CN111909407B - Imprinted thin film material for selectively separating drug-resistant bacillus and preparation and application thereof - Google Patents

Imprinted thin film material for selectively separating drug-resistant bacillus and preparation and application thereof Download PDF

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CN111909407B
CN111909407B CN202010760914.3A CN202010760914A CN111909407B CN 111909407 B CN111909407 B CN 111909407B CN 202010760914 A CN202010760914 A CN 202010760914A CN 111909407 B CN111909407 B CN 111909407B
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resistant
bacillus
bacteria
resistant bacillus
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徐顺清
沈先涛
陈志亮
杨柳倩
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Huazhong University of Science and Technology
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Abstract

The invention discloses a imprinted thin film material for selectively separating drug-resistant bacillus and preparation and application thereof, and relates to the field of new materials. The method comprises the following steps: firstly, preparing a semi-solidified film; taking a drug-resistant bacillus suspension, diluting, dropwise adding the drug-resistant bacillus suspension on a sterilized substrate, standing, and then removing a solvent in an aseptic environment; pressing the substrate carrying the drug-resistant bacillus into a semi-solidified film to enable the drug-resistant bacillus to be used as a template molecule to form an imprint on the semi-solidified film; then heating and completely curing; stripping the imprinted membrane from the substrate to obtain an imprinted membrane precursor, washing off the drug-resistant bacillus serving as template molecules on the precursor, and performing silanization treatment on the surface of the precursor to enhance the hydrophilicity of the membrane; introducing a microporous structure on the blotting membrane to obtain the blotting membrane material for selectively separating the drug-resistant bacillus. On the basis of ensuring the identification capability of the traditional bacterial imprinting material, the invention can avoid the interference of non-drug-resistant bacteria to promote the drug-resistant bacteria to generate selective movement and identification so as to achieve the aim of selectively separating bacteria.

Description

Imprinted thin film material for selectively separating drug-resistant bacillus and preparation and application thereof
Technical Field
The invention relates to the field of new materials, in particular to a blotting membrane material for selectively separating drug-resistant bacilli and preparation and application thereof, and especially relates to preparation and application of a blotting membrane material for selectively separating drug-resistant bacilli based on bacterial chemotaxis.
Background
In recent years, pathogens have gradually developed drug resistance to antibacterial drugs, and the emergence of multi-drug resistant bacteria in particular has brought serious challenges to clinical anti-infective therapy and prevention and control of nosocomial infections. Therefore, it is important to develop more accurate drug-resistant bacteria detection technology and drug-resistant bacteria sterilization technology closely related to food safety, disease prevention and clinical diagnosis and treatment. In the development of accurate drug-resistant bacteria detection and sterilization techniques, highly selective isolation of drug-resistant bacteria is a prerequisite and key step for successful detection and sterilization (Mandal P., Biswas A., Choi K.et., Methods for rapid detection of Food pathogens: an overview. American Journal of Food Technology,2011,6, 87-102). However, as a microorganism having a complicated structure, some biological properties of the drug-resistant bacteria themselves may affect the isolation of the drug-resistant bacteria. For example, the same type of bacteria do not differ in shape, diameter, size, etc., thereby reducing the effectiveness of separation techniques that rely on the shape and size of the bacteria. Meanwhile, the cell walls of different types of bacteria contain peptidoglycan layers with similar structures, and the similar chemical structures increase the difficulty of realizing the drug-resistant bacteria separation technology by relying on the chemical structures of the bacterial cell walls. In addition, during the proliferation and metabolism, the same species of bacteria produce individual variability under the combined action of different internal and external causes (J Davison, Genetic exchange between bacteria in the environment. plasmid,1999,42,73-91), which also increases the difficulty of identifying resistant bacteria. Therefore, the selective separation of the drug-resistant bacteria has important significance in the field of microbial analysis and detection.
At present, the bacteria separation method mainly comprises a traditional culture method, a physical separation method and a biological affinity method. The conventional culture method requires a long time for isolating bacteria and is applicable to only a few bacteria capable of growing in a specific medium. The physical separation method has insufficient selectivity, cannot completely separate other bacteria with the size similar to that of a target bacterium, is difficult to separate bacteria with similar structures, and has a complicated subsequent washing treatment process. The bioaffinity method is not universal, and the separation of bacteria with similar structures (only different intracellular material compositions and contents) is difficult to realize. Molecular imprinting technology has been widely used in the field of bacterial isolation as an emerging molecular recognition technology capable of specifically recognizing a target substance. Researchers have implemented the identification of whole drug-resistant bacteria using a polymer material prepared by surface single-layer imprinting, bacteria-mediated photolithography and other techniques (n.idil, b.material.imprinting of microorganisms for biosensor applications.sensors,2017,17, 708). The sol-gel method, which is an important method for preparing a microbial blot, directly embeds template bacteria in a molecularly imprinted polymer, hinders elution and recognition of the template bacteria, and makes it difficult to selectively separate homologous several bacteria (which only have differences in intracellular material composition and content) (t.cohen, j.starovetsky, u.cheruti, r.armon.white cell immunization in sol-gel thin ms filtration for bacterial retrieval in liquids: Macromolecular finger printing. int.j.mol.sci.,2010,11, 1236-1252). Therefore, it is important to develop a novel material for highly selective isolation of drug-resistant bacteria in response to the need for fine isolation in microbiological assay.
Disclosure of Invention
The invention solves the problems that the selectivity of separating drug-resistant bacteria is insufficient, the elution and identification of template bacteria are difficult, the existing bacteria separation method cannot effectively and finely separate the drug-resistant bacteria and cannot meet the increasingly fine requirements of the existing microorganism inspection analysis and the like, and provides an imprinted film material for selectively separating the drug-resistant bacteria.
According to a first aspect of the present invention, there is provided a method for preparing an imprinted membrane material for selectively isolating drug-resistant bacilli, comprising the steps of:
(1) dissolving a siloxane main agent and a cross-linking agent in a volatile alkane solvent, coating the volatile alkane solvent on a substrate, and heating for semi-curing to obtain a semi-cured film;
(2) taking a drug-resistant bacillus suspension, dropwise adding the drug-resistant bacillus suspension on a sterilized substrate, standing to enable the drug-resistant bacillus to settle, and then removing a solvent in an aseptic environment;
(3) pressing the substrate loaded with the drug-resistant bacillus obtained in the step (2) into the semi-solidified film obtained in the step (1) and keeping for a period of time, wherein the drug-resistant bacillus keeps a complete shape in the process, and groups in the semi-solidified film interact with the surface of the drug-resistant bacillus; then heating and curing to completely cure the semi-cured film;
(4) stripping the blotting membrane obtained in the step (3) from the substrate, washing off the drug-resistant bacillus serving as a template molecule on the blotting membrane, and forming blotting holes matched with the size and the structure of the drug-resistant bacillus; then placing methyl trichlorosilane or hexamethyldisilazane and the blotting membrane into a closed container together, and combining volatilized methyl trichlorosilane or hexamethyldisilazane with the surface of the blotting membrane for silanization treatment so as to enhance the hydrophilicity of the blotting membrane; introducing a microporous structure on the blotting membrane, wherein the microporous structure is used for enabling an attractant and a repellent to permeate from one side of the blotting membrane to the other side of the blotting membrane, and thus obtaining the blotting membrane material for selectively separating the drug-resistant bacilli.
Preferably, in step (1), the siloxane-based main agent is polydimethylsiloxane, tetraethoxysilane or diethyltriethoxysilane, and the volatile alkane solvent is cyclohexane or n-heptane.
Preferably, the semi-curing in the step (1) is carried out for 3-5 min at the temperature of 60-90 ℃; the standing temperature in the step (2) is 3-6 ℃ to reduce the swimming activity of bacteria, so that the bacterial sedimentation is promoted; after the substrate loaded with the drug-resistant bacillus in the step (3) is pressed into a semi-solidified film, keeping the substrate at the temperature of 36.5-38 ℃ for 6-8 h; in the step (3), the curing is carried out for 0.5-2 hours at the temperature of 70-90 ℃.
Preferably, the drug-resistant bacillus in the step (2) is in a logarithmic growth phase, the diluted drug-resistant bacillus is dripped on a sterilized substrate to be kept still, and the optical density value of the diluted drug-resistant bacillus suspension is 0.06-0.08.
Preferably, the drug-resistant bacillus is drug-resistant escherichia coli, drug-resistant pneumobacillus or drug-resistant bacillus.
According to another aspect of the invention, the imprinted membrane material for selectively separating the drug-resistant bacilli prepared by any one of the methods is provided, imprinted holes matched with the drug-resistant bacilli in size and structure are distributed on the surface of the imprinted membrane material, and microporous structures are distributed on the surface of the imprinted membrane material.
According to another aspect of the invention, the imprinted membrane material for selectively separating drug-resistant bacilli is provided for separating drug-resistant bacilli.
Preferably, the application comprises the steps of:
(1) adding a mixed solution of drug-resistant bacillus and non-drug-resistant bacillus into a container, fully filling the container to the opening of the container, and then placing an imprinting film material on the top of the container and contacting with a bacterial solution;
(2) dripping antibiotics which are tolerated by the drug-resistant bacillus and nutrient substances for bacillus growth to the surface of the blotting membrane, and permeating the nutrient substances into the bacteria liquid through a microporous structure on the blotting membrane; the antibiotic which is tolerated by the drug-resistant bacillus serves as a repellent of non-drug-resistant bacillus, nutrient substances for growing the bacillus serve as an attractant of the drug-resistant bacillus, and the repellent and the attractant jointly act to promote the drug-resistant escherichia coli to selectively move and recognize until the drug-resistant bacillus reaches an imprinting hole of an imprinting film, so that the separation of the drug-resistant bacillus is realized.
Preferably, the screening method of the non-drug-resistant bacillus repellent comprises the following steps:
(1) arranging a plurality of repellent groups and a plurality of control groups; each group of repellent groups is prepared by taking suspensions of drug-resistant bacillus and non-drug-resistant bacillus respectively in a buffer solution, adding the same antibiotic, wherein the antibiotics added in each group of repellent groups are different, and then respectively inserting capillaries sucked into the buffer solution into the suspensions added with the antibiotics; the control group corresponding to each repellent group is that the suspension of drug-resistant bacillus and non-drug-resistant bacillus is taken and respectively put in the buffer solution, the buffer solution with the same volume as the antibiotic is added, and then the capillary which is absorbed in the buffer solution is respectively inserted into the suspension solution which is added with the buffer solution;
(2) culturing the bacteria in the repellent group and the control group in the step (1) for a period of time, taking out the capillary, washing the outer wall of the capillary by using buffer solution, transferring suspension in the capillary into a test tube, diluting, taking the suspension with the same volume for plate culture, counting the number of bacterial colonies and calculating a chemotaxis ratio, wherein the calculation formula is as follows: (the number of bacteria in the repellent group-the number of bacteria in the control group)/the number of bacteria in the control group, and selecting the antibiotics in the repellent group with the chemotaxis ratio of the non-drug-resistant bacillus more than 0.5 and the chemotaxis ratio of the drug-resistant bacillus less than 0.5 as the repellent of the non-drug-resistant bacillus.
Preferably, the screening method of the drug-resistant bacillus attractant comprises the following steps:
(1) setting a plurality of groups of attractant groups and a plurality of groups of contrast groups; each group of attractant group is prepared by respectively placing suspension of drug-resistant bacillus and non-drug-resistant bacillus in a buffer solution, and respectively inserting capillaries for sucking the same nutrient substances for bacillus growth into the suspension of the same group of attractant group, wherein the nutrient substances sucked into the capillaries inserted into the suspension of each group of attractant group are different; the control group corresponding to each attractant group is prepared by respectively placing suspension of drug-resistant bacillus and non-drug-resistant bacillus in buffer solution, and then inserting into capillary tube which sucks in buffer solution with the same volume as nutrient substances;
(2) culturing the bacteria in the attractant group and the control group in the step (1) for a period of time, taking out the capillary, cleaning the outer wall of the capillary by using a buffer solution, transferring the suspension in the capillary into a test tube, diluting, taking the suspension with the same volume for plate culture, counting the number of bacterial colonies and calculating a chemotaxis ratio, wherein the calculation formula is as follows: (the number of bacteria in the attractant group-the number of bacteria in the control group)/the number of bacteria in the control group, and selecting nutrient substances added into the attractant group with the difference between the chemotaxis ratio of the drug-resistant bacillus and the chemotaxis ratio of the non-drug-resistant bacillus larger than 0.5 as the attractant of the drug-resistant bacillus.
Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) the bacterial imprinting film material provided by the invention can specifically identify drug-resistant bacilli, particularly can further effectively and selectively identify drug-resistant bacilli under the action of the drug-resistant bacilli attractant and the non-drug-resistant bacteria repellent, effectively avoids the interference of the non-drug-resistant bacilli through the release of the non-drug-resistant bacteria repellent, promotes the selective movement and active identification of the drug-resistant bacilli through the release of the drug-resistant bacilli attractant, and thus achieves the purpose of effectively and finely separating the drug-resistant bacilli.
(2) Compared with the existing bacterial imprinted polymer material, the invention utilizes the recognition capability of bacterial organisms, introduces bacterial chemotaxis into the molecular imprinting technology, designs the bacterial imprinted membrane material capable of passively and actively recognizing drug-resistant bacteria simultaneously, and realizes high-selectivity separation of the drug-resistant bacteria.
(3) Compared with the existing bacterial imprinting material prepared by adopting a surface (interface) imprinting technology, the invention applies the porous structure to the loading and slow release of the attractant and the repellent in the bacterial imprinting material, so that the imprinted polymer material generates chemotaxis to drug-resistant bacteria. In the identification process of the drug-resistant bacteria, the attractant and the repellent are slowly released to form a concentration gradient, so that the interference of non-drug-resistant bacteria can be avoided on the basis of ensuring the identification capability of the traditional bacterial imprinting material, and the drug-resistant bacteria can be promoted to selectively move and be identified so as to achieve the aim of selectively separating the bacteria. The method combines the traditional bacteria passive identification with the active identification induced by the chemotaxis of the drug-resistant bacteria, provides a new method for separating the drug-resistant bacteria by using the imprinted film material, and improves the selectivity of the drug-resistant bacteria.
(4) The invention adopts active recognition induced by bacterial chemotaxis to enhance the separation selectivity of the bacterial imprinting film material to drug-resistant bacteria, and can be applied to the separation of homologous bacteria (such as non-drug-resistant bacteria and drug-resistant bacteria) and other bacteria with similar structures. The separation method for selectively separating homologous bacteria solves the problem to be solved in the field of microbial analysis and detection, and has important biological and clinical application prospects.
Drawings
FIG. 1 is a schematic diagram of the synthesis of a blotting membrane material for selectively separating drug-resistant Escherichia coli according to the present invention.
FIG. 2 is the process of selective adsorption of drug-resistant Escherichia coli by the bacterial blotting membrane material.
FIG. 3 is an electron micrograph of a blotting membrane material for selectively isolating drug-resistant E.coli.
FIG. 4 is an electron micrograph of a non-imprinted thin film material.
FIG. 5 is the effect of porous structure on the adsorption effect of bacterial imprinted membrane material.
FIG. 6 shows the effect of the bacterial blotting membrane material adsorbing drug-resistant E.coli with the help of attractant in a single bacterial liquid.
FIG. 7 shows the effect of the bacterial blotting membrane on the adsorption of drug-resistant E.coli and non-drug-resistant E.coli in the mixed bacterial solution.
FIG. 8 is a screen of drug-resistant E.coli attractants.
FIG. 9 is a screen of non-drug resistant E.coli repellents.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention relates to a preparation method of a blotting membrane material for selectively separating drug-resistant escherichia coli, which comprises the following steps:
the method comprises the following steps: dissolving Polydimethylsiloxane (PDMS) and a cross-linking agent in a proper amount of cyclohexane, coating the solution on a glass plate, and micro-curing at 60-90 ℃ for 3-5 min, wherein the volume ratio of the PDMS to the cross-linking agent is 1: 0.08-0.15, and the volume of the cyclohexane is 1-2 mL. Simultaneously taking a proper amount of drug-resistant escherichia coli in logarithmic growth phase, and diluting to a proper concentration (OD)6000.06-0.08), dripping 1-3 mL of the bacterial suspension on a sterilized glass plate, standing for 2-4 h at 3-6 ℃ to allow bacteria to settle, and removing excessive solvent in a sterile ventilation environment. And pressing the glass plate loaded with the template bacteria into the surface of the micro-cured glass plate loaded with PDMS and a cross-linking agent, keeping the temperature at 37.5 ℃ for 6-8 h, and further curing the glass plate at 70-90 ℃ for 0.5-2 h.
Step two: the blotting membrane was peeled off the glass plate to obtain a bacterial blotting membrane precursor. The precursor is cleaned by deionized water under an ultrasonic condition to remove template bacteria, and then the surface of the precursor is silanized by using a proper amount of methyltrichlorosilane solution under a proper condition to increase the hydrophilicity, wherein the concentration of the methyltrichlorosilane solution is 0.05-0.15%, the volume of the methyltrichlorosilane solution is 5-10 mL, and the reaction condition is 4-6 hours at 50-70 ℃. And further ultrasonically cleaning the precursor by using deionized water for 1-5 min. Dividing the circular thin sheet into circular thin sheets (the diameter is 1-2 cm) with uniform diameters, introducing a porous structure on the surface of each circular thin sheet by using nano-micro needles, wherein each sheet has 150-250 holes, and loading corresponding bacterial attractant or repellent into the micropores to obtain the bacterial imprinting thin film material.
The invention also provides a process method for selectively separating the drug-resistant escherichia coli by selectively separating the imprinted membrane material of the drug-resistant escherichia coli, which comprises the following specific steps:
and (3) taking the bacterial imprinting film material with the same size, and irradiating the bacterial imprinting film material under an ultraviolet lamp for 30-60 min for disinfection. Drug-resistant e.coli was diluted to OD with PBS buffer (pH 7.4)6000.06-0.08. The bacterial suspension is added into an EP tube, and the volume of each tube is 2-3 mL (the EP tube is filled with the suspension). Then placing the sterilized bacterial blotting membrane on the top of an EP (EP) tube and contacting with bacterial liquid, and finally, putting 200-400 mu L of bacterial blotting membrane with the concentration of 10-4～10-6mol L-1The drug-resistant bacteria attractant/non-drug-resistant bacteria repellent solution is dripped on the surface of the blotting membrane and cultured for 1-4 h at 37.5 ℃. And taking out the bacterial blotting membrane material, eluting bacteria adsorbed on the bacterial blotting membrane material by using PBS (phosphate buffer solution) with the pH of 7.4, diluting by 80-200 times, coating a flat plate, culturing at 37.5 ℃ for 15-24 hours, and counting colonies.
Fig. 1 is a synthetic schematic diagram of the blotting membrane material for selectively separating drug-resistant escherichia coli of the present invention.
Example 1
2mL of Polydimethylsiloxane (PDMS) and 0.2mL of crosslinker were dissolved in 1mL of cyclohexane and applied all uniformly to a glass plate at 80 deg.CSlightly curing for 4min under the condition for later use. Simultaneously taking a proper amount of drug-resistant escherichia coli in logarithmic growth phase, washing the drug-resistant escherichia coli by using PBS (phosphate buffer solution) with pH 7.4, and diluting the drug-resistant escherichia coli to OD6000.07. 2mL of the above bacterial suspension was dropped onto a sterilized glass plate, allowed to stand at 4 ℃ for 2h to allow the bacteria to settle, and then excess solvent was removed in a sterile ventilated environment. The bacteria-coated sterile glass plate was pressed into the micro-cured PDMS surface and held at 37.5 ℃ for 7 hours, and further curing was continued at 80 ℃ for 1 hour. The blotting membrane was peeled off the glass plate, and the template bacteria were then washed away with deionized water under ultrasonic conditions. Placing the blotting membrane and 10mL of 0.1% methyltrichlorosilane solution in a closed space together, keeping the closed space at 60 ℃ for 4 hours to finish silanization of the surface of the blotting membrane, further performing ultrasonic cleaning for 1min by using deionized water, and airing. The blotting membrane was then divided into 2cm areas2The round thin sheet is punched on the surface of the cleaned round thin sheet by using nano-micro-needles (36 holes), each sheet of the membrane has 180 holes, and the micro-holes are used for permeating corresponding bacteria attractant or repellent.
As can be seen from the electron microscope images, the imprinted pores in the shape of drug-resistant Escherichia coli exist on the surface of the bacterial imprinted membrane material (see FIG. 3), while the imprinted membrane material does not have the imprinted pores (see FIG. 4). Further discussing the influence of the porous structure prepared by the nano-microneedle on the surface of the material on the adsorption effect of the drug-resistant escherichia coli, as shown in fig. 5, the number of the drug-resistant bacteria adsorbed by the bacterial imprinting film without the porous structure is 58590 CFU/sheet, and the number of the drug-resistant bacteria adsorbed by the bacterial imprinting film with the porous structure is 64410 CFU/sheet. Multiple experiments and statistical analysis show that the porous structure does not influence the adsorption effect of the bacterial imprinting film material on the drug-resistant bacteria.
Example 2
2mL of Polydimethylsiloxane (PDMS) and 0.16mL of cross-linking agent are dissolved in 2mL of cyclohexane, and the solution is uniformly coated on a glass plate and is subjected to micro-curing at 90 ℃ for 3min for later use. Simultaneously taking a proper amount of drug-resistant escherichia coli in logarithmic growth phase, washing the drug-resistant escherichia coli by using PBS (phosphate buffer solution) with pH 7.4, and diluting the drug-resistant escherichia coli to OD6000.08. 1mL of the above bacterial suspension was dropped on a sterilized glass plate and allowed to stand at 3 deg.CThe bacteria were allowed to settle for 3h and excess solvent was removed in a sterile ventilated environment. The bacteria coated sterile glass plate was pressed into the micro-cured PDMS surface and held at 37.5 ℃ for 6h, and further curing was continued at 70 ℃ for 2 h. The blotting membrane was peeled off the glass plate, and the template bacteria were then washed away with deionized water under ultrasonic conditions. Placing the blotting membrane and 5mL of 0.15% methyltrichlorosilane solution in a closed space together, keeping the closed space at 50 ℃ for 6 hours to finish silanization of the surface of the blotting membrane, further performing ultrasonic cleaning for 5min by using deionized water, and airing. The blotting membrane was then divided into 2cm areas2The round thin sheet is punched on the surface of the cleaned round thin sheet by using nano-micro-needles (36 holes), 150 holes are formed in each film, and the micropores are used for permeating corresponding bacteria attractant or repellent.
Example 3
2mL of Polydimethylsiloxane (PDMS) and 0.3mL of a crosslinking agent were dissolved in 1.5mL of cyclohexane, and the solution was uniformly applied to a glass plate and allowed to micro-cure at 60 ℃ for 5min for future use. Simultaneously taking a proper amount of drug-resistant escherichia coli in logarithmic growth phase, washing the drug-resistant escherichia coli by using PBS (phosphate buffer solution) with pH 7.4, and diluting the drug-resistant escherichia coli to OD6000.06. 3mL of the above bacterial suspension was dropped onto a sterilized glass plate, allowed to stand at 6 ℃ for 4h to allow the bacteria to settle, and then excess solvent was removed in a sterile ventilated environment. The bacteria coated sterile glass plate was pressed into the micro-cured PDMS surface and held at 37.5 ℃ for 8h, and further curing was continued at 90 ℃ for 0.5 h. The blotting membrane was peeled off the glass plate, and the template bacteria were then washed away with deionized water under ultrasonic conditions. Placing the blotting membrane and 9mL of 0.05% methyl trichlorosilane solution in a closed space together, keeping the closed space at 70 ℃ for 4.5 hours to finish silanization of the surface of the blotting membrane, further ultrasonically cleaning the blotting membrane for 2min by using deionized water, and airing the blotting membrane. The blotting membrane was then divided into 2cm areas2The round thin sheet is punched on the surface of the cleaned round thin sheet by using nano-micro-needles (36 holes), each piece of membrane has 250 holes, and the micro-holes are used for permeating corresponding bacteria attractant or repellent.
Example 4
2mL of tetraethoxy siliconAlkane and 0.2mL of cross-linking agent were dissolved in 2mL of n-heptane, and the whole was uniformly applied to a glass plate and allowed to micro-cure at 80 ℃ for 4min before use. Simultaneously taking a proper amount of drug-resistant escherichia coli in logarithmic growth phase, washing the drug-resistant escherichia coli by using PBS (phosphate buffer solution) with pH 7.4, and diluting the drug-resistant escherichia coli to OD6000.07. 2mL of the above bacterial suspension was dropped onto a sterilized glass plate, allowed to stand at 5 ℃ for 3 hours to allow the bacteria to settle, and then excess solvent was removed in a sterile ventilated environment. The bacteria-coated sterile glass plate was pressed into the surface of the micro-cured tetraethoxysilane and held at 36.5 ℃ for 8 hours, and further curing was continued at 70 ℃ for 0.5 hour. The blotting membrane was peeled off the glass plate, and the template bacteria were then washed away with deionized water under ultrasonic conditions. Placing the blotting membrane and 5mL of 0.10% hexamethyldisilazane solution in a closed space, keeping the closed space at 60 ℃ for 5 hours to complete silanization of the surface of the blotting membrane, further performing ultrasonic cleaning for 3min by using deionized water, and drying in the air. The blotting membrane was then divided into 2cm areas2The round thin sheet is punched on the surface of the cleaned round thin sheet by using nano-micro-needles (36 holes), 200 holes are formed in each film, and the micropores are used for permeating corresponding bacteria attractant or repellent.
Example 5
2mL of diethyldiethoxysilane and 0.3mL of crosslinker were dissolved in 2mL of n-heptane, applied all uniformly to the glass plate, and allowed to micro-cure at 60 ℃ for 5min for backup. Simultaneously taking a proper amount of drug-resistant escherichia coli in logarithmic growth phase, washing the drug-resistant escherichia coli by using PBS (phosphate buffer solution) with pH 7.4, and diluting the drug-resistant escherichia coli to OD6000.08. 2mL of the above bacterial suspension was dropped onto a sterilized glass plate, allowed to stand at 4 ℃ for 2h to allow the bacteria to settle, and then excess solvent was removed in a sterile ventilated environment. The bacteria-coated sterile glass plate was pressed into the surface of the above-mentioned micro-cured tetraethoxysilane, kept at 37 ℃ for 6 hours and further cured at 90 ℃ for 1 hour. The blotting membrane was peeled off the glass plate, and the template bacteria were then washed away with deionized water under ultrasonic conditions. Placing the blotting membrane and 7mL of 0.15% hexamethyldisilazane solution in a closed space, keeping at 70 deg.C for 4h to complete silanization of the surface of the blotting membrane, and further using deionized waterUltrasonically cleaning for 5min, and air drying. The blotting membrane was then divided into 2cm areas2The round thin sheet is punched on the surface of the cleaned round thin sheet by using nano-micro-needles (36 holes), each piece of membrane has 250 holes, and the micro-holes are used for permeating corresponding bacteria attractant or repellent.
An application of imprinted membrane material for selectively separating drug-resistant Escherichia coli for separating drug-resistant Escherichia coli is shown in FIG. 2, which is a process diagram of selective adsorption of drug-resistant Escherichia coli by bacterial imprinted membrane material.
Example 6
2.3mL of OD600The drug-resistant e.coli suspension was placed in a 2mL EP tube (the EP tube was filled), and then the uv-sterilized bacterial blot membrane material was placed over the bacterial suspension, and 300 μ L of 10 concentration was added-5mol L-1The attractant solution of (a) was added over the bacterial blotting membrane and the device was incubated at 37.5 ℃ for 2 h. The bacterial blotting membrane was removed, the bacteria on the membrane surface were gently washed with PBS (pH 7.4), and then placed in a 3.5cm petri dish, 1mL of PBS (pH 7.4) was added thereto and blown, and the blown suspension was diluted 100-fold, and then 100. mu.L of the solution was smeared and incubated at 37.5 ℃ overnight, followed by colony counting. The amount of the target bacteria adsorbed by the blotting membrane material was 2.82X 10 when the attractant was not loaded (control group)4CFU/tablet, and the adsorption amount of the loaded glycine attractant to the target bacteria is increased to 8.08 multiplied by 104CFU/plate, 5.1 times that of the control (see FIG. 6).
Example 7: application of imprinted membrane material for selectively separating drug-resistant escherichia coli to separation of drug-resistant escherichia coli in mixed bacterial liquid
The bacterial blotting membrane material was placed in a mixed bacterial suspension (drug-resistant E.coli OD)6000.06 and non-drug resistant E.coli OD6000.06), and then 150 μ L was added to a concentration of 10-5mol L-1Glycine solution (attractant for drug-resistant E.coli) and 150. mu.L of 1g L-1Ampicillin solution (repellent to non-drug resistant E.coli) was added on top of the blotting membrane material and incubated at 37.5 ℃ for 2 h. The blotting membrane material was removed, the bacteria on the membrane surface were gently washed with PBS (pH 7.4), and then placed in a 3.5cm dish,after adding 1mL of PBS (pH 7.4) and pipetting, the pipetted suspension was diluted 100-fold, 100 μ L of the pipetted suspension was smeared on a plate and cultured overnight at 37.5 ℃.
The results are shown in fig. 7, when no attractant and repellent were used, the adsorption capacity of the bacterial blotting membrane material to the drug-resistant escherichia coli (control group) was 7950 CFU/sheet, and when attractant glycine was used, the adsorption capacity of the bacterial blotting membrane material to the drug-resistant escherichia coli was increased to 79666 CFU/sheet, which is about 4.6 times that of the non-blotting membrane material; when the repellent ampicillin is used, the adsorption of drug-resistant bacteria and drug-resistant escherichia coli by the bacterial imprinting film material is 47333 CFU/tablet which is about 3.3 times of that of the non-imprinting film material, and the repellent has no repellent effect on the drug-resistant escherichia coli; when glycine serving as an attractant is used in combination with ampicillin serving as a repellent, the adsorption capacity of the bacterial imprinting film material on drug-resistant escherichia coli is 1.01 multiplied by 105The CFU/tablet is about 5.7 times of that of a non-imprinted membrane material, so that the influence of interference bacteria (non-drug-resistant escherichia coli) on the adsorption effect of the drug-resistant escherichia coli can be effectively reduced by using the glycine attractant and the ampicillin repellent in a combined manner. The results further show that the application of the drug-resistant bacteria attractant and the non-drug-resistant bacteria repellent can effectively promote the accurate separation of the bacterial imprinting film on the drug-resistant bacteria in the homologous bacterial mixed solution.
Example 8: screening of drug-resistant escherichia coli attractant and non-drug-resistant escherichia coli repellent
Based on nutrient substances frequently required by drug-resistant bacteria, substances such as glucose, methionine, glycine and the like are selected as drug-resistant escherichia coli attractants. The specific screening method is as follows: respectively taking OD600The drug-resistant e.coli and non-drug-resistant e.coli suspension was centrifuged at 5000rpm for 3min, then washed by centrifugation with PBS buffer pH 7.4, repeated three times, and finally suspended in 2mL PBS buffer. Then 5. mu.L of the solution is inhaled to a concentration of 10-5mol L-1The disposable microcapillary tube (with the tail end sealed) of the attractant was inserted into the bacterial suspension and incubated at 37.5 ℃ for 2 h. The capillary of the control group was replaced with equal amounts of PBS buffer for nutrients, and the rest of the treatment was the same as the attractant group. Taking out the disposable microcapillary, washing the outer wall of the microcapillary with PBS buffer solution, and mixingThe suspension in the capillary was transferred to an EP tube, diluted 1000-fold with PBS buffer, subjected to bacterial plating, counted for bacterial colony number, and calculated for chemotaxis ratio using the formula (number of bacteria in attractant group-number of bacteria in control group)/number of bacteria in control group. A greater chemotactic ratio of the attractant indicates a greater attraction to bacteria. Because the nutrient substances as the attractants have certain attraction effects on both target bacteria (drug-resistant escherichia coli) and interference bacteria (non-drug-resistant escherichia coli), the optimal attractants are screened by utilizing the difference value of the chemotaxis ratio of the target bacteria and the interference bacteria, and when the difference value of the two is more than 0.5, the attraction effect of the attractants on the target bacteria (drug-resistant escherichia coli) is considered to be obviously higher than that of the interference bacteria (non-drug-resistant escherichia coli), so that glycine with the chemotaxis ratio of 1.3 for the drug-resistant escherichia coli, the chemotaxis ratio of 0.7 for the non-drug-resistant escherichia coli and the chemotaxis ratio difference of 0.6 (more than 0.5) is selected as the attractant for the drug-resistant escherichia coli (see fig. 8).
Sulfadiazine, sodium chloride and ampicillin were also selected as non-drug resistant E.coli repellents based on substances that affect the life activities of the bacteria. The screening method of the repellent is similar to that of the attractant, and the specific screening method is as follows: respectively taking OD6000.07 of the drug-resistant E.coli and non-drug-resistant E.coli suspension at 5000rpm for 3min, then using PBS buffer solution with pH 7.4 to carry out centrifugal washing, repeating three times, finally suspending in 2mL PBS buffer solution, then 50 μ L of 10 concentration-5mol L-1The repellent of (a) is added to the bacterial suspension. A disposable microcapillary tube (with the tail sealed) imbibed with 5. mu.L of pH 7.4 PBS buffer was inserted into the bacterial suspension and incubated at 37.5 ℃ for 2 h. The control bacterial suspension was treated with the same amount of PBS buffer instead of antibiotics as the repellent group. Taking out the disposable microcapillary, washing the outer wall of the microcapillary by PBS buffer solution, transferring suspension in the microcapillary to an EP tube, diluting 1000 times by the PBS buffer solution, carrying out bacterial plate culture, counting bacterial colony number, and calculating chemotaxis ratio by using a formula (the bacterial number of a repellent group-the bacterial number of a control group)/the bacterial number of the control group. The larger chemotaxis ratio of the repellent indicates that the repellent has larger repellent effect on bacteria, and when the chemotaxis ratio is more than 0.5, the antibiotic can be considered to beThe antibiotics have obvious repellent effect on bacteria, so the optimal repellent of the bacteria is selected according to chemotactic ratio. The repellent has a repellent effect (chemotaxis ratio is more than 0.5) on non-drug-resistant escherichia coli, and has no obvious repellent effect (chemotaxis ratio is less than 0.5) on target bacteria (drug-resistant escherichia coli), so that ampicillin with the chemotaxis ratio of 4.4 and the chemotaxis ratio of 0.2 on the drug-resistant escherichia coli is selected as the non-drug-resistant escherichia coli repellent (see figure 9).
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (6)

1. A method for selectively separating drug-resistant bacillus by adopting an imprinted membrane material is characterized in that the imprinted membrane material is prepared by the following steps:
(1) dissolving a siloxane main agent and a cross-linking agent in a volatile alkane solvent, coating the volatile alkane solvent on a substrate, and heating for semi-curing to obtain a semi-cured film; the siloxane main agent is tetraethoxysilane or diethyltriethoxysilane;
(2) taking a drug-resistant bacillus suspension, dropwise adding the drug-resistant bacillus suspension on a sterilized substrate, standing to enable the drug-resistant bacillus to settle, and then removing a solvent in an aseptic environment; the drug-resistant bacillus is drug-resistant escherichia coli, drug-resistant pneumobacillus or drug-resistant bacillus;
(3) pressing the substrate loaded with the drug-resistant bacillus obtained in the step (2) into the semi-solidified film obtained in the step (1) and keeping for a period of time, wherein the drug-resistant bacillus keeps a complete shape in the process, and groups in the semi-solidified film interact with the surface of the drug-resistant bacillus; then heating and curing to completely cure the semi-cured film;
(4) stripping the blotting membrane obtained in the step (3) from the substrate, washing off the drug-resistant bacillus serving as a template molecule on the blotting membrane, and forming blotting holes matched with the size and the structure of the drug-resistant bacillus; then placing hexamethyldisilazane and the blotting membrane into a closed container together, and bonding volatilized hexamethyldisilazane on the surface of the blotting membrane for silanization treatment so as to enhance the hydrophilicity of the blotting membrane; introducing a microporous structure on the blotting membrane, wherein the microporous structure is used for enabling an attractant and a repellent to permeate from one side of the blotting membrane to the other side of the blotting membrane, and thus obtaining a blotting membrane material for selectively separating drug-resistant bacilli;
the method for selectively separating the drug-resistant bacillus specifically comprises the following steps:
s1: adding a mixed solution of drug-resistant bacillus and non-drug-resistant bacillus into a container, fully filling the container to the opening of the container, and then placing an imprinting film material on the top of the container and contacting with a bacterial solution;
s2: dripping antibiotics which are tolerated by the drug-resistant bacillus and nutrient substances for bacillus growth to the surface of the blotting membrane, and permeating the nutrient substances into the bacteria liquid through a microporous structure on the blotting membrane; the antibiotic which is tolerated by the drug-resistant bacillus serves as a repellent of non-drug-resistant bacillus, nutrient substances for growing the bacillus serve as an attractant of the drug-resistant bacillus, and the repellent and the attractant jointly act to promote the drug-resistant escherichia coli to selectively move and recognize until the drug-resistant bacillus reaches an imprinting hole of an imprinting film, so that the separation of the drug-resistant bacillus is realized.
2. The method for selectively isolating drug-resistant bacilli using blotting membrane material of claim 1, wherein the volatile alkane solvent is cyclohexane or n-heptane.
3. The method for selectively separating drug-resistant bacilli by adopting imprinted membrane materials as claimed in claim 1, wherein the semi-solidification in the step (1) is heating at 60-90 ℃ for 3-5 min; the standing temperature in the step (2) is 3-6 ℃ to reduce the swimming activity of bacteria, so that the bacterial sedimentation is promoted; after the substrate loaded with the drug-resistant bacillus in the step (3) is pressed into a semi-solidified film, keeping for 6-8 h at 36.5-38 ℃; in the step (3), the curing is carried out for 0.5-2 hours at the temperature of 70-90 ℃.
4. The method for selectively separating drug-resistant bacilli by using blotting membrane material as claimed in claim 1, wherein the drug-resistant bacilli in step (2) are in logarithmic growth phase, and are diluted and dropped on a sterilized substrate for standing, and the optical density value of the diluted drug-resistant bacilli suspension is 0.06-0.08.
5. The method for selectively separating drug-resistant bacilli by using imprinted membrane materials as claimed in claim 1, wherein the screening method of the repellent of non-drug-resistant bacilli comprises:
(1) arranging a plurality of repellent groups and a plurality of control groups; each group of repellent groups is prepared by taking suspensions of drug-resistant bacillus and non-drug-resistant bacillus respectively in a buffer solution, adding the same antibiotic, wherein the antibiotics added in each group of repellent groups are different, and then respectively inserting capillaries sucked into the buffer solution into the suspensions added with the antibiotics; the control group corresponding to each repellent group is that the suspension of drug-resistant bacillus and non-drug-resistant bacillus is taken and respectively put in the buffer solution, the buffer solution with the same volume as the antibiotic is added, and then the capillary which is absorbed in the buffer solution is respectively inserted into the suspension solution which is added with the buffer solution;
(2) culturing the bacteria in the repellent group and the control group in the step (1) for a period of time, taking out the capillary, washing the outer wall of the capillary by using buffer solution, transferring suspension in the capillary into a test tube, diluting, taking the suspension with the same volume for plate culture, counting the number of bacterial colonies and calculating a chemotaxis ratio, wherein the calculation formula is as follows: (the number of bacteria in the repellent group-the number of bacteria in the control group)/the number of bacteria in the control group, and selecting the antibiotics in the repellent group with the chemotaxis ratio of the non-drug-resistant bacillus more than 0.5 and the chemotaxis ratio of the drug-resistant bacillus less than 0.5 as the repellent of the non-drug-resistant bacillus.
6. The method for selectively separating drug-resistant bacilli by adopting imprinted membrane materials as claimed in claim 1, wherein the screening method of the attractant of the drug-resistant bacilli comprises the following steps:
(1) setting a plurality of groups of attractant groups and a plurality of groups of contrast groups; each group of attractant group is prepared by respectively placing suspension of drug-resistant bacillus and non-drug-resistant bacillus in a buffer solution, and respectively inserting capillaries for sucking the same nutrient substances for bacillus growth into the suspension of the same group of attractant group, wherein the nutrient substances sucked into the capillaries inserted into the suspension of each group of attractant group are different; the control group corresponding to each attractant group is prepared by respectively placing suspension of drug-resistant bacillus and non-drug-resistant bacillus in buffer solution, and then inserting into capillary tube which sucks in buffer solution with the same volume as nutrient substances;
(2) culturing the bacteria in the attractant group and the control group in the step (1) for a period of time, taking out the capillary, cleaning the outer wall of the capillary by using a buffer solution, transferring the suspension in the capillary into a test tube, diluting, taking the suspension with the same volume for plate culture, counting the number of bacterial colonies and calculating a chemotaxis ratio, wherein the calculation formula is as follows: (the number of bacteria in the attractant group-the number of bacteria in the control group)/the number of bacteria in the control group, and selecting nutrient substances added into the attractant group with the difference between the chemotaxis ratio of the drug-resistant bacillus and the chemotaxis ratio of the non-drug-resistant bacillus larger than 0.5 as the attractant of the drug-resistant bacillus.
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