CN104730240A - Composite type nanometer fluorescent probe and preparation method and application of composite type nanometer fluorescent probe - Google Patents
Composite type nanometer fluorescent probe and preparation method and application of composite type nanometer fluorescent probe Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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 composite type nanometer fluorescent probe capable of being used for detecting ATP in the fields of biomedicines and bioscience and a preparation method and application of the composite type nanometer fluorescent probe. The invention provides a brand-new composite type nanometer fluorescent probe by combining a goldnano cage, a DNA molecular switch and a DNA amplification technology. The composite type nanometer fluorescent probe is characterized in that the goldnano cage is used as a nano container, and two single-chain DNAs are assembled on the surface of the goldnano cage; a hollow structure of the goldnano cage is filled with a fluorescent dye; by hybrid reaction, a third single-chain DNA is assembled on the surface of the goldnano cage to form the DNA molecular switch. The DNA molecular switch is turned on under controllable unwinding reaction, so that blocked fluorescent molecules in a hole are released. Therefore, according to the composite type nanometer fluorescent probe, high-sensitivity and high-selectivity detection on ATP can be realized, and a new way and a novel method are provided for early diagnosis, treatment, theoretical research and the like on serious diseases such as cancer.
Description
Technical field
The invention belongs to bioanalysis and life science, be specifically related to a kind of compound fluorescent nano probe detecting ATP and its preparation method and application.
Background technology
Atriphos (ATP) is a kind of coenzyme be extensively present in biological cell, is made up of adenosine and three phosphates.From nineteen twenty-nine Lohmann Late Cambrian and since extracting ATP, it has been proved and plays extremely important role in the metabolism of life cells and the energy supply of various biochemical reaction.As " molecule currency " that intracellular energy is transmitted, ATP stores and transmits chemical energy, is the main source of in-vivo tissue cell institute energy requirement, participates in the synthesis of protein, fat, sugar and nucleotide, all has important regulating action to the many metabolic processes of cell.Research shows, ATP is not only widely used in the treatment of malignant tumour, but also obtains as one of the mark of major disease and study widely and pay close attention to.Because ATP is the energy source that living cells is depended on for existence, when after cell damage or death, intracellular ATP content obviously reduces.Therefore, the content of ATP can reflect variation and the damage of cell.By detecting malignant tumour ATP concentration change, can understand its cellular metabolism situation, the external susceptibility being often used to antineoplastic detects, for the heterogeneity of malignant tumour susceptibility and individual chemotherapy provide feasibility therapeutic scheme.In addition, as the energy source that living cells is the most basic, ATP level and living cells numerical value are proportionate linear relationship, therefore, an index of ATP also Chang Zuowei microbial contamination, by measure ATP content number, directly can reflect the quantity of cytoactive, microorganism.Therefore, set up quick, sensitive and accurately ATP analytical approach not only contribute to early diagnosis and the treatment of the major diseases such as cancer, simultaneously also have important practical significance for numerous areas such as clinical medicine, life science, food hygiene, environmental monitoring, medicine and cosmetics.
At present, the detection method of domestic and international ATP is except as except the high performance liquid chromatography of mainstream technology, and other also has electrophoresis, spectrophotometric method and biloluminescence method etc.These methods Shortcomings all more or less in instrument and equipment, time, sensitivity, accuracy etc., especially sensitivity and selectivity wait to improve.For this reason, in the urgent need to developing the detection of detection technique for ATP of highly sensitive, high selectivity.
In recent years, gold nanometer cage obtained as a kind of emerging nano material and paid close attention to widely.This nano material is a kind of structure of hollow porous, and namely the inside of caged particle is hollow-core construction, and smooth cage wall surface is dispersed with aperture.Compared with traditional ball-type gold nano grain, the local surface plasma resonance peak of gold nanometer cage is positioned at near-infrared region 700 ~ 900nm, and this wave band is significant for biomedicine, especially for viviperception.The technology that gold nanometer cage combines with DNA molecular switch and DNA amplifying technique be yet there are no bibliographical information.
Summary of the invention
In order to overcome the deficiency that prior art exists, simultaneously, also in order to more effectively utilize the remarkable advantage of the nano material with special construction, for the rare report of fluorescent nano probe detecting 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, the gold nanometer cage material and DNA amplifying technique with DNA molecular switch are combined, this compound fluorescent nano probe is made not only to have controlled molecular switch, and can after solution chain reaction, discharge a large amount of fluorescence molecule, by realizing the highly sensitive detection to ATP to the detection of fluorescence signal, the second object of the present invention: the preparation method that this compound fluorescent nano probe a kind of is provided, the third object of the present invention: provide this compound fluorescent nano probe of a kind of application to detect the method for ATP.The compound fluorescent nano probe that the present invention proposes is used for the fluoroscopic examination of ATP, effectively can improves sensitivity and the accuracy of ATP detection, significantly reduce amount of samples.
The present invention is achieved through the following technical solutions goal of the invention.Compound fluorescent nano probe of the present invention is using gold nanometer cage as carrier, utilize the architectural characteristic that it is hollow porous, load fluorescence molecule therein, in order to prevent leaking of fluorescence molecule, by at its surface-assembled DNA molecular switch, by the hole shutoff on gold nanometer cage surface; Wherein, described DNA molecular switch is made up of 3 kinds of DNA, and wherein DNA1, DNA2 are complementary with DNA3 respectively, and DNA3 not only also contains the sequence with primer strand DNA4 complementation containing nickase recognition sequence.
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 GCAGCG 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.
Prepare a preparation method for above-mentioned compound fluorescent nano probe, comprise the steps:
(1) in DNA1, DNA2, add DTT solution respectively and carry out activation process 1h;
(2) in the compound of magnetic bead-gold nanometer cage, add DNA1, DNA2 solution activated, shaken at room temperature spends the night, and makes DNA1, DNA2 be assembled into gold nanometer cage surface;
(3) the above-mentioned solution of Magneto separate, add rhodamine B solution with after the cleaning of PBS buffer solution, shaken at room temperature spends the night;
(4) in above-mentioned solution, add DNA3 solution, under room temperature, react 2h, DNA3 and DNA1, DNA2 are hybridized, DNA molecular switch is formed on gold nanometer cage surface, Magneto separate, disperses again with after the cleaning of PBS buffer solution, i.e. obtained compound fluorescent nano probe;
Wherein, the compound of described magnetic bead-gold nanometer cage is prepared from as follows: the mixed solution of magnetic bead and gold nanometer cage is placed in shaken at room temperature and reacts 10h, Magneto separate, with the cleaning of PBS buffer solution, removes supernatant, to obtain final product.
Utilize compound fluorescent nano probe of the present invention for a detection of ATP, method is as follows:
(1) in the fit compound of ATP, add ATP sample solution, 37 DEG C of fit specific bindings of constant temperature oscillation reaction 1h, ATP and ATP, compete primed DNA 4;
(2) the above-mentioned solution of Magneto separate, getting supernatant joins in the PBS suspending liquid of compound fluorescent nano probe of the present invention, make to be competed the primed DNA 4 got off to hybridize with DNA3, Klenow polymerase, dNTPs, Nb.Bpu10I nickase and PBS buffer solution is added again after reaction under room temperature, 2h is reacted under 37 DEG C of conditions, there is the circular response that chain growth, chain replacement and chain are sheared, DNA molecular switch is constantly opened, discharges fluorescence molecule;
(3) the above-mentioned solution of Magneto separate, collects supernatant, detects its fluorescence signal;
Wherein, described ATP is fit, and compound is prepared from as follows: mixed with amido modified ATP aptamer solutions by carboxyl magnetic bead, shaken at room temperature spends the night, Magneto separate, removes supernatant, adds primed DNA 4,37 DEG C of constant temperature oscillation reaction 1h, generate the duplex structure by primed DNA 4 and the fit hybridization of ATP, Magneto separate, to obtain final product.
Beneficial effect of the present invention: nanometer technology combines with molecular biotechnology by the compound fluorescent nano probe that the present invention proposes, and by molecular recognition and specific reaction, can realize detection that is highly sensitive to ATP, high selectivity.This compound fluorescent nano probe is utilized to detect ATP, cause chain growth, chain replaces and chain is sheared circular response by the effect of biology enzyme, DNA molecular switch is constantly opened, discharges fluorescence molecule, achieve the amplification of fluorescence signal, the detection sensitivity of ATP is significantly improved.
Compound fluorescent nano probe structure of the present invention is simple, good stability, and controllability is strong, and fluorescence signal is sensitive, meanwhile, does not affect, has high selectivity, can be applicable to the fluoroscopic examination of ATP in living things system by other common interference material.Experimental result shows, the compound fluorescent nano probe adopting the present invention to propose can 5.0 × 10
-8~ 1.0 × 10
-6realize within the scope of M detecting highly sensitive, the high selectivity of ATP.This probe and detection technique thereof have larger application potential and wide application prospect in fields such as biomedicine, life sciences, can be used for the specific detection of ATP, for the early diagnosis of the major diseases such as cancer and treatment provide new approaches and methods.
Accompanying drawing explanation
Fig. 1 experimental principle figure of the present invention.
The fluorescence signal intensity of the different ATP concentration of Fig. 2.
The linear relationship of Fig. 3 ATP concentration and fluorescence signal intensity.
Embodiment
Be below the specific embodiment that the present invention relates to, technical scheme of the present invention is described further, but protection scope of the present invention is not limited to these embodiments.Everything does not deviate from change of the present invention or equivalent substituting includes within protection scope of the present invention.
Specifically describe the present invention below by embodiment, but the present invention is not by the restriction of following embodiment.
Experimental apparatus: magnetic separation rack (happy chromatographic technique development centre is doubly thought in Tianjin); F-4600 fluorospectrophotometer (Hitachi, Japan); THZ-82A gas bath constant temperature oscillator (medical apparatus and instruments factory of Jintan City).
Experiment reagent: 3-4 μm of carboxyl modified magnetic bead, sulfydryl modification magnetic bead (happy chromatographic technique development centre is doubly thought in Tianjin); Rhodamine B (Aladdin); Atriphos (ATP); Dithiothreitol (DTT) (DTT, Aladdin); Klenow polymerase and buffer (Thermo Scientific, the U.S.) thereof; Nb.Bpu10I nickase and buffer (Thermo Scientific, the U.S.) thereof; DNTPs (match Parkson, Beijing gene technology company limited), DNA artificial sequence synthetic used is buied by match Parkson, Beijing bioengineering company limited, and PBS solution is 0.01M (pH7.4, Na
2hPO
4-NaH
2pO
4).
Embodiment 1:
Prepare a preparation method for the compound fluorescent nano probe of the present invention, comprise the steps:
(1) respectively in 10 μ L1.0 × 10
-510 μ L1.0 × 10 are added in DNA1, DNA2 of mol/L
-3the DTT solution of mol/L, then adds 80 μ LPBS solution respectively, activation 1h;
(2) in the compound of magnetic bead-gold nanometer cage, add DNA1, DNA2 solution activated, shaken at room temperature spends the night, and makes DNA1, DNA2 be assembled into gold nanometer cage surface;
(3) the above-mentioned solution of Magneto separate, adds 100 μ L1.0 × 10 with after the cleaning of PBS buffer solution
-5the PBS solution of mol/L rhodamine B, shaken at room temperature spends the night;
(4) 100 μ L1.0 × 10 are added to above-mentioned solution
-6mol/LDNA3,2h is reacted under room temperature, DNA3 and DNA1, DNA2 are hybridized, DNA molecular switch is formed on gold nanometer cage surface, realize the object of rhodamine B shutoff in gold nanometer cage, Magneto separate, removes supernatant, again be dispersed in 100 μ LPBS solution with after the cleaning of PBS buffer solution, ATP probe preparation completes;
Wherein, the compound of described magnetic bead-gold nanometer cage is prepared from as follows: by 10 μ L sulfydryl magnetic beads and 600 μ L gold nanometer cage Homogeneous phase mixing, shaken at room temperature reaction 10h, and Magneto separate, with PBS solution cleaning, removes supernatant, to obtain final product; Described sulfydryl magnetic bead is the commodity (happy chromatographic technique development centre is doubly thought in Tianjin) bought; Described gold nanometer cage is pressed literature method and is obtained (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:
Utilize compound fluorescent nano probe of the present invention for a detection of ATP, method is as follows:
(1) in the fit compound of ATP, add the ATP solution of 50 μ L variable concentrations, there is the specific binding of fit of ATP and ATP, competed by primed DNA 4 in 37 DEG C of constant temperature oscillation reaction 1h;
(2) the above-mentioned solution of Magneto separate, getting supernatant joins in the PBS suspending liquid of 100 μ L compound fluorescent nano probe of the present invention, there is the hybridization reaction of primed DNA 4 and DNA3,1 μ LKlenow polymerase and 20 μ L buffer, 5 μ LdNTPs, 1 μ L Nb.Bpu10I nickase and 20 μ Lbuffer are added again after room temperature 1h, 200 μ L are diluted to PBS, 2h is reacted under 37 DEG C of conditions, there is the circular response that chain growth, chain replacement and chain are sheared, DNA molecular switch is constantly opened, discharges fluorescence molecule;
(3) Magneto separate cleaning, collects supernatant and washing lotion, is diluted to 2mL, detects its fluorescence signal with redistilled water; Fluoroscopic examination condition: excitation wavelength and emission wavelength are respectively 530,573nm;
Wherein, described ATP is fit, and compound is prepared from as follows: after getting 10 μ L carboxyl magnetic bead PBS cleanings, Magneto separate, removes supernatant, then adds 100 μ L1.0 × 10
-6the fit chain of ATP that mol/L is amido modified, shaken at room temperature spends the night, Magneto separate, removes supernatant, adds 100 μ L1.0 × 10
-6the PBS solution of mol/L primed DNA 4,37 DEG C of constant temperature oscillation reaction 1h, generate the duplex structure by primed DNA 4 and the fit hybridization of ATP, Magneto separate, removing has neither part nor lot in the primer strand of hybridization, to obtain final product.
Fig. 1 is 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 (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, the concentration of fluorescence signal intensity and ATP presents good linear relationship, and its linear equation is: FL=254.87145+46.59514 × C
aTP(10
-7mol/L), linearly dependent coefficient is 0.9925.
Nanometer technology combines with molecular biotechnology by the present invention, by molecular recognition and specific reaction, detection that is highly sensitive to ATP, high selectivity can be realized, meanwhile, the present invention also uses enzyme and cuts cycle signal amplifying technique, and the effect namely passing through biology enzyme causes chain growth, chain replaces and the circular response of chain shearing, DNA molecular switch is constantly opened, discharge fluorescence molecule, the multiplication achieving fluorescence signal is amplified, and the detection sensitivity of ATP is significantly improved.
Compound fluorescent nano probe structure of the present invention is simple, good stability, and controllability is strong, and fluorescence signal is sensitive, meanwhile, does not affect, have high selectivity by other common interference material.This probe and detection technique thereof have larger application potential and wide application prospect in fields such as biomedicine, life sciences, can be used for the specific detection of ATP, for the early diagnosis of the major diseases such as cancer and treatment provide new approaches and methods.
Claims (6)
1. a compound fluorescent nano probe, is characterized in that: using gold nanometer cage as carrier, its inside is loaded with fluorescence molecule, and its assembled upper DNA molecular switch in surface, can, by the shutoff of 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.
2. a compound fluorescent nano probe as claimed in claim 1, is characterized in that: described DNA3 not only also contains the sequence with primer strand DNA4 complementation containing nickase recognition sequence.
3. the compound fluorescent nano probe according to any one of claim 1-2, is characterized in that:
The partial sequence of described 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 TGCGAC GGT GGG GGA-3 ';
The partial sequence of described primer strand DNA4 is: 5 '-AAT ACT CCC CCA CCG-3 '.
4. the compound fluorescent nano probe according to any one of claim 1-3, is characterized in that: described fluorescence molecule is rhodamine B.
5. a preparation method for the compound fluorescent nano probe according to any one of claim 1-4, is characterized in that step is as follows:
(1) in DNA1, DNA2, add DTT solution respectively and carry out activation process 1h;
(2) in the compound of magnetic bead-gold nanometer cage, add DNA1, DNA2 solution activated, shaken at room temperature spends the night, and makes DNA1, DNA2 be assembled into gold nanometer cage surface;
(3) the above-mentioned solution of Magneto separate, add rhodamine B solution with after the cleaning of PBS buffer solution, shaken at room temperature spends the night;
(4) in above-mentioned solution, add DNA3 solution, under room temperature, react 2h, DNA3 is hybridized respectively with DNA1 and DNA2, DNA molecular switch is formed on gold nanometer cage surface, Magneto separate, disperses again with after the cleaning of PBS buffer solution, i.e. obtained compound fluorescent nano probe;
Wherein, the compound of described magnetic bead-gold nanometer cage is prepared from as follows: the mixed solution of magnetic bead and gold nanometer cage is placed in shaken at room temperature and reacts 10h, Magneto separate, with the cleaning of PBS buffer solution, removes supernatant, to obtain final product.
6. an application for the compound fluorescent nano probe according to any one of claim 1-4, it is characterized in that the detection for ATP, method is as follows:
(1) in the fit compound of ATP, add ATP sample solution, 37 DEG C of fit specific bindings of constant temperature oscillation reaction 1h, ATP and ATP, compete primed DNA 4;
(2) the above-mentioned solution of Magneto separate, getting supernatant joins in the PBS suspending liquid of the compound fluorescent nano probe according to any one of claim 1-4, make to be competed the primed DNA 4 got off to hybridize with DNA3, Klenow polymerase, dNTPs, Nb.Bpu10I nickase and PBS buffer solution is added again after reaction under room temperature, 2h is reacted under 37 DEG C of conditions, there is the circular response that DNA chain growth, chain replacement and chain are sheared, DNA molecular switch is constantly opened, discharges fluorescence molecule;
(3) the above-mentioned solution of Magneto separate, collects supernatant, detects its fluorescence signal;
Wherein, described ATP is fit, and compound is prepared from as follows: mixed with amido modified ATP aptamer solutions by carboxyl magnetic bead, shaken at room temperature spends the night, Magneto separate, removes supernatant, adds primed DNA 4,37 DEG C of constant temperature oscillation reaction 1h, generate the duplex structure by primed DNA 4 and the fit hybridization of ATP, Magneto separate, to obtain final product.
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