CN103396786B - Reversibly assembled and decomposable fluorescent magnetic nano material and nano biological probe and preparation methods thereof - Google Patents
Reversibly assembled and decomposable fluorescent magnetic nano material and nano biological probe and preparation methods thereof Download PDFInfo
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Abstract
The invention discloses a reversibly assembled and decomposable fluorescent magnetic nano material and a nano biological probe and preparation methods thereof. A fluorescent magnetic nano material is prepared through incubation of a magnetic nano material and a cationic polymer and then assembly of a water-soluble quantum dot onto the surface thereof, then a sodium alginate modified fluorescent magnetic nano material is prepared through incubation of sodium alginate and the quantum-dot-coated fluorescent magnetic nano material, then multi layers of sodium alginate are assembled onto the surface of the fluorescent magnetic nano material through layer upon layer self-assembly through utilization of bridging effects of a cross-linking agent, and then the reversibly assembled and decomposable fluorescent magnetic nano material can be obtained. The reversibly assembled and decomposable nano biological probe can be obtained by incubation of the fluorescent magnetic nano material activated by the cross-linking agent and a biomolecule-sodium alginate conjugate. The nano biological probe can realize capture and release of biological target molecules through the cross-linking agent and ethylene diamine tetraacetic acid (EDTA). The preparation methods of the fluorescent magnetic nano material and the nano biological probe are simple and practicable, and have strong operability and good repeatability.
Description
Technical field
The invention belongs to biology, chemistry and material science, relate to the fluorescence magnetic nano material of a kind of reversible assembling and decomposition and nanometer bio probe and preparation method thereof.
Background technology
The functions such as fluorescence magnetic nano material has magnetic can be handling, fluorescent tracing, can be for fields such as the identification of the detection of tumor markers, DNA analysis, tumour cell, sorting and imagings at the specific functionalization molecule of its finishing.But current fluorescence magnetic nanometer bio probe can only be used for identifying specifically target, can not realize the reversible of target and catch and discharge, therefore limit its application at biomedical sector.
Sodium alginate, as a kind of natural polysaccharide, has the features such as stability, solvability, viscosity and security.Sodium Alginate Hydrogel Films has formability and biocompatibility has been widely used as in the research of pharmaceutical carrier, cell cultures and biomimetic material because of it.But sodium alginate not yet plays a role for realizing aspect the reversible assembling of nanometer bio probe and decomposition at Surface Modification of Nanosized Materials.
Circulating tumor cell is the tumour cell that enters peripheral blood, the detection of circulating tumor cell can be effectively applied to external early diagnosis, the rapid evaluation of chemotherapeutics, individualized treatment comprises the detection of clinical sieve medicine, resistance, the exploitation of the monitoring of tumor recurrence and tumour novel drugs etc.The not damaged of circulating tumor cell separates, catches and cultivates can provide Research foundation for the gene of circulating tumor cell and protein analysis.Current, the research that the is directed to circulating tumor cell how many detection of circulating tumor cell number in blood that focuses mostly on, less harmless separation and the vitro culture that studies have reported that circulating tumor cell.
Summary of the invention
Primary and foremost purpose of the present invention is to overcome the shortcoming of prior art with not enough, and the preparation method of the coated fluorescence magnetic nano material of the sodium alginate shell of a kind of reversible assembling and decomposition is provided.
Another object of the present invention is to provide the coated fluorescence magnetic nano material of sodium alginate shell of a kind of reversible assembling obtaining by above-mentioned preparation method and decomposition.
A further object of the present invention is to provide the preparation method of the coated nanometer bio probe of the sodium alginate shell of a kind of reversible assembling and decomposition.
The present invention also aims to provide the coated nanometer bio probe of sodium alginate shell of a kind of reversible assembling obtaining by above-mentioned preparation method and decomposition.
Object of the present invention is achieved through the following technical solutions:
A preparation method for the coated fluorescence magnetic nano material of the sodium alginate shell of reversible assembling and decomposition, comprises following steps:
(1) magnetic Nano material of 1～100mg/mL is mixed according to volume ratio 1:0.1～10 with the cationic polymer solution of 1～800mg/mL, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts the magnetic Nano material of cationic polymer coating.The magnetic Nano material surface band positive charge of cationic polymer coating.
(2) water-soluble quantum dot of 1～50 μ M is mixed according to volume ratio 1:0.1～10 with the magnetic Nano material of 1～100mg/mL cationic polymer coating, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, and the magnetic Nano material that quantum dot has been assembled in extraction is fluorescence magnetic nano material.
(3) 1～100mg/mL fluorescence magnetic nano material is mixed according to volume ratio 1:0.1～10 with 1～500mg/mL sodium alginate soln, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts the fluorescence magnetic nano material of modifying through sodium alginate.
(4) the fluorescence magnetic nano material of 1～100mg/mL sodium alginate being modified is mixed according to volume ratio 1:0.1～10 with 10～500mM cross-linking agent solution, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts the fluorescence magnetic nano material of modifying through the sodium alginate of linking agent activation.
(5) the fluorescence magnetic nano material of the sodium alginate of 1～100mg/mL linking agent activation being modified is mixed according to volume ratio 1:0.1～10 with 1～500mg/mL sodium alginate soln, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts two layers of fluorescence magnetic nano material that sodium alginate shell is coated of assembling.
(6) repeating step (4) and (5), assemble the coated fluorescence magnetic nano material of multilayer sodium alginate shell, obtain the coated fluorescence magnetic nano material of sodium alginate shell of reversible assembling and decomposition.
Magnetic Nano material described in step (1) is preferably at the inner embedding γ-Fe of polymer nano-microspheres
2o
3or Fe
3o
4magnetic Nano material, or at polymer nano-microspheres surface-assembled γ-Fe
2o
3or Fe
3o
4the magnetic Nano material of preparation, or at the inner embedding γ-Fe of polymer nano-microspheres
2o
3or Fe
3o
4after at its surface-assembled γ-Fe
2o
3or Fe
3o
4the magnetic Nano material of preparation.
Cationic polymers described in step (1) is preferably polymine (Polyethyleneimine, PEI) or polypropylene ammonium chloride (poly-(allylamine hydrochloride), PAH).
Water-soluble quantum dot described in step (2) is preferably the water-soluble quantum dot of the bear electricity such as CdTe, CdSe, CdS or CdSe/ZnS; Preferred, described water-soluble quantum dot is modified by Thiovanic acid or thiohydracrylic acid.
The molecular weight of the sodium alginate described in step (3) is preferably 4000～500000.
Linking agent described in step (4) is preferably calcium chloride or bariumchloride.
The coated fluorescence magnetic nano material of sodium alginate shell of reversible assembling and decomposition, prepares by aforesaid method.
The assembling number of plies of the sodium alginate of the coated fluorescence magnetic nano material of described reversible assembling and the sodium alginate shell of decomposition is preferably 2～20 layers.
The preparation method of the coated nanometer bio probe of the sodium alginate shell of a kind of reversible assembling and decomposition, comprise the steps: the reversible assembling of 1～100mg/mL linking agent activation and the coated fluorescence magnetic nano material of sodium alginate shell of decomposition to mix according to volume ratio 1:0.1～10 with 1～500mg/mL biomolecules-sodium alginate conjugate, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extract the nanometer bio probe that biomolecules is modified, obtain the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition.
Described biomolecules is preferably for specific recognition and albumen and the nucleic acid of catching, as antibody, Streptavidin, lectin, somatomedin or aptamer etc.
The preparation method of described biomolecules-sodium alginate conjugate preferably includes following steps:
1) 10～5000mg sodium alginate is dissolved in 10mL ultrapure water or PBS, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride of 10～1000mM, EDC.HCl), N-hydroxy-succinamide (N-Hydroxysuccinimide, NHS) activation 10～30min;
2) add 1～10mg biomolecules to hatch 1～10h in the sodium alginate soln of above-mentioned EDC/NHS activation.
The preparation method of the coated nanometer bio probe of described reversible assembling and the sodium alginate shell of decomposition, can also be for comprising the steps:
(1) the reversible assembling of linking agent activation and the coated fluorescence magnetic nano material of sodium alginate shell of decomposition are mixed with Streptavidin-sodium alginate conjugate, hatch, centrifugal, obtain Streptavidin decorated nanometer bioprobe;
(2) biotinylated biomolecules is mixed with Streptavidin decorated nanometer bioprobe, hatch, centrifugal, obtain the nanometer bio probe that biomolecules is modified, obtain the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition.
The preparation method's of the coated nanometer bio probe of the sodium alginate shell of reversible assembling and decomposition mechanism is: biomolecules can be fixed on by the mode that forms sodium alginate conjugate with sodium alginate coupling the surface of the coated fluorescence magnetic nano material of sodium alginate through the bridge linking effect of linking agent (calcium ion or barium ion), can also Streptavidin be first fixed on to the coated nanometer bio probe of sodium alginate shell that fluorescence magnetic nano-material surface that sodium alginate modifies connects biotinylated biomolecules and obtain reversible assembling and decomposition again by Streptavidin-sodium alginate conjugate.
The coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition, prepares by aforesaid method.
Nanometer bio probe coated the sodium alginate shell of reversible 1～100mg/mL assembling and decomposition is mixed according to volume ratio 1:0.1～10 with the EDTA of 50～500mM, hatch 0.5～4 hour, sodium alginate shell is decomposed, and destruction biomolecules is connected with nanometer bio probe.
Magnetic Nano material surface-assembled water-soluble quantum dot is made fluorescence magnetic nano material by the present invention, afterwards sodium alginate and the coated fluorescence magnetic nano material of quantum dot hatched and obtained the fluorescence magnetic nano material that sodium alginate is modified.Subsequently, the surface that utilizes the bridge linking effect of linking agent (calcium ion or barium ion), through self-assembly layer by layer, multilayer sodium alginate is assembled in to fluorescence magnetic nano material makes the coated fluorescence magnetic nano material of sodium alginate.The surface that biological targeting molecule is fixed on fluorescence magnetic nanometer ball by the bridge linking effect of linking agent (calcium ion or barium ion) after sodium alginate is modified obtains the coated fluorescence magnetic nano material of sodium alginate of biological targeting molecular function.
Tool of the present invention has the following advantages and effect:
Coated fluorescence magnetic nano material and the nanometer bio probe of sodium alginate shell of reversible assembling prepared by the present invention and decomposition has excellent magnetic responsiveness, fluorescent characteristic and biological target tropism.The nanometer bio probe of EDTA and biological targeting molecular modification is hatched, sodium alginate shell decomposes, cut off being connected of biological targeting molecule and fluorescence magnetic nano material, thus biological targeting molecule and fluorescence magnetic nano material peel off, realization is caught and discharges the reversible of determinand.The present invention is easy to operation, cost is low, reproducible, all can complete at general chemistry and Biochemistry Experiment chamber.
Brief description of the drawings
Fig. 1 is the fluorescent microscope photo of the coated fluorescence magnetic nano material of the two-layer sodium alginate shell prepared of embodiment 1.
Fig. 2 is the fluorescence spectrum figure of the coated fluorescence magnetic nano material of the sodium alginate of embodiment 1; Wherein, QDs, PEI-QDs, PEI-QDs-AIg, PEI-QDs-AIg-Ca
2+, PEI-QDs-AIg-Ca
2+-Alg represents that respectively quantum dot, the coated magnetic Nano material of quantum dot are the coated fluorescence magnetic nano material of the sodium alginate of the coated fluorescence magnetic nano material of fluorescence magnetic nano material, sodium alginate, calcium ion activation and the fluorescence magnetic nano material that has been coated with two-layer sodium alginate shell.
Fig. 3 be embodiment 1 prepare the fluorescent microscope photo of the coated fluorescence magnetic nanometer bio probe of sodium alginate modified of Streptavidin.
Fig. 4 is the fluorescent microscope photo of the coated fluorescence magnetic nanometer bio probe of the sodium alginate of the epithelial cell adhesion molecule antibody modification prepared of embodiment 2.
Fig. 5 is that the coated fluorescence magnetic nanometer bio probe of sodium alginate of the epithelial cell adhesion molecule antibody modification prepared of embodiment 2 is caught and the result figure of release cycle tumor model cell SK-BR-3.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) adopt the method (with reference to method described in ZL200310111290.9) of embedding by γ-Fe of 10nm
2o
3embedding enters in acrylamide-styrene polymer Nano microsphere to make magnetic Nano material, its median size is 200nm, be scattered in and in ultrapure water, obtain acrylamide-styrene polymer magnetic Nano material that concentration is 10mg/mL, and carried out succinic acid anhydridization: acrylamide-styrene polymer magnetic Nano material of 5 mL 10mg/mL is placed in small beaker, in 10min, add gradually 0.1g Succinic anhydried, and regulate the pH value of reaction with the NaOH of 1 M, make the pH value of reaction system remain at 4 left and right, after adding, continues Succinic anhydried reaction 3h.Afterwards, draw acrylamide-styrene polymer magnetic Nano material of succinic acid anhydridization with magnetic frame, remove unnecessary Succinic anhydried, then add ultrapure water to wash repeatedly, final product is dispersed in ultrapure water, makes the magnetic Nano material that concentration is the C-terminal of 10mg/mL.
(2) get the magnetic Nano material of 1.0mL 10mg/mL, add the PEI(25kDa of 1mL 0.2g/mL) hatch 10h under aqueous solution room temperature, remove unnecessary PEI through the centrifugal 20min of 16000rpm afterwards and obtain the coated magnetic Nano material of PEI.
(3) in the coated magnetic Nano material of PEI, add 1mL 2 × 10
-5the water-soluble CdTe quantum dots that M Thiovanic acid is modified, hatches 30min under room temperature, and the centrifugal 10min of 16000rpm is scattered in centrifugal product that in 1mL ultrapure water, to make the coated magnetic Nano material of CdTe be fluorescence magnetic nano material.
The preparation method of the water-soluble CdTe quantum dots that Thiovanic acid is modified:
1) in three of 250mL mouthfuls of round-bottomed flasks, add the ultrapure water of 50mL to add again 0.1mmol Cd (CH
3cOO)
22H
2after O stirs, add the Thiovanic acid of 8 μ L, use the pH to 10.5 of the NaOH regulation system of 1M, stir more than 5 minutes, add afterwards the K of 0.02mmol
2teO
3(being dissolved in advance in 50 milliliters of ultrapure waters), and in above-mentioned solution, add the NaBH of 40mg
4, continue to stir 5 minutes;
2) there-necked flask is transferred to and also refluxes in pre-heated oil bath pan, the temperature of reaction of the hierarchy of control is 100 DEG C, can obtain by controlling reaction times 4h the CdTe quantum dot that Thiovanic acid is modified.The top condition of synthetic CdTe quantum dot is Cd
2+/ TGA/TeO
3 2-=1: 1.0: 0.2(mol ratio), the pH=10.5～11.0 of reaction system.
(4) sodium alginate (molecular weight approximately 50000) solution and the above-mentioned fluorescence magnetic nano material of the 1mL mixed room temperature of getting 1mL 10mg/mL are hatched 30min, remove unnecessary sodium alginate and centrifugal product is scattered in and in 1mL ultrapure water, obtains the magnetic Nano material that sodium alginate is modified afterwards through the centrifugal 10min of 16000rpm.
(5) get the magnetic Nano material mixed room temperature that the calcium chloride solution of 1mL 100mM and 1mL sodium alginate modify and hatch 30min, remove unnecessary calcium ion and centrifugal product is scattered in and in 1mL ultrapure water, obtains the fluorescence magnetic nano material that the sodium alginate of calcium ion activation is modified through the centrifugal 10min of 16000rpm afterwards.
(6) get the fluorescence magnetic nano material mixed room temperature that the sodium alginate of sodium alginate (the molecular weight approximately 50000) solution of 1mL 10mg/mL and the activation of 1mL calcium ion modifies and hatch 30min, remove unnecessary sodium alginate and centrifugal product is scattered in and in 1mL ultrapure water, obtains the coated fluorescence magnetic nano material of two-layer sodium alginate shell through the centrifugal 10min of 16000rpm afterwards.Its fluorescent microscope photo as shown in Figure 1, shows that the coated fluorescence magnetic nano material of sodium alginate has fabulous dispersiveness and fluorescence property; Fluorescence spectrum figure as shown in Figure 2, quantum dot is assembled in coated its fluorescence spectrum of magnetic Nano material surface of positively charged ion a little red shift, quantum dot its fluorescence property in the assembling process of sodium alginate is not significantly affected, illustrate that Lalgine Sodium/Calcium shell does not have a significant effect to the fluorescence of quantum dot, fluorescence property is not with the number of plies generation considerable change of assembling sodium alginate.
(7) repeating step (5) and (6) thus 3 layers of fluorescence magnetic nano material that sodium alginate shell is coated of assembling so far obtain the coated fluorescence magnetic nano material of sodium alginate shell of reversible assembling and decomposition.
(8) get the coated fluorescence magnetic nano material mixed room temperature of the calcium chloride solution of 1mL 100mM and 3 layers of sodium alginate shell of 1mL and hatch 30min, remove unnecessary calcium ion and centrifugal product is scattered in to the coated fluorescence magnetic nano material of 3 layers of sodium alginate shell that obtains calcium ion activation in 1mL ultrapure water through the centrifugal 10min of 16000rpm afterwards.
(9) get 1mL(10mg/mL) the coated fluorescence magnetic nano material mixed room temperature of 3 layers of sodium alginate shell of Streptavidin-sodium alginate conjugate and 1mL calcium ion activation hatches 30min, remove unnecessary Streptavidin-sodium alginate conjugate and centrifugal product is scattered in 1mL ultrapure water and obtains the coated fluorescence magnetic nano material of sodium alginate that Streptavidin is modified through the centrifugal 10min of 16000rpm afterwards, obtaining the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition.Its fluorescent microscope photo as shown in Figure 3, the coated fluorescence magnetic nano material of sodium alginate that Streptavidin is modified can be shown by the identification of the vitamin H of FITC mark the fluorescence of FITC, illustrate that Streptavidin is successfully fixed on the surface of the coated fluorescence magnetic nano material of sodium alginate, show that the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition is successfully prepared.
The preparation of Streptavidin-sodium alginate conjugate:
1) 10mg sodium alginate (molecular weight approximately 50000) is dissolved in to 1mL ultrapure water or PBS(0.01M, pH7.4) in, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride of 100mM, EDC.HCl), N-hydroxy-succinamide (N-Hydroxysuccinimide, NHS) room temperature activation 30min;
2) add 1mg Streptavidin in the sodium alginate soln of above-mentioned EDC/NHS activation under room temperature shaking table hatch 10h;
3) product after hatching is put in to 4 DEG C of refrigerator overnight, for subsequent use.
(10) the coated fluorescence magnetic nano material of sodium alginate of Streptavidin being modified and the EDTA of 100mM are according to 1:1(volume ratio) ratio mixed room temperature hatch 0.5～4 hour, sodium alginate shell is decomposed, destruction Streptavidin is connected with fluorescence magnetic nano material, discharges Streptavidin molecule.
Embodiment 2
(1) adopt the method (with reference to method described in ZL201010266616.5) of self-assembly by γ-Fe of 10 nm
2o
3assembling multilayer makes magnetic Nano material in acrylamide-styrene polymer Nano microsphere surface of succinic acid anhydridization, and its median size is 400nm, is scattered in and in ultrapure water, obtains the magnetic Nano material that concentration is 10mg/mL.
(2) get the magnetic Nano material of 1.0mL 10mg/mL, add the PEI(750kDa of 1mL 10mg/mL) hatch 10h under aqueous solution room temperature, remove unnecessary PEI through the centrifugal 20min of 16000rpm afterwards and obtain the coated magnetic Nano material of PEI.
(3) in the coated magnetic Nano material of PEI, add 1mL 3 × 10
-5the water-soluble CdTe quantum dots that the Thiovanic acid of M is modified, hatches 30min under room temperature, and the centrifugal 10min of 16000rpm is scattered in centrifugal product that in 1mL ultrapure water, to make the coated magnetic Nano material of CdTe be fluorescence magnetic nano material.
(4) sodium alginate (molecular weight approximately 50000) solution and the above-mentioned fluorescence magnetic nano material of the 1mL mixed room temperature of getting 1mL 10mg/mL are hatched 30min, remove unnecessary sodium alginate and centrifugal product is scattered in and in 1mL ultrapure water, obtains the fluorescence magnetic nano material that sodium alginate is modified afterwards through the centrifugal 20min of 16000rpm.
(5) get the fluorescence magnetic nano material mixed room temperature that the calcium chloride solution of 1mL 100mM and 1mL sodium alginate modify and hatch 30min, remove unnecessary calcium ion and centrifugal product is scattered in and in 1mL ultrapure water, obtains the fluorescence magnetic nano material that the sodium alginate of calcium ion activation is modified through the centrifugal 10min of 16000rpm afterwards.
(6) get the fluorescence magnetic nano material mixed room temperature that the sodium alginate of sodium alginate (the molecular weight approximately 50000) solution of 1mL 10mg/mL and the activation of 1mL calcium ion modifies and hatch 30min, remove unnecessary sodium alginate and centrifugal product is scattered in to the fluorescence magnetic nano material that obtains two-layer sodium alginate shell parcel in 1mL ultrapure water through the centrifugal 10min of 16000rpm afterwards.
(7) repeating step (5) and (6) thus 5 layers of fluorescence magnetic nano material that sodium alginate shell is coated of assembling so far obtain the coated fluorescence magnetic nano material of sodium alginate shell of reversible assembling and decomposition.
(8) get the coated fluorescence magnetic nano material mixed room temperature of the calcium chloride solution of 1mL 100mM and 5 layers of sodium alginate shell of 1mL and hatch 30min, remove unnecessary calcium ion and centrifugal product is scattered in to the coated fluorescence magnetic nano material of 5 layers of sodium alginate shell that obtains calcium ion activation in 1mL ultrapure water through the centrifugal 10min of 16000rpm afterwards.
(9) get 1mL(10mg/mL) the coated fluorescence magnetic nano material mixed room temperature of 5 layers of sodium alginate shell of Streptavidin-sodium alginate conjugate (embodiment 1 is shown in the preparation of Streptavidin-sodium alginate conjugate) and 1mL calcium ion activation hatches 30min, and remove unnecessary Streptavidin-sodium alginate conjugate and centrifugal product is scattered in and in 1mL ultrapure water, obtains the coated fluorescence magnetic nano material of sodium alginate that Streptavidin is modified through the centrifugal 10min of 16000rpm afterwards.
(10) get the biotin labeled epithelial cell adhesion molecule antibody of 5 μ g (eBioscience company, Cat. No.:13-9326-82) the coated fluorescence magnetic nano material mixed room temperature of Na-alginate modified with 1mL Streptavidin hatches 30min, remove unnecessary antibody through the centrifugal 10min of 16000rpm afterwards, centrifugal product is scattered in to 1mL PBS(0.01M, pH 7.4) in obtain the coated fluorescence magnetic nano material (nanometer bio probe) of sodium alginate of epithelial cell adhesion molecule antibody modification.Its fluorescent microscope photo as shown in Figure 4, the coated fluorescence magnetic nano material of sodium alginate of epithelial cell adhesion molecule antibody modification can be shown by the anti-identification of two of FITC mark the fluorescence of FITC, illustrate that epithelial cell adhesion molecule antibody is successfully fixed on the surface of the coated fluorescence magnetic nano material of sodium alginate, show that the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition is successfully prepared.
(11) get 1 × 10
5individual human breast carcinoma SK-BR-3 cell (circulating tumor model cell) is scattered in 400 μ L 1 × PBS(pH 7.4) in, add afterwards the coated fluorescence magnetic nano material of sodium alginate of the above-mentioned epithelial cell adhesion molecule antibody modification of 100 μ L 10mg/mL.Under room temperature, hatch after 30min, through magnetic frame attract 5min, remove the cell of not catching, by the cell of catching in micro-Microscopic observation (Fig. 5 A and B).
(12) cell after catching is scattered in to 400 μ L 1 × PBS(pH 7.4) in, add afterwards the EDTA of 400 μ L 100mM, under room temperature, hatch 10min, under magnetic force, attract afterwards the fluorescence magnetic nano particle of removing, obtain d/d SK-BR-3 cell in micro-Microscopic observation (Fig. 5 C and D).As shown in Figure 5, add the fluorescence (A and B) that shows the coated fluorescence magnetic nano material of the sodium alginate of epithelial cell adhesion molecule antibody modification before EDTA on SK-BR-3 cell, add the destructurized of the coated fluorescence magnetic nano material of the sodium alginate of EDTA epithelium posterius cell adhesion molecule antibody modification, fluorescence magnetic nano material departs from from SK-BR-3 cell, and SK-BR-3 cell surface almost be can't see the fluorescence (C and D) of fluorescence magnetic nano material.The coated nanometer bio probe of sodium alginate that these results suggest that reversible assembling of the present invention and decomposition can catching and discharging for circulating tumor cell.
Above-described embodiment shows that reversible assembling that the inventive method prepares and the coated fluorescence magnetic nano material of sodium alginate shell of decomposition have good fluorescence property and dispersing property, can be used for preparing nanometer bio probe, be applied to biomedical research field.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. a preparation method for the coated fluorescence magnetic nano material of the sodium alginate shell of reversible assembling and decomposition, is characterized in that comprising following steps:
(1) magnetic Nano material of 1～100mg/mL is mixed according to volume ratio 1:0.1～10 with the cationic polymer solution of 10～800mg/mL, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts the magnetic Nano material of cationic polymer coating; Described cationic polymers is polymine or polypropylene ammonium chloride;
(2) water-soluble quantum dot of 1～50 μ M is mixed according to volume ratio 1:0.1～10 with the magnetic Nano material of 1～100mg/mL cationic polymer coating, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, and the magnetic Nano material that quantum dot has been assembled in extraction is fluorescence magnetic nano material;
(3) 1～100mg/mL fluorescence magnetic nano material is mixed according to volume ratio 1:0.1～10 with 1～500mg/mL sodium alginate soln, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts the fluorescence magnetic nano material of modifying through sodium alginate;
(4) the fluorescence magnetic nano material of 1～100mg/mL sodium alginate being modified is mixed according to volume ratio 1:0.1～10 with 10～500mM cross-linking agent solution, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts the fluorescence magnetic nano material of modifying through the sodium alginate of linking agent activation; Described linking agent is calcium chloride or bariumchloride;
(5) the fluorescence magnetic nano material of the sodium alginate of 1～100mg/mL linking agent activation being modified is mixed according to volume ratio 1:0.1～10 with 1～500mg/mL sodium alginate soln, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extracts two layers of fluorescence magnetic nano material that sodium alginate shell is coated of assembling;
(6) repeating step (4) and (5), assemble the coated fluorescence magnetic nano material of multilayer sodium alginate shell, obtain the coated fluorescence magnetic nano material of sodium alginate shell of reversible assembling and decomposition.
2. the preparation method of the coated fluorescence magnetic nano material of the sodium alginate shell of reversible assembling according to claim 1 and decomposition, is characterized in that:
Magnetic Nano material described in step (1) is at the inner embedding γ-Fe of polymer nano-microspheres
2o
3or Fe
3o
4magnetic Nano material, or at polymer nano-microspheres surface-assembled γ-Fe
2o
3or Fe
3o
4the magnetic Nano material of preparation, or at the inner embedding γ-Fe of polymer nano-microspheres
2o
3or Fe
3o
4after at its surface-assembled γ-Fe
2o
3or Fe
3o
4the magnetic Nano material of preparation.
3. the preparation method of the coated fluorescence magnetic nano material of the sodium alginate shell of reversible assembling according to claim 1 and decomposition, is characterized in that:
Water-soluble quantum dot described in step (2) is the water-soluble quantum dot of bear electricity;
The molecular weight of the sodium alginate described in step (3) is 4000～500000.
4. the coated fluorescence magnetic nano material of the sodium alginate shell of reversible assembling and decomposition, is characterized in that: prepare by the method described in claim 1～3 any one.
5. the coated fluorescence magnetic nano material of the sodium alginate shell of reversible assembling according to claim 4 and decomposition, is characterized in that: the assembling number of plies of sodium alginate is 2～20 layers.
6. the preparation method of the coated nanometer bio probe of the sodium alginate shell of a reversible assembling and decomposition, fluorescence magnetic nano material described in the claim 4 or 5 that it is characterized in that comprising the steps: 1～100mg/mL linking agent to activate is mixed according to volume ratio 1:0.1～10 with 1～500mg/mL biomolecules-sodium alginate conjugate, hatch after 0.5～10 hour, 5000～16000rpm is fully centrifugal, extract the nanometer bio probe that biomolecules is modified, obtain the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition.
7. the preparation method of the coated nanometer bio probe of the sodium alginate shell of reversible assembling according to claim 6 and decomposition, is characterized in that:
Described biomolecules is antibody, Streptavidin, lectin, somatomedin or aptamer;
The preparation method of described biomolecules-sodium alginate conjugate comprises the steps:
1) 10～5000mg sodium alginate is dissolved in 10mL ultrapure water or PBS, adds 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, the N-hydroxy-succinamide activation 10～30min of 10～1000mM;
2) add 1～10mg biomolecules to hatch 1～10h in the sodium alginate soln of above-mentioned activation.
8. a preparation method for the coated nanometer bio probe of the sodium alginate shell of reversible assembling and decomposition, is characterized in that comprising the steps:
(1) the fluorescence magnetic nano material described in the claim 4 or 5 of linking agent activation is mixed with Streptavidin-sodium alginate conjugate, hatch, centrifugal, obtain Streptavidin decorated nanometer bioprobe;
(2) biotinylated biomolecules is mixed with Streptavidin decorated nanometer bioprobe, hatch, centrifugal, obtain the nanometer bio probe that biomolecules is modified, obtain the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition.
9. the coated nanometer bio probe of sodium alginate shell of reversible assembling and decomposition, is characterized in that: prepare by the method described in claim 6～8 any one.
10. the coated nanometer bio probe of sodium alginate shell of reversible assembling according to claim 9 and decomposition, it is characterized in that: coated the sodium alginate shell of the reversible assembling described in 1～100mg/mL and decomposition nanometer bio probe is mixed according to volume ratio 1:0.1～10 with the EDTA of 50～500mM, hatch 0.5～4 hour, sodium alginate shell is decomposed, and destruction biomolecules is connected with nanometer bio probe.
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| CN105504314B (en) * | 2014-09-22 | 2017-10-24 | 首都师范大学 | Alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and the application in electro-chemistry immunity probe is prepared |
| CN105623662A (en) * | 2014-11-05 | 2016-06-01 | 北京蛋白质组研究中心 | Lectin-functionalized-hydrophilic-polymer-coated up-conversion fluorescence nanoparticle and application and preparation method thereof |
| CN104388378A (en) * | 2014-12-10 | 2015-03-04 | 江苏三特生物科技有限公司 | Cell reversible separating carrier and application and method of cell reversible separating carrier in cell reversible separation |
| CN104437411A (en) * | 2014-12-15 | 2015-03-25 | 武汉大学 | Reversibly assembled and disassembled Strep-tag polypeptide-labeled biomolecule derivatization matrix and application thereof |
| CN104523753A (en) * | 2015-01-22 | 2015-04-22 | 中国科学院广州生物医药与健康研究院 | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer |
| CN104610912B (en) * | 2015-02-10 | 2016-06-08 | 武汉大学 | Degradable magnetic Nano material and nanometer bio probe and preparation method thereof |
| FR3042874A1 (en) * | 2015-10-21 | 2017-04-28 | Nanobacterie | PARTICLE COMPRISING AT LEAST ONE FERRIIMAGNETIC IRON OXIDE PARTICLE ASSOCIATED WITH AT LEAST ONE COMPOUND FOR MEDICAL OR COSMETIC USE |
| CN111378326B (en) * | 2018-12-28 | 2022-04-01 | Tcl科技集团股份有限公司 | Magnetic quantum dot, preparation method thereof and ink box |
| CN112300994B (en) * | 2019-08-02 | 2024-01-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | Nanometer magnetic bead for capturing circulating tumor cells and preparation method and application thereof |
| CN111100857B (en) * | 2019-12-30 | 2021-09-14 | 武汉大学 | Quantum dot and enzyme-embedded sodium alginate gel microsphere, preparation method thereof and application thereof in biochemical detection |
| JPWO2021193686A1 (en) * | 2020-03-25 | 2021-09-30 | ||
| CN111505287B (en) * | 2020-05-07 | 2023-04-07 | 军事科学院军事医学研究院军事兽医研究所 | Biosensor for detecting food-borne pathogenic bacteria by taking acetaldehyde dehydrogenase as signal transduction system and preparation method and application thereof |
| CN111551722A (en) * | 2020-05-27 | 2020-08-18 | 济南大学 | Preparation and application of magnetic sodium alginate composite material with dual functions of recognition and catalysis |
| CN112858680B (en) * | 2021-01-14 | 2022-10-04 | 四川大学 | Fluorescence resonance energy transfer magnetic sensor for exosome detection and preparation method thereof |
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| CN101601607A (en) * | 2009-05-22 | 2009-12-16 | 东南大学 | A kind of tumor cell is carried out magnetic induction heating, imaging and thermometric method simultaneously |
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