阅读说明：本技术 一种酰胺型交联聚合物及其制备方法和其在生物蛋白质沉淀中的用途 (Amide type cross-linked polymer, preparation method thereof and application thereof in biological protein precipitation ) 是由 王德明 周杰锋 于 2020-07-01 设计创作，主要内容包括：本申请涉及生物蛋白质沉淀用新材料技术领域,尤其涉及一种二元胺二元羧酸交联聚合物及其制备方法和其在生物蛋白质沉淀中的用途。一种二元胺二元羧酸交联聚合物,该交联聚合物采用咪唑基二元胺代替普通二元胺,从而使聚合物具有了较好的热稳定性和抗菌性,可以极大的方便生物检测操作,同时又便于在更宽的温度范围内储存、运输；从而降低生物检测的生产成本。(The application relates to the technical field of new materials for biological protein precipitation, in particular to a diamine dicarboxylic acid cross-linked polymer, a preparation method thereof and application thereof in biological protein precipitation. A diamine dicarboxylic acid cross-linked polymer, the cross-linked polymer adopts imidazolyl diamine to replace common diamine, so that the polymer has better thermal stability and antibacterial property, can greatly facilitate biological detection operation, and is convenient to store and transport in a wider temperature range; thereby reducing the production cost of the biological detection.)

1. An amide-type crosslinked polymer characterized by the formula:

wherein R1 is any one of CH3 and CH2CH 3; r2 is H,Any one of (a); r3 is any one of H, CH 3; n is 5 to 1500.

2. The amide-type crosslinked polymer according to claim 1, wherein n is 50 to 1000.

3. A composition comprising the amide-type crosslinked polymer of claim 1 or 2 and a carrier, wherein the amide-type crosslinked polymer is dispersed in a liquid system to form a stable system with the carrier; preferably, the mass ratio of the amide-type crosslinked polymer to the carrier is 1:2 to 1: 50.

4. A composition according to claim 3, wherein the carrier is a glass, silica, ceramic, plastic, resinous particles, beads, gel, polyelectrolyte or hydrogel.

5. A composition according to claim 3, further comprising a silane coupling agent.

6. A method for precipitating a protein from an aqueous medium containing the protein, which comprises adding the amide-type crosslinked polymer according to claim 1 or 2 or the composition according to any one of claims 3 to 5 to the aqueous medium, and mixing the mixture with stirring; preferably, the amide-type crosslinked polymer is added to the aqueous medium in the form of an emulsion, a suspension, a solution or a dry powder.

7. The method according to claim 6, wherein the mass ratio of the amide-type crosslinked polymer to the protein in the aqueous medium is 1:3 to 3: 1.

8. The method of claim 6, wherein the aqueous medium is a diluted or undiluted biological fluid containing the proteins desired to be removed and includes fluids such as whole blood, plasma, serum, lymph, bile, urine, spinal fluid, sputum, sweat, etc.; excrement excretion; it is also possible to use liquid preparations of human or other animal tissues (e.g. skeletal muscle, heart, kidney, lung, brain), including cell culture extracts or milk or microbial cultures or plant extracts; preferably, the biological fluid is human blood and bacterial cell lysate.

9. The method for producing an amide-type crosslinked polymer according to claim 1 or 2, wherein the reaction equation is as follows:

the method comprises the following steps:

1) respectively dissolving the copolymer II and the imidazolyl diamine III in an organic solvent, dropwise adding the organic solution dissolved with the imidazolyl diamine III into the organic solution dissolved with the copolymer II according to the molar ratio of the copolymer II to the imidazolyl diamine III of 1: 0.5-5, wherein the dropwise adding speed is 3-10.0 ml/min, and the whole dropwise adding time and the subsequent stirring time are 1-5 h;

2) the mixture reacts for 8-24 h at room temperature;

3) after the reaction is completed, primarily purifying and separating the product I by adding deionized water for many times and filtering;

4) and suspending the product I in deionized water to form a relatively uniform suspension with the concentration of 0.05-1.5mol/L, adjusting the pH value of the suspension to 1-2 by using an acidic regulator, adjusting the pH value of the suspension to 8-10 by using an alkaline regulator, finally adjusting the pH value to 7.0, and filtering to obtain the product I with high purity.

10. The method according to claim 9, wherein the organic solvent in step 1) is acetone, dimethyl sulfoxide or dimethylformamide, and the concentration of the copolymer II and the imidazolyl diamine III is 0.05 to 1.5 mol/L.

Technical Field

The application relates to the technical field of new materials for biological protein precipitation, in particular to an amide type cross-linked polymer, a preparation method thereof and application thereof in biological protein precipitation.

Background

The gene amplification detection is based on Polymerase Chain Reaction (PCR) technology, and is an important analysis method in the field of molecular diagnosis, and the standardization requirement of the gene amplification detection is increasingly paid attention to by people. The PCR technology has been widely used in clinical disease diagnosis and genetic disease screening, and blood samples are generally used as detection samples in clinical practice to perform quantitative and qualitative analysis on target DNA by the PCR technology. Hemoglobin in blood cells has an inhibitory effect on the PCR reaction, and therefore, hemoglobin in a blood sample is often removed in nucleic acid detection of the sample. Traditionally, proteins are removed from aqueous media by using reagents or heat, and certain solvents (chloroform, urea) denature the proteins and cause their precipitation. Similarly, increasing the electrolyte level of the medium to a high degree by addition of salt also precipitates proteins. Furthermore, heat not only denatures the protein, but often coagulates the protein, separating it from the aqueous medium. These protein removal methods have three major disadvantages, firstly, the material removed from the medium is not a pure protein, and instead, when the medium comprises a complex chemical mixture (e.g., the medium is a cell lysate), the use of these methods may result in the removal of materials other than proteins. Secondly, proteins removed by these methods are usually irreversibly denatured or renatured only by time-consuming procedures such as dialysis. Third, isolation or purification of proteins typically involves the use of highly toxic solvents (phenol and/or chloroform).

The Chinese patent application (application number: 2020102507995) discloses an integrated adding device for pretreatment reagents of biological samples, which comprises a collecting pipe, a centrifugal column, a test tube cover and reagent solutions, wherein the centrifugal column is arranged in the collecting pipe, the reagent solutions are arranged in the centrifugal column, the test tube cover is arranged at the upper end of the collecting pipe, the test tube cover comprises a cover body, a storage pipe is arranged on the cover body, the bottom of the storage pipe is sealed and extends into the centrifugal column, additives are arranged in the storage pipe, a puncturing device capable of puncturing the bottom of the storage pipe is arranged on the test tube cover, and after the bottom of the storage pipe is punctured, the additives in the storage pipe fall into the centrifugal column. The device adopts the structure of integration, presets reagent solution in the centrifuging tube, is provided with the additive in the storage tube, has avoided on-the-spot configuration work, and during the use, the extract sample can carry out the processing operation through the additive under the reagent solution effect, has avoided reagent solution and additive to influence each other. When a biological sample pretreatment reagent integrated addition device is used, an additive (protein precipitant) needs to be stored in a storage tube, which puts a demand on the thermal stability of the protein precipitant. If a protein precipitation performance which is good, and simultaneously has good thermal stability and storage/transportation stability can be found, biological detection operation can be greatly facilitated, and meanwhile, storage and transportation within a wider temperature range are facilitated; thereby reducing the production cost of the biological detection.

Disclosure of Invention

In order to solve the above-mentioned technical problems, the present application aims to provide a crosslinked polymer bridged by an amide-type crosslinked polymer structure, wherein the crosslinked polymer has a chain skeleton of a copolymer (II) of vinyl ether and maleic anhydride with different branched structures, and an imidazolyl diamine (III) as a crosslinking agent to obtain the amide-type crosslinked polymer (I). The polymer not only has better thermal stability, but also has better hemoglobin separation effect under the condition of being higher than room temperature, and has storage/transportation stability, thereby being greatly convenient for biological detection operation and storage and transportation in a wider temperature range; thereby reducing the production cost of the biological detection.

In order to achieve the above object, the present application adopts the following technical solutions:

an amide-type crosslinked polymer having the formula:

wherein R1 is any one of CH3 and CH2CH 3; r2 is H,Any one of (a); r3 is any one of H, CH 3; n is 5 to 1500.

Preferably, n is 50 to 1000.

Further, the present application discloses a composition comprising an amide-type crosslinked polymer, and a carrier, wherein the amide-type crosslinked polymer is dispersed in a liquid system to form a stable system with the carrier; preferably, the mass ratio of the amide-type crosslinked polymer to the carrier is 1:2 to 1: 50.

Preferably, the carrier is glass, silica, ceramic, plastic, resin particles, beads, gel, polyelectrolyte or hydrogel.

Preferably, the composition further comprises a silane coupling agent, and the silane coupling agent can couple the amide-type crosslinked polymer to the surface and in the pores of the carrier.

Further, the present application discloses a method for precipitating a protein from an aqueous medium containing the protein, which comprises adding the amide-type crosslinked polymer or the composition to the aqueous medium, and mixing with stirring; preferably, the amide-type crosslinked polymer is added to the aqueous medium in the form of an emulsion, a suspension, a solution or a dry powder.

Preferably, the mass ratio of the amide-type crosslinked polymer to the protein in the aqueous medium is 1:3 to 3: 1.

Preferably, the aqueous medium is a diluted or undiluted biological fluid containing the proteins that are desired to be removed, and includes fluids such as whole blood, plasma, serum, lymph, bile, urine, spinal fluid, sputum, sweat, and the like; excrement excretion; it is also possible to use liquid preparations of human or other animal tissues (e.g. skeletal muscle, heart, kidney, lung, brain), including cell culture extracts or milk or microbial cultures or plant extracts; most preferably, the biological fluid is human blood and bacterial cell lysate.

Further, the present application also discloses a method for preparing an amide-type crosslinked polymer, the method having a reaction equation of:

the method comprises the following steps:

1) respectively dissolving the copolymer II and the imidazolyl diamine III in an organic solvent, dropwise adding the organic solution dissolved with the imidazolyl diamine III into the organic solution dissolved with the copolymer II according to the molar ratio of the copolymer II to the imidazolyl diamine III of 1: 0.5-5, wherein the dropwise adding speed is 3-10.0 ml/min, and the whole dropwise adding time and the subsequent stirring time are 1-5 h;

2) the mixture reacts for 8-24 h at room temperature;

3) after the reaction is completed, primarily purifying and separating the product I by adding deionized water for many times and filtering;

4) and suspending the product I in deionized water to form a relatively uniform suspension with the concentration of 0.05-1.5mol/L, adjusting the pH value of the suspension to 1-2 by using an acidic regulator, adjusting the pH value of the suspension to 8-10 by using an alkaline regulator, finally adjusting the pH value to 7.0, and filtering to obtain the product I with high purity.

Preferably, the organic solvent in the step 1) is acetone, dimethyl sulfoxide or dimethylformamide, and the concentration of the copolymer II and the imidazolyl diamine III is 0.05-1.5 mol/L.

By adopting the technical scheme, the cross-linked polymer adopts imidazolyl diamine as a bridging structure, so that the polymer has better thermal stability and storage/transportation stability, the imidazolyl structure has a stable five-membered ring structure, and the cross-linked polymer has better thermal stability so as to be stored and transported in a wider temperature range, the use temperature is properly increased, and the separation speed of the cross-linked polymer on hemoglobin in a biological sample is favorably increased, so that the cross-linked polymer has better application prospect.

Drawings

FIG. 1 shows an IR spectrum of an amide-type crosslinked polymer prepared from a copolymer of methyl vinyl ether and maleic anhydride as a chain skeleton and 1, 2-diaminoimidazole as a crosslinking agent.

FIG. 2 is a graph showing the efficiency of SDS-PAGE protein gel adsorption of Bovine Serum Albumin (BSA) by the amide-type crosslinked polymer.

FIG. 3 is a graph showing the efficiency of adsorption of hemoglobin in whole blood by the amide-type crosslinked polymer.

FIG. 4 is a graph showing a control experiment in which an amide-type crosslinked polymer adsorbs hemoglobin in whole blood.

FIG. 5 is a graph showing the thermal stability of an amide-type crosslinked polymer.

FIG. 6 is a graph showing stability tests of amide-type crosslinked polymers.

Detailed Description

The invention will be described in detail with reference to specific embodiments, which are intended to be illustrative only and not to be limiting of the invention, and any modifications, equivalents and the like based on the invention are intended to be included within the scope of the invention.