CN107988199A - A kind of acetylcholine ester enzyme immobilization carrier and its preparation method and application - Google Patents
A kind of acetylcholine ester enzyme immobilization carrier and its preparation method and application Download PDFInfo
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- C12Y301/01007—Acetylcholinesterase (3.1.1.7)
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Abstract
The invention discloses a kind of acetylcholine ester enzyme immobilization carrier, it is characterised in that the representation of the fixation support is Fe3O4@SiO2‑NH2, it includes the magnetic Fe of spherical spinel structure3O4Nano-particle, particle size are 15 20 nm, are coated on the magnetic Fe3O4The silica of nanoparticle surface, and modification is in Fe3O4@SiO2The amino group of nanoparticle surface, the amino mass percentage are 3.5 4.5%.The magnetic Fe that the present invention is prepared using coprecipitation3O4Nano particle diameter is in 15 20 nm, and superparamagnetism is good, and particle size is uniform.After modified, Fe3O4@SiO2‑NH2Uniform particle diameter is good, and amino content is more, maintains magnetic Fe3O4The structure of nano-particle, and there is good magnetic performance, it can be separated and recovered using magnetic force.The present invention utilizes Fe3O4@SiO2‑NH2Microsphere immobilized carrier immobilized acetylcholinesterase has the high rate of recovery of comparison, and excellent in stability, can reuse and easily store.
Description
Technical field
The present invention relates to a kind of magnetic Nano material for enzyme immobilization carrier, and in particular to one kind is used for acetylcholine
The magnetic Fe of esterase immobilization3O4The application of nanoparticulate carriers and preparation method thereof and immobilised enzymes, belongs to new material preparation
Technical field.
Background technology
Acetylcholinesterase（Acetylcholinesterase, AChE）It is a kind of energy selective degradation（Pass through its hydrolysis
Catalytic activity）Neurotransmitter acetylcholine becomes the enzyme of choline and caproic acid, it has the activity of carboxypeptidase and aminopeptidase, can terminate
Neurotransmitter is to the stimulation of postsynaptic membrane, so as to ensure the normal transmission of nerve signal in vivo.Acetylcholine ester
Enzyme is present in vertebrate cholinergic nerve section, nematode and invertebrate neuromuscular junction, it is also insect god at the same time
Through a kind of important enzyme in conductive process, it is the action target of many never poisons, can makes when acetylcholinesterase is suppressed
Organism produces lethal effect, therefore it is a kind of important target enzymes.
Enzyme is as a kind of catalytic activity albumen, and stability is poor, after departing from cellular environment, in temperature, pH and inorganic ions
Under the action of external factor, activity will be badly damaged or even inactivate.Additionally due to the water solubility of enzyme is stronger, in aqueous
Can cause the separation of enzyme, substrate and product difficult during reaction, though after catalytic process part zymoprotein molecule still have compared with
High activity, it is also difficult to separated and recovered again.Enzyme immobilization technology（immobilization of enzymes）Appearance it is thorough
Solve the problems, such as these.Enzyme immobilization is to fetter enzyme with solid material or be limited in certain area, it is can still provide for spy
Some catalytic reactions, and recyclable and recycling a kind of technology, are one of the research hotspots in modern enzyme engineering field.It is fixed
Change enzyme has the advantages that storage stability is high, conveniently recycling, reuse and operation are continuous controllable compared with resolvase.At present,
Enzyme immobilization technology oneself in Textile Engineering, bioengineering, food industry, fine chemistry industry, medicine, life science and environmental science etc.
Field is widely used.
Research report in recent years on immobilization acetylcholinesteraseelectrochemistry is more, mainly fixes acetylcholinesterase
Change, carry out the development of biology sensor, fixing means is also relatively easy（Chen Junhui, Shi Qian, Chen Chen, wait immobilization acetyl
The preparation of cholinesterase and its characteristic research [J] chemistry journals, 2012,70 (5):624-628；Marinov I,
Gabrovska K, Velichkova J, et al. Immobilization of acetylcholinesterase on
nanostructure polyacrylonitrile membranes.[J]. International Journal of
Biological Macromolecules, 2009, 44(4):338-45；Jeyapragasam T, Saraswathi R.
Electrochemical biosensing of carbofuran based on acetylcholinesterase
immobilized onto iron oxide–chitosan nanocomposite[J]. Sensors & Actuators B
Chemical, 2014, 191(6-7):681-687.）.The special magnetism of magnetic nano-particle, for being recycled for enzyme
With other materials without superiority, but magnetic nano-particle is used to fix the rarely seen report of research of acetylcholinesterase at present
Road, and the method for having studied immobilised enzymes is relatively easy, the magnetic nano-particle ferroso-ferric oxide used is mostly finished product, and four
Fe 3 O is extremely easily denatured because of oxidation.And since ferroferric oxide magnetic nano-particles are more active, bad progress
Utilize, so needing to reduce the surface energy of particle by surface modification, so as to improve its stability and reliability.
How to ensure high efficiency, high activity and high stability after acetylcholine ester enzyme immobilization, be immobilization acetyl
The key and bottleneck of cholinesterase.In view of the method for immobilised enzymes still has various deficiencies at present, acetylcholinesterase is existing
Process for fixation is not met by the demand of practical application.Therefore, constantly propose and improve existing immobilization acetylcholinesteraseelectrochemistry
The technology of performance is extremely urgent.
The content of the invention
There is good magnetic performance, the use that can be separated and recovered using magnetic force the object of the present invention is to provide a kind of
In the magnetic Fe of acetylcholine ester enzyme immobilization carrier3O4Nano-particle, in the magnetism and nanometer particle size for ensureing to keep it good
Under the premise of, being modified by surface reduces its surface energy, reliable and stable magnetic nano-particle is obtained, for immobilization acetylcholine
So as to have the high rate of recovery, excellent in stability, can reuse and easily store esterase.
It is a further object to provide the preparation method of above-mentioned acetylcholine ester enzyme immobilization carrier.
Third object of the present invention is to provide the magnetic Fe of above method preparation3O4Nano-particle is in immobilization acetyl courage
Application on alkali esterase.
To reach above-mentioned purpose, the concrete technical scheme that the present invention takes is：
A kind of acetylcholine ester enzyme immobilization carrier, it is characterised in that the representation of the fixation support is Fe3O4@
SiO2-NH2, it includes the magnetic Fe of spherical spinel structure3O4Nano-particle, particle size are 15-20 nm, are coated on described
Magnetic Fe3O4The silica of nanoparticle surface, and modification is in Fe3O4@SiO2The amino group of nanoparticle surface, it is described
Amino mass percentage is 3.5-4.5%.
The preparation method of above-mentioned acetylcholine ester enzyme immobilization carrier, comprises the following steps：
（1）Prepare magnetic Fe3O4Nano-particle：Prepared, molysite and ferrous salt are mixed, in alkaline solution bar using coprecipitation
Under part, the magnetic Fe of homogeneous black powder is obtained3O4Nano-particle（MNP）；
（2）Magnetic Fe3O4The silanization of nano-particle：By magnetic Fe obtained above3O4Magnetic nano-particle mixes, and adds nothing
Water-ethanol and distilled water, while under nitrogen protection, stir it is uniform to system, then ultrasound, stir and ammonium hydroxide be added dropwise, until pH
Value is adjusted to 9, and ethyl orthosilicate is added under ultrasound（TEOS）, react and obtain the solution of brown, carry out Magnetic Isolation, clean,
Rotary evaporation, vacuum drying obtain the Fe of the powdered silanization of brown3O4Magnetic nano-particle Fe3O4@SiO2（SMNP）；
（3）Amino coats：By step（2）Obtained SMNP is added into toluene, and ultrasound is uniform to system, then under ultrasound
Ammonium hydroxide is slowly added to, will be carried out in whole process under nitrogen protection, be vigorously stirred lower addition 3- aminopropyl triethoxysilanes
（APTES）The lower reaction of ultrasound, carries out Magnetic Isolation, is dried in vacuo after cleaning, that is, obtains the magnetic Fe of black particle shape3O4@
SiO2-NH2Nanoparticle fixation support.
The magnetic Fe3O4Nanoparticle fixation support is used for the application of immobilization acetylcholinesteraseelectrochemistry.
Above-mentioned acetylcholine ester enzyme immobilization carrier be used for immobilization acetylcholinesteraseelectrochemistry method be：By magnetic Fe3O4@
SiO2-NH2Nanoparticle carrier is added in acetylcholinesterase protein liquid, is started concussion and is shaken, is isolated product with magnetic force
Come, you being fixed acetylcholinesterase.
Further, in the method for above-mentioned immobilised enzymes, the optimal conditions of immobilised enzymes is：Immobilization temperature is 35 DEG C,
Zymoprotein concentration is 1.5 U/mL, and immobilization system pH is 8.0, and the immobilization time is 12 h；The optimal conditions of enzyme activity is：It is fixed
It is 30 DEG C to change temperature, and zymoprotein concentration is 1.0 U/mL, and immobilization system pH is 8.0, and the immobilization time is 12 h.
Advantages of the present invention and technique effect：The magnetic Fe that the present invention is prepared using coprecipitation3O4Nano particle diameter
In 15-20 nm, superparamagnetism is good, and particle size is uniform.After modified, Fe3O4@SiO2-NH2Uniform particle diameter is good, amino
Content is more, maintains magnetic Fe3O4The structure of nano-particle, and there is good magnetic performance, it can be separated using magnetic force
Recycling.
The present invention utilizes Fe3O4@SiO2-NH2Microsphere immobilized carrier immobilized acetylcholinesterase has the high recycling of comparison
Rate, excellent in stability, can reuse and easily store.
Brief description of the drawings
Fig. 1 is that the TEM of different magnetic nano-particles schemes.
Fig. 2 is different magnetic nano-particle XRD spectras.
Fig. 3 is the infrared spectrogram of different magnetic nano-particles.
Fig. 4 is the hysteresis curve figure of different magnetic nano-particles.
Fig. 5 is the thermogravimetric analysis figure of different magnetic nano-particles.
Fig. 6 is influence result figure of the different protein concentrations to immobilised enzymes adsorption rate.
Fig. 7 is influence result figure of the different protease concentrations to immobilized enzyme.
Fig. 8 is influence result figure of the immobilization temperature to immobilized enzyme adsorption rate.
Fig. 9 is influence result figure of the immobilization temperature to immobilized enzyme.
Figure 10 is influence result figures of the system pH to immobilized enzyme adsorption rate.
Figure 11 is influence result figures of the system pH to immobilized enzyme.
Figure 12 is influence result figure of the immobilization time to immobilized enzyme adsorption rate.
Figure 13 is influence result figure of the immobilization time to immobilized enzyme.
Embodiment
It is explained further below in conjunction with attached drawing and by specific embodiment and illustrates the present invention.
Embodiment 1：Magnetic Fe3O4@SiO2-NH2The preparation of nanoparticle fixation support
（1）Prepare magnetic Fe3O4Nano-particle：First, under nitrogen protection, the distilled water of 100 mL is boiled to eliminate oxygen
Gas；Then 4.0 g FeCl are accurately weighed2·4H2O and 10.8 g FeCl3·6H2O slowly adds under nitrogen protection, by medicine
Enter；Heating, to temperature to seeing whether after 80 DEG C to produce black even solution, if producing dark solution, is slowly dropped into 50 mL
Ammonium hydroxide, continues to heat 20 min afterwards；After reaction, product sealing is placed on magnet and carries out Magnetic Isolation.After separation
Product distilled water flushing 5-6 times, until washing neutrality, is then washed 2-3 time with absolute ethyl alcohol again, finally by solution with rotating instrument
It is evaporated, obtains the magnetic nano-particle of homogeneous black powder, is dried in vacuo 24 hours, obtains uniform black powder
Magnetic Fe3O4Nano-particle（Abbreviation MNP）.
（2）Silanization：Weigh 1 g MNP to be put into 250 mL two-mouth bottles, 70 mL absolute ethyl alcohols and 30 are added in bottle
ML distilled water, while nitrogen protection is provided, 10 min are stirred, until system is uniform, then after 15 min of ultrasound, start to stir simultaneously
Ammonium hydroxide is added dropwise, until pH value is adjusted to 9, this process will carry out under nitrogen protection.40 DEG C are warming up to, then adds 2 mL
TEOS, reacts 4 h under ultrasound condition, obtains the solution of brown, carries out Magnetic Isolation, is washed with water 5-6 times, then washed with absolute ethyl alcohol
2 times, rotary evaporation is carried out with rotary evaporator, then is dried in vacuo to obtain the Fe of the silanization of brown powder3O4Magnetism is received
Rice corpuscles（SMNP）.
（3）Amino coats：Weigh 0.1 g SMNP to be added in 50 mL toluene, 10 min of ultrasound are uniform to system, then
2.0 mL ammonium hydroxide are slowly added under ultrasound, nitrogen protection will be carried out in whole process, are vigorously stirred 2.0 mL of lower addition
When the lower reaction 24 of APTES ultrasounds is small.Magnetic Isolation is carried out, is first washed with water 5-7 times, then is washed 3 times with absolute ethyl alcohol, vacuum is done
Dry 24 h, obtains black particle shape Fe3O4@SiO2-NH2Nanoparticle fixation support.
（4）Interpretation of result：
（I）The tem analysis of magnetic nano-particle
As shown in Figure 1, wherein (a) is Fe3O4Nano-particle,（b）For Fe3O4@SiO2Nano-particle,（c）For Fe3O4@SiO2-
NH2Nano-particle,（a）（b）（c）Three kinds of Nanoparticle shapes are spherical, and particle size is about 15-20 nm.Due to prepared
Fe3O4For the particle diameter of nano-particle all in nanoscale, influence of the modified functional group to particle diameter is little, is provided for immobilised enzymes
Condition.It can be seen from the figure that（b）With（c）In nano-particle there is the phenomenon wound, do not have（a）In nano-particle that
It is scattered, illustrate modification success.
（II）The XRD spectrum analysis of magnetic nano-particle
As seen from Figure 2, ungroomed Fe3O42 θ of diffraction spectral peak of nano-particle is 35.30O、42.11O、55.23O、
62.12O, do not occur other miscellaneous peak in figure, illustrate Fe3O4Nano-particle is purer spinel structure.Fe3O4@SiO2Nanometer
2 θ of diffraction spectral peak of particle is 35.12O、41.98O、55.01O、62.01O, Fe3O4@SiO2-NH22 θ of diffraction spectral peak of nano-particle
It is 35.13O、42.09O、55.20O、62.11O。Fe3O4@SiO2、Fe3O4@SiO2-NH2Nano-carrier except with Fe3O4Present
There are some particularly weaker peaks outside characteristic peak, pass through comparing result, when finding modified magnetic nano particles, Fe3O4Crystal
Structure change it is little, other Fe3O4@SiO2、Fe3O4@SiO2-NH2There is the diffraction maximum of some widthization in nano material, and intensity is not
Greatly, this is because caused by modification of surfaces activating agent.
（III）Magnetic nano-particle infrared spectrum analysis
As shown in figure 3, figure（a）It can be seen that in Fe3O4The stretching vibration absworption peak of middle Fe-O singly-bounds appears in wave number 572.64
cm-1Left and right, with Fe3O4The stretching vibration absorption frequency of the Fe-O keys of nano-particle is basically identical；Figure（b）、（c）In 802.30
cm-1With 1092.60 cm-1Place corresponds to the stretching vibration absworption peak of the symmetrical keys of O-Si-O and the stretching vibration of asymmetric key respectively
Absworption peak, 464.54 cm-1It is the flexural vibrations absworption peak of Si-O-Si keys, 954.70 cm-1It is the absorption of vibrations of Si-OH keys
Peak.Silica is successfully coated on magnetic nano-particle it can be seen from infrared test result.
Figure（c）In 3960.04 cm-1It is the stretching vibration absworption peak of N-H singly-bounds, 1656.86 cm-1With 1516.46 cm-1
It is-CH2With-CH3Flexural vibrations absworption peak, this is due to Fe3O4@SiO2-NH2Contain substantial amounts of-CH in nano-particle2Group；
1091.20 cm-1It is the stretching vibration absworption peak of C-N singly-bounds, surface stretching vibration absworption peak of Fe-O singly-bounds after modification goes out
Present 586.54 cm-1Place, with the ungroomed Fe in surface3O4Comparatively speaking, the stretching vibration absworption peak wave number of Fe-O singly-bounds
There occurs obvious blue shift, mainly due to APTES and magnetic Fe the reason for blue shift3O4The surface of nano-particle there occurs
Some chemical reactions, reaction generate Fe-O-Si keys.Pass through the electrostatic force inducing action between them so that the bonding force of Fe-O
Constant increases, the group of the Fe-O that can make can frequency can be moved to high wave number, by comparison above can be seen that amino into
The modification of work(is to magnetic Fe3O4On the surface of nano-particle.
（IV）The magnetic property analysis of magnetic nano-particle
Fig. 4 is the hysteresis curve of different magnetic nano-particles.It will be clear that surface modified front and rear magnetic from figure
The coercivity and remanent magnetization of property nano-particle are all almost nil.These nano-particles all show relatively good superparamagnetic
Property, Fe3O4Nano-particle, Fe3O4@SiO2Nano-particle and Fe3O4@SiO2-NH2The saturation magnetization of nano-particle is respectively
The reason for 60.11,38.85,49.89 emu/g, intensity is different is that different functional groups has been coated on the surface of nano-particle.
After amino group is coated, although magnetic weaken, magnetic property is still good, can be separated under the action of externally-applied magnetic field.Magnetic
The SiO of the cladding of property nanoparticle surface2Make that diamagnetism can be shown in its magnetic field, this is the saturated magnetization of magnetic nano-particle
The reason for intensity declines, with single magnetic nano-particle compared with having intuitively in terms of magnetism.In addition, functional group all has
Quality, therefore will also result in the decline of saturation magnetization.
（V）The thermogravimetric analysis of magnetic nano-particle
Fe it can be seen from Fig. 53O4Nano-particle total weight loss is about 4.3 %, Fe3O4@SiO2Nano-particle total weight loss is about
7.5%, Fe3O4@SiO2-NH2Nano-particle total weight loss is about 9.8%.There is a small amount of solvent volatilization to cause one before wherein 100 DEG C
Part is weightless, and between obvious weightlessness appears in 100-200 DEG C for the first time, this part is probably a small amount of water and partial solvent
Mass loss caused by volatilization；Between second of weightlessness appears in 200-700 DEG C, this part weightlessness is probably functional group
Caused by decomposing or volatilizing.After higher than 700 DEG C, thermogravimetric curve afterbody slightly raises up, it is meant that the quality of nano-particle
It increased, this is Fe3O4To α-Fe2O3The process of transformation.The weightlessness of three kinds of particles is incremented by successively, this is because below
Both of which is to continue the process of cladding on the basis of the former.
Embodiment 2：The preparation of immobilised enzymes
Accurately weigh 10 mg magnetic Fes3O4@SiO2-NH2Nanoparticle adds not in 10 mL centrifuge tubes into centrifuge tube
With zymoprotein concentration（0.25、0.5、0.75、1、1.25、1.5、2.0 U/mL）, difference pH 2 mL of lipase solution, if
Condition has been put, then has started to shake with shaking table, product is separated with magnet, obtains supernatant, for calculating protein content,
The vigor, brought calculate immobilised enzymes several times is cleaned again.
1st, the supernatant after immobilised enzymes and cleaning solution are collected together survey absorbance and seek adsorption rate.
The preparation of bovine serum albumin standard protein standard curve：
1）0.9 %NaCl solution：Claim 9 g sodium chloride, pour into the beaker for filling 200 mL distilled water, be chlorination by stirring
Sodium dissolves, and then carries out constant volume with the volumetric flask of 1000 mL.Bovine serum albumin standard protein liquid：Accurately weigh bovine serum albumin
White 0.1 g, pours into the beaker for filling 200 mL sodium chloride solutions, then carries out constant volume with the volumetric flask of 1000 mL.Coomassie
Light blue G-250 dyes liquid making method：0.1 g of Coomassie brilliant G-250 is weighed, pours into the burning for filling 95 % ethanol of 50mL
In cup, by stirring, Coomassie brilliant blue is dissolved, then add the phosphoric acid of 100 mL, 85 %, finally with the volumetric flask of 1000 mL
Constant volume is carried out, is preserved at 4 DEG C.
2）Sequentially added into 10 mL centrifuge tubes by the solution of 3 rows before table 3.It is uniformly mixed, reacts 5 minutes, surveys and inhale
Luminosity, records data.
3）According to data creating standard curve, calculation formula is obtained.Acetylcholinesterase is inhaled for magnetic nano-particle
The calculating of attached rate.Magnet adsorption is used after immobilization, takes supernatant and cleaning solution to survey absorbance, the absorbance measured is brought into
Fitting formula, so as to obtain corresponding protein concentration, according to concentration and molten night volume, obtains the quality of loose enzyme；
The quality for the enzyme being initially added into again with experiment, obtains adsorption rate.
The calculation formula of adsorption rate：
Adsorption rate（Mg/g carriers）=（C1V1-C2V2）/W.
2nd, Acetylcholinesterasein measure compares：
1）The determination of activity of resolvase：According to Ellman methods, in 1 cm cuvettes（It is recommended that use centrifuge tube）In sequentially add 3
MLPBS (0.02 mol/L, pH=7.5), 20 μ L enzyme liquids (1 U/mL), 100 μ lDTNB color developing agents (20 mmol/L) mix.
Add 20 μ LATCh substrate solutions (0.l mol/L) after 25 °C of 15 min of insulation, with the ultraviolet honourable luminosity of UV1102II after mixing
Meter colorimetric at 405 nm.Measure and record in continuous 3 min, every the reading of half a minute.According to internal absorbance in 3min
Changing value, that is, slope seeks the vigor of enzyme.
2) determination of activity of immobilization acetylcholinesteraseelectrochemistry：Immobilised enzymes made from a certain amount of embodiment is accurately weighed,
Reaction system absorbance to be not added with substrate, according to the determination of activity step of solution enzyme, is incited somebody to action as initial value in centrifuge tube
Itself and PBS and DTNB are mixed, and substrate A TCh solution is added after 25 °C of 15 min of insulation, are after mixing placed in cuvette point
In light photometer, the A of record reaction solution every 30 S in 3 min405Nm values.
The calculation formula of vitality test：
Activity recovery=（The total activity of the vigor of immobilised enzymes/addition enzyme liquid）×100%
Relative activity=（Highest enzyme activity force value in the enzyme activity force value/group in every group）×100%
3rd, the condition optimizing of immobilised enzymes
（1）Influence of the zymoprotein concentration to immobilization effect
Accurately weigh 10 mg amino nanoparticles（Fe3O4@SiO2-NH2）In 2 mL zymoprotein concentration be respectively 0.25,0.5,
0.75th, the acetylcholine ester enzyme solutions of 1.0,1.25,1.5,2.0 U/mL, react 10 h in 30 DEG C, 200 rpm shaking tables,
It has studied influence of the enzyme liquid of different protein concentrations to adsorption rate and enzyme activity.
In Fig. 6, protein concentration is continuously increased, and adsorption rate also gradually increases, and adsorption rate increase is rapid before 1.5 U/mL,
Gradually tend towards stability afterwards.In Fig. 7, as protease concentration constantly increases, enzyme rate of recovery first increases and then decreases, reaches in enzyme concentration
During to 1 U/mL, the enzyme rate of recovery reaches maximum, and the rate of recovery of immobilised enzymes is gradually reduced afterwards.
（2）Influence of the reaction temperature to immobilization effect
Accurately weigh 0.01 g amino nano-particles（Fe3O4@SiO2-NH2）In 20,25,30,35,40,50 DEG C of enzyme liquid
（Enzyme liquid protein concentration is 1.0 U/mL, and enzyme liquid volume is 2mL）, 10 h are reacted in shaking table, study different immobilization temperature
Influence to adsorption rate and immobilized enzyme.
In Fig. 8, at different temperature, with the continuous lifting of temperature, adsorption rate fluctuates up and down, is 35 DEG C in temperature
When, adsorption rate is maximum.In Fig. 9, the trend risen, when temperature reaches 30 DEG C, enzyme is presented in the continuous rise of temperature, enzyme activity
Vigor reaches maximum, and temperature continues to increase, and enzyme activity gradually reduces.It can be seen that high temperature can make enzyme lose activity.
（3）Influence of the reaction system pH values to immobilization effect
Accurately weigh 10 mg amino nano-particles（Fe3O4@SiO2-NH2）In pH value of reaction system be respectively 5.0,6.0,7.0,
8.0th, in 9.0,10.0 2mL enzyme liquids（Enzyme liquid protein concentration is 1.0 U/mL）, 10 h are reacted in shaking table（30 DEG C, 200
rpm）, study influence of the different system pHs to adsorption rate and immobilized enzyme.
In Figure 10, as pH becomes larger, adsorption rate first increases and then decreases, when pH is equal to 8, adsorption rate is maximum.In Figure 11, with
The increase of pH, enzyme activity first increases and then decreases, when pH value is 8, enzyme activity is maximum.
（4）Influence of the immobilization time to immobilization effect
Accurately weigh 10 mg amino nano-particles（Fe3O4@SiO2-NH2）It is in the reaction time（Rotating speed is set to 300 rpm, and 30
℃）4th, in 8,12,16,24,32 h enzyme liquids（Enzyme liquid protein concentration is 1.0 U/mL, and enzyme liquid volume is 2mL）Respectively at shaking table
The middle influence for measuring the different immobilization time respectively to adsorption rate and enzyme activity.
In Figure 12, adsorption rate increases over time, and becomes larger, after tend to be constant.In Figure 13, with the immobilization time
Increase, enzyme activity be continuously improved, in the time after 12 h, enzyme activity slowly reduces.
Conclusion：Fe3O4@SiO2-NH2Shadow of the nanoparticle fixation support immobilization acetylcholinesteraseelectrochemistry by different condition
Ring, wherein zymoprotein concentration and system pH have a great influence immobilization effect.Result of study shows, the optimal bar of immobilised enzymes
Part is：Immobilization temperature is 35 DEG C, and zymoprotein concentration is 1.5 U/mL, and immobilization system pH is 8.0, and the immobilization time is 12
h；The optimal conditions of enzyme activity is：Immobilization temperature is 30 DEG C, and zymoprotein concentration is 1.0 U/mL, and immobilization system pH is 8.0,
The immobilization time is 12 h.Immobilization acetylcholinesteraseelectrochemistry prepared by experiment has a high rate of recovery of comparison, excellent in stability, can be with
Recycling and easily storage.
Claims (5)
1. a kind of acetylcholine ester enzyme immobilization carrier, it is characterised in that the representation of the fixation support is Fe3O4@
SiO2-NH2, it includes the magnetic Fe of spherical spinel structure3O4Nano-particle, particle size are 15-20 nm, are coated on described
Magnetic Fe3O4The silica of nanoparticle surface, and modification is in Fe3O4@SiO2The amino group of nanoparticle surface, it is described
Amino mass percentage is 3.5-4.5%.
2. a kind of preparation method of the acetylcholine ester enzyme immobilization carrier described in claim 1, comprises the following steps：
（1）Prepare magnetic Fe3O4Nano-particle：Prepared, molysite and ferrous salt are mixed, in alkaline solution bar using coprecipitation
Under part, the magnetic Fe of homogeneous black powder is obtained3O4Nano-particle；
（2）Magnetic Fe3O4The silanization of nano-particle：By magnetic Fe obtained above3O4Magnetic nano-particle mixes, and adds anhydrous
Ethanol and distilled water, while under nitrogen protection, stir it is uniform to system, then ultrasound, stir and ammonium hydroxide be added dropwise, until pH value
Adjusting to 9, ethyl orthosilicate is added under ultrasound, reaction obtains the solution of brown, carries out Magnetic Isolation, cleaning, rotary evaporation,
Vacuum drying obtains the Fe of the powdered silanization of brown3O4Magnetic nano-particle Fe3O4@SiO2；
（3）Amino coats：By step（2）The Fe of obtained silanization3O4Magnetic nano-particle is added into toluene, ultrasound to body
System is uniform, and ammonium hydroxide is then slowly added under ultrasound, will be carried out in whole process under nitrogen protection, be vigorously stirred lower addition 3-
The lower reaction of aminopropyl triethoxysilane ultrasound, carries out Magnetic Isolation, is dried in vacuo after cleaning, that is, obtains the magnetic of black particle shape
Property Fe3O4@SiO2-NH2Nanoparticle fixation support.
3. acetylcholine ester enzyme immobilization carrier as claimed in claim 1 is used for the application of immobilization acetylcholinesteraseelectrochemistry.
4. acetylcholine ester enzyme immobilization carrier as claimed in claim 1 is used for the method for immobilization acetylcholinesteraseelectrochemistry, its
It is characterized in that including the following steps：By magnetic Fe3O4@SiO2-NH2Nanoparticle carrier is added in acetylcholinesterase protein liquid,
Start concussion to shake, separated product with magnetic force, you being fixed acetylcholinesterase.
5. method as claimed in claim 4, it is characterised in that the condition of the immobilization acetylcholinesteraseelectrochemistry is：Immobilization temperature
Spend for 35 DEG C, zymoprotein concentration is 1.5 U/mL, and immobilization system pH is 8.0, and the immobilization time is 12 h.
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| CN110849857A (en) * | 2019-12-03 | 2020-02-28 | 中国农业科学院农业质量标准与检测技术研究所 | Organophosphorus pesticide detection method based on magnetic nano cholinesterase fluorescent carbon dots |
| CN112680436A (en) * | 2020-12-23 | 2021-04-20 | 陕西科技大学 | Immobilized enzyme for extracting anthocyanin from black rice and preparation and use methods thereof |
| CN117678656A (en) * | 2024-01-16 | 2024-03-12 | 广东广牧动物保健品有限公司 | Composition containing black tea polyphenol and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110849857A (en) * | 2019-12-03 | 2020-02-28 | 中国农业科学院农业质量标准与检测技术研究所 | Organophosphorus pesticide detection method based on magnetic nano cholinesterase fluorescent carbon dots |
| CN110849857B (en) * | 2019-12-03 | 2022-09-02 | 中国农业科学院农业质量标准与检测技术研究所 | Organophosphorus pesticide detection method based on magnetic nano cholinesterase fluorescent carbon dots |
| CN112680436A (en) * | 2020-12-23 | 2021-04-20 | 陕西科技大学 | Immobilized enzyme for extracting anthocyanin from black rice and preparation and use methods thereof |
| CN117678656A (en) * | 2024-01-16 | 2024-03-12 | 广东广牧动物保健品有限公司 | Composition containing black tea polyphenol and preparation method and application thereof |
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