CN117866214A - Macroporous frozen gel medium and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a macroporous frozen gel medium and a preparation method and application thereof. The invention utilizes high-efficiency click reaction to introduce high-performance boric acid ligand into macroporous low-temperature gel for bacterial combination. In order to further increase the binding capacity of the composite cryogel, a nanoparticle-supported hydrophilic, flexible and soft polymer is added to increase the effective inner surface area of the cryogel and the density of the immobilized boric acid. The prepared Kong Lengdong gel medium has a modularized and clicking component and can be applied to rapid detection of bacteria.

Description

A macroporous cryogel medium and its preparation method and application

Technical field:

The invention belongs to the field of bioengineering, and in particular relates to a macroporous cryogel medium and a preparation method and application thereof.

Background technique:

Advances in rapid bacterial detection strategies have enabled the development of bacterial detection in areas such as clinical diagnostics, food safety, and environmental monitoring in terms of speed, specificity, and sensitivity. Advanced technologies can even detect single cells in some cases, revolutionizing bacterial analysis methods. However, these methods still require small volumes of relatively clean samples to achieve sensitivity, which contradicts the characteristics of natural samples, which mostly contain a large amount of excess interfering substances. Based on the above discussion, when using these modern technologies to screen for pathogens in biological samples, separation and concentration of larger volume samples containing complex interfering matrices are essential steps.

Boronate affinity materials have attracted much attention in various research fields due to their remarkable characteristics in recognizing species containing cis-diols. Polysaccharide components composed of cis-diol groups, such as theophyllic acid, lipopolysaccharide and theophyllic acid, form the basis for the identification of bacteria using boronic acid affinity agents. In order to solve the problem of low affinity of single boronic acid ligands in biomacromolecule binding, some studies have used polymer chains or dendrimers as intermediate scaffolds to amplify the density of boronic acid ligands, thereby achieving synergistic enhancement of multiple boronic acid esters, which proves the high efficiency of boronic acid esters in bacterial binding. Among the reported performance enhancement strategies, boronic acid ligand integrated polymers have attracted great interest due to their unique design, innovative functional groups and the effect of reducing structural rigidity.

Composite cryogels have great potential in overcoming the hindrance of interfering matrices in bioseparation processes due to their excellent macroporosity. The interconnected channels and macropores of cryogels are formed during the polymerization of cryogels, with ice crystals acting as pore formers that form a specific structure after melting. This macroporous structure enables rapid mass transfer and high flow rates, allowing cells to pass through the monolith unimpeded even for complex biological samples without any complex pretreatment processes. However, due to the huge physical size of cryogels, bacterial cells cannot enter their internal polymer network. Therefore, it is necessary to immobilize the recognition ligands for bacteria binding on the macroporous exposed surface of cryogels to enhance the recognition effect.

Summary of the invention:

The technical problem to be solved by the present invention is that the physical size of the cryogel is huge and bacterial cells cannot enter its internal polymer network. Therefore, the recognition ligands for bacterial binding must be fixed on the macroporous exposed surface of the cryogel to enhance the recognition effect.

To solve the above problems, the present invention uses efficient click reaction to introduce high-performance boronic acid ligands into macroporous cryogels for bacterial binding. In order to further improve the binding ability of the composite cryogel, a hydrophilic, flexible and soft polymer supported by nanoparticles is added to increase the effective internal surface area of the cryogel and the density of fixed boric acid. The technical effect to be achieved by the present invention is to develop a new affinity medium using the characteristics of these polymer materials.

To achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing a macroporous cryogel medium, comprising the following steps:

(1) Preparation of bromine-functionalized silicon nanoparticles; bromine is modified on the surface of nanosilicon for subsequent introduction of polymers based on ATRP reaction, and there is no structural change on the surface of the nanoparticles.

(2) Preparation of isopropyl acrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles or isopropyl acrylamide-methacrylate glycidyl ester polymer functionalized silicon nanoparticles, soft flexible polymers appear on the surface of nanosilicon spheres, and brush-like substances cover the surface of hard silicon spheres; long-chain polymers on the surface of nanosilicon based on ATRP reaction can increase the modification density of subsequent groups.

(3) Preparation of azide-functionalized silicon nanoparticles; after the introduction of azide, the nanosilica gel can be fixed to the surface of the cryogel through a click reaction, and affinity groups can be introduced subsequently; the nanosilica gel still maintains the state of polymer covering the surface without obvious structural changes.

(4) Preparation of epoxidized cryogel columns; the cryogel preparation process introduces epoxy groups on the surface in one step for subsequent material modification. The cryogel column exhibits a coherent macroporous structure and a coherent surface.

(5) Preparation of alkyne-functionalized cryogel columns; the epoxy groups of the cryogels were modified into acetylene groups, and the azidized nanosilica gel was fixed by a click reaction; the cryogels still maintained a macroporous structure, and the pore continuity did not change significantly.

(6) Prepare a cryogel column modified with isopropylacrylamide-allyl glycidyl ether polymer functionalized silica nanoparticles or prepare a cryogel column modified with isopropylacrylamide-methacrylate glycidyl ether polymer functionalized silica nanoparticles; introduce brush-like polymer modified nanosilica gel on the surface of the cryogel, which can achieve high-density ligand modification on the surface of the cryogel, and can use nanosilica gel to increase the internal specific surface area. After modification, a large amount of nanosilica gel and polymer particles appear in the cryogel, the surface structure is rough, and there is still a macroporous coherent structure.

(7) Preparation of phenylboronic acid functionalized cryogel columns. The cryogel columns introduced affinity groups without significant structural changes and can be used for subsequent target identification.

Furthermore, the preparation method of bromine-functionalized silicon nanoparticles in step (1) is as follows: dispersing silicon nanoparticles in anhydrous ethanol, then adding 3-aminopropyltriethoxysilane and stirring; after the reaction is completed, the product is centrifuged, washed, and dried to obtain amino-functionalized silicon nanoparticles Si@NH ₂ ;

Si@ _NH2 was dispersed in tetrahydrofuran, triethylamine was added, the mixture was cooled in an ice-water bath, and 2-bromoisobutyryl bromide was added dropwise; the system was allowed to react after returning to room temperature; after the reaction, the product was centrifuged, ethanol, and dried to obtain bromine-functionalized silicon nanoparticles Si@Br.

Furthermore, the preparation method of isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles in step (2) is as follows: dissolve Si@Br, N-isopropylacrylamide, allyl glycidyl ether, _CuBr2 , and sodium ascorbate in a flask filled with deionized water; ultrasonicate, after the sample is thoroughly dispersed, blow nitrogen, then add tri[2-(dimethylamino)ethyl]amine to the mixture, continue blowing nitrogen, and react; after the reaction is completed, centrifuge, wash, and dry the product to obtain isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles Si@poly(NIPAm-co-AGE).

Preparation of isopropylacrylamide-glycidyl methacrylate polymer functionalized silicon nanoparticles: Si@Br, N-isopropylacrylamide (NIPAm), glycidyl methacrylate, CuBr ₂ , and sodium ascorbate are dissolved in a flask filled with deionized water; after the sample is thoroughly dispersed by ultrasound, nitrogen is blown, and then tri[2-(dimethylamino)ethyl]amine is added to the mixture, and nitrogen blowing is continued to react; after the reaction is completed, the product is centrifuged, washed, and dried to obtain isopropylacrylamide-glycidyl methacrylate polymer functionalized silicon nanoparticles Si@poly(NIPAm-co-GMA).

Furthermore, the preparation method of the azide-functionalized silicon nanoparticles in step (3) is as follows:

Si@poly(NIPAm-co-AGE) or Si@poly(NIPAm-co-GMA), sodium azide, ammonium chloride and N,N-dimethylformamide were mixed in a flask and sealed, and then ultrasonicated and stirred. After the reaction, the product was centrifuged, washed and dried to obtain azide-functionalized silicon nanoparticles Si@poly(NIPAm-co-AGE)@N ₃ or Si@poly(NIPAm-co-GMA)@N ₃ .

Furthermore, the preparation method of the epoxidized cryogel column in step (4) is as follows: 2-hydroxyethyl methacrylate (HEMA) and allyl glycidyl ether (AGE) are used as comonomers, HEMA:AGE is mixed, and then (HEMA+AGE):N,N'-methylenebisacrylamide (MBAm) is mixed, and the total comonomer concentration in the whole system is maintained at a wt/v ratio of 4% to 25%. By adjusting the ratio of different components, the structural strength, density of active groups and internal pore structure of the cryogel can be changed. If the component ratio is not optimized and screened, the cryogel is difficult to form a solid gel column with a stable structure. Then, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) are added to the system in an amount of 0.5-8% of the mass of the total monomers; HEMA, AGE and MBAm are dissolved in deionized water, the mixture is placed in an ice bath, nitrogen is blown, and TEMED and APS are respectively added to the mixture during the nitrogen blow; after being fully mixed, the mixture is transferred to a precooled glass tube and placed at -15°C for reaction; after the reaction is completed, the sample is thawed at room temperature, and the frozen gel is thoroughly washed with deionized water, and after drying, an epoxidized frozen gel column HA is obtained;

Or 2-hydroxyethyl methacrylate and glycidyl methacrylate are used as copolymer monomers, HEMA:GMA are mixed according to the molar ratio of 5.5:4.5, 6.5:3.5, 7.5:2.5, 8.5:1.5, 9.5:0.5, and then (HEMA+GMA):N,N'-methylenebisacrylamide (MBAm) are mixed according to the molar ratio of 15:1 and 6:1, and the total monomer concentration in the whole system is maintained at a wt/v ratio of 5-60%; then persulfate is added to the system HEMA, GMA and MBAm are dissolved in deionized water, the mixture is placed in an ice bath, nitrogen is blown, and TEMED and APS are respectively added to the mixture during nitrogen blowing; after being fully mixed, the mixture is transferred to a precooled glass tube and placed at -30°C for reaction; after the reaction is completed, the sample is thawed, and the cryogel is thoroughly washed with deionized water, and then dried to obtain the epoxidized cryogel column HA.

Furthermore, the preparation method of the alkynyl functionalized cryogel column in step (5) is as follows: HA is immersed in a carbonate buffer solution containing propargylamine and 2-aminoethanol and sealed; after the reaction is completed, the cryogel column is washed and then dried to obtain the alkynyl functionalized cryogel column HA-alkyne.

Furthermore, the preparation method of the cryogel column modified with isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles in step (6) is as follows: Si@poly(NIPAm-co-AGE)@N ₃ is dispersed in a methanol aqueous solution, and then a copper sulfate solution and sodium ascorbate are added; dried HA-alkyne is placed in a glass tube and circulated with the above Si@poly(NIPAm-co-AGE)@N ₃ solution in the glass tube; sodium ascorbate is added to the mixture; after the reaction is completed, HA-alkyne is washed and then dried in a vacuum to obtain a cryogel column modified with isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles HA-alkyne@Si@polymer@N _3. The alkyne group (alkyne) of HA-alkyne and the azide group (-N ₃ ) of Si@poly(NIPAm-co-AGE)@N ₃ are fixed together through a click reaction.

The preparation method of the cryogel column modified with isopropylacrylamide-glycidyl methacrylate polymer functionalized silicon nanoparticles is as follows: Si@poly(NIPAm-co-GMA)@N ₃ is dispersed in an acetonitrile aqueous solution, and then a cupric chloride solution and sodium ascorbate are added; dried HA-alkyne is placed in a glass tube, and then the dried HA-alkyne and the Si@poly(NIPAm-co-GMA)@N ₃ solution are circulated in the glass tube; after the reaction is completed, the HA-alkyne is washed and dried to obtain a cryogel column HA-alkyne@Si@polymer@N ₃ modified with isopropylacrylamide-glycidyl methacrylate polymer functionalized silicon nanoparticles.

Furthermore, the preparation method of the phenylboronic acid functionalized cryogel column in step (7) is as follows: immersing HA-alkyne@Si@polymer@N ₃ in a methanol aqueous solution containing 108 mg of 3-(prop-2-ynyloxycarbonylamino)-phenylboronic acid; after nitrogen blowing, adding copper sulfate and sodium ascorbate; washing the obtained product, and then drying it to obtain a phenylboronic acid functionalized cryogel column HA-alkyne@Si@polymer@pBA;

Alternatively, HA-alkyne@Si@polymer@N ₃ was immersed in an acetonitrile aqueous solution containing 3,5-difluoro-4-formylphenylboronic acid (DFFPBA); nitrogen was blown, and copper chloride and sodium ascorbate were added. The obtained product was washed and then dried to obtain a phenylboronic acid functionalized cryogel column HA-alkyne@Si@polymer@dBA.

A macroporous cryogel medium prepared by the above preparation method has modularization and click components. The method prepares cryogel and polymer functionalized nanoparticles respectively, and modifies the nanoparticles with azide groups to prepare modular components. Composite cryogel is prepared by an azidation reaction between azide groups and alkyne groups of cryogel, and affinity cryogel is prepared by a click reaction between alkyne groups of phenylboronic acid and residual azide groups of fixed nanosilica gel in the dark. The macroporous composite cryogel has the advantages of macroporous structure and high group density, and can effectively overcome the influence of complex matrices such as physical particles and proteins in samples, and realize efficient separation and capture of bacteria.

An application of the above macroporous cryogel medium in rapid bacterial detection.

The beneficial effects of the present invention are:

The composite cryogel material prepared by the present invention using a modular solution exhibits significant affinity for Staphylococcus aureus and Salmonella. In addition, the composite cryogel also has good matrix tolerance and can achieve direct adsorption and separation of bacteria in complex biological samples.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 shows the infrared spectra of silicon nanoparticles functionalized to different degrees. (a) Silicon nanoparticles; (b) Amino-functionalized silicon nanoparticles (Si@NH ₂ ); (c) Bromine-functionalized silicon nanoparticles (Si@Br); (d) Isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles (Si@poly(NIPAm-co-AGE)); (e) Azide-functionalized silicon nanoparticles (Si@poly(NIPAm-co-AGE)@N ₃ ).

Figure 2 shows the infrared spectra of cryogel columns functionalized to different degrees. (a) Epoxidized cryogel column (HA); (b) Alkyne-functionalized cryogel column (HA-alkyne); (c) Azide-functionalized cryogel column (HA@N ₃ ); (d) Monophenylboronic acid-functionalized cryogel column (HA-BA); (e) Isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticle-modified cryogel column (HA-alkyne@Si@polymer@N ₃ ); (f) Phenylboronic acid-functionalized cryogel column (HA-alkyne@Si@polymer@pBA).

FIG3 is an image of cryogel columns synthesized with different ratios of monomers in swollen (A) and dry (B) form.

Figure 4 shows the evaluation and optimization of the bacterial binding performance of the composite material phenylboronic acid functionalized cryogel column. (A) Binding effect of different cryogel composite materials on Staphylococcus aureus and Salmonella at pH 8.0. (B) Effect of pH and (C) temperature on the adsorption of bacteria by phenylboronic acid functionalized cryogel columns. Dilute with 0.01mmol/L PBS and adjust the initial _OD600 value of the bacterial suspension to 1.2. (D) Changes in DNA concentration in the binding system of Staphylococcus aureus and Salmonella. (E) Binding isotherms of phenylboronic acid functionalized cryogel columns with Staphylococcus aureus and Salmonella at pH8.0. (F) Effects of different elution media on the desorption of Staphylococcus aureus and Salmonella from phenylboronic acid functionalized cryogel columns: (a) 0.01-PBS (0.01 mol/L, pH 8.0), 37°C; (b) 0.2 mol/L fructose-PBS (0.01 mol/L, pH 9.0), 25°C; (c) 0.5 mol/L fructose-PBS (0.01 mol/L, pH 9.0), 25°C; (d) 0.5 mol/L fructose-PBS (0.01 mol/L, pH 9.0), 37°C. (G) Effects of coexisting cis-dihydroxy compounds on the ability of bacteria to bind to phenylboronic acid functionalized cryogel columns. (H) Recovery and reuse of phenylboronic acid functionalized cryogel columns.

Figure 5 shows SEM images of phenylboronic acid functionalized cryogel columns binding to Staphylococcus aureus (A) and Salmonella (B). The scale bar is 10 μm.

Figure 6 shows the separation of Salmonella and Staphylococcus aureus from spiked tap water, 40% milk (v/v) and sea cucumber hydrolysate using a phenylboronic acid functionalized cryogel column. Salmonella in tap water (A), 40% milk (v/v) (B) and sea cucumber hydrolysate (C); the content of Salmonella in tap water (D), 40% milk (v/v) (E) and sea cucumber hydrolysate (F) treated with a phenylboronic acid functionalized cryogel column; Staphylococcus aureus in tap water (G), 40% milk (v/v) (H) and sea cucumber hydrolysate (I); the content of Staphylococcus aureus in tap water (J), 40% milk (v/v) (K) and sea cucumber hydrolysate (L) treated with a phenylboronic acid functionalized cryogel column.

Detailed ways:

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

Large yellow croaker muscle stem cells were prepared in the laboratory using conventional methods. The muscle tissue of large yellow croaker fry was isolated and hydrolyzed with 0.2% collagenase I and 0.1% trypsin solution, respectively. The solution was filtered through 100 μm gauze and centrifuged. After being resuspended in complete culture medium, it was inoculated into a T25 bottle and cultured in a humid sterile environment at 28°C.

A method for preparing a fetal bovine serum substitute for culturing large yellow croaker muscle stem cells. The fetal bovine serum substitute comprises a substitute and an additive, wherein the substitute does not contain fetal bovine serum.

Example 1 A method for preparing a macroporous cryogel medium comprises the following steps:

(1) Preparation of bromine-functionalized silicon nanoparticles: 600 mg of silicon nanoparticles were dispersed in 98 mL of anhydrous ethanol, and then 2 mL of LAPTES (3-aminopropyltriethoxysilane) was added and stirred at 40°C for 3 h. After the reaction, the product was centrifuged, washed twice with ethanol, and dried in a vacuum environment at 60°C to obtain amino-functionalized silicon nanoparticles (Si@NH ₂ );

500 mg Si@NH ₂ was dispersed in 36 mL of tetrahydrofuran (THF), 1.2 mL of triethylamine was added, the mixture was cooled in an ice-water bath, and 0.91 mL of 2-bromoisobutyryl bromide (BIBB) was added dropwise. After the system returned to room temperature, the reaction was continued for 12 h. After the reaction was completed, the product was centrifuged, washed twice with anhydrous ethanol, and dried in a vacuum environment at 60 ° C to obtain bromine-functionalized silicon nanoparticles (Si@Br).

(2) Preparation of isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles: 180 mg Si@Br, 849 mg N-isopropylacrylamide (NIPAm), 890 μL allyl glycidyl ether (AGE), 6.6 mg CuBr ₂ , and 14.85 mg sodium ascorbate were dissolved in a 100 mL round-bottom flask filled with 55 mL deionized water. After ultrasonication for 15 min to completely disperse the sample, nitrogen was blown for 15 min, and then 139 μL tris[2-(dimethylamino)ethyl]amine was added to the mixture. After nitrogen was blown for another 15 min, the mixture was reacted for 24 h. After the reaction was completed, the product was centrifuged, washed with 5% disodium ethylenediaminetetraacetic acid (EDTA-Na ₂ ), deionized water, and anhydrous ethanol, respectively, and dried in a vacuum environment at 60°C to obtain isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles (Si@poly(NIPAm-co-AGE)).

(3) Preparation of azide-functionalized silicon nanoparticles: 120 mg Si@poly(NIPAm-co-AGE), 156 mg sodium azide, 129 mg ammonium chloride and 12 mL N,N-dimethylformamide (DMF) were mixed and sealed in a 25 mL round-bottom flask. After ultrasonication for 15 min, the sample was stirred at 45 °C for 24 h. After the reaction, the product was centrifuged, washed with deionized water and anhydrous ethanol, respectively, and dried in a vacuum environment at 60 °C to obtain azide-functionalized silicon nanoparticles (Si@poly(NIPAm-co-AGE)@N ₃ ).

(4) Preparation of epoxidized cryogel column: 2-Hydroxyethyl methacrylate (HEMA) and AGE were used as comonomers. HEMA:AGE was mixed at a molar ratio of 5:5, 6:4, 7:4, and 9:1. Then (HEMA+AGE):N,N'-methylenebisacrylamide (MBAm) was mixed at a molar ratio of 20:1 and 8:1. The total monomer concentration in the whole system was kept at 10% (wt/v). Then, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) were added to the system in an amount of 2% (w/w) of the total monomers. HEMA, AGE, and MBAm were dissolved in deionized water, and the mixture was placed in an ice bath and nitrogen purged for 15 minutes. During the nitrogen purging, TEMED and APS were added to the mixture respectively. After thorough mixing, 0.5 mL of the mixture was transferred to a precooled glass tube (inner diameter 7 mm) and placed at -15°C for reaction for 12 hours. After the reaction, the sample was thawed at room temperature, and the cryogel was thoroughly washed with deionized water and then dried in an oven at 60°C to obtain the epoxidized cryogel column (HA).

(5) Preparation of alkynyl functionalized cryogel column: 4 pieces of HA were immersed in carbonate buffer (8 mL, 0.1 mol/L, pH 11.0) containing 160 μL of propargylamine and 240 μL of 2-aminoethanol and sealed. After the reaction was completed, the cryogel column was thoroughly washed with deionized water and then dried in a vacuum to obtain an alkynyl functionalized cryogel column (HA-alkyne).

(6) Preparation of cryogel column modified with isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles: 150 mg Si@poly(NIPAm-co-AGE)@N ₃ was dispersed in 20 mL methanol: water (1:1, v/v) solution, and then 80 μL copper sulfate solution (0.2 mol/L) and 40 mg sodium ascorbate were added. The dried HA-alkyne was placed in a glass tube (inner diameter 7 mm), and then the prepared solution was circulated in the glass tube at a flow rate of 0.5 mL/min using a peristaltic pump for 24 h. Every 6 h, the flow direction was reversed to avoid clogging of the HA-alkyne, and 10 mg sodium ascorbate was added to the mixture to compensate for the loss caused by oxygen oxidation. After the reaction, the HA-alkyne was thoroughly washed with deionized water and then dried in a vacuum to obtain a cryogel column modified with isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles (HA-alkyne@Si@polymer@N ₃ ).

(7) Preparation of phenylboronic acid functionalized cryogel columns: Four pieces of HA-alkyne@Si@polymer@N ₃ were immersed in a methanol:water (12 mL, 1:1, v/v) solution containing 108 mg of 3-(prop-2-ynyloxycarbonylamino)-phenylboronic acid (PCAPBA). After nitrogen blowing for 20 min, 40 μL of copper sulfate (0.1 mol/mL) and 4 mg of sodium ascorbate were added. The obtained products were thoroughly washed with 50% ethanol aqueous solution, 5% EDTA-Na ₂ and deionized water, respectively, and then dried in vacuum to obtain phenylboronic acid functionalized cryogel columns (HA-alkyne@Si@polymer@pBA).

Example 2: A method for preparing a macroporous cryogel medium, comprising the following steps:

(1) Preparation of bromine-functionalized silicon nanoparticles: 2 g of silicon nanoparticles were dispersed in 200 mL of anhydrous ethanol, and then 5.5 mL of APTES (3-aminopropyltriethoxysilane) was added and stirred at 55°C for 2 h. After the reaction, the product was centrifuged, washed with ethanol and deionized water three times, and dried in a vacuum environment at 40°C to obtain amino-functionalized silicon nanoparticles (Si@NH ₂ );

2 mg Si@NH ₂ was dispersed in 150 mL of tetrahydrofuran (THF), 5 mL of ethylenediamine was added, the mixture was cooled in an ice-water bath, and 3.5 mL of 2-bromoisobutyryl bromide (BIBB) was added dropwise. After the system returned to 25 ° C, the reaction was continued for 18 h. After the reaction was completed, the product was centrifuged, washed with anhydrous ethanol and acetonitrile three times respectively, and dried in a vacuum environment at 45 ° C to obtain bromine-functionalized silicon nanoparticles (Si@Br).

(2) Preparation of isopropylacrylamide-glycidylmethacrylate polymer functionalized silicon nanoparticles: 300 mg Si@Br, 2.7 g N-isopropylacrylamide (NIPAm), 2.1 mL glycidylmethacrylate (GMA), 12.66 mg CuBr ₂ , and 30.5 mg sodium ascorbate were dissolved in a 200 mL round-bottom flask filled with 175 mL deionized water. After ultrasonication for 30 min to completely disperse the sample, nitrogen was blown for 20 min, and then 365 μL tris[2-(dimethylamino)ethyl]amine was added to the mixture. After nitrogen blowing for 30 min, the mixture was reacted for 36 h. After the reaction was completed, the product was centrifuged, washed with 5% ethylenediaminetetraacetic acid (EDTA), deionized water, and anhydrous ethanol, respectively, and dried in a vacuum environment at 50°C to obtain isopropylacrylamide-glycidylmethacrylate polymer functionalized silicon nanoparticles (Si@poly(NIPAm-co-GMA)).

(3) Preparation of azide-functionalized silicon nanoparticles: 300 mg Si@poly(NIPAm-co-GMA), 505 mg sodium azide, 356 mg ammonium chloride and 34 mL N,N-dimethylformamide (DMF) were mixed and sealed in a 100 mL round-bottom flask. After ultrasonication for 25 min, the sample was stirred at 40 °C for 30 h. After the reaction, the product was centrifuged, washed with deionized water and anhydrous ethanol, respectively, and dried in a vacuum environment at 55 °C to obtain azide-functionalized silicon nanoparticles (Si@poly(NIPAm-co-GMA)@N ₃ ).

(4) Preparation of epoxidized cryogel column: 2-Hydroxyethyl methacrylate (HEMA) and GMA were used as copolymer monomers. HEMA:GMA were mixed at a molar ratio of 5.5:4.5, 6.5:3.5, 7.5:2.5, 8.5:1.5, and 9.5:0.5. Then (HEMA+GMA):N,N'-methylenebisacrylamide (MBAm) were mixed at a molar ratio of 15:1 and 6:1. The total monomer concentration in the whole system was kept at 12% (wt/v). Then, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) were added to the system in an amount of 2.5% (w/w) of the total monomers. HEMA, GMA, and MBAm were dissolved in deionized water, and the mixture was placed in an ice bath and nitrogen purged for 30 minutes. During the nitrogen purging, TEMED and APS were added to the mixture respectively. After thorough mixing, 1.2 mL of the mixture was transferred into a precooled glass tube (inner diameter 7 mm) and placed at -30 ° C for reaction for 20 h. After the reaction, the sample was thawed at 25 ° C, and the cryogel was thoroughly washed with deionized water and then dried in a vacuum dryer at 40 ° C to obtain an epoxidized cryogel column (HA).

(5) Preparation of alkynyl functionalized cryogel column: 8 pieces of HA were immersed in carbonate buffer (19 mL, 0.1 mol/L, pH 11.0) containing 400 μL of propargylamine and 1 mL of 2-aminoethanol and sealed. After the reaction was completed, the cryogel column was thoroughly washed with deionized water and then dried in a vacuum to obtain an alkynyl functionalized cryogel column (HA-alkyne).

(6) Preparation of cryogel column modified with isopropylacrylamide-glycidylmethacrylate polymer functionalized silica nanoparticles: 200 mg Si@poly(NIPAm-co-GMA)@N ₃ was dispersed in 20 mL acetonitrile:water (1:1, v/v) solution, and then 150 μL copper chloride solution (0.2 mol/L) and 70 mg sodium ascorbate were added. The dried HA-alkyne was placed in a glass tube (inner diameter 7 mm) and then circulated in the glass tube with the above Si@poly(NIPAm-co-GMA)@N ₃ solution. Every 2 h, the flow direction was reversed to avoid clogging of HA-alkyne, and 6 mg sodium ascorbate was added to the mixture to compensate for the loss caused by oxygen oxidation. After the reaction, HA-alkyne was thoroughly washed with deionized water and then dried in vacuum to obtain cryogel column modified with isopropylacrylamide-glycidylmethacrylate polymer functionalized silica nanoparticles (HA-alkyne@Si@polymer@N ₃ ).

(7) Preparation of phenylboronic acid functionalized cryogel columns: 7 pieces of HA-alkyne@Si@polymer@N ₃ were immersed in an acetonitrile:water (12 mL, 1:1, v/v) solution containing 240 mg of 3,5-difluoro-4-formylphenylboronic acid (DFFPBA). After nitrogen blowing for 30 min, 55 μL of copper chloride (0.1 mol/mL) and 7 mg of sodium ascorbate were added. The obtained product was thoroughly washed with 75% aqueous ethanol, 7% EDTA and deionized water, respectively, and then dried in vacuum to obtain a phenylboronic acid functionalized cryogel column (HA-alkyne@Si@polymer@dBA).

Comparative Example 1 A method for preparing a monophenylboronic acid functionalized cryogel column comprises the following steps:

(1) Preparation of bromine-functionalized silicon nanoparticles: 600 mg of silicon nanoparticles were dispersed in 98 mL of anhydrous ethanol, and then 2 mL of LAPTES (3-aminopropyltriethoxysilane) was added and stirred at 40°C for 3 h. After the reaction, the product was centrifuged, washed twice with ethanol, and dried in a vacuum environment at 60°C to obtain amino-functionalized silicon nanoparticles (Si@NH ₂ );

500 mg Si@NH ₂ was dispersed in 36 mL of tetrahydrofuran (THF), 1.2 mL of triethylamine was added, the mixture was cooled in an ice-water bath, and 0.91 mL of 2-bromoisobutyryl bromide (BIBB) was added dropwise. After the system returned to room temperature, the reaction was continued for 12 h. After the reaction was completed, the product was centrifuged, washed twice with anhydrous ethanol, and dried in a vacuum environment at 60 ° C to obtain bromine-functionalized silicon nanoparticles (Si@Br).

(2) Preparation of isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles: 180 mg Si@Br, 849 mg N-isopropylacrylamide (NIPAm), 890 μL allyl glycidyl ether (AGE), 6.6 mg CuBr ₂ , and 14.85 mg sodium ascorbate were dissolved in a 100 mL round-bottom flask filled with 55 mL deionized water. After ultrasonication for 15 min to completely disperse the sample, nitrogen was blown for 15 min, and then 139 μL tris[2-(dimethylamino)ethyl]amine was added to the mixture. After nitrogen was blown for another 15 min, the mixture was reacted for 24 h. After the reaction was completed, the product was centrifuged, washed with 5% disodium ethylenediaminetetraacetic acid (EDTA-Na ₂ ), deionized water, and anhydrous ethanol, respectively, and dried in a vacuum environment at 60°C to obtain isopropylacrylamide-allyl glycidyl ether polymer functionalized silicon nanoparticles (Si@poly(NIPAm-co-AGE)).

(3) Preparation of azide-functionalized silicon nanoparticles: 120 mg Si@poly(NIPAm-co-AGE), 156 mg sodium azide, 129 mg ammonium chloride and 12 mL N,N-dimethylformamide (DMF) were mixed and sealed in a 25 mL round-bottom flask. After ultrasonication for 15 min, the sample was stirred at 45 °C for 24 h. After the reaction, the product was centrifuged, washed with deionized water and anhydrous ethanol, respectively, and dried in a vacuum environment at 60 °C to obtain azide-functionalized silicon nanoparticles (Si@poly(NIPAm-co-AGE)@N ₃ ).

(4) Preparation of epoxidized cryogel column: 2-Hydroxyethyl methacrylate (HEMA) and AGE were used as comonomers. HEMA:AGE was mixed at a molar ratio of 5:5, 6:4, 7:4, and 9:1. Then (HEMA+AGE):N,N'-methylenebisacrylamide (MBAm) was mixed at a molar ratio of 20:1 and 8:1. The total monomer concentration in the whole system was kept at 10% (wt/v). Then, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) were added to the system in an amount of 2% (w/w) of the total monomers. HEMA, AGE, and MBAm were dissolved in deionized water, and the mixture was placed in an ice bath and nitrogen purged for 15 minutes. During the nitrogen purging, TEMED and APS were added to the mixture respectively. After thorough mixing, 0.5 mL of the mixture was transferred to a precooled glass tube (inner diameter 7 mm) and placed at -15°C for reaction for 12 hours. After the reaction, the sample was thawed at room temperature, and the cryogel was thoroughly washed with deionized water and then dried in an oven at 60°C to obtain the epoxidized cryogel column (HA).

(5) Preparation of an azide-functionalized cryogel column: 4 pieces of HA, 156 mg of sodium azide, 129 mg of ammonium chloride and 18 mL of DMF were mixed, ultrasonicated for 15 min, and stirred at 45° C. for 24 h. After the reaction, the cryogel column was thoroughly washed with deionized water and then dried in a vacuum to obtain an azide-functionalized cryogel column (HA@N ₃ ).

(6) Preparation of phenylboronic acid functionalized cryogel column: Four pieces of HA@N ₃ were immersed in a methanol:water (12 mL, 1:1, v/v) solution containing 108 mg of 3-(prop-2-ynyloxycarbonylamino)-phenylboronic acid (PCAPBA). After nitrogen blowing for 20 min, 40 μL of copper sulfate (0.1 mol/mL) and 4 mg of sodium ascorbate were added. The obtained product was thoroughly washed with 50% ethanol aqueous solution, 5% EDTA-Na ₂ and deionized water, respectively, and then dried in vacuum to obtain a single phenylboronic acid functionalized cryogel column (HA-BA).

Comparative Example 2: A method for preparing a cryogel column functionalized with monophenylboronic acid, comprising the following steps:

(1) Preparation of bromine-functionalized silicon nanoparticles: 2 g of silicon nanoparticles were dispersed in 200 mL of anhydrous ethanol, and then 5.5 mL of APTES (3-aminopropyltriethoxysilane) was added and stirred at 55°C for 2 h. After the reaction, the product was centrifuged, washed with ethanol and deionized water three times, and dried in a vacuum environment at 40°C to obtain amino-functionalized silicon nanoparticles (Si@NH ₂ );

2 mg Si@NH ₂ was dispersed in 150 mL of tetrahydrofuran (THF), 5 mL of ethylenediamine was added, the mixture was cooled in an ice-water bath, and 3.5 mL of 2-bromoisobutyryl bromide (BIBB) was added dropwise. The system was allowed to react for 18 h after returning to 25 °C. After the reaction, the product was centrifuged, washed with anhydrous ethanol and acetonitrile three times respectively, and dried in a vacuum environment at 45 °C to obtain bromine-functionalized silicon nanoparticles (Si@Br).

(2) Preparation of isopropylacrylamide-glycidylmethacrylate polymer functionalized silicon nanoparticles: 300 mg Si@Br, 2.7 g N-isopropylacrylamide (NIPAm), 2.1 mL glycidylmethacrylate (GMA), 12.66 mg CuBr ₂ , and 30.5 mg sodium ascorbate were dissolved in a 200 mL round-bottom flask filled with 175 mL deionized water. After ultrasonication for 30 min to completely disperse the sample, nitrogen was blown for 20 min, and then 365 μL tris[2-(dimethylamino)ethyl]amine was added to the mixture. After nitrogen blowing for 30 min, the mixture was reacted for 36 h. After the reaction was completed, the product was centrifuged, washed with 5% ethylenediaminetetraacetic acid (EDTA), deionized water, and anhydrous ethanol, respectively, and dried in a vacuum environment at 50°C to obtain isopropylacrylamide-glycidylmethacrylate polymer functionalized silicon nanoparticles (Si@poly(NIPAm-co-GMA)).

(3) Preparation of azide-functionalized silicon nanoparticles: 300 mg Si@poly(NIPAm-co-GMA), 505 mg sodium azide, 356 mg ammonium chloride and 34 mL N,N-dimethylformamide (DMF) were mixed and sealed in a 100 mL round-bottom flask. After ultrasonication for 25 min, the sample was stirred at 40 °C for 30 h. After the reaction, the product was centrifuged, washed with deionized water and anhydrous ethanol, respectively, and dried in a vacuum environment at 55 °C to obtain azide-functionalized silicon nanoparticles (Si@poly(NIPAm-co-GMA)@N ₃ ).

(4) Preparation of epoxidized cryogel column: 2-Hydroxyethyl methacrylate (HEMA) and GMA were used as copolymer monomers. HEMA:GMA were mixed at a molar ratio of 5.5:4.5, 6.5:3.5, 7.5:2.5, 8.5:1.5, and 9.5:0.5. Then (HEMA+GMA):N,N'-methylenebisacrylamide (MBAm) were mixed at a molar ratio of 15:1 and 6:1. The total monomer concentration in the whole system was kept at 12% (wt/v). Then, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) were added to the system in an amount of 2.5% (w/w) of the total monomers. HEMA, GMA, and MBAm were dissolved in deionized water, and the mixture was placed in an ice bath and nitrogen purged for 30 minutes. During the nitrogen purging, TEMED and APS were added to the mixture respectively. After thorough mixing, 1.2 mL of the mixture was transferred into a precooled glass tube (inner diameter 7 mm) and placed at -30 ° C for reaction for 20 h. After the reaction, the sample was thawed at 25 ° C, and the cryogel was thoroughly washed with deionized water and then dried in a vacuum dryer at 40 ° C to obtain an epoxidized cryogel column (HA).

(5) Preparation of an azide-functionalized cryogel column: 7 pieces of HA, 330 mg of sodium azide, 285 mg of ammonium chloride and 45 mL of DMF were mixed, ultrasonicated for 45 min, and stirred at 55° C. for 30 h. After the reaction, the cryogel column was thoroughly washed with deionized water and then dried in a vacuum to obtain an azide-functionalized cryogel column (HA@N ₃ ).

(6) Preparation of phenylboronic acid functionalized cryogel column: 7 pieces of HA@N ₃ were immersed in acetonitrile: water (12 mL, 1:1, v/v) solution containing 240 mg 3,5-difluoro-4-formylphenylboronic acid (DFFPBA). After nitrogen blowing for 30 min, 55 μL copper chloride (0.1 mol/mL) and 7 mg sodium ascorbate were added. The obtained product was thoroughly washed with 75% ethanol aqueous solution, 7% EDTA and deionized water, respectively, and then dried in vacuum to obtain a monophenylboronic acid functionalized cryogel column (HA-dBA).

Verification results:

1. The prepared boron affinity materials were characterized by FT-IR.

As shown in Figure 1, the prepared composite nano-silica material was characterized by FT-IR. Obvious adsorption bands were observed near the Si-O-Si asymmetric stretching vibration at 1057 cm ^-1 , the Si-OH asymmetric vibration at 945 cm ^- 1, and the Si-O symmetric vibration at 794 cm ^-1 . After the introduction of APTES, no obvious changes in the infrared spectrum were observed. After the acylation reaction with BIBB, two adsorption bands related to amide I and amide II were observed at 1641 cm ^-1 and 1531 cm ^-1 (Figure 1c). After grafting the polymer, two new bands appeared at 1390 cm ^-1 and 1471 cm ^-1 for Si@poly(NIPAm-co-AGE), which were the stretching vibration bands of the ester carbonyl group and the shear bands of the methylene group, respectively (Figure 1d). All these bands indicate that the polymer was successfully grafted to the particle surface. After reacting Si@poly(NIPAm-co-AGE) particles with NaN ₃ , a new adsorption band of Si@poly(NIPAm-co-AGE)@N ₃ particles was observed at 2059 cm ⁻¹ (Figure 1e).

No obvious adsorption band of AGE segment epoxy groups was observed on HA cryogel (Figure 3a). After reaction with sodium azide and propargylamine, new infrared spectral bands appeared at 2100 cm ^-1 and 2140 cm ^-1 , which can be attributed to the stretching vibration of the azide group and the alkyne group, respectively (Figure 2b, c). After the introduction of alkyne-labeled boronic acid on HA- _N3 , the strong azide signal disappeared completely (Figure 2d). After conjugation of Si@poly(NIPAm-co-AGE)@ _N3 with HA-alkyne, a new adsorption band appeared at 1386 cm ^-1 , which was caused by the asymmetric stretching of the carbonyl group (Figure 2e). The characteristic azide band appearing at 2100 cm ^-1 is generated by the remaining azide group after click conjugation of the nanoparticle-supported polymer with the cryogel. All these adsorption bands indicate the successful incorporation of silica chimeric polymers on the cryogel surface. After the final introduction of alkynyl-labeled boronic acid on HA-alkyne@Si@polymer-N ₃ cryogel by click-reaction, the azide band completely disappeared, and characteristic bands corresponding to phenyl hydrogen on boronic acid were observed at 785 cm ^-1 and 1065 cm ^-1 (Figure 2f), indicating the successful introduction of boronic acid ligands. Elemental analysis showed that the boron contents in HA-BA and HA-alkyne@Si@polymer-pBA were 0.029 and 0.834 mg/g, respectively. Based on the boron content, the density of PCAPBA ligands immobilized on HA-BA and HA-alkyne@Si@polymer-pBA was calculated to be 0.0027 mmol/g and 0.077 mmol/g, respectively.

Cryogels have large and interconnected channels and have great potential for processing particle-containing fluids such as complex biological samples at low pressure without clogging problems. The water-soluble monomers are selected based on their physicochemical properties, molecular interactions with recognition ligands, and biocompatibility requirements. The cryogel matrix HA used in this study was synthesized by copolymerization of HEMA, AGE, and MBA in water at -15°C. In order to investigate the effect of the monomer ratio of the copolymer on the structure and stability of the prepared cryogels, a series of composite cryogels were synthesized. Table 1, Table 2, and Figure 3 list the cryogels polymerization and cryogels yields using different monomer compositions. When the molar ratio of HEMA-AGE was greater than 6:4, the polymerization yield was higher.

Table 1 Synthesis ratio and characterization of physical properties of phenylboronic acid functionalized cryogel columns

Table 2 Amounts of reagents required for the synthesis of different phenylboronic acid functionalized cryogel columns

2. Evaluation of HA-alkyne@Si@polymer@N ₃ ’s binding ability to bacteria

The HA-alkyne@Si@polymer@N ₃ composite cryogel was inserted into a glass tube (inner diameter 7 mm) and 15 mL of PBS (pH 6.5-8.0, 0.01 mol/L) was added for activation. Subsequently, a peristaltic pump was used to circulate 4 mL of S. aureus and Salmonella spp. suspension (OD ₆₀₀ = 1.2) in the cryogel at a flow rate of 0.5 mL/min for 40 min. Every 10 min, the peristaltic pump was adjusted to reverse the flow to avoid clogging. After completion, the gel column was washed with 10 mL of PBS to remove nonspecifically bound bacteria. The number of bacteria bound to the cold gel was calculated by measuring the bacterial content in the effluent and washing solution at OD ₆₀₀ .

Take the frozen gel column that has completed bacterial adsorption, wash off the non-specifically bound bacteria, and then elute the bacteria adsorbed on the composite material under the following different conditions: 3mL 0.2mol/L acetate buffer (pH 4.0, containing 0.3mol/LNaCl, 25℃), 3mL 0.2mol/L acetate buffer (pH 4.0, containing 0.3mol/LNaCl, 40℃), 3mL 0.1mol/L fructose-PBS solution (0.01mol/L, pH 9.0, containing 0.3mol/LNaCl), 3mL 0.2mol/L fructose-PBS solution (0.01mol/L, pH 9.0, containing 0.3mol/LNaCl). During the elution process, close the outlet of the column, let the bacterial cells desorb for 15min, and then squeeze the frozen gel to collect the eluate. The bacterial concentration was determined by spectrophotometry.

result:

To demonstrate the important role of immobilized boronic acid and the effect of anchored silica integrated polymers in enhancing bacterial binding, we used composite cryogels prepared at different modification stages to study their ability to bind S. aureus and Salmonella spp. As shown in Figure 4A, the composites HA-BA and HA-alkyne@Si@polymer-pBA functionalized with boronic acid ligands showed significantly higher specific binding to model bacteria than the composite cryogels without boronic acid ligands, indicating that the specific recognition interaction between boronic acid ligands and cis-diols present on the bacterial cell wall is dominant. Compared with HA-BA with boronic acid directly immobilized on the surface, HA-alkyne@Si@polymer-pBA showed much higher bacterial binding ability.

pH-mediated recognition is one of the notable features of boronate affinity. Generally speaking, boronate affinity recognition needs to be performed at a higher pH (>8.0) to ensure the efficiency of covalent binding. Strong alkaline conditions are not suitable for pH-labeled biomacromolecules. Since alkyne-labeled boronic acid ligand chimeric polymers have been shown to have biorecognition potential under lower pH conditions (pH < 7.0), we expect that bacteria can effectively bind to HA-alkyne@Si@polymer-pBA under near-neutral pH conditions. As shown in Figure 4B, the binding affinity of S. aureus was insensitive to changes in pH in the range of pH = 6.5-8.5, while the binding affinity of Salmonella spp. was highest at pH 8.0.

The addition of pNIPAm fragments not only improves the hydrophilicity of the final composite, but also provides thermal responsiveness. As shown in Figure 4C, when the temperature increases to the lower critical solution temperature (LCST) of pNIPAm, the binding ability of HA-alkyne@Si@polymer-pBA to S. aureus and Salmonella spp. decreases. However, compared with the protein binding using pNIPAm integrated polymer, the composite cryogel did not show obvious temperature responsiveness.

SEM images showed that no obvious destruction of bacterial cells was observed during the binding process (Figure 5). In addition, to further demonstrate the effect of HA-alkyne@Si@polymer-pBA on bacterial viability, the concentration of DNA at different binding times was also measured. As shown in Figure 4D, during the binding of composite cryogels to S. aureus and Salmonella spp., the concentration of DNA remained stable (P>0.05), respectively, indicating that the residual chemical groups (including boronic acid groups, azide groups, benzene ring groups, and amino groups) did not cause bacterial apoptosis.

Figure 4E shows the equilibrium binding isotherms of HA-alkyne@Si@polymer-pBA with two model bacteria. The results show that the binding capacity of S. aureus and Salmonella spp. to the composite cryogel increases nonlinearly with the increase of equilibrium concentration. The final binding capacity of cryogel to HA-alkyne@Si@polymer-pBA is 91.6×10 ⁷ CFU/g and 241.3×10 ⁷ CFU/g, respectively.

Given the pH-directed reversible binding and release properties of boronate affinity materials when capturing cis-diol-containing species, acidic solutions, especially acetic acid solutions, are a general strategy for desorption of targets, however, lower pH is always unfavorable for sensitive biological species. To avoid bacterial decomposition in acidic media, a competitive elution strategy using D-fructose was tested. In addition, the temperature responsiveness of the pNIPAm fragment as a bacterial desorption method was also investigated. As shown in Figure 4F, when the system temperature was increased to 37°C, only about 30% of the bound Salmonella spp. and about 23% of the bound S. aureus could be released. 0.2 mol/L and 0.5 mol/L fructose-PBS (0.01 mol/L, pH 9.0) solutions could elute about 70% of S. aureus and Salmonella spp., respectively, which is consistent with the diversity of HA-alkyne@Si@polymer-pBA's affinity for S. aureus and Salmonella spp. Combining the established fructose-PBS elution with a temperature-based release method resulted in approximately 80% loading of S. aureus and Salmonella spp., respectively.

Several competing compounds were tested, including 3.5% D-fructose, 3.5% glucose, 3.5% lactose, 3.5% sucrose, 3.5% soluble starch, and 5% ovalbumin, to measure the competitive effect of HA-alkyne@Si@polymer-pBA binding to model bacteria. As shown in Figure 4G, D-fructose had the strongest competitive effect on the binding of model bacteria. In this study, glucose and lactose had weaker competitive effects on model bacteria, indicating that the structure of small molecule compounds plays a key role in the affinity of boronate esters. At the same time, Gram-positive bacteria (S. aureus) and Gram-negative bacteria (Salmonella spp.) showed obvious differences in D-fructose and glucose.

In this study, after eluting the model bacteria bound to the composite cryogels with an optimized elution buffer, the composite cryogels were rinsed with 100 mmol/L acetic acid solution at room temperature to remove fructose, and then the composite cryogels were rinsed with PBS binding buffer to reactivate them. The recyclability of the cryogels was measured by normalizing the bacterial binding capacity with the binding capacity measured in the first run. As shown in Figure 4H, the binding capacity of the composite cryogels remained essentially unchanged during the first five bacterial separations. After five separations, the binding capacity slowly decreased to nearly 90%.

3. Verification of the adsorption effect of cryogel medium on bacteria using composite samples

Sea cucumber (Stichopus japonicus) was mixed with dispase (0.3% by weight of the raw material), and enzymolysis was performed at 55°C for 6 hours. The enzymolysis solution was centrifuged at room temperature for 45 minutes, and the supernatant was collected as a sample.

10 mL of aliquots of tap water, 40% milk (v/v), and sea cucumber hydrolysate were adjusted to pH 8.0 and then spiked with 10 ³ CFU/mL S. aureus and Salmonella spp., respectively. Unbound bacteria were collected and quantified. A spiked milk sample without cryogel treatment was used as a control.

result:

To further demonstrate the feasibility and practicality of HA-alkyne@Si@polymer-pBA in isolating bacteria from complex biological samples, S. aureus and Salmonella spp. were added to tap water, 40% milk (v/v), and sea cucumber enzymatic hydrolysate at a concentration of approximately 10 ³ CFU/mL, and the pH was adjusted to 8.0. The samples were circulated in the cryogel column at a flow rate of 0.5 mL/min for 40 min. The number of bacteria in the crude sample and the combined solution was quantified using the traditional viable count method. As shown in Figure 6, most of the bacteria in the spiked samples were separated by cryogel. In view of the presence of a large number of interfering matrices in the test samples, such as ions, carbohydrates, peptides, proteins, lipids, etc., cryogel showed an ideal effect in removing bacteria.

The same experiment was performed on a cryogel column modified with isopropylacrylamide-glycidyl methacrylate polymer functionalized silica nanoparticles. The experimental results showed that its performance was similar to that of the cryogel column modified with isopropylacrylamide-allyl glycidyl ether polymer functionalized silica nanoparticles, with no significant difference.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (10)

1. The preparation method of the macroporous frozen gel medium is characterized by comprising the following steps:

(1) Preparing bromine functionalized silicon nano-particles;

(2) Preparing isopropyl acrylamide-allyl glycidyl ether polymer functionalized silicon nano-particles or preparing isopropyl acrylamide-glycidyl methacrylate polymer functionalized silicon nano-particles;

(3) Preparing azide functionalized silicon nano-particles;

(4) Preparing an epoxidation frozen gel column;

(5) Preparing alkynyl functional frozen gel columns;

(6) Preparing a frozen gel column modified by isopropyl acrylamide-allyl glycidyl ether polymer functionalized silicon nano-particles or preparing a frozen gel column modified by isopropyl acrylamide-glycidyl methacrylate polymer functionalized silicon nano-particles;

(7) Preparing a phenylboronic acid functionalized frozen gel column.

2. The method of claim 1, wherein: the preparation method of the bromine functionalized silicon nano-particles in the step (1) comprises the following steps: dispersing silicon nano particles in absolute ethyl alcohol, then adding 3-aminopropyl triethoxysilane, and stirring; after the reaction is finished, the product is centrifuged and cleaned,after drying, obtaining amino functionalized silicon nano particles Si@NH ₂ ；

Si@NH ₂ Dispersing in tetrahydrofuran, adding triethylamine, cooling the mixture in ice water bath, and dropwise adding 2-bromoisobutyryl bromide; after the system is restored to room temperature, reacting; after the reaction is finished, the product is centrifuged, ethanol and dried to obtain the bromine functionalized silicon nano particles Si@Br.

3. The method of claim 1, wherein: the preparation method of the isopropyl acrylamide-allyl glycidyl ether polymer functionalized silicon nano-particles in the step (2) comprises the following steps: si@Br, N-isopropylacrylamide, allyl glycidyl ether and CuBr ₂ Dissolving sodium ascorbate in a flask filled with deionized water; ultrasonic treatment, nitrogen blowing after thoroughly dispersing the sample, followed by addition of tris [2- (dimethylamino) ethyl group to the mixture]Amine, continuing nitrogen blowing and reacting; after the reaction is finished, centrifuging, cleaning and drying a product to obtain isopropyl acrylamide-allyl glycidyl ether polymer functionalized silicon nano particles Si@poly (NIPAm-co-AGE);

preparation of isopropyl acrylamide-glycidyl methacrylate polymer functionalized silicon nanoparticles: si@Br, N-isopropylacrylamide (NIPAm), glycidyl methacrylate, cuBr ₂ Dissolving sodium ascorbate in a flask filled with deionized water; after the sample is thoroughly dispersed by sonication, nitrogen is blown, followed by addition of tris [2- (dimethylamino) ethyl group to the mixture]Amine, continuing nitrogen blowing and reacting; after the reaction is finished, centrifuging, cleaning and drying the product to obtain the isopropyl acrylamide-glycidyl methacrylate polymer functionalized silicon nanoparticle Si@poly (NIPAm-co-GMA).

4. The method of claim 1, wherein: the preparation method of the azido functionalized silicon nano-particles in the step (3) comprises the following steps: mixing Si@poly (NIPAm-co-AGE) or Si@poly (NIPAm-co-GMA), sodium azide, ammonium chloride and N, N-dimethylformamide in a flask, sealing, and stirring after ultrasonic treatment; after the reaction is finished, the product is centrifuged Cleaning and drying to obtain azido functionalized silicon nano particles Si@poly (NIPAm-co-AGE) @N ₃ Or Si@poly (NIPAm-co-GMA) @ N ₃ 。

5. The method of claim 1, wherein: the preparation method of the epoxidation frozen gel column in the step (4) comprises the following steps: HEMA was prepared using 2-hydroxyethyl methacrylate, allyl glycidyl ether as comonomer: AGE mix, followed by (hema+age): mixing N, N ' -methylene bisacrylamide, keeping the total monomer concentration in the whole system at a weight/v ratio of 4-25%, and then adding ammonium persulfate and N, N, N ', N ' -tetramethyl ethylenediamine into the system, wherein the addition amount is 0.5-8% of the total monomer mass; dissolving HEMA, AGE and MBAm in deionized water, placing the mixture in an ice bath, nitrogen blowing, and adding TEMED and APS into the mixture respectively during nitrogen blowing; after fully mixing, transferring the mixture into a precooled glass tube, and placing the mixture into a-15 ℃ for reaction; after the reaction is finished, thawing the sample, thoroughly cleaning the frozen gel with deionized water, and drying to obtain an epoxidized frozen gel column HA;

or 2-hydroxyethyl methacrylate and glycidyl methacrylate are used as comonomers, HEMA is prepared by the following steps: GMA mixing, followed by (hema+gma): mixing N, N' -methylene bisacrylamide, wherein the total monomer concentration in the whole system is kept at a weight/v ratio of 5-60%; then ammonium persulfate and N, N, N ', N' -tetramethyl ethylenediamine are added into the system, wherein the addition amount is 0.5-8% of the mass of the total monomer; HEMA, GMA and MBAm are dissolved in deionized water, the mixture is placed in an ice bath, nitrogen is blown, and TEMED and APS are respectively added into the mixture during nitrogen blowing; after fully mixing, transferring the mixture into a precooled glass tube, and placing the mixture into a-30 ℃ for reaction; after the reaction is finished, thawing the sample, thoroughly cleaning the frozen gel with deionized water, and drying to obtain the epoxidized frozen gel column HA.

6. The method of claim 1, wherein: the preparation method of the alkynyl functional frozen gel column in the step (5) comprises the following steps: immersing HA in carbonate buffer solution containing propynylamine and 2-amino ethanol and sealing; after the reaction is finished, the frozen gel column is washed and then dried, and the alkynyl functional frozen gel column HA-alkyne is obtained.

7. The method of claim 1, wherein: the preparation method of the frozen gel column modified by the isopropyl acrylamide-allyl glycidyl ether polymer functionalized silicon nano-particles in the step (6) comprises the following steps: si@poly (NIPAm-co-AGE) @ N ₃ Dispersing in methanol water solution, and then adding copper sulfate solution and sodium ascorbate; the dried HA-alkyne was placed in a glass tube and was then heated with the above Si@poly (NIPAm-co-AGE) @ N ₃ The solution circulates in the glass tube; adding sodium ascorbate to the mixture; after the reaction is finished, cleaning HA-alkyne, and then drying in vacuum to obtain a frozen gel column HA-alkyne@Si@polymer@N modified by isopropyl acrylamide-allyl glycidyl ether polymer functionalized silicon nano particles ₃ ；

The preparation method of the isopropyl acrylamide-glycidyl methacrylate polymer functionalized silicon nanoparticle modified cryogel column comprises the following steps: si@poly (NIPAm-co-GMA) @ N ₃ Dispersing in acetonitrile water solution, and then adding copper chloride solution and sodium ascorbate; the dried HA-alkyne was placed in a glass tube and then reacted with Si@poly (NIPAm-co-GMA) @ N as described above ₃ The solution circulates in the glass tube; after the reaction is finished, cleaning HA-alkyne, drying to obtain a frozen gel column HA-alkyne@Si@polymer@N modified by isopropyl acrylamide-glycidyl methacrylate polymer functionalized silicon nano particles ₃ 。

8. The method of claim 1, wherein: the preparation method of the phenylboronic acid functionalized cryogel column in the step (7) comprises the following steps: HA-alkyne@Si@Polymer@N ₃ Immersing in an aqueous methanol solution containing 108mg of 3- (prop-2-ynyloxycarbonylamino) -phenylboronic acid; after nitrogen blowing, adding copper sulfate and sodium ascorbate; cleaning the obtained product, and then drying to obtain a phenylboronic acid functionalized frozen gel column HA-alkyne@Si@polymer@pBA;

or HA-alkyne@Si@Polymer@N ₃ Immersing in a solution containing 3, 5-diAqueous acetonitrile of fluoro-4-formylphenylboronic acid (DFFPBA); nitrogen blowing, adding copper chloride and sodium ascorbate. And cleaning and drying the obtained product to obtain the phenylboronic acid functionalized frozen gel column HA-alkyne@Si@polymer@dBA.

9. A macroporous cryogel medium prepared by the method of any one of claims 1-9.

10. Use of the macroporous cryogel medium of claim 9 in the rapid detection of bacteria.