Magnetic nano crystal dry powder after the carboxyl of finishing is activated and preparation method thereof
Technical field
Magnetic nano crystal dry powder after the functional group that the present invention relates to finishing is activated, particularly the carboxyl of finishing is activated into magnetic nano crystal dry powder of N-hydroxy-succinamide ester and preparation method thereof.Through this dry powder and biomolecule are mixed, can directly obtain the covalency coupling matter of magnetic nano crystal and biomolecule in aqueous solution.
Background technology
Magnetic nano crystal is widely used in biomedical sector, comprising: NMR contrast agent, the separation of cell, albumen, nucleic acid and labelling, magnetic target medicine, the magnetic thermal therapeutical agent of tumor etc.Realize the above-mentioned application of magnetic nano crystal, the coupling couplet that solves effectively between magnetic nano crystal and the biomolecule is one of problem of most critical.
At present, the method that nanocrystal and biomolecule are carried out the coupling couplet mainly contains two kinds: (1) comprises Electrostatic Absorption based on the weak interaction between nanocrystal and the biomolecule, hydrophobic interaction, and the coordination between metal ion and the histidine residues etc.; (2) utilize the chemical reaction between nanocrystal and the biomolecule to prepare the covalency coupling matter.Compare with weak interaction, the chemical stability of the resulting coupling matter of covalency coupling linked method strengthens greatly, and therefore application prospect is widely arranged.At present; Most widely used covalency coupling linked method is to adopt EDC (1-(3-dimethylamino-propyl)-3-ethyl carbodiimide) and Sulfo-NHS (N-hydroxy thiosuccinimide) activator as hydroxy-acid group; Change hydroxy-acid group into the Sulfo-NHS ester, this Sulfo-NHS ester can form amido link with amine groups under temperate condition, thereby the covalency coupling of realizing nanocrystal and biomolecule joins (Bioconjugate Techniques; AcademicPress; New York, 1996, p173-176; Adv.Mater., 2006,18,2553-2556).The advantage of this method is under the condition of gentleness, to prepare the covalency coupling matter of nanocrystal and biomolecule easily.In the actual use of this method, generally be earlier hydroxy-acid group to be modified at nanocrystal surface, and then it is carried out activation.Yet, because there is the low shortcoming of activation efficiency in the easy hydrolysis of nanocrystal activation products; In addition; The facile hydrolysis character of nanocrystal activation products also causes these activation products can't long preservation; Therefore; When adopting EDC and Sulfo-NHS to carry out the coupling couplet as activator, the priming reaction and the coupled reaction between activation products and the biomolecule of the carboxyl that nanocrystal surface is modified must carry out continuously.
The present invention has developed above-mentioned technology; Adopt DCC (dicyclohexylcarbodiimide) and NHS (N-hydroxy-succinamide) or DIC (N; N '-DIC) and NHS be activator; As reaction medium, the hydroxy-acid group activation that nanocrystal surface is modified becomes the N-hydroxy-succinamide ester with organic solvent.Because the priming reaction of carboxyl carries out in organic solvent, has avoided the hydrolysis of activation products, therefore, activation efficiency improves greatly; On the other hand; After priming reaction in the organic solvent finishes; Can also adopt the method for evacuation that organic solvent is drained; Obtaining can long preservation and the activation products dry powder being convenient to transport, separates and carries out thereby the activation step of the carboxyl that can nanocrystal surface be modified and activation products and coupling between the biomolecule join step, helps realizing that nano material uses in biomedical research and clinical diagnosis widely.
Summary of the invention
Magnetic nano crystal dry powder after the carboxyl that one of the object of the invention provides finishing is activated.
The carboxyl that two of the object of the invention provides finishing is activated into the magnetic nano crystal dry powder of N-hydroxy-succinamide ester.
The method for preparing of the magnetic nano crystal dry powder after the carboxyl that three of the object of the invention provides finishing is activated.
The carboxyl that four of the object of the invention provides finishing is activated into the method for preparing of the magnetic nano crystal dry powder of N-hydroxy-succinamide ester.
The magnetic nano crystal dry powder that the carboxyl of the finishing that five of the object of the invention provides is activated into the N-hydroxy-succinamide ester can directly obtain the covalency coupling matter of magnetic nano crystal and biomolecule through mixing in aqueous solution with the biomolecule that has amido.
The magnetic nano crystal dry powder that the carboxyl of the finishing that six of the object of the invention provides is activated into the N-hydroxy-succinamide ester still can keep carrying out the reactivity that the covalency coupling joins with the biomolecule that has amido after long-term the placement.
Finishing described in the present invention has the magnetic nano crystal of carboxyl to utilize prior art for preparing to obtain; As according to Chinese patent 03136273.7,200710062721.5 or document (Adv.Mater.; 2006,18,2553-2556) disclosed method is synthesized.
Magnetic nano crystal dry powder after the carboxyl of finishing of the present invention is activated, the carboxyl of this magnetic nano crystal finishing is activated into the N-hydroxy-succinamide ester.The magnetic nano crystal dry powder that the carboxyl of finishing is activated into the N-hydroxy-succinamide ester further with the biomolecule that has amido mix homogeneously in aqueous solution; Can realize that the covalency coupling between them joins; Directly obtain the covalency coupling matter of magnetic nano crystal and biomolecule, the magnetic nano crystal dry powder that the carboxyl of finishing is activated into the N-hydroxy-succinamide ester is 1: 0.01~1: 1000 with the molar feed ratio that the biomolecule that has amido is carried out the covalency coupled reaction.
The carboxyl of described finishing is activated into the magnetic nano crystal dry powder of N-hydroxy-succinamide ester and under 4 ℃ of dry lucifuge conditions, deposits in the one-year age, still keeps carrying out the reactivity that the covalency coupling joins with the biomolecule that has amido.
The described biomolecule that has amido is selected from a kind of in the amido derivative of aminoacid, polypeptide, albumen, carbohydrate or nucleic acid.
It is magnetic transition metal and oxide thereof, the magnetic lanthanide rare metallic oxide that the finishing of 1~60nm has carboxyl that the magnetic nano crystal of described finishing carboxyl is selected from particle diameter; Transition and rare earth metal doping type magnetic oxide; Comprise ferrum, cobalt, nickel, manganese or their oxide; Gadolinia., terbia. Diterbium trioxide, dysprosia, holmia, Erbia and Dithulium trioxide, or above-mentioned these metal-doped type magnetic oxides.
Magnetic Nano described in the present invention is brilliant, and its most important architectural feature is that nanocrystal surface is modified with Polyethylene Glycol (PEG) or Polyethylene Glycol segment.PEG modifies and makes that magnetic nano crystal both can be water-soluble, can be dissolved in organic solvent again, thereby guarantees that the hydroxy-acid group that carry on the magnetic nano crystal surface can carry out activation in organic facies.
The activation of magnetic nano crystal finishing carboxyl can be carried out priming reaction through adding the method for activator among the present invention in organic solvent, and activated carboxylic is become the N-hydroxy-succinamide ester.
The method for preparing that the carboxyl of finishing of the present invention is activated into the magnetic nano crystal dry powder of N-hydroxy-succinamide ester may further comprise the steps:
(1) there is the magnetic nano crystal of carboxyl to be dissolved in finishing and forms solution in the organic solvent; The organic solvent solution and activator dicyclohexylcarbodiimide (DCC) or the activator N that add activator N-hydroxy-succinamide (NHS) then respectively; The organic solvent solution of N '-DIC (DIC); The carboxyl of magnetic nano crystal finishing is carried out activation, and (15 ℃~30 ℃) reaction is 40 minutes~18 hours under the room temperature, or reacts 2~24 hours down at 4 ℃; Wherein, Finishing has magnetic nano crystal and the dicyclohexylcarbodiimide or the N of carboxyl; The mol ratio of N '-DIC is 1: 2~1: 2000; Dicyclohexylcarbodiimide or N, the mol ratio of N '-DIC and N-hydroxy-succinamide is 1: 0.9~1: 10;
Or have magnetic nano crystal and the activator N-hydroxy-succinamide of carboxyl to be dissolved in finishing to form solution in the organic solvent; Dropwise add activator dicyclohexylcarbodiimide or activator N then; The organic solvent solution of N '-DIC; The carboxyl of magnetic nano crystal finishing is carried out activation, and (15 ℃~30 ℃) reaction is 40 minutes~18 hours under the room temperature, or reacts 2~24 hours down at 4 ℃; Wherein, Finishing has magnetic nano crystal and the dicyclohexylcarbodiimide or the N of carboxyl; The mol ratio of N '-DIC is 1: 2~1: 2000; Dicyclohexylcarbodiimide or N, the mol ratio of N '-DIC and N-hydroxy-succinamide is 1: 0.9~1: 10;
(2) reactant liquor in the step (1) is centrifugal; Get the supernatant, will precipitate, merge the supernatant and cleaning mixture with discarding precipitate behind the organic solvent washing; Vacuum is drained organic solvent, and the carboxyl that obtains finishing is activated into the magnetic nano crystal dry powder of N-hydroxy-succinamide ester.
Described organic solvent is selected from a kind of or wherein any two kinds mixture in dichloromethane, chloroform, oxolane, ethyl acetate, glycol dimethyl ether, dioxane, pyridine, dimethyl formamide, the dimethyl sulfoxide.
The carboxyl of finishing of the present invention is activated into the magnetic nano crystal dry powder of N-hydroxy-succinamide ester and the covalency coupling couplet of the biomolecule that has amido; Its step is following: the magnetic nano crystal dry powder and the biomolecule mix homogeneously in aqueous solution that has amido that the carboxyl of finishing are activated into the N-hydroxy-succinamide ester; (15 ℃~30 ℃) reaction is 30 minutes~4 hours under the room temperature; Or under 4 ℃, reacted 2 hours~24 hours, can prepare the covalency coupling matter of magnetic nano crystal and biomolecule.Wherein, the carboxyl of finishing magnetic nano crystal dry powder that is activated into the N-hydroxy-succinamide ester and the mol ratio of biomolecule in formed coupling matter that has amido is 1: 0.01~1: 1000.
The described biomolecule that has amido is selected from a kind of in the amido derivative of aminoacid, polypeptide, albumen, carbohydrate or nucleic acid.
The present invention adopts DCC and NHS or DIC and the NHS activator as hydroxy-acid group, through in organic solvent with the hydroxy-acid group activation of magnetic nano crystal finishing, prepared the magnetic nano crystal dry powder that the surface has the N-hydroxy-succinamide ester.This dry powder and biomolecule are mixed in aqueous solution, can directly generate the covalency coupling matter of magnetic nano crystal and biomolecule.The magnetic nano crystal dry powder that the carboxyl of this finishing is activated into the N-hydroxy-succinamide ester still keeps carrying out the reactivity that the covalency coupling joins with biomolecule after long-term the placement.The carboxyl of this finishing is activated into the magnetic nano crystal dry powder of N-hydroxy-succinamide ester can long preservation and be convenient to transportation, and the covalency coupling between the biomolecule joins simple and easy to doly, is suitable for scale and commercially produces.
Description of drawings
Fig. 1. Fe in the embodiment of the invention 1
3O
4The electromicroscopic photograph of magnetic nano crystal before priming reaction carries out.
Fig. 2. the carboxyl of finishing is activated into the Fe of N-hydroxy-succinamide ester in the embodiment of the invention 1
3O
4The photo of magnetic nano crystal dry powder under the action of a magnetic field.
Fig. 3. the surface of the embodiment of the invention 1 preparation has the Fe of N-hydroxy-succinamide ester group
3O
4The photo of the aqueous solution that magnetic nano crystal dry powder forms in phosphate buffer under the action of a magnetic field.
Fig. 4. coupling matter and to the protein staining photo after impinging upon electrophoresis and finishing in the embodiment of the invention 2.
1#:Fe
3O
4Coupling matter with anti-CEA chimeric antibody rch 24;
2#:Fe
3O
4Mixture with anti-CEA chimeric antibody rch 24;
3#:Fe
3O
4;
4#: anti-CEA chimeric antibody rch 24.
Fig. 5. Fe in the embodiment of the invention 7
3O
4T after hatching with the covalency coupling matter of anti-CEA chimeric antibody rch 24 and tumor cell
2Weighting picture and control experiment result.From left to right, the echo time is followed successively by 25ms, 75ms, 125ms, 175ms.Wherein, the first stock layout article are and Fe
3O
4The tumor cell sample that-(rch 24mAb) coupling matter obtains after hatching jointly; Second row is and Fe
3O
4The tumor cell sample that-IgG coupling matter obtains after hatching jointly; The 3rd row is and Fe
3O
4The tumor cell sample that nanocrystal obtains after hatching jointly; The 4th row is the control sample of tumor cell.
Fig. 6. the molecular structural formula of used lactose in the embodiment of the invention 11.
Fig. 7. the molecular structural formula of used biotin in the embodiment of the invention 13.
Reference numeral
1. Magnet
The specific embodiment
Preparation embodiment
1.06g ferric acetyl acetonade, the two carboxyl PEG2000 of 12g (are pressed document Adv.Mater., 2005,17 (8); 1001 methods preparations), the 3.87mL oleyl amine is dissolved in the 50mL phenylate, then above-mentioned solution changed in the there-necked flask of 100mL, feeds nitrogen deoxygenation 30 minutes; Back flow reaction liquid 20 hours; Reaction system is cooled to room temperature, goes out the surface with ether sedimentation and have the biological compatibility magnetic nano crystal of carboxyl and wash three times, centrifugalize obtains biocompatibility Fe
3O
4Magnetic nano crystal.The gained nanoparticle is dissolved in the deionized water, dialysed 24 hours, gained solution is precipitated with the mixed liquor (volume ratio is 3: 1) of ether and acetone and washs, drying in vacuum obtains the magnetic nano crystal that the surface has hydroxy-acid group.This magnetic nano crystal both can be dissolved in organic solvents such as chloroform, again can water-soluble both normal saline.Adopt the method for record in said method and the Chinese patent 200710062721.5, can prepare the magnetic nano crystal that the various finishinges of using in following examples have carboxyl.
Embodiment 1
The Fe that the 0.32g finishing is had carboxyl
3O
4Magnetic nano crystal (10nm) and N-hydroxy-succinamide (Aldrich 130672 for 17.3mg, 0.15mmol, and purity is greater than 98%) are dissolved in the 50mL dimethyl formamide; (Fluka 36650 for 30.9mg, 0.15mmol in this solution, dropwise to add the 1mL dicyclohexylcarbodiimide then; Purity is greater than 99%) dimethyl formamide solution, under the room temperature (25 ℃) reaction 12 hours, centrifugal; Get the supernatant, will precipitate with after the dimethyl formamide washing three times and discard precipitate, merge the supernatant and cleaning mixture; Vacuum is drained organic solvent, obtains magnetic nano crystal dry powder, and the carboxyl of this dry powder finishing has been activated and has been the N-hydroxy-succinamide ester.Fig. 1 is Fe
3O
4The electromicroscopic photograph of magnetic nano crystal before priming reaction carries out.Fig. 2 is activated into the Fe of N-hydroxy-succinamide ester for the carboxyl of finishing
3O
4The photo of magnetic nano crystal dry powder under the action of a magnetic field.
Embodiment 2
The dry powder that obtains among the title 10mg embodiment 1, and the anti-CEA chimeric antibody rch24 phosphate buffer of adding 10mL 1mg/mL (PBS, pH=7.4), mixing, (20 ℃) reaction is 4 hours under the room temperature condition, obtains Fe
3O
4Covalency coupling matter with anti-CEA chimeric antibody rch 24.Fig. 3 is activated into the Fe of N-hydroxy-succinamide ester for the carboxyl of the finishing that obtains among the embodiment 1
3O
4The photo of aqueous solution under the action of a magnetic field that magnetic nano crystal dry powder and anti-CEA chimeric antibody rch 24 covalency couplings form in phosphate buffer before joining.After coupled reaction finishes, adopt the native polyacrylamide gel electrophoresis of 5% (w/v) to detect coupled reaction.Fig. 4 is for coupling matter and to impinging upon the protein staining photo after electrophoresis finishes.1# is Fe
3O
4Coupling matter with antibody; 2# is Fe
3O
4Mixture with antibody; 3# is Fe
3O
44# is an antibody.Band through behind the comparison antibody staining is found Fe
3O
4Almost do not have difference with the migration velocity and the pure antibody of antibody in the mixtures of antibodies in swimming lane, and the migration velocity of antibody is much faster than pure antibody in the coupling matter, this is owing to the Fe with a large amount of negative charges
3O
4Nanoparticle coupling couplet causes on the antibody surface; Simultaneously, can infer antibody and Fe in the coupling matter through the migration velocity that compares antibody in coupling matter and the mixture
3O
4Be to carry out coupling through covalent bond to join, rather than non-specific interaction.
Embodiment 3
The 1.18g finishing is had magnetic Nano cobalt crystal (20nm) and the N-hydroxy-succinamide of carboxyl, and (67.9mg 0.59mmol) is dissolved in the 30mL oxolane, in this solution, dropwise adds 2mL dicyclohexylcarbodiimide (134mg then; 0.65mmol) tetrahydrofuran solution, 4 ℃ the reaction 24 hours, centrifugal; Get the supernatant; To precipitate with after the oxolane washing three times and discard precipitate, and merge the supernatant and cleaning mixture, vacuum is drained organic solvent; Obtain magnetic nano crystal dry powder, the carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester.Claim 10mg this dry powder, add PBS (pH=7.4) solution of 2mL L-lysine (1.64mg), mixing, 4 ℃ were reacted 2 hours, obtained the covalency coupling matter of cobalt nanocrystal body and L-lysine.Utilize the uv-visible absorption spectra method, detect the not amount of the lysine of coupling couplet, confirm that the number of the lysine that each magnetic nano crystal surface coupling joins is 800 through adopting the 1,2,3-indantrione monohydrate chromogenic reaction.
Embodiment 4
There is Ni (30nm) magnetic nano crystal of carboxyl to be dissolved in the 80mL dichloromethane 3.75g finishing, then, in this solution, adds 1mL N-hydroxy-succinamide (138mg; 1.2mmol) dimethyl formamide solution, mixing dropwise adds 1mL N; (Fluka 38370 for 37.8mg, 0.3mmol for N '-DIC; Purity is greater than 98%) dichloromethane solution, under the room temperature condition (15 ℃) reaction 18 hours, centrifugal; Get the supernatant, will precipitate with after the washed with dichloromethane three times and discard precipitate, merge the supernatant and cleaning mixture; Evacuation obtains magnetic nano crystal dry powder, and the carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester.Claim 10mg this dry powder, add the single stranded DNA (NH that contains 18 bases of 5mL terminal amino groupization
2-ssDNA, 5 '-Amine-C
6TA CAG GTC ATGTAA CTT-3c) (4.67mg) TE (Tris-disodiumedetate) buffer soln; Mixing; Room temperature (28 ℃) reaction 30 minutes; Obtain the covalency coupling matter of nickel nanocrystal and DNA, the mobile result (8 nanometer) of the surface plasma body resonant vibration absworption peak of magnetic nano crystal before and after coupling joins shows that above-mentioned coupling has joined successfully realization.
Embodiment 5
The Mn that the 2.29g finishing is had carboxyl
3O
4(2nm) (11.5mg 0.1mmol) is dissolved in the 50mL dioxane, and mixing dropwise adds 1mL dicyclohexylcarbodiimide (30.9mg for magnetic nano crystal and N-hydroxy-succinamide; 0.15mmol) dioxane solution, 4 ℃ the reaction 2 hours, centrifugal; Get the supernatant, will precipitate with after the dioxane washing three times and discard precipitate, merge the supernatant and cleaning mixture; Evacuation obtains magnetic nano crystal dry powder, and the carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester.Claim 10mg this dry powder, and adding 5mL human IgG (immunoglobulin, PBS solution 10mg), mixing, 4 ℃ were reacted 24 hours, and obtained Mn
3O
4With the covalency coupling matter of IgG, and adopt among the embodiment 2 similar electrophoresis method to prove the formation of above-mentioned coupling matter.
Embodiment 6
The Fe that the 2.32g finishing is had carboxyl
3O
4(10nm) (18.4mg 0.16mmol) is dissolved in the 50mL dimethyl formamide, in this solution, dropwise adds 1mL dicyclohexylcarbodiimide (30.9mg then for magnetic nano crystal and N-hydroxy-succinamide; 0.15mmol) dichloromethane solution, under the room temperature condition (30 ℃) reaction 40 minutes, centrifugal; Get the supernatant; To precipitate with after the dimethyl formamide washing three times and discard precipitate, merge the supernatant and cleaning mixture, evacuation; Obtain magnetic nano crystal dry powder, the carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester.Claim 10mg this dry powder, add the PBS solution of 10mL insulin human (4mg), mixing, (25 ℃) reaction is 2 hours under the room temperature condition, obtains Fe
3O
4With the covalency coupling matter of insulin human, and adopt among the embodiment 2 similar electrophoresis method to prove the formation of above-mentioned coupling matter.
Embodiment 7
With the Fe that obtains among the 50 μ L embodiment 2
3O
4PBS solution and 1mL (2 with anti-CEA chimeric antibody rch 24 covalency coupling matters; 000; 000) the antigenic human colon cancer cell LS 180 of surface expression CEA hatches 1 hour at 37 ℃, and is centrifugal, after PBS buffer solution washing three times; SDS (dodecyl sodium sulfate) the solution cell lysis that adds 200 μ L 0.1% obtains cell solution after the cracking.According to the method described above, with Fe
3O
4Covalency coupling matter and Fe with human IgG (immunoglobulin)
3O
4Nanocrystal respectively with LS 180 cells of equivalent hatch under the same condition, wash and cracking after, the lysate of above-mentioned three kinds of cell pyrolysis liquids and untreated LS 180 cells is carried out the magnetic resonance radiography experiment.Fig. 5 is the T2 weighting picture of magnetic resonance radiography experiment gained.Can find out by figure, compare Fe with untreated LS 180 cells
3O
4After hatching with the coupling matter of anti-CEA chimeric antibody rch 24 and cell, the relaxation time of proton obviously shortens, and this just shows Fe
3O
4Nanocrystal has been enriched in LS 180 cell surfaces through anti-CEA chimeric antibody rch 24.And with Fe
3O
4Coupling matter or Fe with human IgG
3O
4After nanocrystal and LS 180 cells were hatched, the relaxation time of proton and untreated LS 180 cells did not have significant difference, and this just shows, Fe
3O
4Interaction between the coupling matter of nanocrystal and anti-CEA chimeric antibody rch 24 and LS 180 cells has specificity.
Embodiment 8
There are the Gadolinia. nanocrystal (3.9nm) of carboxyl and the N-hydroxy-succinamide of 11.5mg (0.1mmol) to be dissolved in the 50mL chloroformic solution 0.35g finishing; Slowly splash into 1mL then and be dissolved with the chloroformic solution of 8.24mg (0.04mmol) DCC; 25 ℃ reaction is after 12 hours down, and centrifugal removal deposition is with the supernatant vacuum drying that obtains; Obtain magnetic nano crystal dry powder, the carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester.Take by weighing the above-mentioned dry powder of 10mg, be dissolved in the 5mL PBS buffer, add 10mg human IgG (immunoglobulin), 4 ℃ were reacted 24 hours down, obtain Gadolinia. (Gd
2O
3) the covalency coupling matter of nanocrystal and human IgG, and adopt among the embodiment 2 similar electrophoresis method to prove the formation of above-mentioned coupling matter.
Embodiment 9
10mg has been activated the magnetic Fe into the N-hydroxy-succinamide ester according to the finishing carboxyl that embodiment 1 similar approach obtains
3O
4Nano crystal dry powder is dissolved in 5mL PBS buffer, mixes with the PBS buffer (pH=7.4) that 10mL is dissolved with 10mg L-lysine then, and 20 ℃ reaction is after 4 hours down, and the reactant liquor of dialysing again obtained magnetic Fe in 7 hours
3O
4The covalency coupling matter of nanocrystal and L-lysine.Utilize the uv-visible absorption spectra method, detect the not amount of the lysine of coupling couplet, confirm that the number of the lysine that each magnetic nano crystal surface coupling joins is 300 through adopting ninhydrin reaction.
Embodiment 10
The 0.81g finishing is had magnetic oxygenated erbium nanocrystal (3.2nm) and the N-hydroxy-succinamide of carboxyl, and (67.9mg 0.59mmol) is dissolved in the 30mL dichloromethane, in this solution, dropwise adds 2mL dicyclohexylcarbodiimide (49.5mg then; 0.24mmol) dichloromethane solution, 25 ℃ the reaction 8 hours, centrifugal; Get the supernatant; Vacuum is drained organic solvent, obtains magnetic nano crystal dry powder, and the carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester.Claim 10mg this dry powder; Adding 5mL is dissolved with PBS (pH=7.4) solution of the anti-McAb against gastric carcinoma 3H11 of 4mg; Mixing; 4 ℃ of reactions 6 hours obtain the covalency coupling matter of Erbia nanocrystal and anti-McAb against gastric carcinoma 3H11, and adopt similar electrophoresis method among the embodiment 2 to prove the formation of above-mentioned coupling matter.
Embodiment 11
The Fe that the 0.23g finishing is had carboxyl
3O
4Nanocrystal (8.2nm) and 57.7mg (0.5mmol) NHS are dissolved in the 100mL chloroformic solution, dropwise add the chloroformic solution that 1mL is dissolved with 41.2mg (0.2mmol) DCC then.25 ℃ were reacted 12 hours down, and centrifugal removal deposition with the supernatant vacuum drying, obtains the magnetic Fe that the surface has the N-hydroxy-succinamide ester group
3O
4Nano crystal dry powder.This dry powder of 10mg and 1mg lactose (molecular structural formula is seen accompanying drawing 6) are dissolved in the 20mL water, and 25 ℃ were reacted 40 minutes, and obtained magnetic Fe
3O
4The coupling matter of nanocrystal and lactose.The examination of infrared spectrum result of coupling matter demonstrates the characteristic absorption peak of amido link, as: corresponding to the peak (1690cm of C=O vibration
-1) and corresponding to C-N stretching vibration peak (1260cm
-1).
Embodiment 12
The 0.58g finishing is had magnetic oxygenated dysprosium nanocrystal (2.3nm) and the N-hydroxy-succinamide of carboxyl, and (138mg 1.2mmol) is dissolved in the 30mL dichloromethane, in this solution, dropwise adds 2mL N then; (25 ℃ were reacted 6 hours N '-DIC for 37.8mg, dichloromethane solution 0.3mmol); Centrifugal; Get the supernatant, vacuum is drained organic solvent, obtains magnetic nano crystal dry powder; The carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester, and has obtained the checking of infrared spectrum.
Embodiment 13
10mg has been activated the magnetic Fe into the N-hydroxy-succinamide ester according to the finishing carboxyl that embodiment 1 similar approach obtains
3O
4The soluble derivative of nano crystal dry powder and 0.5mg biotin (molecular structural formula is seen accompanying drawing 7) is dissolved in the 20mL water, and 25 ℃ were reacted 1 hour, and obtained magnetic Fe
3O
4The coupling matter of nanocrystal and biotin, and obtained the checking of infrared spectrum, the infrared spectrum of coupling matter demonstrates the amido link carbonyl at 1680cm
-1With the N-H key at 3340cm
-1The characteristic absorption at place.
Embodiment 14
The 0.8g finishing is had the adulterated magnetic ferric oxide nano crystal of manganese (6.8nm) and the N-hydroxy-succinamide of carboxyl, and (1 38mg 1.2mmol) is dissolved in the 30mL chloroform, in this solution, dropwise adds 2mL N then; N '-DIC (37.8mg; 0.3mmol) chloroformic solution, 25 ℃ the reaction 6 hours, centrifugal; Get the supernatant; Vacuum is drained organic solvent, obtains the adulterated magnetic nano crystal dry powder of manganese, and the carboxyl of this dry powder finishing has been activated into the N-hydroxy-succinamide ester.Adulterated magnetic ferric oxide nano crystal of manganese that the reaction condition of employing embodiment 2 prepares and the coupling matter of anti-CEA chimeric antibody rch 24; And adopting among the embodiment 2 similar electrophoresis method to prove the formation of above-mentioned coupling matter, electrophoresis result explains that also the N-hydroxy-succinamide ester that is modified at the magnetic nano crystal surface has further reactivity.