Microorganism surface imprinted polymer as well as synthesis method and application thereof

Abstract

The invention relates to a microbial surface imprinted polymer as well as a synthesis method and application thereof. Sulfydryl is introduced to the surface of a microorganism through a Traut or SPDP reagent, and efficient grafting of an initiator (BiBOEDS) is ensured in combination with a TCEP / DTT reduction reaction. By adopting an in-situ polymerization technology, in-situ polymerization of functional monomers (DMC, SBMA and the like) on the surfaces of microorganisms is realized through a synergistic effect of metal halides (CuBr2 and the like), ligands (PMDETA and the like) and ascorbic acid under an anaerobic condition, and a complementary topological structure is formed. Through disulfide bond breakage, dialysis purification and freeze drying, specific recognition sites of the polymer are reserved, and interference of residual reagents is avoided. Compared with a free copolymerization method of a traditional solution phase, the method has the advantages of being high in affinity, good in dispersity, high in specific recognition capacity and the like, the advantages of integral imprinting and epitope imprinting are combined, different microorganism requirements are met, and development and application of a microorganism surface imprinting technology can be effectively promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and more specifically relates to a microbial surface imprinted polymer, a synthesis method thereof, and an application thereof. Background Art

[0002] Molecular imprinting is a cavity-structured material synthesized with a target as a template through a functional material monomer, an initiator, and a cross-linking agent, which has a complementary structure to the target template and can specifically recognize the target. This cavity structure can specifically bind to the target template through size and shape matching and various interaction forces. In the early stage, most molecular imprinting templates were small molecules such as pesticides and explosives, as well as biological macromolecules such as proteins. In recent years, molecular imprinting templates have been extended to cells, viruses, etc. The microbial imprint obtained by using the target microorganism as a template has an affinity recognition ability with the target microorganism. This property of the microbial imprint can be used in the rapid identification and killing of the target microorganism, providing a new method for dealing with microbial infections.

[0003] Microbial molecular imprinting can be divided into two types: whole imprinting and epitope imprinting according to the imprinting object. The former uses intact cells as templates, which are rigid and have poor water medium compatibility. In practical applications, they generally need to be loaded on the material surface. Due to the dynamic growth state of the live cell template, the topological binding efficiency and recognition selectivity of the whole imprinting method are not high. Epitope imprinting mainly targets some highly expressed biomolecules on the bacterial surface, such as lipopolysaccharide, peptidoglycan, etc. This method lacks information such as the bacterial surface morphology, charge, and hydrophobicity, and faces problems such as poor discrimination of bacteria with similar epitopes by single epitope imprinting and difficulty in synthesis. Therefore, it is urgent to develop a microbial imprinted material with good water solubility that can combine the complementary advantages of the whole imprinting and epitope imprinting methods and apply its high affinity ability to the rapid identification and killing of microorganisms. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides a microbial surface imprinted polymer, a synthesis method thereof, and an application thereof, aiming to obtain a microbial surface imprinted polymer by in-situ imprinting polymerization on the microbial surface through an in-situ polymerization method. Compared with the microbial surface imprinted polymer obtained by the traditional solution-phase free copolymerization method, the microbial surface imprinted polymer has characteristics such as high affinity and good dispersibility, and can be more widely applied to directions such as rapid identification of microorganisms and drug targeted delivery.

[0005] To achieve the above object, in the first aspect, the present invention provides a synthesis method of a microbial surface imprinted polymer, including the following steps:

[0006] Microbial pretreatment: Microorganisms in the exponential growth phase are centrifuged and washed for the first time, then 2-iminothiolane hydrochloride and / or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester are added, followed by centrifugation and washing for the second time. Tris(2-carboxyethyl)phosphine hydrochloride or dithiothreitol is added for reaction to obtain microorganisms with thiol groups on the surface;

[0007] Initiator grafting: The microorganisms with thiol groups on the surface are reacted with an initiator containing a disulfide bond, and the initiator containing a disulfide bond is a disulfide with a 2'-bromo-isobutyryloxy structural unit, to obtain a suspension of microorganisms grafted with the initiator;

[0008] Monomer polymerization: The suspension of microorganisms grafted with the initiator is mixed with a functional monomer. The functional monomer is selected from two or more combinations of methacryloyloxyethyltrimethylammonium chloride, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, fluorescein-O-acrylate, and 2-hydroxyethyl methacrylate. Under anaerobic conditions, metal halides, ligands, and reducing agents are added for surface in-situ polymerization reaction to obtain polymerized microorganisms;

[0009] Post-treatment: The polymerized microorganisms are subjected to disulfide bond cleavage, centrifugation, dialysis, and freeze-drying to obtain a microbial surface imprinted polymer.

[0010] Different from the prior art, the above technical solution uses specific initiators and functional monomers, and is an in-situ polymerization preparation technology for obtaining a highly affinity microbial surface imprinted polymer that highly fits the template microorganism on the microbial surface. Specifically, 2-iminothiolane hydrochloride (Traut) or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) reagents are used to introduce thiol groups on the microbial surface, combined with the reduction reaction of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT) to ensure the efficient grafting of the initiator bis[2-(2'-bromo-isobutyryloxy)ethyl] disulfide (BiBOEDS). Then, an in-situ polymerization technology is adopted, and under anaerobic conditions, through the synergistic effect of metal halides, ligands, and reducing agents, the in-situ polymerization of functional monomers methacryloyloxyethyltrimethylammonium chloride (DMC) and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) on the microbial surface is realized to form a complementary topological structure. Then, through disulfide bond cleavage, dialysis purification, and freeze-drying, the specific recognition sites of the polymer are retained to avoid interference from residual reagents.

[0011] Compared with the traditional solution-phase free copolymerization method, it has the characteristics of high affinity, good dispersibility, strong specific recognition ability, etc., combines the advantages of overall imprinting and epitope imprinting, adapts to the needs of different microorganisms, and can effectively promote the development and application of microbial surface imprinting technology.

[0012] In a further preferred synthesis method of the present invention, the disulfide with a 2'-bromoisobutyryloxy structural unit is bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide. More preferably, the mass concentration of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide is 0.1-10 mg / mL. Preferably, in the above preparation method, the addition concentration of the 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) solution is 0.5-20 mg / mL; the addition concentration of the 2-iminothiolane hydrochloride (Traut) solution is 0.2-10 mg / mL; the addition concentration of the tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT) solution used is 1-50 mg / mL; the addition concentration of the initiator bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (BiBOEDS) is 0.1-10 mg / mL. In the actual chemical polymerization process of the microbial surface imprinted polymer, different experimental operations, differences in raw material purity, environmental temperature and humidity, and stirring speed, etc. will all cause differences in the physical and chemical properties of the microbial surface imprinted polymer. Through a large number of experiments, it is found that within the above range, the microbial surface imprinted polymer of the present invention can ensure more beneficial effects.

[0013] In some preferred synthesis methods, the mass concentrations of the methacryloyloxyethyl trimethylammonium chloride (DMC) and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (SBMA) are both 5-50 mg / mL. More preferably, the addition concentration of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (SBMA) is 5-50 mg / mL, the addition concentration of methacryloyloxyethyl trimethylammonium chloride (DMC) is 5-50 mg / mL, the addition concentration of fluorescein-O-acrylate (FOA) is 0-10 mg / mL, and the addition concentration of 2-hydroxyethyl methacrylate (HEMA) is 0-10 mg / mL. Among them, the positively charged monomer DMC adsorbs mutually with the electronegative surface of bacteria; the zwitterionic monomer SBMA is the source of the selectivity of the imprinted polymer due to its property of resisting non-specific protein adsorption; HEMA acts as a spacer unit to adjust the physical and chemical properties of the polymer; and FOA serves as the fluorescence recognition element of the system.

[0014] In some other preferred synthesis methods, the metal halide includes but is not limited to at least one of metal halides such as CuBr2, CuCl2 or FeCl3, and the total addition concentration is 5-100 mg / mL.

[0015] There are also some more preferred synthesis methods, where the ligand is NNN’N”N”-pentamethyldiethylenetriamine or tris(2-dimethylaminoethyl)amine (ME6TREN), and the addition concentration is 5 - 100 mg / mL.

[0016] The reducing agent described in the present invention can reduce a high-valent metal to a low valence state. In some of the more preferred embodiments, the reducing agent is ascorbic acid, and the concentration is 5 - 100 mg / mL.

[0017] For the present invention, the microorganism is a bacterium or a fungus, and the bacterium or fungus includes, but is not limited to, Gram-positive bacteria such as Staphylococcus aureus, Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and fungi such as Pichia pastoris, and probiotics such as Escherichia coli and Lactobacillus plantarum.

[0018] Preferably, the dialysis uses a dialysis bag with a molecular weight cut-off of 3000 - 5000. In this way, compared with most traditional molecularly imprinted polymers, the microbial surface imprinted polymer obtained in the present invention has a lower molecular weight. More preferably, a dialysis bag with a molecular weight of 3500 is used. Of course, in other different embodiments, the ultrafiltration method can also achieve the technical purpose of the present invention. However, the microbial surface imprinted polymer obtained by dialysis with a dialysis bag having a molecular weight cut-off of 3000 - 5000 has been proven to have better solubility through experiments and is a water-soluble polymer.

[0019] In the second aspect, the present invention provides a microbial surface imprinted polymer synthesized by using the synthesis method described in the first aspect of the present invention. After the microbial surface imprinted polymer is released by the cleavage of disulfide bonds, it has specific recognition sites on its surface that can specifically bind to the microorganism through charge, hydrophobicity, and shape matching.

[0020] In the third aspect, the present invention provides at least one application of the microbial surface imprinted polymer synthesized by using the synthesis method described in the first aspect of the present invention in the rapid identification, capture, tracing, and drug delivery of bacteria and / or fungi, etc. Correspondingly, the bacteria and / or fungi include, but are not limited to, Gram-positive bacteria such as Staphylococcus aureus, Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and fungi such as Pichia pastoris, and probiotics such as Escherichia coli and Lactobacillus plantarum.

[0021] Different from the prior art, the above technical solution can introduce reversible cleavage initiation sites on the surfaces of various template microorganisms, utilize the unique surface physicochemical environment of bacteria and / or fungi, guide the selective enrichment and adsorption of aptamer monomers onto the surfaces of bacteria and / or fungi, induce polymerization to form polymers highly adapted to the surfaces of bacteria and / or fungi, and after reduction excision and elution, obtain microbial surface imprinted polymers that can specifically bind to the template microorganisms with high affinity. The synthesis method provided by the present invention has a simple process and low cost. Compared with the traditional method, the imprinted polymers prepared by in-situ polymerization on the microbial surface have a strong affinity for the template microorganisms and can be used for rapid identification, capture, tracing, drug delivery, etc. of bacteria.

[0022] The above relevant description of the invention content is only an overview of the technical solution of this application. In order to enable those of ordinary skill in the art to more clearly understand the technical solution of this application, and thus can be implemented according to the content recorded in the description and the drawings, and in order to make the above objects, other objects, features, and advantages of this application more easily understood, the following is described in conjunction with the specific embodiments and drawings of this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings are only used to illustrate the principles, implementation methods, applications, features, and effects of the specific embodiments of this application and other related contents, and should not be considered as a limitation to this application.

[0024] In the accompanying drawings of the specification:

[0025] Figure 1 It is a particle size diagram of the original Escherichia coli described in the specific embodiment and after recognition with the corresponding imprinted polymer;

[0026] Figure 2 It is a Zeta potential diagram of the original Escherichia coli described in the specific embodiment and after recognition with the corresponding imprinted polymer;

[0027] Figure 3 It is a scanning electron microscope image of the original Escherichia coli described in the specific embodiment;

[0028] Figure 4 It is a scanning electron microscope image of the original Escherichia coli and after recognition with the corresponding microbial surface imprinted polymer described in the specific embodiment;

[0029] Figure 5 It is a particle size diagram of the original Staphylococcus aureus and after recognition with the corresponding imprinted polymer described in the specific embodiment;

[0030] Figure 6 It is a Zeta potential diagram of the original Staphylococcus aureus and after recognition with the corresponding imprinted polymer described in the specific embodiment;

[0031] Figure 7SEM image of Staphylococcus aureus as described in the specific implementation manner;

[0032] Figure 8 SEM image of Staphylococcus aureus after recognition by the corresponding imprinted polymer as described in the specific implementation manner;

[0033] Figure 9 Fluorescence image of Escherichia coli after binding to its corresponding imprinted polymer as described in the specific implementation manner;

[0034] Figure 10 Fluorescence image of Staphylococcus aureus after binding to its corresponding imprinted polymer as described in the specific implementation manner;

[0035] Figure 11 Fluorescence image of Pichia pastoris after binding to its corresponding imprinted polymer as described in the specific implementation manner;

[0036] Figure 12 Recognition result of Escherichia coli and its imprinted polymer in fetal bovine serum as described in the specific implementation manner;

[0037] Figure 13 Fluorescence microscopy image of Escherichia coli imprinted polymer in the Escherichia coli intestinal colonization model (positive matching model) of zebrafish;

[0038] Figure 14 Fluorescence microscopy image of Escherichia coli imprinted polymer in the Lactobacillus plantarum intestinal colonization model (mismatch model) of zebrafish. Specific implementation manner

[0039] To illustrate in detail the possible application scenarios, technical principles, specific implementable solutions, achievable objectives and effects of this application, etc., the following will be described in detail with reference to the specific examples listed and in conjunction with the accompanying drawings. The examples described herein are only used to more clearly illustrate the technical solutions of this application, so they are only examples and cannot be used to limit the protection scope of this application.

[0040] Referring to "embodiment" in this context means that the specific features, structures or characteristics described in conjunction with the embodiment can be included in at least one embodiment of this application. The term "embodiment" appearing in various positions in the specification does not necessarily refer to the same embodiment, nor does it particularly define its independence or relevance to other embodiments. In principle, in this application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0041] Unless otherwise defined, the technical terms used herein have the same meanings as those commonly understood by those skilled in the technical field to which this application belongs; the use of relevant terms herein is only for describing specific embodiments and is not intended to limit this application.

[0042] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that there can be three relationships. For example, A and / or B means: there is A, there is B, and there is both A and B at the same time. In addition, the character " / " herein generally represents an "or" logical relationship between the associated objects before and after.

[0043] In this application, terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantitative, primary-secondary or sequential relationships between these entities or operations.

[0044] Without further limitations, in this application, the expressions "including", "comprising", "having" or other similar expressions used in the statements are intended to cover non-exclusive inclusion. These expressions do not exclude that there may be additional elements in the process, method or product including the said elements, so that the process, method or product including a series of elements may not only include those defined elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0045] The same as the understanding in the "Examination Guidelines", in this application, expressions such as "greater than", "less than", "exceeding" are understood not to include the number itself; expressions such as "above", "below", "within" are understood to include the number itself. In addition, in the description of the embodiments of this application, the meaning of "multiple" is two or more (including two). Similar expressions related to "many" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise specifically defined.

[0046] In the description of the embodiments of this application, the spatially related expressions used, such as "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "perpendicular", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the specific embodiment or the drawings, and is only for the convenience of describing the specific embodiments of this application or for the convenience of readers' understanding, rather than indicating or implying that the device or component referred to must have a specific position, a specific orientation, or be constructed or operated in a specific orientation. Therefore, it cannot be understood as a limitation to the embodiments of this application.

[0047] Unless otherwise clearly specified or defined, in the description of the embodiments of the present application, terms such as "installation", "connection", "linkage", "fixation", "setting", etc. shall be understood in a broad sense. For example, the "connection" may be a fixed connection, a detachable connection, or an integral setting; it may be a mechanical connection, an electrical connection, or a communication connection; it may be a direct connection or an indirect connection through an intermediate medium; it may be the communication inside two components or the interaction relationship between two components. For those skilled in the art to which the present application pertains, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0048] The existing microbial imprinting technology mainly has the following two defects:

[0049] 1. Whole-cell imprinting: Using intact microorganisms as templates, but limited by the dynamic growth characteristics of living cells, resulting in low topological binding efficiency, poor water solubility, relying on carrier materials for loading, low recognition selectivity, and limited applications.

[0050] 2. Epitope imprinting: Only targeting specific molecules on the surface of microorganisms (such as lipopolysaccharides, peptidoglycans), ignoring the overall morphology, charge, and hydrophobicity characteristics of microorganisms, insufficient ability to distinguish microorganisms with similar epitopes, and complex synthesis processes.

[0051] It can be seen from this that the existing technology has not effectively combined the advantages of whole-cell imprinting and epitope imprinting. The present invention uses a microbial surface in-situ polymerization technology, with specific initiators and functional monomers, to directly form an imprinted polymer on the surface of microorganisms that highly matches the template microorganisms, having high affinity, water solubility, and specific recognition ability, which greatly improves the development level of microbial imprinting technology and is expected to play a greater role in fields such as rapid microbial identification, capture, tracing, and drug delivery.

[0052] The steps of the synthesis method of the present invention include:

[0053] (1) After activating the microbial broth, perform streak plating, culture overnight at 37 °C in an incubator, pick single colonies on the plate and place them in a liquid medium, amplify and culture in a shaker, centrifuge and resuspend 3 times at 5000 rpm with PBS buffer to wash away impurities in the culture solution, add SPDP and react for 30 min, then resuspend 3 times, add TCEP or DTT and react for 30 min to introduce sulfhydryl groups to the surface of the microorganisms; or directly add Traut reagent to the broth and react for 30 min to introduce sulfhydryl groups to the surface of the microorganisms, wash with PBS buffer, add DMSO and the initiator to the precipitate and shake well, and react in the dark for 30 min to obtain a microbial suspension coupled with the initiator.

[0054] (2) Put the microbial suspension obtained in step (1) into a round-bottom flask, add two monomers such as SBMA and DMC, or three monomers such as SBMA, DMC, and HEMA, or four monomers such as SBMA, DMC, FOA, and HEMA according to the dosage requirements, seal it with a rubber stopper and continuously inject nitrogen to remove dissolved oxygen, maintain an anaerobic state in the flask, and carry out a one-pot reaction.

[0055] (3) Put metal halides CuBr2, or CuCl2, or FeCl3 and ligand PMDETA or ME6TREN into another round-bottom flask containing UP water, seal it with a rubber stopper and continuously inject nitrogen to remove dissolved oxygen.

[0056] (4) Inject the solution obtained in step (3) into the flask of step (2) with a syringe, react at low temperature and in the dark for 30 min, then add the reducing agent ascorbic acid. After reacting for 20 min, expose the reaction to air to terminate the reaction.

[0057] (5) Take out the polymerized bacterial solution obtained in step (4), centrifuge it at 3000 rpm for 3 min, discard the supernatant, resuspend the precipitate with UP water, add TCEP, fully oscillate and react for 30 min, centrifuge it at 3000 rpm for 3 min to obtain the supernatant, place the supernatant in a dialysis bag with a molecular weight cut-off of 3500 and dialyze for one day, changing the dialysis solution four times. After dialysis, freeze-dry the solution to obtain the corresponding powder, which is the microbial surface imprinted polymer.

[0058] Unless otherwise specified, all raw materials in the present invention, including Escherichia coli, Staphylococcus aureus (ATCC33591), the fungus Pichia pastoris, fetal bovine serum, zebrafish, and the reagents used, are commercially available. Reagents not specifically mentioned, such as LB solid medium, LB liquid medium, PBS buffer, etc., are all well-known and commonly used reagents in the art. Among them, the sources of some reagents and supplies used in the examples are shown in Table 1.

[0059] Table 1 Some reagents and supplies used in the examples

[0060]

[0061] Example 1 Surface Imprinted Polymer of Gram-Negative Bacterium Escherichia coli and Its Synthesis Method

[0062] Take out Escherichia coli from the glycerol preservation tube, streak it on the LB solid medium, and then place it in a constant temperature incubator at 37 °C for overnight incubation. Pick a single colony and inoculate it into the LB liquid medium, and activate and culture it in a constant temperature shaker at 37 °C and 180 rpm for 12 h. Take 200 μL of the activated Escherichia coli and inoculate it into 5 mL of the LB liquid medium, and culture it in a constant temperature shaker at 37 °C and 180 rpm. When Escherichia coli is in the exponential growth phase, transfer the bacterial solution into a 10 mL centrifuge tube, adjust the centrifuge speed to 5000 rpm, and centrifuge for 5 min. Discard the supernatant, add 5 mL of PBS buffer (pH = 7.4) to the precipitate, shake it well, and centrifuge and wash three times to thoroughly wash away impurities such as extracellular polymers on the cell surface. Add Traut reagent to the washed bacterial suspension and react for 60 min. After centrifugation and washing, add DMSO, add the initiator BiBOEDS, and mix well by shaking in the dark. Add the treated bacterial suspension, four functional material monomers DMC, SBMA, FOA, and HEMA to reaction flask 1, and continuously introduce nitrogen. Add CuBr2, PMDTEA / ME6TREN, and UP (ultrapure) water to reaction flask 2, and continuously introduce nitrogen to remove dissolved oxygen. Inject the solution in reaction flask 2 into reaction flask 1 with a syringe, add the reducing agent ascorbic acid and react for 60 min, and introduce air to end the reaction. Centrifuge the reaction solution at 3000 rpm for 5 min, discard the supernatant, add UP water to the precipitate and shake it well to obtain a bacterial solution with polymerized polymers on the surface. Add TCEP to the bacterial solution with polymerized polymers on the surface to cut the disulfide bond, shake and react for 30 min, centrifuge at 3000 rpm for 5 min, take the supernatant and place it in a dialysis bag with a molecular weight cut-off of 3500 and dialyze for 1 day, changing the dialysis fluid every 3 hours. After dialysis is completed, take out the liquid in the dialysis bag, and obtain the microbial surface imprinted polymer after freeze-drying.

[0063] Measure the particle size and Zeta potential of the original Escherichia coli and Escherichia coli + Escherichia coli surface imprinted polymer by dynamic light scattering, and the measurement results are shown in Figure 1 and Figure 2 . From Figure 1 and Figure 2 , it can be seen that the Zeta potential of the original Escherichia coli is about -40 mV, and after polymerization, the Zeta potential increases to ~ -22 mV. Correspondingly, the initial hydrated particle size of Escherichia coli is ~ 2000 nm, and the particle size becomes ~ 2600 nm after polymerization. After co-incubating Escherichia coli with the Escherichia coli surface imprinted polymer, take a scanning electron microscope image. The scanning electron microscope image of the original Escherichia coli can be seen in Figure 3 , and the scanning electron microscope image after co-incubating Escherichia coli with the surface imprinted polymer can be seen in Figure 4 . Figure 3 and Figure 4It shows that the surface of the original Escherichia coli is smoother. After co-incubation with the corresponding microbial surface imprinted polymer, the surface of the bacteria becomes rough and has obvious granularity, further indicating the successful preparation of the Escherichia coli surface imprinted polymer.

[0064] Example 2 Preparation of a surface imprinted polymer for Gram-positive bacterium Staphylococcus aureus and its preparation

[0065] Take out Staphylococcus aureus (ATCC33591) from the glycerol stock culture tube. After streaking on the LB solid medium plate, place it in an incubator and culture overnight at 37 °C. Pick a single colony and inoculate it into the LB liquid medium, and activate and culture it on a shaker at 37 °C and 180 rpm for 12 h. Take 200 μL of the activated Staphylococcus aureus and inoculate it into 5 mL of LB liquid medium, and culture it in a constant temperature shaker at 37 °C and 180 rpm. When Staphylococcus aureus is in the exponential growth phase, put the bacterial solution into a 10 mL centrifuge tube, adjust the centrifuge speed to 5000 rpm and centrifuge for 5 min. Discard the supernatant, add 5 mL of PBS buffer, shake well, and centrifuge and wash three times to thoroughly wash away impurities such as extracellular polymers on the cell surface. Add SPDP to the bacterial suspension and react for 30 min, then centrifuge and wash three times with PBS. Add TCEP and react for 30 min. After centrifuging and washing, add DMSO. Add the initiator BiBOEDS and shake well. Add the treated bacterial suspension to reaction flask 1, add three functional material monomers DMC, FOA, and HEMA and mix evenly. Pass nitrogen to remove dissolved oxygen. Add CuBr2, PMDTEA, and UP water to reaction flask 2, pass nitrogen to remove dissolved oxygen, and inject the solution in reaction flask 2 into reaction flask 1 with a syringe. Add the reducing agent ascorbic acid and react for 60 min, then open the reaction flask mouth to end the reaction. Centrifuge the reaction solution at 3000 rpm for 5 min, discard the supernatant, add UP water to the precipitate and shake well to obtain a bacterial solution with a polymer surface polymerization. Add DTT to the bacterial solution with a polymer surface polymerization to cut off the disulfide bond, shake well and react for 30 min, centrifuge at 3000 rpm for 5 min, take the supernatant and place it in a dialysis bag with a cut-off molecular weight of 3500 and dialyze for 1 day, changing the dialysis solution every 3 hours. After dialysis is completed, take out the liquid in the dialysis bag, and freeze-dry it to obtain the Staphylococcus aureus surface imprinted polymer.

[0066] Measure the particle size and Zeta potential of the original Staphylococcus aureus and Staphylococcus aureus + Staphylococcus aureus surface imprinted polymer by dynamic light scattering. For the measurement results, please refer to Figure 5 and Figure 6 .

[0067] After co-incubation of Staphylococcus aureus with the Staphylococcus aureus surface imprinted polymer, a scanning electron microscope is used for shooting. For the scanning electron microscope image of the original Staphylococcus aureus, please refer to Figure 7, please refer to the scanning electron microscope image after co-incubation with the surface imprinted polymer of Staphylococcus aureus Figure 8 , from Figures 5 - 8 The presented results show that the Zeta potential of the original Staphylococcus aureus is approximately -33 mV. After polymerization, the Zeta potential increases to ~ -10 mV. Correspondingly, the initial hydrodynamic diameter of Escherichia coli is ~1300 nm, and after polymerization, the diameter becomes ~1800 nm. The electron microscope shows that the surface of the original Staphylococcus aureus is smoother, and after co-incubation with its corresponding surface imprinted polymer, the bacterial surface is rough and has obvious granularity. This further indicates the successful preparation of the surface imprinted polymer of Staphylococcus aureus, as well as the precise matching of the surface topology of the Staphylococcus aureus surface imprinted polymer in this example to that of Staphylococcus aureus microorganisms.

[0068] Example 3: Surface Imprinted Polymer of Pichia pastoris and Its Preparation

[0069] Take out Pichia pastoris (GS115) from the glycerol stock tube, streak plate on a solid medium (a common medium for culturing Pichia pastoris), and place it in an incubator at 30 °C for overnight culture. Pick a single colony and inoculate it into a liquid medium, and activate and culture it in a constant temperature shaker at 30 °C and 180 rpm for 12 h. Take 200 μL of the activated Pichia pastoris and inoculate it into 5 mL of BMGY liquid medium, and culture it in a constant temperature shaker at 30 °C and 180 rpm. When Pichia pastoris is in the exponential growth phase, transfer the bacterial solution into a 10 mL centrifuge tube, adjust the centrifuge speed to 5000 rpm, and centrifuge for 5 min. Discard the supernatant, add 5 mL of PBS buffer, shake well, and centrifuge and wash three times to thoroughly wash away impurities such as extracellular polymers on the cell surface. Add SPDP to the bacterial suspension and react for 30 min, then centrifuge and wash three times with PBS. Add TCEP and react for 30 min. After centrifugation and washing, add DMSO. Add the initiator BiBOEDS and shake well. Add the treated bacterial suspension into reaction flask 1, and add two functional monomer materials DMC and FOA. Purge with nitrogen to remove dissolved oxygen. Add CuBr2, PMDTEA, and UP water into reaction flask 2, and purge with nitrogen to remove dissolved oxygen. Inject the solution in reaction flask 2 into reaction flask 1 with a syringe, add the reducing agent ascorbic acid and react for 60 min, then open the reaction flask mouth to end the reaction. Centrifuge the reaction solution at 3000 rpm for 5 min, discard the supernatant, add UP water to the precipitate and shake well to obtain a bacterial solution with a polymer on the surface. Add TCEP to the bacterial solution with a polymer on the surface to cut the disulfide bond, shake well and react for 30 min, centrifuge at 3000 rpm for 5 min, take the supernatant and place it in a dialysis bag with a molecular weight cut-off of 3500 and dialyze for 1 day, changing the dialysis solution 4 times a day. After dialysis is completed, take out the liquid in the dialysis bag, and freeze-dry it to obtain the surface imprinted polymer of Pichia pastoris.

[0070] Specific recognition ability of Escherichia coli surface - imprinted polymer in Example 4

[0071] After redissolving the Escherichia coli surface - imprinted polymer prepared in Example 1 with UP water and co - incubating it with Escherichia coli, due to the addition of monomer FOA, please refer to Figure 9 , under the characterization at 480 nm of an inverted fluorescence microscope, it can be observed that after Escherichia coli binds to its surface - imprinted polymer, there is obvious fluorescence on the surface and partial aggregation phenomenon.

[0072] This specific recognition ability of Escherichia coli surface - imprinted polymer can be specifically applied in aspects such as rapid identification, capture, tracing, and drug delivery of Escherichia coli.

[0073] Specific recognition ability of Staphylococcus aureus surface - imprinted polymer in Example 5

[0074] After redissolving the Staphylococcus aureus surface - imprinted polymer prepared in Example 2 with UP water and co - incubating it with Staphylococcus aureus, please refer to Figure 10 , under the characterization at 480 nm of an inverted fluorescence microscope, it can be observed that after Staphylococcus aureus binds to its surface - imprinted polymer, there is obvious fluorescence on the surface and partial aggregation phenomenon.

[0075] This specific recognition ability of Staphylococcus aureus surface - imprinted polymer can be specifically applied in aspects such as rapid identification, capture, tracing, and drug delivery of Staphylococcus aureus.

[0076] Specific recognition ability of Pichia pastoris surface - imprinted polymer in Example 6

[0077] After redissolving the Pichia pastoris surface - imprinted polymer prepared in Example 3 with UP water and co - incubating it with Pichia pastoris, please refer to Figure 11 , under the characterization at 480 nm of an inverted fluorescence microscope, it can be observed that after Pichia pastoris binds to its surface - imprinted polymer, there is obvious fluorescence on the surface and partial aggregation phenomenon.

[0078] This specific recognition ability of Pichia pastoris surface - imprinted polymer can be specifically applied in aspects such as rapid identification, capture, tracing, and drug delivery of Pichia pastoris.

[0079] Specific recognition of microbial surface - imprinted polymer in serum environment in Example 7

[0080] Mix 50 μL of Escherichia coli in the logarithmic growth phase with 50 μL of fetal bovine serum. After redissolving the Escherichia coli - imprinted polymer prepared in Example 1 with UP water and injecting it into the serum - bacteria solution for co - incubation for 30 min, take 10 μL for making a slide and observe under a laser confocal microscope. Part of the Escherichia coli aggregates and emits strong green fluorescence (please refer to Figure 12) It shows that the prepared Escherichia coli surface - imprinted polymer still has the ability of recognition and specific binding in a complex serum environment, indicating that the microbial surface - imprinted polymer is applicable to the rapid screening of clinical samples.

[0081] Example 8: Colonization of Escherichia coli - imprinted polymer on Escherichia coli and Lactobacillus plantarum in zebrafish intestine

[0082] A cross - test was carried out using the zebrafish model. Two different strains, namely Escherichia coli and Lactobacillus plantarum, were colonized in the intestine of this model. Zebrafish larvae were treated with the Escherichia coli surface - imprinted polymer. Observation was carried out 24 h after administration (please refer to Figure 13 and Figure 14 ).

[0083] It can be seen from the figure that in the correctly paired larval group of zebrafish larvae colonized with Escherichia coli, the fluorescent - labeled Escherichia coli surface - imprinted polymer MIPs significantly aggregated in the foregut and midgut regions ( Figure 13 ). In the group of zebrafish larvae colonized with Lactobacillus plantarum, the fluorescent - labeled Escherichia coli surface - imprinted polymer MIPs could not stay in the foregut and midgut regions and were excreted out of the body ( Figure 14 ). Through verification by the zebrafish model, the results confirmed that the Escherichia coli surface - imprinted polymer MIPs had strong specific binding ability. The polymer could be accurately colonized in the target microbial enrichment area and could play an extremely promising application prospect in in - vivo microbial detection, tracking and intestinal flora regulation.

[0084] In addition, when the microbial surface - imprinted polymer provided by the present invention is loaded on the surface of a sensor, it can also selectively adsorb target pathogens (such as Escherichia coli) in water.

[0085] Compared with the existing imprinting polymerization technology, the present invention synergistically combines the advantages of whole - microbial imprinting and bacterial - template polymerization: (1) Surface - confined polymerization ensures that the polymer is only guided by the microbial template; (2) The method of the present invention can imprint the physicochemical information (such as surface charge and hydrophobicity) and specific epitopes on the cell surface; (3) The obtained microbial surface - imprinted polymer can be separated from the microbial surface through redox - cleavable disulfide bonds, which is convenient for efficient and flexible purification. Therefore, the microbial surface - imprinted polymer of the present invention exhibits high affinity and selectivity, enabling it to distinguish the template microorganism from other microorganisms in vitro and in vivo. Combining the overall morphology and epitope characteristics of microorganisms, the recognition efficiency of the microbial surface - imprinted polymer provided by the present invention is more than 3 times higher than that of traditional methods and can adapt to the needs of different microorganisms. The application of the microbial surface - imprinted polymer of the present invention can cover multiple fields such as medical diagnosis, targeted therapy, and environmental monitoring, and has excellent industrialization potential.

[0086] Finally, it should be noted that although the above embodiments have been described in the text and drawings of the specification of this application, the patent protection scope of this application cannot be limited thereby. Any technical solutions resulting from equivalent structure or equivalent process substitution or modification made by using the content recorded in the text and drawings of the specification of this application based on the essential concept of this application, as well as the direct or indirect implementation of the technical solutions of the above embodiments in other related technical fields, etc., are all included within the patent protection scope of this application.

Claims

1. A method for synthesizing a microbial surface imprinted polymer, characterized in that, It includes the following steps: Microbial pretreatment: After centrifugally washing the microorganisms in the exponential growth phase for the first time, 2-iminothiolane hydrochloride and / or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester are added, followed by centrifugal washing for the second time. Then, tris(2-carboxyethyl)phosphine hydrochloride or dithiothreitol is added for reaction to obtain microorganisms with thiol groups on the surface; Initiator grafting: React the microorganisms with thiol groups on the surface with an initiator containing a disulfide bond. The initiator containing a disulfide bond is a disulfide with a 2'-bromo-2-methylpropanoyl group structural unit to obtain a suspension of microorganisms grafted with the initiator; Monomer polymerization: Mix the suspension of microorganisms grafted with the initiator with a functional monomer. The functional monomer is selected from two or more combinations of methacryloyloxyethyltrimethylammonium chloride, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, fluorescein-O-acrylate, and 2-hydroxyethyl methacrylate. Under anaerobic conditions, a mixed solution containing a metal halide, a ligand, and a reducing agent is added for surface in-situ polymerization reaction to obtain polymerized microorganisms; Post-treatment: Subject the polymerized microorganisms to disulfide bond cleavage, centrifugation, dialysis, and freeze-drying to obtain a microbial surface imprinted polymer.

2. The synthesis method according to claim 1, wherein The disulfide with a 2'-bromo-2-methylpropanoyl group structural unit is bis[2-(2'-bromo-2-methylpropanoyl)ethyl]disulfide. Preferably, the mass concentration of bis[2-(2'-bromo-2-methylpropanoyl)ethyl]disulfide is 0.1-10 mg / mL.

3. The synthesis method according to claim 1, characterized in that, The mass concentrations of methacryloyloxyethyltrimethylammonium chloride and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide are both 5-50 mg / mL.

4. The synthesis method according to claim 1, characterized in that, The metal halide includes at least one of CuBr2, CuCl2, or FeCl3.

5. The synthesis method according to claim 1, characterized in that, The ligand is NNN’N”N”-pentamethyldiethylenetriamine or tris(2-dimethylaminoethyl)amine.

6. The synthesis method according to claim 1, wherein The reducing agent is ascorbic acid.

7. The synthesis method according to claim 1, wherein The microorganism is a bacterium or a fungus.

8. The synthesis method according to claim 1, wherein The dialysis uses a dialysis bag with a molecular weight cut-off of 3000-5000.

9. A microbial surface imprinted polymer, characterized in that, Synthesized by the synthesis method described in any one of claims 1-8, after the polymer is released by disulfide bond cleavage, its surface has specific recognition sites that can specifically bind to the microorganism through charge, hydrophobicity, and shape matching.

10. At least one application of the microbial surface imprinted polymer synthesized by the synthesis method described in any one of claims 1-8 in the aspects of rapid identification, capture, tracing, and drug delivery of bacteria and / or fungi.

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