CN104195042A - Preparation method and application of nano material monolithic column immobilized enzyme biological micro-reactor - Google Patents

Preparation method and application of nano material monolithic column immobilized enzyme biological micro-reactor Download PDF

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CN104195042A
CN104195042A CN201410361654.7A CN201410361654A CN104195042A CN 104195042 A CN104195042 A CN 104195042A CN 201410361654 A CN201410361654 A CN 201410361654A CN 104195042 A CN104195042 A CN 104195042A
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nano material
integral post
enzyme
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reactor
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吕永琴
谭天伟
王红霞
朱正礼
林祥华
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Beijing University of Chemical Technology
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings

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Abstract

The invention discloses a preparation method and an application of a nano material monolithic column immobilized enzyme biological micro-reactor. The preparation method comprises the following steps of firstly, preparing a porous organic polymer monolithic column by using a mixed solution of a functional monomer, a crosslinking agent, a pore forming agent and an initiating agent through in-situ thermal-initiated or light-initiated polymerization in the column, and then bonding a nano material after functional modification to obtain a nano material monolithic column; and secondly, realizing immobilization of the enzyme on the monolithic column by using the nano material as an intermediate ligand to obtain the nano material monolithic column immobilized enzyme biological micro-reactor. The biological micro-reactor is successfully applied to proteomic analysis, medicament chiral resolution and catalyzed ester exchange reactions. The biological micro-reactor disclosed by the invention has the following advantages that the preparation method is simple, the immobilization amount of the enzyme is large, the catalytic activity is high, the enzymolysis speed is high, the efficiency is high, the service life is long, and the biological micro-reactor can be reused.

Description

A kind of preparation and application thereof of nano material integral post enzyme immobilization micro-bioreactor
Technical field
The present invention relates to preparation and the application thereof of integral post enzyme immobilization micro-bioreactor, a preparation method for the porous organo polysilica compound integral post enzyme immobilization micro-bioreactor of part in the middle of based on nano material being specifically, and study its application in proteome analysis, medicine chiral separation and catalyzed transesterification.
Background technology
Enzyme immobilization technology is a cross discipline technology.Immobilized enzyme in keeping its efficient single-minded and gentle enzymic catalytic reaction characteristic, have again higher enzyme/substrate than and enzymolysis efficiency, faster enzymolysis speed, can use continuously and can reduce self degrading and the advantage such as cross infection of enzyme; And enzyme digestion reaction is controlled, enzyme is easy to separate with product, and product is easily purified, and the space of reacting required is little, can realize the coupling with separation identification systems.The solid support material that the performance of immobilized enzyme depends primarily on process for fixation and uses, the two can directly affect the catalytic activity of immobilized enzyme.But fixation support and the method for report all have certain limitation both at home and abroad at present, as lower to the supported quantity of enzyme in carriers such as silicon-dioxide, synthetic particulate, mesopore molecular sieve, macroporous resin and synthon, and mass transfer velocity is slow, be unfavorable for the enzymic catalytic reaction that carries out rapidly and efficiently.And conventional enzyme immobilization method, as entrapping method, crosslinking, covalent attachment, ionic bond and physical adsorption etc. all have certain defect.For example, the preparation of ionic bond method and physisorphtion is simple, activity recovery is high, carrier can be regenerated, however the immobilization degree of enzyme a little less than, in catalytic process, enzyme easily comes off.Covalent coupling method, crosslinking and entrapping method are strong to the immobilization degree of enzyme, but preparation is more difficult, activity recovery is low, carrier can not be regenerated.Therefore, at present in the urgent need to exploitation enzyme immobilization carrier and process for fixation, strengthen the catalytic activity of enzyme, increase work-ing life and recyclability, improve speed of reaction and the efficiency of enzyme digestion reaction.
Organic polymer integral post has that preparation is simple, good penetrability, back pressure is low, mass transfer velocity is fast, be easy to modification and chemical stability advantages of higher, can provide convective mass transfer performance fast for enzyme and substrate, because of but one of ideal carrier of immobilized enzyme.The such as people such as Krenkova and Urban (Anal.Chem.2009,81,2004-2012; Biotechnology and Bioengineering, 2012,109,371-380) apply respectively epoxide group or azlactone group reacts with the amino of enzyme, by covalent method enzyme immobilization in integral post, be respectively used to proteome analysis and catalyzed transesterification.Spross (Anal.Chem.2010,82,1434-1443) utilizes glutaraldehyde cross-linking method enzyme immobilization in integral post, for protein digestion.Ma, the people such as Wu and Zhang (Proteomics 2011,11,991-995; J.Chromatogr.A 2012,1256,136-143; Anal.Bioanal.Chem.2012,402,703-710) respectively by metal ion-chelant and ionic linkage enzyme immobilization, enzymolysis protein matter, carries out proteome analysis.Although integral post enzyme immobilization micro-bioreactor can provide good convective mass transfer performance for enzyme and substrate, improve enzyme digestion reaction speed, but in use procedure, still have two problems, the one, integral post is owing to having the continuous macropore that runs through, its specific surface area is less, and therefore enzyme supported quantity is lower.The 2nd, traditional integral post enzyme immobilization method, as covalent method and ion exchange method etc. exist certain drawback, is unfavorable for improving stability and the work-ing life of enzyme.
Nano material, due to its unique size and physical properties, such as having small particle size effect, surface effects and interfacial effect etc., has the plurality of advantages such as specific surface area is large.Be used for the immobilization of enzyme as carrier, be conducive to keep activity and the stability of enzyme, greatly improve supported quantity and the catalytic efficiency of enzyme.In this patent, nano material and integral post are combined, first integral post polymkeric substance is carried out to functional modification, bonding nano material; Taking nano material as middle part, realize the immobilization of enzyme by the affine supramolecular recognition of exploitation nano material and enzyme, nano materials integral post immobilized enzyme micro-bioreactor again.Organic Polymer Monolithic Columns chemical stability is high, and permeability is good, can provide convective mass transfer performance fast for enzyme and substrate.The specific surface area that nano material is huge and volume ratio, be conducive to greatly improve the supported quantity of enzyme; Affinity interaction power is strong, can prevent enzyme coming off from carrier; And reaction conditions gentleness, can keep three-dimensional structure and the biological activity of enzyme.The two is organically combined, and the problem such as can solve on the one hand that traditional enzyme immobilization micro-bioreactor rate of mass transfer is slow, supported quantity is low and catalytic efficiency is low, improves stability, work-ing life, catalytic rate and the efficiency of enzyme immobilization greatly; Affine supramolecule reactive force is reversible on the other hand, and enzyme can wash-out after repeatedly using inactivation, the new enzyme of bonding again, and enzyme immobilization reactor is renewable, greatly reduces cost, for the preparation of enzyme immobilization micro-bioreactor provides novel method.
Summary of the invention:
One of object of the present invention is a kind of nano material integral post enzyme immobilization microorganism microreactor of research, by by nano material and the two combination of integral post, solve the problems such as traditional enzyme immobilization micro-bioreactor rate of mass transfer is slow, enzyme supported quantity is low, catalytic efficiency is low and differ from work-ing life, thereby obtain the renewable enzyme immobilization micro-bioreactor that catalytic rate is fast, efficiency is high.
Two of object of the present invention is the application in catalyzed transesterification, medicine chiral separation and proteome analysis of research nano material integral post enzyme immobilization micro-bioreactor, realize enzymic catalytic reaction fast, efficiently carry out.
The present invention realizes by following technical scheme measure: in organic polymer capillary monolithic column hole surface bonding one deck queueing discipline of functional modification, densification, continuously, the high-density nano material of different-grain diameter, taking nano material as middle part, enzyme is fixed in integral post, thereby obtains nano material integral post enzyme immobilization micro-bioreactor.
A preparation for nano material integral post enzyme immobilization micro-bioreactor, comprising: steps A, preparation nano material; Step B, Organic Polymer Monolithic Columns synthetic: by the mixing solutions of function monomer, linking agent, pore-creating agent and initiator, by original position thermal initiation or light-initiated synthesizing porous Organic Polymer Monolithic Columns; Step C, nano materials integral post: integral post is carried out to functional modification, introduce highdensity specific function group, by the specificity affinity interaction of functional group and nano material, bonding nano material on the hole surface of integral post; Step D, the preparation of enzyme immobilization micro-bioreactor: taking nano material as middle part, by the specificity affinity interaction of nano material and enzyme, the immobilization of realize target enzyme in integral post, design preparation nano material enzyme immobilization micro-bioreactor.
In steps A, synthetic nano material comprises nanometer gold, nanometer silver, nano silicon, football alkene C 60, carbon nanotube, Graphene, magnetic ferroferric oxide, metal-organic framework materials MOF or covalency organic backbone COF.Nano material colloid is spherical, and particle diameter is at 5～100nm.
In step B, synthetic integral post function monomer used comprises glycidyl methacrylate, methyl methacrylate, butyl methacrylate, lauryl methacrylate(LMA), vinylformic acid, acrylamide, NIPA, methacrylic acid proyl ester, glycidyl propargyl ether, 2-vinyl-4,4-dimethyl-2-oxazoline-5-ketone, 2-methyl-2-vinylformic acid-2,3-dihydroxyl propyl ester, vinylbenzene or 4-vinylpridine.Linking agent comprises ethylene glycol dimethacrylate, iso-cyanuric acid triallyl ester, Vinylstyrene, N, N '-methylene-bisacrylamide etc.Pore-creating agent comprises the polyoxyethylene glycol of toluene, normal heptane, hexalin, lauryl alcohol, n-propyl alcohol, BDO or different molecular weight.Initiator comprises Diisopropyl azodicarboxylate, 2,2'-Azobis(2,4-dimethylvaleronitrile), 2,2-dimethoxy-2-phenyl methyl phenyl ketone or 2-hydroxy-2-methyl-1-phenyl-acetone.5～30% (w/w) function monomer, 5～30% (w/w) linking agent, 30～80% (w/w) pore-creating agent, and the quality summation of function monomer, linking agent and pore-creating agent is 100%.Adding is in addition 0.2～5% initiator of function monomer and linking agent quality, and thermal booster reaction temperature is 45～85 DEG C, reaction times 6～48h.Light-initiated wavelength is 254nm or 365nm, and light intensity is 6-30mw/cm 2, light application time is 5-60min.Integral post is at stainless steel chromatogram post (internal diameter 3-6mm, length 6-30cm), synthetic in kapillary (internal diameter 50-600 μ m, length 5-50cm), polypropylene-base rifle head (volume 0.01-10mL), polypropylene-base syringe (volume 0.1-10mL), high-boron-silicon glass plate or silicon wafer.
In step C by porous organo polysilica compound integral post is carried out to surface-functionalized modification, bonding one deck queueing discipline, densification, continuous high-density nano material.
In step D, enzyme solution used is the phosphate buffer solution preparation with pH 6.5～8.0, and its concentration is 5～50mg/mL.
Brief description of the drawings
Transmission electron microscope (TEM) figure of the nm gold particles of Fig. 1 different-grain diameter.(a)15nm，(b)17nm，(c)30nm，(d)?40nm，(e)50nm。
Fig. 2 magnetic ferroferric oxide (Fe 3o 4) the transmission electron microscope figure of Nano microsphere.
Fig. 3 Fe 3o 4@SiO 2the transmission electron microscope figure of nuclear shell structured nano-microballoon.
Scanning electronic microscope (SEM) figure of Fig. 4 Poly (GMA-co-EDMA) capillary monolithic column section.
Scanning electronic microscope (SEM) figure of Fig. 5 Poly (MS-co-CMS-co-DVB) capillary monolithic column section.
(poly (GMA-co-EDMA) (a) for scanning electronic microscope (SEM) figure of the polyalcohol integral column section of Fig. 6 based on polypropylene syringe, (b), poly (LMA-co-EDMA) (c) for poly (BMA-co-EDMA)).
Scanning electronic microscope (SEM) figure of poly (GMA-co-EDMA) the capillary monolithic column section of Fig. 7 bonding 15nm nanometer gold.
Resolvase and high performance liquid chromatography (HPLC) comparison diagram of immobilized enzyme to cytochrome C enzymolysis at 37 DEG C of Fig. 8.(1) cytochrome C, (2) nanometer gold integral post immobilized enzyme catalysis 1.6min, (3) resolvase catalysis 24h.
Fig. 9 nanometer gold integral post immobilized enzyme carries out the MALDI-TOD-MS figure of the polypeptide fragment of enzymolysis to cytochrome C.
Resolvase and high performance liquid chromatography (HPLC) comparison diagram of immobilized enzyme to bovine serum albumin (BSA) enzymolysis at 37 DEG C of Figure 10.(1) bovine serum albumin (BSA), (2) nanometer gold integral post immobilized enzyme catalysis 1.6min, (3) resolvase catalysis 24h.
Figure 11 nanometer gold integral post immobilized enzyme carries out the MALDI-TOD-MS figure of the polypeptide fragment of enzymolysis to bovine serum albumin (BSA).
Embodiment
Describe the present invention in detail below in conjunction with embodiment, these examples only play illustrative effect, are not limited to range of application of the present invention.
Embodiment
Embodiment 1: nanometer gold synthetic
(1) nanometer gold of different-grain diameter is synthetic
To the hydrochloro-auric acid that adds 50mg in the Erlenmeyer flask of 2000mL, the ultrapure water of 500mL, is heated to 98 DEG C under magnetic agitation, then adds a certain amount of Trisodium Citrate, holding temperature is reacted at 98 DEG C, reaction solution color becomes indigo plant blackening then in several seconds, then becomes redness, continues to stop heating after heating 10min, continue to stir 0.5h, prepare nanometer gold colloidal solution, the cooling rear high speed centrifugation purifying of room temperature, saves backup.Change the size of synthetic nanometer gold by changing the consumption of Trisodium Citrate, as shown in table 1.Utilize transmission electron microscope (TEM) to study size and the pattern (Fig. 1) of synthetic nanometer gold colloid.
Embodiment 2:Fe 3o 4@SiO 2-NH 2synthesizing of magnetic nanoparticle
(1) solvent-thermal method synthesizing magnetic Fe 3o 4nano particle
By FeCl 3.6H 2o (1.35g, 5mmol) and NaAc (3.6g) be dissolved in ethylene glycol (50mL), ultrasonic 30min makes solid fully dissolve dispersion, dispersion liquid is transferred in band teflon-lined stainless steel autoclave and sealed, at 200 DEG C, react 8h, reaction finishes rear naturally cooling, alternately washes 4 times with dehydrated alcohol and water by magnet, and 60 DEG C of vacuum-drying 6h obtain magnetic Fe 3o 4nano particle, its transmission electron microscope photo as shown in Figure 2.
(2) Fe 3o 4@SiO 2synthesizing of nuclear shell structured nano-particle
By the magnetic Fe of preparation in (1) 3o 4nano particle 350mg is dispersed in the ethanol of 160mL, supersound process 0.5h.Add successively 1mL ammoniacal liquor (25wt%), 40mL H 2o and 1mL tetraethoxy, mechanical stirring 6h under room temperature, cleans 3 times with 50mL ethanol by magnet, obtains Fe 3o 4@SiO 2the magnetic nanoparticle of nucleocapsid structure, its transmission electron microscope photo as shown in Figure 3.
(3) Fe 3o 4@SiO 2-NH 2synthesizing of magnetic nanoparticle
By Fe 3o 4@SiO 2nuclear shell structured nano-particle is dispersed in 60mL Virahol, and 1mL 3-aminopropyl triethoxysilane is added drop-wise in dispersion liquid, and mechanical stirring 24h under room temperature, washes 4-5 time with dehydrated alcohol by magnet, and 50 DEG C of vacuum-drying 6h obtain Fe 3o 4@SiO 2-NH 2magnetic nanoparticle.
Embodiment 3: metal organic frame material MIL-101's is synthetic
Utilize hydrothermal method to synthesize MIL-101, concrete step is as follows: the Cr (NO that takes 2.0g 3) 3.6H 2o is dissolved in 25mL deionized water, adds 0.83g terephthalic acid, then add 410 μ L hydrofluoric acid after fully dissolving.Then mixing solutions is moved into 100mL with in teflon-lined stainless steel autoclave, be positioned in the baking oven of 220 DEG C and react 8h.After cooling, product is filtered, washing, find time to be dried at 60 DEG C, obtain the mixture of green powder and white needle-like crystals.Product utilizes N successively, and N '-dimethyl formamide, trichloromethane and dehydrated alcohol, to remove white needle-like crystals and to be blocked in the unreacted inorganic and organic impurity in duct, obtain the MIL-101 that particle diameter is about 500nm.Utilizing BET method to measure the specific surface area of MIL-101, is 3000m 2/ g.
Embodiment 4: Organic Polymer Monolithic Columns synthesizing in kapillary
(1) activation of capillary tube inner wall
Be that 100 μ m, external diameter are the quartz capillary of 365 μ m by internal diameter, its inwall adopts acetone and water to clean first successively, and recycling 2M NaOH activates 3h at 120 DEG C.Capillary tube inner wall utilization is washed to after neutrality, and the hydrochloric acid of recycling 0.25M at room temperature activates 0.5h, washes with water to neutrality, cleans afterwards with acetone, and nitrogen dries up, and is placed on dry 2h in the vacuum drying oven of 120 DEG C.
(2) the two keys of capillary tube inner wall bonding
3-(trimethoxysilyl) propyl group acrylate is dissolved in dry toluene, and concentration is 20% (v/v).Utilize syringe pump, with the flow velocity of 0.5 μ L/min, the above-mentioned solution of 30 μ L is continuously pumped into the capillary inner reaction tube after activation.With acetone, capillary tube inner wall is rinsed well afterwards, after placement 24h, used.
(3) Poly (GMA-co-EDMA) capillary monolithic column is synthetic
By the glycidyl methacrylate of 24% (wt%) (GMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the hexalin of 30% (wt%), ultrasonic the mixing of mixing solutions of the initiator of the lauryl alcohol of 30% (wt%) and 1% (being the quality of function monomer and linking agent), after logical nitrogen 5min, pack in the quartz capillary of the two keys of inwall bonding, by original position thermal initiation or light-initiated synthetic capillary monolithic column.Wherein, it is initiator that thermal initiation adopts Diisopropyl azodicarboxylate (AIBN), and reaction conditions for to react 24h at 60 DEG C.Light-initiated employing 2,2-dimethoxy-2-phenyl methyl phenyl ketone (DMPA) is initiator, and light-initiated wavelength is 365nm, and light intensity is 12mw/cm 2, light application time is 15min.After having reacted, utilize washed with methanol capillary column 2h, to remove pore-creating agent and the unreacted material in post, thereby make poly (GMA-co-EDMA) capillary monolithic column.The scanning electronic microscope (SEM) in synthetic integral post cross section is analyzed as shown in Figure 4.
(4) Poly (BMA-co-EDMA) capillary monolithic column is synthetic
By the butyl methacrylate of 24% (wt%) (BMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the n-propyl alcohol of 40% (wt%), 20% (wt%) 1, ultrasonic the mixing of mixing solutions of the initiator of 4-butyleneglycol and 1% (being the quality of function monomer and linking agent), after logical nitrogen 5min, pack in the quartz capillary of the two keys of inwall bonding, pass through original position thermal initiation or light-initiated synthetic poly (BMA-co-EDMA) capillary monolithic column according to the method in (3).
(5) Poly (LMA-co-EDMA) capillary monolithic column is synthetic
By the lauryl methacrylate(LMA) of 24% (wt%) (LMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the n-propyl alcohol of 40% (wt%), 20% (wt%) 1, ultrasonic the mixing of mixing solutions of the initiator of 4-butyleneglycol and 1% (being the quality of function monomer and linking agent), after logical nitrogen 5min, pack in the quartz capillary of the two keys of inwall bonding, pass through original position thermal initiation or light-initiated synthetic poly (LMA-co-EDMA) capillary monolithic column according to the method in (3).
(6) Poly (MS-co-CMS-co-DVB) capillary monolithic column is synthetic
By the vinyl toluene of 21% (wt%) (MS), the 1-chloro-4-methyl-benzene (CMS) of 7% (wt%), 12% Vinylstyrene (DVB), the toluene of 13% (wt%), ultrasonic the mixing of mixing solutions of Diisopropyl azodicarboxylate (AIBN) initiator of the lauryl alcohol of 47% (wt%) and 1% (being the quality of function monomer and linking agent), after logical nitrogen 5min, pack in the quartz capillary of the two keys of inwall bonding, synthesize poly (MS-co-CMS-co-DVB) capillary monolithic column according to the method in (3) by original position thermal initiation.The scanning electronic microscope (SEM) in synthetic integral post cross section is analyzed as shown in Figure 5.
Embodiment 5: Organic Polymer Monolithic Columns synthesizing in polypropylene syringe
(1) the two keys of polypropylene syringe inwall bonding
Be the polypropylene syringe that 5.6mm, length are 5.5cm by internal diameter, its inwall adopts ethanol and acetone to clean first successively, and after nitrogen dries up, adding 0.5mL concentration is the methanol solution of the deoxidation benzophenone of 5wt%, illumination 3min under UV 365nm, light intensity is 20mw/cm 2.After adopting methyl alcohol thoroughly to clean, utilize nitrogen to dry up, then to add 0.5mL concentration be the methanol solution of the Ethylene glycol dimethacrylate of 15wt%, illumination 3min under UV 365nm, light intensity is 20mw/cm 2.Finally utilize methyl alcohol thoroughly to clean syringe inwall, adopt nitrogen to dry up.
(2) Poly (GMA-co-EDMA) syringe integral post is synthetic
By the glycidyl methacrylate of 24% (wt%) (GMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the hexalin of 20% (wt%), 2 of the lauryl alcohol of 40% (wt%) and 1% (being the quality of function monomer and linking agent), ultrasonic the mixing of mixing solutions of 2-dimethoxy-2-phenyl methyl phenyl ketone (DMPA), after logical nitrogen 5min, pack in the polypropylene syringe of the two keys of inwall bonding, by the light-initiated synthetic syringe integral post of original position, light-initiated wavelength is 365nm, and light intensity is 20mw/cm 2, light application time is 6min.After having reacted, utilize washed with methanol syringe integral post, to remove pore-creating agent and the unreacted material in post, thereby make poly (GMA-co-EDMA) syringe integral post, the scanning electronic microscope (SEM) in its cross section is as shown in Fig. 6 (a).
(3) Poly (BMA-co-EDMA) syringe integral post is synthetic
By the butyl methacrylate of 24% (wt%) (BMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the n-propyl alcohol of 26% (wt%), 34% (wt%) 1,2 of 4-butyleneglycol and 1% (being the quality of function monomer and linking agent), ultrasonic the mixing of mixing solutions of 2-dimethoxy-2-phenyl methyl phenyl ketone (DMPA), after logical nitrogen 5min, pack in the polypropylene syringe of the two keys of inwall bonding, by the light-initiated synthetic syringe integral post of original position, light-initiated wavelength is 365nm, and light intensity is 20mw/cm 2, light application time is 6min.After having reacted, utilize washed with methanol syringe integral post, to remove pore-creating agent and the unreacted material in post, thereby make poly (BMA-co-EDMA) syringe integral post, the scanning electronic microscope (SEM) in its cross section is as shown in Fig. 6 (b).
(4) Poly (LMA-co-EDMA) syringe integral post is synthetic
By the lauryl methacrylate(LMA) of 24% (wt%) (LMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the n-propyl alcohol of 26% (wt%), 34% (wt%) 1,2 of 4-butyleneglycol and 1% (being the quality of function monomer and linking agent), ultrasonic the mixing of mixing solutions of 2-dimethoxy-2-phenyl methyl phenyl ketone (DMPA), after logical nitrogen 5min, pack in the polypropylene syringe of the two keys of inwall bonding, by the light-initiated synthetic syringe integral post of original position, light-initiated wavelength is 365nm, and light intensity is 20mw/cm 2, light application time is 6min.After having reacted, utilize washed with methanol syringe integral post, to remove pore-creating agent and the unreacted material in post, thereby make poly (LMA-co-EDMA) syringe integral post, the scanning electronic microscope (SEM) in its cross section is as shown in Fig. 6 (c).
Embodiment 6: Organic Polymer Monolithic Columns synthesizing in polypropylene liquid-transfering gun rifle head
(1) the two keys of the inwall bonding of polypropylene liquid-transfering gun rifle head
By the polypropylene liquid-transfering gun rifle head of 10～200 μ L, its inwall adopts ethanol and acetone to clean first successively, after nitrogen dries up, adds the methanol solution of the deoxidation benzophenone that 5 μ L concentration are 5wt%, illumination 3min under UV 365nm, and light intensity is 20mw/cm 2.After adopting methyl alcohol thoroughly to clean, utilize nitrogen to dry up, then add the methanol solution of the Ethylene glycol dimethacrylate that 5 μ L concentration are 15wt%, illumination 3min under UV 365nm, light intensity is 20mw/cm 2.Finally utilize the thorough cleaning device head of methyl alcohol inwall, adopt nitrogen to dry up.
(2) Poly (GMA-co-EDMA) rifle head integral post is synthetic
By the glycidyl methacrylate of 24% (wt%) (GMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the hexalin of 20% (wt%), 2 of the lauryl alcohol of 40% (wt%) and 1% (being the quality of function monomer and linking agent), ultrasonic the mixing of mixing solutions of 2-dimethoxy-2-phenyl methyl phenyl ketone (DMPA), after logical nitrogen 5min, pack in the polypropylene liquid-transfering gun rifle head of the two keys of inwall bonding, by the light-initiated synthetic rifle head integral post of original position, light-initiated wavelength is 365nm, and light intensity is 20mw/cm 2, light application time is 6min.After having reacted, utilize washed with methanol rifle head integral post, to remove pore-creating agent and the unreacted material in post, thereby make poly (GMA-co-EDMA) rifle head integral post.
(3) Poly (BMA-co-EDMA) rifle head integral post is synthetic
By the butyl methacrylate of 24% (wt%) (BMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the hexalin of 20% (wt%), 2 of the lauryl alcohol of 40% (wt%) and 1% (being the quality of function monomer and linking agent), ultrasonic the mixing of mixing solutions of 2-dimethoxy-2-phenyl methyl phenyl ketone (DMPA), after logical nitrogen 5min, pack in the polypropylene liquid-transfering gun rifle head of the two keys of inwall bonding, by the light-initiated synthetic rifle head integral post of original position, light-initiated wavelength is 365nm, and light intensity is 20mw/cm 2, light application time is 6min.After having reacted, utilize washed with methanol rifle head integral post, to remove pore-creating agent and the unreacted material in post, thereby make poly (BMA-co-EDMA) rifle head integral post.
(4) Poly (LMA-co-EDMA) rifle head integral post is synthetic
By the lauryl methacrylate(LMA) of 24% (wt%) (LMA), the ethylene glycol dimethacrylate (EDMA) of 16% (wt%), the hexalin of 20% (wt%), 2 of the lauryl alcohol of 40% (wt%) and 1% (being the quality of function monomer and linking agent), ultrasonic the mixing of mixing solutions of 2-dimethoxy-2-phenyl methyl phenyl ketone (DMPA), after logical nitrogen 5min, pack in the polypropylene liquid-transfering gun rifle head of the two keys of inwall bonding, by the light-initiated synthetic rifle head integral post of original position, light-initiated wavelength is 365nm, and light intensity is 20mw/cm 2, light application time is 6min.After having reacted, utilize washed with methanol rifle head integral post, to remove pore-creating agent and the unreacted material in post, thereby make poly (LMA-co-EDMA) rifle head integral post.
The surface graft modification of embodiment 7:Poly (BMA-co-EDMA) and Poly (LMA-co-EDMA) integral post
Respectively Poly (BMA-co-EDMA) and Poly (LMA-co-EDMA) integral post are carried out to surface grafting glycidyl methacrylate, bonding epoxide group, so that follow-up functional modification.Concrete steps are as follows: in integral post, be full of the methanol solution that concentration is the deoxidation benzophenone of 5wt%, and illumination 15min under UV 365nm, light intensity is 20mw/cm 2.After utilizing methyl alcohol that integral post is thoroughly cleaned, then be full of the methanol solution that concentration is the glycidyl methacrylate of 15wt%, illumination 15min under UV 365nm, light intensity is 20mw/cm 2, finally utilize methyl alcohol thoroughly to clean integral post.
Embodiment 8: nanometer gold organic polymer capillary monolithic column synthetic
By poly (GMA-co-EDMA) capillary monolithic column synthetic in example 4 (3) first with the cystamine solution reaction of 1mol/L, temperature of reaction is 50 DEG C, reaction times 2h.After having reacted, water is extremely neutral by integral post punching.Afterwards, integral post is at room temperature reacted to 2h with dithiothreitol (DTT) (DTT) or three (2-carbonyl ethyl) phosphorus (TCEP) solution of 0.25mol/L, can obtain SH-poly (GMA-co-EDMA) integral post of finishing sulfydryl.It is 7.23wt% that energy Mass spectrum (EDS) is measured the S constituent content showing in integral post, has shown in integral post successful bonding sulfydryl (SH).Finally, the nanometer gold colloid aqueous solution that is 15nm by particle diameter synthetic in example 1 pumps into syringe pump in the integral post of finishing sulfydryl, and bonding nanometer gold in integral post, until saturated, obtain nanometer gold integral post, the scanning electronic microscope (SEM) in its cross section as shown in Figure 7.It is 52.70wt% that energy Mass spectrum (EDS) is measured the Au constituent content showing in nanometer gold integral post, the nanometer gold that shown in integral post successful bonding.
Embodiment 9: the preparation method of the immobilized trypsinase micro-bioreactor of nanometer gold integral post
By the AuNPs-poly preparing in embodiment 8 (GMA-co-EDMA) integral post, after phosphate buffer solution flushing balance with 50mmol/L pH 7.2, the trypsinase that is 50mmol/L by 1mL concentration pumps in AuNPs-poly (GMA-co-EDMA) integral post, enzyme is fixed on integral post nano surface gold carrier to saturated, rinse this integral post with the phosphoric acid buffer of 50mmol/L pH 7.2, remove loose enzyme, obtain the immobilized trypsinase micro-bioreactor of nanometer gold integral post.
Embodiment 10: the immobilized trypsinase micro-bioreactor of nanometer gold integral post enzymolysis cytochrome C
Immobilized nanometer gold integral post synthetic in embodiment 9 trypsinase micro-bioreactor is applied to enzymolysis cytochrome C.Concrete steps are as follows: in the ammonium bicarbonate soln of 100mmol/L (pH 8.2), and the cytochrome C that configuration concentration is 1.5mg/mL.Adopt syringe pump under the flow velocity of 0.5 μ L/min, cytochrome c solution is pumped in the immobilized trypsinase micro-bioreactor of nanometer gold integral post synthetic in embodiment 9, at 37 DEG C, enzyme digestion reaction occurs.Flowing liquid is collected in the sample bottle of 1.5mL, carry out liquid chromatography (HPLC) and analyze the polypeptide fragment (Fig. 8) after enzymolysis, meanwhile, utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF-MS) to analyze the polypeptide fragment (Fig. 9) after enzymolysis.
The cytochrome C that is 1.5mg/mL by the concentration of above-mentioned configuration, be diluted to 0.15mg/mL with the ammonium acetate solution of pH=8.75, be that 50: 1 (w/w) adds trypsin solution by material mass ratio, in shaking table, at 37 DEG C, react 24h, add afterwards the acetic acid solution termination reaction of 1mL, by solution filter, carry out liquid chromatography mass coupling (HPLC-MS) and analyze the polypeptide fragment (Fig. 8) after enzymolysis.
Embodiment 11: the immobilized trypsinase micro-bioreactor of nanometer gold integral post enzymolysis bovine serum albumin (BSA)
Immobilized nanometer gold integral post synthetic in example 2 trypsinase micro-bioreactor is applied to enzymolysis bovine serum albumin (BSA).Concrete steps are as follows:
(1), in the ammonium bicarbonate soln that contains 8mol/L urea that is 8.2 in pH value, obtaining concentration is the BSA solution of 5mg/mL.
(2) get the BSA solution 1mL configuring in step (1), add dithiothreitol (DTT) (DTT) solution of the 45mM of 1mL, at 60 DEG C, react 45min.
(3) solution obtaining in step (2) is taken out, return to room temperature.Adding 1mL concentration is iodo-acid amide (IAA) solution of 100mM again, reacts 30min in dark surrounds.
(4) solution obtaining in step (3) being added to concentration is the ammonium acetate solution (pH=8.75) of 50mM, the ultra-filtration centrifuge tube that is 30000 with molecular weight cut-off under the rotating speed of 6000rpm centrifugal 3 times, each 30min.
(5) ammonium acetate solution (pH=8.75) that is 50mM by the solution after centrifugal in step (4) by concentration is diluted to 0.15mg/mL.
(6) adopt syringe pump under the flow velocity of 0.5 μ L/min, the BSA solution obtaining in step (5) is pumped in the immobilized trypsinase micro-bioreactor of nanometer gold integral post synthetic in example 2, at 37 DEG C, enzyme digestion reaction occurs.Flowing liquid is collected in the sample bottle of 1.5mL, carry out liquid chromatography (HPLC) and analyze the polypeptide fragment (Figure 10) after enzymolysis, meanwhile, utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF-MS) to analyze the polypeptide fragment (Figure 11) after enzymolysis.
(7) the BSA solution that is 0.15mg/mL by the concentration of configuration in step (5), be that 50: 1 (w/w) adds trypsin solution by material mass ratio, in shaking table, at 37 DEG C, react 24h, add afterwards the acetic acid solution termination reaction of 1mL, by solution filter, carry out liquid chromatography (HPLC) and analyze the polypeptide fragment (Figure 10) after enzymolysis.
Embodiment 12: the preparation method of the immobilized rice of nanometer gold integral post root miehei lipase micro-bioreactor
Rice root miehei lipase is dissolved in the phosphate buffer solution of pH 7.6, and concentration is 50mmol/L.Utilize the flow velocity of syringe pump with 0.5 μ L/min, under room temperature, rice root miehei lipase solution is pumped in nanometer gold integral post synthetic in example 1, until saturated.Afterwards, rinse this integral post with the phosphate buffer solution of 0.5mL 50mmol/L pH 7.2, remove the not rice root miehei lipase of bonding, obtain nanometer gold immobilization rice root miehei lipase micro-bioreactor.
Example 13: the immobilized rice of nanometer gold integral post root miehei lipase micro-bioreactor chiral separation Ibuprofen BP/EP propyl ester
Immobilized nanometer gold integral post synthetic in example 10 rice root miehei lipase micro-bioreactor is applied to chiral separation Ibuprofen BP/EP.At 40 DEG C, the racemize Ibuprofen BP/EP of 0.03mol/L and the n-propyl alcohol of 0.06mol/L are dissolved in normal hexane, with the flow velocity of 0.5uL/min, carry out continuous catalysis, and externally racemic ibuprofen carries out chiral separation, and transformation efficiency is 46, and selectivity E value is 9.And the selectivity E value of rice root miehei lipase under unbound state is only 2.
The hydrochloro-auric acid that the nanometer gold colloid of the synthetic different-grain diameter size of table 1 is used and the consumption of Trisodium Citrate.
The polypeptide fragment that table 2 nanometer gold integral post immobilized enzyme carries out enzymolysis to cytochrome C is compared correct polypeptide fragment after secondary analysis.
The polypeptide fragment that table 3 nanometer gold integral post immobilized enzyme carries out enzymolysis to bovine serum albumin (BSA) is compared correct polypeptide fragment after secondary analysis.
Table 1
Table 2
Table 3

Claims (3)

1. a preparation method for nano material integral post enzyme immobilization micro-bioreactor, is characterized in that comprising: steps A, preparation nano material; Nano material comprises nanometer gold, nanometer silver, nano silicon, football alkene C 60, carbon nanotube, Graphene, magnetic ferroferric oxide, metal-organic framework materials MOF or covalency organic backbone COF; Wherein, the particle diameter of nano material is at 5～1000nm;
Step B, synthesizing of Organic Polymer Monolithic Columns: comprise that mass percent is 5～30% function monomers, 5～30% linking agents and 30～80% pore-creating agents, and the quality summation of function monomer, linking agent and pore-creating agent is 100%, also add in addition the mixing solutions of 0.2～5% initiator of function monomer and linking agent quality, by original position thermal initiation or light-initiated in stainless steel chromatogram post, kapillary, polypropylene-base rifle head, polypropylene-base syringe, high-boron-silicon glass plate and silicon wafer synthesizing porous Organic Polymer Monolithic Columns; Wherein the internal diameter of stainless steel chromatogram post is 3-6mm, and length is 6-30cm; Internal diameter capillaceous is 50-600 μ m, and length is 5-50cm; The volume of polypropylene-base rifle head is 0.01-10mL; The volume of polypropylene-base syringe is 0.1-10mL; Thermal booster reaction temperature is 45～85 DEG C, reaction times 6～48h; Light-initiated wavelength is 254nm or 365nm, and light intensity is 6-30mw/cm 2, light application time is 5-60min; Function monomer comprises glycidyl methacrylate, methyl methacrylate, butyl methacrylate, lauryl methacrylate(LMA), vinylformic acid, acrylamide, NIPA, methacrylic acid proyl ester, glycidyl propargyl ether, 2-vinyl-4,4-dimethyl-2-oxazoline-5-ketone, 2-methyl-2-vinylformic acid-2,3-dihydroxyl propyl ester, vinylbenzene or 4-vinylpridine; Linking agent comprises ethylene glycol dimethacrylate, iso-cyanuric acid triallyl ester, Vinylstyrene, N, N '-methylene-bisacrylamide; Pore-creating agent comprises the polyoxyethylene glycol of toluene, normal heptane, hexalin, lauryl alcohol, n-propyl alcohol, BDO or different molecular weight; Initiator comprises Diisopropyl azodicarboxylate, 2,2'-Azobis(2,4-dimethylvaleronitrile), 2,2-dimethoxy-2-phenyl methyl phenyl ketone or 2-hydroxy-2-methyl-1-phenyl-acetone;
Step C, nano materials integral post: porous organo polysilica compound integral post is carried out to functional modification;
Step D taking nano material as middle part, realizes the immobilization of enzyme in integral post, design preparation nano material integral post enzyme immobilization micro-bioreactor.
2. the preparation method of nano material integral post enzyme immobilization micro-bioreactor as claimed in claim 1, is characterized in that: enzyme solution is the phosphate buffer solution preparation with pH 6.5～8.0, and its concentration is 5～50mg/mL.
3. the nano material integral post enzyme immobilization micro-bioreactor of preparing by the preparation method described in claim 1 or 2.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102391947A (en) * 2011-11-03 2012-03-28 广西师范大学 Preparation method for porous monolithic column immobilized enzyme micro-reactor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102391947A (en) * 2011-11-03 2012-03-28 广西师范大学 Preparation method for porous monolithic column immobilized enzyme micro-reactor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
任华华，刘丽娜，柳晗等: "金纳米粒子修饰有机聚合物整体柱的制备及其在柱SERS检测研究", 《 湖北大学学报(自然科学版》 *
何丽，冯海建，李静杰，刘欢等: "有机聚合物整体柱的改性与应用进展", 《分析测试学报 》 *
吕永琴，谭天伟，FRANTISEK SVEC: "新型纳米金整体柱的制备及在HPLC分离和酶反应器中的应用", 《2013中国化工学会年会论文集》 *
谢柏盛等: "金属框架结构材料MOF-199对漆酶的固定化及其性质", 《生物加工过程》 *

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