CN102373192A - Immobilization method of proteinase molecule by using nano-material and application thereof - Google Patents
Immobilization method of proteinase molecule by using nano-material and application thereof Download PDFInfo
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Abstract
The invention discloses an immobilization method of a proteinase molecule by using a nano-material, namely a carbon nano-tube. The method comprises the following steps: (1) dispersing the carbon nano-tube to form a uniform solution; (2) acidifying the carbon nano-tube, wherein the carbon nano-tube obtained in the step (1) is acidified with mixed acid of concentrated nitric acid and concentrated sulfuric acid at a volume ratio of 1:2-1:5; and (3) functionalizing the carbon nano-tube, wherein a -NH2 functional group is functionally connected to the wall of the carbon nano-tube by utilizing NHS (N-hydroxy-succinamide) and EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and reaction is carried out with -NH2 of enzyme to immobilize the enzyme on the tube wall. The preparation process has convenient and quick procedure, stable and firm bonding and high efficiency. The carbon nano-tube for bio-catalysis or transformation in energy, environmental and biochemical pharmacy and other fields has low price, is easy for scale utilization, and is a nano-enzyme catalysis means with practical application prospect.
Description
Technical field
The present invention relates to biological nano preparation and biological nano enzyme catalysis field, more particularly, the present invention relates to the method and the application thereof of the immobilized protease molecule of nano material.
Background technology
For the protein stabilization Journal of Sex Research, as far back as middle 1990s, people just begin protein stability has been carried out systematic research.At present; Improvement to protein stability mainly is that it is modified and the various stablizers of interpolation in solution both at home and abroad; The former is because the higher modification pharmaceutical protein that is mainly used in of cost; For example polyoxyethylene glycol (PEG) is modified protein medicaments such as various cytokines, and the latter is mainly used in various zymins.Through stablizer come stabilizing protein comparison its to carry out chemically modified simple, cost is also lower; And protein is after process is modified, and change has all taken place for structure, activity and antigenicity, and still, this change is disadvantageous to proteinic reality utilization often, so study the stability that the method for attempting the interpolation stablizer realizes proteolytic enzyme both at home and abroad.
The unstable of proteolytic enzyme is a problem that always exists, and it is seriously restricting the practical application of enzyme.Because proteolytic enzyme is a kind of macromolecular substance with complex spatial three-dimensional arrangement; Be subject to the sex change that influences of ambient conditions; Each proteolytic enzyme all has own distinctive space structure or three-dimensional structure simultaneously, so be difficult to find a kind of stable agent to be fit to all protein.This causes developing the prescription not a duck soup that is fit to the prolonged preservation protein formulation.This mainly is because the physics of proteolytic enzyme and chemical instability determine; Different with low-molecular-weight compound; Want to keep protease activities just must keep its three-dimensional structure constant, in addition, the amino-acid residue of constitutive protein matter skeleton is easy to take place chemical degradation; Therefore be familiar with the unstable of proteolytic enzyme from molecular level, the stable problem that solves numerous protease preparations has great realistic significance.The unstable of proteolytic enzyme mainly is because of its congregation, and the gathering of proteolytic enzyme mainly is the change because of its conformation, and protein conformation changes and increases along with reducing of solvent polarity.Because protein contains wetting ability and hydrophobic group simultaneously, hydrophobic part extends sex change easily, so the protein hydrophobic site is many more, and easy more the gathering.The traditional viewpoint of protein aggregation is thought with proteinic stretching mode relevant, but according to up-to-date research, think protein aggregation be with folding and stretch between a kind of middle model relevant.Protein all folding or that all open is difficult for assembling, and do not contact with water or random exposure because the chain of their hydrophobic sides wraps up fully, and the protein of intermediate state has caused the accumulative generation in order to cover up contiguous hydrophobic grouping.Other has reported in literature, and protein aggregation is all produced by βZhe Die basically, and the α spiral is little to the accumulative influence, and this possibly be because the dipole moment of α spiral strong than βZhe Die.Proteinic degradation pathway can be divided into two types; Be chemical instability and physical instability. chemical instability is because the modification/variation of amino acid whose residue; Mainly contain several kinds of reactions: oxygenizement, reductive action, desamination reaction, hydrolytic action, l-arginine transform, β eliminates and racemization. and physical instability relates to proteinic secondary, three grades, the variation of quaternary structure; Do not relate to the variation of protein covalent linkage, comprise sex change, surface adsorption, cohesion and deposition.Relevant in addition research surface, secondary protein structure is controlled proteinic gathering, stability and toxicity.
There is certain obstacle in resolvase to the Industrial Catalysis process.The enzyme catalysis process has obtained more application in industry; But still the following obstacle of existence; Having limited enzyme uses in industry widely: (1) enzyme, often is difficult to keep secular stability owing to broken away from intracellular environment in the extracellular, loses activity easily; Especially at oil phase or oil-water interface, lose activity more easily; (2) speed of reaction is slow than chemical reaction; (3) because enzyme require extracts and purifying, and repeating utilization factor is low simultaneously, cost is high, cost further increases when relating to coenzyme system; (4) existing industrial application is confined to single enzyme catalysis system mostly, and the multi-enzyme system application difficult has limited the exploitation of more high value added products.The enzyme of occurring in nature has 700,000 kinds at least, has only 10,000 kinds structure and chemical property known at present, and realize industrial application only hundreds of.Therefore, aspect development and use enzyme resource, we can say just at the early-stagely, have a high potential, press for and overcome the obstacle that influence the enzyme industrial applications, the efficient utilization of realization enzyme.This not only need make single enzyme bring into play its high reactivity in each individual system (water, oil phase, oil-water interface etc.), and need to realize enzyme recycling, realize the concerted reaction and the regenerating coenzyme of multienzyme.In recent years, along with developing rapidly of nano science and nanotechnology, be expected to utilize nanotechnology to realize the breakthrough of above-mentioned gordian technique.
The tradition enzyme immobilization also exists certain defective.The immobilization of enzyme is with solid material enzyme to be fettered or is limited in certain zone, makes it still can carry out its distinctive catalyzed reaction, and recyclable and reusable one type of technology.By its character branch; Mainly contain four kinds; Be entrapping method, crosslinking, absorption method and covalent method; Compare with resolvase, immobilized enzyme when keeping its efficient, single-minded and gentle enzymic catalytic reaction characteristic, also appear package stability height, Separation and Recovery easily, can be repeatedly used, operate continuously and controlled, series of advantages such as technology is easy.The catalytic efficiency (of enzyme and selectivity depend on the specific surface area and the geometrical shape of the enzyme that uses to a great extent; As do not consider other factors, and then particle diameter is more little, and specific surface area is big more, and the efficient of catalyzer is just high more.Traditional immobilized enzyme particle diameter is generally (0.1-10) mm.Because receive the few and influence of resistance to mass transfer greatly of immobilized enzyme amount, the catalytic efficiency (of this type immobilized enzyme is more limited.Specific surface area is big, resistance to mass transfer is little, the immobilized enzyme amount many and the catalytic efficiency (advantages of higher and the immobilized enzyme of nano level has, and this makes the nano level immobilization technology of enzyme receive concern more and more widely.
Modern nanotechnology is expected to realize the held stationaryization and the stabilization of enzyme.Enzyme immobilization technology can solve enzyme stability and recycling problem to a certain extent, is utilized in the enzyme immobilization technology on the micron order carrier, can improve the stability of enzyme and realize the recycling of enzyme.But; Enzyme is tending to cause enzyme allosteric to a certain degree with the immobilization on the phase interface, the active reduction, and the contact frequency that is fixed on simultaneously between the enzyme-to-substrate on the carrier reduces; Cause catalytic efficiency (further to reduce, limited more carrying out in a deep going way of enzyme immobilization technology research.After being fixed on enzyme on the nano particle, find that nano particle has shown significant dimensional effect, the particle diameter of nano particle is more little, and the catalytic efficiency (of institute's immobilized enzyme is high more.Find that through theoretical investigation the pedesis of this and nano particle is relevant, particle is more little, and the collision frequency of raising of pedesis speed and substrate improves rapidly, thereby has improved catalytic efficiency (.The immobilized enzyme on the nano particle and the collision frequency of substrate are between resolvase and traditional micron order carrier immobilized enzyme; Both stablized the structure of enzyme through enzyme immobilization; Overcome the unsettled shortcoming of resolvase, overcome the low problem of catalytic efficiency (that the micron order carrier exists again.Simultaneously; The size of discovery nano particles such as Dordick has very big influence to the fixed sturcture of enzyme; They have compared particle diameter and have been respectively the influence of the nano SiO 2 particle of 4nm, 20nm, 100nm to activity of the immobilized enzyme, find that the nano particle diameter is more little, are not easy to make allostery more.The particle diameter of nano particle is more little, and the curvature of nano grain surface is big more, and the contact area of enzyme and nano grain surface is just more little, just is not easy to make the allostery inactivation; And the diameter of nano particle is when big, and the surface of nano particle becomes smooth, increases with the site that contacts of enzyme, makes the allostery inactivation easily.Therefore, the combination of nanotechnology and zymotechnic has broken through the notion of traditional enzyme with fixedization, for crucial solution has been found in the held stationary research of enzyme.Kim etc. have prepared the nano particle (single enzyme nanoparticle) of the single enzyme of embedding with nanotechnology embedding enzyme, and the nanometer restriceted envelope (microenvironment) in the nano particle makes that enzyme can't free allosteric, thereby has highly kept enzymic activity.
Summary of the invention
The object of the present invention is to provide the method for the immobilized protease molecule of nano material, said nano material refers to carbon nanotube.
Second purpose of the present invention provides the application of method in fixing, the stable and utilization of enzyme of the immobilized protease molecule of nano material.
For realizing above purpose, the present invention discloses following technical scheme: the method for the immobilized protease molecule of a kind of nano material, it is characterized in that said nano material refers to carbon nanotube, and comprise SWCN and multi-walled carbon nano-tubes, said method comprising the steps of:
(1) carbon nanotube disperses, to the solution that forms homogeneous;
(2) acidifying of carbon nanotube: be the carbon nanotube that the nitration mixture acidification step (1) of concentrated nitric acid and the vitriol oil of 1:2-1:5 obtains with volume ratio;
(3) functionalization of carbon nanotube: utilize NHS to connect being connected-NH of functionalization at the carbon nanotube tube wall with EDC
2Functional group, last and enzyme-NH
2The reaction, with enzyme immobilization at tube wall.
During functionalization, pH value is 7.0-7.5.
Said protease molecule; Be meant the superoxide dismutase (SOD), acetyl-CoA carboxylase (the Acetyl-CoA carboxylase that produce through genetically engineered; AACase), lypase (lipase), phosphatidic acid phosphatase (phosphatidic acid phosphatase; PAP) and diacylglycerol acyltransferase (diacylglycerol acyltransferase, DGAT), 1, one or more in ammediol oxydo-reductase, the people's Pseudocholinesterase.
The application of the method for the immobilized protease molecule of nano material in fixing, the stable and utilization of enzyme, said enzyme is meant and is used for the enzyme that biocatalysis or conversion are carried out in the energy, environment and biochemical pharmacy field.
The invention has the advantages that: the present invention disclose a kind of utilize single wall or carbon multi-wall nano tube loaded, fix, transport and utilize its contained protease molecule; And be applied to specific catalyzed reaction and catalystsystem, play effect stable, that transport and assist performance biopharmacy function.According to different carbon nanotubes, different protease molecule and different target purposes; Adopt specific technology that carbon nanotube is combined with protease molecule, for this type of protease molecule less stable, be difficult for the actual biomacromolecule technology of utilizing efficient performance its should have biological function that feasible technology is provided.Manufacture craft program of the present invention is easy, quick, and in conjunction with firmly stable, and efficient is high.The carbon nanotube that carries out biocatalysis or conversion especially for fields such as the energy, environment and biochemical pharmacies is cheap, is easy to the mass-producing utilization, is the nano enzyme catalysis means with actual application prospect.
Description of drawings
Fig. 1 is that multi-walled carbon nano-tubes is connected and fixing means with the concrete of SOD enzyme.
Fig. 2 is the fixedly schema of SWCN to the SOD enzyme.
Fig. 3 is a carbon nanotube immobilized enzyme quality determination table.
Fig. 4 is a zymoprotein bioassay standard curve.
Embodiment
Below in conjunction with accompanying drawing the present invention is elaborated, the effect of embodiment only is to explain and non-limiting the present invention.
Embodiment one: the activation of multi-walled carbon nano-tubes (MWNTs) and the immobilization of SOD enzyme.
0.5g commodity multi-walled carbon nano-tubes is poured in the nitration mixture of the 15ml concentrated nitric acid and the 45mL vitriol oil into container closure, the UW 3h that vibrates respectively; With big water gaging dilution, hold over night, 0.22 μ m millipore filtration; To neutral, weigh by 100 ℃ of vacuum dryings with the deionized water repetitive scrubbing for the multi-walled carbon nano-tubes that filtration obtains.Get the acid-treated MWNTs of exsiccant 50 mg, add in the PH=7.0 phosphate buffered saline buffer of 15ml, ultra-sonic dispersion is even; Again to carbodiimide that wherein adds 200 mg (EDC) and N-hydroxy-succinamide (NHS) 250 mg, ultrasonic 30 min; Get a certain amount of enzyme liquid and add normal temperature concussion 24h in the above-mentioned solution; Use the polycarbonate membrane filtration of 0.22 μ m then; The solid that obtains washs with phosphoric acid buffer, and dried in vacuum obtains final immobilized enzyme then.
Earlier on the carbon nanotube tube wall, generate carboxyl with nitration mixture, down auxiliary at carbon diamines (EDC) then, carboxyl and N-hydroxy-succinamide (NHS) form ester bond, with proteinic free ammonia cardinal extremity formation amido linkage, enzyme are fixed then.Concrete connection is as shown in Figure 1 with fixing means.
SOD connects and fixing front and back activity determination method; Adopt pyrogallol generation autoxidation to measure: the Tris-HCl of 50mM (PH=8.2) damping fluid 3ml; Be preheated to 250 ℃, add the pyrogallol of l0ul l50mM, fully shake up; Do blank with damping fluid, every at a distance from absorbancy of half a minute survey.Similarity condition adds l0ul SOD liquid to be measured, reads its absorbancy.
The mensuration of standard pyrogallol autoxidation curve; 50mmol/LTris-HCl pH8.2 damping fluid 3mI; Be preheated to 25 ℃; Add l0ul 50mmol/L pyrogallol and fully shake up, it is every at a distance from absorbancy of half a minute (adding timing from pyrogallol) survey to make blank with damping fluid, and making it autoxidation speed, to reach PM A325 be 0.070.
Standard SOD vitality test.The definition of enzyme activity unit: in the 1ml reaction solution, PM suppresses the enzyme amount that pyrogallol autoxidation speed reaches at 50% o'clock and is decided to be a unit of activity.
50mmol/L Tris-HCIpH8.2 damping fluid 3ml is preheated to 25 ℃, adds 5ommol/L pyrogallol (amount that autoxidation speed has reached) and each 10ul of sample simultaneously, fully shakes up, and does empty from contrast with damping fluid.Every separated half a minute (adding timing from biphenyl 3 phenol and sample) is surveyed an A325 value reaches about 50% inhibition pyrogallol autoxidation speed.The result shows that the mass ratio of homemade SWNTs and sample SOD is about 4:1.
Embodiment two: the functionalization multi-walled carbon nano-tubes is used for lipase immobilization.
(the multi-wall carbon nano-tube tube-surface after the nitration mixture of the concentrated nitric acid of volume ratio 1:2 ~ 1:5) and the vitriol oil is handled is connected with carboxyl through certain proportion; Use sulfur oxychloride and N again; Dinethylformamide carries out the acyl group chlorization and obtains chloride acyl group multi-walled carbon nano-tubes, and then chloride acyl group multi-walled carbon nano-tubes being reacted the multi-walled carbon nano-tubes that obtains the polyamino terminal with hexanediamine in chloroform is CNT-NH.Acidifying carbon nanotube and hexadecyl bromine etc. react under certain condition and obtain CNT-NH.
It is centrifugal that functionalized carbon nano-tube ultrasonic back in the Tris-HCl damping fluid adds the lypase liquid concussion of genetically engineered preparation.The immobilized enzyme amount utilizes Brandford to measure protein content in the supernatant.Adopt simultaneously ordinary method detect fixing before and after the activity change of lypase, and CNT – COOH shows typical band when adopting FTIR spectrum and Raman spectrum to characterize multi-walled carbon nano-tubes fixed fat enzyme.Through comparative analysis CNT-COOH, CNT-NH, CNT-R immobilization efficiency, find that CNT-COOH is most effective, tonburden is also maximum.
Through detect fixing before and after the activity change of lypase, find equivalent amounts of enzyme after adopting functionalized carbon nano-tube fixing, it is active significantly not to increase, but playing an active hold-time prolongs a lot, is up to 5-7 by doubly.For the repeatedly recycling and the raising service efficiency of this enzyme are laid a good foundation.
Embodiment three: SWCN (SWNTs) is to the immobilization of SOD enzyme.
6mgSWNTs is suspended in 6mlBMIM-BF4 (a kind of ionic liquid) gets liquid A; In 12mlDMF (N), add 60mgN-hydroxysuccinimide eater 1-pyrene butyric acid then and get liquid B; A liquid is added B liquid at room temperature mix concussion 12h; Add 30ml methyl alcohol then, functionalization SWNTs will precipitate; To precipitate with pure methanol wash three times, each 50ml removes unnecessary reductive agent; Dry under vacuum, obtain functionalization SWNTs; Getting 5mg functionalization SMNTs is suspended in and adds SOD solution (it is 7.2 phosphoric acid buffer that the 54mg enzyme is dissolved in 10ml20mM pH) among the 5ml BMIM-BF4 then; Above-mentioned heterogeneous mixture is at room temperature shaken 24h; Use the polycarbonate membrane filtration of 0.1 um then; The solid that obtains at last is with phosphoric acid buffer (25mlx2) washing, and dried in vacuum obtains final immobilized enzyme then.Concrete SWCN is as shown in Figure 2 to the fixedly schema of SOD enzyme.
In order to verify fixed enzyme amount, standard enzyme, sample enzyme and immobilized enzyme are carried out deciding albumen, carried out the preparation of Bradford working fluid: Coomassie brilliant blue G250 100mg is dissolved in the ethanol of 50ml 95%, adds the H of 100ml 85%
3PO
4, with distilled water diluting to 1000ml, filter paper filtering, brown bottle keeps in Dark Place.
In different condition, comprise aspects such as ph optimum, optimum temperuture, metals ion and storage stability, resolvase and immobilized enzyme Study on Properties are studied, find in optimum condition: there are FeSO in 37 ℃, pH7.0,3h
4, MnCl
2Perhaps CuCl
2The time, Fe
2+, Mn
2+, Cu
2+The final concentration of ion in system is lxl0
-3Mol/L ~ lxl0
-4Mol/L.The relative enzyme activity difference of resolvase and immobilized enzyme is not too remarkable, and just activity slightly descends after the immobilization.Influential according to the active site group of bibliographical information SOD and active site and immobilization.
Though the activity of enzyme does not reduce, aspect amount, immobilized amount is fewer, and its condition is still waiting further optimization.Its reason possibly be because in the operating process, SWNTs has loss.But aspect storage stability, the enzyme after the immobilization is carried out the repetition catalyst operation, see the enzyme activity changing conditions through after the multi-pass operations.With the enzyme after the immobilization and free enzyme in refrigerator 4 ℃, 37 ℃ and normal temperature are placed, and whenever survey an enzyme activity at a distance from 10 days, and enzyme work changes all enzyme height of specific ionization, is up to 7 times.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (4)
1. the method for the immobilized protease molecule of nano material is characterized in that, said nano material refers to carbon nanotube, comprises SWCN and multi-walled carbon nano-tubes, said method comprising the steps of:
(1) carbon nanotube disperses, to the solution that forms homogeneous;
(2) acidifying of carbon nanotube: be the carbon nanotube that the nitration mixture acidification step (1) of concentrated nitric acid and the vitriol oil of 1:2-1:5 obtains with volume ratio;
(3) functionalization of carbon nanotube: utilize NHS to connect being connected-NH of functionalization at the carbon nanotube tube wall with EDC
2Functional group, last and enzyme-NH
2The reaction, with enzyme immobilization at tube wall.
2. the method for the immobilized protease molecule of a kind of nano material according to claim 1 is characterized in that, during functionalization, pH value is 7.0-7.5.
3. the method for the immobilized protease molecule of a kind of nano material according to claim 1; It is characterized in that; Said protease molecule; Be meant superoxide dismutase SOD, acetyl-CoA carboxylase, lypase, phosphatidic acid phosphatase and the diacylglycerol acyltransferase, 1 produced through genetically engineered, one or more in ammediol oxydo-reductase or the people's Pseudocholinesterase.
4. the application of the method for the immobilized protease molecule of the described nano material of claim 1 in fixing, the stable and utilization of enzyme, said enzyme is meant and is used for the enzyme that biocatalysis or conversion are carried out in the energy, environment and biochemical pharmacy field.
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CN104109662A (en) * | 2014-06-23 | 2014-10-22 | 华中科技大学 | Immobilized Burkholderia cepacia lipase and preparation method thereof |
CN104404027A (en) * | 2014-12-10 | 2015-03-11 | 南京工业大学 | Method for treating multi-walled carbon nanotube immobilized enzyme by plasma |
CN106105816A (en) * | 2016-06-21 | 2016-11-16 | 天津师范大学 | A kind of method using CNT regulation and control lawn composting substrate enzymatic activity |
CN107543933A (en) * | 2016-06-24 | 2018-01-05 | 江苏雷森生物科技有限公司 | A kind of preparation method of carbon nano-particle of antibody labeling and the early pregnancy test strips using its preparation |
CN107988196A (en) * | 2017-12-21 | 2018-05-04 | 华北电力大学 | A kind of preparation method of the carboxyl carbon nanotubes immobilization laccase coated with polymethyl methacrylate |
CN108018281A (en) * | 2017-12-21 | 2018-05-11 | 华北电力大学 | A kind of application of immobilization laccase in methylene blue decoloration |
CN108217972A (en) * | 2017-12-21 | 2018-06-29 | 华北电力大学 | A kind of application of immobilization laccase in gold orange II is decolourized |
CN110527660A (en) * | 2019-08-15 | 2019-12-03 | 西安交通大学 | A kind of cell membrane magnetic carbon nano-tube drug screening material and preparation method and application |
CN111499757A (en) * | 2020-04-13 | 2020-08-07 | 鲁东大学 | Novel material based on carbon nanotube coupled phycoerythrin and application |
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CN104109662A (en) * | 2014-06-23 | 2014-10-22 | 华中科技大学 | Immobilized Burkholderia cepacia lipase and preparation method thereof |
CN104404027A (en) * | 2014-12-10 | 2015-03-11 | 南京工业大学 | Method for treating multi-walled carbon nanotube immobilized enzyme by plasma |
CN104404027B (en) * | 2014-12-10 | 2018-01-05 | 南京工业大学 | Method for treating multi-walled carbon nanotube immobilized enzyme by plasma |
CN106105816A (en) * | 2016-06-21 | 2016-11-16 | 天津师范大学 | A kind of method using CNT regulation and control lawn composting substrate enzymatic activity |
CN107543933A (en) * | 2016-06-24 | 2018-01-05 | 江苏雷森生物科技有限公司 | A kind of preparation method of carbon nano-particle of antibody labeling and the early pregnancy test strips using its preparation |
CN107988196A (en) * | 2017-12-21 | 2018-05-04 | 华北电力大学 | A kind of preparation method of the carboxyl carbon nanotubes immobilization laccase coated with polymethyl methacrylate |
CN108018281A (en) * | 2017-12-21 | 2018-05-11 | 华北电力大学 | A kind of application of immobilization laccase in methylene blue decoloration |
CN108217972A (en) * | 2017-12-21 | 2018-06-29 | 华北电力大学 | A kind of application of immobilization laccase in gold orange II is decolourized |
CN110527660A (en) * | 2019-08-15 | 2019-12-03 | 西安交通大学 | A kind of cell membrane magnetic carbon nano-tube drug screening material and preparation method and application |
CN111499757A (en) * | 2020-04-13 | 2020-08-07 | 鲁东大学 | Novel material based on carbon nanotube coupled phycoerythrin and application |
CN111499757B (en) * | 2020-04-13 | 2022-03-11 | 鲁东大学 | Material based on carbon nanotube coupled phycoerythrin and application thereof |
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