CN104027824A - Preparation method of antibody-mediated optomagnetic double-mode meso-porous silicon nanoparticles - Google Patents
Preparation method of antibody-mediated optomagnetic double-mode meso-porous silicon nanoparticles Download PDFInfo
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Abstract
The invention belongs to the field of nano composite materials and discloses a preparation method of antibody-mediated optomagnetic double-mode meso-porous silicon nanoparticles. The preparation method comprises the steps of by taking nano ferroferric oxide as a core, applying silicon dioxide to package, labeling by fluorescein isothiocyanate, carrying out surface modification on 3-aminopropyl triethoxysilane to realize amination; then, grafting lactobionic acid by carboxyl-ammonia crosslinking; and finally, activating carboxyl and prostate stem cell antigen (PSCA) antibody. The preparation method disclosed by the invention is simple, and convenient to operate; the obtained nanoparticles have the advantages of active targeting, good biocompatibility, small toxic and side effect, good monodispersity and stability, stable imagnetic and fluorescence performance and the like.
Description
Technical field:
The preparation method that the present invention relates to have antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle, belongs to field of biomedicine technology.
Background technology:
Fe304 nano-particle is a kind of typical magnetic material, because Fe304 nanoparticle has quantum size effect and skin effect and the distinctive magnetic responsiveness of magnetic material of nanoparticle concurrently, make it there is the not available property of conventional particle: (1) Fe304 nanoparticle has good magnetic susceptibility.Magnetic Fe 304 nanoparticles have very strong magnetic, can in very weak externally-applied magnetic field, obtain very strong magnetic induction, and can displacement; (2) in certain size distribution, there is superparamagnetism.In the time that the particle diameter of magnetic nano-particle is less than 16nm, its anisotropy is reduced to can be comparable with energy of thermal motion, and direction of easy axis presents irregular variation, so just produced superparamagnetism; (3) under the effect of additional alternating electromagnetic field, can produce heat etc.Magnetic Fe 304 nanoparticles, under the induction of alternating magnetic field, can produce high temperature, high heat.
Fe304 nanoparticle has excellent magnetic property and good biocompatibility, has broad application prospects in fields such as cell separation, target administration, cancer thermotherapy, nuclear magnetic resonances, and be one of study hotspot of current nano biological medical domain.Magnetic Fe 304 nanoparticles are owing to having the quantum size effect of nanometer materials and the magnetic dipole graviational interaction of magnetic material concurrently, make magnetic nano-particle reunite serious, poor chemical stability, oxidizable, surface hydroxyl is not enough, these shortcomings have directly limited the application of Fe304 nanoparticle.And by finishing, not only can improve dispersive property and the stability of Fe304 nanoparticle in solution, can also regulate the compatibility and the response characteristic of nanoparticle and other materials.Must there is following feature through the magnetic nano-particle of modifying, just can be for medical domain: (1) hydrophilic, the hydrophobic group of magnetic nano particle sub-surface must be replaced by hydrophilic group, otherwise can seriously reunite in vivo, this is also the first step to modification to the Hang Ji of magnetic nano-particle Jin simultaneously; (2) biocompatibility, nanoparticle itself must have good biocompatibility, can is not so just that harmful substance is got rid of by the immune system recognition of organism; (3) toxicity, the magnetic nano-particle that is applied to biomedical sector must be nontoxic or low toxicity, so just can not damage patient; (4) shape, there are some researches show, for example nanoparticle bar-shaped, child worm shape of out-of-shape is more difficult than nano spherical particle is antigen by the immune system recognition of organism.Improve the Aspect Ratio of nanoparticle and can significantly optimize the long-range cycle performance of nano material in blood.Similarly, the magnetic nano-particle of high aspect ratio also has longer blood circulation time than spherical magnetic nano-particle.These results show, the Pharmacokinetic effect maximum of further definite which kind of Aspect Ratio of researcheres needs to magnetic nano-particle.(5) surface charge, different electric charges can affect the combination of magnetic nano-particle and some protein or cell; (6) magnetic, magnetic nano-particle should be handled by externally-applied magnetic field easily.So select desirable clad material, be also important research.
Metaporous silicon dioxide material because of have bigger serface, high pore volume, evenly adjustable aperture, avirulence, good chemical stability, easily tissue and the structural behaviour of the uniqueness such as modification become a kind of desirable clad material.Fe304@Si02 composite nanoparticle, has the magnetic property of Fe304 nanoparticle uniqueness and Si02 nanoparticle good biocompatibility, the easy advantage of finishing concurrently.Coated to Fe304 nanoparticle with silicon dioxide, not only can improve the oxidation resistance of magnetic nano-particle and the dispersive property in liquid medium, and due to very easily functionalization of Si02 nanoparticle surface, can easily realize and the linking of each organic molecular species and biomolecule, for further finishing and application are laid a good foundation.
As everyone knows, fluorescent labeling be monitoring live body Chinese medicine transmission route one in real time, simple and efficient way.Therefore, the functional mesoporous silicon dioxide drug controlled release system of light has identification, follows the tracks of and monitor the ability of drug release efficiency and disease treatment effect, has become gradually another study hotspot of medicament slow release field.At present, mainly include organic dye, quantum dot and fluorescent RE powder for the synthesis of the fluorescent material of composite.Organic dyestuff, such as fluorescein isothiocyanate (FITC), rhodamine (RITC) etc., be a kind of traditional fluorescent material.Maximum shortcoming is that photobleaching and optical quenching easily occur, and is not suitable for responsive detection and real-time tracking.But the synthetic above-mentioned drawback that has not only overcome organic dyestuff of the design of the silicon dioxide composite material of organic dyestuff doping, and single dye molecule corresponding to fluorescence intensity ratio obviously strengthens.
At present, the research of optomagnetic functional mesoporous earth silicon material mainly comprises following three aspects: mesoporous silicon oxide/atresia silicon dioxide composite material of (1) ferrum oxide and organic dyestuff functionalization; (2) mesoporous silicon oxide/atresia silicon dioxide composite material of ferrum oxide and quantum dot functionalization; (3) the atresia silicon dioxide composite material of ferrum oxide and fluorescent RE powder functionalization; Multi-functional mesoporous silicon dioxide composite material not only can be for targeting drug delivery system but also can be for imaging and photo-thermal therapy, and multi-functional atresia silicon dioxide composite material is difficult to be used as slow releasing carrier of medication.
A lot of patents all relate to preparation and the application about multi-functional mesoporous silicon dioxide.As: Chinese patent literature " based on the preparation of fluorescence mesoporous silicon oxide yolk-eggshell Nano capsule "
(CN 102350281 A) disclose a kind of taking fluorescence mesoporous silicon oxide as eggshell, magnetic Nano material is the Nano capsule of egg yolk: Chinese patent literature " a kind of multifunctional nanoparticles and preparation thereof " (CN 103566381 A) discloses a kind of preparation of magnetic nanoparticle, and in the finishing of silicon dioxide hydrophilic group and targeting agent; It is that core silicon dioxide is the nano-particle of shell by microemulsion method preparation parcel fluorescent dye and magnetic nanoparticle that Chinese patent literature " a kind of multifunctional tumor preparation preparation method and application " (CN 102614532 A) discloses a kind of, finishing polypeptide; Chinese patent literature " preparation method of multi-functional eccentric mesoporous silicon dioxide nano particle " (CN103211767A) discloses a kind of silica surface that polyacrylic acid is connected to amido modified mistake, and the control drug delivery system of a PH response that set it as vector construction; Chinese patent literature " multifunctional nuclear shell structure drug carrier and preparation thereof " (CN 101670107 A) discloses one and has crossed coated ferroferric oxide by the double-deck titanium dioxide of preparation of sol-gel, finally adds fluorescence; Chinese patent literature " Multifunctional core-shell structure fluorescent coding magnetic microspheres and preparation method thereof " (CN 102120168 A) discloses a kind of by regulating the different proportionings of two kinds of fluoresceins to reach the multiple fluorescence-encoded magnetic microsphere of preparation.And about by taking nano ferriferrous oxide as core, use Silica-coated, by marked by fluorescein isothiocyanate, make its amination by finishing 3-aminopropyl triethoxysilane, then by carboxylic ammonia cross-linked graft lactobionic acid, finally, by activated carboxyl and prostate cancer stem cells antigen-antibody (PSCA), obtain the initiatively patent of the bimodal mesoporous silicon nanoparticle implementation method of target function, also similarly do not report.
Summary of the invention:
The object of the present invention is to provide a kind of preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle, taking nano ferriferrous oxide as core, use Silica-coated, by marked by fluorescein isothiocyanate, make its amination by finishing 3-aminopropyl triethoxysilane, then by carboxylic ammonia cross-linked graft lactobionic acid, finally by activated carboxyl and prostate cancer stem cells antigen-antibody (PSCA), the method is simple, easy to operate, the nano-particle obtaining has initiatively targeting, good biocompatibility, toxic and side effects is little, monodispersity and good stability, the advantages such as magnetic and fluorescence property are stable.
For solving the problems of the technologies described above, the technical solution used in the present invention is: the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle: first prepare the 3-aminopropyl triethoxysilane that Fluorescein isothiocyanate is modified, again using ammonia as catalyst, in ferriferrous oxide nano solution, the 3-aminopropyl triethoxysilane that adds tetraethoxysilane and Fluorescein isothiocyanate to modify, reaction obtains having the mesoporous silica nano-particle of double-functional group, modify again amino with 3-aminopropyl triethoxysilane, and then provide functional group for nano-particle modified lactobionic acid, after crosslinked lactobionic acid, utilize the carboxyl of carbodiimide hydrochloride and N-hydroxy thiosuccinimide and lactobionic acid to form the active NHS ester of semistable ammonia, the active NHS ester of semistable ammonia and the anti-generation condensation reaction of prostate stem cell antigen form amido link, realize antibody linked, obtain the initiatively bimodal mesoporous silicon nanoparticle of target function.
Further, the preparation method of described optomagnetic bimodal mesoporous silicon nano-particle specifically comprises the following steps:
(1) the 3-aminopropyl triethoxysilane of the Fluorescein isothiocyanate of 0.01g～10g0.01mmol～10mmol and 0.10g～10g0.1mmol～10mmol is joined in the ethanol of 10ml～100ml, at 10 DEG C～60 DEG C, the logical nitrogen 0.1mol～5mol of lucifuge reacts 10h～28h, obtain the 3-aminopropyl triethoxysilane that Fluorescein isothiocyanate is modified, note is FITC-APTES;
(2) the ferroferric oxide nano granules aqueous solution 0.1ml～10ml0.001g/ml～10g/ml of citric acid protection is dispersed in the mixed liquor of 40ml～130ml second alcohol and water, ultrasonic 20min～120min, add ammonia 0.1ml～10ml, concentration is that 0.1mol/l～20mol/l is as catalyst, at 10 DEG C～60 DEG C, vigorous stirring 10min～6.5h, mixing speed is 60r/s, add the tetraethoxysilane of 0.01ml～5ml, at 10 DEG C～60 DEG C, reaction 2h～24h, the 3-aminopropyl triethoxysilane 0.1ml～20ml that adds the synthetic Fluorescein isothiocyanate of step (1) to modify, continue to stir 2h～24h, in 1h～10h, dropwise add the tetraethoxysilane of 0.01ml～10ml, lucifuge vigorous stirring 1h～10h, mixing speed is 60r/s, obtain the nano-particle of double-functional group, note is FMNPs, do centrifuge washing for the first time with the water of 1ml～10ml, the ethanol of 1ml～10ml does centrifuge washing for the second time, the water of 1ml～10ml does for the third time centrifuge washing three times, kept dry,
(3) the nano-particle FMNPs of the double-functional group of 0.01g～10g is dispersed in the mixed liquor of 40ml～130ml second alcohol and water, ultrasonic 20min～120min fully dissolves it, in 30min～5h, dropwise add the 3-aminopropyl triethoxysilane of 0.1ml～20ml, at 50 DEG C～240 DEG C, vigorous stirring 1h～24h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 1ml～10ml, the ethanol of 1ml～10ml does centrifuge washing for the second time, and the water of 1ml～10ml does for the third time centrifuge washing three times;
(4) get 0.1ml～10ml1mg/ml～10mg/ml containing the lactobionic acid molecule of galactosyl, under ice-water bath, add wherein 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and the 0.1mL～10ml0.1mg/ml～10mg/ml N-maloyl imines of 0.1ml～10ml0.1mg/ml～10mg/ml, ultrasonic reaction 20min～2h, make the hydroxy-acid group activation of lactobionic acid, then the amido modified FMNPs solution of the lactobionic acid solution of activation being prepared with step (3) mixes, continue to stir 1h～19h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified,
(5) by 0.10mg～10mg carbodiimide hydrochloride and 0.01mg～20mg N-hydroxy thiosuccinimide, be blended in the MES buffer of 1ml～10ml, buffer concentration is 0.01mol/l～10mol/l, pH is 4～9.5, the amino functional FMNPs that finally adds 1ml～50ml to have lactobionic acid to modify, stir 20h～40h, mixing speed 60r/s 10 DEG C～60 DEG C lucifuges, the carboxyl of carbodiimide hydrochloride and N-hydroxy thiosuccinimide and lactobionic acid forms the active NHS ester of semistable ammonia, it is 4～8.5 that dialysis joins pH after separating, concentration is in the phosphate buffer of 1ml～50ml of 0.001mol/l～10mol/l, add again the prostate stem cell antigen of the 10 μ g/ml～100 μ g/ml of 0.1 μ l～100 μ l, at 10 DEG C～60 DEG C lucifuge reaction 4h～22h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linked, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Further, the aminopropyl triethoxysilane that prepared Fluorescein isothiocyanate is modified is kept at 4 DEG C.
Further, in described step (2), the interval that dropwise drips tetraethoxysilane is with every 10 times in microlitre counting period, drips at 1h～10h.
Further, in described step (2), in the mixed liquor of second alcohol and water, the volume ratio of second alcohol and water is 5:2.
Further, in described step (3), the interval that dropwise drips 3-aminopropyl triethoxysilane is with every 10 times in microlitre counting period, drips at 30min～5h.
Further, in described step (3), in the mixed liquor of second alcohol and water, the volume ratio of second alcohol and water is 5:1.
Further, in described step (4), ice-water bath temperature be zero degrees celsius;
Further, in described step (4), the mixed proportion of amido modified FMNPs solution prepared by the lactobionic acid solution of activation and step (3) is 1:3.
Beneficial effect of the present invention:
1, the inventive method is simple, fast, easy to operate, reproducible, has universally, and experiment condition is easily realized;
2, the nanoparticulate dispersed that the present invention obtains is good, good stability, the mesoporous medicine needing that loads;
3, nano-particle of the present invention has several functions, has both possessed the function of magnetic nanoparticle, possesses again the function of fluorescent probe;
4, the nano-particle that the present invention obtains has good biocompatibility, inanimate object toxicity;
5, the finishing of nano-particle of the present invention APTES, there is abundant amino, be conducive to modify again, and can be widely used in biological medicine carrying;
6, the present invention's step condition is simple, gentleness, natural environmental protection.
Brief description of the drawings:
Fig. 1 is the Principle of Process figure that the present invention prepares the optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated.
Fig. 2 is the transmission electron microscope picture of the optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated.
Fig. 3 be prostate cancer stem cells Antigen-antibody mediated optomagnetic bimodal mesoporous silicon nano-particle at body nuclear magnetic resonance figure, A-first day in figure, B-second day, C-the 3rd day, D-the 4th day, E-the 5th day.
Fig. 4 is the fluorescence spectrum figure of the optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated.
Fig. 5 is the fluorescence microscope images of the optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated.
Detailed description of the invention:
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
As shown in Figure 1, this law is bright for having the preparation method of antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle, comprises the following steps:
(1) the 3-aminopropyl triethoxysilane of the Fluorescein isothiocyanate of 0.01g～10g0.01mmol～10mmol and 0.10g～10g0.1mmol～10mmol is joined in the ethanol of 10ml～100ml, at 10 DEG C～60 DEG C, the logical nitrogen 0.1mol～5mol of lucifuge reacts 10h～28h, obtain the 3-aminopropyl triethoxysilane that Fluorescein isothiocyanate is modified, note is FITC-APTES;
(2) the ferroferric oxide nano granules aqueous solution 0.1ml～10ml0.001g/ml～10g/ml of citric acid protection is dispersed in the mixed liquor of 40ml～130ml second alcohol and water, ultrasonic 20min～120min, add ammonia 0.1ml～10ml, concentration is that 0.1mol/l～20mol/l is as catalyst, at 10 DEG C～60 DEG C, vigorous stirring 10min～6.5h, mixing speed is 60r/s, add the tetraethoxysilane of 0.01ml～5ml, at 10 DEG C～60 DEG C, reaction 2h～24h, the 3-aminopropyl triethoxysilane 0.1ml～20ml that adds the synthetic Fluorescein isothiocyanate of step (1) to modify, continue to stir 2h～24h, in 1h～10h, dropwise add the tetraethoxysilane of 0.01ml～10ml, lucifuge vigorous stirring 1h～10h, mixing speed is 60r/s, obtain the nano-particle of double-functional group, note is FMNPs, do centrifuge washing for the first time with the water of 1ml～10ml, the ethanol of 1ml～10ml does centrifuge washing for the second time, the water of 1ml～10ml does for the third time centrifuge washing three times, kept dry,
(3) the nano-particle FMNPs of the double-functional group of 0.01g～10g is dispersed in the mixed liquor of 40ml～130ml second alcohol and water, ultrasonic 20min～120min fully dissolves it, in 30min～5h, dropwise add the 3-aminopropyl triethoxysilane of 0.1ml～20ml, at 50 DEG C～240 DEG C, vigorous stirring 1h～24h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 1ml～10ml, the ethanol of 1ml～10ml does centrifuge washing for the second time, and the water of 1ml～10ml does for the third time centrifuge washing three times;
(4) get 0.1ml～10ml1mg/ml～10mg/ml containing the lactobionic acid molecule of galactosyl, under ice-water bath, add wherein 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and the 0.1mL～10ml0.1mg/ml～10mg/ml N-maloyl imines of 0.1ml～10ml0.1mg/ml～10mg/ml, ultrasonic reaction 20min～2h, make the hydroxy-acid group activation of lactobionic acid, then the amido modified FMNPs solution of the lactobionic acid solution of activation being prepared with step (3) mixes, continue to stir 1h～19h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified,
(5) by 0.10mg～10mg carbodiimide hydrochloride and 0.01mg～20mg N-hydroxy thiosuccinimide, be blended in the MES buffer of 1ml～10ml, buffer concentration is 0.01mol/l～10mol/l, pH is 4～9.5, the amino functional FMNPs that finally adds 1ml～50ml to have lactobionic acid to modify, stir 20h～40h, mixing speed 60r/s 10 DEG C～60 DEG C lucifuges, the carboxyl of carbodiimide hydrochloride and N-hydroxy thiosuccinimide and lactobionic acid forms the active NHS ester of semistable ammonia, it is 4～8.5 that dialysis joins pH after separating, concentration is in the phosphate buffer of 1ml～50ml of 0.001mol/l～10mol/l, add again the prostate stem cell antigen of the 10 μ g/ml～100 μ g/ml of 0.1 μ l～100 μ l, at 10 DEG C～60 DEG C lucifuge reaction 4h～22h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linked, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
As embodiment more specifically of the present invention, the aminopropyl triethoxysilane that above-mentioned prepared Fluorescein isothiocyanate is modified should be kept under four degrees Celsius.
As embodiment more specifically of the present invention, in step (2), the interval that dropwise drips tetraethoxysilane is with every 10 times in microlitre counting period, drips in 1h～10.
As embodiment more specifically of the present invention, in step (2), the volume ratio of ethanol and water mixed liquid is 5:2.
As embodiment more specifically of the present invention, in step (3), the interval that dropwise drips 3-aminopropyl triethoxysilane is with every 10 times in microlitre counting period, drips at 30min～5h.
As embodiment more specifically of the present invention, in step (3), the volume ratio of ethanol and water mixed liquid is 5:1.
As embodiment more specifically of the present invention, in step (4), ice-water bath temperature be zero degrees celsius;
As embodiment more specifically of the present invention, in step (4), the mixed proportion of amido modified FMNPs solution prepared by the lactobionic acid solution of activation and step (3) is 1:3.
For easy, following compound uses English abbreviation:
1, Fluorescein isothiocyanate: FITC; 2,3-aminopropyl triethoxysilane: APTES; 3, tetraethoxysilane: TEOS; 4, the 3-aminopropyl triethoxysilane that Fluorescein isothiocyanate is modified: FITC-APTES; 5, lactobionic acid: LA; 6,1-(3-dimethylaminopropyl)-3-ethyl carbodiimide: EDC; 7, N-maloyl imines: NHS; 8, optomagnetic multi-functional mesoporous silicon: FMNPs; 9, carbodiimide hydrochloride: EDAC; 10, N-hydroxy thiosuccinimide: Sulfo-NHS; 11, MES: MES; 12, prostate stem cell antigen: PSCA.
Embodiment 1
The APTES of the FITC of 0.01g0.01mmol and 0.10g0.1mmol is joined in the ethanol of 10ml, at 10 DEG C, lucifuge is led to nitrogen 0.1mol, and reaction 10h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 0.1ml0.001g/ml of citric acid protection is dispersed in the mixed liquor of 10ml second alcohol and water, mixed proportion is 5:2, ultrasonic 20min, add the ammonia 0.1ml of 0.1mol/l as catalyst, at 10 DEG C, vigorous stirring 10min, mixing speed 60r/s, add the TEOS of 0.01ml, at 10 DEG C, reaction 2h, add FITC-APTES0.1ml, continue to stir 2h, in 1h, dropwise add the TEOS of 0.01ml, lucifuge vigorous stirring 10h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 1ml, the ethanol of 1ml does centrifuge washing for the second time, the water of 1ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 0.01g is dispersed in the mixed liquor of 10ml second alcohol and water, mixed proportion is 5:1, ultrasonic 20min fully dissolves it, in 30min, dropwise add the APTES of 0.1ml, at 50 DEG C, vigorous stirring 1h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 1ml, the ethanol of 1ml does centrifuge washing for the second time, the water of 1ml does for the third time centrifuge washing three times.Get the LA of 0.1ml1mg/ml, under ice-water bath, add wherein EDC and the 0.1mL0.1mg/ml NHS of 0.1ml0.1mg/ml, ultrasonic reaction 20min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 1h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 0.10mg and 0.01mg, be blended in the MES buffer of 1ml, buffer concentration is 0.01mol/l, and pH is 4, and the amino functional FMNPs that finally adds 1ml to have lactobionic acid to modify stirs 20h, mixing speed 60r/s 10 DEG C of lucifuges; Dialysis joins 1ml pH after separating is that 4 concentration are in 0.001mol/l phosphate buffer, add again the PSCA of the 10 μ g/ml of 0.1 μ l, at 10 DEG C of lucifuge reaction 4h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 2
The APTES of the FITC of 0.1g0.1mmol and 0.20g0.5mmol is joined in the ethanol of 20ml, at 20 DEG C, lucifuge is led to nitrogen 0.5mol, and reaction 12h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 0.2ml0.01g/ml of citric acid protection is dispersed in the mixed liquor of 20ml second alcohol and water, mixed proportion is 5:2, ultrasonic 30min, add the ammonia 0.5ml of 1mol/l as catalyst, at 20 DEG C, vigorous stirring 30min, mixing speed 60r/s, add the TEOS of 0.1ml, at 20 DEG C, reaction 5h, add FITC-APTES0.5ml, continue to stir 5h, in 2h, dropwise add the TEOS of 0.1ml, lucifuge vigorous stirring 12h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 2ml, the ethanol of 2ml does centrifuge washing for the second time, the water of 2ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 0.1g is dispersed in the mixed liquor of 20ml second alcohol and water, mixed proportion is 5:1, ultrasonic 30min fully dissolves it, in 60min, dropwise add the APTES of 0.5ml, at 80 DEG C, vigorous stirring 3h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 2ml, the ethanol of 2ml does centrifuge washing for the second time, the water of 2ml does for the third time centrifuge washing three times.Get the LA of 1ml2mg/ml, under ice-water bath, add wherein EDC and the 0.5mL1mg/ml NHS of 1ml1mg/ml, ultrasonic reaction 30min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 3h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 1mg and 0.1mg, be blended in the MES buffer of 2ml, buffer concentration is 0.1mol/l, and pH is 5, and the amino functional FMNPs that finally adds 5ml to have lactobionic acid to modify stirs 24h, mixing speed 60r/s 20 DEG C of lucifuges; Dialysis joins 5ml pH after separating is that 4.5 concentration are in the phosphate buffer of 0.01mol/l, add again the PSCA of the 20 μ g/ml of 1 μ l, at 20 DEG C of lucifuge reaction 6h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 3
The APTES of the FITC of 1g1mmol and 1g2mmol is joined in the ethanol of 30ml, at 25 DEG C, lucifuge is led to nitrogen 1mol, and reaction 14h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 0.5ml0.1g/ml of citric acid protection is dispersed in the mixed liquor of 30ml second alcohol and water, mixed proportion is 5:2, ultrasonic 40min, add the ammonia 1ml of 3mol/l as catalyst, at 25 DEG C, vigorous stirring 50min, mixing speed 60r/s, add the TEOS of 0.5ml, at 25 DEG C, reaction 7h, add FITC-APTES1ml, continue to stir 8h, in 3h, dropwise add the TEOS of 1ml, lucifuge vigorous stirring 14h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 3ml, the ethanol of 3ml does centrifuge washing for the second time, the water of 3ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 1g is dispersed in the mixed liquor of 30ml second alcohol and water, mixed proportion is 5:1, ultrasonic 40min fully dissolves it, in 90min, dropwise add the APTES of 1ml, at 100 DEG C, vigorous stirring 5h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 3ml, the ethanol of 3ml does centrifuge washing for the second time, the water of 3ml does for the third time centrifuge washing three times.Get the LA of 2ml3mg/ml, under ice-water bath, add wherein EDC and the 1mL2mg/ml NHS of 2ml2mg/ml, ultrasonic reaction 40min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 5h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 2mg and 1mg, be blended in the MES buffer of 3ml, buffer concentration is 1mol/l, and pH is 6, and the amino functional FMNPs that finally adds 10ml to have lactobionic acid to modify stirs 26h, mixing speed 60r/s 25 DEG C of lucifuges; It is that 5 concentration are in the phosphate buffer of 0.1mol/l that dialysis adds the pH of 10ml after separating, add again the PSCA of the 30 μ g/ml of 10 μ l, at 25 DEG C of lucifuge reaction 8h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 4
The APTES of the FITC of 2g2mmol and 2g3mmol is joined in the ethanol of 40ml, at 30 DEG C, lucifuge is led to nitrogen 1.5mol, and reaction 16h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 1ml1g/ml of citric acid protection is dispersed in the mixed liquor of 40ml second alcohol and water, mixed proportion is 5:2, ultrasonic 50min, add the ammonia 2ml of 5mol/l as catalyst, at 30 DEG C, vigorous stirring 70min, mixing speed 60r/s, add the TEOS of 1ml, at 30 DEG C, reaction 9h, add FITC-APTES5ml, continue to stir 11h, in 4h, dropwise add the TEOS of 2ml, lucifuge vigorous stirring 16h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 4ml, the ethanol of 4ml does centrifuge washing for the second time, the water of 4ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 2g is dispersed in the mixed liquor of 40ml second alcohol and water, mixed proportion is 5:1, ultrasonic 50min fully dissolves it, in 2h, dropwise add the APTES of 3ml, at 120 DEG C, vigorous stirring 7h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 4ml, the ethanol of 4ml does centrifuge washing for the second time, the water of 4ml does for the third time centrifuge washing three times.Get the LA of 3ml4mg/ml, under ice-water bath, add wherein EDC and the 3mL3mg/ml NHS of 3ml3mg/ml, ultrasonic reaction 50min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is, 1:3, continue to stir 7h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 3mg and 3mg, be blended in the MES buffer of 4ml, buffer concentration is 2mol/l, and pH is 6.5, and the amino functional FMNPs that finally adds 15ml to have lactobionic acid to modify stirs 28h, mixing speed 60r/s 30 DEG C of lucifuges; It is that 5.5 concentration are in the phosphate buffer of 1mol/l that dialysis adds the pH of 15ml after separating, add again the PSCA of the 40 μ g/ml of 20 μ l, at 30 DEG C of lucifuge reaction 10h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 5
The APTES of the FITC of 3g3mmol and 3.5g4mmol is joined in the ethanol of 50ml, at 35 DEG C, lucifuge is led to nitrogen 2mol, and reaction 18h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 3ml2g/ml of citric acid protection is dispersed in the mixed liquor of 50ml second alcohol and water, mixed proportion is 5:2, ultrasonic 60min, add the ammonia 3ml of 7mol/l as catalyst, at 35 DEG C, vigorous stirring 90min, mixing speed 60r/s, add the TEOS of 1.5ml, at 35 DEG C, reaction 11h, add FITC-APTES7ml, continue to stir 14h, in 5h, dropwise add the TEOS of 3ml, lucifuge vigorous stirring 18h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 5ml, the ethanol of 5ml does centrifuge washing for the second time, the water of 5ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 3g is dispersed in the mixed liquor of 50ml second alcohol and water, mixed proportion is 5:1, ultrasonic 60min fully dissolves it, in 2.5h, dropwise add the APTES of 5ml, at 140 DEG C, vigorous stirring 9h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 5ml, the ethanol of 5ml does centrifuge washing for the second time, the water of 5ml does for the third time centrifuge washing three times.Get the LA of 4ml5mg/ml, under ice-water bath, add wherein EDC and the 4mL4mg/ml NHS of 4ml4mg/ml, ultrasonic reaction 60min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 9h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 4mg and 5mg, be blended in the MES buffer of 5ml, buffer concentration is 3mol/l, and pH is 7, and the amino functional FMNPs that finally adds 20ml to have lactobionic acid to modify stirs 30h, mixing speed 60r/s 35 DEG C of lucifuges; Dialysis joins 20ml pH after separating is that 6 concentration are in the phosphate buffer of 2mol/l, add again the PSCA of the 50 μ g/ml of 30 μ l, at 35 DEG C of lucifuge reaction 12h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 6
The APTES of the FITC of 4g4mmol and 5g5mmol is joined in the ethanol of 60ml, at 40 DEG C, lucifuge is led to nitrogen 2.5mol, and reaction 20h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 5ml3g/ml of citric acid protection is dispersed in the mixed liquor of 70ml second alcohol and water, mixed proportion is 5:2, ultrasonic 70min, add the ammonia 5ml of 9mol/l as catalyst, at 40 DEG C, vigorous stirring 110min, mixing speed 60r/s, add the TEOS of 2ml, at 40 DEG C, reaction 13h, add FITC-APTES9ml, continue to stir 17h, in 6h, dropwise add the TEOS of 4ml, lucifuge vigorous stirring 20h, mixing speed is 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 6ml, the ethanol of 6ml does centrifuge washing for the second time, the water of 6ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 4g is dispersed in the mixed liquor of 70ml second alcohol and water, mixed proportion is 5:1, ultrasonic 70min fully dissolves it, in 3h, dropwise add the APTES of 7ml, at 160 DEG C, vigorous stirring 13h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 6ml, the ethanol of 6ml does centrifuge washing for the second time, the water of 6ml does for the third time centrifuge washing three times.Get the LA of 5ml6mg/ml, under ice-water bath, add wherein EDC and the 5mL5mg/mlNHS of 5ml5mg/ml, ultrasonic reaction 70min, makes the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3; Continue to stir 11h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 5mg and 7mg, be blended in the MES buffer of 6ml, buffer concentration is 4mol/l, and pH is 7.5, and the amino functional FMNPs that finally adds 25ml to have lactobionic acid to modify stirs 32h, mixing speed 60r/s 40 DEG C of lucifuges; Dialysis joins 25ml pH after separating is that 6.5 concentration are in the phosphate buffer of 3mol/l, add again the PSCA of the 60 μ g/ml of 40 μ l, at 40 DEG C of lucifuge reaction 14h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 7
The APTES of the FITC of 5g5mmol and 6g6mmol is joined in the ethanol of 70ml, at 45 DEG C, lucifuge is led to nitrogen 3mol, and reaction 22h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 6ml5g/ml of citric acid protection is dispersed in the mixed liquor of 90ml second alcohol and water, mixed proportion is 5:2, ultrasonic 80min, add ammonia 6ml as catalyst, at 45 DEG C, vigorous stirring 130min, mixing speed 60r/s, add the TEOS of 2.5ml, at 45 DEG C, reaction 15h, add FITC-APTES13ml, continue to stir 20h, in 7h, dropwise add the TEOS of 5ml, lucifuge vigorous stirring 22h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 7ml, the ethanol of 7ml does centrifuge washing for the second time, the water of 7ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 5g is dispersed in the mixed liquor of 90ml second alcohol and water, mixed proportion is 5:1, ultrasonic 80min fully dissolves it, in 3.5h, dropwise add the APTES of 10ml, at 180 DEG C, vigorous stirring 15h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 7ml, the ethanol of 7ml does centrifuge washing for the second time, the water of 7ml does for the third time centrifuge washing three times.Get the LA of 6ml7mg/ml, under ice-water bath, add wherein EDC and the 7mL7mg/mlNHS of 7ml7mg/ml, ultrasonic reaction 80min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 13h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 6mg and 10mg, the MES that is blended in 7ml rushes in liquid, and buffer concentration is 6mol/l, and pH is 8, and the amino functional FMNPs that finally adds 30ml to have lactobionic acid to modify stirs 34h, mixing speed 60r/s 45 DEG C of lucifuges; Dialysis joins 30mol/l pH after separating is 7, concentration is in the phosphate buffer of 5mol/l, add again the PSCA of the 70 μ g/ml of 50 μ l, at 45 DEG C of lucifuge reaction 16h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 8
The APTES of the FITC of 6g6mmol and 7.5g7mmol is joined in the ethanol of 80ml, at 50 DEG C, lucifuge is led to nitrogen 3.5mol, and reaction 24h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 7ml7g/ml of citric acid protection is dispersed in the mixed liquor of 110ml second alcohol and water, mixed proportion is 5:2, ultrasonic 90min, add the ammonia 7ml of 15mol/l as catalyst, at 50 DEG C, vigorous stirring 150min, mixing speed 60r/s, add the TEOS of 3ml, at 50 DEG C, reaction 17h, add FITC-APTES15ml, continue to stir 22h, in 8h, dropwise add the TEOS of 7ml, lucifuge vigorous stirring 24h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 8ml, the ethanol of 8ml does centrifuge washing for the second time, the water of 8ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 6g is dispersed in the mixed liquor of 110ml second alcohol and water, mixed proportion is 5:1, ultrasonic 90min fully dissolves it, in 4h, dropwise add the APTES of 14ml, at 200 DEG C, vigorous stirring 18h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 8ml, the ethanol of 8ml does centrifuge washing for the second time, the water of 8ml does for the third time centrifuge washing three times.Get the LA of 7ml8mg/ml, under ice-water bath, add wherein EDC and the 8mL8mg/ml NHS of 8ml8mg/ml, ultrasonic reaction 90min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 15h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 7mg and 15mg, be blended in the MES buffer of 8ml, buffer concentration is 8mol/l, and pH is 8.5, and the amino functional FMNPs that finally adds 40ml to have lactobionic acid to modify stirs 36h, mixing speed 60r/s 50 DEG C of lucifuges; Dialysis joins 35ml pH after separating is 7.5, concentration is in the phosphate buffer of 7mol/l, add again the PSCA of the 80 μ g/ml of 70 μ l, at 50 DEG C of lucifuge reaction 18h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 9
The APTES of the FITC of 8g8mmol and 9g9mmol is joined in the ethanol of 90ml, at 55 DEG C, lucifuge is led to nitrogen 4mol, and reaction 26h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 9ml9g/ml of citric acid protection is dispersed in the mixed liquor of 130ml second alcohol and water, mixed proportion is 5:2, ultrasonic 100min, add the ammonia 9ml of 18mol/l as catalyst, at 55 DEG C, vigorous stirring 170min, mixing speed 60r/s, add the TEOS of 4ml, at 55 DEG C, reaction 20h, add FITC-APTES18ml, continue to stir 23h, in 9h, dropwise add the TEOS of 9ml, lucifuge vigorous stirring 26h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 9ml, the ethanol of 9ml does centrifuge washing for the second time, the water of 9ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 8g is dispersed in the mixed liquor of 130ml second alcohol and water, mixed proportion is 5:1, ultrasonic 100min fully dissolves it, in 4.5h, dropwise add the APTES of 18ml, at 220 DEG C, vigorous stirring 20h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 9ml, the ethanol of 9ml does centrifuge washing for the second time, the water of 9ml does for the third time centrifuge washing three times.Get the LA of 8ml9mg/ml, under ice-water bath, add wherein EDC and the 9mL9mg/ml NHS of 9ml9mg/ml, ultrasonic reaction 100min, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 17h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 9mg and 18mg, be blended in the MES buffer of 9ml, buffer concentration is 9mol/l, and pH is 9, and the amino functional FMNPs that finally adds 45ml to have lactobionic acid to modify stirs 38h, mixing speed 60r/s 55 DEG C of lucifuges; Dialysis joins 40mol/l pH after separating is 8, concentration is in the phosphate buffer of 9mol/l, add again the PSCA of the 90 μ g/ml of 90 μ l, at 55 DEG C of lucifuge reaction 20h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
Embodiment 10
The APTES of the FITC of 10g10mmol and 10g10mmol is joined in the ethanol of 100ml, at 60 DEG C, lucifuge is led to nitrogen 5mol, and reaction 28h, obtains FITC-APTES, the ferroferric oxide nano granules aqueous solution 10ml10g/ml of citric acid protection is dispersed in the mixed liquor of 150ml second alcohol and water, mixed proportion is 5:2, ultrasonic 2h, add the ammonia 10ml of 20mol/l as catalyst, at 60 DEG C, vigorous stirring 210min, mixing speed 60r/s, add the TEOS of 5ml, at 60 DEG C, reaction 24h, add FITC-APTES20ml, continue to stir 24h, in 10h, dropwise add the TEOS of 10ml, lucifuge vigorous stirring 28h, mixing speed 60r/s, obtain the nano-particle of functional group, note is FMNPs, do centrifuge washing for the first time with the water of 10ml, the ethanol of 10ml does centrifuge washing for the second time, the water of 10ml does for the third time centrifuge washing three times, kept dry, the nano-particle FMNPs of the double-functional group of 10g is dispersed in the mixed liquor of 150ml second alcohol and water, mixed proportion is 5:1, ultrasonic 2h fully dissolves it, in 5h, dropwise add the APTES of 20ml, at 240 DEG C, vigorous stirring 24h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 10ml, the ethanol of 10ml does centrifuge washing for the second time, the water of 10ml does for the third time centrifuge washing three times.Get the LA of 10ml10mg/ml, under ice-water bath, add wherein EDC and the 10mL10mg/ml NHS of 10ml10mg/ml, ultrasonic reaction 2h, make the hydroxy-acid group activation of lactobionic acid, then the lactobionic acid solution of activation is mixed with amido modified FMNPs solution, mixed proportion is 1:3, continues to stir 19h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified; By the Sulfo-NHS of the EDAC of 10mg and 20mg, be blended in the MES buffer of 10ml, buffer concentration is 10mol/l, and pH is 9.5, and the amino functional FMNPs that finally adds 50ml to have lactobionic acid to modify stirs 40h, mixing speed 60r/s 60 DEG C of lucifuges; Dialysis joins 50mol/l pH after separating is 8.5, concentration is in the phosphate buffer of 10mol/l, add again the PSCA of the 100 μ g/ml of 100 μ l, at 60 DEG C of lucifuge reaction 22h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linkedly, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
The transmission electron microscope picture of the optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated of the present invention as shown in Figure 2; The optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated at body nuclear magnetic resonance figure as shown in Figure 3; The fluorescence spectrum figure of the optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated as shown in Figure 4; The fluorescence microscope images of the optomagnetic bimodal mesoporous silicon nano-particle of prostate cancer stem cells Antigen-antibody mediated as shown in Figure 5.Above-mentioned image shows that nanoparticulate dispersed is good, good stability, and the function of the nano-particle that is magnetic, and also fluorescence property strengthens.
Claims (9)
1. there is the preparation method of antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle, it is characterized in that: this preparation method is: first prepare the 3-aminopropyl triethoxysilane that Fluorescein isothiocyanate is modified, again using ammonia as catalyst, in ferriferrous oxide nano solution, the 3-aminopropyl triethoxysilane that adds tetraethoxysilane and Fluorescein isothiocyanate to modify, reaction obtains having the mesoporous silica nano-particle of double-functional group, modify again amino with 3-aminopropyl triethoxysilane, and then provide functional group for nano-particle modified lactobionic acid, after crosslinked lactobionic acid, utilize the carboxyl of carbodiimide hydrochloride and N-hydroxy thiosuccinimide and lactobionic acid to form the active NHS ester of semistable ammonia, the active NHS ester of semistable ammonia and the anti-generation condensation reaction of prostate stem cell antigen form amido link, realize antibody linked, obtain the initiatively bimodal mesoporous silicon nanoparticle of target function.
2. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 1, is characterized in that: the method specifically comprises the following steps:
(1) the 3-aminopropyl triethoxysilane of the Fluorescein isothiocyanate of 0.01g～10g0.01mmol～10mmol and 0.10g～10g0.1mmol～10mmol is joined in the ethanol of 10ml～100ml, at 10 DEG C～60 DEG C, the logical nitrogen 0.1mol～5mol of lucifuge reacts 10h～28h, obtain the 3-aminopropyl triethoxysilane that Fluorescein isothiocyanate is modified, note is FITC-APTES;
(2) the ferroferric oxide nano granules aqueous solution 0.1ml～10ml0.001g/ml～10g/ml of citric acid protection is dispersed in the mixed liquor of 40ml～130ml second alcohol and water, ultrasonic 20min～120min, add ammonia 0.1ml～10ml, concentration is that 0.1mol/l～20mol/l is as catalyst, at 10 DEG C～60 DEG C, vigorous stirring 10min～6.5h, mixing speed is 60r/s, add the tetraethoxysilane of 0.01ml～5ml, at 10 DEG C～60 DEG C, reaction 2h～24h, the 3-aminopropyl triethoxysilane 0.1ml～20ml that adds the synthetic Fluorescein isothiocyanate of step (1) to modify, continue to stir 2h～24h, in 1h～10h, dropwise add the tetraethoxysilane of 0.01ml～10ml, lucifuge vigorous stirring 1h～10h, mixing speed is 60r/s, obtain the nano-particle of double-functional group, note is FMNPs, do centrifuge washing for the first time with the water of 1ml～10ml, the ethanol of 1ml～10ml does centrifuge washing for the second time, the water of 1ml～10ml does for the third time centrifuge washing three times, kept dry,
(3) the nano-particle FMNPs of the double-functional group of 0.01g～10g is dispersed in the mixed liquor of 40ml～130ml second alcohol and water, ultrasonic 20min～120min fully dissolves it, in 30min～5h, dropwise add the 3-aminopropyl triethoxysilane of 0.1ml～20ml, at 50 DEG C～240 DEG C, vigorous stirring 1h～24h, mixing speed 60r/s, obtain amido modified FMNPs, do centrifuge washing for the first time with the water of 1ml～10ml, the ethanol of 1ml～10ml does centrifuge washing for the second time, and the water of 1ml～10ml does for the third time centrifuge washing three times;
(4) get 0.1ml～10ml1mg/ml～10mg/ml containing the lactobionic acid molecule of galactosyl, under ice-water bath, add wherein 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and the 0.1mL～10ml0.1mg/ml～10mg/ml N-maloyl imines of 0.1ml～10ml0.1mg/ml～10mg/ml, ultrasonic reaction 20min～2h, make the hydroxy-acid group activation of lactobionic acid, then the amido modified FMNPs solution of the lactobionic acid solution of activation being prepared with step (3) mixes, continue to stir 1h～19h, make the carboxyl of lactobionic acid activation and the amido modified amino combination of FMNPs, thereby obtain the amino functional FMNPs that lactobionic acid is modified,
(5) by 0.10mg～10mg carbodiimide hydrochloride and 0.01mg～20mg N-hydroxy thiosuccinimide, be blended in the MES buffer of 1ml～10ml, buffer concentration is 0.01mol/l～10mol/l, pH is 4～9.5, the amino functional FMNPs that finally adds 1ml～50ml to have lactobionic acid to modify, stir 20h～40h, mixing speed 60r/s 10 DEG C～60 DEG C lucifuges, the carboxyl of carbodiimide hydrochloride and N-hydroxy thiosuccinimide and lactobionic acid forms the active NHS ester of semistable ammonia, it is 4～8.5 that dialysis joins pH after separating, concentration is in the phosphate buffer of 1ml～50ml of 0.001mol/l～10mol/l, add again the prostate stem cell antigen of the 10 μ g/ml～100 μ g/ml of 0.1 μ l～100 μ l, at 10 DEG C～60 DEG C lucifuge reaction 4h～22h, the active NHS ester of semistable ammonia forms amido link with the amino generation condensation reaction on prostate stem cell antigen antibody surface and is connected, thereby realize antibody linked, obtaining finishing has the bifunctional meso-porous silicon dioxide of antibody prostate stem cell antigen.
3. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 2, is characterized in that: the aminopropyl triethoxysilane that prepared Fluorescein isothiocyanate is modified is kept at 4 DEG C.
4. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 2, it is characterized in that: in described step (2), the interval that dropwise drips tetraethoxysilane is with every 10 times in microlitre counting period, drips at 1h～10h.
5. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 2, is characterized in that: in described step (2), in the mixed liquor of second alcohol and water, the volume ratio of second alcohol and water is 5:2.
6. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 2, it is characterized in that: in described step (3), the interval that dropwise drips 3-aminopropyl triethoxysilane is with every 10 times in microlitre counting period, drips at 30min～5h.
7. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 2, is characterized in that: in described step (3), in the mixed liquor of second alcohol and water, the volume ratio of second alcohol and water is 5:1.
8. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 2, is characterized in that: in described step (4), ice-water bath temperature be zero degrees celsius.
9. the preparation method with antibody-mediated optomagnetic bimodal mesoporous silicon nano-particle according to claim 2, it is characterized in that: in described step (4), the mixed proportion of amido modified FMNPs solution prepared by the lactobionic acid solution of activation and step (3) is 1:3.
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