CN104730240B - A kind of compound fluorescent nano probe and its preparation method and application - Google Patents
A kind of compound fluorescent nano probe and its preparation method and application Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
- G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP
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Abstract
The invention provides a kind of compound fluorescent nano probe that can be used for biomedicine and life science detection ATP and its preparation method and application.Gold nanometer cage is combined by the present invention with DNA molecular switch and DNA amplifying technique, proposes a kind of brand-new compound fluorescent nano probe.The feature of this compound fluorescent nano probe is: using gold nanometer cage as nano container, at 2 kinds of single stranded DNAs of its surface-assembled, fluorescent dye is filled in the hollow structure of gold nanometer cage, utilizes hybridization that the third single stranded DNA is assembled into gold nanometer cage surface, form DNA molecular switch.Once occurring controlled solution chain reaction that DNA molecular switch will be caused to be opened, the fluorescence molecule blocked in making hole is released.Therefore, this compound fluorescent nano probe is utilized, it is possible to realize the selective enumeration method highly sensitive, high to ATP.The early diagnosis of the major diseases such as it is cancer, treatment, theoretical research etc. provide new approaches and methods.
Description
Technical field
The invention belongs to bioanalysis and life science, be specifically related to a kind of compound fluorescence nano detecting ATP
Probe and its preparation method and application.
Background technology
Adenosine triphosphate (ATP) is a kind of coenzyme being widely present in biological cell, basis set by adenosine and three phosphoric acid
Become.Since nineteen twenty-nine Lohmann finds first and extracts ATP, it has been found at the metabolism of life cells and each
Plant in the energy supply of biochemical reaction and play extremely important role.As " the molecule currency " of intracellular energy transmission, ATP
Store and transmission chemical energy, be the main source of energy needed for in-vivo tissue cell, participate in protein, fat, sugar and nucleotide
Synthesis, cell many metabolic processes are all had important regulation effect.Research shows, ATP is not only in the treatment of malignant tumor
In be widely used, but also one of the mark as major disease has obtained studying widely and paying close attention to.Due to
ATP is the energy source that living cells is depended on for existence, when, after cell damage or death, intracellular ATP content substantially reduces.Cause
This, the changes of contents of ATP can reflect variation and the damage of cell.By detection malignant tumor ATP concentration change, it will be appreciated that
Its cellular metabolism situation, is commonly used for the external susceptibility detection of antitumor drug, for heterogeneity and the individuality of malignant tumor susceptibility
Chemotherapy provides feasibility therapeutic scheme.It addition, as the most basic energy source of living cells, ATP level and living cells numerical value in
Positive correlation linear relationship, therefore, ATP also frequently as microorganism pollute an index, by measure ATP content number, can
Directly to reflect the quantity of cytoactive, microorganism.Therefore, set up quick, sensitive and accurately ATP analyze method not only help
In early diagnosis and the treatment of the major diseases such as cancer, simultaneously for clinical medicine, life sciences, food hygiene, environmental monitoring,
The numerous areas such as medicine and cosmetics also have important practical significance.
At present, the detection method of domestic and international ATP is in addition to as the high performance liquid chromatography of mainstream technology, and other also has
Electrophoresis method, spectrophotography and biloluminescence method etc..These methods are at aspects such as instrument and equipment, time, sensitivity, accuracys all
Shortcomings more or less, especially sensitivity and selectivity wait to improve.To this end, in the urgent need to developing highly sensitive, Gao Xuan
The detection technique of selecting property is for the detection of ATP.
In recent years, gold nanometer cage had obtained paying close attention to widely as a kind of emerging nano material.This nano material is
The structure of a kind of hollow porous, the i.e. inside of caged granule are hollow-core construction, and smooth cage wall surface is dispersed with aperture.With biography
The ball-type gold nano grain of system is compared, and the local surface plasma resonance peak of gold nanometer cage is positioned near infrared region 700～900nm,
This wave band is significant, especially for viviperception for biomedicine.Gold nanometer cage is put with DNA molecular switch and DNA
The technology that big technology combines yet there are no document report.
Summary of the invention
For the deficiency overcoming prior art to exist, meanwhile, also for more efficiently utilizing, there is receiving of special construction
The remarkable advantage of rice material, for the rare report of fluorescent nano probe of detection ATP, therefore, the first object of the present invention: build
And prepare a kind of novel compound fluorescent nano probe that can be used for detecting ATP, will there is the gold nanometer cage of DNA molecular switch
Material combines with DNA amplifying technique, makes this compound fluorescent nano probe not only have controlled molecular switch, and
A large amount of fluorescence molecule can be discharged, by fluorescence signal being detected the highly sensitive inspection realized ATP after solving chain reaction
Survey；The second object of the present invention: the preparation method of this compound fluorescent nano probe a kind of is provided；The third object of the present invention:
The method that this compound fluorescent nano probe of a kind of application detection ATP is provided.The compound fluorescence nano present invention proposed is visited
Pin is for the fluoroscopic examination of ATP, it is possible to is effectively improved sensitivity and the accuracy of ATP detection, significantly reduces amount of samples.
The present invention is achieved through the following technical solutions goal of the invention.The compound fluorescent nano probe of the present invention be with
Gold nanometer cage, as carrier, utilizes the architectural characteristic that it is hollow porous, loads fluorescence molecule therein, in order to prevent fluorescence from dividing
Leaking of son, by switching at its surface-assembled DNA molecular, blocks the hole on gold nanometer cage surface；Wherein, described DNA divides
Son switch is made up of 3 kinds of DNA, and wherein DNA1, DNA2 are complementary with DNA3 respectively, and DNA3 not only contains nickase recognition sequence also
Containing the sequence complementary with primer strand DNA4.
Preferably, above-mentioned compound fluorescent nano probe, the partial sequence of wherein said DNA1 is: 5 '-GGT CCA
GCT GGC TTT TTT---SH-3’；
The partial sequence of described DNA2 is: 5 '-SH---TTT TTT CCG TCG CAC GCT TTT-3 '；
The partial sequence of described DNA3 is: 5 '-GCC AGC TGG ACC TG GCT AAG GCA GCG TGC GAC
GGT GGG GGA-3’；
The partial sequence of described primer strand DNA4 is: 5 '-AAT ACT CCC CCA CCG-3 '.
Preferably, above-mentioned compound fluorescent nano probe, described fluorescence molecule is rhodamine B.
A kind of preparation method preparing above-mentioned compound fluorescent nano probe, comprises the steps:
(1) in DNA1, DNA2, add DTT solution respectively and carry out activation processing 1h；
(2) in the complex of magnetic bead-gold nanometer cage, add DNA1, DNA2 solution activated, be shaken at room temperature overnight, make
DNA1, DNA2 are assembled into gold nanometer cage surface；
(3) the above-mentioned solution of Magneto separate, adds rhodamine B solution, is shaken at room temperature overnight after cleaning with PBS buffer solution；
(4) in above-mentioned solution, add DNA3 solution, react 2h under room temperature, make DNA3 Yu DNA1, DNA2 hybridize, Jenner
Rice cage surface forms DNA molecular switch, Magneto separate, again disperses, i.e. prepare compound fluorescence and receive after cleaning with PBS buffer solution
Rice probe；
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 compound fluorescent nano probe detection for ATP utilizing the present invention, 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 spies of 1h, ATP and ATP
Anisogamy, primed DNA 4 is competed；
(2) the above-mentioned solution of Magneto separate, takes the PBS suspension that supernatant joins the compound fluorescent nano probe of the present invention
In, make to be competed the primed DNA 4 got off and hybridize with DNA3, under room temperature reaction after add Klenow polymerase, dNTPs,
Nb.Bpu10I nickase and PBS buffer solution, react 2h, the circulation that chain increases, chain replaces and chain is sheared occur under the conditions of 37 DEG C
Reaction, makes DNA molecular switch 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 carboxyl magnetic bead and amido modified ATP
Aptamer solutions mixes, and is shaken at room temperature overnight, Magneto separate, removes supernatant, adds 4,37 DEG C of constant temperature oscillation reaction 1h of primed DNA,
Generate by the duplex structure of the fit hybridization of primed DNA 4 and ATP, Magneto separate, to obtain final product.
Beneficial effects of the present invention: the compound fluorescent nano probe that the present invention proposes is by nanotechnology and molecular biosciences skill
Art combines, and by molecular recognition and specific reaction, can realize selective detection highly sensitive to ATP, high.Utilize this to be combined
Type fluorescent nano probe detection ATP, causes, by the effect of enzyme, the circular response that chain increases, chain replaces and chain is sheared, makes
DNA molecular switch is constantly opened, and discharges fluorescence molecule, it is achieved that the amplification of fluorescence signal, makes the detection sensitivity of ATP obtain
To significantly improving.
The compound fluorescent nano probe simple in construction of the present invention, good stability, controllability is strong, and fluorescence signal is sensitive, with
Time, do not affected by other common interference material, be there is high selectivity, be can be applicable to the fluoroscopic examination of ATP in living things system.Real
Testing result to show, the compound fluorescent nano probe using the present invention to propose can be 5.0 × 10-8～1.0 × 10-6In the range of M in fact
The now selective enumeration method highly sensitive, high to ATP.This probe and detection technique thereof have in fields such as biomedicine, life sciences
Bigger application potential and wide application prospect, can be used for the specific detection of ATP, and the early stage for major diseases such as cancers examines
Disconnected and treatment provides new approaches and methods.
Accompanying drawing explanation
The experimental principle figure 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.Everything is included in the present invention without departing substantially from change or the equivalent replacement of 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: magnetic separation rack (Tianjin thinks happy chromatographic technique development centre again)；F-4600 spectrofluorophotometer
(Hitachi, Japan)；THZ-82A gas bath constant temperature oscillator (medical apparatus and instruments factory of Jintan City).
Experiment reagent: 3-4 μm carboxyl modified magnetic bead, sulfydryl modification magnetic bead (think again in the exploitation of happy chromatographic technique by Tianjin
The heart)；Rhodamine B (Aladdin)；Adenosine triphosphate (ATP)；Dithiothreitol, DTT (DTT, Aladdin)；Klenow polymerase and
Buffer (Thermo Scientific, the U.S.)；Nb.Bpu10I nickase and buffer (Thermo Scientific, U.S.
State)；DNTPs (Beijing SBS Genetech gene technology company limited), DNA artificial sequence synthetic used is by Beijing SBS Genetech biology work
Journey company limited buys, and PBS solution is 0.01M (pH7.4, Na2HPO4-NaH2PO4)。
Embodiment 1:
A kind of preparation method preparing the compound fluorescent nano probe of the present invention, comprises the steps:
(1) respectively in 10 μ L1.0 × 10-5DNA1, DNA2 of mol/L adds 10 μ L1.0 × 10-3The DTT of mol/L is molten
Liquid, is then respectively adding 80 μ LPBS solution, activates 1h；
(2) in the complex of magnetic bead-gold nanometer cage, add DNA1, DNA2 solution activated, be shaken at room temperature overnight, make
DNA1, DNA2 are assembled into gold nanometer cage surface；
(3) the above-mentioned solution of Magneto separate, adds 100 μ L1.0 × 10 after cleaning with PBS buffer solution-5Mol/L rhodamine B
PBS solution, is shaken at room temperature overnight；
(4) 100 μ L1.0 × 10 are added to above-mentioned solution-6Mol/LDNA3, under room temperature react 2h, make DNA3 Yu DNA1,
DNA2 hybridizes, and forms DNA molecular switch on gold nanometer cage surface, it is achieved the purpose blocked in gold nanometer cage by rhodamine B, magnetic
Separate, remove supernatant, be again dispersed in 100 μ LPBS solution after cleaning with PBS buffer solution, prepared by ATP probe；
Wherein, the complex of described magnetic bead-gold nanometer cage is prepared as follows forming: by 10 μ L sulfydryl magnetic beads with
600 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 gold nanometer cage presses document side
Method obtains (G.D.Moon, S.W.Choi, X.Cai, W.Y.Li, E.C.Cho, U.Jeong, L.V.Wang
andY.N.Xia.J.Am.Chem.Soc.2011,133,4762–4765)。
Embodiment 2:
A kind of compound fluorescent nano probe detection for ATP utilizing the present invention, method is as follows:
(1) in the fit complex of ATP, add the ATP solution of 50 μ L variable concentrations, 37 DEG C of constant temperature oscillation reaction 1h, occur
Specific binding between ATP and ATP is fit, competes primed DNA 4；
(2) the above-mentioned solution of Magneto separate, takes the PBS that supernatant joins the compound fluorescent nano probe of the 100 μ L present invention
In suspension, there is the hybridization of primed DNA 4 and DNA3, after room temperature 1h, add 1 μ L Klenow polymerase and 20 μ L
Buffer, 5 μ LdNTPs, 1 μ L Nb.Bpu10I nickase and 20 μ L buffer, be diluted to 200 μ L with PBS, under the conditions of 37 DEG C
, there is the circular response that chain increases, chain replaces and chain is sheared in reaction 2h, makes DNA molecular switch constantly be opened, discharge fluorescence
Molecule；
(3) Magneto separate cleaning, collects supernatant and washing liquid, is diluted to 2mL with redistilled water, detects its fluorescence letter
Number；Fluoroscopic examination condition: excitation wavelength and launch wavelength be respectively 530,573nm；
Wherein, described ATP is fit, and complex is prepared as follows forming: after taking 10 μ L carboxyl magnetic bead PBS,
Magneto separate, removes supernatant, adds 100 μ L1.0 × 10-6ATP amido modified for mol/L is fit chain, is shaken at room temperature overnight, magnetic
Separate, remove supernatant, add 100 μ L1.0 × 10-6The PBS solution of mol/L primed DNA 4,37 DEG C of constant temperature oscillation reaction 1h are raw
Become by the duplex structure of the fit hybridization of primed DNA 4 and ATP, Magneto separate, remove the primer strand having neither part nor lot in hybridization, to obtain final product.
Fig. 1 is the experimental principle figure of the present invention.Fig. 2 is the fluorescence signal intensity of different ATP concentration, and the concentration of ATP is respectively
For (0,5.0 × 10-8,1.0×10-7,2.0×10-7,5.0×10-7,8.0×10-7,1.0×10-6,5.0×10-6,1.0×
10-5mol/L).Fig. 3 is the linear relationship of ATP concentration and fluorescence signal intensity.Result shows, ATP concentration is 5.0 × 10-8~
1.0×10-6During mol/L, fluorescence signal intensity presents good linear relationship with the concentration of ATP, and its linear equation is: FL=
254.87145+46.59514×CATP(10-7Mol/L), linearly dependent coefficient is 0.9925.
Nanotechnology is combined by the present invention with molecular biotechnology, by molecular recognition and specific reaction, can realize
Selective detection highly sensitive to ATP, high, meanwhile, the present invention also uses enzyme action cycle signal amplifying technique, i.e. by biology
The effect of enzyme causes the circular response that chain increases, chain replaces and chain is sheared, and makes DNA molecular switch constantly be opened, discharges glimmering
Optical molecule, it is achieved that the multiplication of fluorescence signal is amplified, and makes the detection sensitivity of ATP be significantly improved.
The compound fluorescent nano probe simple in construction of the present invention, good stability, controllability is strong, and fluorescence signal is sensitive, with
Time, do not affected by other common interference material, be there is high selectivity.This probe and detection technique thereof are at biomedical, life
The fields such as science have bigger application potential and wide application prospect, can be used for the specific detection of ATP, for weights such as cancers
The early diagnosis of big disease and treatment provide new approaches and methods.
Claims (5)
1. a compound fluorescent nano probe, it is characterised in that: using gold nanometer cage as carrier, it is loaded with fluorescence inside it and divides
Son, the assembled upper DNA molecular in its surface switchs, can be blocked in gold nanometer cage hole, prevent fluorescence molecule from leaking；Wherein, described
DNA molecular switch is made up of 3 kinds of DNA, and wherein DNA1 and DNA2 is complementary with DNA3 respectively, and described DNA3 not only contains nickase
Recognition sequence is possibly together with the sequence complementary with primer strand DNA4.
2. a compound fluorescent nano probe as claimed in claim 1, it is characterised in that:
The sequence of described DNA1 is: 5 '-GGT CCA GCT GGC TTT TTT---SH-3 ';
The sequence of described DNA2 is: 5 '-SH---TTT TTT CCG TCG CAC GCT TTT-3 ';
The sequence of described DNA3 is: 5 '-GCC AGC TGG ACC TG GCT AAG GCA GCG TGC GAC GGT GGG
GGA-3’；
The sequence of described primer strand DNA4 is: 5 '-AAT ACT CCC CCA CCG-3 '.
3. a compound fluorescent nano probe as claimed in claim 1, it is characterised in that: described fluorescence molecule is Luo Dan
Bright B.
4. the preparation method of a compound fluorescent nano probe as claimed in claim 1, it is characterised in that step is as follows:
(1) in DNA1, DNA2, add DTT solution respectively and carry out activation processing 1h；
(2) in the complex of magnetic bead-gold nanometer cage, add DNA1, DNA2 solution activated, be shaken at room temperature overnight, make
DNA1, DNA2 are assembled into gold nanometer cage surface；
(3) the above-mentioned solution of Magneto separate, adds rhodamine B solution, is shaken at room temperature overnight after cleaning with PBS buffer solution；
(4) in above-mentioned solution, add DNA3 solution, under room temperature, react 2 h, make DNA3 hybridize, at gold with DNA1 and DNA2 respectively
Nanocages surface forms DNA molecular switch, Magneto separate, again disperses, i.e. prepare compound fluorescence after cleaning with PBS buffer solution
Nano-probe；
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 and reacts 10 h, Magneto separate, cleans with PBS buffer solution, removes supernatant, to obtain final product；
The sequence of described DNA1 is: 5 '-GGT CCA GCT GGC TTT TTT---SH-3 ';
The sequence of described DNA2 is: 5 '-SH---TTT TTT CCG TCG CAC GCT TTT-3 ';
The sequence of described DNA3 is: 5 '-GCC AGC TGG ACC TG GCT AAG GCA GCG TGC GAC GGT GGG
GGA-3’。
5. the application of a compound fluorescent nano probe as claimed in claim 1, it is characterised in that for the detection of ATP,
Method is as follows:
(1) adding ATP sample solution in the fit complex of ATP, it is fit special that 37 DEG C of constant temperature oscillations react 1 h, ATP and ATP
Property combine, primed DNA 4 is competed；
(2) the above-mentioned solution of Magneto separate, takes supernatant and joins the PBS of compound fluorescent nano probe as claimed in claim 1
In suspension, make to be competed the primed DNA 4 got off and hybridize with DNA3, under room temperature reaction after add Klenow polymerase,
DNTPs, Nb.Bpu10I nickase and PBS buffer solution, react 2 h under the conditions of 37 DEG C, occurs DNA growth, chain to replace and chain
The circular response sheared, makes DNA molecular switch 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 4,37 DEG C of constant temperature oscillations of primed DNA and reacts 1 h, generates
By the duplex structure of the fit hybridization of primed DNA 4 and ATP, Magneto separate, to obtain final product.
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| CN110609020B (en) * | 2019-08-15 | 2021-10-01 | 济南大学 | Biosensor for detecting ATP based on palindromic molecular beacon and preparation method and application thereof |
| CN113252622A (en) * | 2020-02-12 | 2021-08-13 | 青岛科技大学 | Hg simultaneous detection based on single excitation2+And Ag+Method (2) |
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| CN103157118A (en) * | 2011-12-09 | 2013-06-19 | 沈阳工业大学 | Composite nano-grade novel material based on cancer early-stage integrated detection, diagnoses, and treatment, and preparation method thereof |
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