CN111087472A

CN111087472A - Preparation method of biological active diatom biomineralization silicon

Info

Publication number: CN111087472A
Application number: CN201811234358.5A
Authority: CN
Inventors: 冯超; 穆愈之; 陈西广
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2020-05-01

Abstract

The invention discloses a preparation technology of a bioactive marine silicon-based porous material, namely a preparation method of bioactive biomineralization silicon. Can be used for food processing, medical biomaterials and drug carriers, and has good research, development and application prospects.

Description

Preparation method of biological active diatom biomineralization silicon

Technical Field

The invention belongs to a preparation method of biological activity diatom biomineralization silicon in the field of biology.

Background

Diatoms, a unicellular photosynthetic algae, of the marine diatom type over 10 million, have cell walls composed of silica, are called frustules, and are also called biomineralization. The frustules have a highly ordered pore structure and a hierarchical pore organization, and have unique mechanical properties, molecular transport properties, and optical properties. Is a high-quality resource with high activity, large-scale culture and environmental protection. At present, diatom biomineralization silicon has applications in the aspects of optics, biosensing, micro-processing, protein separation and drug delivery. The biomineralization silicon mainly comprises silicon dioxide which has stable chemical property, so that chemical modification of the biomineralization silicon has certain difficulty, and meanwhile, the biomineralization silicon of diatom is difficult to load substances with biological activity, such as enzyme, antibody, functional polypeptide and the like. At present, the biological active substance loading modes for biomineralization of silicon by diatoms mainly comprise two modes: 1. physical adsorption, which adsorbs biological molecules through the porous structure on the surface of the diatom biomineralization silicon. 2. Chemically modifying, the surface of the diatom biomineralization silicon contains silanol groups which can react with a silane coupling agent, so that the surface of the diatom biomineralization silicon is chemically combined with biomolecules after chemical groups such as carboxyl, amino and the like are attached to the surface of the diatom biomineralization silicon. Both the two modes have certain defects, and the physically adsorbed biomolecules have weak binding force and are easy to fall off; in the chemical modification mode, because the content of silanol groups on the surface of the diatom biomineralization silicon is low and the modification difficulty is high, the degree of surface chemical modification is low, and the binding capacity of biomolecules is limited. Compounds containing catechol groups, such as dopamine, a major component of Mussel Adhesive Proteins (MAP), undergo self-polymerization under alkaline conditions to form a polydopamine coating on the surface of the material. Compounds containing catechol groups may convert themselves to the quinone form, and catechol compounds in the quinone form are highly reactive to other functional groups, including thiols, amines and other catechol-quinones, by Schiff base substitution or 1, 4-Micheal type addition. Therefore, catechol-based compounds such as polydopamine derived from mussels have become organic macromolecules which form adhesive layers on various substrates to anchor compounds, biological substances, and the like. According to the invention, the poly-catechol coating is constructed on the surface of the diatom biomineralization silicon, and the bioactive substances are further loaded, so that the loading capacity of the bioactive substances on the surface of the diatom biomineralization silicon can be increased, the bioactive substances are prevented from falling off from the diatom biomineralization silicon, a large amount of diatom biomineralization silicon with various biological activities can be constructed, and the diatom biomineralization silicon has very important theoretical significance and clinical application value.

Disclosure of Invention

The invention aims to provide a preparation method of biological mineralized silicon from biological active diatom, which is used for overcoming the defects of the prior art.

The invention takes diatom biomineralization silicon and a compound containing catechol group as raw materials, prepares biomineralization silicon with a bioadhesive polymer coating by an alkaline self-polymerization method, and obtains bioactive diatom biomineralization silicon by mixing with active protein molecules, and the specific steps are as follows:

(1) preparation of bio-adhesive coating diatom biomineralization silicon: dispersing the purified diatom biomineralization silicon in a weakly alkaline solution, wherein the pH value of the solution is 7.0-9.0, adding a compound solution containing catechol groups, wherein the concentration of the compound solution containing the catechol groups is 0.05% -10% (M/V), the mass ratio of the diatom biomineralization silicon to the compound containing the catechol groups is 0.1-1.2, stirring and reacting at 10-50 ℃ for 0.5-16 hours, washing with deionized water for multiple times to remove unreacted catechol compounds, and drying to obtain polymer coating diatom biomineralization silicon;

(2) preparing biological active diatom biomineralization silicon: uniformly dispersing the bio-adhesive coating diatom biomineralization silicon in the solution, and then adding an active protein factor solution, wherein the active protein factor added per milligram of the bio-adhesive coating diatom biomineralization silicon is 0.1 muM-1000 mM. The mixture was stirred for reaction overnight. And after the reaction, washing the mixture with deionized water for multiple times, and freeze-drying to obtain the bioactive diatom biomineralization silicon.

The method has the advantages of simple and convenient operation, simple preparation technology process, low manufacturing cost and the like, the content of the catechol can be adjusted by changing the feeding ratio and the reaction time, the load capacity to bioactive molecules is further changed, and the formed bioactive diatom biomineralization silicon has complete appearance and clear surface aperture structure. The invention has the important significance that the formed bioactive diatom biomineralization silicon can increase the loading capacity of bioactive substances on the surface of the diatom biomineralization silicon, prevent the bioactive substances from falling off from the diatom biomineralization silicon, can construct a large amount of diatom biomineralization silicon with various bioactivities, and has good development and application potentials.

Example 1

30 mg of purified diatomic biomineralized silica was weighed out and dispersed in 1 mL of NaOH solution with pH 8.5. 10 mg of dopamine hydrochloride is dissolved in 3mL of deionized water, added into the Tris-HCl dispersion liquid of diatom biomineralization silicon, and stirred for reaction for 4 hours at 37 ℃ in a dark place. Fully washing with deionized water, and freeze-drying to obtain polydopamine-diatom biomineralization silicon; weighing 10 mg of polydopamine-diatom biomineralization silicon, dispersing the polydopamine-diatom biomineralization silicon into 1.5 mL of PBS buffer solution with pH 7.4, adding 0.1 mL of thrombin solution (enzyme activity is 50U), stirring at 4 ℃ for reaction overnight, fully washing with deionized water, and freeze-drying to obtain the thrombin active polydopamine-diatom biomineralization silicon.

Example 2

60 mg of purified diatomic biomineralization silica was weighed out and dispersed in 1.5 mL of PBS solution with pH 7.2. Dissolving 5mg of levodopa in 2.5 mL of deionized water, adding the solution into a Tris-HCl dispersion of diatom biomineralization silicon, and stirring the solution at room temperature in the dark for reaction for 14 hours. Fully washing with deionized water, and freeze-drying to obtain poly-levodopa amine-diatom biomineralization silicon; weighing 20mg of poly-levodopa amine-diatom biomineralization silicon, dispersing the poly-levodopa amine-diatom biomineralization silicon into 2.5 mL of physiological saline, adding 0.5 mL of antibacterial peptide solution with the concentration of 0.5mg/mL, stirring and reacting overnight at room temperature, fully washing with deionized water, and freeze-drying to obtain the antibacterial peptide active poly-levodopa amine-diatom biomineralization silicon.

Example 3

70 mg of purified diatom biomineralization silicon is weighed and dispersed in 1 mL of Tris-HCl solution with pH 8.0. 35 mg of norepinephrine was dissolved in 3mL of deionized water, and the solution was added to a Tris-HCl dispersion of diatom biomineralization silicon, and the reaction was stirred at 37 ℃ for 4 hours in the dark. Fully washing with deionized water, and freeze-drying to obtain the polynnorepinephrine-diatom biomineralization silicon; weighing 10 mg of the polynnorepinephrine-diatom biomineralization silicon, dispersing the polynnorepinephrine-diatom biomineralization silicon into 1.5 mL of PBS buffer solution with the pH value of 7.4, adding 0.02 mL of Anti-EpCAM/CD326 epithelial cell adhesion molecule antibody solution, stirring and reacting overnight at the temperature of 4 ℃, fully washing with deionized water, and freeze-drying to obtain the polynnorepinephrine-diatom biomineralization silicon with antibody activity.

Claims

1. A preparation method of bioactive diatom biomineralization silicon is characterized in that a compound containing catechol groups is oxidized and crosslinked on the surface of diatom biomineralization silicon to form a polymer coating, the polymer coating is mixed with an active protein factor solution, and then the active protein factor in the solution is adhered and immobilized by utilizing the bioadhesion of the catechol groups in the polymer coating to obtain the bioactive diatom biomineralization silicon, and the preparation method comprises the following specific steps:

(1) preparation of bio-adhesive coating diatom biomineralization silicon: dispersing the purified diatom biomineralization silicon in a weakly alkaline solution, wherein the pH value of the solution is 7.0-9.0, adding a catechol group-containing compound solution, wherein the concentration of the catechol group-containing compound solution is 0.05% -10% (M/V), the mass ratio of the diatom biomineralization silicon to the catechol group-containing compound is 0.1-1.2, stirring and reacting at 10-50 ℃ for 0.5-16 hours, washing with deionized water for multiple times to remove unreacted catechol compounds, and drying to obtain the bioadhesive coating diatom biomineralization silicon;

(2) preparing biological active diatom biomineralization silicon: uniformly dispersing the biological adhesive coating diatom biomineralization silicon in the solution, then adding an active protein factor solution, wherein the active protein factor added per milligram of the biological adhesive coating diatom biomineralization silicon is 0.1 mu M-1000 mM, stirring the mixture for reacting overnight, washing the reacted mixture with deionized water for multiple times, and freeze-drying to obtain the biological active diatom biomineralization silicon.

2. The method for preparing bio-active diatom biomineralized silicon as claimed in claim 1, wherein the source of the diatom biomineralized silicon used in the preparation of the bio-adhesive coated diatom biomineralized silicon is selected from diatoms and diatomaceous earth of class Celite or class Lutraria; the compound containing catechol group includes dopamine, dopamine hydrochloride, levodopa, norepinephrine, 3, 4-dihydroxyphenylpropionic acid, caffeic acid, etc.; the alkalescent solution can be NaOH solution, phosphate buffer solution or Tris-HCl buffer solution; in the preparation of the biological mineralized silicon of the biological activity diatom, the solution required by the reaction can be phosphate buffer solution, Tris-HCl buffer solution, normal saline, distilled water and the like; the protein active factors used include enzymes (thrombin, amylase, pectinase, etc.), polypeptides, antibodies, etc.

3. The use of the bioactive diatomic biomineralization of silicon of claim 1 including the following:

(1) a bait additive for aquatic or livestock animals;

(2) adding protein active factors with different biological functions, and can be applied in the fields of food processing, biomedicine, cosmetics, textile processing, health products, etc.