Magnetic epitope imprinted gel polymer capable of specifically adsorbing beta-lactamase SHV family as well as preparation method and application of magnetic epitope imprinted gel polymer
By synthesizing SHV family-specific magnetic epitope blot gel polymers on the surface of Fe3O4 particles, the problem of difficult to quickly isolate β-lactamase SHV family members in the prior art is solved, and efficient selective separation from complex matrix is achieved The effect of this method provides a new strategy for bacterial drug resistance detection.
Patent Information
- Application Number
- CN202411509698.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art is difficult to quickly and efficiently identify and isolate the β-lactamase SHV family and its members, especially in complex bacterial lysates.
Through surface epitope blotting technology, a SHV family-specific magnetic epitope blotting gel polymer was prepared, and submicron Fe3O4 particles were used as solid phase carrier, combined with the SHV family-specific epitope VDAGDEQLER to achieve selective separation of SHV family members. .
This method can efficiently and selectively isolate SHV family members from bacterial lysate, significantly improving the efficiency and accuracy of drug resistance detection, and providing a solid theoretical and practical basis for new bacterial drug resistance detection strategies.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of nanocomposite materials, and specifically relates to a magnetic epitope imprinting gel polymer capable of selectively separating beta-lactamase SHV family from complex matrices such as bacterial lysate, and a preparation method and application thereof. Background Art
[0002] β-lactam antibiotics bind to bacterial penicillin-binding proteins to form stable acyl-enzyme intermediates, blocking the cross-linking of bacterial cell wall peptidoglycan, thereby producing an antibacterial effect. With the widespread use of this class of antibiotics in clinical practice, bacterial resistance to them is becoming more and more common: among them, the conformational change or up-regulated expression of penicillin-binding proteins is the most important resistance mechanism for Gram-positive bacteria; although the loss of outer membrane porins and the expression of efflux pumps are unique resistance mechanisms for Gram-negative bacteria, the production of β-lactamase (hydrolyzing antibiotics) is the most popular resistance mechanism for this class of bacteria and has significant clinical significance.
[0003] To date, more than 4,900 β-lactamases have been discovered. Based on the structural homology of the enzymes, they are divided into four major categories (A, B, C, and D), which include hundreds of different families. These enzymes have different hydrolysis characteristics (i.e., hydrolysis spectrum and anti-enzyme inhibitor activity) against β-lactam antibiotics. In any particular enzyme family, all members of the family have similar amino acid sequences: (i) The first reported enzyme is named "-1", such as SHV-1, TEM-1, and CTX-M-1. Other members are derived from enzyme-1 after several amino acid substitutions. They are named "-2", "-3", "-4", etc. according to the order of discovery, and so on; (ii) Amino acid substitutions usually occur in certain specific regions of the protein sequence. These mutations give the family members new hydrolysis characteristics; (iii) Certain conserved amino acid sequences present in family members can serve as an important basis for the identification of the family. Therefore, it is of great practical significance to develop a novel drug resistance detection strategy that complements the existing genetic detection technology and realizes the rapid identification of a specific enzyme family and all its members based on the recognition and capture of specific peptides.
[0004] Epitope imprinting technology is a material preparation process developed on the basis of traditional molecular imprinting. This technology uses the specific polypeptide (i.e., epitope) of the target protein as a template. Under the action of a cross-linking agent, the non-covalent effect of the functional monomer on the template molecule is memorized in the polymer. After the template is eluted, an imprinted hole that completely matches the epitope polypeptide appears in the polymer, and the functional monomer's action site is exposed in the hole. The imprinted polymer prepared in this way can specifically adsorb the target protein through epitope recognition. Compared with protein imprinting, epitope imprinting has many advantages: the template molecule can be obtained through artificial synthesis; small molecule polypeptides can tolerate organic solvents and are stable during the imprinting process; the template is easy to elute and the imprinted holes are uniform. In recent years, epitope imprinting technology has been widely used in the field of protein separation and purification.
[0005] In epitope imprinting, the length of the template polypeptide is a key factor affecting the polymer imprinting effect and recognition characteristics. Generally speaking, the ideal epitope molecule is usually composed of 9 to 15 amino acids. The imprinted polymer prepared with this as a template shows good selectivity and high affinity for the epitope molecule and the target protein. The magnetic epitope imprinting technology uses Fe3O4 nanoparticles as a solid phase carrier to synthesize an imprinted polymer film on its surface: because the adsorption sites are uniformly distributed on the surface of the material, it not only accelerates the mass transfer rate between the target molecule and the imprinted pores, but also ensures the high-capacity adsorption of the material to the target molecule; coupled with the easy magnetic separation of Fe3O4, it simplifies the material extraction process and improves the efficiency of protein detection.
[0006] The present invention selects the SHV family of beta-lactamase as the research object (the SHV family is a typical representative of class A beta-lactamase, which includes more than 240 enzymes, and these enzymes have different hydrolysis characteristics), uses the SHV family-specific epitope as a template, uses submicron Fe3O4 particles as a solid phase carrier, and uses surface epitope imprinting technology to prepare a magnetic epitope imprinted gel polymer specific to the SHV family, and makes a detailed evaluation of its adsorption performance. The present invention also verifies that the polymer has the performance of selectively separating the SHV family from complex matrices such as bacterial lysates, thereby laying a solid theoretical and practical foundation for the development of new strategies for bacterial resistance detection. Summary of the invention
[0007] The key to the successful preparation of SHV family-specific magnetic epitope imprinted gel polymer is to determine the specific epitopes of the SHV family. The steps are as follows:
[0008] S1: The present invention utilizes Expasy( https: / / www.expasy.org / ) Powerful protein analysis function, simulating the digestion and cleavage of SHV-1 (Expasy\UniProtKB, ID: P0AD64) by protease (Expasy\PeptideCutter), and selecting peptide fragments containing 9 to 15 amino acids as candidate fragments among many cleavage fragments;
[0009] S2: Analyze the characteristics of the candidate fragments (Expasy\ProtParam), and select stable and hydrophilic peptide fragments for local sequence comparison analysis (Expasy\UniProt BLAST);
[0010] S3: By comparing all proteins in the UniProtKB database, SHV-specific and conserved polypeptide fragments were selected, which are the specific epitopes of the SHV family.
[0011] Because different proteases are selected during simulated digestion and cleavage, the specific epitopes selected in the end are also different. The present invention takes the simulated trypsin digestion and cleavage of SHV-1 as an example, and selects the polypeptide fragment VDAGDEQLER as the specific epitope of the SHV family.
[0012] The present invention adopts a simple, efficient, green and environmentally friendly synthesis method for preparing a SHV family-specific magnetic epitope imprinting gel polymer, comprising the following steps:
[0013] S1: hydrophilic modification of submicron Fe3O4 particles;
[0014] S2: hydrophilic Fe3O4 particles are modified with silanol groups to obtain Fe3O4@SiO2;
[0015] S3: Synthesis of epitope-imprinted gel polymer on Fe3O4@SiO2 surface.
[0016] S1 is to modify the carboxyl groups on the surface of Fe3O4 particles. Specifically, self-synthesized or commercialized submicron Fe3O4 particles (particle size ≈ 300-400nm) are dispersed in the water phase by ultrasonic stirring, and polyacrylic acid (M w ≈3,000~100,000Da) aqueous solution is added dropwise therein, and ultrasonic stirring is continued during the dropwise addition. After the dropwise addition is completed, ultrasonic stirring is continued to complete the modification; the molar ratio of acrylic acid to Fe ion is (0.5~4):1, and the whole process is completed under N2 protection, lasting for 2~4h, and the reaction temperature is controlled between 25~40℃.
[0017] S2 is SiO2 coated on the outer surface of Fe3O4 particles modified by carboxyl obtained in S1. Specifically, Fe3O4 particles modified by carboxyl are uniformly dispersed in ethanol / water, to which a 1-2% volume fraction of ammonia solution is added, and tetraethyl orthosilicate is added to the above system in 6 times, each reaction lasts 1-2 hours; the molar ratio of tetraethyl orthosilicate to Fe ion is (0.6-2.4):1, and the whole process is completed under continuous stirring conditions, and the reaction temperature is controlled between 25 and 40°C.
[0018] S3 is an epitope-imprinted gel polymer synthesized on the surface of Fe3O4@SiO2 obtained in S2. Specifically, the SHV-specific epitope is used as a template molecule, dissolved in ethanol / 1,2-propylene glycol (1:1, v / v), and three functional monomers are added in sequence: (3-aminopropyl) triethoxysilane, (3-ureapropyl) triethoxysilane and (3-bis(2-hydroxyethyl)aminopropyl) triethoxysilane. Each time a functional monomer is added, the template molecule and the functional monomer are preassembled for 1 hour; after 3 hours, ethyl orthosilicate (i.e., cross-linking agent), uniformly dispersed Fe3O4@SiO2 colloidal solution and deionized water are added in sequence. Under continuous stirring conditions, the polymer film is self-assembled on the surface of Fe3O4@SiO2 particles. The reaction lasts for 24 to 72 hours, and the reaction temperature is controlled between 25 and 40°C. After the synthesis, the template molecule is washed away to obtain a magnetic epitope-imprinted gel polymer with imprinted holes. The molar ratio of the template molecule, the functional monomer and the cross-linking agent is (0.5-2): (x·8): (y·4): (z·4): ((x+y+z)·4), wherein x, y, and z represent the number of -COOH, -CONH- and -NH2 in the template molecule, respectively. This ratio is also applicable to other families of β-lactamases. The present invention takes VDAGDEQLER as an example of a specific epitope of SHV: x, y, and z are 5, 9, and 3, respectively.
[0019] One of the advantages of the present invention is that: through a series of adsorption experiments, it is confirmed that the magnetic epitope imprinted gel polymer prepared above has good selectivity, high adsorption capacity and fast adsorption rate for template molecules (i.e. SHV specific epitopes) and SHV family members, as shown in the following example: Figure 7 and Figure 8 As shown, Figure 7 and Figure 8 For a detailed description, please refer to the description in the specific implementation manner.
[0020] Another advantage of the present invention is that: through practical application, it is verified that the magnetic epitope imprinting gel polymer prepared above can selectively separate SHV family members from complex matrices such as bacterial lysate. Fig. 9 As shown, Fig. 9 For a detailed description, please refer to the description in the specific implementation manner.
[0021] According to the strategy provided by the present invention, epitope-imprinted gel polymers prepared using other family-specific epitopes of β-lactamase as templates can also exhibit good recognition, adsorption and separation performance for the corresponding enzyme family members.
[0022] Therefore, the present invention lays a solid theoretical and practical foundation for the development of new strategies for bacterial resistance detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 :Preparation process of SHV family-specific magnetic epitope imprinting gel polymer and its recognition mechanism for SHV.
[0024] Figure 2 : Transmission electron micrograph of SHV family-specific magnetic epitope imprinted gel polymer (c), with carboxyl-modified Fe3O4 particles (a), Fe3O4@SiO2 (b) and magnetic non-imprinted gel polymer (the preparation method is the same as that of magnetic epitope imprinted gel polymer, the difference is that no template molecule is added in the preparation system) (d) as controls.
[0025] Figure 3 : Fourier transform-infrared spectra of SHV family-specific magnetic epitope imprinted gel polymer (c), with carboxyl-modified Fe3O4 particles (a), Fe3O4@SiO2 (b) and magnetic non-imprinted gel polymer (d) as controls.
[0026] Figure 4 : X-ray photoelectron spectroscopy of SHV family-specific magnetic epitope-imprinted gel polymer (c), with carboxyl-modified Fe3O4 particles (a), Fe3O4@SiO2 (b) and magnetic non-imprinted gel polymer (d) as controls.
[0027] Figure 5 : X-ray diffraction spectrum of SHV family-specific magnetic epitope imprinted gel polymer (c), with carboxyl-modified Fe3O4 particles (a), Fe3O4@SiO2 (b) and magnetic non-imprinted gel polymer (d) as controls.
[0028] Figure 6 : Magnetization curves of SHV family-specific magnetic epitope imprinted gel polymer (c), with carboxyl-modified Fe3O4 particles (a), Fe3O4@SiO2 (b) and magnetic non-imprinted gel polymer (d) as controls.
[0029] Figure 7: Isothermal adsorption curves of SHV family-specific magnetic epitope imprinted gel polymer for template molecule VDAGDEQLER (i.e. SHV specific epitope) (Aa) and SHV family members (taking SHV-1 (Ba) and SHV-18 (Bc) as examples), with TEM family-specific epitopes VDAGQEQLGR (Ac) and TEM-1 (TEM family member) (Be) as reference molecules, and the adsorption of the above-mentioned epitopes (Ab and Ad) and protein molecules (Bb, Bd and Bf) by magnetic non-imprinted gel polymer as control.
[0030] Figure 8 : Adsorption kinetic curves of SHV family-specific magnetic epitope imprinted gel polymer to template molecules VDAGDEQLER (ie, SHV-specific epitope) (A) and SHV-1 (B). The obtained data were fitted by the pseudo-second-order kinetic model.
[0031] Fig. 9 : SHV family-specific magnetic epitope imprinting gel polymer was used to detect SHV-1 (produced from HFK417, a strain preserved in our laboratory and isolated from clinical samples) and SHV-18 (produced from 700603, which is a standard strain), the extract was detected by MALDI-TOF MS, and the obtained mass spectra are shown in Bb and Cb respectively. The extraction of 25922 (the standard strain that does not produce SHV) (Ab), direct detection of bacterial lysate (Aa, Ba and Ca) and extraction of these three strains by magnetic non-imprinting gel polymer (Ac, Bc and Cc) were used as controls. DETAILED DESCRIPTION
[0032] In order to make the technical route of the present invention clearer, the implementation and application of the present invention are further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, not to limit the present invention; relevant technical personnel can still modify or replace the specific implementation of the present invention, but as long as it is in accordance with the spirit and scope of the present invention, it should be covered by the claims of the present invention.
[0033] The technical route of the present invention is described in detail below in conjunction with specific embodiments.
[0034] like Figure 1 As shown, the preparation method of the SHV family-specific magnetic epitope imprinting gel polymer provided in this embodiment comprises the following steps:
[0035] S1: hydrophilic modification of submicron Fe3O4 particles;
[0036] S2: hydrophilic Fe3O4 particles are modified with silanol groups to obtain Fe3O4@SiO2;
[0037] S3: Synthesis of epitope-imprinted gel polymer on Fe3O4@SiO2 surface.
[0038] S1 is to modify the carboxyl groups on the surface of submicron Fe3O4 particles. Specifically, 2.7g (0.01mol) FeCl3·6H2O was ultrasonically dissolved in 80mL ethylene glycol, then 3.6g sodium acetate and 2g polyethylene glycol 2000 were added in sequence, and the solution was gently stirred for 30min after ultrasonic dissolution. The above solution was transferred to a reactor and reacted at 200℃ for 10h. After the temperature was cooled to room temperature, the contents of the reactor were transferred to a three-necked flask, and after ultrasonic stirring and dispersion for 30min, polyacrylic acid (M w ≈3,000Da) aqueous solution (1.17mL polyacrylic acid 3000 (≈0.01mol acrylic acid) dissolved in 40mL deionized water) was added dropwise to the above system. The addition was completed after 1h and ultrasonic stirring was continued for 30min. The whole process was completed under N2 protection. Finally, carboxyl-modified submicron Fe3O4 particles were obtained. After rinsing three times with deionized water, a 25mg / mL uniform colloidal solution was prepared.
[0039] S2 is SiO2 coated on the outer surface of carboxyl-modified Fe3O4 particles obtained in S1. Specifically: 10mL of carboxyl-modified Fe3O4 colloidal solution is evenly mixed with 100mL of ethanol / water (4:1, v / v), 1mL of ammonia water (25-28wt%) is added thereto, and 0.672mL (3mmol) of ethyl orthosilicate is added to the above system in 6 times at 30°C and under continuous stirring, and each reaction lasts for 1h; after 6h, the reaction is terminated to obtain Fe3O4@SiO2 particles with a mononuclear core-shell structure, which are rinsed three times with ethanol / water (1:1, v / v) to prepare a 30mg / mL stable colloidal solution.
[0040] S3 is an epitope-imprinted gel polymer synthesized on the surface of Fe3O4@SiO2 obtained in S2. Specifically, 5.65mg (0.005mmol) of the template molecule VDAGDEQLER (i.e., SHV-specific epitope, synthesized by BGI) was fully dissolved in 80mL of ethanol / 1,2-propylene glycol (1:1, v / v), and three functional monomers were added in sequence: 0.2mmol (3-aminopropyl) triethoxysilane, 0.18mmol (3-ureapropyl) triethoxysilane and 0.06mmol (3-bis(2-hydroxyethyl)aminopropyl) triethoxysilane. Each time a functional monomer was added, the template molecule was preassembled with the functional monomer for 1h; after 3h, 0.4mmol of ethyl orthosilicate (i.e., crosslinker), 5mL Fe3O4@SiO2 colloidal solution and 20mL deionized water were added at 30°C and continuously stirred to form a polymer film on the surface of Fe3O4@SiO2 particles. After 24 hours, the reaction was completed and the template molecules were washed away with ammonia (25-28wt%) / ethanol / water (1:2:2, v / v / v) to obtain a magnetic epitope imprinted gel polymer with imprinted pores. After rinsing three times with ethanol / water (1:1, v / v), a 35mg / mL stable colloidal solution was prepared.
[0041] For comparison purposes, a magnetic non-imprinted gel polymer was also prepared in this example. The preparation method was the same as above, except that no template molecule was added to the preparation system.
[0042] like Figure 1 As shown in the figure, the SHV family-specific magnetic epitope imprinting gel polymer can specifically adsorb SHV family members through epitope recognition. The recognition of the epitope by the polymer is based on the electrostatic attraction and hydrogen bonding between the imprinted holes and the epitope molecules, and the perfect fit between the two in shape, size and functional group position is the key to epitope-specific recognition. Due to the different selected specific epitopes, the pre-treatment of the target protein before material adsorption is also different: if the selected epitope is located on the surface of the protein, because it is exposed and mostly linear, the target protein can be adsorbed by the material without treatment; if the specific epitope is located inside the protein, the target protein needs to be denatured to expose the epitope before it can be adsorbed by the material.
[0043] In this example, the SHV-specific epitope VDAGDEQLER was used as a template molecule to prepare a magnetic epitope imprinted gel polymer specific to the SHV family. Before the polymer adsorbs SHV, the following two points need to be considered: (1) Artificially synthesized polypeptides are mostly present in a linear form, and thus the recognition pores of the imprint are also linear, while the above polypeptide (i.e., VDAGDEQLER) has a unique secondary structure (α-helix and Ω-loop, Figure 1 ); (2) This polypeptide fragment is not completely exposed, but is partially hidden in the globular structure of SHV ( Figure 1 ); Because of this, the polymer cannot directly adsorb SHV. For this reason, SHV must first be denatured into a soluble linear structure to fully expose its epitope before it can be specifically recognized and adsorbed by the material. Therefore, this embodiment provides a simple and rapid protein denaturation method, that is, adding 2% (w / v) SDS to the protein aqueous solution and heating and boiling at 100°C for 10 minutes, SHV is denatured into a linear structure, because the denatured SHV is surrounded by a large number of DSˉ ions ( Figure 1 ), thus giving it good water solubility characteristics.
[0044] The following is a characteristic description of the materials prepared in this example in combination with material characterization.
[0045] like Figure 2 As shown in the figure, these four materials (carboxyl-modified Fe3O4 (a), Fe3O4@SiO2 (b), magnetic epitope imprinted gel polymer (c) and magnetic non-imprinted gel polymer (d)) are all nearly spherical, with uniform size distribution (340±20nm), and can be evenly dispersed in the aqueous phase; except for Fe3O4 particles, the other three materials have an obvious single-core core-shell structure with a shell thickness of ≤10nm.
[0046] like Figure 3 As shown, 1715cm -1 The C=O stretching vibration peak at 1635 cm indicates the presence of -COOH on the Fe3O4 surface. -1 The obvious absorption peak at 1564cm is attributed to the deformation vibration of -OH on the surface of these four materials. -1 The deformation vibration peak of -NH2 at -NH2 indicates the successful synthesis of polymer film on the Fe3O4@SiO2 surface; in addition, Figure 3 Obvious Si-O-Si and Fe-O stretching vibration peaks can also be seen at 1095 and 580 cm -1 Place.
[0047] like Figure 4 As shown in the figure, after Fe3O4 is modified with carboxyl groups, its obvious O1s (531.3 eV) and C1s (288.3 eV) energy spectrum peaks can be seen; due to the encapsulation of SiO2, the other three materials can also show obvious Si 2s (154.4 eV) and Si 2p (103.3 eV) peaks; compared with Fe3O4@SiO2, the imprinted polymer has a significantly raised C1s peak (285.1 eV) and a newly emerged N1s peak (399.7 eV), confirming the successful synthesis of the polymer.
[0048] like Figure 5As shown, the X-ray diffraction patterns of these four materials are the same. The seven diffraction peaks at 2θ angles of 30.1°, 35.5°, 37.1°, 43.1°, 53.5°, 57.0° and 62.6° correspond to the seven groups of crystal plane constants
[220] ,
[311] ,
[222] ,
[400] ,
[422] ,
[511] and
[440] of inverse spinel Fe3O4, respectively. The positions and relative intensities of these diffraction peaks are highly consistent with the diffraction peaks of Fe3O4 (ICDD Ref. Code: 01-075-0033) in the standard card library, indicating that the functional modification of Fe3O4 in each step in this embodiment has not changed its original crystal properties.
[0049] like Figure 6 As shown, the hysteresis curves of the four materials are completely closed and centrosymmetric about the origin. The remanence of each material is less than 1emu / g, and the coercive force is less than 20Oe, which confirms their superparamagnetic characteristics. Figure 6 It can be clearly seen that the saturation magnetization of the imprinted polymer is as high as 52.9emu / g, indicating that the polymer can be quickly separated by an external magnetic field.
[0050] The adsorption performance of the magnetic epitope imprinted gel polymer prepared in this example is evaluated in detail below in combination with adsorption experiments.
[0051] The adsorption experiment of the imprinted polymer includes its adsorption on the template molecule VDAGDEQLER (i.e., SHV-specific epitope) and on SHV family members (taking SHV-1 and SHV-18 as examples), with TEM family-specific epitopes VDAGQEQLGR and TEM-1 (TEM family member) as reference molecules, respectively, and the adsorption of the non-imprinted polymer on the above-mentioned epitopes and protein molecules as a control.
[0052] The template molecule and TEM-specific epitope were dissolved in ethanol / water (1:1, v / v) to prepare a series of standard solutions (C o =0.02~1.5mg / mL). 20mg of imprinted polymer was mixed with 1mL of standard solution of each concentration and adsorbed under oscillation for 24h at room temperature (25±0.5℃); an external magnetic field was applied to separate the material from the solution, and the remaining epitope molecules in the solution were quantitatively detected by HPLC-UV. The equilibrium adsorption amount of the material corresponding to each initial concentration was used as the data point to draw the isothermal adsorption curve of the imprinted polymer for the epitope molecule ( Figure 7 A).
[0053] SHV-1, SHV-18 and TEM-1 were dissolved in 1,2-propylene glycol / water (1:1, v / v) to prepare a series of standard solutions (C o=0.02~1.5mg / mL), 2% (w / v) SDS was added respectively, and heated and boiled at 100℃ for 10min. 20mg of imprinted polymer was mixed with 1mL of denatured protein solution, and adsorbed under oscillation at room temperature for 24h; the material was separated from the solution, and the remaining protein molecules in the solution were quantitatively detected by a commercial kit provided by Shenggong Bioengineering, and the adsorption data obtained were used to draw the isothermal adsorption curve ( Figure 7 B).
[0054] like Figure 7 As shown in Aa, the imprinted polymer exhibited a significantly higher adsorption capacity for SHV-specific epitopes compared to the non-imprinted polymer (Q m =37.71 mg / g), and is much higher than its adsorption to TEM specific epitopes (Q m <10mg / g)( Figure 7 Ac), which indicates that the polymer has good selectivity for the template epitope, confirming that the perfect fit between the target epitope and the imprinted pore in terms of shape, size and functional group position is the key to epitope-specific recognition. Based on this, the gel polymer imprinted with SHV-specific epitopes can effectively distinguish SHV from other families of β-lactamases, such as Figure 7 As shown in B, the polymer has an effect on SHV-1 ( Figure 7 Ba) and SHV-18( Figure 7 The adsorption amount (Q m =27.41~28.66mg / g) is much higher than its adsorption to TEM-1 (Q m <15mg / g)( Figure 7 In addition, when SHV (composed of 286 amino acids) was captured by a specific adsorption site (imprinted with a polypeptide composed of 10 amino acids) based on epitope recognition, the adjacent adsorption sites were masked by the amino acid sequence outside the template epitope, resulting in a decrease in the adsorption amount of SHV by the imprinted polymer ( Figure 7 Ba and 7Bc) are lower than their adsorption to the template epitope ( Figure 7 Aa).
[0055] Since there are no imprinted holes on the surface of non-imprinted polymers, there is almost no difference in the adsorption amount of target molecules and reference molecules ( Figure 7 Ab and 7Ad and Figure 7 Bb, 7Bd and 7Bf).
[0056] 20 mg of imprinted polymer was mixed with 1 mL of template epitope and SHV-1 standard solution (C o=0.5 mg / mL) and adsorbed under oscillation at room temperature; 8 (2-120 min) and 13 (2-300 min) adsorption time nodes were selected, and the concentration of the remaining target molecule in the solution was measured at each time node. The adsorption amount of the material corresponding to each time node was used as the data point to draw the adsorption kinetic curves of the imprinted polymer for the template epitope and SHV-1, respectively ( Figure 8 ).
[0057] like Figure 8 As shown in Figure A, the imprinted polymer exhibits a very fast adsorption rate for the template epitope, and the adsorption reaches equilibrium within only 2 minutes. This is due to the perfect match between the template epitope and the imprinted pores, and the surface adsorption accelerates the mass transfer rate between the template molecule and the imprinted pores. In contrast, the adsorption of SHV-1 by the polymer takes 30 minutes to reach the initial equilibrium ( Figure 8 B), which is attributed to the time consumption generated by epitope recognition and the size exclusion effect between protein macromolecules (competing for binding to the adsorption sites imprinted by small molecule polypeptides). In addition, when SHV-1 is captured by the adsorption sites, there is a weak van der Waals interaction between it and the free protein molecules in the solution, which further contributes to the adsorption of SHV-1 by the imprinted polymer, such as Figure 8 As shown in B, the adsorption amount of SHV-1 by the material showed a slow upward trend after 30 minutes.
[0058] The pseudo-second-order kinetic model fitting results ( Figure 8 ) showed that the adsorption of the imprinted polymer to the template epitope and SHV was based on chemical force adsorption rather than simple physical adhesion.
[0059] The following is a detailed evaluation of the matrix interference removal ability of the prepared magnetic epitope imprinting gel polymer in combination with practical applications.
[0060] In this example, two strains were selected for the selective extraction of SHV produced by bacteria: one was HFK417, which was a strain preserved in this laboratory and isolated from clinical samples. This strain produces SHV-1; the other was 700603, which is a standard strain purchased by our laboratory and used for quality control of routine testing in our laboratory. This strain produces SHV-18. This example also selects another standard strain 25922, which does not produce SHV, was used as a control for the extraction of this strain using the imprinted polymer.
[0061] 300 mg of pure cultured strain was evenly dispersed in 1 mL of sterile water, and the bacteria were broken by ultrasound until the bacterial suspension was clear; the bacterial lysate was centrifuged at 10,000 rpm for 10 min, and the obtained protein supernatant was used for selective extraction of SHV.
[0062] 0.5 mL of protein supernatant was evenly mixed with 0.5 mL of 1,2-propylene glycol, 2% (w / v) SDS was added, and the mixture was heated and boiled at 100°C for 10 min; 0.4 mL of denatured protein solution was mixed with 10 mg of imprinted polymer, and oscillated and adsorbed at room temperature for 30 min; an external magnetic field was applied to separate the material, and under ultrasonic-assisted conditions, 0.2 mL of deionized water was used to elute the material twice to remove non-specific adsorption; SHV specifically adsorbed by the imprinted polymer was eluted with 0.1 mL of ammonia (25-28 wt%) / water (1:4, v / v) and then detected by MALDI-TOF MS.
[0063] like Fig. 9 Bb and 9Cb, and direct detection with bacterial lysate ( Fig. 9 Ba and 9Ca), after the protein solution was extracted with the imprinted polymer, the matrix interference was effectively removed, and the mass spectrum baseline became very smooth, so that the SHV signal was significantly enhanced and clearly presented in the mass spectrum. Fig. 9 As shown in Ab, After 25922 was extracted with the imprinted polymer, no SHV peak was found in the mass spectrum, indicating that the polymer can effectively distinguish between enzyme-producing and non-enzyme-producing strains. After the two enzyme-producing strains were extracted with the non-imprinted polymer, SHV was not detected ( Fig. 9 Bc and 9Cc), confirming that epitope-specific recognition is the key factor determining the selective extraction of SHV.
[0064] The above practical application organically combines the specificity of epitope imprinting technology with the sensitivity of mass spectrometry detection, so that the bacterial resistance mechanism is intuitively presented in the mass spectrum; according to this strategy, other families of β-lactamase can also be specifically detected. According to the detected molecular weight, the enzyme-producing type of pathogens and the hydrolysis characteristics of the enzyme can be quickly determined, and then the dosing regimen of β-lactam antibiotics can be preliminarily formulated, which is of great practical significance for shortening the time of clinical experience medication and effectively curbing the development of bacterial resistance. Therefore, the present invention has laid a solid theoretical and practical foundation for the development of new strategies for bacterial resistance detection.
[0065] The present invention and its implementation methods are described in detail above. Such description is not restrictive. The accompanying drawings only show one of the specific implementation methods of the present invention, and the actual effect is not limited to this. It should be understood that if relevant technical personnel are inspired by it and do not deviate from the purpose of the invention, they can creatively design implementation methods similar to the technical solution, which should fall within the scope of protection of the present invention.
Claims
1. A method for preparing a magnetic epitope imprinting gel polymer for specific adsorption of β-lactamase SHV family, characterized in that: The preparation method comprises the following steps: S1: hydrophilic modification of submicron Fe3O4 particles; S2: The hydrophilic Fe3O4 particles obtained in S1 are modified with silanol groups to obtain Fe3O4@SiO2; S3: The template molecules and functional monomers self-assemble into a polymer film on the Fe3O4@SiO2 surface under the action of a cross-linking agent, and the template molecules are washed away to obtain a magnetic epitope imprinted gel polymer.
2. The method for preparing a magnetic epitope imprinting gel polymer for specific adsorption of β-lactamase SHV family according to claim 1, characterized in that: The hydrophilic modification described in S1 is to modify carboxyl groups on the surface of Fe3O4 particles, wherein: The Fe3O4 particles are submicron-sized, and the carboxyl matrix is polyacrylic acid; The molar ratio of acrylic acid to Fe ion is (0.5-4):1; The hydrophilic modification is carried out in water phase, the reaction time is 2 to 4 hours, and the reaction temperature is 25 to 40°C; The hydrophilic modification is completed under N2 protection, ultrasound assistance and continuous stirring conditions.
3. The method for preparing a magnetic epitope imprinting gel polymer for specific adsorption of β-lactamase SHV family according to claim 1, characterized in that: The silanol modification in S2 is to coat SiO2 on the outer surface of the Fe3O4 particles modified with carboxyl groups obtained in S1, wherein: The silicon-based precursor is ethyl orthosilicate; The molar ratio of ethyl orthosilicate to Fe ion is (0.6-2.4):1; The silanol modification is carried out in an ethanol / water system containing 1 to 2% by volume of aqueous ammonia, the reaction time is 6 to 12 hours, and the reaction temperature is 25 to 40° C.; The silanol modification is performed under continuous stirring conditions.
4. The method for preparing a magnetic epitope imprinting gel polymer for specific adsorption of β-lactamase SHV family according to claim 1, characterized in that: The epitope-imprinted gel polymer in S3 is self-assembled on the Fe3O4@SiO2 surface obtained in S2, wherein: The SHV family-specific epitope is used as the template molecule; Three silane coupling agents: (3-aminopropyl)triethoxysilane, (3-ureapropyl)triethoxysilane and (3-bis(2-hydroxyethyl)aminopropyl)triethoxysilane are used as functional monomers; Tetraethyl orthosilicate is used as the crosslinking agent; Under continuous stirring conditions, the polymer film self-assembled on the surface of Fe3O4@SiO2 particles through the cross-linking effect of ethyl orthosilicate; After the template molecules are washed away, a magnetic epitope-imprinted gel polymer with imprinted pores is obtained.
5. The method for preparing a magnetic epitope imprinting gel polymer for specific adsorption of β-lactamase SHV family according to claim 4, characterized in that: The determination of SHV family-specific epitopes includes the following steps: S3.1: Computer simulation of the digestion and cleavage of SHV-1 by protease, and selection of a stable and hydrophilic polypeptide fragment containing 9 to 15 amino acids from among the numerous cleavage fragments; S3.2: In the protein database, the above-mentioned preliminary screening fragments are subjected to sequence comparison analysis to select SHV-specific and conserved polypeptide fragments, which are the specific epitopes of the SHV family.
6. The method for preparing a magnetic epitope imprinting gel polymer for specific adsorption of β-lactamase SHV family according to claim 4, characterized in that: The molar ratio of the template molecule, the functional monomer and the cross-linking agent is (0.5-2): (x·8): (y·4): (z·4): ((x+y+z)·4), wherein: x, y, and z represent the number of -COOH, -CONH-, and -NH2 in the template molecule, respectively; The preparation system uses ethanol / 1,2-propylene glycol (1:1, v / v) as a co-solvent, water as a proton donor, a reaction time of 24 to 72 hours, and a reaction temperature of 25 to 40°C.
7. A magnetic epitope imprinted gel polymer that specifically adsorbs the β-lactamase SHV family, prepared by the preparation method according to claim 1 and any combination of conditions according to claims 2 to 6.
8. Use of the magnetic epitope imprinting gel polymer according to claim 7 for specific adsorption of SHV family members based on epitope recognition.
9. Use of the magnetic epitope imprinting gel polymer according to claim 7 for selectively separating SHV family members from complex matrices such as bacterial lysates.
10. The method for preparing the magnetic epitope imprinted gel polymer according to claims 1 to 7, characterized in that: The preparation method is applicable to all families of β-lactamases, and the key lies in the replacement of family-specific epitopes.