CN109609336B

CN109609336B - A catch sieve for catching biomolecule, cell or bacterium

Info

Publication number: CN109609336B
Application number: CN201811375355.3A
Authority: CN
Inventors: 颜菁; 方欣
Original assignee: Jiangsu Huixian Pharmaceutical Technology Co ltd
Current assignee: Jiangsu Huixian Pharmaceutical Technology Co ltd
Priority date: 2018-11-19
Filing date: 2018-11-19
Publication date: 2021-05-18
Anticipated expiration: 2038-11-19
Also published as: WO2020103422A1; CN109609336A

Abstract

The invention discloses a capture sieve for capturing biomolecules, cells or bacteria, which has higher capture efficiency. A capture sieve for capturing biomolecules, cells or bacteria comprises a substrate layer, a protective layer formed on the substrate layer and a macromolecule layer formed on the protective layer, and comprises the substrate layer, the protective layer formed on the substrate layer and the macromolecule layer formed on the protective layer, wherein the macromolecule layer comprises the following raw materials: polyvinyl alcohol and/or polyethylene glycol; hyaluronic acid; hydroxyethyl cellulose; agarose and/or dextran.

Description

A catch sieve for catching biomolecule, cell or bacterium

Technical Field

The invention relates to the technical field of biology, in particular to a capture sieve for capturing biomolecules, cells or bacteria.

Background

The microfluidic chip technology integrates basic operation processes of laboratories such as biology, chemistry and the like into one chip, has the advantages of integration, high flux, rapid detection, convenient operation, small required sample volume and low energy consumption, and has more and more wide application prospects in many scientific research and living fields such as pharmaceutical research and the like in recent years. Means for on-chip cell separation and capture involve numerous fields of optical, electrical, acoustic, magnetic, hydrodynamic, mechanical processing, and chemical methods. Micromachining techniques combined with hydrodynamic control for the capture of whole and single cell, bacterial samples are currently the most effective means of immobilization. The technology is often used for capturing cells by processing geometric traps or obstacles such as micro wells, micro holes, micro dams, micro slits, micro pipelines and the like with the sizes matched with the cells, so that an open array system can be formed, and the cells can be controlled in a micro channel.

With the continuous progress of micro-processing technology, the advantages of the micro-fluidic chip in biological analysis are more prominent, and the micro-fluidic chip has great development potential and application value in a plurality of fields such as disease diagnosis, drug screening, cell molecular biology research and the like. In the field of biological analysis, the polymer used in the capture screen mainly includes dimethyl siloxane (PDMS) and Polyethylene (PC), among which PDMS is one of the longest used materials due to its good biocompatibility, light permeability and easy processing. However, the existing capture sieve is prone to non-specific adsorption of biological macromolecules such as proteins, bacteria, cells and other substances in a biological sample, and affects the bioanalysis performance of the chip. And the polymer has low surface energy, lacks active groups required by the functionalization reaction and has low capture efficiency.

Disclosure of Invention

It is an object of the present invention to provide a capture sieve for capturing biomolecules, cells or bacteria, which has a high capture efficiency.

In order to achieve the purpose, the invention adopts the technical scheme that:

a capture sieve for capturing biomolecules, cells or bacteria, which comprises a substrate layer, a protective layer formed on the substrate layer and a macromolecule layer formed on the protective layer, wherein the macromolecule layer comprises the following raw materials: polyvinyl alcohol and/or polyethylene glycol; hyaluronic acid; hydroxyethyl cellulose; agarose and/or dextran.

Further, the raw material of the polymer layer further includes a degradation liquid. The polymer layer added with the degradation liquid can be rapidly degraded, and the 'lossless' desorption and re-culture of the captured substance are realized.

Furthermore, the raw material of the macromolecule layer also comprises at least one of allyl alcohol, sodium alginate, polylactic acid, polyamide and chitosan.

Still further, the degradation liquid is selected from one or a combination of two of hyaluronidase and cellulase.

Preferably, the raw materials of the high polymer comprise one or a mixture of two of polyvinyl alcohol, polyethylene glycol and allyl alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and dextran and chitosan in a mass ratio of 3-10:8-15:0.1-6:4-12:4-18:0.5-5: 10-20.

Preferably, the raw materials of the polymer comprise one or a mixture of two of polyvinyl alcohol and polyethylene glycol and allyl alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and dextran, and one or a mixture of two of chitosan and chitosan, and a degradation liquid in a mass ratio of 3-10:8-15:0.1-6:4-12:4-18:0.5-5:10-20:0.1-6, wherein the degradation liquid is selected from one or a combination of two of hyaluronidase and cellulase.

Furthermore, the raw materials of the macromolecule layer also comprise branched polymers, and the branched polymers and other raw materials of the macromolecule layer form a cross-linked network structure, so that the capture efficiency is further improved. And can increase the species of capture, such as an antibody, antigen, aptamer, protein, or a combination thereof.

Still further, the branched polymer is selected from the group consisting of one or more of polyacrylamide, acrylamide, polyetheramine, polyamidoamine, polyesteramide, poly (amide-ester), polyphenylene ether, polyethylene glycol.

Further, the capture screen further comprises a branched polymer layer formed on the polymer layer. The species of capture objects, such as antibodies, antigens, aptamers, proteins, or combinations thereof, can be increased.

Still further, the raw material of the branched polymer layer is selected from one or more of polyacrylamide, acrylamide, polyetheramine, polyamide-amine, polyesteramide, poly (amide-ester), polyphenylene oxide, and polyethylene glycol.

Further, the material of the substrate layer is stainless steel; the protective layer is made of noble metal or alloy thereof.

According to the capture sieve provided by the invention, the degradable high polymer layer is covered on the surface of the protective layer, and polyethylene glycol, polyvinyl alcohol, transparent grease, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose or glucan or chitosan and the like are adopted for polymerization to activate the surface of the material, so that the high polymer material film has good hydrophilicity. The degradation liquid is added into the copolymer, so that lossless elution or re-culture can be realized at the stage of eluting target molecules. On one hand, the capture efficiency of target molecules is improved, and on the other hand, the elution efficiency of target capture substances is improved by adding degradation liquid into the high polymer material.

Compared with the prior art, the invention has the following advantages by adopting the scheme:

the polymer coating is covered on the surface of the protective layer, polyethylene glycol and/or polyvinyl alcohol, hyaluronic acid, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and/or glucan and the like are introduced for polymerization, and the surface of the material is activated, so that the polymer material film has good hydrophilicity, and the capture efficiency of target molecules is improved (can reach more than 93%). The capture chip material made of the high polymer material has low price, is easy to process, is suitable for industrial mass production, consumes less sample reagent, can carry out various biochemical modifications according to the biochemical analysis requirement, meets the biochemical analysis requirement, and can meet the process requirement of the microfluidic chip according to the proportion condition.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a schematic cross-sectional view of a capture screen according to the present invention;

figure 2 is a schematic cross-sectional view of another capture screen according to the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the invention may be more readily understood by those skilled in the art. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

The present embodiment provides a capture sieve, which can be used in a microfluidic chip to capture molecules (especially biomacromolecules), cells, bacteria, and the like in a biological sample. Figure 1 shows a partial cross-sectional view of the capture screen. Referring to fig. 1, the capture screen is composed of a substrate layer 1, a protective layer 2, a polymer layer 3, a branched polymer layer 4, and a functional material layer (not shown). When fluid containing biomolecules, bacteria or cells flows through the capture sieve, the capture sieve fixes the captured substances on the capture sieve through physical action, so that the purpose of capture is achieved.

The material of the substrate layer 1 is stainless steel, a plurality of micron-sized pore channels for fluid to pass through are formed on the substrate layer, the pore diameter of each pore channel is micron-sized, and the pore channels are parallel or parallel to each other.

The protective layer 2 is formed on the substrate layer 1, for example, on the outer surface of the substrate layer 1, specifically, on the hole walls of the respective channels. The protective layer 2 is a metal protective layer made of noble metal or alloy thereof, specifically, noble metal such as gold, nickel, iron, or alloy thereof.

The polymer layer 3 is formed on the protective layer 2, for example, covering the outer surface of the protective layer 2. The starting materials for the polymer layer 3 include: one or more of polyvinyl alcohol, polyethylene glycol and allyl alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose, dextran, chitosan, and the like, and mixtures of one or more of the foregoing materials. The raw material of the high molecular layer also comprises degradation liquid, and the degradation liquid is selected from one or the combination of two of hyaluronidase and cellulase.

The branched polymer layer 4 is formed on the polymer layer 3, for example, covering the outer surface of the polymer layer 3. The material of the branched polymer layer 4 is selected from one or more of polyacrylamide, acrylamide, polyetheramine, polyamide, polyesteramide, poly (amide-ester), polyphenylene oxide, and polyethylene glycol.

The functional material layer is formed on the branched polymer layer 4, and specifically includes a capture substance, such as an antibody, a phage, or the like, coupled to the branched polymer layer 4.

The preparation method of the capture screen comprises the following steps:

step 1, preparing a substrate layer: providing stainless steel, forming a plurality of parallel micron-sized fluid pore channels on the stainless steel to form a sieve-shaped structure, and obtaining a stainless steel substrate layer with the micron-sized fluid pore channels;

step 2, preparing a plating layer (namely the protective layer): plating metal or metal alloy on the wall of each pore channel of the substrate layer by adopting a Physical Vapor Deposition (PVD) or Chemical Vapor Deposition (CVD) method, and covering the wall of each pore channel of the substrate layer to form a metal protective layer;

step 3, wrapping a biological macromolecule layer (namely the macromolecule layer): coupling the raw material of the polymer layer to the surface of the metal protective layer in the pore channel by adopting a physical adsorption or chemical reaction method;

step 4, preparing a branched polymer layer: the raw material of the branched polymer layer is chemically or physically coupled to the surface of the polymer layer in the pore channel.

Step 5, preparing a functional material layer: coupling capture objects (e.g. antibodies, phage, etc.) to the branching layer by physical or chemical means

The step 3 is specifically as follows:

3-1, dissolving one or two of mixture of polyvinyl alcohol and polyethylene glycol and allyl alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and dextran and chitosan, and degradation liquid (hyaluronidase and/or cellulase) in PBS-EDTA solution with pH of 8 to prepare solution A, wherein the mass percentages of the components are 3-10%, 8-15%, 0.1-6%, 4-12%, 4-18%, 0.5-5%, 10-20% and 0.1-6%, respectively.

And 3-2, preparing 0.8mM DTT (dithiothreitol) or TECP (tris (2-carboxyethyl) phosphine) solution B by adopting a PBS-EDTA solution with the pH value of 8.

And 3-3, mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain a solution C.

3-4, placing the cleaned capture sieve prepared in the step 2 in the solution C, and incubating for 12h on a shaking table;

and 3-5, cleaning the capture sieve by adopting a PBS-EDTA solution to obtain the capture sieve wrapped by the bio-macromolecule layer.

The step 4 is specifically as follows:

step 4-1, branched polymer preparation: dissolving a mixture of polyacrylamide, acrylamide, polyetheramine, polyamide-amine, polyesteramide, poly (amide-ester), polyphenyl ether and polyethylene glycol in PBS-EDTA buffer solution with the pH value of 8 to prepare solution D, wherein the mass percentages of the components are 5-20%, 1-16%, 0.1-8%, 2-16%, 2.5-10%, 3-15%, 0.1-3% and 2-12%.

And 4-2, preparing 0.8mM DTT (dithiothreitol) or TECP (tris (2-carboxyethyl) phosphine) solution E by using a PBS-EDTA solution with the pH value of 8.

And 4-3, preparing a 1mM biotin PEG solution F by using the solution E, mixing the solution D and the solution F according to a ratio of 1:1 to obtain a solution G, and incubating the solution G at room temperature for 12 hours.

4-4, adding streptavidin into the solution G, and incubating at room temperature for 1H to obtain a solution H;

4-5, placing the semi-finished product of the capture sieve prepared in the step 3-5 in a solution H, and incubating for 3H in a shaking table;

and 4-6, cleaning the capture sieve by using a PBS-EDTA solution to obtain the capture sieve wrapped by the branched polymer layer.

Example 2

The present embodiment provides a capture sieve, which can be used in a microfluidic chip to capture molecules (especially biomacromolecules), cells, bacteria, and the like in a biological sample. Figure 2 shows a partial cross-sectional view of the capture screen. Referring to fig. 2, the capture screen differs from the capture screen of example 1 in that: the capture screen is composed of a substrate layer 1, a protective layer 2, a polymer layer 3 'and a functional material layer (not shown in the figure), and a branched polymer is added into the raw material of the polymer layer 3'. The base material layer 1 and the protective layer 2 were the same as in example 1.

The starting materials for the polymer layer 3' include: one or more of polyvinyl alcohol, polyethylene glycol, and propylene glycol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose, dextran, and chitosan, and the like, formed from a combination of one or more of the foregoing materials. The raw material of the high molecular layer 3' also comprises degradation liquid, and the degradation liquid is selected from one or the combination of two of hyaluronidase and cellulase. The raw material of the polymer layer 3' further comprises a branched polymer, and the branched polymer is selected from one or more of polyacrylamide, acrylamide, polyether amine, polyamide, polyester amide, poly (amide-ester), polyphenyl ether and polyethylene glycol.

The functional material layer is formed on the polymer layer 3 ', and specifically includes a capture substance, such as an antibody, a phage, etc., coupled to the polymer layer 3'.

The preparation method of the capture screen comprises the following steps:

step 3, preparation of bio-macromolecule layer and branched polymer layer (namely, the macromolecule layer): the raw material of the macromolecule layer is coupled to the surface of the metal protective layer in the pore channel through physical adsorption or chemical reaction.

Step 4, preparing a functional material layer: coupling capture objects (e.g. antibodies, phage, etc.) to the branching layer by physical or chemical means

The step 3 is specifically as follows:

step 3-1, stirring and dissolving one or two mixture of polyvinyl alcohol and polyethylene glycol and propylene glycol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and one or two mixture of dextran and chitosan, degradation liquid (hyaluronidase and/or cellulase) and branched polymer (polyacrylamide, acrylamide and polyether amine) in PBS-EDTA solution with pH of 8 to prepare copolymer solution A, wherein the mass percentage of each component is 3-10%, 8-15%, 0.1-6%, 4-12%, 4-18%, 0.5-5%, 10-20%, 0.1-6% and 0.1-20%;

step 3-2, preparing 0.8mM DTT (dithiothreitol) or TECP (tris (2-carboxyethyl) phosphine) solution B by adopting a PBS-EDTA solution with the pH value of 8;

3-3, preparing a 1mM biotin PEG solution C by using the solution B, mixing the solution A and the solution C according to a ratio of 1:1 to obtain a solution D, and incubating the solution D at room temperature for 12 hours;

step 3-4, adding streptavidin into the solution D, and incubating for 1h at room temperature to obtain a solution E;

3-5, placing the cleaned semi-finished product of the capture sieve prepared in the step 2 into the solution E, and incubating for 3 hours by using a shaking table;

and 3-6, cleaning the capture sieve by adopting a PBS-EDTA solution to obtain a biological macromolecule layer and a capture sieve wrapped by branched molecules.

According to the capture sieve provided by the invention, the degradable high polymer coating is coated on the surface of the metal protective layer, and polyethylene glycol, polyvinyl alcohol, transparent grease, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose or glucan or chitosan and the like are adopted for polymerization to activate the surface of the material, so that the high polymer material film has good hydrophilicity. The degradation liquid is added into the copolymer, so that lossless elution or re-culture can be realized at the stage of eluting target molecules. On one hand, the capture efficiency of target molecules is improved, and on the other hand, the elution efficiency of target capture substances is improved by adding degradation liquid into the high polymer material.

In addition, the invention coats the surface of the macromolecule layer with a supported polymer coating, or adds a branched polymer into the macromolecule layer, the functional group on the surface of the branched polymer increases with the relative molecular mass and the molecular size, the dispersing ability of the nano particles in the polymer is improved, the interface bonding force of the nano particles and the polymer is increased, the surface energy of the particles is reduced, the surface charge of the particles is eliminated, the affinity of the particles and an organic phase is improved, the surface polarity of the particles is weakened, and the branched polymer is introduced to show better toughness than the modified organic silicate. The inner core or outer shell of the core-shell structure of the branched polymer is used as a site for small molecule reaction, so that the performance of the capture sieve is improved. By introducing the branched polymer comprising materials such as polyacrylamide, acrylamide, polyether amine and the like, the nonspecific adsorption and toxicity of the material are reduced, the hydrophilicity and the modification of the material are improved, the material and liquid are easy to separate, centrifugal operation is not needed, cells, bacteria and target biomolecules can be efficiently captured, and the activity of the captured cells is not obviously damaged.

The capture screen of the invention has the following characteristics: (1) the capture sieve of the invention connects target molecules (biological small molecules, DNA, RNA, antibody protein and the like) on a high molecular polymer capture sieve carrier through photocrosslinking and covalence, compared with the conventional capture sieve, the capture sieve has greatly improved nonspecific resistance, greatly improves the capture success rate of drug targets, and reaches 93 percent. (2) The high molecular copolymer and the branched polymer adopted by the invention can increase the types of capture objects, such as antibodies, antigens, aptamers, proteins or combinations thereof. (3) The whole hybridization process has small nonspecific adsorption and no obvious mutual cross interference. (4) The capture chip material made of the high polymer material has low price, easy processing, suitability for industrial mass production, less sample reagent consumption, capability of carrying out various biochemical modifications according to the biochemical analysis requirement, meeting the biochemical analysis requirement, low cost and batch manufacturing, and the composition proportion of the high polymer material is non-single material, and can meet the process requirement of the microfluidic chip according to the proportion condition. (5) The polymer layer added with the degradation liquid adopted by the capture sieve can be rapidly degraded, so that 'lossless' desorption and re-culture of the captured objects are realized, and the elution efficiency is over 85 percent.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and are preferred embodiments, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention. All equivalent changes or modifications made according to the principles of the present invention should be covered within the protection scope of the present invention.

Claims

1. A capture sieve for capturing biomolecules, cells or bacteria, comprising a base layer, a protective layer formed on the base layer, and a polymer layer formed on the protective layer, characterized in that: the raw materials of the macromolecule layer comprise polyvinyl alcohol, polyethylene glycol, allyl alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose, glucan, chitosan and degradation liquid, wherein the degradation liquid is selected from hyaluronidase and cellulase; the capture screen further includes a branched polymer layer formed on the macromolecule layer.

2. The capture screen of claim 1, wherein: the raw material of the branched polymer layer is selected from one or more of polyacrylamide, acrylamide, polyether amine, polyamide-amine, polyester amide, poly (amide-ester), polyphenyl ether and polyethylene glycol.

3. The capture screen of claim 1, wherein: the base layer is made of stainless steel; the protective layer is made of noble metal or alloy thereof.