CN110152571A - A kind of environment sensitive type magnetic microsphere and its preparation method and application for isolating and purifying labelled protein - Google Patents
A kind of environment sensitive type magnetic microsphere and its preparation method and application for isolating and purifying labelled protein Download PDFInfo
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Abstract
The invention discloses a kind of environment sensitive type magnetic microspheres and its preparation method and application for isolating and purifying labelled protein.The magnetic microsphere forms sulfhydrylation shell using magnetic nanoparticle as kernel, on its surface layer, then the JA(junction ambient) sensitive group on sulfhydrylation shell, and is coupled click-reaction group, the as described magnetic microsphere by environment sensitive group.Magnetic microsphere of the present invention can be promptly combined with labelled protein by efficient click-reaction, and under the action of external source magnetic field, it is separated from detection sample solution, then labeled albumen is discharged under certain environment stimulation, so that labeled albumen can be separated quickly, simply, and purity is higher, subsequent detection result is more acurrate；After labelled protein separated simultaneously, labeled albumen is not also recyclable in test sample to be checked and repeats to apply, and modifies/unmodified albumen, the especially protein of antibody class for recycle, the waste of albumen can be greatly reduced, reduce cost.
Description
Technical field
The present invention relates to polymer chemistry and biomedical engineering technology field, more particularly, to one kind for separating
Environment sensitive type magnetic microsphere of purification tag albumen and its preparation method and application.
Background technique
Fast inspection technology based on immune lateral chromatography technology has the advantages that easy to operate, immediately quick and cost is relatively low,
Early pregnancy detection, heart infarction early diagnosis and in terms of be widely applied, fast inspection by bed is realized, for improving
Medical diagnosis on disease efficiency plays positive effect, has broad application prospects and market value.
However, in the industrialized production for inspecting paper slip fastly based on immune lateral chromatography technology, there are still problems.At this
In technology, fluorescent microsphere, quantum dot, fluorescent dye etc. are inevitably tagged to antibody surface, issue letter in the detection
Number instruction sample in whether contain target detection thing.Currently, antibody is reacted with the label of signaling molecule is typically based on N- hydroxyl amber
The reaction of amber acid imide (NHS) and amino, maleimide (Mal) and sulfydryl.Since click chemistry reacts proposition and development,
Because its efficient reaction efficiency and its environmentally protective advantage, are applied in biochemical reaction, more and more in egg
It is also widely used in the label of bletilla antibody.But after reaction, there are still many not labeled antibody eggs in solution
It is white to be difficult to separate, recycle, not only make production cost high, very big influence can be also caused to detection sensitivity.
And traditional method for purifying proteins is mostly chromatography, such as affinity chromatography, Hydroxylapatite chromatography, fiber
Column chromatography, ion-exchange chromatography etc.；Its purification process needs to select suitable packing material size, the suitable eluent of proportion, and
Filler price is higher, eluting solvent dosage is larger, leads to higher cost；In addition, chromatography also has the used time longer, inconvenient
The problems such as.
In addition, easily being degraded by the enzyme in body fluid when the protein medicaments such as insulin convey in vivo, it is unable to reach treatment effect
Fruit.Therefore, the PEG of activation need to be coupled on drug by protein medicaments surface PEG by chemical method, with protection
Protein medicaments are not digested.However, there is likely to be poly glycol monomethyl ethers, intersection in solution after protein medicaments are PEGylated
The substances such as the PEG glycol of connection or polymerization, it is difficult to isolate and purify, limit the application of protein medicaments PEG modification technique.
It is easy and fast to therefore, it is necessary to provide one kind, is precisely separating purification tag albumen, while not labeled albumen is also
The method that can effectively recycle.
Summary of the invention
It is an object of the invention to overcome the shortcomings of the detection process marker protein purification process of existing labelled protein, with
And the defect that not labeled albumen can not be recycled effectively, it provides a kind of for isolating and purifying the environment sensitive type magnetic of labelled protein
Property microballoon.Environment sensitive type magnetic microsphere surface modification click-reaction group of the present invention, albumen to be detected use and magnetism
Identical click-reaction group label on microballoon, then connects signaling molecule corresponding with the click-reaction group of protein labeling
Click-reaction group, then signaling molecule can respectively with labelled protein and the efficient click-reaction of magnetic microsphere, by labelled protein with
Magnetic microsphere coupling, in conjunction with the superparamagnetism of magnetic bead itself, under the action of outer source magnet, with magnetism after labelled protein coupling
Microballoon is separated reaction system due to magnetic attraction, thereafter under certain environmental stimulus, labelled protein and magnetic microsphere
Between coupling be disconnected, labelled protein is released.By the above method, the purity is high of obtained labelled protein, purification process
Simply, purification efficiency is high；Meanwhile remaining unlabelled albumen (especially antibody) is also recyclable and sharp again in reaction system
With reducing production cost.
Another object of the present invention is to provide described for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein
Preparation method.
A further object of the present invention is to provide described for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein
Application.
It is described for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein a further purpose of the present invention is to provide
Application method.
Above-mentioned purpose of the invention is achieved by following scheme:
It is a kind of for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein, using magnetic nanoparticle as kernel,
Its surface layer forms sulfhydrylation shell, then the JA(junction ambient) sensitive group on sulfhydrylation shell, and even by environment sensitive group
Join click-reaction group, it is as described for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein.
Magnetic microsphere of the present invention is the click-reaction in the surface modification JA(junction ambient) sensitivity key of nanometer magnetic bead particle
Group, by needing albumen to be separated in efficient click-reaction combination anchor, using the superparamagnetism of magnetic bead, outside
Under the magnetic field of source can fast enriching, it is simple and efficient to handle；After being stimulated by certain environment, target protein can quick release, purity is high；
The environment sensitive type magnetic microsphere not only can replace the methods of traditional column chromatography, and to reach purifying destination protein, recycling not anti-
The purpose of albumen is answered, purification efficiency can also be improved, simplifies operating process, there is very big advantage.
Preferably, the magnetic nanoparticle is ferroferric oxide nano granules, the ferroso-ferric oxide of doping manganese element is received
One of rice grain or γ-di-iron trioxide nano particle are a variety of.
Preferably, the environment sensitive group is one of acylhydrazone key, hydrazone bond, imine linkage, original acid ester key or acetal bonds
Or it is a variety of.
Preferably, the click-reaction group is azepine dibenzo cyclooctyne (DBCO), nitrine (N3), end-group alkyne, Malaysia
Acid imide (Mal), sulfydryl or furans it is one or more.
Preferably, the magnetic nanoparticle is ferroferric oxide nano granules；The environment sensitive group is acylhydrazone key；
The click-reaction group is azepine dibenzo cyclooctyne (DBCO) or nitrine (N3)。
Also protection is described for isolating and purifying the preparation side of the environment sensitive type magnetic microsphere of labelled protein simultaneously by the present invention
Method, comprising the following steps:
S1., magnetic nanoparticle is obtained to the magnetic nanoparticle (Fe of sulfhydrylation through hydrosulphonyl silane reagent3O4@SiO2-
SH)；
S2. by the substance containing environment sensitive group and the substance reaction containing click-reaction group, preparation contains simultaneously
The substance of environment sensitive group and click-reaction group；
S3. by the magnetic nanoparticle of the step S1 sulfhydrylation prepared and step S2 prepare containing environment sensitive group and
Under the action of photoinitiator, through 365nm ultraviolet light, surface modification is prepared in reaction has the substance of click-reaction group
The environment sensitive magnetic microsphere of click-reaction group.
Preferably, magnetic nanoparticle described in step S1 is ferroferric oxide nano granules.
Preferably, the substance containing environment sensitive group is allyl aldehyde radical polyethylene glycol or alkene butyl aldehyde radical in step S2
Polyvinyl alcohol；The substance containing click-reaction group is nitrine propionyl hydrazine, nitrine acethydrazide or azepine dibenzo cyclooctyne
Acethydrazide.
Preferably, photoinitiator described in step S3 is 2- hydroxyl -4'- (2- hydroxy ethoxy) -2- methyl phenyl ketone, 2- hydroxyl
Base-2- methyl-1-phenyl-1- acetone, 2,4,6- trimethylbenzoy-dipheny phosphine oxide or 2- dimethylamino-2- benzyl-
One of 1- [4- (4- morpholinyl) phenyl] -1- butanone.
It is highly preferred that photoinitiator described in step S3 is 2- hydroxyl -4'- (2- hydroxy ethoxy) -2- methyl phenyl ketone.
It is highly preferred that the detailed process of step S2 are as follows:
S21. the preparation of the substance containing environment sensitive group:
It take hydrazine hydrate, methyl bromide c, sodium azide etc. as the nitrine propionyl hydrazine of Material synthesis small molecule；
Or, using hydrazine hydrate, DBCO-NHS as Material synthesis azepine dibenzo cyclooctyne propionyl hydrazine (DBCO)；
S22. the preparation of the substance containing click-reaction group:
With allyl polyglycol (APEG3k- OH) it is raw material, by TEMPO selective oxidation, synthesis of allyl aldehyde radical is poly-
Ethylene glycol (APEG3k-CHO)；
S23. the preparation of the substance simultaneously containing environment sensitive group and click-reaction group:
APEG3k- CHO and nitrine propionyl hydrazine are raw material, and schiff base reaction occurs, synthesizes the poly- second of nitrine allyl of acid-sensitive
Glycol (APEG3k-pH-N3), the as substance containing environment sensitive group and click-reaction group simultaneously；
Or, APEG3kSchiff base reaction occurs for-CHO and azepine dibenzo cyclooctyne propionyl hydrazine, synthesizes azepine dibenzo ring
Octyne allyl polyglycol (APEG3k- pH-DBCO), the as object containing environment sensitive group and click-reaction group simultaneously
Matter.
Preferably, in step S22, the catalyst of the allyl polyglycol oxidation is one of following system: 1. tetramethyl
Piperidine nitroxide (TEMPO), sodium bromide, sodium hypochlorite；2. tetrapropyl ammonium crosses rhodate (TPAP), potassium carbonate；3. vinegar
Sour palladium, pyridine,Molecular sieve.
It is highly preferred that the detailed process of step S3 are as follows:
Fe3O4@SiO2-SH、APEG3k-pH-N3Or APEG3k- pH-DBCO is under photoinitiator I2959 existence condition, warp
365nm ultraviolet light, reacts, and Fe can be prepared respectively3O4@SiO2-PEG-pH-N3Or Fe3O4@SiO2-PEG-
pH-DBCO。
The environment sensitive type magnetic microsphere for isolating and purifying labelled protein is in separation, purification tag albumen, recycling
For labelled protein detection process application also within protection scope of the present invention.
Preferably, applicable albumen includes: antibody, such as Troponin I antibody, myoglobins antibody, hepatitis B core antibody
IgM etc.；And protein medicaments, such as insulin, apoptosis element, trichosanthin.
Also protection is described for isolating and purifying the application side of the environment sensitive type magnetic microsphere of labelled protein simultaneously by the present invention
Method comprises the following processes:
S4. the substance containing click-reaction group will be added in sample to be tested, is marked, obtains containing labelled protein
Detection sample；
S5. by click-reaction genetic material corresponding in signaling molecule surface modification, the corresponding point of modification is prepared
Hit the signaling molecule of reactive group；
S6. the step S5 signaling molecule prepared is added in the detection sample of step S4, claims 1 to 3 is then added
Any magnetic microsphere mixes reaction；
S7. to separate magnetic microsphere from detection sample under the action of externally-applied magnetic field after reaction；So
It is placed under acid condition and discharges albumen, albumen after purification can be obtained.
Preferably, the click-reaction group in the substance containing click-reaction group described in step S4 and magnetic microsphere table
The click-reaction group of face modification is identical.
Preferably, the click-reaction group of corresponding click-reaction genetic material described in step S5 and institute in step S4
The click-reaction group of labelled protein surface modification is stated as a pair of of click-reaction group.
Preferably, step S4~S6 is carried out under the conditions of 7.3~7.5 pH；Acid condition described in step S7 is pH5.0
~6.5.
It is highly preferred that step S4~S6 is carried out under the conditions of pH is 7.4；Acid condition described in step S7 is that pH is 6.0.
Preferably, the click-reaction group is azepine dibenzo cyclooctyne (DBCO), nitrine (N3), end-group alkyne, Malaysia
Acid imide (Mal), sulfydryl or furans.
Wherein click-reaction group is to for azepine dibenzo cyclooctyne (DBCO)/nitrine (N3), end-group alkyne/nitrine, Malaysia
Acid imide (Mal)/sulfydryl, diels-Alder (Diels-Alder) reaction are to (such as furans and maleimide).A pair of of point
Two groups for hitting reactive group centering can expeditiously occur click-reaction and be combined.
Compared with prior art, the invention has the following advantages:
Magnetic microsphere provided by the invention can be promptly combined with labelled protein by efficient click-reaction, and
Under the action of external source magnetic field, is separated from detection sample solution, then discharge labeled albumen in acid condition, so that
Labeled albumen quickly, can be separated simply, and purity is higher, and subsequent detection result is more acurrate；
After labelled protein is separated simultaneously, labeled albumen is not also recyclable in test sample to be checked and repeats to apply,
For recycling modification/unmodified albumen, the especially protein of antibody class, the waste of albumen can be greatly reduced, reduce at
This.
Magnetic microsphere of the present invention is in application process, high with the Percentage bound of labelled protein, the collection of labelled protein and
Purification effect is good, and application method is simple, quick, convenient for applying in clinically coherent detection.
Detailed description of the invention
Fig. 1 is the nuclear magnetic spectrogram of bromo propionyl hydrazine, nitrine propionyl hydrazine and azepine dibenzo cyclooctyne propionyl hydrazine.
Fig. 2 is allyl aldehyde radical polyethylene glycol (APEG3k- CHO), the allyl nitrine polyethylene glycol (APEG of acid-sensitive3k-
) and the allyl nitrine polyethylene glycol (APEG of acid-sensitive pH-N33k- pH-DBCO) nuclear magnetic spectrogram.
Fig. 3 is ferroso-ferric oxide (Fe3O4), the ferroso-ferric oxide (Fe of sulfhydrylation3O4@SiO2- SH), the nitrine magnetic of acid-sensitive
Property ferroso-ferric oxide (Fe3O4@SiO2-pH-N3), the DBCO magnetic ferroferric oxide (Fe of acid-sensitive3O4@SiO2-pH-DBCO)
Infrared spectrum.
Fig. 4 is that the TEM of magnetic microsphere schemes, unused staining reagent, (A) Fe3O4, (B) Fe3O4@SiO2- SH, (C) Fe3O4@
SiO2-pH-N3, (D) Fe3O4@SiO2-pH-DBCO。
Fig. 5 is the hysteresis loop figure of magnetic microsphere, and measurement temperature is 300K.
Fig. 6 is accumulation ability comparison diagram of the magnetic microsphere under external source magnetic fields and gravity.
Fig. 7 is the schematic illustration that magnetic microsphere plays a role.
Fig. 8 is two kinds of acid-sensitive magnetic microspheres (surface modification nitrine and surface modification DBCO) at pH 7.4, pH 6.0
Under part in conjunction with fluorescent molecule after acid-sensitive release figure.
Fig. 9 is the magnetic microsphere (Fe of surface modification nitrine3O4@SiO2-pH-N3) to two kinds of albumen of surface modification DBCO
The combination of (Cas9 and BSA) discharges figure.
Figure 10 is the magnetic microsphere (Fe of surface modification DBCO3O4@SiO2- pH-DBCO) to the two of the combination of nitrine fluorescent microsphere
The release figure of kind albumen (Cas9 and BSA).
Specific embodiment
The present invention is made combined with specific embodiments below and further being elaborated, the embodiment is served only for explaining this
Invention, is not intended to limit the scope of the present invention.Test method as used in the following examples is normal unless otherwise specified
Rule method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
Embodiment 1
It is a kind of for isolating and purifying the environment sensitive type magnetic microsphere Fe of labelled protein3O4@SiO2-PEG-pH-N3, specifically
Preparation process it is as follows:
S1. the preparation of sulfhydrylation magnetic ferroferric oxide microballoon:
S11. the preparation of magnetic ferroferric oxide microballoon:
4.8-5.2g sodium hydroxide (NaOH) is dissolved in 250mL deionized water, above-mentioned sodium hydroxide solution is placed in
In 500mL three-necked flask, deoxygenation half an hour is vacuumized at room temperature, is replaced into nitrogen atmosphere.By 6.4-6.6g ferric chloride hexahydrate
(FeCl3·6H2) and 2.4-2.6g Iron dichloride tetrahydrate (FeCl O2·4H2O) it is dissolved in 12mL 0.4molL-1HCl solution
In, deoxygenation is vacuumized, nitrogen atmosphere is replaced into.Above-mentioned sodium hydroxide solution is heated to 40 DEG C, by the above-mentioned (Fe containing ferrous iron2+)、
Iron (Fe3+) solion is slowly dropped in sodium hydroxide solution.After being added dropwise, reaction temperature is improved to 80 DEG C, is continued
Reaction one hour.After fully reacting, restore to room temperature, black solid is collected to bottom of bottle by magnet magnetic suck, removes supernatant
Liquid.300mL dehydrated alcohol is continuously added, is washed ten minutes under ultrasound, magnetic separation is continued.It is so repeated four times, finally will
Product is dried in vacuo at 60 DEG C, obtains product ferroso-ferric oxide 2.52-2.54g, yield about 87%-92%.
S12. the preparation of the mercapto-modified Fe 3 O 4 magnetic microballoon in surface:
By 0.9-1.1g ferroso-ferric oxide ultrasonic disperse in 300mL ethyl alcohol, under nitrogen protection, continue that 0.8- is slowly added dropwise
1.2mL mercaptopropyltriethoxysilane and 45-50 μ L ammonium hydroxide continue ultrasonic reaction 3 hours at room temperature.It is separated with magnet magnetic,
It discards supernatant liquor and continuously adds 50mL dehydrated alcohol, washed ten minutes under ultrasound, continue magnetic separation.So repeat four
It is secondary, product is dried in vacuo at 60 DEG C finally, obtains the ferroso-ferric oxide (Fe of product sulfydryl siloxanes modification3O4@SiO2-
SH) 0.7-0.9g, yield about 78%-82%.
S2. the preparation of the substance containing environment sensitive group:
S21. the preparation of nitrine propionyl hydrazine:
Reaction route is as follows:
Synthetic bromide is for propionyl hydrazine first: 0.8-1.0g hydrazine hydrate is weighed in bis- mouthfuls of bottles of 250mL, addition 100mL ethyl alcohol, and 80
After DEG C being heated to reflux, the 50mL ethyl alcohol dissolved with 1.4-1.6g methyl bromide c is instilled, stirring is heated to reflux about 4h to solution in light
Yellow clear solution；After being restored to room temperature, faint yellow to colourless flocculent deposit is precipitated in -40 DEG C of freezing 0.5h.It filters, solid is used
Ethyl alcohol recrystallization, filter, be dried in vacuo white powder bromo propionyl hydrazine 1.24-1.26g, yield about 80%-83%.
Then it synthesizes nitrine propionyl hydrazine: weighing 0.8-1.0g bromo propionyl hydrazine in the two-mouth bottle of 100mL, 30mL is added and steams
Distilled water dissolution；By 1.2-1.4g sodium azide (NaN3) be dissolved in 3mL distilled water after, be added to above-mentioned containing the molten of bromo propionyl hydrazine
In liquid, stirring, the heated overnight at reflux at 80 DEG C, until solution is in lavender clear solution.After reaction, solution is cooled to
Water phase is extracted with ethyl acetate, in triplicate in room temperature.Merge organic phase, with anhydrous magnesium sulfate (MgSO4) dry, filter off drying
Agent, organic phase is through concentrated by rotary evaporation.Crude product methylene chloride (CH2Cl2): methanol (CH3OH)=9:1 crosses column, obtains colorless oil
Liquid 0.42-0.43g, yield about 57%-68%.
S22. the synthesis of the allyl polyglycol of acid-sensitive:
Aldehyde radical allyl polyglycol (APEG3k- CHO) preparation, reaction route is as follows:
It takes 2.9-3.1g allyl polyglycol in 100mL Shrek bottle, the dissolution of 50mL methylene chloride is added.By 90-
100 μ L aqueous sodium hypochlorite solutions are added in 5mL saturated sodium bicarbonate aqueous solution, 1.9-2.1mg tetramethyl piperidine nitrogen oxides
(TEMPO), 9-11mg sodium bromide is dissolved in the sodium bicarbonate aqueous solution containing sodium hypochlorite.It will be dissolved with allyl polyglycol
Shrek bottle is placed in ice-water bath, is vigorously stirred down and above-mentioned solution is slowly added dropwise, and is maintained in -4 DEG C of next hours and is dripped, after
Continuous reaction 4h.After reaction, liquid separation removes water phase, and organic phase is dry with anhydrous magnesium sulfate, after filtering off desiccant, rotates dense
Contracting, is deposited in anhydrous ether, and suction filtration obtains white solid powder shape crude product.Powder is re-dissolved with anhydrous methylene chloride,
Continue to be deposited in anhydrous ether, repeated precipitation, filters, drain, obtain white powdery solids 1.9-2.1g, yield is about
65%-67%.
S23. the acid-sensitive nitrine polyethylene glycol (APEG of allyl3k-pH-N3) preparation:
Reaction route is as follows:
Take 0.9-1.0g allyl aldehyde radical polyethylene glycol (APEG3k- CHO), it is dissolved in 30mL anhydrous methylene chloride, is added
48-52mg nitrine propionyl hydrazine, reaction is stayed overnight at room temperature.After reaction, concentrated by rotary evaporation is deposited in anhydrous ether, is obtained white
Color powder.Crude product continues to be dissolved in methylene chloride, continues to be deposited in anhydrous ether, so repeats four or five times, obtains white
Powder 0.74-0.76g, yield about 73%-79%.
S3. for isolating and purifying the preparation of the environment sensitive type magnetic microsphere of labelled protein:
Take the ferroso-ferric oxide (Fe of the above-mentioned sulfydryl siloxanes modification of 0.49-0.51g3O4@SiO2- SH) ultrasonic disperse in
In 200mL dehydrated alcohol, the acid-sensitive nitrine polyethylene glycol (APEG of allyl of above-mentioned synthesis is added3k-pH-N3) 0.24-0.26g,
4.8-5.2mg photoinitiator I2959 is added, under ultraviolet light (365nm), stirring, illumination reaction 2 hours.After reaction, it uses
The separation of magnet magnetic, discards supernatant liquor.50mL dehydrated alcohol is continuously added, is washed ten minutes under ultrasound, magnetic point is continued
From.So it is repeated four times.Finally product is dried in vacuo at 60 DEG C, the acid-sensitive magnetism for obtaining the modification of surface azido group is micro-
Ball product (Fe3O4@SiO2-PEG-pH-N3) 0.48-0.52g, yield about 90%.
The nuclear-magnetism of the bromo propionyl hydrazine, nitrine propionyl hydrazine and the azepine dibenzo cyclooctyne propionyl hydrazine that prepare in the above process is composed
Figure is as shown in Figure 1.
Embodiment 2
It is a kind of for isolating and purifying the environment sensitive type magnetic microsphere Fe of labelled protein3O4@SiO2The system of-PEG-pH-DBCO
Standby, wherein for step S1 with embodiment 1, step S2~S3 is as follows:
The preparation of S21 azepine dibenzo cyclooctyne (DBCO) hydrazides:
Reaction route is as follows:
It takes 0.098-0.102g azepine dibenzo cyclooctyne propionyl-N- succinimide ester in 100mL Shrek bottle, adds
Enter 30mL methylene chloride, while 0.04-0.06g hydrazine hydrate is added, is stirred overnight at room temperature.After reaction, revolving removes
Solvent, crude product methylene chloride: methanol=1:1 crosses column purification, obtains white powder 73-76mg, yield about 91%-95%.
The acid-sensitive azepine dibenzo cyclooctyne polyethylene glycol (APEG of S22 allyl3k- pH-DBCO) synthesis
Reaction route is as follows:
Take 0.98-1.02g allyl aldehyde radical polyethylene glycol (APEG3k- CHO), it is dissolved in 30mL anhydrous methylene chloride, is added
98-100mg azepine dibenzo cyclooctyne propionyl hydrazine (DBCO), reaction is stayed overnight at room temperature.After reaction, concentrated by rotary evaporation, precipitating
In anhydrous ether, white powder is obtained, crude product continues to be dissolved in methylene chloride, be deposited in anhydrous ether, so repeats
Four or five times, obtain white powder 0.75-0.85g, yield about 77%-85%.
Step S3 is with embodiment 1, the difference is that by the acid-sensitive nitrine polyethylene glycol (APEG of allyl3k-pH-N3) use
The acid-sensitive azepine dibenzo cyclooctyne polyethylene glycol (APEG of allyl3k- pH-DBCO) substitution, Fe is prepared3O4@SiO2-PEG-
PH-DBCO, detailed process are as follows:
Take the ferroso-ferric oxide (Fe of the above-mentioned sulfydryl siloxanes modification of 0.49-0.51g3O4@SiO2- SH) ultrasonic disperse in
In 200mL dehydrated alcohol, the acid-sensitive azepine dibenzo cyclooctyne polyethylene glycol (APEG of allyl of above-mentioned synthesis is added3k-pH-
DBCO 4.8-5.2mg photoinitiator I2959 is added in) 0.24-0.26g, under ultraviolet light (365nm), stirs, illumination reaction 2 is small
When.After reaction, it is separated with magnet magnetic, discards supernatant liquor.50mL dehydrated alcohol is continuously added, washs ten under ultrasound
Minute, continue magnetic separation, is so repeated four times.Product is dried in vacuo at 60 DEG C finally, obtains surface azepine dibenzo
The acid-sensitive magnetic microsphere product (Fe of cyclooctyne (DBCO) base group modification3O4@SiO2- PEG-pH-DBCO) 0.48-0.52g, it produces
Rate about 90%.
In the preparation process of Examples 1 and 2, the allyl aldehyde radical polyethylene glycol (APEG of preparation3k-CHO)、APEG3k-pH-
N3And APEG3kThe nuclear magnetic spectrogram of-pH-DBCO is as shown in Figure 2.
The morphology characterization of 3 magnetic microsphere of embodiment
It is characterized with the magnetic microsphere that Fourier-infrared spectrometer prepares Examples 1 and 2, takes two kinds of magnetism respectively
Microballoon about 1mg ground and mixed in 0.18-0.22g potassium bromide (KBr) is uniform, takes a little tabletting, is placed in 670 Fu of Nexus
It is tested in leaf-infrared spectrometer, the variation of detection reaction front and back ferroso-ferric oxide surface group, such as Fig. 3.
The pattern of magnetic microsphere is observed with transmission electron microscope (TEM), brief operation is as follows: 2 μ L samples (0.2mg/mL) being taken to drip
On pure carbon film copper mesh, dry at room temperature.It is observed at 120kV with transmission electron microscope (described to operate using this field routine skill
Art is detected)；Testing result is as shown in Figure 4, wherein Fig. 4 (A) is magnetic ferroferric oxide, it can be seen that partial size is about
250nm；Fig. 4 (B) is the ferroso-ferric oxide of surface sulfydryl, it can be seen that partial size is about in 500nm；Fig. 4 (C) is the folded of acid-sensitive
Magnetic microsphere (the Fe of nitrogen groups modification3O4@SiO2-PEG-pH-N3), it can be seen that partial size is about in 500nm；Fig. 4 (D) is table
The acid-sensitive magnetic microsphere product (Fe of face azepine dibenzo cyclooctyne (DBCO) base group modification3O4@SiO2- PEG-pH-DBCO),
It can be seen that partial size is about at 1 μm.
The magnetic behavior of 4 magnetic microsphere of embodiment and the Assembling Behavior under external source magnetic fields
To prove that Examples 1 and 2 prepare resulting two kinds of magnetic microspheres with good superparamagnetism and its response external source
The ability of aggregation in magnetic field takes 0.98-1.02mg respectively to walk magnetic microsphere, at room temperature, uses Quantum Design MPMS XL-
7 magnetic property measuring systems, measurement Examples 1 and 2 prepare resulting two kinds of magnetic microspheres and ferroso-ferric oxide and sulfhydrylation
Ferroso-ferric oxide hysteresis loop, measurement result such as Fig. 5.
From figure 5 it can be seen that all magnetic microspheres all have superparamagnetism well, moreover, with four oxidations three
The microballoon magnetism of the modification on ferromagnetic bilayers surface, phase homogenous quantities reduces, the reason is that the mass fraction of ferroso-ferric oxide is reducing.
Meanwhile can be quickly and effectively enriched under the action of external source magnetic field to verify prepared magnetic microsphere, it selects
Embodiment 2 prepares the acid-sensitive magnetic microsphere Fe of resulting surface azepine dibenzo cyclooctyne (DBCO) base group modification3O4@SiO2-
PEG-pH-DBCO is verified, and it is 0.1T externally-applied magnetic field, the complete natural subsidence of control group that experimental group, which applies a magnetic field strength,.Knot
Fruit is as shown in Figure 6.
From fig. 6 it can be seen that under the action of external source magnetic field, Fe3O4@SiO2- PEG-pH-DBCO magnetic microsphere can
Rapidly in one lateral enrichment of magnet, solution clear after enrichment in 5s；Relatively, natural subsidence needs 2min, and is enriched with
Micro magnetic microsphere particle is still had in solution afterwards；Experiment shows prepared magnetic microsphere under the action of external source magnetic field
It can quickly and effectively be enriched with, can play the role of being rapidly separated enrichment destination protein.
5 Fe of embodiment3O4@SiO2The acid-sensitive release behavior of-PEG-pH-DBCO magnetic microsphere
The principle that the environment sensitive type magnetic microsphere for isolating and purifying labelled protein plays a role is as shown in Figure 7.
The environment sensitive type magnetic microsphere surface modification click-reaction group of embodiment preparation, albumen to be detected uses and magnetic
Property microballoon on identical click-reaction group label, then signaling molecule is connected opposite with the click-reaction group of protein labeling
(the click-reaction group on click-reaction group and magnetic microsphere connected on signaling molecule is a pair to the click-reaction group answered
Efficient click-reaction can occur for click-reaction group pair), then signaling molecule can respectively with labelled protein and magnetic microsphere
Labelled protein and magnetic microsphere are coupled, in conjunction with the superparamagnetism of magnetic bead itself, in the work of outer source magnet by efficient click-reaction
Under, with magnetic microsphere after labelled protein coupling due to magnetic attraction, it is separated reaction system, thereafter in certain environment
Under stimulation, the coupling between labelled protein and magnetic microsphere is disconnected, and labelled protein is released, the purity of obtained labelled protein
Height, purification process is simple, and purification efficiency is high；Meanwhile remaining unlabelled albumen (especially antibody) can also return in reaction system
It receives and utilizes again, reduce production cost.
1, in order to verify the acid-sensitive release behavior of acylhydrazone key, respectively by N3The fluorescent molecule FITC and DBCO of modification are modified
Magnetic microsphere combine, by DBCO modify fluorescent molecule FITC and N3The magnetic microsphere of modification combines, in different pH conditions
Under, it detects the concentration for the fluorescent molecule that release combines, obtains acid-sensitive release figure, testing result is as shown in Figure 8.
Specifically detection process are as follows: 19-21mg embodiment 2 is taken to prepare resulting surface azepine dibenzo cyclooctyne (DBCO)
The acid-sensitive magnetic microsphere Fe of base group modification3O4@SiO2- PEG-pH-DBCO is scattered in 4mL pH7.4PBS solution, is added
1mg nitrine isothiocyanates fluorescein (N3- FITC), magnetic is separated off unreacted N3- FITC, by resulting isothiocyanic acid
The magnetic microsphere that ester fluorescein combines is dispersed in respectively in 4mL pH 7.4PBS solution and 4mL pH 6.0PBS solution；Equally
Ground, the acid-sensitive magnetic microsphere Fe of surface azido group modification3O4@SiO2-PEG-pH-N3It is magnetic with the acid-sensitive of DBCO modification
Microballoon Fe3O4@SiO2- PEG-pH-DBCO reaction, is dispersed in 4mL pH 7.4PBS solution and 4mL pH 6.0PBS solution respectively
In.0.5h, 1h, 2h, 4h, 8h, 12h, for 24 hours, 48h take 1mL solution respectively, and the corresponding buffer solution of 1mL is added after taking, is taken
Release after the solution containing FITC is using Hitachi F-4500 fluorescence spectrophotometer measurement fluorescence content and to calculate its dense
Degree, does the curve graph of sample time and cumulative release concentration, such as Fig. 8.
As can be seen from Figure 8,2 kinds of synthesized acid-sensitive magnetic microspheres have good acid-sensitive releasability, can
Discharge combined FITC substantially in 12h.
2, in order to verify acid-sensitive release behavior of the acid-sensitive magnetic microsphere to albumen of synthesis, DBCO is utilized to modify fluorescence
Two kinds of albumen (DBCO-Cas9-FITC and DBCO-BSA-FITC) and the magnetism of corresponding base group modification of molecule FITC label
It after microballoon combination under the conditions of different pH, detects the concentration for the fluorescent molecule that release combines, obtains acid-sensitive release figure, such as scheme
9。
Specifically detection process are as follows: 19-21mg embodiment 1 is taken to prepare the acid-sensitive magnetic of resulting surface azido group modification
Property microballoon Fe3O4@SiO2-PEG-pH-N3, it is scattered in 4mL pH 7.4PBS solution, 0.98-1.02mg DBCO modification is added
Two kinds of albumen (DBCO-Cas9-FITC and DBCO-BSA-FITC) of fluorescent molecule FITC label, magnetic is separated off unreacted
Albumen, by resulting isothiocyanates fluorescein labelled protein combine magnetic microsphere be dispersed in 4mL pH 7.4PBS respectively
In solution and 4mL pH 6.0PBS solution.0.5h, 1h, 2h, 4h, 8h, 12h, for 24 hours, 48h, 72h take 1mL solution respectively, take
After add the corresponding buffer solution of 1mL, after the release of acquirement containing FITC label albumen solution use Hitachi F-
Its fluorescence content of 4500 fluorescence spectrophotometer measurements simultaneously calculates concentration, does the curve graph of sample time and cumulative release concentration,
Such as Fig. 9.
It can be seen in figure 9 that synthesized acid-sensitive magnetic microsphere, which has, well modifies protein binding with surface DBCO
Ability, and the albumen of combination can be released effectively under the conditions of slightly sour (pH 6.0).In addition, measuring knot by microplate reader
The BSA protein concentration of conjunction is about 250 μ g/mL, so, the protein concentration in conjunction with release is about 245 μ g/mL, therefore synthesized
Acid-sensitive magnetic microsphere is about 98% to albumen (BSA) separative efficiency, shows there is very high separative efficiency.
3, it in order to verify acid-sensitive release behavior of the acid-sensitive magnetic microsphere to fluorescent microsphere labelled protein of synthesis, utilizes
DBCO modifies two kinds of albumen (DBCO-Cas9-MS and DBCO-BSA-MS) and corresponding the group (- N of fluorescent microsphere label3)
After the magnetic microsphere combination of modification under the conditions of different pH, detects the concentration for the fluorescent microsphere that release combines, obtain acid-sensitive
Release figure, such as Figure 10.
Specifically detection process are as follows: 19-21mg embodiment 4 is taken to prepare the acid-sensitive magnetic of resulting surface azido group modification
Property microballoon, be scattered in 4mL pH 7.4PBS solution, be added 0.98-1.02mg DBCO modification fluorescent microsphere label two hatching eggs
White (DBCO-Cas9-MS and DBCO-BSA-MS), magnetic is separated off unreacted albumen, and resulting fluorescent microsphere is marked egg
The magnetic microsphere of white combination is dispersed in respectively in 4mL pH 7.4PBS solution and 4mL pH 6.0PBS solution.0.5h, 1h, 2h,
4h, 8h, 12h, for 24 hours, 48h take 1mL solution respectively, and the corresponding buffer solution of 1mL, containing after the release of acquirement are added after taking
The solution for the albumen for having fluorescent microsphere to mark is using its fluorescence content of Hitachi's F-4500 fluorescence spectrophotometer measurement and calculates dense
Degree, does the curve graph of sample time and cumulative release concentration, such as Figure 10.
From fig. 10 it can be seen that synthesized acid-sensitive magnetic microsphere has the knot of good recovery fluorescent microsphere labelled protein
Conjunction ability, and it can be released effectively the albumen of combination under the conditions of low acid (pH 6.0), it can be effective in process of production
In conjunction with the albumen marked with fluorescent microsphere, play the role of isolating and purifying.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art
Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all of the invention
Made any modifications, equivalent replacements, and improvements etc., should be included in the protection of the claims in the present invention within spirit and principle
Within the scope of.
Claims (10)
1. a kind of for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein, which is characterized in that with magnetic nanoparticle
For kernel, sulfhydrylation shell is formed on its surface layer, then the JA(junction ambient) sensitive group on sulfhydrylation shell, and quick by environment
Sensitive group is coupled click-reaction group, as described for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein.
2. according to claim 1 for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein, which is characterized in that institute
Stating magnetic nanoparticle is ferroferric oxide nano granules, the ferroferric oxide nano granules for adulterating manganese element or γ-three oxidation
One of two iron nano-particles are a variety of；
The environment sensitive group is one of acylhydrazone key, hydrazone bond, imine linkage, original acid ester key or acetal bonds or a variety of；
The click-reaction group is the one of azepine dibenzo cyclooctyne, nitrine, end-group alkyne, maleimide, sulfydryl or furans
Kind is a variety of.
3. according to claim 2 for isolating and purifying the environment sensitive type magnetic microsphere of labelled protein, which is characterized in that institute
Stating magnetic nanoparticle is ferroferric oxide nano granules；The environment sensitive group is acylhydrazone key；The click-reaction group
For azepine dibenzo cyclooctyne or nitrine.
4. special for isolating and purifying the preparation method of the environment sensitive type magnetic microsphere of labelled protein described in claims 1 to 3
Sign is, comprising the following steps:
S1., magnetic nanoparticle is obtained to the magnetic nanoparticle of sulfhydrylation through hydrosulphonyl silane reagent；
S2. by the substance containing environment sensitive group and the substance reaction containing click-reaction group, preparation contains environment simultaneously
The substance of sensitive group and click-reaction group；
S3. contain environment sensitive group and click for prepared by the magnetic nanoparticle of the step S1 sulfhydrylation prepared and step S2
For the substance of reactive group under the action of photoinitiator, through 365nm ultraviolet light, surface modification, which is prepared, in reaction click
The environment sensitive magnetic microsphere of reactive group.
5. according to claim 4 for isolating and purifying the preparation method of the environment sensitive type magnetic microsphere of labelled protein,
It is characterized in that, magnetic nanoparticle described in step S1 is ferroferric oxide nano granules；
Substance containing environment sensitive group in step S2 is allyl aldehyde radical polyethylene glycol or alkene butyl aldehyde radical polyvinyl alcohol；Institute
Stating the substance containing click-reaction group is nitrine propionyl hydrazine, nitrine acethydrazide or azepine dibenzo cyclooctyne acethydrazide；
Photoinitiator described in step S3 is 2- hydroxyl -4'- (2- hydroxy ethoxy) -2- methyl phenyl ketone, 2- hydroxy-2-methyl -
1- phenyl -1- acetone, 2,4,6- trimethylbenzoy-dipheny phosphine oxide or 2- dimethylamino -2- benzyl -1- [4- (4-
One of quinoline base) phenyl] -1- butanone.
6. any environment sensitive type magnetic microsphere for isolating and purifying labelled protein of claims 1 to 3 is in separation, purifying
Labelled protein is recovered as the application of the detection process of labelled protein.
7. applying according to claim 6, which is characterized in that applicable albumen includes antibody and protein medicaments.
8. a kind of claims 1 to 3 is any described for isolating and purifying the application of the environment sensitive type magnetic microsphere of labelled protein
Method, which is characterized in that comprise the following processes:
S4. the substance containing click-reaction group will be added in sample to be tested, is marked, obtains the inspection containing labelled protein
Test sample；
S5. by click-reaction genetic material corresponding in signaling molecule surface modification, it is anti-that the corresponding click of modification is prepared
Answer the signaling molecule of group；
S6. the step S5 signaling molecule prepared is added in the detection sample of step S4, it is any that claims 1 to 3 is then added
The magnetic microsphere mixes reaction；
S7. to separate magnetic microsphere from detection sample under the action of externally-applied magnetic field after reaction；Then it sets
Albumen is discharged under acid condition, albumen after purification can be obtained.
9. application method according to claim 8, which is characterized in that the substance containing click-reaction group described in step S4
In click-reaction group it is identical as the click-reaction group of magnetic microsphere surface modification；
The click-reaction group of corresponding click-reaction genetic material described in step S5 and labelled protein described in step S4
The click-reaction group of surface modification is a pair of of click-reaction group.
10. application method according to claim 8, which is characterized in that step S4~S6 is carried out under the conditions of pH7.3~7.5；
Acid condition described in step S7 is pH5.0~6.5.
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