CN103333295B - Preparation method of thymopentin molecularly-imprinted magnetic microspheres - Google Patents
Preparation method of thymopentin molecularly-imprinted magnetic microspheres Download PDFInfo
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Abstract
The invention provides a preparation method of thymopentin molecularly-imprinted magnetic microspheres, which comprises the following steps: preparing oleic-acid-modified Fe3O4 magnetic nanoparticles, and preparing carboxylated Fe3O4 magnetic nanoparticles with methyl methacrylate and methacrylic acid by using sodium lauryl sulfate as a surfactant, potassium persulfate as a initiator and ethylene glycol dimethacrylate as a crosslinking agent; and finally, successfully preparing the thymopentin molecularly-imprinted magnetic microspheres by using thymopentin as a template molecule, acrylamide as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent and carboxylated Fe3O4 as a magnetic carrier. The preparation method has the advantages of mild reaction conditions, high imprinting efficiency, controllable magnetic microsphere size, high adsorption capacity and easy separation. By integrating the single recognition characteristic of the molecularly-imprinted polymer and the easy separation characteristic of the magnetic microspheres under the action of an external magnetic field, the preparation method has wide application prospects in the fields of separation, enrichment, detection, medicine targeted therapy and the like.
Description
Technical field
The invention belongs to the preparation method of peptide molecule trace magnetic microsphere, be specially the preparation method of thymopeptide-5 molecular blotting magnetic microsphere, belong to the preparing technical field of functional materials.
Background technology
The research in the fields such as biology, medical science shows that in many physiological processs, requisite regulatory factor is all less peptide class.In recent years along with the increase to AIDS, cancer class pharmaceutical requirements, accelerate the research speed of peptide medicament and peptide class reagent in the world, biological extraction peptide can not meet the demand of market and medicament research and development, and therefore synthetic peptide technology obtains and develop rapidly.But synthetic peptide technology, due to its special synthesis strategy, brings certain difficulty to product separation purifying.
Thymopoietin (Thymopoietin, TP) be isolated a kind of 49 amino acid whose polypeptide from people's thymic tissue, relative molecular mass is the single polypeptide compound of 5562, it has and promotes thymocyte and periphery T cell and B cell differentiation and development, the biological activitys such as conditioner body immunity function.Wherein, what play immunologic function is the 32-36 amino acids of TP.Thymopeptide-5 (Arg-Lys-Asp-Va1-Tyr, TP5) is the pentapeptide of synthetic, and its amino-acid sequence forms identical with structure with the 32-36 amino acids of TP, and biologic activity is identical.
In the process of synthetic thymopeptide-5, along with the prolongation of peptide chain, be linked at the reactive amino acid side chain on peptide chain, if do not protected (closing), a series of by product will be produced in the reaction, difficulty is brought to product purification.At present, mostly the thick peptide utilizing solid phase synthesis technique to prepare is to adopt inverse technique (RP-HPLC) to be separated, and this technology needs to carry out under elevated pressure conditions, and this must cause, and equipment energy consumption increases, cost improves.Therefore, find a kind of purification process that is efficient, low cost and have huge economic worth.
Molecular imprinting is that preparation fast has the effective of the polymer materials of special identification function and the method for cheapness.The imprinted polymer prepared based on this technology has affinity and selectivity is good, anti-adverse environment ability is strong, good stability, the advantage such as long service life and applied range, be widely used in chemical catalysis, sepn process (comprises film analysis and Solid-Phase Extraction, and chromatographic separation), Clinical Application Analysis (comprises slowly released and controlled-drug delivery system, SCL administration, pharmaceutical carrier, activity regulates drug delivery system, targeting drug delivery system and capture systems), sensing technology (comprises electrochemical sensor, Optochemical sensor and Mass sen-sitivity sensor) etc. field.
The method adopting molecular imprinting to combine with magnetic microsphere prepares thymopeptide-5 molecular engram polymer magnetic microballoon, molecular engram microsphere both magnetic and specific recognition capability can be made, by this compound, specific recognition is reached to thymopeptide-5 molecular structure distinctive " memory ", under additional the action of a magnetic field, can sharp separation be reached again simultaneously, greatly improve the separation efficiency of thymopeptide-5.
At present, amino acid and derivative thereof, sugar, steroid, Nucleotide, hormone, the engram technology of the biological micromolecules such as VITAMIN is comparatively ripe, investigator is just being devoted to molecular imprinting to be applied to biomacromolecule, the biomacromolecule being used successfully to molecular imprinting has ribonuclease A, Fibrinogen, N,O-Diacetylmuramidase, glutamine synthetase, streptavidin, urease, albumin, myosin, oxyphorase, cytochrome C, lactoperoxidase, horseradish peroxidase, glucose oxidase, Uteracon, antidiuretic hormone.By literature search, not yet find to adopt molecular imprinting to prepare the relevant report of thymopeptide-5 molecular blotting magnetic microsphere.
Summary of the invention
The technical problem solved
In order to avoid the deficiencies in the prior art part, the present invention proposes a kind of preparation method of thymopeptide-5 molecular blotting magnetic microsphere.
Technical scheme
A preparation method for thymopeptide-5 molecular blotting magnetic microsphere, is characterized in that step is as follows:
Step 1: according to mol ratio 1:2 by FeCl
24H
2o and FeCl
36H
2o is dissolved in distilled water, and at 30 DEG C, mix and blend under nitrogen protection, the NaOH solution then dripping 1mol/L makes pH reach 11, obtains black turbid solution; Adopt hydrochloric acid black turbid solution pH=7, add the oleic acid of distilled water volume 5%, be warming up to 80 DEG C, at 800rmpmin
-1under rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material; Adopt distilled water and dehydrated alcohol cross washing repeatedly, vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle;
Step 2: by the Fe of oleic acid modification
3o
4magnetic nano-particle adds ultrasonic disperse in normal hexane according to 0.5g/mL, forms oil-based magnetic fluids;
Step 3: be the lauryl sodium sulfate aqueous solution of 3.5mg/mL by concentration, join in oil-based magnetic fluids, form magnetic template emulsion, add methyl methacrylate MMA and methacrylic acid MAA more successively, at 60 ~ 70 DEG C, mechanical stirring 2h under 800r/min rotating speed, continue to add the Potassium Persulphate KPS of 0.01g, the Ethylene glycol dimethacrylate EGDMA of 0.4 ~ 0.6mL, thermal-initiated polymerization 20h, obtain the Fe of carboxyl-functional
3o
4magnetic nano-particle; The volume ratio of described methyl methacrylate MMA and methacrylic acid MAA is 2:1 ~ 4:1;
Step 4: be dissolved in distilled water by thymopeptide-5 TP-5 and acrylamide AM, at 25 DEG C after prepolymerization 20 ~ 60min, adds the Fe of the carboxyl-functional of 1.2% of this distilled water quality
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, forms aqueous phase mixed solution; The mol ratio of described thymopeptide-5 TP-5 and acrylamide AM is 1:20 ~ 1:28;
Step 5: under 800r/min stirring action, the potassium persulfate solution 1mL of EGDMA, 1.5mg/mL of 0.3 ~ 0.5mL is added successively in aqueous phase mixed solution, in 30 DEG C of water-baths after thermal-initiated polymerization 5 ~ 7h, be cooled to room temperature, adopt distilled water and dehydrated alcohol cross washing repeatedly, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere;
Step 6: joined by molecular blotting magnetic microsphere in the mixing solutions of phosphate buffered saline buffer and methyl alcohol, at 25 DEG C, 120rmpmin
-1under rotating speed, shaking table vibration 10h is with wash-out thymopeptide-5 TP-5; Again with distilled water wash repeatedly, vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere; The volume ratio of described phosphate buffered saline buffer and methyl alcohol is 60:40.
The pH=7 of described phosphate buffered saline buffer.
Distilled water and dehydrated alcohol cross washing 6 times is adopted in step 1.
Step 5 adopts distilled water and dehydrated alcohol cross washing 3 times.
Step 6 distilled water wash 6 times.
Beneficial effect
The present invention proposes a kind of preparation method of thymopeptide-5 molecular blotting magnetic microsphere.First the Fe of oleic acid modification is prepared
3o
4magnetic nano-particle, then under sodium lauryl sulphate makees tensio-active agent, take Potassium Persulphate as initiator, Ethylene glycol dimethacrylate is linking agent, utilizes methyl methacrylate and methacrylic acid to prepare the Fe of carboxyl-functional
3o
4magnetic nano-particle.Last is template molecule with thymopeptide-5, acrylamide is function monomer, Ethylene glycol dimethacrylate is linking agent, the Fe of carboxyl-functional
3o
4for magnetic carrier, successfully prepare thymopeptide-5 molecular blotting magnetic microsphere.Preparation method's reaction conditions of the present invention is gentle, and imprinting efficiency is high, and magnetic microsphere particle diameter is controlled, and adsorptive capacity is high and easily separated.The characteristic that the single-minded evident characteristics of molecularly imprinted polymer and magnetic microsphere are easily separated under extraneous the action of a magnetic field unites two into one by this preparation method, therefore has a wide range of applications in fields such as separation, enrichment, detection, drug targeting treatments.
Embodiment
Now the invention will be further described in conjunction with the embodiments:
Embodiment 1: by 0.01moL FeCl
24H
2o and 0.02moLFeCl
36H
2o is dissolved in 30mL distilled water, 30 DEG C, drip 1mol/L NaOH solution to pH value of solution=11, Keep agitation 2h after mechanical stirring 1h under nitrogen protection, obtain black turbid solution.Adjust this pH value of solution=7 with hydrochloric acid, add 1.5mL oleic acid, be warming up to 80 DEG C, 800rmpmin
-1under rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material.It is washed 6 times with distilled water, each 40mL of dehydrated alcohol successively, and vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle.Take the Fe of 0.6g oleic acid modification
3o
4magnetic nano-particle ultrasonic disperse, in 1.2mL normal hexane, forms oil-based magnetic fluids.Configuration concentration is the lauryl sodium sulfate aqueous solution 20mL of 3.5mg/mL, is proceeded to oil-based magnetic fluids, forms magnetic template emulsion, adds 0.9mLMMA and 0.3mLMAA successively, at 70 DEG C, 800rmpmin
-12h under lower mechanical stirring, continues to add 0.01gKPS, 0.6mLEGDMA, and thermal-initiated polymerization 20h obtains the Fe of carboxyl-functional
3o
4magnetic nano-particle.0.029gTP-5 and 1mmoLAM is dissolved in respectively in 10mL distilled water, at 25 DEG C after prepolymerization 40min.Add the Fe of 0.12g carboxyl-functional
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, at 800rmpmin
-1under stirring action, add 0.4mLEGDMA, 1.5mg/mL potassium persulfate solution 1mL successively, at 30 DEG C in water-bath after thermal-initiated polymerization 6h.Be cooled to room temperature, wash 3 times with distilled water, each 40mL of ethanol successively, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere.
Molecular blotting magnetic microsphere is added 50mL phosphate buffered saline buffer (pH=7) with the mixing solutions of methyl alcohol, both amount ratios are 60:40 (volume ratio).At 25 DEG C, 120rmpmin
-1shaking table vibration 10h wash-out TP-5 under rotating speed.By the 40mL distilled water wash 6 times of molecular blotting magnetic microsphere after wash-out.Vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere.
After testing, prepared thymopeptide-5 molecular blotting magnetic microsphere median size is 2.6 μm.Saturated extent of adsorption is 15.63mg/g, and imprinting factor is 1.46.
Embodiment 2: by 0.01moL FeCl
24H
2o and 0.02moLFeCl
36H
2o is dissolved in 30mL distilled water, 30 DEG C, drip 1mol/L NaOH solution to pH value of solution=11, Keep agitation 2h after mechanical stirring 1h under nitrogen protection, obtain black turbid solution.Adjust this pH value of solution=7 with hydrochloric acid, add 1.5mL oleic acid, be warming up to 80 DEG C, 800rmpmin
-1under rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material.It is washed 6 times with distilled water, each 40mL of dehydrated alcohol successively, and vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle.Take the Fe of 0.6g oleic acid modification
3o
4magnetic nano-particle ultrasonic disperse is in 1.2mL normal hexane, and form oil-based magnetic fluids, configuration concentration is the lauryl sodium sulfate aqueous solution 20mL of 3.5mg/mL, proceeded to oil-based magnetic fluids, form magnetic template emulsion, add 0.8mLMMA and 0.2mLMAA successively, at 60 DEG C, 800rmpmin
-12h under lower mechanical stirring, continues to add 0.01gKPS, 0.4mLEGDMA, and thermal-initiated polymerization 20h obtains the Fe of carboxyl-functional
3o
4magnetic nano-particle.0.024gTP-5 and 1mmoLAM is dissolved in respectively in 10mL distilled water, at 25 DEG C after prepolymerization 60min.Add the Fe of 0.12g carboxyl-functional
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, at 800rmpmin
-1under stirring action, add 0.5mLEGDMA, 1.5mg/mL potassium persulfate solution 1mL successively, at 30 DEG C in water-bath after thermal-initiated polymerization 7h.Be cooled to room temperature, wash 3 times with distilled water, each 40mL of ethanol successively, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere.
Molecular blotting magnetic microsphere is added 50mL phosphate buffered saline buffer (pH=7) with the mixing solutions of methyl alcohol, both amount ratios are 60:40 (volume ratio).At 25 DEG C, 120rmpmin
-1shaking table vibration 10h wash-out TP-5 under rotating speed.By the 40mL distilled water wash 6 times of molecular blotting magnetic microsphere after wash-out.Vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere.
After testing, prepared thymopeptide-5 molecular blotting magnetic microsphere median size is 2.3 μm.Saturated extent of adsorption is 15.5mg/g, and imprinting factor is 1.37.
Embodiment 3: by 0.01moL FeCl
24H
2o and 0.02moLFeCl
36H
2o is dissolved in 30mL distilled water, 30 DEG C, drip 1mol/L NaOH solution to pH value of solution=11, Keep agitation 2h after mechanical stirring 1h under nitrogen protection, obtain black turbid solution.Adjust this pH value of solution=7 with hydrochloric acid, add 1.5mL oleic acid, be warming up to 80 DEG C, 800rmpmin
-1under rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material.It is washed 6 times with distilled water, each 40mL of dehydrated alcohol successively, and vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle.Take the Fe of 0.6g oleic acid modification
3o
4magnetic nano-particle ultrasonic disperse is in 1.2mL normal hexane, and form oil-based magnetic fluids, configuration concentration is the lauryl sodium sulfate aqueous solution 20mL of 3.5mg/mL, proceeded to oil-based magnetic fluids, form magnetic template emulsion, add 0.8mLMMA and 0.4mLMAA successively, at 65 DEG C, 800rmpmin
-12h under lower mechanical stirring, continues to add 0.01gKPS, 0.5mLEGDMA, and thermal-initiated polymerization 20h obtains the Fe of carboxyl-functional
3o
4magnetic nano-particle.0.029gTP-5 and 1mmoLAM is dissolved in respectively in 10mL distilled water, at 25 DEG C after prepolymerization 40min.Add the Fe of 0.12g carboxyl-functional
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, at 800rmpmin
-1under mechanical agitation, add 0.4mLEGDMA, 1.5mg/mL potassium persulfate solution 1mL successively, in 30 DEG C of water-baths after thermal-initiated polymerization 6h.Be cooled to room temperature, wash 3 times with distilled water, each 40mL of ethanol successively, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere.
Molecular blotting magnetic microsphere is added 50mL phosphate buffered saline buffer (pH=7) with the mixing solutions of methyl alcohol, both amount ratios are 60:40 (volume ratio).At 25 DEG C, 120rmpmin
-1shaking table vibration 10h wash-out TP-5 under rotating speed.By the 40mL distilled water wash 6 times of molecular blotting magnetic microsphere after wash-out.Vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere.
After testing, prepared thymopeptide-5 molecular blotting magnetic microsphere median size is 2.8 μm.Saturated extent of adsorption is 15.52mg/g, and imprinting factor is 1.42.
Embodiment 4: by 0.01moL FeCl
24H
2o and 0.02moLFeCl
36H
2o is dissolved in 30mL distilled water, 30 DEG C, drip 1mol/L NaOH solution to solution PH=11, Keep agitation 2h after mechanical stirring 1h under nitrogen protection, obtain black turbid solution.Adjust this solution PH=7 with hydrochloric acid, add 1.5mL oleic acid, be warming up to 80 DEG C, 800rmpmin
-1under rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material.It is washed 6 times with distilled water, each 40mL of dehydrated alcohol successively, and vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle.Take the Fe of 0.6g oleic acid modification
3o
4magnetic nano-particle ultrasonic disperse is in 1.2mL normal hexane, and form oil-based magnetic fluids, configuration concentration is the lauryl sodium sulfate aqueous solution 20mL of 3.5mg/mL, proceeded to oil-based magnetic fluids, form magnetic template emulsion, add 0.8mLMMA and 0.2mLMAA successively, at 60 DEG C, 800rmpmin
-12h under lower mechanical stirring, continues to add 0.01gKPS, 0.4mLEGDMA, and thermal-initiated polymerization 20h obtains the Fe of carboxyl-functional
3o
4magnetic nano-particle.0.029gTP-5 and 1mmoLAM is dissolved in respectively in 10mL distilled water, at 25 DEG C after prepolymerization 40min.Add the Fe of 0.12g carboxyl-functional
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, at 800rmpmin
-1under stirring action, add 0.4mLEGDMA, 1.5mg/mL potassium persulfate solution 1mL successively, in 30 DEG C of water-baths after thermal-initiated polymerization 6h.Be cooled to room temperature, wash 3 times with distilled water, each 40mL of ethanol successively, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere.
Molecular blotting magnetic microsphere is added 50mL phosphate buffered saline buffer (pH=7) with the mixing solutions of methyl alcohol, both amount ratios are 60:40 (volume ratio).At 25 DEG C, 120rmpmin
-1shaking table vibration 10h wash-out TP-5 under rotating speed.By the 40mL distilled water wash 6 times of molecular blotting magnetic microsphere after wash-out.Vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere.
After testing, prepared thymopeptide-5 molecular blotting magnetic microsphere median size is 2.6 μm.Saturated extent of adsorption is 14.1mg/g, and imprinting factor is 1.35.
Embodiment 5: by 0.01moL FeCl
24H
2o and 0.02moLFeCl
36H
2o is dissolved in 30mL distilled water, 30 DEG C, drip 1mol/L NaOH solution to pH value of solution=11, Keep agitation 2h after mechanical stirring 1h under nitrogen protection, obtain black turbid solution.Adjust this pH value of solution=7 with hydrochloric acid, add 1.5mL oleic acid, be warming up to 80 DEG C, 800rmpmin
-1under rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material.It is washed 6 times with distilled water, each 40mL of dehydrated alcohol successively, and vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle.Take the Fe of 0.6g oleic acid modification
3o
4magnetic nano-particle ultrasonic disperse is in 1.2mL normal hexane, and form oil-based magnetic fluids, configuration concentration is the lauryl sodium sulfate aqueous solution 20mL of 3.5mg/mL, proceeded to oil-based magnetic fluids, form magnetic template emulsion, add 0.9mLMMA and 0.3mLMAA successively, at 70 DEG C, 800rmpmin
-12h under lower mechanical stirring, continues to add 0.01gKPS, 0.6mLEGDMA, and thermal-initiated polymerization 20h obtains the Fe of carboxyl-functional
3o
4magnetic nano-particle.0.033gTP-5 and 1mmoLAM is dissolved in respectively in 10mL distilled water, at 25 DEG C after prepolymerization 20min.Add the Fe of 0.12g carboxyl-functional
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, at 800rmpmin
-1under stirring action, add 0.3mLEGDMA, 1.5mg/mL potassium persulfate solution 1mL successively, at 30 DEG C in water-bath after thermal-initiated polymerization 5h.Be cooled to room temperature, wash 3 times with distilled water, each 40mL of ethanol successively, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere.
Molecular blotting magnetic microsphere is added 50mL phosphate buffered saline buffer (pH=7) with the mixing solutions of methyl alcohol, both amount ratios are 60:40 (volume ratio).At 25 DEG C, 120rmpmin
-1shaking table vibration 10h wash-out TP-5 under rotating speed.By the 40mL distilled water wash 6 times of molecular blotting magnetic microsphere after wash-out.Vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere.
After testing, prepared thymopeptide-5 molecular blotting magnetic microsphere median size is 2.2 μm.Saturated extent of adsorption is 12.6mg/g, and imprinting factor is 1.32.
Embodiment 6: by 0.01moL FeCl
24H
2o and 0.02moLFeCl
36H
2o is dissolved in 30mL distilled water, 30 DEG C, drip 1mol/L NaOH solution to pH value of solution=11, Keep agitation 2h after mechanical stirring 1h under nitrogen protection, obtain black turbid solution.Adjust this pH value of solution=7 with hydrochloric acid, add 1.5mL oleic acid, be warming up to 80 DEG C, 800rmpmin
-1under rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material.It is washed 6 times with distilled water, each 40mL of dehydrated alcohol successively, and vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle.Take the Fe of 0.6g oleic acid modification
3o
4magnetic nano-particle ultrasonic disperse is in 1.2mL normal hexane, and form oil-based magnetic fluids, configuration concentration is the lauryl sodium sulfate aqueous solution 20mL of 3.5mg/mL, proceeded to oil-based magnetic fluids, form magnetic template emulsion, add 0.9mLMMA and 0.3mLMAA successively, at 70 DEG C, 800rmpmin
-12h under lower mechanical stirring, continues to add 0.01gKPS, 0.6mLEGDMA, and thermal-initiated polymerization 20h obtains the Fe of carboxyl-functional
3o
4magnetic nano-particle.0.024gTP-5 and 1mmoLAM is dissolved in respectively in 10mL distilled water, at 25 DEG C after prepolymerization 60min.Add the Fe of 0.12g carboxyl-functional
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, at 800rmpmin
-1under stirring action, add 0.5mLEGDMA, 1.5mg/mL potassium persulfate solution 1mL successively, at 30 DEG C in water-bath after thermal-initiated polymerization 7h.Be cooled to room temperature, wash 3 times with distilled water, each 40mL of ethanol successively, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere.
Molecular blotting magnetic microsphere is added 50mL phosphate buffered saline buffer (pH=7) with the mixing solutions of methyl alcohol, both amount ratios are 60:40 (volume ratio).At 25 DEG C, 120rmpmin
-1shaking table vibration 10h wash-out TP-5 under rotating speed.By the 40mL distilled water wash 6 times of molecular blotting magnetic microsphere after wash-out.Vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere.
After testing, prepared thymopeptide-5 molecular blotting magnetic microsphere median size is 2.5 μm.Saturated extent of adsorption is 15.2mg/g, and imprinting factor is 1.36.
Claims (2)
1. a preparation method for thymopeptide-5 molecular blotting magnetic microsphere, is characterized in that step is as follows:
Step 1: according to mol ratio 1:2 by FeCl
24H
2o and FeCl
36H
2o is dissolved in distilled water, and at 30 DEG C, mix and blend under nitrogen protection, the NaOH solution then dripping 1mol/L makes pH reach 11, obtains black turbid solution; Adopt hydrochloric acid to adjust black turbid solution pH=7, add the oleic acid of distilled water volume 5%, be warming up to 80 DEG C, under 800r/min rotating speed after mechanical stirring 2h, under outside magnetic field effect, isolate black solid material; Adopt distilled water and dehydrated alcohol cross washing 6 times, vacuum-drying 24h at 30 DEG C, obtains the Fe of oleic acid modification
3o
4magnetic nano-particle;
Step 2: by the Fe of oleic acid modification
3o
4magnetic nano-particle adds ultrasonic disperse in normal hexane according to 0.5g/mL, forms oil-based magnetic fluids;
Step 3: be the lauryl sodium sulfate aqueous solution of 3.5mg/mL by concentration, join in oil-based magnetic fluids, form magnetic template emulsion, add methyl methacrylate MMA and methacrylic acid MAA more successively, at 60 ~ 70 DEG C, mechanical stirring 2h under 800r/min rotating speed, continue to add the Potassium Persulphate KPS of 0.01g, the Ethylene glycol dimethacrylate EGDMA of 0.4 ~ 0.6mL, thermal-initiated polymerization 20h, obtain the Fe of carboxyl-functional
3o
4magnetic nano-particle; The volume ratio of described methyl methacrylate MMA and methacrylic acid MAA is 2:1 ~ 4:1;
Step 4: be dissolved in distilled water by thymopeptide-5 TP-5 and acrylamide AM, at 25 DEG C after prepolymerization 20 ~ 60min, adds the Fe of the carboxyl-functional of 1.2% of this distilled water quality
3o
4magnetic nano-particle, the ultrasonic 15min of room temperature, forms aqueous phase mixed solution; The mol ratio of described thymopeptide-5 TP-5 and acrylamide AM is 1:20 ~ 1:28;
Step 5: under 800r/min stirring action, the potassium persulfate solution 1mL of EGDMA, 1.5mg/mL of 0.3 ~ 0.5mL is added successively in aqueous phase mixed solution, in 30 DEG C of water-baths after thermal-initiated polymerization 5 ~ 7h, be cooled to room temperature, adopt distilled water and dehydrated alcohol cross washing 3 times, at 30 DEG C, vacuum-drying 24h, obtains molecular blotting magnetic microsphere;
Step 6: molecular blotting magnetic microsphere is joined in the mixing solutions of phosphate buffered saline buffer and methyl alcohol, 25 DEG C, under 120r/min rotating speed shaking table vibration 10h with wash-out thymopeptide-5 TP-5; Use distilled water wash again 6 times, vacuum-drying 24h at 25 DEG C, obtains thymopeptide-5 molecular blotting magnetic microsphere; The volume ratio of described phosphate buffered saline buffer and methyl alcohol is 60:40.
2. the preparation method of thymopeptide-5 molecular blotting magnetic microsphere according to claim 1, is characterized in that: the pH=7 of described phosphate buffered saline buffer.
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| KR100861452B1 (en) * | 2007-03-05 | 2008-10-02 | 성균관대학교산학협력단 | Method for preparing surface-imprinted polyacrylate microsphere in the form of core-shell for the selective separation of heavy metal ion |
| CN102585119B (en) * | 2012-02-23 | 2014-04-09 | 宁波市疾病预防控制中心 | Preparation method of magnetic nanometer molecular imprinting composite material related to estrogen |
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