CN108569772A - A kind of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles and its preparation method and application - Google Patents
A kind of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to inorganic material and analysis technical field, it is related to a kind of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles and its preparation method and application；The present invention is added AABOB, N allyl imidazole, crosslinking agent MBAA and initiator azodiisobutyronitrile, heats initiated polymerization first by Fe3O4@MPS microballoons with after acetonitrile mixing ultrasonic disperse；Products therefrom is dried in vacuo after washing repeatedly；Then synergistic effect that products therefrom of the present invention is passed through to nickel ion carries out the immobilization of horseradish peroxidase, and obtained immobilised enzymes is used for the degradation of phenolic comp ' ds pollution；There is the immobilised enzymes of materials synthesis of the present invention recycling ability well, opposite enzyme activity can still reach 83% after reusing 5 times, be far above current document report；Its pH stability, temperature stability are compared with the material of unlockedization with storage-stable and are all increased significantly.
Description
Technical field
The invention belongs to inorganic material and analysis technical field, are related to a kind of Wulff types phenyl boric acid AABOB functional magnetics
Nanoparticle and its preparation method and application.
Background technology
Phenol wastewater be in the world industrial wastewater pollution most serious the problem of one of.Phenol wastewater is mainly derived from steel work
Industry, paper industry and plastics processing industry etc., if this waste water is directly discharged to without processing in river, environment, meeting
Very serious pollution is caused, serious harm is brought to human body, water body, fish body and crops.Phenol-Containing Wastewater Treatment at present
Method integrate mainly include Physical, chemical method and biochemical process three categories.Physical treatment process is to treatment conditions requirement
It is very high, and somewhat expensive needed for processing procedure；Method of chemical treatment may will produce the case where secondary pollution；Microbial degradation
Phenols wastewater needs to consume a large amount of time, and phenols wastewater can generate toxic action to microorganism.
Enzymic catalytic reaction is a kind of green, environmental protection chemical reaction process, uses horseradish peroxidase in recent years（HRP）
The research of processing phenols and amino benzene analog waste water receives the extensive research of researcher.Traditional solution enzyme is useless containing phenol in processing
It in water, is easy by other pollutant effects, and enzyme cannot recycle, easy in inactivation, stability are poor.And enzyme passes through immobilization
Afterwards, the problems such as difficult recycling, recycling difference, can effectively be solved, and the stability of enzyme can be improved, so the immobilization to HRP
Research is particularly important.Include mainly following four classes at present to the carrier material of HRP immobilizations：（1）Inorganic carrier is commonly used
Have Fe3O4, activated carbon；（2）Natural polymer subcarrier, such as chitosan, cellulose and alginic acid；（3）Macromolecule is synthesized to carry
Body reports that more carrier has polyacrylamide, polyaniline, polyketone, polyvinyl alcohol etc. at present；（4）Complex carrier, when single
Carrier cannot be met the requirements, and complex carrier also generates therewith, can be supplied a gap by the combination of different storerooms, to expire
To the requirement of carrier under sufficient specified conditions.These the carrier material reported at present needs further the immobilization effect of HRP
Optimize, the carrying capacity that major defect is embodied in enzyme is high, and recycle ability have it is to be strengthened.
Utilize carrier（Fe3O4）The metal ion Ni of upper grafting2+, synergistic effect can be generated with the amide group in HRP
And generate enzyme-inorganic hybrid nano flower that there is high enzyme to live.But for Ni2+The HRP of immobilization, catalysis reaction should be in
It is carried out under conditions of property or weak base, because of Ni3(PO4)2There is unstability in acid medium；Simultaneously because HRP does not organize ammonia
Acidity scale label, so the Ni on carrier granular2+Synergistic effect with the amide group on enzyme is not very strong, this can cause undesirable
Immobilization effect.Currently, the hybridized nanoparticle of boric acid functionalization be widely used in selectively be enriched with glycoprotein,
In dynamic glucose monitoring, controlled release and drug conduction, but but seldom had been reported that in enzyme immobilizatio about boric acid.
Invention content
It is an object of the invention to overcome, current immobilized HRP supported quantity is relatively low, recycling ability
The some shortcomingss such as poor, it is peppery to provide a kind of magnetic Nano microsphere immobilization of Wulff types phenyl boric acid AABOB functionalization
Root peroxidase.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
Present invention firstly provides a kind of novel magnetic Nano microsphere by Wulff type phenyl boric acid AABOB functionalization, the microballoons
A diameter of 150 nm is integrally uniformly dispersed and has superparamagnetism, can realize quick separating under externally-applied magnetic field.
The present invention also provides a kind of preparation method of the magnetic Nano microsphere by Wulff type phenyl boric acid AABOB functionalization, tools
Preparation step is as follows：
（1）5- nitros -2-（Methylol）Phenyl boric acid ring-type monoesters（NBOB）Synthesis：
A certain amount of fuming nitric aicd is added dropwise to a certain amount of 2- while stirring in -40 DEG C（Methylol）Phenyl boric acid ring-type monoesters
（BOB）In, completion of dropwise addition continues to be stirred to react 20 min.After the completion of the reaction measured by TLC contact plates, mixture is shifted
To be vigorously stirred in ice water a period of time.After reaction, separation product is filtered, is washed with deionized several times, then 40
It is dried in vacuum overnight at DEG C.
Wherein, the fuming nitric aicd addition is 5-15 mL；
The BOB additions are 1-2.2 g；
The mixture is vigorously stirred the time as 1.5-2.5 h in ice water.
（2）5- amino -2-（Methylol）Phenyl boric acid ring-type monoesters（ABOB）Synthesis：
By a certain amount of NBOB and a certain amount of（50 %）Raney's nickel is dissolved in 90 mL methanol, is existed under nitrogen protection later
The hydrazine hydrate of certain volume is added in mixture while stirring, control system temperature is 0 DEG C in entire reaction process.It is logical
It crosses after the completion of TLC measurement reactions, by mixture by suction filtered through kieselguhr, takes filtrate to carry out rotary evaporation and obtain product.It obtains
Product is used directly for next step synthetic reaction.
Wherein, the NBOB meltages are 3-3.8 g；Raney's nickel meltage is 2-4 g；
The hydrazine hydrate addition is 5-8.2 ml.
（3）Wulff type phenyl boric acids（AABOB）Synthesis：
The ABOB of 3 g is dissolved in the in the mixed solvent of the tetrahydrofuran containing 35 mL distilled water and certain volume, then by one
The acryloyl chloride of quantitative sodium bicarbonate and certain volume is successively added in above-mentioned solution, when being stirred to react one section at 0 DEG C
Between.After the completion of measuring reaction by TLC, evaporation removes tetrahydrofuran, is obtained after 2 h are extracted with ethyl acetate in remaining product
Crude product.Abstraction purification is carried out with 50 mL distilled water, 50 mL saturated sodium carbonate solutions, 50 mL distilled water successively later, will be had
Machine layer is filtered, and revolving obtains final product.
Wherein, amount of the tetrahydrofuran in mix reagent is 30-40 ml；
The sodium bicarbonate addition is 6.4-7.2 g；Acryloyl chloride addition is 2.8-3.6 ml；
Described, mixing time is 3-5 h at 0 DEG C.
（4）Fe3O4The synthesis of microballoon：
It is prepared using a kind of improved hydro-thermal method.By a certain amount of FeCl3·6H2O, a certain amount of NH4AC and a certain amount of
Two citric acid monohydrate trisodiums be dissolved in the single-necked flask (250 ml) for the ethylene glycol for filling certain volume, the oil bath of certain temperature
It heats and stirs 1 h and obtain the uniform system of black, be transferred to polytetrafluoroethyllining lining stainless steel autoclave (100 later
Ml), the baking oven for being put into certain temperature reacts 16 h, is cooled to room temperature, Magneto separate black product is washed several times extremely with absolute ethyl alcohol
After supernatant does not have color, dry 24 h are placed in 30 DEG C of vacuum drying chambers.
Wherein, the ferric chloride hexahydrate addition is 1.050-1.650 g；
The NH4AC additions are 3.454-4.254 g；The two citric acid monohydrate trisodiums of addition are 0.3-0.5 g；It is added
Ethylene glycol is 60-80 mL；
The temperature being stirred to react in the oil bath is 80-120 DEG C；
The temperature reacted in stainless steel autoclave is 180-220 DEG C.
（5）Fe3O4The synthesis of@MPS microballoons：
Weigh a certain amount of Fe3O440 mL ethyl alcohol, 10 mL water and 1.5 mL are added in the there-necked flask of 250 mL in nano-particle
Ammonium hydroxide, 1 h of ultrasonic disperse is to bottom without obvious sediment.Then under conditions of certain temperature while stirring slowly into system
The MPS of certain volume is added dropwise.After reacting 24 h of lasting progress, product is detached from solution with permanent magnet, it is anti-with ethyl alcohol
After backwashing is to neutrality, while the solution after cleaning will not become cloudy, and product is finally placed in 30 DEG C of vacuum drying chambers dry 24
h。
Wherein, the Fe3O4Nano-particle addition is 0.1-0.5 g；
The temperature when reaction is 60-80 DEG C；
The MPS additions are 0.4-0.6 mL.
（6）Fe3O4The synthesis of@p (AABOB-co-AIM) microballoon：
Weigh a certain amount of Fe3O4The acetonitrile of 40 mL, ultrasonic disperse to bottom is added in 100 mL there-necked flasks in@MPS microballoons
Without obvious sediment, backward system in a certain amount of AABOB, a certain amount of N- allyl imidazoles is added（AIM）, a certain amount of friendship
Join agent N, N '-di-2-ethylhexylphosphine oxide acrylamide（MBAA）With the initiator azodiisobutyronitrile of 10 mg（AIBN）.Add thermal-initiated polymerization
It reacts, flask is placed in oil bath device in reaction process, above connects still, condenser pipe and reception device.It is adjusted in reaction process
Section temperature makes system boil in 0.5 h and acetonitrile is made to start to condense, and the time of 1 h of control or so collects about 20 mL acetonitriles, this
When stop polymerization, remove oil bath device and be cooled to room temperature.Gained particle is washed with absolute ethyl alcohol repeatedly, wash away extra monomer and
Product is placed in 45 DEG C of vacuum drying chambers dry 24 h by oligomer until after supernatant liquid clarification.
Wherein, the Fe3O4The@MPS microballoon amounts of weighing are 0.03-0.07 g；AABOB additions are 50-70 mg；
The AIM additions are 20-60 mg；
The crosslinking agent MBAA additions are 30-50 mg.
The obtained Fe of the present invention3O4@P (AABOB-co-AIM) magnetic Nano microsphere is used for horseradish peroxidating
Object enzyme immobilizatio, steps are as follows for specific immobilization：
（1）Solution is prepared：
The preparation of phosphate buffer solution：Prepare 0.2 M Na2HPO4Solution and with 0.2 M, 100 mL NaH2PO4
Adjust pH to 7.0.
（2）Immobilization of Horseradish Peroxidase process：
By a certain amount of composite magnetic nanoparticle（Fe3O4@p(AABOB-co-AIM)）With the NiCl of 0.2 M of certain volume into
Row mixing, being stirred to react at 25 DEG C makes Ni2+It is fixed by the N atoms on AIM imidazole rings.Magneto separate salvage material later, washing
Drying overnight, obtains Fe3O4@P(AABOB-co-AIM)-Ni2+Magnetic Nano microsphere.
Wherein, the Fe3O4@p (AABOB-co-AIM) addition is 90-110 mg；
The NiCl additions are 7-13 mL.
The Fe that will be obtained3O4@P(AABOB-co-AIM)-Ni2+Magnetic Nano microsphere is configured to certain density suspension,
And mix the certain horseradish peroxidase solution of concentration with the solution of carrier material, ensure that solution carrier material is dense after mixing
Degree is 0.4 mgmL-1, a concentration of 0.004 mgmL of horseradish peroxidase-1 .System is in room temperature（25 ℃）It is lower to be incubated one
The section time detaches the carrier material after immobilization with permanent magnet, it is to be measured to retain supernatant.
With phosphate buffer solution flushing material twice to remove loose horseradish peroxidase.Using ultraviolet/visible
Spectrophotometer detects remaining horseradish peroxidase concentration in adsorption liquid, calculates supported quantity.
The supported quantity of horseradish peroxidase（Q）It can be calculated with following formula：
Q= (m 1- m 2)/m (mg·g-1)
In formula,m 1 It indicates that protein content in enzyme solution is added（mg）,m 2 Indicate protein content in remaining supernatant（mg）,m Indicate carrier
The quality of material（g）.
The present invention also provides one kind by Ni2+With the magnetic Nano microsphere immobilization of Wulff type phenyl boric acid AABOB functionalization
Horseradish peroxidase.The present invention is another object is that by the novel magnetic nanoparticle immobilization horseradish peroxidase of above-mentioned synthesis
Enzyme is for the degradation to pollutant phenol.
Compared with prior art, the invention has the advantages that：
（1）This invention has synthesized a kind of novel composite magnetic nanoparticle Fe3O4@P (AABOB-co-AIM), and to it
Property and pattern characterized.The magnetic bulb diameter is 150 nm, is integrally uniformly dispersed and has superparamagnetism, can add outside
Quick separating is realized under magnetic field.The immobilization of horseradish peroxidase may be implemented in the abundant functional groups in its surface.
（2）Using Fe3O4@P(AABOB-co-AIM)-Ni2+Immobilized HRP, optimum optimizing condition
For, when crosslinking agent MBAA be 40 mg, monomer AABOB dosages be 60 mg, monomer AIM dosages be 40 mg, immobilization pH 6,
The supported quantity of immobilization can reach 174.75 mg g-1, significantly larger than current reported supported quantity.
（3）As a kind of typical glycoprotein, functionalized with glycols group is prevalent on HRP, therefore can be transferred on carrier
The Wulff type phenyl boric acid AABOB that the present invention synthesizes are met, to form stable five yuan or hexa-atomic cyclic ester with the glycol in HRP, most
Realize that HRP is firmly combined with carrier eventually.Finally, magnetic carrier material（Fe3O4）Application enable to immobilised enzymes easily by again
Secondary Collection utilization increases its reuse ability.
The present invention has inquired into the recycling ability and zymologic property of immobilized HRP, and by its
Resolvase under recycling ability, pH stability, temperature stability and storage-stable and equal conditions has been done accordingly
Comparison, the results showed that, there is the immobilised enzymes that the present invention synthesizes good recycling ability, opposite enzyme activity to repeat profit
With can still reach 83% after 5 times, it is far above current document report；Its pH stability, temperature stability and storage-stable and not
The material of immobilization is compared and is all increased significantly.
（4）The immobilized HRP of synthesis is applied in degradation of phenol waste water, as a result, it has been found that, with resolvase
And pure magnetic Nano bead is compared, the degradation rate of immobilised enzymes Pyrogentisinic Acid has reached 93 %, has and is obviously improved, shows
Preferable application prospect.
Description of the drawings
Fig. 1 is sample F e3O4 (a) and Fe3O4The transmission electron microscope figure of@p (AABOB-co-AIM) (b)；
Fig. 2 is sample F e3O4（a）, Fe3O4@MPS（b）And Fe3O4@p(AABOB-co-AIM)（c）X-ray diffractogram；
Fig. 3 is sample F e3O4（a）, Fe3O4@MPS（b）, Fe3O4@pAABOB（c）And Fe3O4@p(AABOB-co-AIM) （d）
Hysteresis loop figure；
Fig. 4 is sample F e3O4 (a), Fe3O4@MPS (b), Fe3O4@pAABOB (c), Fe3O4@pAIM (d) and Fe3O4@
The infrared spectrogram of p (AABOB-co-AIM) (e)；
Fig. 5 is the reaction principle schematic diagram of AABOB and horseradish peroxidase；
Fig. 6 is influence result figure of the crosslinking agent MBAA additions to horseradish peroxidase fixed amount under condition of different pH；
Influence result figure of ratios of the Fig. 7 between monomer AABOB and AIM to horseradish peroxidase fixed amount；
Fig. 8 is the circulation ability shadow of the magnetic composite microsphere immobilized HRP of different AABOB and AIM ratios synthesis
Ring result figure；
Fig. 9 is the influence result figure of temperature (a), pH value (b) and storage time (c) to resolvase and immobilised enzymes；
Figure 10 be composite magnetic microballoon, resolvase, immobilised enzymes Pyrogentisinic Acid degradation results figure.
Specific implementation mode
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with attached drawing to the present invention
Technical solution is clearly and completely described, and embodiment described in the invention is a part of the embodiment in the present invention, without
It is whole embodiment, based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work
Under the premise of the every other embodiment that is obtained, shall fall within the protection scope of the present invention.
Embodiment 1：The preparation of Fe3O4@p (AABOB-co-AIM) microballoon
（1）The synthesis of NBOB：
The fuming nitric aicd of 5 mL is added dropwise to the 2- of 1 g while stirring in -40 DEG C（Methylol）Phenyl boric acid ring-type monoesters（BOB）
In, completion of dropwise addition continues to be stirred to react 20 min.After the completion of the reaction measured by TLC contact plates, mixture is transferred to ice
1.5 h are vigorously stirred in water.After reaction, separation product is filtered, is washed with deionized several times, it is then true at 40 DEG C
Sky is dried overnight.
（2）The synthesis of ABOB：
By the NBOB of 3 g and 2 g（50 %）Raney's nickel is dissolved in 90 mL methanol, later under nitrogen protection by 5 mL's
Hydrazine hydrate is added in mixture while stirring, and control system temperature is 0 DEG C in entire reaction process.It is measured by TLC anti-
After the completion of answering, by mixture by suction filtered through kieselguhr, takes filtrate to carry out rotary evaporation and obtain product.Obtained product can be direct
For next step synthetic reaction.
（3）The synthesis of AABOB：
The ABOB of 3 g is dissolved in the in the mixed solvent containing 35 mL distilled water and 30 mL tetrahydrofurans, then by 6.4 g's
The acryloyl chloride of sodium bicarbonate and 2.8 mL are successively added in above-mentioned solution, and 3 h are stirred to react at 0 DEG C.It is surveyed by TLC
After the completion of fixed reaction, evaporation removes tetrahydrofuran, and crude product is obtained after 2 h are extracted with ethyl acetate in remaining product.Later
Successively 50 mL distilled water, 50 mL saturated sodium carbonate solutions, 50 mL distilled water carry out abstraction purification, and organic layer was carried out
Filter, revolving obtain final product.
（4）The synthesis of Fe3O4 microballoons：
It is prepared using a kind of improved hydro-thermal method.By 1.050 g FeCl3·6H2O, the NH of 3.454g4AC and 0.3 g
Two citric acid monohydrate trisodiums are dissolved in the single-necked flask (250 ml) for filling 60 mL ethylene glycol, and 80 DEG C of oil bath heatings simultaneously stir 1
H obtains the uniform system of black, is transferred to polytetrafluoroethyllining lining stainless steel autoclave (100 ml) later, is put into baking oven
180 DEG C of 16 h of reaction, are cooled to room temperature, and Magneto separate black product is washed with absolute ethyl alcohol after not having color to supernatant several times,
Dry 24 h are placed in 30 DEG C of vacuum drying chambers.
（5）The synthesis of Fe3O4@MPS microballoons：
Fe in this experiment3O4 Microsphere surface increases double bond using silane coupling agent MPS modifications.Weigh the Fe of 0.1 g3O4Nanoparticle
In the there-necked flask of 250 mL 40 mL ethyl alcohol, 10 mL water and 1.5 mL ammonium hydroxide are added, 1 h of ultrasonic disperse is to bottom without bright in son
Aobvious precipitation.Then the MPS of 0.4 mL is slowly added dropwise into system while stirring under conditions of 60 DEG C.It waits reacting lasting progress
After 24 h, product is detached from solution with permanent magnet, neutrality is washed till repeatedly with ethyl alcohol, while the solution after cleaning will not become
Product is finally placed in 30 DEG C of vacuum drying chambers dry 24 h by muddiness.
（6）The synthesis of Fe3O4@p (AABOB-co-AIM) microballoon：
Weigh the Fe of 0.03 g3O440 mL acetonitriles, 15 min of ultrasonic disperse is added in the there-necked flask of 100 mL in@MPS microballoons
To bottom without obvious sediment, 50 mg AABOB, 20 mg N- allyl imidazoles are added later（AIM）, 30 mg crosslinking agents MBAA
With the initiator A IBN of 10 mg.Heat initiated polymerization, flask is placed in oil bath device in reaction process, above connect still,
Condenser pipe and reception device.Temperature is adjusted in reaction process to be made system boil in 0.5 h and acetonitrile is made to start to condense, and is controlled
The time of 1 h or so collects about 20 mL acetonitriles, stops polymerization at this time, removes heat source and be cooled to room temperature.Gained particle is repeatedly with nothing
Water-ethanol washs, and washes away the monomer and oligomer of more Shes, until after supernatant liquid clarification, product is placed in 45 DEG C of vacuum drying chambers
24 h of interior drying.
Embodiment 2：The preparation of Fe3O4@p (AABOB-co-AIM) microballoon
（1）The synthesis of NBOB：
The fuming nitric aicd of 15 mL is added dropwise to the 2- of 2.2 g while stirring in -40 DEG C（Methylol）Phenyl boric acid ring-type monoesters
（BOB）In, completion of dropwise addition continues to be stirred to react 20 min.After the completion of the reaction measured by TLC contact plates, mixture is turned
It moves on in ice water and is vigorously stirred 2.5 h.After reaction, separation product is filtered, is washed with deionized several times, then 40
It is dried in vacuum overnight at DEG C.
（2）The synthesis of ABOB：
By the NBOB of 3.8 g and 4 g（50 %）Raney's nickel is dissolved in 90 mL methanol, later under nitrogen protection by 8.2
The hydrazine hydrate of mL is added in mixture while stirring, and control system temperature is 0 DEG C in entire reaction process.It is surveyed by TLC
After the completion of fixed reaction, by mixture by suction filtered through kieselguhr, takes filtrate to carry out rotary evaporation and obtain product.Obtained product can be with
It is directly used in next step synthetic reaction.
（3）The synthesis of AABOB：
The ABOB of 3 g is dissolved in the in the mixed solvent containing 35 mL distilled water and 40 mL tetrahydrofurans, then by 7.2 g's
The acryloyl chloride of sodium bicarbonate and 3.6 mL are successively added in above-mentioned solution, and 5 h are stirred to react at 0 DEG C.It is surveyed by TLC
After the completion of fixed reaction, evaporation removes tetrahydrofuran, and crude product is obtained after 2 h are extracted with ethyl acetate in remaining product.Later
Successively 50 mL distilled water, 50 mL saturated sodium carbonate solutions, 50 mL distilled water carry out abstraction purification, and organic layer was carried out
Filter, revolving obtain final product.
（4）The synthesis of Fe3O4 microballoons：
It is prepared using a kind of improved hydro-thermal method.By 1.650 g FeCl3·6H2O, the NH of 4.254g4AC and 0.5 g
Two citric acid monohydrate trisodiums are dissolved in the single-necked flask (250 ml) for filling 80 mL ethylene glycol, and 120 DEG C of oil bath heatings simultaneously stir 1
H obtains the uniform system of black, is transferred to polytetrafluoroethyllining lining stainless steel autoclave (100 ml) later, is put into baking oven
220 DEG C of 16 h of reaction, are cooled to room temperature, and Magneto separate black product is washed with absolute ethyl alcohol after not having color to supernatant several times,
Dry 24 h are placed in 30 DEG C of vacuum drying chambers.
（5）The synthesis of Fe3O4@MPS microballoons：
Fe in this experiment3O4 Microsphere surface increases double bond using silane coupling agent MPS modifications.Weigh the Fe of 0.5 g3O4Nanoparticle
In the there-necked flask of 250 mL 40 mL ethyl alcohol, 10 mL water and 1.5 mL ammonium hydroxide are added, 1 h of ultrasonic disperse is to bottom without bright in son
Aobvious precipitation.Then the MPS of 0.6 mL is slowly added dropwise into system while stirring under conditions of 80 DEG C.It waits reacting lasting progress
After 24 h, product is detached from solution with permanent magnet, neutrality is washed till repeatedly with ethyl alcohol, while the solution after cleaning will not become
Product is finally placed in 30 DEG C of vacuum drying chambers dry 24 h by muddiness.
（6）The synthesis of Fe3O4@p (AABOB-co-AIM) microballoon：
Weigh the Fe of 0.07 g3O440 mL acetonitriles, 15 min of ultrasonic disperse is added in the there-necked flask of 100 mL in@MPS microballoons
To bottom without obvious sediment, 70 mg AABOB, 60 mg N- allyl imidazoles are added later（AIM）, 50 mg crosslinking agents MBAA
With the initiator A IBN of 10 mg.Heat initiated polymerization, flask is placed in oil bath device in reaction process, above connect still,
Condenser pipe and reception device.Temperature is adjusted in reaction process to be made system boil in 0.5 h and acetonitrile is made to start to condense, and is controlled
The time of 1 h or so collects about 20 mL acetonitriles, stops polymerization at this time, removes heat source and be cooled to room temperature.Gained particle is repeatedly with nothing
Water-ethanol washs, and washes away the monomer and oligomer of more Shes, until after supernatant liquid clarification, product is placed in 45 DEG C of vacuum drying chambers
24 h of interior drying.
Embodiment 3：The preparation of Fe3O4@p (AABOB-co-AIM) microballoon
（1）The synthesis of NBOB：
The fuming nitric aicd of 10 mL is added dropwise to the 2- of 1.6 g while stirring in -40 DEG C（Methylol）Phenyl boric acid ring-type monoesters
（BOB）In, completion of dropwise addition continues to be stirred to react 20 min.After the completion of the reaction measured by TLC contact plates, mixture is turned
It moves on in ice water and is vigorously stirred 2 h.After reaction, separation product is filtered, is washed with deionized several times, then at 40 DEG C
Under be dried in vacuum overnight.
（2）The synthesis of ABOB：
By the NBOB of 3.4 g and 3 g（50 %）Raney's nickel is dissolved in 90 mL methanol, later under nitrogen protection by 6.6
The hydrazine hydrate of mL is added in mixture while stirring, and control system temperature is 0 DEG C in entire reaction process.It is surveyed by TLC
After the completion of fixed reaction, by mixture by suction filtered through kieselguhr, takes filtrate to carry out rotary evaporation and obtain product.Obtained product can be with
It is directly used in next step synthetic reaction.
（3）The synthesis of AABOB：
The ABOB of 3 g is dissolved in the in the mixed solvent containing 35 mL distilled water and 35 mL tetrahydrofurans, then by 6.8 g's
The acryloyl chloride of sodium bicarbonate and 3.2 mL are successively added in above-mentioned solution, and 4 h are stirred to react at 0 DEG C.It is surveyed by TLC
After the completion of fixed reaction, evaporation removes tetrahydrofuran, and crude product is obtained after 2 h are extracted with ethyl acetate in remaining product.Later
Successively 50 mL distilled water, 50 mL saturated sodium carbonate solutions, 50 mL distilled water carry out abstraction purification, and organic layer was carried out
Filter, revolving obtain final product.
（4）The synthesis of Fe3O4 microballoons：
It is prepared using a kind of improved hydro-thermal method.By 1.350 g FeCl3·6H2O, the NH of 3.854g4AC and 0.4 g
Two citric acid monohydrate trisodiums are dissolved in the single-necked flask (250 ml) for filling 70 mL ethylene glycol, and 100 DEG C of oil bath heatings simultaneously stir 1
H obtains the uniform system of black, is transferred to polytetrafluoroethyllining lining stainless steel autoclave (100 ml) later, is put into baking oven
200 DEG C of 16 h of reaction, are cooled to room temperature, and Magneto separate black product is washed with absolute ethyl alcohol after not having color to supernatant several times,
Dry 24 h are placed in 30 DEG C of vacuum drying chambers.
（5）Fe3O4The synthesis of@MPS microballoons：
Fe in this experiment3O4Microsphere surface increases double bond using silane coupling agent MPS modifications.Weigh the Fe of 0.3 g3O4Nanoparticle
In the there-necked flask of 250 mL 40 mL ethyl alcohol, 10 mL water and 1.5 mL ammonium hydroxide are added, 1 h of ultrasonic disperse is to bottom without bright in son
Aobvious precipitation.Then the MPS of 0.5 mL is slowly added dropwise into system while stirring under conditions of 70 DEG C.It waits reacting lasting progress
After 24 h, product is detached from solution with permanent magnet, neutrality is washed till repeatedly with ethyl alcohol, while the solution after cleaning will not become
Product is finally placed in 30 DEG C of vacuum drying chambers dry 24 h by muddiness.
（6）The synthesis of Fe3O4@p (AABOB-co-AIM) microballoon：
Weigh the Fe of 0.05 g3O440 mL acetonitriles, 15 min of ultrasonic disperse is added in the there-necked flask of 100 mL in@MPS microballoons
To bottom without obvious sediment, 60 mg AABOB, 40 mg N- allyl imidazoles are added later（AIM）, 40 mg crosslinking agents MBAA
With the initiator A IBN of 10 mg.Heat initiated polymerization, flask is placed in oil bath device in reaction process, above connect still,
Condenser pipe and reception device.Temperature is adjusted in reaction process to be made system boil in 0.5 h and acetonitrile is made to start to condense, and is controlled
The time of 1 h or so collects about 20 mL acetonitriles, stops polymerization at this time, removes heat source and be cooled to room temperature.Gained particle is repeatedly with nothing
Water-ethanol washs, and washes away the monomer and oligomer of more Shes, until after supernatant liquid clarification, product is placed in 45 DEG C of vacuum drying chambers
24 h of interior drying.
TEM figures, XRD diagram, VSM figures and the FT-IR of the magnetic microsphere of the method synthesis of the present invention scheme as follows：
Fig. 1 is sample F e3O4 (a) and Fe3O4The TEM of@p (AABOB-co-AIM) (b) schemes；From Fig. 1's（a）Middle discovery, synthesis
Fe3O4Microballoon is in uniform-spherical and particle diameter distribution is uniform, and preferably, average diameter is about 120 nm to dispersibility；Fig. 1（b）In it is micro-
Spherolite diameter is gradually increased to 150nm or so, and the grey shell of wherein 15nm or so is clearly uniform, and entire microballoon presents more perfect
Nucleocapsid, and synthesize Fe3O4@p (AABOB-co-AIM) composite magnetic nanoparticle is uniformly dispersed, and can be effective
Fixed horseradish peroxidase（HRP）Important leverage.
Fig. 2 is sample F e3O4（a）, Fe3O4@MPS（b）And Fe3O4@p(AABOB-co-AIM)（c）XRD diagram；In fig. 2
It was found that 6 notable 2 θ peak values are respectively 30.3 °, 35.2 °, 43.5 °, 53.1 °, 57.0 ° and 62.9 ° and JCSD numbers in figure
According to card（74-748）The crystal form value of upper ferroso-ferric oxide（220）（311）（400）（422）（511）（440）Mutually correspond.This can
To find out after carrying out a series of synthetic reaction, the crystal form of the ferroso-ferric oxide of composite magnetic microballoon does not occur significantly
Change, is demonstrated by ferroso-ferric oxide and maintains larger integrality.
Fig. 3 is sample F e3O4（a）, Fe3O4@MPS（b）, Fe3O4@pAABOB（c）And Fe3O4@p(AABOB-co-AIM)
（d）Hysteresis loop figure；It can be found that Fe from Fig. 33O4 、Fe3O4@MPS、Fe3O4@pAABOB and Fe3O4@p(AABOB-co-
AIM saturation magnetic strength angle value) is respectively 53.47,45.41,31.82 and 28.27 emu g-1.Although be saturated magnetic intensity with
The gradually modification of magnetic microsphere surfacing and continuously decrease, but finally obtained composite magnetic nanoparticle still have it is higher
Saturation magnetic intensity；As shown in the illustration of the upper lefts Fig. 3, magnetic composite microsphere can be effective from solution by externally-applied magnetic field in 20 s
Separation, in addition to this, the magnetic microspheres of all synthesis all shows superparamagnetism, and without apparent remanent magnetism and coercivity.
Fig. 4 is sample F e3O4 (a), Fe3O4@MPS (b), Fe3O4@pAABOB (c), Fe3O4@pAIM (d) and
Fe3O4The infrared spectrogram of@p (AABOB-co-AIM) (e)；From fig. 4, it can be seen that upper 588 cm of curve a-1With 3380 cm-1
It is the peaks Fe-O and Fe respectively3O4The peaks-OH of microsphere surface.In addition to this, 1641 cm-1With 1414 cm-1The absorption peak at place is
The characteristic peak of microsphere surface carboxyl；In 1641 cm on curve b-1The absorption peak of left and right is the characteristic absorption peak of upper C=C of MPS,
In 1157 cm-1The absorption peak at place is the characteristic absorption peak of Si-O-Si；In 3114 cm on curve c−1, 1228 cm−1, 899
cm−1With 712 cm−1The absorption peak at place is the vibration absorption peak of imidazole ring on AIM；In 1685 cm-1With 1527 cm-1The suction at place
The characteristic peak that peak is respectively the C=O and N H on MBAA is received, these characteristic peaks can show polymeric shell layer, and successfully modification exists
Fe3O4The surfaces@MPS；The 1473cm in curve d−1With 1324 cm−1It is that the feature of phenyl ring and boron oxygen key is inhaled on AABOB respectively
It receives, also shows the success of polymerisation, these characteristic peaks can embody in curve e, it can be seen that Fe3O4@p(AABOB-
Co-AIM successful synthesis).
Embodiment 4：The preparation of immobilized HRP
（1）Solution is prepared
The preparation of phosphate buffer solution：Prepare 0.2 M Na2HPO4Solution and with 0.2 M, 100 mL NaH2PO4Adjust pH to
7.0。
（2）The immobilization process of horseradish peroxidase
The 90 mg composite magnetic nanoparticles synthesized are mixed with the NiCl of 7 ml, 0.2 M, are stirred to react at 25 DEG C
Make Ni2+It is fixed by the N atoms on AIM imidazole rings.Magneto separate salvage material later, washing and drying is overnight, the compound magnetic that will be obtained
Property nanoparticle is configured to certain density suspension, and by the certain horseradish peroxidase solution of concentration and carrier material
Solution mixes, and ensures that solution carrier material concentration is 0.4 mgmL after mixing-1, horseradish peroxidase a concentration of 0.004
mg·mL-1 .System is in room temperature（25 °C）Lower incubation a period of time, the carrier material after immobilization is detached with permanent magnet, is retained
Supernatant is to be measured.With phosphate buffer solution flushing material twice to remove loose horseradish peroxidase.Using it is ultraviolet/can
See remaining horseradish peroxidase concentration in spectrophotometer detection adsorption liquid, calculates supported quantity.
The supported quantity of horseradish peroxidase（Q）It can be calculated with following formula：
Q= (m 1- m 2)/m (mg·g-1)
In formula,m 1 It indicates that protein content in enzyme solution is added（mg）,m 2 Indicate protein content in remaining supernatant（mg）,m Indicate carrier
The quality of material（g）.
Embodiment 5：The preparation of immobilized HRP
（1）Solution is prepared
The preparation of phosphate buffer solution：Prepare 0.2 M Na2HPO4Solution and with 0.2 M, 100 mL NaH2PO4Adjust pH to
7.0。
（2）The immobilization process of horseradish peroxidase
The 110 mg composite magnetic nanoparticles synthesized are mixed with the NiCl of 13 ml, 0.2 M, are stirred at 25 DEG C anti-
It should make Ni2+It is fixed by the N atoms on AIM imidazole rings.Magneto separate salvage material later, washing and drying is overnight, compound by what is obtained
Magnetic Nano microsphere is configured to certain density suspension, and by concentration certain horseradish peroxidase solution and carrier material
Solution mixing, after mixing ensure solution carrier material concentration be 0.4 mgmL-1, horseradish peroxidase a concentration of 0.004
mg·mL-1 .System is in room temperature（25 °C）Lower incubation a period of time, the carrier material after immobilization is detached with permanent magnet, is retained
Supernatant is to be measured.With phosphate buffer solution flushing material twice to remove loose horseradish peroxidase.Using it is ultraviolet/can
See remaining horseradish peroxidase concentration in spectrophotometer detection adsorption liquid, calculates supported quantity.
The supported quantity of horseradish peroxidase（Q）It can be calculated with following formula：
Q= (m 1- m 2)/m (mg·g-1)
In formula,m 1 It indicates that protein content in enzyme solution is added（mg）,m 2 Indicate protein content in remaining supernatant（mg）,m Indicate carrier
The quality of material（g）.
Embodiment 6：The preparation of immobilized HRP
（1）Solution is prepared
The preparation of phosphate buffer solution：Prepare 0.2 M Na2HPO4Solution and with 0.2 M, 100 mL NaH2PO4Adjust pH to
7.0。
（2）The immobilization process of horseradish peroxidase
The 100 mg composite magnetic nanoparticles synthesized are mixed with the NiCl of 10 ml, 0.2 M, are stirred at 25 DEG C anti-
It should make Ni2+It is fixed by the N atoms on AIM imidazole rings.Magneto separate salvage material later, washing and drying is overnight, compound by what is obtained
Magnetic Nano microsphere is configured to certain density suspension, and by concentration certain horseradish peroxidase solution and carrier material
Solution mixing, after mixing ensure solution carrier material concentration be 0.4 mgmL-1, horseradish peroxidase a concentration of 0.004
mg·mL-1 .System is in room temperature（25 °C）Lower incubation a period of time, the carrier material after immobilization is detached with permanent magnet, is retained
Supernatant is to be measured.With phosphate buffer solution flushing material twice to remove loose horseradish peroxidase.Using it is ultraviolet/can
See remaining horseradish peroxidase concentration in spectrophotometer detection adsorption liquid, calculates supported quantity.
The supported quantity of horseradish peroxidase（Q）It can be calculated with following formula：
Q= (m 1- m 2)/m (mg·g-1)
In formula,m 1 It indicates that protein content in enzyme solution is added（mg）,m 2 Indicate protein content in remaining supernatant（mg）,m Indicate carrier
The quality of material（g）.
Embodiment 7：The immobilization effect of the horseradish peroxidase prepared under different condition
（1）Influence of the crosslinking agent MBAA contents to enzyme immobilization under different pH value
The pH that common phenyl boric acid is combined with the molecule containing cis- o-dihydroxy is generally relatively high, this is to horseradish peroxidase
Activity have bigger influence, therefore, it is monomer to select Wulff type phenyl boric acids AABOB, as shown in figure 5, intramolecular B-O
Bond energy enough effectively reduces most suitable in conjunction with pH, this can enable material more effectively to apply in biosystem.Such as institute in Fig. 6
Show, due to used in synthesis Wulff type phenyl boric acids AABOB be monomer, immobilized HRP it is fixed most
Good pH is 6, this is beneficial for maintaining the vigor of enzyme.It can also be found when the amount of crosslinking agent reaches 40mg, to peppery from Fig. 6
The fixation of root peroxidase has reached highest.This is because when content of crosslinking agent is very few, it can not be effectively by monomer AABOB
It is aggregated in magnetic ball surface, it is weaker to the immobilization ability of horseradish peroxidase；And when content of crosslinking agent is excessively high, it can make
The polymeric layer for obtaining magnetic ball surface is excessively fine and close, and steric hindrance is larger, and effectively object cannot be fixed.
（2）Ratio between AABOB and AIM ligands and Ni2+Modify the influence to enzyme immobilization
As can be seen from Figure 7 pass through Ni2+The carrier of modification will be significantly larger than by Ni2+The carrier of modification is to horseradish peroxidating
The supported quantity of object enzyme, to Ni2+Synergistic effect between AABOB can significantly improve the supported quantity of horseradish peroxidase.
When synthesizing magnetic material, the ratio (gross mass 100mg) being added between monomer AABOB and monomer AIM becomes influence horseradish mistake
One key factor of oxide enzyme crystallized ability.As shown in fig. 7, when the ratio between monomer AABOB and AIM reaches 3:When 2,
The magnetic Nano microsphere Fe of synthesis3O4@p (AABOB-co-AIM) reach maximum to the supported quantity of horseradish peroxidase, are
174.75 mg g-1.Opposite, the Fe synthesized with single monomer3O4@AIM and Fe3O4@AABOB magnetic Nano microspheres are to peppery
The fixed amount of root peroxidase is only 139.54 mg g-1 and 131.28 mg g-1 , adequately demonstrate both lists
The importance of bluk recombination.
（3）The circulation ability of immobilized HRP prepared by the ratio between different AABOB and AIM
It is 100mg to keep monomer AABOB and monomer AIM gross masses, and changes the amount ratio between them, after 8 cycles,
As shown in figure 8, curve a（100 % AABOB）With curve f（0 % AABOB）The opposite enzyme activity of representative immobilised enzymes is only
20 % and 30 % of initial enzyme activity.These are statistics indicate that by the synthesized immobilized HRP of single monomer effect
Recycling ability it is weaker, in recycled for multiple times enzyme activity loss it is larger.Likewise, present invention discover that curve c was represented
The rate that the enzyme activity of immobilised enzymes reduces will be significantly lower than other immobilised enzymes, it is with respect to enzyme after recycling 8 times
Work can still reach 60%.This is the result shows that when the ratio between monomer AABOB and monomer AIM is 3:When 2, material is solid
Surely the recycling ability for changing horseradish peroxidase is most strong.The immobilized HRP synthesized in report document at present
Ability is relatively low recycling, and the enzyme activity highest after cycle 5 times is also only capable of reaching 62%, however what is synthesized in the present invention consolidates
Surely change horseradish peroxidase Fe3O4@p(AABOB-co-AIM)-Ni2+The enzyme activity of-HRP can reach 83% after recycling 5 times,
Only there was only 17% enzyme activity loss, it is prominent to prove its excellent performance.
Embodiment 8：The zymologic property of immobilized HRP
The present invention is studied and has been measured to the zymologic property of resolvase and obtained immobilised enzymes respectively, and the result of measurement is such as
Shown in Fig. 9.It is found from Fig. 9 a, the optimum temperature of resolvase and immobilised enzymes enzyme activity is 50 DEG C, when temperature range is arrived 20
When between 60 DEG C, immobilised enzymes shows better stability and activity.Such as at 30 °C, the opposite enzyme activity energy of immobilised enzymes
Enough reach 90 %, however the enzyme activity of resolvase only only has 77 %.Fig. 9 b show the opposite enzyme for the immobilised enzymes that the present invention synthesizes
Living to reach highest when pH is 6.5, being compared with the optimal pH of free horseradish peroxidase has certain reduction.It will synthesis
Immobilised enzymes and resolvase store the different time in the environment of 4 DEG C, then under optimum temperature and suitable environment（50
DEG C, pH 6.5）The sample that these have been stored is used for the measurement of enzyme activity.From in Fig. 9 c it can be found that storage 30 days after, Gu
Surely the opposite enzyme activity of change horseradish peroxidase, which is compared for 82.7 % with the enzyme activity of 53.1 % of resolvase, increases significantly.
After storage reaches 50 days, free horseradish peroxidase largely inactivates, and opposite enzyme activity only has 30.9 %, however fixed
The enzyme activity for changing enzyme still keeps preferable, and enzyme activity reaches 71.1 %, this series of result all shows free horseradish peroxidating
Its storage-stable has preferable promotion to object enzyme after fixation.
Embodiment 9：Immobilized HRP degradation of phenol effect
As shown in Figure 10, pure ferroso-ferric oxide composite magnetic microballoon Fe3O4@P(AABOB-co-AIM) -Ni2+The drop of Pyrogentisinic Acid
Solution ability is very low, is not achieved 20%, this is because there is only very weak catalytic capabilities and certain object by magnetic Nano microsphere Pyrogentisinic Acid
Adsorption capacity is managed, the ability of degradation of phenol itself is very low.Opposite, free horseradish peroxidase Pyrogentisinic Acid has certain urge
Change degradation capability, the maximum catalysis degradation modulus of Pyrogentisinic Acid can reach 50% in 20 minutes, but be a lack of the energy of recycling
Power.However, found when investigating the degradation capability of immobilized HRP Pyrogentisinic Acid, it can be in 10min energy
Phenol in effective degraded solutions, the degradation rate of phenol have reached 93%.These are the result shows that magnetic Nano bead and horseradish mistake
There is apparent synergistic effect between oxide enzyme, effectively raises immobilized HRP entirety degradation of phenol
Ability, the material have good actual application ability.
Claims (10)
1. a kind of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles（Fe3O4@P (AABOB-co-AIM) magnetic Nano is micro-
Ball）, which is characterized in that the microsphere diameter is 150 nm, is integrally uniformly dispersed and has superparamagnetism, can be under externally-applied magnetic field
Realize quick separating.
2. a kind of preparation method of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles, which is characterized in that according to as follows
Method carries out：
Weigh a certain amount of Fe3O4In there-necked flask a certain amount of acetonitrile is added, ultrasonic disperse to bottom is without apparent in@MPS microballoons
Precipitation, backward system in be added a certain amount of AABOB, a certain amount of N- allyl imidazoles, a certain amount of crosslinking agent MBAA and
Initiator azodiisobutyronitrile；Heat initiated polymerization, flask is placed in oil bath device in reaction process, above connect still,
Condenser pipe and reception device；Temperature is adjusted in reaction process to be made system boil within a certain period of time and acetonitrile is made to start to condense,
Stop polymerization at this time, removes oil bath device and be cooled to room temperature；Gained particle is washed with absolute ethyl alcohol repeatedly, washes away extra monomer
And oligomer dries product vacuum until after supernatant liquid clarification.
3. a kind of preparation method of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles according to claim 2,
It is characterized in that, the Fe3O4@MPS microballoon dosages are 0.03-0.07 g.
4. a kind of preparation method of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles according to claim 2,
It is characterized in that, the AABOB additions are 50-70 mg；
The AIM additions are 20-60 mg；
The crosslinking agent MBAA additions are 30-50 mg；
The dosage of initiator azodiisobutyronitrile is 10mg.
5. a kind of preparation method of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles according to claim 2,
It is characterized in that, the boiling time is in 0.5h；It is described stop polymerization opportunity in order to control 1 h or so time collect about 20
ML acetonitriles.
6. a kind of preparation method of Wulff types phenyl boric acid AABOB functional magnetic nanoparticles according to claim 2,
It is characterized in that, the drying condition is dry 24 h in 45 DEG C of vacuum drying chambers.
7. Wulff types phenyl boric acid AABOB functional magnetic nanoparticles described in claim 1（Fe3O4@P(AABOB-co-AIM)
Magnetic Nano microsphere）Immobilization for horseradish peroxidase.
8. the Wulff type phenyl boric acid AABOB functional magnetic nanoparticles described in claim 7 are for horseradish peroxidase
The specific method of immobilization is：
By a certain amount of composite magnetic nanoparticle（Fe3O4@p(AABOB-co-AIM)）It is mixed with the NiCl of certain volume,
Being stirred to react at 25 DEG C makes Ni2+It is fixed by the N atoms on AIM imidazole rings；Magneto separate salvage material later, washing and drying mistake
Night obtains Ni2+With Wulff type phenyl boric acids AABOB synergistic effect functional magnetic nanoparticle magnetic Nano microspheres；By the microballoon
It is configured to certain density suspension, and the solution of certain density horseradish peroxidase solution and carrier material is mixed into
Row enzyme immobilizatio.
9. a kind of horseradish peroxidase of immobilization, which is characterized in that the horseradish peroxidase of the immobilization is by right
It is required that the Ni described in 82+Magnetic Nano microsphere immobilization with Wulff type phenyl boric acid AABOB functionalization obtains.
10. the horseradish peroxidase of the immobilization described in claim 9 is for the degradation to pollutant phenol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269365A (en) * | 2019-04-15 | 2020-06-12 | 中国农业科学院农业质量标准与检测技术研究所 | Temperature-sensitive Wulff type boron affinity nano-particles and preparation method and application thereof |
| CN113214440A (en) * | 2021-04-30 | 2021-08-06 | 江苏大学 | UCST type nonionic water-soluble polymer and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558463A (en) * | 2012-01-04 | 2012-07-11 | 福州大学 | One-pot synthesis phenylboronic acid polymer magnetic nano composite material and preparation method and application thereof |
| JP2015057956A (en) * | 2013-09-17 | 2015-03-30 | 学校法人上智学院 | Structure and bacterium collection and detection method using the same |
| CN104841403A (en) * | 2015-05-06 | 2015-08-19 | 西北大学 | High-capacity boron affinity separation material and preparation method and application thereof |
| CN107418950A (en) * | 2017-08-30 | 2017-12-01 | 江苏大学 | Multi-arm magnetic composite microsphere immobilized HRP and its preparation method and application |
-
2018
- 2018-03-30 CN CN201810291359.7A patent/CN108569772A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558463A (en) * | 2012-01-04 | 2012-07-11 | 福州大学 | One-pot synthesis phenylboronic acid polymer magnetic nano composite material and preparation method and application thereof |
| JP2015057956A (en) * | 2013-09-17 | 2015-03-30 | 学校法人上智学院 | Structure and bacterium collection and detection method using the same |
| CN104841403A (en) * | 2015-05-06 | 2015-08-19 | 西北大学 | High-capacity boron affinity separation material and preparation method and application thereof |
| CN107418950A (en) * | 2017-08-30 | 2017-12-01 | 江苏大学 | Multi-arm magnetic composite microsphere immobilized HRP and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| XIHAO ZHANG等: "Tailor-Made Boronic Acid Functionalized Magnetic Nanoparticles with a Tunable Polymer Shell-Assisted for the Selective Enrichment of Glycoproteins/Glycopeptides", 《ACS APPL. MATER. INTERFACES》 * |
| YUTING ZHANG等: "Fe3O4/PVIM-Ni2+ Magnetic Composite Microspheres for Highly Specific Separation of Histidine-Rich Proteins", 《ACS APPL. MATER. INTERFACES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269365A (en) * | 2019-04-15 | 2020-06-12 | 中国农业科学院农业质量标准与检测技术研究所 | Temperature-sensitive Wulff type boron affinity nano-particles and preparation method and application thereof |
| CN113214440A (en) * | 2021-04-30 | 2021-08-06 | 江苏大学 | UCST type nonionic water-soluble polymer and preparation method and application thereof |
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Application publication date: 20180925 |