CN115806973A - Method for rapidly extracting biomass by utilizing diatom biological silicon and application - Google Patents
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Abstract
The invention belongs to the technical field of biological materials, and relates to a method for quickly extracting biomass by utilizing diatom biological silicon and an application thereof, wherein the method comprises the following steps: 1) Metal mineralization of diatom bio-silicon; 2) And (4) rapidly extracting biomass. According to the invention, a complex reaction container and a solvent are not needed, the structure of the biomass is not damaged, adsorption collection and desorption collection are realized by changing the adsorption strength of the diatom biological silicon mineralized by the metal to different biomasses, and the purity of the biomasses is further improved; biomass is rapidly extracted and separated from a biological sample by introducing metal mineralized diatom biological silicon and buffer solution, metal elements are selectively solidified on the surface of the diatom biological silicon, metal residues in the separated biomass are reduced, the operation is simple, and the extraction efficiency is high; the method is simple, convenient, rapid and stable to extract the biomass, reduces the extraction cost of the biomass, and is particularly suitable for biological diagnosis and detection and the research of downstream molecular biology.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a method for quickly extracting biomass by utilizing diatom biological silicon and application thereof.
Background
The continuous expansion of the Nucleic Acid Amplification Tests (NAATs) industry has led to the increasing attention of people in the field of biological diagnosis and detection. The problem of how to extract biomass quickly and efficiently has received much attention from researchers as an important component and an essential process in biological diagnostic tests.
At present, biomass extraction methods widely used in the field are mainly classified into liquid phase extraction and solid phase extraction. Liquid phase extraction is to separate biomass compounds according to different relative solubilities of the compounds, wherein an organic solvent extraction method is the most classical liquid phase extraction method, but is gradually eliminated due to the fact that toxic reagents are more involved and the extraction period is long. Compared with liquid phase extraction, the solid phase extraction of biomass has the characteristics of high separation efficiency, low pollution risk and less sample requirement. The most common solid phase extraction methods used today are the centrifugal column method and the magnetic bead method. The magnetic bead method has the defects of high material price and small sample extraction amount, and has more application limitations. Besides magnetic beads, the silica material is also often used as a solid phase matrix for biomass extraction, has relatively low price, is mostly used as a substrate of a centrifugal column at present, and is matched with a buffer solution system containing metal salt to complete biomass extraction. However, the activity of the metal salt in the buffer system used in the prior art on the biomass can bring about subsequent adverse effects, for example, chinese patent application with publication number CN114450419a of bayocardia bio-ltd discloses a method for extracting nucleic acid by using a silica solid support, which can efficiently enrich nucleic acid molecules from a solution, but the extraction buffer introduces a plurality of metal ions, which causes the inhibition on the downstream nucleic acid amplification reaction, and increases the difficulty of subsequent detection. Therefore, how to extract and enrich the biomass in the sample quickly and efficiently, improve the concentration of the biomass, reduce the damage to the structure of the biomass, and do not hinder the subsequent detection process is the key of the biomass extraction technology.
Diatom is a unicellular photosynthetic algae, which is a kind of unicellular photosynthetic algae, and is widely distributed, and the silicified cell walls of the diatom have a 3D hierarchical porous structure which is mutually communicated, and the diatom is named as diatom biological silicon (DOI: 10.1021/acsami.6b12317). The applicant team filed the Chinese invention patent application with the publication number of CN111087472A, namely 'a manufacturing method of 3D structure level porous silica micro-particles', and discloses an extraction method of diatom biological silicon, wherein the purification process is simple and easy to implement, and the obtained diatom biological silicon is complete in shape and low in organic matter content (figure 1), and is a source of the diatom biological silicon used by the invention. The 3D hierarchical porous structure spanning from nanometer to micrometer scale enables the diatom biological silicon to have huge specific surface area and excellent adsorption performance, and the diatom biological silicon is applied to the fields of drug delivery, catalysis, adsorbents, electrode materials and the like. Based on the excellent adsorbability, hemophilicity and capability of activating an endogenous coagulation pathway of diatom bio-silicon, the applicant developed a series of novel hemostatic materials (DOI: 10.1039/d0bm02116 d) and found that the hemostatic materials have adsorption effect on biomass such as enzyme, coagulation factor, serum protein and the like. Research teams at home and abroad have related reports on the biomass adsorption effect of diatom biological silicon, but due to the lack of effective separation means for adsorbed biomass, reports that diatom biological silicon is used as a biomass extraction tool are rare.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for rapidly extracting biomass by utilizing diatom biological silicon and application thereof.
The specific technical scheme of the invention is as follows:
a method for rapidly extracting biomass by utilizing diatom biological silicon comprises the following steps:
1) Metal mineralization of diatom bio-silicon: uniformly dispersing diatom biological silicon in distilled water, then adding metal ion salt and reaction liquid, uniformly mixing, carrying out heating treatment, cooling to room temperature, centrifuging, collecting precipitate, rinsing with distilled water for 3-4 times, and drying;
2) And (3) rapidly extracting biomass: extracting and separating biomass in a biological sample by using the metal mineralized diatom biological silicon prepared in the step 1) and a buffer solution.
In the method, the metal mineralization of the diatom biological silicon is the key of the technical scheme, and the adsorption strength of the metal mineralized diatom biological silicon on different biomasses is adjusted by changing the pH value so as to realize the targets of adsorption collection and desorption collection and further improve the purity of the biomasses; the biomass can be quickly extracted and separated from the biological sample only by introducing the metal mineralized diatom biological silicon and the buffer solution, metal elements can be selectively solidified on the surface of the diatom biological silicon, metal residues in the separated biological material are reduced, the operation is simple, and the extraction efficiency is high.
Further, the step 2) adopts a method I or a method II;
the method I comprises the following steps: adding a biological sample and metal mineralized diatom bio-silicon into a first buffer solution of a centrifuge tube, adjusting the pH value of the first buffer solution to 4-10, centrifuging by using a centrifuge to collect bottom metal mineralized diatom bio-silicon precipitate, adding a second buffer solution for heavy suspension, centrifuging by using the centrifuge again and collecting a supernatant;
the second method comprises the following steps: adding the biological sample into a chromatographic column filled with metal mineralized diatom biological silicon, eluting with a buffer solution III with the pH value of 4-10 under the gravity flow rate, and collecting biological sample components absorbed on the chromatographic column.
Further, the diatom biological silicon comprises diatomite, diatom shells and the like.
Furthermore, the metal ions are one or two or more of metal ions such as iron, zinc, copper, cerium, calcium and the like, and are derived from corresponding metal salts.
Further, the mol ratio of the diatom biological silicon to the metal ions is 1: (0 to 20).
Further, the reaction solution is a weak base solution, including ammonia water, potassium hydroxide solution and the like, wherein the ammonia concentration of the ammonia water solution is 0.01-12 mol/L, the sodium hydroxide concentration is 0.01-12 mol/L, and the potassium hydroxide concentration is 0.01-12 mol/L.
Further, in the step 1), the heating temperature is 90-250 ℃, and the heating time is 0.01-24 hours.
Furthermore, the biological sample contains protein, nucleic acid, polysaccharide and other biological macromolecules, and can be expanded to monomers such as ribonucleic acid, deoxyribonucleic acid, amino acid, fatty acid, monosaccharide and the like.
Further, in the step 2), the concentration of the metal mineralized diatom bio-silicon is 1 × 10 -1 ～1×10 6 μg/mL。
Further, the pH value of the buffer solution II is 4-10, the rotating speed of the centrifugal machine is 1000-5000 rpm, and the time for centrifugal collection is 1-60s.
Further, in the step 2), the buffer I, the buffer II and the buffer III each independently include, but are not limited to, an ammonium formate + -formic acid buffer, a phosphate buffer, a citrate buffer, a carbonate buffer, an acetate buffer, a barbiturate buffer, and a Tris-hydroxymethyl-aminomethane (Tris-HCl) buffer.
The method for rapidly extracting biomass by utilizing diatom biological silicon can be applied to the fields of biological medicine, environmental engineering, food science, engineering materials, chemical engineering and the like.
The invention has the beneficial effects that:
the invention provides a simple, convenient, rapid and stable method for extracting biomass, reduces the cost of biomass extraction, and is particularly suitable for biological diagnosis and detection and the research of downstream molecular biology. According to the invention, a complex reaction container and a solvent are not needed, the structure of the biomass is not damaged, the adsorption strength of the metal mineralized diatom biological silicon to different biomasses is adjusted by changing the pH value so as to realize the purposes of adsorption collection and desorption collection, and the purity of the biomass is further improved; can follow biological sample and extract and separate living beings fast through introducing diatom biological silicon and buffer, the alternative solidifies metallic element on diatom biological silicon surface, reduces the metal residue in the biological material after the separation, easy operation, extraction efficiency is high.
Drawings
FIG. 1 is a scanning electron micrograph of diatom bio-silicon used in an example of the present invention;
A-5.00μm；B-1.00μm。
FIG. 2 is a graph showing the results of detection in example 1 of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The purification process of the diatom bio-silicon extraction method in the patent of CN111087472A by the applicant team is simple and easy to implement, and the obtained diatom bio-silicon has complete shape and low organic matter content, as shown in FIG. 1, which is a source of diatom bio-silicon used in the embodiment of the invention.
Example 1:
a method for rapidly extracting biomass by utilizing diatom biological silicon comprises the following steps:
1) Metal mineralization of diatom bio-silicon: uniformly dispersing 30mg of diatom bio-silicon in 5mL of distilled water, then adding 45mg of copper sulfate and 1mL of ammonia water into a reaction kettle, uniformly mixing, heating the reaction solution at high temperature of 250 ℃ for 2 hours with the ammonia concentration of 2mol/L, cooling, centrifuging, collecting precipitates, rinsing the precipitates collected by centrifuging for 4 times by using distilled water, and drying to obtain the copper mineralized diatom bio-silicon.
2) Fast extraction of biomass: adding 100 μ g of copper-mineralized diatom bio-silicon into 100 μ L of bovine serum albumin solution with concentration of 0.5mg/mL, adjusting pH to 6 and shaking for 60s on a vortex shaker, then centrifuging for 20s at 3000rpm, collecting bottom copper-mineralized diatom bio-silicon, adding 100 μ L of carbonate buffer solution with pH of 8 into a centrifuge tube to resuspend the copper-mineralized diatom bio-silicon, centrifuging for 20s at 3000rpm, and collecting supernatant.
As shown in FIG. 2, the concentration of bovine serum albumin detected in the supernatant was 0.711. + -. 0.006mg/mL, i.e., the adsorption amount of copper-mineralized diatom bio-silicon to bovine serum albumin was 71.1. + -. 6.2mg/g.
Example 2:
a method for rapidly extracting biomass by utilizing diatom biological silicon comprises the following steps:
1) Metal mineralization of diatom bio-silicon: similar to the first embodiment, further description is omitted here.
2) Fast extraction of biomass:
adding the bovine blood biological sample into a chromatographic column, wherein the filler of the chromatographic column is copper mineralized diatom biological silicon, slowly dropwise adding ammonium formate +/-formic acid buffer solution to elute the components of the bovine blood biological sample adsorbed on the chromatographic column, wherein the pH value of the ammonium formate +/-formic acid buffer solution is 6, replacing a collection bottle at intervals of 10 seconds, and detecting the target protein in the collection bottle.
Example 3:
a method for rapidly extracting biomass by utilizing diatom biological silicon comprises the following steps:
1) Metal mineralization of diatom bio-silicon: uniformly dispersing 20mg of diatom bio-silicon in 9mL of distilled water, adding 30mg of zinc sulfate and 1mL of ammonia water into a reaction kettle, uniformly mixing, keeping the ammonia concentration of reaction liquid at 1.2mol/L at 110 ℃ for 24 hours, cooling, rinsing the precipitate obtained by centrifugal collection with distilled water for 4 times, and drying to obtain the zinc-mineralized diatom bio-silicon.
2) Fast extraction of biomass: 10 μ g Zinc mineralized Diatom biosilica was added to 100 μ L of Staphylococcus aureus broth (1X 10) 5 CFU/mL), adjusting pH to 5, shaking for 60s on a vortex shaker, then centrifuging for 20s at 2000rpm, collecting zinc mineralized diatom bio-silicon at the bottom, adding 20 mu L of Tris-HCl buffer solution with pH of 9 into the centrifuge tube to resuspend the zinc mineralized diatom bio-silicon, centrifuging for 20s at 3000rpm, collecting supernatant, then adding 0.2mM dNTPs, TB green and 0.5U Taq DNA polymerase to carry out nucleic acid amplification reaction detection, and then detecting nuc gene of staphylococcus aureus in the amplification solution.
Therefore, the method for rapidly extracting the biomass by using the diatom biological silicon does not need a complex reaction container and a solvent and does not damage the structure of the biomass, and the adsorption strength of the metal mineralized diatom biological silicon to different biomasses is adjusted by changing the pH value so as to realize the targets of adsorption collection and desorption collection, thereby further improving the purity of the biomass; the biomass can be quickly extracted and separated from the biological sample by introducing the diatom biological silicon and the buffer solution; the method is simple, convenient, rapid and stable to extract the biomass and has obvious effect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for rapidly extracting biomass by utilizing diatom biological silicon is characterized by comprising the following steps:
1) Metal mineralization of diatom bio-silicon: uniformly dispersing the diatom bio-silicon in distilled water, then adding metal ion salt and reaction liquid, uniformly mixing, heating, cooling to room temperature, centrifuging, collecting precipitate, rinsing with distilled water for 3-4 times, and drying to obtain metal mineralized diatom bio-silicon;
2) And (3) rapidly extracting biomass: the metal mineralized diatom bio-silicon prepared in the step 1) and the buffer are used for extracting and separating biomass in a biological sample.
2. The method for rapidly extracting the biomass by utilizing the diatom bio-silicon as claimed in claim 1, wherein the step 2) adopts method one or method two;
the first method comprises the following steps: adding a biological sample and metal mineralized diatom bio-silicon into a first buffer solution of a centrifuge tube, adjusting the pH value of the first buffer solution to 4-10, centrifuging by using a centrifuge to collect bottom metal mineralized diatom bio-silicon precipitate, adding a second buffer solution for heavy suspension, centrifuging by using the centrifuge again and collecting a supernatant;
the second method comprises the following steps: adding the biological sample into a chromatographic column filled with metal mineralized diatom biological silicon, eluting by using a buffer solution III with the pH value of 4-10 under the gravity flow rate, and collecting the components of the biological sample absorbed on the chromatographic column.
3. The method for rapidly extracting biomass by using the diatom bio-silicon as claimed in claim 1, wherein the diatom bio-silicon comprises diatomaceous earth, frustules; the metal ions are one or two or more of iron, zinc, copper, cerium or calcium ions; the mol ratio of the diatom biological silicon to the metal ions is 1:0 to 20.
4. The method for rapidly extracting biomass by using diatom biosilica according to claim 1, wherein the reaction solution is an alkaline solution, the alkaline solution is an ammonia water, sodium hydroxide or potassium hydroxide solution, the ammonia water solution has an ammonia concentration of 0.01-12 mol/L, the sodium hydroxide concentration is 0.01-12 mol/L, and the potassium hydroxide concentration is 0.01-12 mol/L.
5. The method for rapidly extracting biomass by using diatom bio-silicon according to claim 2, wherein in step 1), the heating temperature is 90-250 ℃ and the heating time is 0.01-24 hours.
6. The method for rapid extraction of biomass using diatom bio-silicon according to claim 1, wherein the biological sample is a biological macromolecule, ribonucleic acid, deoxyribonucleic acid, amino acid or monosaccharide sugar.
7. The method for rapidly extracting biomass by using diatom bio-silicon as claimed in claim 5, wherein in step 2), the concentration of said metal mineralized diatom bio-silicon is 1x10 -1 ～1×10 6 μg/mL。
8. The method for rapidly extracting biomass by using the diatom bio-silicon as claimed in claim 6, wherein the pH of the second buffer solution is 4-10, the rotation speed of the centrifuge is 1000-5000 rpm, and the time for collecting by centrifugation is 1-60s.
9. The method for rapidly extracting biomass by using diatom biosilica according to claim 1, wherein in step 2), the buffer is ammonium formate ± formic acid buffer, phosphate buffer, citrate buffer, carbonate buffer, acetate buffer, barbiturate buffer or tris buffer.
10. The use of the method for rapid extraction of biomass from diatom biosilica according to any of claims 1-9 in the fields of biomedicine, environmental engineering, food science, engineering materials or chemical engineering.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676503A (en) * | 2012-05-16 | 2012-09-19 | 亚能生物技术（深圳）有限公司 | Method for quickly extracting DNA (deoxyribonucleic acid) |
| CN106011131A (en) * | 2016-07-29 | 2016-10-12 | 北京旌准医疗科技有限公司 | Method for separating free nuclei free nucleic acids from plasma |
| CN106902753A (en) * | 2017-03-03 | 2017-06-30 | 安徽金培因科技有限公司 | A kind of preparation method of sorbing material |
| CN107881577A (en) * | 2017-12-11 | 2018-04-06 | 东莞市联洲知识产权运营管理有限公司 | A kind of renewable biomass nanofiber prepared using diatom as raw material and preparation method thereof |
| CN111085165A (en) * | 2018-10-23 | 2020-05-01 | 中国海洋大学 | Preparation method of surface mineralized diatom biomineralized silicon |
| CN111087472A (en) * | 2018-10-23 | 2020-05-01 | 中国海洋大学 | Preparation method of biological active diatom biomineralization silicon |
| CN112972752A (en) * | 2021-02-04 | 2021-06-18 | 中国海洋大学 | Method for stopping bleeding by mixing frustules with different structural characteristics |
| CN114107282A (en) * | 2021-11-24 | 2022-03-01 | 青岛科技大学 | Method for extracting nucleic acid by modified diatomite and application |
-
2022
- 2022-12-16 CN CN202211622325.4A patent/CN115806973B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676503A (en) * | 2012-05-16 | 2012-09-19 | 亚能生物技术（深圳）有限公司 | Method for quickly extracting DNA (deoxyribonucleic acid) |
| CN106011131A (en) * | 2016-07-29 | 2016-10-12 | 北京旌准医疗科技有限公司 | Method for separating free nuclei free nucleic acids from plasma |
| CN106902753A (en) * | 2017-03-03 | 2017-06-30 | 安徽金培因科技有限公司 | A kind of preparation method of sorbing material |
| CN107881577A (en) * | 2017-12-11 | 2018-04-06 | 东莞市联洲知识产权运营管理有限公司 | A kind of renewable biomass nanofiber prepared using diatom as raw material and preparation method thereof |
| CN111085165A (en) * | 2018-10-23 | 2020-05-01 | 中国海洋大学 | Preparation method of surface mineralized diatom biomineralized silicon |
| CN111087472A (en) * | 2018-10-23 | 2020-05-01 | 中国海洋大学 | Preparation method of biological active diatom biomineralization silicon |
| CN112972752A (en) * | 2021-02-04 | 2021-06-18 | 中国海洋大学 | Method for stopping bleeding by mixing frustules with different structural characteristics |
| CN114107282A (en) * | 2021-11-24 | 2022-03-01 | 青岛科技大学 | Method for extracting nucleic acid by modified diatomite and application |
Non-Patent Citations (2)
| Title |
|---|
| BANSI GHADIA ET AL.: "An improved method of DNA purification from secondary metabolites rich medicinal plants using certain chaotropic agents", 《ACTA PHYSIOL PLANT》 * |
| CHAO FENG ET AL.: "Chitosan-Coated Diatom Silica as Hemostatic Agent for Hemorrhage Control", 《ACS APPLIED MATERIALS & INTERFACES》 * |
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