescence signal △ F and ATP
Good linear relationship is presented, linear equation is：△ F=64.1209+54.0283CATP(10-8M), linearly dependent coefficient R=
0.9933。
The present invention mutually ties gold nanoparticle, DNA hybridization technology, biological digestion cycle signal amplifying technique with nano-carrier
It closes, proposes a kind of new nano fluorescent biosensor based on gold nanoparticle for hole cap switch, realize the Gao Ling of ATP
Quick, highly selective detection.The present invention is reacted particular by the specific recognition of biomolecule, is cut using primer strand and biology
The effect of enzyme cutting causes the circular response of chain hybridization and chain shearing, so that cap switch in gold nanoparticle hole is constantly opened, releases
Fluorescent molecular realizes the multiplication amplification of fluorescence signal, 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 excellent choosing
Selecting property can be applied to the fluoroscopic examination of ATP in biosystem.The experimental results showed that being given birth to using nano fluorescent proposed by the present invention
Object sensor can be 1.0 × 10-9~1.0 × 10-7The highly sensitive detection to ATP is realized in M concentration ranges, and there is sensitive, warp
Ji, simple operation and other advantages, before the fields such as biomedicine, life science have larger application potential and wide application
Scape is the early diagnosis and treatment of major disease, the especially control based on nano-carrier such as targeted drug, photo-thermal therapy of tumour
The research and application for releasing technology provide new approaches and methods.
Claims (6)
1. a kind of nano fluorescent biosensor, the hole cap using gold nanoparticle as gold nanometer cage, for blocking gold nanometer cage
Interior substance, prevents it from leaking, it is characterised in that：The gold nanoparticle is to be assembled into Jenner by DNA hybridization technology
Rice cage surface.
2. a kind of nano fluorescent biosensor as described in claim 1, it is characterised in that：Object in the gold nanometer cage
Matter can be fluorescent molecular.
3. a kind of nano fluorescent biosensor as described in claim 1, it is characterised in that：
The gold nanoparticle is to be assembled into gold nanometer cage with the complementation that hybridizes of single stranded DNA S1 by single stranded DNA S2
Surface, wherein single stranded DNA S1 is modified gold nanometer cage surface by the effect of Au-S keys.
4. a kind of nano fluorescent biosensor as claimed in claim 3, 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';
The gold nanoparticle is combined by the effect of Au-S keys with the ends 5' of DNAS2.
5. a kind of preparation method of nano fluorescent biosensor as described in claim 1, it is characterised in that steps are as follows：
(1) DNAS1 and DTT solution is added in the compound of magnetic bead-gold nanometer cage, is shaken at room temperature overnight, S1 modifications are arrived
Gold nanometer cage surface；
(2) the above-mentioned solution of Magneto separate is added rhodamine B solution after being cleaned with PBS buffer solutions, is shaken at room temperature overnight；
(3) mixed solution of gold nanoparticle and DNA S2 and DTT is placed in and is shaken at room temperature overnight；
(4) (3) step acquired solution is mixed with (2) step acquired solution, the hybridization reaction of DNAS2 and DNAS1 occurs, makes gold nano
Particle is assembled into gold nanometer cage surface, and leaking for rhodamine B, magnetic point can be prevented by the hole cap that gold nanometer cage hole blocks by being formed
From removal supernatant disperses after being cleaned with PBS, obtains nano fluorescent biosensor again；
Wherein, the compound of the magnetic bead-gold nanometer cage is prepared as follows：By the mixing of magnetic bead and gold nanometer cage
Solution is placed in shaken at room temperature and reacts 10 h, and Magneto separate clean with PBS buffer solutions, removal supernatant to get.
6. a kind of application of nano fluorescent biosensor as described in any one of claim 1-4, it is characterised in that be used for
The detection of ATP, method are as follows：
(1) ATP sample solutions are added in ATP aptamer compounds, 37 DEG C of constant temperature oscillations react 1 h, and ATP and ATP aptamers are special
Property combine, primed DNA S3 is competed；
(2) the above-mentioned solution of Magneto separate takes the nano fluorescent biology that supernatant is added to as described in any one of claim 1-4 to pass
In the PBS suspension of sensor, makes to be competed the primer S3 generation hybridization reactions to get off, add excision enzyme Exo III, digestion occurs
Reaction, makes hole cap constantly be opened, releases fluorescent molecular；
(3) the above-mentioned solution of Magneto separate collects supernatant, detects its fluorescence signal；
Wherein, the ATP aptamer compounds are prepared as follows：By carboxyl magnetic bead and amido modified ATP aptamers
Solution mixes, and is shaken at room temperature overnight, Magneto separate, removes supernatant, and primed DNA S3 solution, 37 DEG C of constant temperature oscillation reactions 1 are added
H, generate by primer S3 and ATP aptamer hybridize made of duplex structure, Magneto separate to get；Wherein, the sequence of primed DNA S3 is：
5'-CCT CCG CAA T-3'。
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| CN107132354B (en) * | 2017-05-24 | 2019-04-30 | 青岛科技大学 | A method of detection salmonella typhimurium |
| CN108660185A (en) * | 2018-03-09 | 2018-10-16 | 山东师范大学 | A kind of biosensor and preparation method based on golden selenium key |
| CN110819695B (en) * | 2018-08-13 | 2022-11-08 | 青岛科技大学 | Method for detecting silver ions |
| CN112094886A (en) * | 2020-08-05 | 2020-12-18 | 青岛科技大学 | Method for detecting urine biomarkers of tumor patients |
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