CN110028586A - A kind of large biological molecule microreactor fixed based on protein film isolation - Google Patents

A kind of large biological molecule microreactor fixed based on protein film isolation Download PDF

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Publication number
CN110028586A
CN110028586A CN201910334485.0A CN201910334485A CN110028586A CN 110028586 A CN110028586 A CN 110028586A CN 201910334485 A CN201910334485 A CN 201910334485A CN 110028586 A CN110028586 A CN 110028586A
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biological molecule
caco
large biological
microreactor
particle
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杨鹏
刘瑞瑞
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Shaanxi Normal University
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Shaanxi Normal University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
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  • Molecular Biology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Inorganic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
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Abstract

The invention discloses a kind of microreactors that fixed large biological molecule is isolated based on protein film, the functional biomacromolecules being isolated will be needed to be supported on CaCO by the method being co-precipitated first3Inside particle and surface, or the method by directly adsorbing are supported on CaCO3Then protein film is wrapped in CaCO by particle surface3Particle surface removes internal CaCO finally by EDTA or acid solution etching3Template realizes that functional biomacromolecules are fixed in the isolation of microcosmic point, and then the performance for the large biological molecule physiological function that is independent of each other.The preparation method of microreactor of the present invention is simple, low in cost, environmentally protective, the microcosmic isolation to various functional biomacromolecules can be achieved, including various protein, enzyme, antibody, lipid, polypeptide, DNA, RNA etc., with extensive universality, it easily realizes large-scale industrial application, and size, the shape of the microreactor that large biological molecule is isolated can be regulated and controled by template.

Description

A kind of large biological molecule microreactor fixed based on protein film isolation
Technical field
The present invention relates to one kind to be realized to various functional biomacromolecules based on kind of starch sample protein film in microcosmic Level carries out isolation fixation, and then is independent of each other and plays the large biological molecule microreactor of respective physiological function.
Background technique
In organism, most material conversions are realized by many kinds of enzyme sequential catalysts.In order to guarantee The high efficiency of sequential catalyst, the enzyme of these Collaboration are usually concentrated in certain cells or organelle.This physiological structure Scattering and disappearing for intermediate product not only can be effectively reduced, can also prevent the by-product in material conversion process from making to the murder by poisoning of body With.Therefore just seem very important for the package of large biological molecule and fixation.Gold with special colloform texture and property Belong to, inorganic or protein tridimensional capsule or microreactor are since it has the space being isolated and controllable permeability etc. multi-functional With potential application, has in the different fields such as medicine/gene delivering, catalysis, sensor, medicine, cosmetics and be widely applied And it is particularly concerned.Up to the present, extensive hollow structure has been developed, such as condensate, multilayer capsule and tiny balloon Etc. the preparation method of various microreactors.However its most preparation method is complicated, with high costs, biocompatibility is poor, such as Layer-by-layer (LBL), be difficult to meet it is industrial there is an urgent need to.Therefore it provides a kind of simply and effectively realize biology The microreactor preparation method of the microcosmic isolation of macromolecular seems urgent need.
Summary of the invention
The purpose of the present invention is in view of the above shortcomings of the prior art, provide a kind of easy to operate, low in cost, green ring It protects, there is extensive universality, realize and isolation fixation is carried out in microcosmic point to various functional biomacromolecules, and then mutually not Influence the large biological molecule microreactor of the respective physiological function of performance.
To achieve the above object, large biological molecule microreactor of the present invention is prepared by the following method to obtain:
1, by Na2CO3And CaCl2It is added to the water, and large biological molecule is added, stir 15~60 seconds, stands 2~10 minutes Afterwards, by deionized water eccentric cleaning, the CaCO that load has large biological molecule is obtained3Particle；Or by CaCO3Particle is scattered in It in large biological molecule aqueous solution, cultivates 12~24 hours at room temperature, obtains the CaCO that load has large biological molecule3Particle.
2, by the phosphate buffer of 1~200mmol/L tri- (2- carboxyethyl) phosphine with NaOH be adjusted to pH value be 6.5~ 7.5, then it is uniformly mixed with the phosphate buffer of 1~50mg/mL protein, and the above-mentioned load prepared is added to have The CaCO of large biological molecule3Particle is cultivated 30~60 minutes at room temperature, and eccentric cleaning removes reaction impurities, and it is thin to obtain protein The load of film package has the CaCO of large biological molecule3Particle.
3, the load that protein film is wrapped up is had to the CaCO of large biological molecule3Particle is immersed in 0.001~0.1mol/L EDTA solution or pH be 1~6 acid solution in, etch away CaCO3, it is big to obtain the fixed biology of protein film isolation Molecule microreactor.
In above-mentioned steps 1, by Na2CO3And CaCl2It is added to the water, preferably Na in control water2CO3Concentration be 0.1~ 0.6moml/L、CaCl2Concentration be 0.1~0.6moml/L, Na2CO3And CaCl2Molar ratio be 1:1.
In above-mentioned steps 1, the large biological molecule is protein, enzyme, antibody, lipid, polypeptide, any in DNA, RNA One or more, preferably the addition quality of large biological molecule is CaCO3The 0.01%~1% of granular mass.
In above-mentioned steps 2, preferably the phosphate buffer of 8~40mmol/L tri- (2- carboxyethyl) phosphine is adjusted to NaOH PH value is 7.0, is then uniformly mixed it for 1:1 by volume with the phosphate buffer of 2~10mg/mL protein, and is added Enter the CaCO that the above-mentioned load prepared has large biological molecule3Particle is cultivated 30~60 minutes at room temperature.
In above-mentioned steps 2, the protein is lysozyme, bovine serum albumin(BSA), insulin, any in α-lactalbumin It is a kind of.
In above-mentioned steps 3, the load that protein film is wrapped up preferably is had to the CaCO of large biological molecule3Particle is immersed in In the acid solution that the EDTA solution or pH of 0.01~0.1mol/L is 3~4, CaCO is etched away3。
Beneficial effects of the present invention are as follows:
1, it is realized the present invention is based on protein film and various functional biomacromolecules be isolated admittedly in microcosmic point It is fixed, and then the large biological molecule microreactor for the respective physiological function of performance being independent of each other.The present invention can both be realized to each The microcosmic isolation of kind large biological molecule can also wrap up a variety of large biological molecules into the micro- reaction of protein film simultaneously as desired Device is isolated.
2, the preparation process of large biological molecule microreactor of the present invention is simple and efficient, is easily operated, while low in cost, green Colour circle is protected, and can be used for realizing the microcosmic isolation to various large biological molecules, including various protein, enzyme, antibody, lipid, polypeptide, DNA, RNA etc. have extensive universality, Yi Shixian large-scale industrial application.
3, the protein film in large biological molecule microreactor of the present invention has good biocompatibility and contains in itself There is functional group abundant, these functional groups ensure that the subsequent various functionalizations to microreactor, and in various substrates The fixing process on surface.
4, the protein film in large biological molecule microreactor of the present invention has extraordinary isolation effect.For 3nm Small molecule below may be implemented freely to circulate, and the macromolecular of 3nm or more may be implemented 100% barrier.
5, the present invention is by adjusting Na2CO3And CaCl2Concentration and the two hybrid reaction time and revolving speed can control CaCO processed3The size and shape of particle, and then can control the shapes and sizes of microreactor.
Detailed description of the invention
Fig. 1 is the CaCO that the load that embodiment 1 obtains has bovine serum albumin(BSA)3The optical photograph of particle.
Fig. 2 is CaCO3The load that particle and embodiment 1 obtain has the CaCO of bovine serum albumin(BSA)3Particle (CaCO3@BSA- FITC fluorogram analysis).
Fig. 3 is that embodiment 1 obtains the CaCO that load has bovine serum albumin(BSA)3The fluorescence (left side) and optics (right side) of particle are micro- Figure.
Fig. 4 is the CaCO that the load that embodiment 1 obtains has bovine serum albumin(BSA)3The scanning electron microscopy of particle.
Fig. 5 is CaCO3The load that particle and embodiment 1 obtain has the CaCO of bovine serum albumin(BSA)3Particle (CaCO3@BSA- FITC infrared spectrum analysis).
Fig. 6 is the CaCO that the load that embodiment 1 obtains has bovine serum albumin(BSA)3The fluorescence intensity profile analysis of particle.
Fig. 7 is that the load for the lysozyme film package that embodiment 1 obtains has the CaCO of bovine serum albumin(BSA)3The fluorescence of particle (left side) and optics (right side) micrograph.
Fig. 8 is that the load for the lysozyme film package that embodiment 1 obtains has the CaCO of bovine serum albumin(BSA)3The scanning of particle Electron micrograph.
Fig. 9 is the fluorescence (left side) of the bovine serum albumin(BSA) microreactor for the lysozyme film isolation fixation that embodiment 1 obtains With optics (right side) micrograph.
Figure 10 is the scanning electron of the bovine serum albumin(BSA) microreactor for the lysozyme film isolation fixation that embodiment 1 obtains Micrograph.
Figure 11 is the fluorescence intensity of the bovine serum albumin(BSA) microreactor for the lysozyme film isolation fixation that embodiment 1 obtains Profile analysis.
Figure 12 is the CaCO that the load that embodiment 2 obtains has bovine serum albumin(BSA)3The fluorescence (left side) and optics (right side) of particle Micrograph.
Figure 13 is the CaCO that the load that embodiment 2 obtains has bovine serum albumin(BSA)3The scanning electron microscopy of particle.
Figure 14 is the microreactor of lysozyme film package bovine serum albumin(BSA) and human serum albumins that embodiment 4 obtains Optics (left side) and fluorescence (right side) micrograph.
Figure 15 is the microreactor optics of PTL film package bovine serum albumin(BSA) and chicken ovalbumin that embodiment 4 obtains (left side) and fluorescence (right side) micrograph.
Specific embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to These embodiments.
Embodiment 1
1,5mL 0.33mol/L Na is prepared respectively2CO3Aqueous solution and 0.33mol/L CaCl2Aqueous solution, then by 100 μ The Bovine Serum Albumin in Aqueous Solution of L 10mg/mL fluorescent marker is added to CaCl2In solution aqueous solution, Na is added2CO3It is water-soluble Liquid, and 650rpm is kept to stir 30 seconds, it stands after five minutes, by deionized water eccentric cleaning, obtains internal and area load There is the CaCO of bovine serum albumin(BSA)3Particle.As shown in figs. 1 to 6, being successfully prepared internal and area load has bovine serum albumin White CaCO3Particle.
2, the PBS buffer solution of 1mL 10mmol/L tri- (2- carboxyethyl) phosphine is adjusted to pH value with NaOH is 7.0, then will It is uniformly mixed with the PBS buffer solution of 1mL 2mg/mL lysozyme, and the above-mentioned load prepared is added has bovine serum albumin(BSA) CaCO3Particle is cultivated 50 minutes at room temperature；Reaction impurities are then removed by eccentric cleaning, obtain lysozyme film package Load have the CaCO of bovine serum albumin(BSA)3Particle.As shown in Fig. 7~8, successfully there is ox blood in load by a step Aqueous phase The CaCO of pure albumen3Particle surface wraps up lysozyme film.
3, the load for wrapping up lysozyme film has the CaCO of bovine serum albumin(BSA)3Particle is immersed in 0.05mmol/L's In EDTA aqueous solution, CaCO is etched away3, obtain the fixed bovine serum albumin(BSA) microreactor of lysozyme film isolation.As Fig. 9~ Shown in 11, by by template CaCO3After particle etching, it is micro- anti-successfully to obtain the fixed bovine serum albumin(BSA) of lysozyme film isolation Answer device.
Embodiment 2
In the step 1 of the present embodiment, 5mL 0.33mol/L Na is prepared respectively2CO3Aqueous solution and 0.33mol/L CaCl2 Aqueous solution, then by Na2CO3Aqueous solution is added to CaCl2In aqueous solution, and 650rpm is kept to stir 30 seconds, stands 5 minutes Afterwards, by deionized water eccentric cleaning, CaCO is obtained3Particle.The CaCO that will be obtained3Particle is scattered in 100 μ L 10mg/mL fluorescence In the Bovine Serum Albumin in Aqueous Solution of label, cultivates 24 hours at room temperature, obtain the CaCO that area load has bovine serum albumin(BSA)3 Particle, as shown in Figure 12~13.Other steps are same as Example 1, obtain the fixed bovine serum albumin of lysozyme film isolation White microreactor.
Embodiment 3
In the step 1 of the present embodiment, 5mL 0.33mol/L Na is prepared respectively2CO3Aqueous solution and 0.33mol/L CaCl2 Aqueous solution, then by the Bovine Serum Albumin in Aqueous Solution and 100 μ L10mg/mL fluorescent markers of 100 μ L 10mg/mL fluorescent markers Human serum albumins aqueous solution be added to CaCl2In solution aqueous solution, other steps are same as Example 1, obtain lysozyme The microreactor of film isolation fixed bovine serum albumin(BSA) and human serum albumins (see Figure 14).
Embodiment 4
In the step 1 of the present embodiment, 5mL 0.33mol/L Na is prepared respectively2CO3Aqueous solution and 0.33mol/L CaCl2 Aqueous solution, then by the Bovine Serum Albumin in Aqueous Solution and 100 μ L10mg/mL fluorescent markers of 100 μ L 10mg/mL fluorescent markers Chicken ovalbumin aqueous solution be added to CaCl2In solution aqueous solution, other steps are same as Example 1, and it is thin to obtain lysozyme The microreactor of film isolation fixed bovine serum albumin(BSA) and chicken ovalbumin (see Figure 15).
Embodiment 5
In the present embodiment, with the lysozyme in bovine serum albumin(BSA) alternative embodiment 1, other steps are same as Example 1, Obtain the fixed bovine serum albumin(BSA) microreactor of bovine serum albumin white film isolation.
Embodiment 6
In the present embodiment, with the lysozyme in insulin alternative embodiment 1, other steps are same as Example 1, obtain pancreas The fixed bovine serum albumin(BSA) microreactor of island element film isolation.
Embodiment 7
In the present embodiment, with the lysozyme in α-lactalbumin alternative embodiment 1, other steps are same as Example 1, obtain The bovine serum albumin(BSA) microreactor fixed to the isolation of α-lactalbumin film.

Claims (7)

1. a kind of large biological molecule microreactor fixed based on protein film isolation, it is characterised in that the microreactor is under The method of stating is prepared:
(1) by Na2CO3And CaCl2It is added to the water, and large biological molecule is added, stirs 15~60 seconds, standing 2~after ten minutes, By deionized water eccentric cleaning, the CaCO that load has large biological molecule is obtained3Particle；Or by CaCO3Particle is scattered in biology It in macromolecular aqueous solution, cultivates 12~24 hours at room temperature, obtains the CaCO that load has large biological molecule3Particle；
(2) phosphate buffer of 1~200mmol/L tri- (2- carboxyethyl) phosphine is adjusted to pH value with NaOH is 6.5~7.5, Then it is uniformly mixed with the phosphate buffer of 1~50mg/mL protein, and the above-mentioned load prepared is added biology The CaCO of macromolecular3Particle is cultivated 30~60 minutes at room temperature, and eccentric cleaning removes reaction impurities, obtains protein film packet The load wrapped up in has the CaCO of large biological molecule3Particle；
(3) load that protein film is wrapped up is had to the CaCO of large biological molecule3Particle is immersed in 0.001~0.1mol/L's In the acid solution that EDTA solution or pH are 1~6, CaCO is etched away3, obtain big point of the fixed biology of protein film isolation Sub- microreactor.
2. the large biological molecule microreactor fixed based on protein film isolation according to claim 1, feature exist In: in step (1), by Na2CO3And CaCl2It is added to the water, controls Na in water2CO3Concentration be 0.1~0.6moml/L, CaCl2 Concentration be 0.1~0.6moml/L, Na2CO3And CaCl2Molar ratio be 1:1.
3. the large biological molecule microreactor fixed based on protein film isolation according to claim 1, feature exist In: in step (1), the large biological molecule is protein, enzyme, antibody, lipid, polypeptide, any one in DNA, RNA or more Kind.
4. the large biological molecule microreactor fixed based on protein film isolation according to claim 3, feature exist In: the addition quality of the large biological molecule is CaCO3The 0.01%~1% of granular mass.
5. the large biological molecule microreactor fixed based on protein film isolation according to claim 1, feature exist In: in step (2), it is 7.0 that the phosphate buffer of 8~40mmol/L tri- (2- carboxyethyl) phosphine, which is adjusted to pH value with NaOH, Then it is uniformly mixed by volume for 1:1 with the phosphate buffer of 2~10mg/mL protein, and above-mentioned preparation is added Good load has the CaCO of large biological molecule3Particle is cultivated 30~60 minutes at room temperature.
6. the large biological molecule microreactor fixed based on protein film isolation according to claim 1 or 5, feature Be: in step (2), the protein is lysozyme, bovine serum albumin(BSA), insulin, any one in α-lactalbumin.
7. the large biological molecule microreactor fixed based on protein film isolation according to claim 1, feature exist In: in step (3), the load that protein film is wrapped up is had to the CaCO of large biological molecule3Particle is immersed in 0.01~0.1mol/ In the acid solution that the EDTA solution or pH of L is 3~4, CaCO is etched away3。
CN201910334485.0A 2019-04-24 2019-04-24 A kind of large biological molecule microreactor fixed based on protein film isolation Pending CN110028586A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106479997A (en) * 2016-11-28 2017-03-08 陕西师范大学 Lysozyme nanocrystalline colloidal sol and the protein polycrystalline hydrogel prepared using which and preparation method
CN108451924A (en) * 2017-12-13 2018-08-28 温州生物材料与工程研究所 The method that one step absorption method prepares protein microcapsules
CN108751126A (en) * 2018-06-08 2018-11-06 陕西师范大学 The method for preparing three-dimensional self-supporting film based on lysozyme nano thin-film

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106479997A (en) * 2016-11-28 2017-03-08 陕西师范大学 Lysozyme nanocrystalline colloidal sol and the protein polycrystalline hydrogel prepared using which and preparation method
CN108451924A (en) * 2017-12-13 2018-08-28 温州生物材料与工程研究所 The method that one step absorption method prepares protein microcapsules
CN108751126A (en) * 2018-06-08 2018-11-06 陕西师范大学 The method for preparing three-dimensional self-supporting film based on lysozyme nano thin-film

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RUIRUI LIU ET AL.: "One-Step Assembly of a Biomimetic Biopolymer Coating for Particle Surface Engineering", 《ADV. MATER.》 *
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