CN101750449A - Lipoprotein capillary coating and the preparation method thereof - Google Patents

Abstract

The invention relates to a lipoprotein capillary coating and a preparation method thereof. The coating can be prepared by physical adsorption and chemical bonding. In the physical adsorption method, a lipoprotein solution with a concentration of 0.01-10 mg/mL interacts non-covalently with the inner wall of the capillary, and self-assembles to the inner surface to form a coating. The chemical bonding method is to first modify the capillary with aminopropyl siloxane and introduce amino groups; -3-(3-Dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide as a coupling agent, at buffer pH (4-9) and lipoprotein concentration (0.1-10mg/mL ) conditions, covalently link the amino acid residues on the capillary wall with the amino acid residues on the lipoprotein to obtain a lipoprotein coating. The coating retains biological activity and can be used to inhibit capillary surface adsorption and electroosmotic regulation, and can also be used for biochemical separation analysis.

Description

Lipoprotein capillary coating and preparation method thereof

technical field

The invention relates to a lipoprotein capillary coating and a preparation method thereof, which are suitable for biochemical separation analysis, clinical diagnosis and environmental pollutant toxicity analysis by capillary electrophoresis and electrochromatography.

Background technique

Capillary electrophoresis (CE) and capillary electrochromatography (CEC) technologies are the products of the combination of classic electrophoresis technology and modern microcolumn chromatography separation technology. Molecular analysis becomes possible. In recent years, with the rapid development of life sciences and environmental health, capillary electrophoresis and electrochromatography have been widely used in the analysis and identification of biological macromolecules (such as protein separation, DNA fragment and sequence analysis, etc.), clinical disease diagnosis and clinical drug monitoring, Metabolic research, pathological research and other aspects have shown unique application advantages, and have won the favor of scientific researchers.

Although CE and CEC technologies have achieved rapid development at present, with the continuous deepening of scientific research purposes and the continuous upgrading of people's requirements for information accuracy and precision, this technology is also facing more severe technical challenges. For example, the irreversible adsorption of biological samples or complex matrices on capillaries is one of the key difficulties often encountered in the study of biological macromolecules. Due to the irreversible adsorption of biological macromolecules, the zeta potential of the capillary wall surface changes, causing problems such as reduced separation effect, poor reproducibility, and reduced accuracy, and even leads to the loss of biological samples and capillary damage in severe cases.

In order to overcome this problem, some effective improvement measures have been proposed, such as using high ionic strength electrophoresis buffer solution and selecting extreme pH conditions (pH < 2.5 or > 10), but the most commonly accepted solution is capillary coating. Capillary coating technology mainly refers to the method of forming one or more layers of coating materials on the inner wall of capillary by physical coating or chemical bonding. According to the preparation method and application mode of the coating, capillary coating can be divided into dynamic coating and permanent coating. Surfactants and some polymers have attracted much attention as capillary coating materials. However, most of the reported methods will cause protein denaturation or activity loss to varying degrees, which has become a fly in the ointment in many studies. Canadian chemist Lucy et al. have developed a semi-permanent phospholipid coating in recent years, which not only ensures satisfactory separation performance and stability, but also achieves good biocompatibility, injecting new vitality into this research field. Phospholipid is a coating material with good biocompatibility, which has been intensively studied and reported in recent years. However, the construction of a good phospholipid bilayer coating structure is highly dependent on the coating conditions, such as the size and type of phospholipid vesicles formed by self-assembly, which greatly limits the practical application of this method.

Protein is a natural endogenous, biocompatible biomacromolecule. Using protein as capillary coating material, in addition to the function of inhibiting adsorption and regulating electroosmosis, will also produce a new and valuable function, that is, it can be used as a test tool for antibody and drug screening and clinical diagnosis.

Based on the above purpose, the present invention uses lipoprotein as a coating material to prepare a capillary coating with lipoprotein activity and biocompatibility by means of physical self-assembly and chemical bonding. Compared with the phospholipid bilayer, the construction of the lipoprotein coating is simpler, more direct, and more stable, and the coating has a wider range of functions.

Lipoproteins are a class of transport proteins in blood and cell membranes, mainly composed of liposomes and apolipoproteins. Due to the difference in structure and density, plasma lipoproteins are commonly divided into chylomicrons (CM), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) by ultracentrifugation. The function of chylomicrons is to transport exogenous fat; the function of very low density lipoprotein is to transport endogenous fat; the function of low density lipoprotein is to transport cholesterol; the function of high density lipoprotein is to transport endogenous cholesterol and phospholipids. The function of lipoprotein is closely related to its structure. Most lipoproteins are composed of phospholipids, apolipoproteins (such as Apo B and Apo E, etc.), cholesterol and cholesteryl lipids. Taking low-density lipoprotein as an example, phospholipids and apolipoprotein (Apo B) distributed on the surface of the molecule each account for 1/4 of the structural composition.

Therefore, the present invention immobilizes different lipoproteins on the capillary, which can not only effectively inhibit the irreversible adsorption caused by silanol, but also carry out applied research related to lipoprotein functions. Through experiments, it is preliminarily shown that the lipoprotein coating has certain application value in the biochemical separation and analysis of proteins, the transport mechanism of environmental pollutants in vivo and the protein damage caused by free radicals. Correspondingly, it is believed that it will also have good application prospects in clinical diagnosis, antibody and drug screening, and environmental health effect research.

Contents of the invention

The present invention relates to a kind of lipoprotein capillary coating and its preparation method, and its purpose is to develop a kind of macromolecule coating, and retain certain biological activity and biocompatibility, make it can be used for the suppression of the capillary surface adsorption and the electric charge It can also be used in specific applications such as biochemical analysis and clinical detection. Lipoproteins include very low-density lipoproteins, low-density lipoproteins, high-density lipoproteins, and chylomicrons.

Another object of the present invention is to provide a method for preparing the lipoprotein coating, which is simple to operate and easy to maintain the activity and stability of the lipoprotein.

The preparation of the lipoprotein capillary coating involved in the present invention can be realized by two methods, namely physical adsorption method (or called self-assembly method) and chemical bonding method.

The principle of preparing lipoprotein capillary coating by physical adsorption method (self-assembly method): under the conditions of a certain concentration of buffer solution (0.1-100mM, pH 4-9) and temperature (4-37°C), lipoprotein (concentration 0.01 -10mg/mL) has non-covalent interactions such as hydrophobic and electrostatic attraction with the silanol on the inner wall of the quartz or glass capillary, thereby self-assembling to the surface of the inner wall to form a binding layer; Lipoprotein capillary coating.

The principle of preparing lipoprotein capillary coating by chemical bonding: firstly, according to the sol-gel technique, the capillary is modified with silylating reagents such as γ-aminopropyltriethoxysilane or γ-aminopropyltrimethoxysilane, Amino active groups are introduced to prepare amino-modified capillaries; then excess glutaraldehyde or N, N-succinyl carbonate (DSC) or disuccinimidyl suberate (DSS) or 1-ethane Base-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide system (EDC/NHS) as coupling agent, in a certain buffer solution pH value (pH 4-9) , temperature (4-37°C) and lipoprotein concentration (0.1-10mg/mL), covalently link the amino group on the capillary to the amino acid residue on the lipoprotein, and fix the lipoprotein to the inner wall of the capillary; finally use buffer The unbound lipoproteins were washed away with liquid to obtain lipoprotein capillary coatings.

The lipoprotein capillary coating and preparation method involved in the present invention have the following characteristics: good biocompatibility, suitable for the separation and analysis of biological macromolecules; retaining the activity of lipoproteins, and can simulate biomembrane for its interaction with The research on the interaction between biological macromolecules and small molecules can be used in clinical diagnosis and pharmacological research; according to the isoelectric point (pI5-6) of lipoproteins, the electrophoresis buffer can be selected to regulate electrophoresis; with phospholipids and Active components such as apolipoproteins can provide research tools for the design of drug carriers and the mechanism of exogenous substances in vivo transport process.

Description of drawings

Fig. 1 is a schematic diagram of the synthesis route of lipoprotein coating prepared by chemical bonding method.

Figure 2 is a graph showing the relationship between the pH of the buffer and the electroosmosis produced by the low-density lipoprotein coating.

Figure 3 is a chromatogram of a lipoprotein capillary coating used for protein separation.

Detailed ways

Lipoprotein capillary coatings and methods for their preparation are exemplified by the following embodiments.

Before preparing the lipoprotein coating, the capillary should be pretreated first. Wash the capillary with chromatographically pure methanol for 1 hour, then wash it with 1mol/L sodium hydroxide for 1-2 hours, wash the capillary with deionized water for 15 minutes, then wash it with 1mol/L hydrochloric acid for 1-2 hours, and finally wash it with deionized water After 15 minutes, blow dry with high-purity nitrogen for later use. After the pretreatment is completed, the preparation of the lipoprotein coating is carried out.

Embodiment 1 physical adsorption method prepares low-density lipoprotein capillary coating

Weigh 5.0 mg of low-density lipoprotein freeze-dried powder, fully dissolve in 5.0 mL of 25 mmoL phosphate buffer solution (pH=7.4), and prepare a coating solution of 1.0 mg/mL. Pass the coating solution through the pretreated capillary under 10psi pressure for 15 minutes, then seal both ends of the capillary with silicone rubber, fill the capillary with the above-mentioned low-density lipoprotein coating solution, and let it stand at room temperature for 2-4 hours . Fill the coating solution again, seal both ends of the capillary, and let it stand at room temperature for more than 2 hours. After completing the above operations, rinse the capillary with 25mmoL phosphate buffer solution (pH=7.4) for 30 minutes to remove the low-density lipoprotein not bound to the tube wall, seal both ends of the capillary with silicone rubber, and store in a refrigerator at 4°C.

The synthesis scheme of high-density lipoprotein, very low-density lipoprotein, chylomicron and mixed lipoprotein coating is the same as the above operation, only the low-density lipoprotein coating solution needs to be replaced with the corresponding lipoprotein solution.

The preparation of the capillary of embodiment 2 aminopropyl modifications

Pipette 0.1mL of γ-aminopropyltrimethoxysilane, dissolve it in 1.0mL of water (dilute acetic acid to adjust pH 5)-methanol (2:8, volume ratio) mixed solution, mix well and quickly fill into In the pretreated capillary, seal both ends of the capillary with silicon rubber, and react at room temperature for 2-4 hours. After the reaction is completed, the capillary is washed with methanol and water for 30 minutes each, and then the capillary is aged and dried in an oven at 105-120° C. for more than 2 hours under the protection of high-purity nitrogen. Aminopropyl-modified capillaries were obtained through the above-mentioned treatment steps.

Embodiment 3 uses glutaraldehyde as the chemical bonding method of coupling agent to prepare low-density lipoprotein capillary coating

Pipette 0.5mL of 50% glutaraldehyde aqueous solution, join in the 25mmoL phosphate buffered saline solution (pH=7.4) of 4.5mL, after fully mixing, fill in by the aminopropyl group prepared by embodiment 2 under the pressure of 5psi After modifying the capillary, seal both ends of the capillary with silicone rubber after 1 hour, and let it stand at room temperature for 2-6 hours. Then, 25 mmoL phosphate buffer solution (pH=7.4) was used to flush out the unreacted glutaraldehyde solution to obtain a glutaraldehyde-modified capillary.

Prepare a 1.0 mg/mL low-density lipoprotein coating solution according to the method described in Example 1, fill the solution into the above-mentioned capillary treated with glutaraldehyde, seal both ends after 30 minutes, and continue the reaction at room temperature 2-4 hours. During this reaction, lipoproteins covalently interact with the aldehyde groups of glutaraldehyde to form Schiff base compounds. The unreacted lipoprotein coating solution was flushed out with phosphate buffer solution, and then washed with 0.5 mg/mL sodium cyanoborohydride solution (in 25 mmoL phosphate buffer, pH 6.4) for 20 minutes. Seal both ends of the capillary with silicon rubber, and continue to react at room temperature for 4-8 hours to reduce the unstable Schiff base structure to a relatively stable imine structure (see accompanying drawing 1). Finally, the reaction solution was flushed out with 25 mmoL phosphate buffer solution (pH=7.4) to obtain a capillary coating of low-density lipoprotein. If not used immediately, store in refrigerator at 4°C.

The synthesis scheme of high-density lipoprotein, very low-density lipoprotein, chylomicron and mixed lipoprotein coating is the same as the above operation, only the low-density lipoprotein coating solution needs to be replaced with the corresponding lipoprotein solution.

Example 4 Preparation of high-density lipoprotein capillary coating with N, N-succinylaminocarbonate (DSC) as coupling agent

Weigh 5.0 mg of DSC and dissolve it in 5 mL of acetonitrile, and mix well. Fill the above solution into the aminopropyl-modified capillary prepared in Example 2, seal both ends of the capillary with silicon rubber after 20 minutes, and place it in a 45° C. water bath for 2-4 hours to react. After the reaction was completed, the capillary was rinsed with acetonitrile, deionized water, and 25 mmoL phosphate buffer (pH 7.4) for 10 minutes.

According to the method described in Example 1, a 1.0 mg/mL high-density lipoprotein coating solution was prepared, and the solution was filled into the above-mentioned DSC-treated capillary, and reacted at room temperature for 2-4 hours. After the reaction was completed, the capillary was rinsed with 25mmoL phosphate buffer (pH 7.4) for 30 minutes to obtain a capillary coating of high-density lipoprotein (see Figure 1). If not used immediately, store in refrigerator at 4°C.

The synthesis scheme of low-density lipoprotein, very low-density lipoprotein, chylomicron and mixed lipoprotein coating is the same as above, only the high-density lipoprotein coating solution needs to be replaced with the corresponding lipoprotein solution.

Example 5 Preparation of high-density lipoprotein capillary coating with disuccinimidyl suberate (DSS) as coupling agent

The operation of using DSS as the coupling agent to prepare the VLDL capillary coating is the same as in Example 4, except that N, N-succinylaminocarbonate is replaced by DSS (see Figure 1).

The synthesis scheme of low-density lipoprotein, very low-density lipoprotein, chylomicron and mixed lipoprotein coating is the same as above, only the high-density lipoprotein coating solution needs to be replaced with the corresponding lipoprotein solution.

Example 6 Preparation of very low-density lipoprotein capillary coating by chemical bonding using EDC/NHS as coupling agent

Weigh 5.0 mg of very low-density lipoprotein and dissolve it in 5 mL of 25 mmoL phosphate buffer (pH 6.0); add 2.0 mg of EDC and 3.0 mg of NHS to the solution in turn, vortex for 5 minutes at room temperature, and let stand for 25 minute. Fill the above solution into the aminopropyl-modified capillary prepared in Example 2, seal both ends of the capillary with silicone rubber after 15 minutes, and react at 4°C-25°C for more than 4 hours. After the reaction was completed, the capillary was rinsed with 25mmoL phosphate buffer (pH 7.4) for 30 minutes to obtain a capillary coating of very low-density lipoprotein (see Figure 1).

The synthesis scheme of high-density lipoprotein, low-density lipoprotein, chylomicron and mixed lipoprotein coating is the same as the above operation, only the very low-density lipoprotein coating solution needs to be replaced with the corresponding lipoprotein solution.

Embodiment 7 lipoprotein capillary coating is for the control of electroosmosis

The physically adsorbed (self-assembled) lipoprotein capillary coating prepared in Example 1 has similar electroosmotic control behavior to the chemically bonded lipoprotein capillary coating prepared in Examples 3-5. The lipoprotein capillary coating prepared in the above embodiments can realize the control of electroosmosis according to the different electrophoresis buffers used. The formula for calculating electroosmotic mobility is:

Electroosmosis (cm ² /Vs) = total capillary length (cm) × effective length (cm) / [voltage (V) × migration time (s)]

Taking physical adsorption and chemical bonding low-density lipoprotein coatings as examples, the selected experimental conditions are: the inner diameter of the coating capillary is 50 μm, the total length is 50 cm, the effective length is 40 cm, and the applied voltage is 15 kV. Amide (DMF) is an electroosmotic marker, the injection pressure is 0.5 psi, and the injection time is 5 seconds. Under these conditions, if the pH value of the electrophoresis buffer (25mmoL phosphate buffer) is gradually changed from 4 to pH 8, the direction and absolute value of the electroosmotic mobility will change (see accompanying drawing 2). Moreover, the intersection point of the linear fitting curve and the X-axis (pH value) is between 5.3-5.5, which is close to the isoelectric point of low-density lipoprotein (pI is about 5.5).

The experimental results verified the controllability of lipoprotein capillary coating on electroosmosis. This feature can provide flexible and simple operating parameters for different analysis and separation purposes, thereby improving separation efficiency and analysis speed.

Example 8 Lipoprotein capillary coating is used for biochemical separation and analysis of proteins

The low-density lipoprotein coating prepared by chemical bonding is effective for four kinds of cytochrome c (Cyt c), lysozyme (Lys), ribonuclease A (RNase A) and α-chymotrypsinogen A (α-Chy A) Basic proteins were separated. Electrophoresis conditions: capillary inner diameter 50μm, total length 50cm, effective length 40cm, applied voltage +15kV, 25mmoL phosphate solution (pH 7.4) as electrophoresis buffer, protein concentration in the mixed sample 0.2mg/mL, injection pressure 0.5psi, The injection time is 5 seconds. The results showed that under the above electrophoresis conditions, the above four basic proteins were completely separated by electrophoresis, and the separation degree was greater than 1.5 (see accompanying drawing 3). The separation efficiency of protein is 8700-124000 theoretical plates/meter, and the rate of recovery is between 82%-96%, and the relative standard deviation (RSD) of precision between day and day is 1.2%-5.7% (see Table 1), Meet the requirements of the above basic protein biochemical separation and qualitative and quantitative analysis.

In addition to hydrophobic and electrostatic interactions, there may also be a certain affinity between the lipoprotein coating and the analyte. Lipoprotein coating has good structural characteristics and biocompatibility, can inhibit the irreversible adsorption of protein and other biomacromolecules on the capillary wall, and is suitable for biochemical separation and analysis of proteins.

Table 1 Properties of proteins separated by low-density lipoprotein capillary coating electrophoresis

Example 9 Low Density Lipoprotein Capillary Coating Used in Research on the Mechanism of Free Radical Oxidative Damage

The low-density lipoprotein-coated capillary was used to study the free radical oxidative damage of low-density lipoprotein induced by divalent copper ions (Cu ²⁺ ). Electrophoresis conditions: capillary inner diameter 50 μm, total length 50 cm, effective length 40 cm, applied voltage +15 kV, 25 mmoL phosphate solution (pH 7.4) as the electrophoresis buffer, 0.2% DMF as the electroosmotic marker, and the concentration of each protein in the mixed sample was 0.2 mg/mL, injection pressure 0.5psi, injection time 5 seconds. Preparation of CuSO ₄ solution: Weigh 8.0 mg (50 μmol) of anhydrous copper sulfate, dissolve it in 10 mL of 25 mmoL phosphate buffer (pH 7.4), and prepare 5 μmol of CuSO ₄ solution. Oxidative modification of the low-density lipoprotein coating: the above CuSO ₄ solution was washed into three low-density lipoprotein-coated capillaries of the same size, sealed at both ends, and incubated in a 37°C water bath for 2, 6 and 12 hours respectively. After the reaction is completed, wash out the solution in the tube with electrophoresis buffer, and continue to wash for 20-30 minutes, and finally connect to a capillary electrophoresis instrument for electroosmotic determination and protein separation.

Electroosmosis was measured according to the method described in Example 7, and protein separation was performed according to the method described in Example 8. The reduced oxidation rate of lipoprotein is expressed by the relative change of electroosmotic flow, that is, [(electroosmosis after oxidation of Cu ²⁺ - electroosmosis without oxidation)/electroosmosis without oxidation]×100%. The results show that divalent copper ions (Cu ²⁺ ) can induce free radical oxidative damage to low-density lipoprotein, and the equivalent oxidation rate increases with the increase of treatment time, the stability of lipoprotein coating decreases, and the protein The efficiency of the separation was also significantly reduced (see Table 2).

The oxidative modification of lipoprotein is one of the main causes of atherosclerosis, and the existence of free radicals will promote the oxidative damage of lipoprotein. Lipoprotein capillary coating can be used to study the oxidative damage of lipoprotein free radicals and the pathogenesis of atherosclerosis.

Table 2 Relationship between changes in coating properties caused by Cu ²⁺ and free radical oxidation of lipoproteins

Claims (5)

1. a lipoprotein capillary coating is characterized in that, capillary tube inner wall is fixed with one or more layers lipoprotein; Lipoprotein comprises high-density lipoprotein (HDL), low-density lipoprotein, very low density lipoprotein (VLDL) and chylomicron.

2. the preparation method of a lipoprotein capillary coating is characterized in that, can lipoprotein be fixed to capillary tube inner wall by the physisorption (or claiming self-assembly method) and the method for chemical bonding, forms lipoprotein capillary coating.

3. lipoprotein capillary coating according to claim 1 is characterized in that, has components such as phosphatide, apolipoprotein, cholesterol ester, remains with the activity of lipoprotein.

4. the method for lipoprotein capillary coating according to claim 2, it is characterized in that, the principle of physisorphtion (or claiming self-assembly method) is: at buffer solution (0.1-100mM, pH 4-9) and under temperature (4-37 ℃) condition, make noncovalent interactions such as lipoprotein (concentration 0.01-10mg/ml) is hydrophobic with the silicon hydroxyl generation of quartz or glass capillary inwall, electrostatic attraction, thereby self-assemble to inner wall surface, make lipoprotein capillary coating.

5. the method for lipoprotein capillary coating according to claim 2, it is characterized in that, the chemical bonding ratio juris is: adopt silylating reagents such as gamma-aminopropyl-triethoxy-silane or γ-An Bingjisanjiayangjiguiwan that kapillary is carried out modification earlier, introduce amino reactive group; Then with excessive glutaraldehyde or N, N-succinyl amino-carbon acid esters (DSC) or disuccinimidyl suberate (DSS) or 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides/N-hydroxy-succinamide system (EDC/NHS) are coupling agent, under the condition of pH value of buffer solution (pH 4-9), temperature (4-37 ℃) and lipoprotein concentration (0.1-10mg/mL), amino on the covalently bound kapillary and the amino acid residue on the lipoprotein, thus lipoprotein is fixed to capillary tube inner wall.

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