阅读说明：本技术 一种聚合物、其制备方法及作为检测细菌及抗菌材料的应用 (Polymer, preparation method thereof and application of polymer as bacteria detection and antibacterial material ) 是由 张晓梅 李德康 荆路 于 2019-09-18 设计创作，主要内容包括：本公开属于抗菌纳米材料技术领域,具体涉及一种聚合物、其制备方法及作为检测细菌及抗菌材料的应用。抗菌纳米材料由于稳定、不易产生耐药性等优点成为抗菌领域的研究热点。本公开提供了一种基于铁卟啉的多孔有机金属聚合物,以吡咯和化合物BFPB为原料,通过水热法合成,制备方法简单。该聚合物具有良好的化学和热稳定性,其3D互连多孔结构提供了丰富的催化活性位点,具有良好类过氧化物酶活性。在近红外光照条件下,该聚合物对金黄色葡萄球菌具有杀灭作用,其制备的Ab<Sub>2</Sub>@Au@FePPOP<Sub>BFPB</Sub>应用于金黄色葡萄球菌的检测具有良好的特异性和灵敏度。(The disclosure belongs to the technical field of antibacterial nano materials, and particularly relates to a polymer, a preparation method thereof and application of the polymer as a bacteria detection and antibacterial material. The antibacterial nano material becomes a research hotspot in the antibacterial field due to the advantages of stability, difficult generation of drug resistance and the like. The disclosure provides a porous organic metal polymer based on ferriporphyrin, pyrrole and a compound BFPB are used as raw materials, and the porous organic metal polymer is synthesized by a hydrothermal method, so that the preparation method is simple. The polymer has good chemical and thermal stability, and the 3D interconnected porous structure of the polymer provides rich catalytic active sites and has good peroxidase-like activity. Under the condition of near-infrared illumination, the polymer has a killing effect on staphylococcus aureus, and Ab prepared from the polymer 2 @Au@FePPOP BFPB The kit has good specificity and sensitivity when being applied to the detection of staphylococcus aureus.)

1. Polymer FePPOP_BFPBCharacterised in that the polymer FePPOP_BFPBHas a repeating structural unit shown as the following formula I,

2. the polymer FePPOP as claimed in claim 1_BFPBCharacterized in that the polymer FePPOP_BFPBPyrrole and a compound BFPB as raw materials are synthesized by a hydrothermal method, the structure of the compound BFPB is shown as the following formula II,

3. the polymer FePPOP as claimed in claim 2_BFPBThe preparation method is characterized by comprising the following specific steps: redistilled pyrrole, BFPB and FeCl₃·6H₂Dispersing O in an organic solvent, stirring under the atmosphere of inert gas, transferring to a high-pressure reaction kettle, heating and reacting to obtain a precipitate, namely the polymer FePPOP_BFPB；

Preferably, the redistilled pyrrole, BFPB and FeCl₃·6H₂The molar ratio of O is 10-14: 16-20: 7.5-8.5;

preferably, the organic solvent is propionic acid;

preferably, the stirring temperature is 22-27 ℃, or the stirring time is 3.5-4.5 hours;

preferably, the heating reaction temperature is 170-190 ℃, or the heating time is 70-80 hours;

preferably, the preparation method further comprises the processes of washing and drying the precipitate.

4. Complex Au @ FePPOP_BFPBCharacterized in thatThe complex is formed by gold nanoparticles and FePPOP as claimed in claim 1_BFPBAnd compounding.

5. The complex Au @ FePPOP as set forth in claim 4_BFPBThe method for preparing the FePPOP-containing composite, wherein the FePPOP-containing composite is prepared by the method of claim 1_BFPBWith HAuCl₄Is prepared by a light deposition method; preferably, the preparation method comprises the following specific steps:

FePPOP (FePPOP)_BFPBMixing with alkali solution and ethanol to obtain solution, and slowly adding HAuCl into the solution₄Obtaining a mixture, irradiating the mixture by using a xenon lamp, and stirring until the solution becomes reddish brown, wherein the precipitate is Au @ FePPOP_BFPBThe composite.

6. Ab₂@[email protected]_BFPBA conjugate, wherein Ab is a rabbit anti-staphylococcus aureus chemotaxis antibody.

7. Ab according to claim 6₂@[email protected]_BFPBA method for preparing a conjugate by placing the complex of claim 4 in a container containing Ab₂Is obtained by ultrasonic treatment in the solution; preferably, the Ab is contained₂The solution of (A) contains Ab₂In PBS.

8. The polymer FePPOP as claimed in claim 1_BFPBApplication as a peroxide mimic enzyme.

9. The polymer FePPOP as claimed in claim 1_BFPBThe application in preparing a bacteria detection biosensor.

10. A sterilization method, characterized in that it comprises the steps of: adding a sample to be treated containing the FePPOP of claim 1_BFPBAnd irradiating the sample to be treated with near infrared light.

Technical Field

The disclosure belongs to the technical field of porous organic polymers, and particularly relates to a porous organic polymer based on ferriporphyrin, a preparation method of the polymer, and application of the polymer as a material for detecting bacteria and resisting bacteria.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Bacterial infections are one of the major threats to human health and are a major cause of death in the population of many developing and less developed countries. The development of the nano material technology provides an opportunity for developing novel antibacterial drugs, and compared with the traditional antibiotics, the nano material has a multi-mode antibacterial effect and is not easy to cause bacterial drug resistance. Among them, nanoenzymes (enzyme mimics based on nanomaterials) are a current research hotspot, and they draw inspiration from the natural self-defense system, namely H₂O₂Converted into Reactive Oxygen Species (ROS) in the organism to inhibit bacterial viability. For example, the dawn research group reported that graphene quantum dots are capable of converting H to H₂O₂Catalyzing into hydroxyl free radical (OH) with higher antibacterial activity, and can be used for wound disinfection. High profit, etc. to discover Fe₃O₄The nanoparticles have excellent antibacterial activity, and can eliminate biofilms by in-situ generated free radicals. However, further applications of nanoenzymes are still limited because of their poor catalytic activity and low antibacterial efficiency in physiological environments (usually at neutral pH) relative to the native enzyme. The problem of low efficiency of the nano enzyme can be overcome by establishing a composite antibacterial system to achieve positive synergistic effect. For example, the Zhaoyangliang topic group established a synergistic antimicrobial system combining peroxidase-like catalytic activity with PEG-MoS₂NFs, OH generated by the system firstly destroys the integrity of microbial membranes, and then the subsequent thermotherapy caused by 808nm laser irradiation further accelerates the oxidation of Glutathione (GSH) to cause more efficient bacterial death. In addition, Au/g-C₃N₄、 MoS₂Composites of @ PDA-Ag, nFeS, etc. also have similar positive synergistic effects for combating bacteria.

On the other hand, detection of bacteria is currently carried out by methods such as colony counting, Polymerase Chain Reaction (PCR), and Flow Cytometry (FCM). However, these methods require intensive operations, require long incubation times, and are highly demanding for the skilled worker to operate. Other methods, e.g. surface enhanced Raman scattering, fluorescence resonance energy transferElectrochemiluminescence, immunological assays, have been developed for detecting pathogenic bacteria. Among them, sandwich enzyme-linked immunosorbent assay (ELISA) has wide applications due to its low cost, high selectivity, simplicity and rapidity. Since different kinds of enzymes are used as catalysts in ELISA, such as horseradish peroxidase. However, in general, natural enzymes have low physical/chemical stability, high cost, and difficulty in recovery and recycling, and nanoenzymes having adjustable catalytic activity and high stability are gradually becoming an alternative product to natural enzymes. Recently, some nanomaterials (Fe) have been used in colorimetric biosensors for detecting pathogenic bacteria₃O₄Nanoparticle clusters and Cu-MOF nanoparticles) were reported as promising alternatives to natural enzymes.

In recent years, porphyrin-based porous organic polymers (PPOPs) constructed from porphyrins or metalloporphyrins through strong covalent bonds have received considerable attention, and their high activity and their close biological relevance to natural enzymes have determined their usefulness as good photocatalysts, electrocatalysts and biomimetic catalysts. The inherent advantages of Porous Organic Polymers (POPs) are, for example, large surface area, tunable pore structure, functionalizability, and high thermal and chemical stability. Due to proper exposure of modifiable catalytic active sites and a pre-enrichment effect, the porphyrin is integrated into the porous skeleton, so that the catalytic activity is further improved as a biomimetic catalyst, and compared with natural Horse Radish Peroxidase (HRP), PPAP has excellent peroxidase-like activity. PPOP also shows a significantly narrow band gap and strong optical absorption within the desired therapeutic window (700-1000nm) due to extended π - π conjugation on the framework. These characteristics make them a good choice for constructing new antibacterial and antimicrobial materials.

Disclosure of Invention

However, against the above research background, the present disclosure reports a bifunctional porphyrin-based porous organic polymer, FePPOP_BFPBFor bacterial detection and elimination. By reaction of pyrrole with 4-2, 2-bis [ (4-formylphenoxy) methyl]FePPOP synthesis by aromatic substitution reaction between-3- (4-formylphenoxy) propoxy } benzaldehyde (BFPB)_BFPB. C-centered tetrahedral structure of BFPBHas a 3D interconnected porous structure, high chemical and thermal stability, high density of catalytically active sites and outstanding Near Infrared (NIR) absorption properties. FePPOP benefiting from these characteristics_BFPBThe method has the advantages of being excellent in double application in quantitative, selective and rapid detection of staphylococcus aureus (S. aureus) and effective inhibition thereof.

In a first aspect of the disclosure, a polymer FePPOP is provided_BFPBThe polymer FePPOP_BFPBHas a repeating structural unit shown as the following formula I,

the compound has a tetrahedral structure with C as a center, and has good chemical and thermal stability. And the structure enables the nano material to have a 3D interconnected porous structure, and provides high density of catalytic active sites and prominent NIR absorption sites. Through thermogravimetric analysis, the polymer has the mass loss of less than 10 percent when being heated to 300 ℃, and has excellent thermal stability. The polymer is proved to be capable of providing abundant reaction sites as a mimic enzyme, is expected to have good reaction efficiency, and can be suitable for the application of bacteria detection and bacteria resistance under severe conditions.

In a second aspect of the present disclosure, the polymer FePPOP of the first aspect is provided_BFPBThe polymer FePPOP_BFPBPyrrole and a compound BFPB as raw materials are synthesized by a hydrothermal method, the structure of the compound BFPB is shown as the following formula II,

preferably, the preparation method comprises the following specific steps: redistilled pyrrole, BFPB and FeCl₃·6H₂Dispersing O in an organic solvent, stirring under the atmosphere of inert gas, transferring to a high-pressure reaction kettle, heating and reacting to obtain a precipitate, namely the polymer FePPOP_BFPB。

Further preferably, said redistilled pyrrole, BFPB and FeCl₃·6H₂Molar ratio of O10 to 14:16 to 20:7.5 to 8.5.

Further preferably, the organic solvent is propionic acid.

Further preferably, the stirring temperature is 22-27 ℃, or the stirring time is 3.5-4.5 hours.

Further preferably, the heating reaction temperature is 170-190 ℃, or the heating time is 70-80 hours.

Further preferably, the preparation method further comprises the steps of washing and drying the precipitate.

In a third aspect of the disclosure, a complex Au @ FePPOP is provided_BFPBThe complex is formed by gold nanoparticles and the FePPOP of the first aspect_BFPBAnd compounding.

Preferably, the FePPOP_BFPBThe surface is loaded with gold nanoparticles.

In a fourth aspect of the present disclosure, there is provided the complex Au @ FePPOP of the third aspect_BFPBThe composite is the FePPOP of the first aspect_BFPBWith HAuCl₄Is prepared by a light deposition method.

Preferably, the preparation method comprises the following specific steps:

FePPOP (FePPOP)_BFPBMixing with alkali solution and ethanol to obtain solution, and slowly adding HAuCl into the solution₄Obtaining a mixture, irradiating the mixture by using a xenon lamp, and stirring until the solution becomes reddish brown, wherein the precipitate is Au @ FePPOP_BFPBThe composite.

Further preferably, the xenon lamp is 580-620W.

In a fifth aspect of the disclosure, an Ab is provided₂@[email protected]_BFPBThe conjugate, wherein Ab₂Rabbit anti-staphylococcus aureus chemotactic antibody.

The staphylococcus aureus (S.aureus) has abundant protein A on the surface, and can be specifically combined with rabbit anti-staphylococcus aureus chemotaxis antibody (bs-4582R). By using the aggregate as a sensor for biological detection of bacteria, the bs-4582R and the protein A on the surface of a target cell have strong specific binding to form a complex, so that the target bacteria can be screened and detected.

In a sixth aspect of the disclosure, there is provided Ab as described in the fifth aspect₂@[email protected]_BFPBA method for preparing a conjugate by placing the complex of the third aspect in a solution containing Ab₂Is sonicated in the solution of (1).

Preferably, the Ab is contained₂The solution of (A) contains Ab₂In PBS.

In a seventh aspect of the present disclosure, there is provided the compound of the first aspect, which is FePPOP_BFPBApplication as a peroxide mimic enzyme.

In an eighth aspect of the present disclosure, the polymer FePPOP of the first aspect is provided_BFPBThe application in preparing a bacteria detection biosensor.

In a ninth aspect of the present disclosure, there is provided a sterilization method comprising the steps of: adding the FePPOP to a sample to be treated_BFPBAnd irradiating the sample to be treated with near infrared light.

Compared with the prior art, the beneficial effect of this disclosure is:

the present disclosure provides a porphyrin-based porous organic polymer FePPOP_BFPBThe polymer has good thermal stability and wider pore size distribution; the 3D structure of the photocatalyst provides abundant catalytic sites, and the photocatalyst has good light absorption capacity and excellent photo-thermal performance under 808nm irradiation. The structural characteristics are applied to the antibacterial field, and researches show that the polymer has good stability and can obviously improve the repeated utilization rate. The polymer has excellent peroxidase-like activity, and can be used as a biosensor and a bactericide for S.aureus, and is used for quantitative detection and sterilization.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 shows FePPOP from example 1_BFPBThermogravimetric analysis of (a);

FIG. 2 shows BFPB and FePPOP in example 1_BFPBA structural characterization result graph of (1);

wherein, FIG. 2(A) is (a) BFPB and (b) FePPOP_BFPBFT-IR spectrum of (1);

FIG. 2(B) is FePPOP_BFPBIs/are as follows¹³C CP/MAS NMR spectra;

FIG. 2(C) is FePPOP_BFPBSEM image of (a);

FIG. 2(D) shows FePPOP_BFPBThe nitrogen adsorption-desorption isotherm diagram of (a);

FIG. 2(E) is FePPOP_BFPBThe aperture distribution map of (a);

FIG. 2(F) shows (a) FePPOP_BFPBUV-Vis-NIR diffuse reflectance spectra of powders and (b) iron porphyrin monomer [ Fe 5,10,15, 20-tetra- (4' -bromophenyl) porphyrin, FeTBrPP]UV-Vis absorption spectrum in chloroform.

FIG. 3 shows FePPOP from example 1_BFPBThe photo-thermal property verification result chart;

wherein, FIG. 3A shows FePPOP under 808nm laser irradiation_BFPBTemperature profile of concentration;

FIG. 3B shows different FePPOPs_BFPBExposure to 808nm laser light at concentration (1.2W/cm)²) A temperature delta map of time;

FIG. 3C shows the irradiation of FePPOP with laser_BFPBThe monitored temperature curve of natural cooling is obtained under the condition that the laser is turned off after 850 seconds, and the power density of 808nm laser is 1.2W/cm²；

FIG. 3D is a graph of the time constant (τ) for heat transfer from the system determined by applying the linear time data from the cooling period of (C) versus the negative natural logarithm of the driving force temperature_s) Is 319.67 s.

FIG. 4 shows FePPOP in example 1_BFPBResults plot of peroxidase-like catalytic activity on TMB;

wherein, FIG. 4A is the UV-Vis spectrum of the TMB reaction mixture, inset: corresponding photograph of TMB reaction mixture: (a) TMB + H₂O₂+FePPOP_BFPB，(b)FePPOP_BFPB+TMB，(c)H₂O₂+ TMB and (d) TMB;

FIG. 4(B) is TMB (0.1mM), FePPOP_BFPB(10μg mL^-1) And H₂O₂(100mM) pH-dependent activity.

FIG. 4(C) is a graph showing temperature change; the pH in experiment C was set to 3.8;

FIG. 4(D) is H₂O₂Concentration-dependent peroxidase-like activity and FePPOP_BFPB(10μg mL^-1) And TMB (0.1 mM). The temperature in experiment D was set to 25 ℃.

FIG. 5 shows FePPOP from example 1_BFPBAnd a normal ferriporphyrin monomer cyclic catalytic oxidation TMB performance diagram;

FIG. 6 shows FePPOP in example 1_BFPBPerforming a steady state dynamics determination result graph;

wherein, FIG. 6A shows TMB and 40mM H at different concentrations₂O₂Under the condition, FePPOP is subjected to Michaelis-Menten model_BFPBA graph of kinetic measurements;

FIG. 6B shows TMB and 40mM H at different concentrations₂O₂Under the condition, a Lineweaver-Burk double reciprocal model is adopted to couple FePPOP_BFPBA graph of kinetic measurements;

FIG. 6C shows 0.2mM TMB and different concentrations of H₂O₂Under the condition, FePPOP is subjected to Michaelis-Menten model_BFPBA graph of kinetic measurements;

FIG. 6D is 0.2mM TMB and different concentrations of H₂O₂Under the condition, a Lineweaver-Burk double reciprocal model is adopted to couple FePPOP_BFPBAnd (4) a kinetic measurement result graph.

FIG. 7 is Au @ FePPOP in example 1_BFPBSEM picture of (1);

FIG. 8 is Au @ FePPOP in example 1_BFPBSchematic of the antibiotic affinity strategy for determination of staphylococcus aureus;

FIG. 9 shows FePPOP in example 1_BFPBThe staphylococcus aureus measured dose-response relationship of the biosensor of (a);

wherein, FIG. 9A is based on FePPOP_BFPBThe staphylococcus aureus measured dose-response relationship of the biosensor of (a);

FIG. 9B is based on FePPOP_BFPBThe statistical result of the staphylococcus aureus assay of the biosensor of (1);

FIG. 9C shows the results of absorbance of different strains, E.coli Escherichia coli BL21(E.coli BL21), E.coli DH 5. alpha., E.coli XL1 and S.aureus at a concentration of 6.0X 10⁷CFU/mL。

FIG. 10 shows FePPOP in example 1_BFPBHistogram of cytotoxicity results on s.aureus;

FIG. 11 shows FePPOP concentrations in example 1_BFPBThe result graph of the effect of the laser irradiation at 808nm on staphylococcus aureus;

in which FIG. 11(A) was irradiated with 808nm laser light at different concentrations of FePPOP_BFPBWith or without H₂O₂(100 μ M) relative bacterial viability histogram of Staphylococcus aureus incubated together;

FIG. 11(B) is FePPOP_BFPBTime-concentration dependent antibacterial activity against staphylococcus aureus;

FIG. 11(C) is a photograph of Staphylococcus aureus in different experimental groups;

staphylococcus aureus exposure to (1) PBS formed bacterial colony photographs, (2) FePPOP_BFPB， (3)FePPOP_BFPB+NIR，(4)H₂O₂，(5)H₂O₂+FePPOP_BFPB，(6) H₂O₂+FePPOP_BFPB+ NIR. Concentration: FePPOP_BFPB500μg mL^-1，H₂O₂100 μ M, NIR Power Density 1.2W cm^-2For 20 minutes.

FIG. 12 is a photograph showing Staphylococcus aureus cultured at 37 ℃ for 12 hours in example 1 after incubation;

wherein, FIG. 12(1) is PBS at 37 ℃; FIG. 12(2) is 52.5 ℃ PBS.

FIG. 13 shows FePPOP in example 1_BFPBA bacteriostatic performance fluorescence detection result graph;

wherein, FIG. 13A shows NBT + NADH + H₂O₂+ NIR and NBT + NADH + FePPOP_BFPB+H₂O₂+ UV-Vis spectra of NIR system;

FIG. 13B is H₂O₂+ FePPOP Presence and absence in NIR System_BFPBGraph of temporal DPBF content;

FIG. 13C is a graph showing fluorescence spectra of different reaction systems;

FIG. 13D is a 425nm fluorescence spectrum histogram;

FIG. 13E shows the utilization of FePPOP_BFPB+H₂O₂Schematic diagram of process s.

FIG. 14 is SEM images of bacteria in different experimental groups of example 1;

wherein, fig. 14A is an SEM image of bacteria; FIG. 14B shows bacteria + FePPOP_BFPBThe image of (a); FIG. 14C is bacteria + FePPOP_BFPB+ NIR image; FIG. 14D is bacterium + H₂O₂SEM image of (a); FIG. 14E is bacterium + H₂O₂+FePPOP_BFPBThe image of (a); FIG. 14F is bacterium + H₂O₂+FePPOP_BFPB+ NIR image; concentration: FePPOP_BFPB500μg mL^-1，H₂O₂100 mu M; NIR Power Density 1.2W cm^-2For 20 minutes.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background art, the synergistic antibacterial system constructed by the nano antibacterial material can improve individual treatment by multi-mode antibacterial and synergistic effectLow efficacy of the treatment. The present disclosure provides an antibacterial active material FePPOP_BFPBExcellent performance in quantitative, selective and rapid detection and effective inhibition of s.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.