CN106497978B - A kind of magnetic composite nano particle and its preparation method and application - Google Patents

A kind of magnetic composite nano particle and its preparation method and application Download PDF

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CN106497978B
CN106497978B CN201610811557.2A CN201610811557A CN106497978B CN 106497978 B CN106497978 B CN 106497978B CN 201610811557 A CN201610811557 A CN 201610811557A CN 106497978 B CN106497978 B CN 106497978B
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赵美萍
翟筠秋
刘艺斌
黄山
方思敏
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Abstract

The invention discloses a kind of magnetic composite nano particles and its preparation method and application, the magnetic composite nano particle uniform particle diameter, diameter 40-60nm, the DNA double chain containing abasic site that surface is connected by Avidin-Biotin specifically can be identified and be cut by the abasic endonuclease I (APE1) of target protein, release fluorophor or drug molecule；Meanwhile present invention magnetic composite nano particle obtained has good dispersibility, stability and biocompatibility, so as to the fluorescence in situ imaging for APE1 in living cells.It is usually overexpressed APE1 additionally, due to cancer cell, above-mentioned magnetic composite nano particle can more effectively discharge drug in cancer cell, be expected to realize the accurate treatment for being directed to cancer cell.

Description

A kind of magnetic composite nano particle and its preparation method and application
Technical field
The present invention relates to a kind of Multifunctional composite nanometer particles and its preparation method and application.More specifically to one The nucleic acid function magnetic composite nano particle and its preparation method and application of kind Avidin modification.The nano particle can be used as base Because of carrier, the in situ imaging and pharmaceutical carrier of nucleic acid in living cell repair enzyme, belong to nano particle preparation technical field.
Background technique
Nano material has excellent electricity, optics, mechanical property, morphology controllable and surface is easily modified.Biomolecule tool There is the features such as high specificity, binding site are abundant (Eugenii Katz, Itamar Willner.Integrated Nanoparticle-Biomolecule Hybrid Systems:Synthesis,Properties,and Applications.Angew.Chem.Int.Ed(2004),43,6042–6108).It is fluffy with nanotechnology and biotechnology Nano material is combined with biomolecule and various composite nano materials is made by the exhibition of breaking out, can be used to organism into Row diagnoses, treats, repairs or replaces its disease damage tissue, and cell imaging, in terms of show good application Prospect.
Combination between Avidin and biotin is most firm articulated system between presently found biomolecule (Buller,HR.,Gallus,AS.,Pillion,G.,Prins,MH.,Raskob,GE.Enoxaparin followed by once-weekly idrabiotaparinux versus enoxaparin plus warfarin for patients with acute symptomatic pulmonary embolism:a randomised,double-blind,double- Dummy, non-inferiority trial.Lancet (2012), 379,123-129), organic solvent can be resisted, surface is lived (Green, the N.Michael.Avidin.Advances in such as property agent, the influence of proteolytic enzyme, extreme temperature and pH Protein Chemistry Volume(1975),29,85-133).Avidin is total to the nano silicon particles for having modified carboxyl Valence connection, then by biotinylated DNA by the modification of affinity interaction between Avidin and biotin to being covalently attached parent With the nano silicon particles surface of element, the quantity of nano grain surface DNA can be greatly improved, and enhances DNA and nanometer load The fastness of body.
DNA or drug transduction, which refer to, imported into DNA or drug in cancer cell or target cell by external source mediation.Mesh Though provirus carrier can achieve higher transfection efficiency, there are strongly immunogenic, high mutagenesis risk, low capacities etc. to lack Point limits its application in biology and clinical treatment.And non-virus carrier, especially nano particle can not only overcome with Upper disadvantage, and have many advantages, such as convieniently synthesized, repeated high, a variety of physicochemical properties and easy surface modification, realize nanometer The multifunction of carrier.
Magnetic nano particle is not only easily modified, also have magnetic field effect and Fluorescence quenching effect, by magnetic nano particle by other Particle combines and modifies biomolecule, can solve the problems, such as that nano particle disengaging time is long and transfection time is long (Liu,YB.,Fang,SM.,Zhai,JQ.,Zhao,MP.Construction of antibody-like nanoparticles for selective protein sequestration in living cells.NANOSCALE (2015),7(16):7162-7167).Nano silicon particles not only have the advantages of good biocompatibility, porosity, Yi Fanying, also Can protect modification nano grain surface DNA not by the interference of DNase I enzyme (Roy, I., Ohulchanskyy, TY., Bharali,DJ.,Pudavar,HE.,Mistretta,RA.,Kaur,N.,Prasad,PN.Optical tracking of organically modified silica nanoparticles as DNA carriers:A nonviral, Nanomedicine approach for gene delivery.PNAS (2005), 102 (2): 279-284), therefore magnetic is received Rice grain is combined with nano silicon particles, not only realizes transfection efficiency rapidly and efficiently, is reduced bio-toxicity, is also protected The DNA being modified can be completely transfected in cell.
At present some using nano silicon particles by DNA import cell method, have covalent linkage DNA (Cai L, Chen Z Z,Chen M Y,et al.MUC-1aptamer-conjugated dye-doped silica Nanoparticles for MCF-7cells detection [J] .Biomaterials, 2013,34 (2): 371-381.) and (Qian R, Ding L, Ju H.Switchable fluorescent imaging of is connected using electrostatic interaction intracellular telomerase activity using telomerase-responsive mesoporous silica nanoparticle.Journal of the American Chemical Society,2013,135(36): The methods of 13282-13285.).Such method is mainly used for hybridizing (Zhang P, Cheng with intracellular miRNA or mRNA F,Zhou R,et al.DNA-hybrid-gated multifunctional mesoporous silica nanocarriers for dual-targeted and microRNA-responsive controlled drug Delivery.Angewandte Chemie International Edition, 2014,53 (9): 2371-2375.), due to Protective effect (He X, Wang K, Tan W, et al.Bioconjugated nanoparticles of the nano silicon particles to DNA for DNA protection from cleavage.Journal of the American Chemical Society, 2003,125 (24): 7168-7169.), these nano particles usually all inhibit reacting for DNA and intracellular nucleic acid enzyme, thus It is difficult to use in activity analysis and the in situ imaging of nuclease.In addition, importing miRNA drug currently with nano particle, lack needle To release regulating and controlling effect (Mura S, Nicolas J, the Couvreur P.Stimuli-responsive of cancer cell specificity nanocarriers for drug delivery.Nature materials,2013,12(11):991-1003.)。
Summary of the invention
The object of the present invention is to provide a kind of preparation method and application of magnetic composite nano particle.The magnetic composite nano Particle can not only carry out the identification and quantitative analysis of specificity to the target protein in solution in vitro, it is even more important that It can be rapidly introduced into living cells under magnetic fields, fluorescence imaging in situ is carried out to target protein therein, passes through fluorescence Microscope carries out tracer to its distribution in cell.If fluorescence group is replaced with anticancer drug, can also realize to cancer Carry out immunotherapy targeted autoantibody.
To achieve the goals above, the present invention uses following technical scheme.
A kind of magnetic composite nano particle successively includes magnetic core, silica shell and biomolecule modification from inside to outside Layer, the magnetic core are ferromagnetic nanoparticle, and the silica shell is the silica that surface modification has carboxyl, the life Object molecular modification layer is Avidin and the biotinylated double-stranded DNA containing abasic vacancy that (i.e. Avidin) combines therewith (biotinylated-AP-DNA)。
Further, the ferromagnetic nanoparticle is ferroferric oxide nano granules or γ-di-iron trioxide nanometer Grain.
Further, the TEM partial size of the magnetic composite nano particle is 40-60nm, and DLS partial size is 65-75nm.
Further, the diameter of the magnetic core be 20-30nm, the silica shell with a thickness of 10-15nm, it is described The thickness of biomolecule decorative layer is less than or equal to 20nm.
Further, the expression formula of the magnetic composite nano particle is Fe3O4@SiO2@AVD-AP-DNA or γ-Fe2O3@ SiO2@AVD-AP-DNA.Wherein Fe3O4Represent ferroso-ferric oxide magnetic core, γ-Fe2O3Then represent γ-di-iron trioxide magnetic core； SiO2Represent silica shell；AVD represents Avidin layer；AP-DNA represents the biotinylated double-strand containing abasic vacancy DNA (biotinylated-AP-DNA) layer.
Further, the biotinylated double-stranded DNA (biotinylated-AP-DNA) containing abasic vacancy Two single-stranded sequences are respectively as follows: 5 '-GGGGACGACTCCATCGACCTCAAGCAGTTGATCCTTTGGAAAAAAA- Biotin-3 ' and 5 '-GTCGATGGAGXCGTCCCC-FAM-3 ', wherein X indicates that abasic site, Biotin indicate biotin Label, FAM are Fluoresceincarboxylic acid label.
The present invention also provides the preparation methods of above-mentioned magnetic composite nano particle, comprising: first in silica shell Outer layer is covalently attached upper Avidin, then passes through the strong affinity interaction of Avidin and biotin, by the biotinylated base containing U Double-stranded DNA (dsDNA) modification finally recycles UDG enzyme to cut U base on the ferromagnetic nanoparticle surface of coated with silica It removes, generates the double-stranded DNA (AP-DNA) containing abasic vacancy.
Further, specific step is as follows for above-mentioned preparation method:
1) the ferromagnetic nanoparticle ultrasonic disperse for coating the silica shell of carboxyl modified is in pure water, respectively plus Enter EDC and NHS, oscillating reactions under room temperature (referring both to 15-28 DEG C below), as solution A.
2) Avidin is added in solution A, at room temperature oscillating reactions.Magneto separate washing removes unreacted substance, then By obtained reactant again ultrasonic disperse in pure water, as solution B.
3) double-stranded DNA of the biotinylated base containing U is added in solution B, is incubated for 30-90 minutes at room temperature.Magneto separate And wash, unreacted substance is removed, then surpasses the magnetic nanoparticle that the double-stranded DNA of the obtained base containing U is modified again Sound is dispersed in PBS solution, as solution C.
4) solution C is added in 1.1 buffer solution of buffer, UDG digestion is added except the U base in DNA double chain, obtains To magnetic composite nano particle.
Further, in step 1), the ferromagnetic nanoparticle ultrasonic disperse of the silica shell cladding of carboxyl modified Final concentration of 0.5-2mg/mL in pure water.
Further, ultrasonic time requires more than 10 minutes in step 1).
Further, the concentration of EDC is 0.5-2mg/mL in step 1), and the concentration of NHS is 1-5mg/mL, EDC, NHS with The weight ratio of the ferromagnetic nanoparticle of the silica shell cladding of carboxyl modified is 4:10:1.
Further, to activate the carboxyl of silica shell layer surface sufficiently, the amount of skipping over will be added in EDC and NHS, activation Time should not be too short or too long, and according to the difference of room temperature, preferably activation time is 15-45 minutes.
Further, the concentration of Avidin is 0.1-0.2mg/mL, preferably 0.1mg/mL in step 2).Avidin, which is added, to be made Nano particle and Avidin (weight) ratio of final concentration in mixed solution are 5:1, at room temperature 8-12 hour of oscillating reactions, are surpassed Sound jitter time is 5 minutes.
Wherein, outside silicon dioxide layer be covalently attached Avidin when, to keep nano particle fully dispersed, nano particle it is dense Degree should not be excessively high, preferred concentration 0.5-1mg/mL.
Step 2) after reaction, in order to remove the unreacted reactant in reaction system, can be washed with deionized water 1-3 times, Then re-ultrasonic dispersion is in pure water.
Further, in step 3), nano particle is arrived into double-stranded DNA (dsDNA) modification of the biotinylated base containing U When on surface, to keep the conformation of dsDNA more stable consistent, heating annealing is carried out first and (90s is incubated at 95 DEG C, at 75 DEG C It is incubated for 90s, 90s is incubated at 50 DEG C, is finally incubated for 600s at 37 DEG C), then dsDNA is added in solution B.
Further, in step 3), the double-stranded DNA of the biotinylated base containing U of addition is final concentration of in solution B 0.2-1μM。
It is most for the quantity that makes dsDNA be connected to nano grain surface, its concentration can be optimized, make 1 μM of ultimate density.
Further, in step 3), the magnetic nanoparticle that the double-stranded DNA of the obtained base containing U is modified is again ultrasonic It is dispersed in the PBS solution of 0.1M, the ultrasonic disperse time is 5 minutes, if do not used immediately, then should be placed in 4 DEG C of preservations.
Further, in step 4), the composition of 1.1 buffer solution of buffer are as follows: 10mM Bis TrisPropane- HCl,10mM MgCl2, 100 μ g/ml BSA, pH 7.0 (25 DEG C).
Final concentration 1.0-100U/mL of the UDG enzyme of addition in 1.1 buffer solution of buffer comprising solution C, reaction Time range is 0.5-10min.
The present invention also provides the enzyme assays, nucleic acid in living cell repair enzyme in vitro of above-mentioned magnetic composite nano particle In situ imaging in application.
The invention has the benefit that
Ferroso-ferric oxide or γ-kernel of the di-iron trioxide magnetic core as composite particles, its main feature is that: to some fluorescent bases Group has quenching effect, helps to separate particle in particulate production, wash and when importing cell, can Enter the process of cell with the effect acceleration nanoparticle by externally-applied magnetic field.Surface modification has the silicon dioxide layer of carboxyl can The hydrophily and biocompatibility for improving nano particle provide carboxyl and are covalently attached with Avidin generation and react, and guarantee when glimmering Fluorescence can be discharged when light blob is far from particle surface.Avidin (AVD) has with the biotin on biontinylated-ds DNA Ds DNA can be zoomed in nano grain surface by strong affinity interaction, this effect, so that ds DNA is succeeded and effectively repaired Decorations are on nano particle.Most importantly, we have found and confirm under study for action, AVD and abasic endonuclease I (APE1) there is the affinity interaction of specificity between, this interaction can make AVD that the APE1 in solution is attracted to nanometer Particle surface acts on the abasic site on DNA, the phosphodiester bond of 5 ' side of abasic site is effectively cut, to discharge The fluorophor or drug being connected on DNA.DNA can not only connect fluorophor herein as the molecular arm of nano-carrier For carrying out fluorescence detection or intracellular imaging to APE1, drug molecule can also be connected, and can guarantee only and exist in APE1 In the case where just release fluorophor or drug molecule etc., to ensure that composite nanometer particle can expressed in APE1 It effectively plays a role in the cancer cell of amount, and reduces the side effect even avoided to normal cell.
The present invention has synthesized a kind of magnetic, multi-functional composite nanometer particle, uniform particle diameter, diameter 40-60nm, surface connection DNA double chain containing abasic site specifically can be identified and be cut by the abasic endonuclease I (APE1) of target protein, release Discharging fluorescence group or drug molecule.Nano particle obtained has good dispersibility, stability and biocompatibility, from And it can be used for the fluorescence in situ imaging of APE1 in living cells.APE1, above-mentioned nano particle are usually overexpressed additionally, due to cancer cell Drug can be more effectively discharged in cancer cell, be expected to realize the accurate treatment for being directed to cancer cell.
Detailed description of the invention
Fig. 1 is the preparation process and schematic illustration of magnetic composite nano particle.
Fig. 2 is nano-probe A (Fe3O4@SiO2@AVD-AP-DNA nano particle) transmission electron microscope picture.
Fig. 3 is MNPs (Fe3O4@SiO2), MNP@AVD (Fe3O4@SiO2@AVD) and nano-probe A (Fe3O4@SiO2@AVD- AP-DNA) zeta potential of three kinds of particles in 0.1mg/mL aqueous solution.
Fig. 4 a is the time graph that nano-probe A responds various concentration APE1；Fig. 4 b is that nano-probe A detection APE1 is living The linear fit curve of property.
Fig. 5 is selectivity of the nano-probe A to various common nucleic acid enzymes.
Fig. 6 is the fluorogram changed over time after nano-probe A magnetic transfects 30 minutes into HeLa cell.
The variation diagram of Fig. 7 intracellular Fluorescence signal for the fluorogram of intracellular control probe and under different pharmaceutical effect.
Fig. 8 is toxicity of the nano-probe A to cell.
Specific embodiment
It elaborates with reference to the accompanying drawings and detailed description to the present invention.Following specific embodiments will be helpful to Understand invention, but is not intended to limit the contents of the present invention.
Embodiment 1 prepares magnetic composite nano particle Fe3O4@SiO2@AVD-AP-DNA (nano-probe A)
The present invention prepare the process of magnetic composite nano particle as shown in Figure 1, specifically includes the following steps:
By 1.0mg Fe3O4@SiO2Under room temperature, 40KHz (it is all made of the supersonic frequency in the present invention, is adjusted as needed For different ultrasonic times) ultrasound 20 minutes, it is dispersed in the solution for being made into 0.5mg/mL in 2.0mL pure water, 4.0mg EDC is added With 10.0mg NHS activated carboxyl, at room temperature oscillating reactions 30 minutes.The Avidin that 0.1mL concentration is 2.0mg/mL, room is added Warm lower 8 hours of oscillating reactions.Magneto separate removes unreacted substance, is washed with deionized water three times, ultrasonic disperse exists again In 2.0mL pure water.The double-stranded DNA of final concentration of 1 μM of the biotinylated base containing U is added, is incubated for 1 hour at room temperature.Magnetic point From, it is washed with PBS (pH 7.4,0.1M) and removes unreacted DNA three times, the magnetism that the double-stranded DNA of the obtained base containing U is modified Again ultrasonic disperse continues to use or is stored in 4 DEG C in 2.0mL 0.1M PBS (pH 7.4) to nano particle.In 50 μ L PCR Guan Zhong, the magnetic nanoparticle that the above-mentioned double-stranded DNA modification for being dispersed in the base containing U in 0.1M PBS of 5 μ L 1.0mg/mL is added are molten Liquid, 5 μ L10 × Buffer1.1 buffers add deionized water and total volume are made to be 47 μ L, are then added 1 μ L 500U/mL's UDG enzyme reacts 2min, obtains Multifunctional composite nanometer particle Fe3O4@SiO2@AVD-AP-DNA (nano-probe A), is immediately available for The determination of activity of APE1 imports cell experiment.
Composite nanometer particle Fe is characterized by transmission electron microscope3O4@SiO2@AVD-AP-DNA is as shown in Figure 2, it is seen that Composite nanometer particle size is uniform, and good dispersion, partial size is in 60nm or so.From former nano particle Fe3O4@SiO2(MNPs), in Mesosome Fe3O4@SiO2@AVD (MNP@AVD) and composite nanometer particle Fe3O4@SiO2The zeta of@AVD-AP-DNA (nano-probe A) Known to potential value (Fig. 3), former nano particle successfully links up AVD and biotinylated-AP-DNA.
Embodiment 2 directly detects the APE1 activity in solution
In this embodiment, the DNA probe sequence of design is as follows:
5’-GGGGACGACTCCATCGACCTCAAGCAGTTGATCCTTTGGAAAAAAA-Biotin-3’
5’-GTCGATGGAGXCGTCCCC-FAM-3’
Wherein X indicates that abasic site, Biotin indicate that biotin labeling, FAM are carboxyl Fluoresceincarboxylic acid label.
Experimental procedure is as follows:
1. the magnetic composite nano particle that 10 μ L 0.5mg/mL embodiments 1 are prepared is added in 200 μ L PCR pipes Solution, 5 μ L 10 × Buffer1.1 buffers, add deionized water make total volume be 48 μ L, it is dense that 2 μ L differences are then added The APE1 solution of degree, making the final concentration of APE1 is respectively 0,0.01,0.05,0.1,0.2,0.5,1U/mL, detects composite Nano The response of grain.
Using Stratagene Mx3000P fluorescent PCR instrument, every 5s acquires fluorescence, excitation wavelength 470nm, In at 37 DEG C Transmitting signal is surveyed under 510nm, and is fitted the working curve for reacting fluorescence climbing speed and APE1 concentration in initial 50s.
As shown in fig. 4 a, fitting result is as shown in Figure 4 b for testing result, and composite nanometer particle can detecte 0.01U/mL APE1, there is sensitive response.With the increase of APE1 concentration, fluorescence climbing speed and APE1 concentration are in a linear relationship, R2=0.996, working range is 0.01-1.0U/mL.
2. the magnetic composite nano particle that 10 μ L 0.5mg/mL embodiments 1 are prepared is added in 200 μ LPCR pipes Solution, 5 μ L 10 × DNase I buffers add deionized water and total volume are made to be 48 μ L, 2 μ L 125U/mL DNase are added I solution makes the final concentration of 5U/mL of DNase I, detects the response of composite nanometer particle.
Using Stratagene Mx3000P fluorescent PCR instrument, every 5s acquires fluorescence, excitation wavelength 470nm, In at 37 DEG C Transmitting signal is surveyed under 510nm.Magnetic composite nano particle is identical with this response detection process of other enzymes, only by buffer Replace with its most suitable condition.
Testing result is as shown in figure 5, the final concentration of various enzymatic determinations is respectively APE1:2.0U/mL；DNase I:5.0U/ mL；Exo III:4.0U/mL；Lambda exo:66.7U/mL；Exo I:12.5U/mL；T5:5U/mL；T7 50U/mL).It can To find composite nanometer particle in the presence of APE1, the abasic site in AP-DNA can be released distal end by cutting Nucleotide with FAM so that fluorescence climbing speed is very fast, and then responds other enzymes, especially DNase I etc. very slow. Illustrate that composite nanometer particle has selectivity well to APE1.It is worth noting that Exo III is by bacterial expression, together When with 3 ' is exo-acting and the nucleases of abasic site endo-activity, it can also be seen that magnetic composite nano from Fig. 5 Grain has certain response to it.
Embodiment 3, composite nanometer particle quickly introduce cell under magnetic fields and discharge fluorescence signal
The magnetic composite nano particle being prepared has preferable stability, biocompatibility, leads under magnetic fields Hela cell can be quickly introduced and APE1 is imaged by crossing endocytosis.
Experimental procedure is as follows:
1. cell is cultivated in dual anti-containing 1% Pen .- Strep and 10% inactivation fetal calf serum DMEM culture medium, Incubator condition is 37 DEG C and 5%CO2/ 95% air.As the 70%-80% of the long extremely covering culture bottle of cell, it is added 0.25% pancreatin handles 4min at 37 DEG C and then shifts cell on 96 hole flat bottom glass plates, and it is thin that 0.1mL is contained in each hole Born of the same parents' culture solution, there are about 104A cell.Make its adherent for 24 hours cell culture before experiment.
2. being washed with DPBS buffer to cell, the magnetic composite nano that embodiment 1 is prepared then is added The solution of grain, makes its final concentration of 50 μ g/mL.96 orifice plates are placed in the MagnetoFACTOR-96 magnetic sheet of Chemicell company On, at 37 DEG C and 5%CO230min is placed in the incubator of/95% air.After removing magnetic sheet, siphon away molten in 96 orifice plates Liquid is washed cell 5 times with DPBS buffer, sufficiently washes away the magnetic nano particle for not entering into cell.
3. being added in the cell for importing magnetic composite nano particle and containing colourless DMEM culture medium, at 37 DEG C and 5% CO2Cultivated in/95% online culture apparatus, use be equipped with Evolve-EMCCD 71 fluorescence microscope of Olympus IX with And cell is shot under 100 times of object lens, the fluorescence picture (time for exposure of corresponding different probe is shot with corresponding fluorescence grating 100) 10ms, transmitting gain value EM gain are.Result treatment is carried out with ImageJ software, as shown in Figure 6.It is tied as magnetic transfects The region of the increase of incubation time after beam, (green) fluorescence distribution in HeLa cell increases, and fluorescence intensity also increases, 120 Reach maximum at minute.Fluorescence distribution region and fluorescence intensity gradually decrease after 180 minutes.Illustrate that nano-probe A is entered In HeLa cell.
4. further to prove the fluorescence signal generated into the cell actually from intracellular APE1 to magnetic composite nano The fixed point of abasic site is cut in DNA sequence dna on grain, we modify the double-stranded DNA of the base containing U without UDG enzymatic treatment Magnetic nanoparticle is introduced directly into cell and carries out control experiment.In addition, also select that two kinds of drugs handle cell, wherein Tert-butyl hydroperoxide (TBHP) is APE1 zymoexciter, and 7- nitroindoline -2- carboxylic acid (NCA) is APE1 activity inhibitor, Observe the situation of change of the intracellular enzyme activity under drug effect.It will be seen in fig. 7 that will directly contain U without UDG enzymatic treatment The magnetic nanoparticle of the double-stranded DNA modification of base imports cell, does not occur fluorescence signal yet after 120min.And it will contain de- After the nano-probe A of base position imports cell, apparent fluorescence signal is seen by the same time.Show that the present invention is made Standby magnetic composite nano particle also has good selectivity in the cell.Inhibited in addition, can further be seen that from Fig. 7 with NCA Agent treated cell, is hardly visible fluorescence signal after importing nano-probe A, and with the processed cell of TBHP, fluorescence Signal is remarkably reinforced, and with the increase of TBHP additional amount, fluorescence signal is further enhanced.The above results further confirm, carefully The fluorescence signal of appearance intracellular is to produce specificity for the DNA abasic site in magnetic composite nano particle by APE1 enzyme It is generated after dissection.
Embodiment 4, the Study of cytotoxicity of magnetic composite nano particle
The nano-probe A of final concentration of 25-100 μ g/mL is subjected to magnetic transfection experiment to HeLa cell, is incubated for 120 minutes. Then CCK-8 reagent is added to be incubated for 1 hour, as a control group by the group for being added without nano-probe, will both be added without nano-probe Also the group without DPBS buffer washing operation is as blank group.The UV absorption in culture solution is detected at 450 nm, then Calculate the activity of cell.As can be seen from Figure 8, when the concentration of nano-probe is 100 μ g/mL, 80% cell still protects Hold activity.Demonstrate this composite nanometer particle i.e. hypotoxicity of nano-probe A.

Claims (9)

  1. It from inside to outside successively include magnetic core, silica shell and biomolecule decorative layer 1. a kind of magnetic composite nano particle, The magnetic core is ferromagnetic nanoparticle, and the silica shell is the silica that surface modification has carboxyl, the biology Molecular modification layer is Avidin and the biotinylated double-stranded DNA containing abasic vacancy in combination, the biotinylation Two single-stranded sequences of the double-stranded DNA containing abasic vacancy be respectively as follows:
    5 '-GGGGACGACTCCATCGACCTCAAGCAGTTGATCCTTTGGAAAAAAA-Biotin-3 ' and 5 '- GTCGATGGAGXCGTCCCC-FAM-3 ', wherein X indicates that abasic site, Biotin indicate biotin labeling, and FAM is carboxyl Fluorescein label.
  2. 2. magnetic composite nano particle as described in claim 1, which is characterized in that the ferromagnetic nanoparticle is four oxidations Three iron nano-particles or γ-di-iron trioxide nano particle.
  3. 3. magnetic composite nano particle as described in claim 1, which is characterized in that the TEM of the magnetic composite nano particle Partial size is 40-60nm, and DLS partial size is 65-75nm.
  4. 4. magnetic composite nano particle as described in claim 1, which is characterized in that the expression of the magnetic composite nano particle Formula is Fe3O4@SiO2@AVD-AP-DNA or γ-Fe2O3@SiO2@AVD-AP-DNA, wherein Fe3O4Ferroso-ferric oxide magnetic core is represented, γ-Fe2O3Then represent γ-di-iron trioxide magnetic core；SiO2Represent silica shell；AVD represents Avidin layer；AP-DNA generation The biotinylated double-stranded DNA layer containing abasic vacancy of table.
  5. 5. the preparation method of magnetic composite nano particle described in claim 1-4 any one, comprising: first in silica shell The outer layer of layer is covalently attached upper Avidin, then passes through the strong affinity interaction of Avidin and biotin, by biotinylated alkali containing U The double-stranded DNA modification of base finally recycles UDG enzyme by U base excision on the ferromagnetic nanoparticle surface of coated with silica, Generate the double-stranded DNA containing abasic vacancy.
  6. 6. preparation method as claimed in claim 5, which is characterized in that specific step is as follows:
    1) the ferromagnetic nanoparticle ultrasonic disperse for coating the silica shell of carboxyl modified is separately added into pure water EDC and NHS, oscillating reactions at room temperature, as solution A；
    2) Avidin is added in solution A, at room temperature concussion reaction, Magneto separate washing removes unreacted substance, then incites somebody to action To reactant ultrasonic disperse is in pure water again, as solution B；
    3) double-stranded DNA of the biotinylated base containing U is added in solution B, is incubated for 30-90 minutes at room temperature, Magneto separate is simultaneously washed It washs, removes unreacted substance, the magnetic nanoparticle for then modifying the double-stranded DNA of the obtained base containing U again divide by ultrasound It is dispersed in PBS solution, as solution C；
    4) solution C is added in 1.1 buffer solution of buffer, UDG digestion is added except the U base in DNA double chain, obtains magnetic Property composite nanometer particle, wherein 1.1 buffer solution of the buffer is by 10mM Bis Tris Propane-HCl, 10mM MgCl2, 100 μ g/ml BSA composition, pH 7.0.
  7. 7. preparation method as claimed in claim 6, which is characterized in that in step 1), the silica shell packet of carboxyl modified Final concentration of 0.5-2mg/mL of the ferromagnetic nanoparticle ultrasonic disperse covered in pure water；The concentration of EDC is 0.5-2mg/mL, The concentration of NHS is 1-5mg/mL, the weight of the ferromagnetic nanoparticle of the silica shell cladding of EDC, NHS and carboxyl modified Than for 4:10:1.
  8. 8. preparation method as claimed in claim 6, which is characterized in that the concentration of Avidin is 0.1-0.2mg/ in step 2) ML, Avidin, which is added, makes the ratio 5:1 of nano particle and the Avidin final concentration in mixed solution, at room temperature oscillating reactions 8-12 A hour；In step 3), final concentration of 0.2-1 μM in solution B of the double-stranded DNA of the biotinylated base containing U of addition；Step It is rapid 4) in, final concentration 1.0-100U/mL of the UDG enzyme of addition in 1.1 buffer solution of buffer comprising solution C, when reaction Between range be 0.5-10min.
  9. 9. magnetic composite nano particle described in claim 1-4 any one in vitro survey by abasic endonuclease I activity Application in fixed, abasic endonuclease I in living cells in situ imaging.
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