CN103467603B - A kind of method utilizing polyelectrolyte brush ankyrin - Google Patents

Abstract

The invention discloses a method for fixing protein by using a polyelectrolyte brush. The present invention uses a polyelectrolyte brush with a 3D structure as a carrier, absorbs a large amount of protein under the condition that it is favorable for protein physical adsorption, and then stably binds the protein to the polyelectrolyte brush carrier by chemical bonds through coupling chemical means. The invention greatly increases the binding amount of the protein, and realizes the stable binding of the polyelectrolyte brush to the protein, so that it can be efficiently applied to various downstream biotechnologies.

Description

A Method for Immobilizing Proteins Using Polyelectrolyte Brushes

technical field

The invention relates to the technical field of biological materials, in particular to a protein immobilization method.

Background technique

The immobilization process of proteins plays an important role in many biotechnologies such as biosensors, protein separation and purification, diagnostic detection and analysis, and immobilized enzyme catalysis. In many applications, it is required that the carrier has a high protein binding capacity to improve the application performance of the carrier in the processes of separation, detection, analysis, and catalysis. The polyelectrolyte brush is a new type of carrier, which is composed of a carrier and a polyelectrolyte segment grafted on the surface of the carrier. Due to the extremely high density of the polyelectrolyte segment grafted on the surface of the carrier, in a good solvent, the polyelectrolyte chain The segments stretch out on the surface of the carrier to present a brush-like structure. According to the shape of the carrier, it can be divided into polyelectrolyte plate brush and polyelectrolyte ball brush. Polyelectrolyte plate brushes were first synthesized by Rühe et al. in 1999 by means of surface-initiated polymerization, and polyelectrolyte ball brushes were also synthesized by Ballauff et al. in the same year. In this carrier, the monomer grows on the surface of the microsphere to form a polyelectrolyte brush structure with a "3D" structure, which provides a large number of sites for protein binding, which can realize multi-layer protein binding and greatly improve protein binding. Quantitative potential. In addition, polyelectrolyte brushes can also be formed by first synthesizing polyelectrolyte segments and then grafting onto the surface of the carrier.

Literature search of the prior art found that "Adsorption of proteins on spherical polyelectrolyte brushes in" (Adsorption of proteins on spherical polyelectrolyte brushes in aqueous solution) published by Ballauff et al. aqueoussolution, Phys.Chem.Chem.Phys., 2003, 5, 1671–1677) proved for the first time that polyelectrolyte ball brushes can bind a large number of proteins by electrostatic adsorption. Bruening et al. published in "Langmuir" in 2006 the paper "High-Capacity Binding of Proteins by Poly(Acrylic Acid) Brushes and Their Derivatives, Langmuir, 2006, 22,42744281) reported that negatively charged polyacrylic acid brushes could bind a positively charged protein, lysozyme, by electrostatic adsorption, and the binding amount was as high as 80 layers. The above studies demonstrate physical adsorption as a simple and effective method to unleash the potential of polyelectrolyte brushes to bind proteins in large quantities. However, the nature of physical adsorption determines that this combination is unstable. The above-mentioned literature also shows that a large amount of protein is released from the polyelectrolyte brush carrier under the condition of higher salt concentration (such as physiological environment) and the change of environmental pH. However, in practical applications such as biosensors, diagnostic testing and analysis, and immobilized enzyme catalysis, it is usually required that the complex of protein and carrier has a high degree of stability in physiological conditions and various complex environments and operating processes, which makes it difficult to achieve a simple Carriers that physically adsorb binding proteins are not adequate for some applications. The chemical combination can just make up for this deficiency.

For the chemical covalent immobilization of proteins on polyelectrolyte ball brushes, the amount of protein immobilization obtained varies greatly with different proteins and polyelectrolyte brushes in existing technical solutions. For example, also using the coupling method based on water-soluble carbodiimide EDC, Bruening et al. published in the paper "Langmuir" (High-Capacity Binding of Proteins by Poly(Acrylic Acid) Brushes and Their Derivatives, Langmuir, 2006, 22,42744281) mentioned that the immobilization efficiency of polyacrylic acid brushes on BSA was low, and its binding capacity was less than 2 times that of monolayer protein binding. Another literature report uses polyacrylic acid brushes with a degree of polymerization of more than 1000 to bind RNase (RNase A) in the same way. Kinetics and Activity, Langmuir, 2008, 24, 913-920), although the amount of bonding has improved, but for such a high-length polyacrylic brush, the utilization rate of the internal space of the brush is still at a low level. Therefore, in this field, there is an urgent need to establish a simple, efficient and universal method for chemically covalently immobilizing proteins, so that people can fully increase the number of positions in the internal space of polyelectrolyte brushes in a controllable manner. In order to improve the utilization of dots, a large amount of protein is stably bound to the polyelectrolyte brush carrier to improve its application performance.

Contents of the invention

Aiming at the above-mentioned deficiencies in the prior art, the present invention invents a chemical covalent immobilization method of polyelectrolyte brushes with high binding capacity to proteins.

First, the protein is adsorbed on the polyelectrolyte brush under favorable conditions for physical adsorption. For a certain polyelectrolyte brush and protein, by controlling the pH, salt concentration and temperature of the system, the protein can be adsorbed to the polyelectrolyte brush in large quantities through electrostatic, hydrogen bond, hydrophobic interaction or van der Waals force. Secondly, wash under favorable conditions for physical adsorption to remove excess protein in the system and protein loosely bound to the surface of the polyelectrolyte brush. Thirdly, under the favorable condition of physical adsorption, the protein is firmly bound to the brush by chemical bond through coupling chemical reaction. Finally, washing removes excess reactants and transfers the polyelectrolyte brush-protein complexes individually to the environment required for their respective downstream applications. Compared with immobilizing protein by physical adsorption on the polyelectrolyte brush, the protein chemical covalent immobilization method proposed by the present invention can ensure the stable combination of protein and polyelectrolyte brush, and the protein will not be damaged due to changes in the external environment such as pH and salt concentration. Shedding or dissociation from polyelectrolyte brushes. In addition, compared with the existing technology of direct chemical covalent immobilization of proteins, the amount of protein bound on the surface of polyelectrolyte brushes by using the method of chemical covalent binding of proteins proposed by the present invention is several times to tens of times the amount of single-layer adsorption on the surface of the carrier. times, which can effectively overcome the problems of low protein loading and poor experimental reproducibility on polyelectrolyte brushes immobilized by direct chemical covalent immobilization.

The present invention solves the above technical problems through the following technical solutions:

The present invention provides a method for immobilizing proteins with polyelectrolyte brushes, which can realize chemical covalent immobilization of ultra-high protein loads on polyelectrolyte brushes, comprising the following steps,

Step 1. Disperse the polyelectrolyte brush in the buffer solution, add the protein-dissolved buffer solution into the polyelectrolyte brush dispersion, mix and adsorb for 2 hours, and the protein is adsorbed on the polyelectrolyte brush;

Step 2, centrifuge and wash the polyelectrolyte brush-protein complex for 2 to 3 times, remove excess protein in the system and protein loosely bound to the surface of the polyelectrolyte brush, and resuspend the polyelectrolyte brush-protein complex in buffer solution to prepare Polyelectrolyte brush-protein complex system;

Step 3, adding a chemical cross-linking reagent to the system containing the polyelectrolyte brush-protein complex, and reacting at room temperature for 2 hours after mixing;

Step 4, washing the polyelectrolyte brush-protein complex 2 to 3 times after centrifugation to remove excess chemical cross-linking agent.

In the present invention, the buffer is preferably a buffer that is beneficial to the physical adsorption of the polyelectrolyte brush used and the protein to be adsorbed. The buffer that is beneficial to the physical adsorption of the polyelectrolyte brush used and the protein to be adsorbed refers to, but is not limited to,

Taking the isoelectric point of the adsorbed protein less than 6.0 as an example, the selection rules of the buffer are as follows:

For polyelectrolyte brushes containing carboxylic acid functional groups, the buffer solution can choose MES buffer solution with pH=4.0~6.0;

For polyelectrolyte brushes containing amino groups, the buffer can be phosphate buffer with pH=7.0-8.0;

For polyelectrolyte brushes containing hydroxyl groups, the buffer can choose pH=6.0-8.0 phosphate buffer;

For polyelectrolyte brushes containing sulfhydryl groups, the buffer solution can be phosphate buffer solution with pH=6.0-8.0.

Taking the isoelectric point of the adsorbed protein greater than 6.0 as an example, the selection rules of the buffer are as follows:

For polyelectrolyte brushes containing carboxylic acid functional groups, the buffer can be MES buffer or phosphate buffer with pH=5.0-7.0;

For polyelectrolyte brushes containing amino groups, borate buffers with a pH of 7.0 to 9.0 can be selected as the buffer;

For polyelectrolyte brushes containing hydroxyl groups, the buffer can choose pH=6.0-8.0 phosphate buffer;

For polyelectrolyte brushes containing sulfhydryl groups, the buffer solution can be phosphate buffer solution with pH=6.0-8.0.

Preferably, the concentration of the buffer solution is 5mM-10mM, and 0.05% w/v Tween-20 is added to the buffer solution.

In the first step, the polyelectrolyte brush is composed of a solid phase carrier and polyelectrolyte segments grafted on the surface of the carrier. One end of the polyelectrolyte segment is grafted on the surface of the carrier and stretches outward to present a brush structure. Wherein, the carrier can be a flat plate, or a microsphere with a particle diameter of 5 nanometers to 1000 nanometers, and the polyelectrolyte is a polymer with charges and reactive groups.

Preferably, in step 1, the polyelectrolyte brush is a polyacrylic acid ball brush or a poly N-(2-aminoethyl)acrylamide ball brush.

Preferably, in step one, the adsorption temperature is 25-37°C.

Preferably, in step 3, the selection rules of the chemical cross-linking agent are as follows: for polyelectrolyte brushes containing carboxylic acid functional groups such as polyacrylic acid, the preferred chemical cross-linking agent is 1-20 mM 1-ethyl-(3-dimethyl Aminopropyl) carbodiimide hydrochloride (EDC) aqueous solution; for polyelectrolyte brushes containing amino groups, the preferred chemical crosslinking agent is glutaraldehyde aqueous solution; for polyelectrolyte brushes containing hydroxyl groups, the preferred chemical crosslinking agent It is an aqueous solution of sodium periodate; for polyelectrolyte brushes containing mercapto groups, the preferred chemical crosslinking agent is a maleimide reagent.

In step 4, the washing solution for washing the polyelectrolyte brush-protein complex is preferably, but not limited to, an isotonic solution similar to a physiological environment, such as PBS, physiological saline, Tris-HCl, and the like.

A preferred implementation route of the present invention is to place the polyelectrolyte brush in an aqueous solution with a suitable pH and salt concentration that is beneficial to protein physical adsorption, and dissolve excess protein in the same aqueous solution and add it to the above-mentioned polyelectrolyte brush system , and mix at a suitable temperature for 2 hours to make the protein adsorb to the polyelectrolyte brush. Wash the polyelectrolyte brush-protein complex for 2 to 3 times with the aqueous solution used for protein adsorption by the polyelectrolyte ball brush to remove excess protein in the system and protein loosely bound to the surface of the polyelectrolyte brush. To the system containing the polyelectrolyte brush-protein complex, add an aqueous solution containing a chemical cross-linking reagent that allows the functional groups on the polyelectrolyte brush to undergo a chemical covalent cross-linking reaction with the protein, mix and react at room temperature for 2 hours. The aqueous solution is the same as that used for protein adsorption by the polyelectrolyte brush. Wash the polyelectrolyte brush-protein complex 2 to 3 times with washing liquid to remove excess chemical cross-linking agent, and finally disperse the polyelectrolyte brush-protein complex in the required amount to adapt to their respective downstream applications according to the requirements of their downstream applications. environment.

Preferably, but not limited to, the polyelectrolyte brush adopts the surface-initiated RAFT polymerization method published in "Journal of Colloid and Interface Science" in 2013, volume 398, pages 82-87, to obtain polyelectrolyte brush segments with controllable thickness, Polyelectrolyte brushes with narrow molecular weight distribution of polyelectrolyte segments and small particle size distribution of ball brushes.

In a preferred embodiment of the present invention, the favorable conditions for physical adsorption refer to a solution with suitable pH, salt concentration and suitable adsorption temperature. The preferred solution is MES buffer with a concentration of 10 mM and pH=4.0-6.0. The adsorption temperature is preferably 25°C-37°C.

In a specific embodiment of the present invention, the chemical crosslinking reagent refers to, but is not limited to, for polyelectrolyte brushes containing carboxylic acid functional groups such as polyacrylic acid, preferably 1-20 mM 1-ethyl-(3- Dimethylaminopropyl) carbodiimide hydrochloride (EDC) aqueous solution; for polyelectrolyte brushes containing amino groups, aqueous solutions such as glutaraldehyde can be used; for polyelectrolyte brushes containing hydroxyl groups, periodic acid can be used Sodium aqueous solution; for polyelectrolyte brushes containing mercapto groups, maleimide reagents, etc. can be used.

In a specific embodiment of the present invention, the required application environment refers to the environment required in the subsequent application of the protein-bound polyelectrolyte brush. It can be phosphate buffered saline (PBS) physiological saline or serum, plasma, whole blood and other physiological environments that simulate the body fluid environment.

In the above process, the amount of protein chemical binding on the final polyelectrolyte brush can be determined by the BCA protein quantification method, respectively, to measure the amount of protein in the polyelectrolyte suspension system added and the reaction system supernatant that is not bound to the polyelectrolyte ball brush and The protein content in the washing liquid during the protein immobilization washing process is calculated by the difference before and after adding the protein.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example. In the examples, the reagents used are commercially available unless otherwise stated.

Example 1

Chemical immobilization of bovine serum albumin (BSA protein) was performed using polyacrylic acid ball brushes.

First, the surface-initiated RAFT polymerization method published in "Journal of Colloid and Interface Science" 2013, Vol. Polyacrylic acid electrolyte ball brushes with branch density of 0.24nm ^-2 .

By centrifugation-resuspension, the above-mentioned ball brush was washed twice with 10mM MES buffer (pH=5.0, containing 0.05% w/v Tween-20), and ultrasonically dispersed in the above-mentioned buffer. At the same time, the buffer was used to dissolve the BSA protein to prepare a protein solution, which was added to the dispersion containing the ball brush and mixed evenly. The final ball brush concentration was 0.8 mg/ml, and the protein concentration was 1 mg/ml.

Incubate at a constant temperature in an oven at 25°C for 2 hours to fully adsorb the protein on the surface of the polyelectrolyte brush segment.

Centrifuge, discard the supernatant, and wash the product 2 times with the above MES buffer. At the same time, the EDC was dissolved in the buffer solution to prepare an EDC solution with a concentration of 5 mM, and added to the centrifuge tube containing the product, and mixed evenly. Continue to incubate at 25° C. for 2 hours, so that the protein adsorbed on the ball brush is connected to the ball brush through chemical bonds.

Centrifuge, discard the supernatant, wash the product twice with the above MES buffer to remove excess EDC, then wash the product twice with 10mM PBS buffer (pH=7.2), and disperse the product in PBS.

Tested by BCA method, the protein binding amount of the polyacrylic acid ball brush-BSA complex obtained by the above method is 790 μg BSA/mg ball brush, which is 6 times of the saturated binding amount of a single layer, and the complex can be stable under conditions such as PBS Existence and absence of protein release.

Example 2

Lysozyme protein was chemically immobilized using polyacrylic acid ball brushes.

The polyelectrolyte brush used was the same as in Example 1.

By centrifugation-resuspension, the above-mentioned ball brush was washed twice with 10mM MES buffer (pH=6.0, containing 0.05% w/v Tween-20), and ultrasonically dispersed in the above-mentioned buffer. At the same time, dissolve the lysozyme protein with the buffer solution to prepare a protein solution, add it into the dispersion liquid containing the ball brush, and mix evenly. The final ball brush concentration was 0.5 mg/ml and the protein concentration was 1.5 mg/ml.

Incubate at a constant temperature in an oven at 37°C for 2 hours, so that the protein is fully adsorbed on the surface of the polyelectrolyte brush.

Centrifuge, discard the supernatant, and wash the product 2 times with the above MES buffer. At the same time, the EDC was dissolved in the buffer solution to prepare an EDC solution with a concentration of 2 mM, and added to the centrifuge tube containing the product, and mixed evenly. Continue to incubate at 37° C. for 2 hours, so that the protein adsorbed on the ball brush is connected to the ball brush through chemical bonds.

Centrifuge, discard the supernatant, wash the product twice with the above MES buffer to remove excess EDC, then wash the product twice with 10mM PBS buffer (pH=7.2), and disperse the product in PBS.

Tested by BCA method, the protein binding amount of the polyacrylic acid ball brush-BSA complex obtained by the above method is 2400 μg BSA/mg ball brush, which is 35 times of the saturated binding amount of a single layer, and the complex can be stable under conditions such as PBS Existence and absence of protein release.

Example 3

BSA proteins were chemically immobilized using poly-N-(2-aminoethyl)acrylamide ball brushes.

The synthesis method of the polyelectrolyte ball brush is the same as in Examples 1 and 2, wherein the monomer is replaced by N-(2-aminoethyl)acrylamide by acrylic acid to obtain a ball core of 100 nanometer silicon oxide, and the polyelectrolyte brush chain Polyacrylic acid electrolyte ball brushes with a segment length of 60 nm and a graft density of 0.15 nm ^-2 .

The chemical immobilization method of the ball brush is the same as in Example 2, and the protein binding amount of the obtained poly N-(2-aminoethyl)acrylamide ball brush-BSA complex is 1500 μg BSA/mg ball brush, tested by the BCA method, It is 11 times of the monolayer saturation binding capacity, and the complex can exist stably under conditions such as PBS, and there is no protein release phenomenon.

Example 4

BSA protein was chemically immobilized using a polyacrylic acid brush grafted on a silicon plate.

The synthesis method of the polyelectrolyte scrub brush is basically the same as that of Examples 1-3. Among them, the polyacrylic acid segment immobilized carrier was changed from 100nm silicon oxide microspheres to silicon slabs to obtain a polyacrylic acid scrub brush with a polyelectrolyte segment length of 100 nm and a graft density of 0.2 nm ⁻² .

The chemical immobilization method of the polyacrylic acid scrub brush to protein is the same as that in Example 2. The protein immobilization density of the obtained polyacrylic acid scrub brush-BSA composite is 6.0 μg/cm ² , which is 15 times of the saturated binding capacity of a single layer. , and the complex can exist stably under conditions such as PBS, and there is no protein release phenomenon.

Example 5

BSA protein was chemically immobilized using a polyacrylic acid ball brush with a core of 1000 nm.

The synthesis method of the polyelectrolyte ball brush is basically the same as that of Examples 1-4. Among them, the ball core in Examples 1-3 was changed from 100nm silicon oxide microspheres to 1000nm silicon oxide microspheres to obtain a polyacrylic acid ball brush with a polyelectrolyte segment length of 120nm and a graft density of 0.55nm ⁻² .

The chemical immobilization method of the protein by the ball brush is the same as that in Example 2. After testing by the BCA method, the protein immobilization density of the obtained polyacrylic acid plate brush-BSA composite is 450 μg BSA/mg, which is 36 times of the saturated binding capacity of the single layer, and The complex can exist stably under conditions such as PBS, and there is no protein release phenomenon.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims (9)

1. utilize a method for polyelectrolyte brush ankyrin, comprise the steps:

Step one, be scattered in damping fluid by polyelectrolyte brush, added by the damping fluid being dissolved with albumen in above-mentioned polyelectrolyte brush dispersion liquid, mixing and absorption 2 hours, protein adsorption brushes to polyelectrolyte;

Step 2, centrifuge washing polyelectrolyte brush-albumen composition 2 ~ 3 times, albumen excessive in removing system and the albumen be combined with polyelectrolyte brush surface porosity, by polyelectrolyte brush-albumen composition Eddy diffusion obtained polyelectrolyte brush-albumen composition system in damping fluid;

Step 3, in the system containing polyelectrolyte brush-albumen composition, add chemical cross-linking agent, room temperature reaction 2 hours after mixing;

Step 4, centrifugal rear washing polyelectrolyte brush-albumen composition 2 ~ 3 times, to remove excessive chemical cross-linking agent;

In described step one, polyelectrolyte brush is made up of solid phase carrier and the polyelectrolyte segment being grafted on carrier surface, one end of polyelectrolyte segment is grafted on carrier surface and outwards stretches and presents brush structure, wherein, carrier is dull and stereotyped, or be the microballoon of particle diameter 5nm-1000nm, polyelectrolyte segment is with electric charge and have can the polymkeric substance of reactive group;

In described step one and step 2, damping fluid is be the favourable damping fluid of physical adsorption to polyelectrolyte brush used and the albumen of intending absorption thereof.

2. the method for claim 1, wherein described damping fluid system of selection is as follows,

When the iso-electric point of adsorbed albumen is less than 6.0:

For the polyelectrolyte brush containing function perssad carboxylate, select the MES damping fluid of pH=4.0 ~ 6.0;

For containing amino polyelectrolyte brush, select the phosphate buffered saline buffer of pH=7.0 ~ 8.0;

Or

When the iso-electric point of adsorbed albumen is greater than 6.0:

For the polyelectrolyte brush containing function perssad carboxylate, select MES damping fluid or the phosphate buffered saline buffer of pH=5.0 ~ 7.0;

For containing amino polyelectrolyte brush, select the borate buffer solution of pH=7.0 ~ 9.0.

3. method as claimed in claim 2, wherein, described buffer concentration is 5mM-10mM, adds the Tween-20 of 0.05%w/v in described damping fluid.

4., the method for claim 1, wherein in step one, polyelectrolyte brush is, polyacrylic acid brush or poly-N-(2-amino-ethyl) acrylamide brush.

5., the method for claim 1, wherein in step one, adsorption temp is 25-37 DEG C.

6., the method for claim 1, wherein in step 3, the selective rule of chemical cross-linking agent is as follows:

For the polyelectrolyte brush containing function perssad carboxylate, chemical cross-linking agent is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) aqueous solution;

For the polyelectrolyte brush containing amino, chemical cross-linking agent is glutaraldehyde water solution.

7., the method for claim 1, wherein in step 4, the washings of washing polyelectrolyte brush-albumen composition is the isotonic solution similar to physiological environment.

8. method as claimed in claim 7, wherein, described isotonic solution is PBS damping fluid, physiological saline or Tris-HCl damping fluid.

9. the method for claim 1, wherein described albumen is bSA or antalzyme protein.