Quaternary ammonium salt micelle antibacterial agent as well as preparation method and application thereof

Abstract

The invention discloses a quaternary ammonium salt micelle antibacterial agent and a preparation method and application thereof, the quaternary ammonium salt micelle antibacterial agent is obtained by doping and introducing metal ions into spherical quaternary ammonium salt micelles, and the spherical quaternary ammonium salt micelles are formed by crosslinking amphiphilic quaternary ammonium salt molecular monomers; the metal ions are any one of bivalent copper ions, bivalent zinc ions and monovalent silver ions. The quaternary ammonium salt micelle structure can stably enter a biological membrane through cross-linking, the permeability of medicament molecules is improved, and quaternary ammonium salt positive charges are distributed on the spherical surface to improve the adsorption performance of the micelle and bacteria; due to the spherical shape, the diffusion speed of the micelle in the membrane is increased, and the antibacterial effect is improved. The microenvironment of sulfate reducing bacteria can be responded, quaternary ammonium salt oligomer and metal ions can be dissociated, and a biological membrane can be efficiently removed through synergistic sterilization of the two components.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacterial control in oil and gas field exploitation, and particularly relates to a quaternary ammonium salt micelle antibacterial agent, a preparation method thereof, and an application thereof. Background Art

[0002] During the exploitation of oil and gas fields, bacteria generally exist in the produced fluid. Under the conditions suitable for bacterial growth, the corrosion is quite serious. Therefore, the corrosion caused by microorganisms such as sulfate-reducing bacteria, saprophytic bacteria, and iron bacteria has received great attention. Currently, adding antibacterial agents is an economical and effective corrosion protection measure. However, traditional antibacterial agents commonly used in oil and gas fields, such as 1227, due to microbial variation, especially the variation of sulfate-reducing bacteria, make microorganisms gradually develop drug resistance, thus affecting the use effect of the antibacterial agent, and the generation of drug resistance is related to the formation of biofilms. In addition, the formation of biofilms also affects the use effect of antibacterial agents.

[0003] The existing antibacterial agents have poor stripping effects on biofilms, so they cannot kill the bacteria in the biofilms. It is generally believed that the microorganisms mainly causing serious corrosion of oil and gas well strings, surface system equipment, and pipelines in oil and gas fields are the fixed sulfate-reducing bacteria, iron bacteria, etc. under the biofilms. Therefore, the prevention and control of corrosion caused by bacteria by the existing antibacterial agents are limited in practical applications.

[0004] The existing patent CN115850185A introduces the preparation of an asymmetric bisquaternary ammonium salt compound and an asymmetric bisquaternary ammonium salt antibacterial agent, including a low molecular alcohol solvent containing the asymmetric bisquaternary ammonium salt compound. This antibacterial agent is used for the corrosion protection of oil and gas wells, surface gathering pipelines, and water treatment system equipment pipelines containing microbial corrosion in oil and gas fields. This patent utilizes the excellent water solubility and high surface activity of the bisquaternary ammonium salt-type surfactant, which is easy to adsorb on the biofilm to make the biofilm easy to peel off, enhancing its bactericidal effect. However, the uneven distribution of the agent during actual use of a single quaternary ammonium salt results in a low local agent concentration, usually far lower than the minimum inhibitory concentration value. In such an environment, bacteria will induce physiological modifications in bacteria, including inducing the biodegradation of quaternary ammonium salts, enhancing biofilm formation, obtaining efflux genes, and overexpression of extracellular efflux pumps to promote the internal removal of antibacterial agents, thereby causing bacteria to develop drug resistance to quaternary ammonium salts, resulting in the bactericidal effect still unable to meet the bactericidal requirements in the existing oil and gas field exploitation process.

[0005] In view of this, this patent application is proposed. Summary of the Invention

[0006] The purpose of this patent application is to solve the problem that bacteria develop resistance to existing quaternary ammonium salt antibacterial agents, resulting in the inability to effectively prevent and control corrosion caused by bacteria. The present invention provides a quaternary ammonium salt micelle antibacterial agent, and also provides a preparation method and application of the quaternary ammonium salt micelle antibacterial agent. The obtained spherical quaternary ammonium salt micelle antibacterial agent doped with metal ions can stably enter the biofilm through crosslinking, increasing the adsorption performance of the micelles to bacteria, improving the diffusion rate of the micelles in the membrane, and enhancing its antibacterial effect.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a quaternary ammonium salt micelle antibacterial agent, which is obtained by doping metal ions into spherical quaternary ammonium salt micelles, and the spherical quaternary ammonium salt micelles are formed by crosslinking amphiphilic quaternary ammonium salt molecular monomers;

[0009] The metal ions are any one of divalent copper ions, divalent zinc ions, and monovalent silver ions.

[0010] In the embodiments of the present invention, metal ions are doped into the quaternary ammonium salt micelles. By using the effect that metal ions can inhibit the formation of sulfate-reducing bacteria biofilms, the metal ions can denature proteins and cause their functions to be lost; at the same time, the presence of metal ions will affect the substrates in the metabolic process of sulfate-reducing bacteria, such as affecting the mass transfer efficiency and mass transfer flux of sulfate ions and lactate ions.

[0011] The antibacterial agent provided in the embodiments of the present invention can efficiently penetrate the sulfate-reducing bacteria biofilm and accumulate at the bottom, and then release quaternary ammonium oligomers and loaded metal ions in response to the microenvironment of the biofilm. First, the synergistic bactericidal effect of quaternary ammonium oligomers and metal ions can not only efficiently remove the sulfate-reducing bacteria biofilm, but also significantly reduce the dosage of the antibacterial agent; second, metal ions can affect the mass transfer of substrates required for the formation of sulfate-reducing bacteria biofilms, thereby affecting the formation of biofilms. Finally, the multi-drug combination strategy of quaternary ammonium oligomers and metal ions can reduce the risk of the generation of drug-resistant strains of sulfate-reducing bacteria.

[0012] The spherical quaternary ammonium salt micelle antibacterial agent doped with metal ions can stably enter the biofilm through crosslinking, improving its antibacterial effect. On the one hand, the positive charges of the quaternary ammonium are distributed on the spherical surface, increasing the adsorption performance of the micelles to bacteria. On the other hand, the spherical shape increases the diffusion rate of the micelles in the membrane, increasing the permeability of the membrane, and significantly improving the bactericidal performance.

[0013] In an optional embodiment, the cationic structure of the amphiphilic quaternary ammonium salt molecular monomer is:

[0014]

[0015] Among them, R1, R2, R3, and R4 are non-polar tail chains, and at least one of R1, R2, R3, and R4 is a terminal structure containing cross-linking activity. The terminal with cross-linking activity can cross-link with the non-polar tail chains of other quaternary ammonium salt amphiphilic molecular monomers to form cross-linking bonds;

[0016] The number of carbon atoms in any one of the non-polar tail chains of R1, R2, R3, and R4 is greater than or equal to 7 compared to the other three non-polar tail chains, and the longest non-polar tail chain contains any one of the structures -NH-, -O-, and -S-.

[0017] In the embodiments of the present invention, the number of carbon atoms in one tail chain of the amphiphilic quaternary ammonium salt molecule monomer is significantly higher than that of the other three tail chains, and the longest tail chain of R1, R2, R3, and R4 contains structures such as -NH-, -O-, and -S-, which can increase the metal ion loading capacity of the spherical quaternary ammonium salt micelles and further improve the bactericidal effect.

[0018] In an alternative embodiment, the cross-linking bond includes any one of disulfide bonds, thioether bonds, diselenide bonds, orthoester bonds, R-CH=N-NH2, and RRC=N-NH2 bonds. The above cross-linking bonds are chemical bonds that can respond to the microenvironment of bacteria and biofilms.

[0019] In an alternative embodiment, the structure of the amphiphilic quaternary ammonium salt molecule monomer is any one of the following structures:

[0020]

[0021] In an alternative embodiment, the source of the metal ion is any one of copper chloride, zinc sulfate, and silver nitrate.

[0022] The second object of the present invention is to provide a preparation method of a quaternary ammonium salt micelle antibacterial agent described in any one of the above, including the following steps:

[0023] (1) Prepare amphiphilic quaternary ammonium salt molecule monomers, where the number of carbon atoms in one tail chain of each molecule monomer is 7 or more greater than that in other chains, and the longest tail chain contains any one of the structures -NH-, -O-, and -S-;

[0024] (2) Prepare uncrosslinked quaternary ammonium salt micelles from the amphiphilic quaternary ammonium salt molecule monomers at a certain critical micelle concentration, and crosslink the quaternary ammonium salt micelles to obtain spherical quaternary ammonium salt micelles;

[0025] (3) Introduce metal ions into the spherical crosslinked quaternary ammonium salt micelles to obtain a quaternary ammonium salt micelle antibacterial agent.

[0026] In an alternative embodiment, the preparation method of the uncrosslinked quaternary ammonium salt micelles in step (2) is:

[0027] Dissolve the prepared amphiphilic quaternary ammonium salt molecular monomer in water, and add dithiothreitol and a photoinitiator to prepare a mixed solution;

[0028] Carry out photocrosslinking in a UV reactor;

[0029] Remove unreacted monomers and impurities by dialysis to obtain crosslinked spherical quaternary ammonium salt micelles.

[0030] In an optional embodiment, the photoinitiator is PI2959, and photocrosslinking is carried out in a UV reactor with a wavelength of 254 nm for 5 h;

[0031] Preferably, the concentration of the added amphiphilic quaternary ammonium salt molecular monomer is 1 to 10 times the critical micelle concentration of the monomer.

[0032] In an optional embodiment, the method for introducing metal ions into the spherical crosslinked quaternary ammonium salt micelles in step (3) is as follows:

[0033] Drop the metal ion solution into the spherical quaternary ammonium salt micelle solution;

[0034] Slowly add the NaBH4 solution to the above solution until its color changes from colorless to light brown;

[0035] Remove unreacted monomers and impurities by dialysis to obtain spherical quaternary ammonium salt micelles loaded with metal nanoparticles.

[0036] The third object of the present invention is to provide an application of a quaternary ammonium salt micelle antibacterial agent described in any one of the above, which is used to kill microorganisms that corrode mining equipment during oil and gas field exploitation.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] (1) The metal ion-doped spherical quaternary ammonium salt micelle antibacterial agent obtained in the embodiment of the present invention can stably enter the biofilm through crosslinking to improve the molecular permeability of the medicament and its antibacterial effect. The positive charge of the quaternary ammonium salt is distributed on the spherical surface to increase the adsorption performance of the micelle to bacteria; the spherical shape also increases the diffusion rate of the micelle in the membrane and improves its antibacterial effect.

[0039] (2) The metal-doped quaternary ammonium salt micelle antibacterial agent obtained in the embodiment of the present invention can respond to the microenvironment of sulfate-reducing bacteria and dissociate quaternary ammonium salt oligomers and metal ions. The synergistic sterilization of these two components can not only efficiently remove the biofilm but also significantly reduce the dosage of the antibacterial agent.

[0040] The dissociated metal ions can affect the mass transfer of the substrates required for bacterial biofilm formation, thereby affecting biofilm formation. The multi-drug combination strategy of quaternary ammonium salt oligomers and metal ions can reduce the risk of the emergence of drug-resistant strains of sulfate-reducing bacteria.

[0041] (3) The metal ion-doped spherical quaternary ammonium salt micelle antibacterial agent obtained in the embodiments of the present invention further defines the chain length of the quaternary ammonium salt molecule, and the carbon chain containing the NH / O / S structure is used at the chain end, which can increase the metal ion loading amount and further improve the bactericidal effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings. In the drawings:

[0043] Figure 1 It is a process schematic diagram for preparing amphiphilic quaternary ammonium salt molecular monomers using the embodiments of the present invention;

[0044] Figure 2 It is the NMR spectrum of the amphiphilic quaternary ammonium salt molecular monomer prepared in Example 1.

[0045] Figure 3 It is a comparison chart of the metal copper loading rates of the antibacterial agents obtained in Example 1, Example 2, Comparative Example 1, and Comparative Example 2.

[0046] Figure 4 It is a comparison chart of the anti-biofilm rates of the antibacterial agents obtained in Example 1, Example 2, Comparative Example 1, and Comparative Example 2.

[0047] Figure 5 It is a schematic diagram of the results of studying the biofilm removal ability using the antibacterial agent obtained in Example 1, the molecular monomer of Comparative Example 3, and the spherical micelles of Comparative Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0049] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is obvious to those of ordinary skill in the art that the present invention does not have to be practiced with these specific details. In other embodiments, well-known methods have not been described in detail in order to avoid obscuring the present invention.

[0050] Throughout the specification, references to "one embodiment", "an embodiment", "one example" or "an example" mean that the specific features, structures, or characteristics described in connection with that embodiment or example are included in at least one embodiment of the present invention. Thus, the phrases "one embodiment", "an embodiment", "one example" or "an example" appearing in various places throughout the specification do not necessarily all refer to the same embodiment or example. In addition, the specific features, structures, or characteristics may be combined in any suitable combination and / or sub-combination in one or more embodiments or examples.

[0051] In the description of the present invention, the orientation or positional relationships indicated by terms such as "front", "rear", "left", "right", "upper", "lower", "vertical", "horizontal", "high", "low", "inner", "outer", etc. are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the protection scope of the present invention.

[0052] In the embodiments of the present invention, the following method is used to prepare the quaternary ammonium salt micelle antibacterial agent:

[0053] The first step is to synthesize or obtain amphiphilic quaternary ammonium salt molecular monomers:

[0054] 1) Acryloyl chloride and N-methyldiethanolamine (molar ratio 3:1 to 4:1) are stirred at room temperature for 24 h in a dichloromethane solution of triethylamine. The concentrated reaction mixture is purified by silica gel column chromatography to obtain Compound 5 (yellow transparent).

[0055] 2) Potassium carbonate is added to a DMF solution containing 4-hydroxybenzaldehyde and stirred at 60 °C for 1 h. Then 1-bromododecane is slowly added to the above mixed solution and reacted under nitrogen at 80 °C for 24 h. Finally, the above reaction solution is extracted (extracted with ethyl acetate), dried (using MgSO4), and concentrated to obtain a yellow oily liquid (Compound 2). The molar ratio of 4-hydroxybenzaldehyde to 1-bromododecane is 1:1 to 1:1.2.

[0056] 3) The tetrahydrofuran solution containing Compound 2 is added dropwise to the tetrahydrofuran solution (30 mL) containing NaBH4 under ice bath conditions and stirred at room temperature for 24 h. Then the above reaction solution is filtered and concentrated. Next, the concentrated product is dissolved in dichloromethane and washed three times with saturated brine. After drying and concentrating, Compound 3 (white powder) is obtained.

[0057] 4) Dissolve Compound 3 in 75 mL of anhydrous dichloromethane. Then, gradually add the phosphorus tribromide solution to the anhydrous dichloromethane containing Compound 3 under an ice bath condition. Stir the above mixed solution for 3 hours, and then add it to distilled water. Finally, extract the above reaction solution (with dichloromethane), wash it (with saturated brine), dry it (with Na2SO4), and concentrate to obtain a yellow solid (Compound 4).

[0058] 5) Dissolve Compound 5 in acetonitrile, and then slowly add it to the acetonitrile solution containing Compound 4. After stirring at room temperature for 3 days, remove the acetonitrile under vacuum. The residue is purified by silica gel column chromatography to obtain Compound 1, which is the amphiphilic quaternary ammonium salt molecular monomer (white powder).

[0059] Second step, preparation of uncrosslinked quaternary ammonium salt micelles and crosslinking thereof:

[0060] Dissolve Compound 1 in the first step in water (the added concentration of Compound 1 is 1 to 10 times the CMC concentration, and the CMC concentration is the critical micelle concentration), add dithiothreitol and PI2959 to prepare a mixed solution. Then, perform photocrosslinking in a UV reactor with a wavelength of 254 nm for 5 h. Finally, remove the unreacted monomers and other small molecule impurities by dialysis (1 kDa) to obtain crosslinked spherical quaternary ammonium salt micelles.

[0061] Third step, introducing metal ions into the crosslinked quaternary ammonium salt micelles to obtain a metal ion-doped spherical quaternary ammonium salt micelle antibacterial agent:

[0062] Drop the metal ion solution into the crosslinked spherical quaternary ammonium salt micelle solution. Then, slowly add the NaBH4 solution to the above solution until its color changes from colorless to light brown. Finally, remove the unreacted monomers and other small molecule impurities by dialysis (using a 1 kDa dialysis bag) to obtain quaternary ammonium salt micelles loaded with metal nanoparticles.

[0063] The following is described in detail through specific examples.

[0064] Example 1:

[0065] A preparation method of a quaternary ammonium salt micelle antibacterial agent is prepared according to the following method:

[0066] 1. Synthesize the amphiphilic quaternary ammonium salt molecular monomer (prepared according to the process as in Figure 1 ):

[0067] 1) Add acryloyl chloride (2.35 g, 26.0 mmol) to a dichloromethane solution (20 mL) containing n-methyldiethanolamine (0.98 g, 8.2 mmol) and triethylamine (3.4 g, 33.6 mmol), and stir at room temperature for 24 h. Purify the concentrated reaction mixture by silica gel column chromatography to obtain Compound 5 (yellow transparent).

[0068] 2) Potassium carbonate (12.4 g, 90 mmol) was added to a solution of 4-hydroxybenzaldehyde (1.83 g, 15 mmol) in DMF (45 mL). The mixture was stirred at 60 °C for 1 h, and then 1-bromododecane (4.35 mL, 18 mmol) was slowly added to the above mixed solution. The reaction was carried out under nitrogen at 80 °C for 24 h. Finally, the reaction solution (ethyl acetate) was extracted, dried (MgSO4), and concentrated to obtain a yellow oily liquid (Compound 2).

[0069] 3) A solution of Compound 2 (4 g, 14 mmol) in tetrahydrofuran (40 mL) was added dropwise to a solution of NaBH4 (1.1 g, 28 mmol) in tetrahydrofuran (30 mL) under ice bath conditions. The mixture was stirred at room temperature for 24 h. Then, the reaction solution was filtered and concentrated. Then, the concentrated product was dissolved in dichloromethane (25 ml) and washed three times with saturated brine. After drying and concentration, Compound 3 (white powder) was obtained.

[0070] 4) Compound 3 (3.0 g, 10 mmol) was dissolved in 75 mL of anhydrous dichloromethane. Then, phosphorus tribromide solution (2.0 mL, 20 mmol) was gradually added to the anhydrous dichloromethane containing Compound 3 under ice bath conditions. Then, the above mixed solution was stirred for 3 h and added to distilled water (600 mL). Finally, the reaction solution was extracted (dichloromethane), washed (saturated brine), dried (Na2SO4), and concentrated to obtain a yellow solid (Compound 4).

[0071] 5) Compound 5 (2.05 g, 9 mmol) was dissolved in 6 mL of acetonitrile, and then slowly added to a 10 mL acetonitrile solution containing Compound 4 (3.01 g, 8.5 mmol). After stirring at room temperature for 3 days, acetonitrile was removed under vacuum, and the residue was purified by silica gel column chromatography to obtain Compound 1 (white powder).

[0072] The structural formula of the obtained amphiphilic quaternary ammonium salt molecular monomer (Compound 1) is as follows:

[0073]

[0074] The NMR spectrum of the amphiphilic quaternary ammonium salt molecular monomer is shown in Figure 2 as shown below.

[0075] 2. Obtaining spherical quaternary ammonium salt micelles

[0076] Dissolve compound 1 (5.8 mg, 10 μM) in 10 mL of water, add dithiothreitol (DTT, 1.5 mg, 10 μL) and PI2959 (4.5 mg / mL, 10 μL) to prepare a mixed solution. Then, perform photocrosslinking in a UV reactor at a wavelength of 254 nm for 5 h. Finally, remove unreacted monomers and other small molecule impurities by dialysis (1 kDa) to obtain crosslinked quaternary ammonium salt micelles.

[0077] 3. Doping with metal ions to obtain an antibacterial agent

[0078] First, add CuCl2 solution (113 μL, 1 mg / mL) dropwise to the quaternary ammonium salt micelle solution (5 mL, 0.5 mg / mL). Then, slowly add NaBH4 solution (200 ml, 0.01 mol / L) to the above solution until its color changes from colorless to light brown. Finally, remove unreacted monomers and other small molecule impurities by dialysis (1 kDa) to obtain a quaternary ammonium salt micelle antibacterial agent loaded with copper nanoparticles, designated as (Cu@QAS@CM)1.

[0079] (QAS represents the amphiphilic quaternary ammonium salt molecule monomer, and CM represents the micelle).

[0080] Example 2:

[0081] A preparation method of a quaternary ammonium salt micelle antibacterial agent is prepared according to the following method:

[0082] The difference from Example 1 is that 1-bromododecane in step 2) of Example 1 is replaced with 6-bromo-1-hexanol, and the chemical formula structure of the new compound 1 obtained finally is as follows:

[0083]

[0084] The steps of obtaining micelles and metal doping are the same as in Example 1 to obtain (Cu@QAS@CM)2.

[0085] Comparative Example 1:

[0086] A preparation method of a quaternary ammonium salt micelle antibacterial agent is prepared according to the following method:

[0087] The difference from Example 1 is that 1-bromododecane in Example 1 is replaced with 1-bromohexane, and the chemical formula structure of the new compound 1 obtained finally is as follows:

[0088]

[0089] The steps of obtaining micelles and metal doping are the same as in Example 1 to obtain (Cu@QAS@CM)3.

[0090] Comparative Example 2:

[0091] In the process of obtaining spherical quaternary ammonium salt micelles in Step 2 of Example 1, the concentration of Compound 1 was increased to 20 times the critical micelle concentration. After crosslinking, non-spherical quaternary ammonium salt micelles were obtained. After loading with Cu ions, (Cu@QAS@CM)4 was obtained.

[0092] Determination of the Cu loading ratio:

[0093] For the dialyzed Cu@QAS@CM, after repeatedly calcining a fixed weight of the micelle bactericide in a muffle furnace at 500 °C, the mass before and after calcination was measured. The Cu loading ratio was the mass after calcination divided by the mass before calcination multiplied by 100%.

[0094] The Cu loading ratios in Examples 1-2, Comparative Examples 1 and 2 were measured as shown in Figure 3 and the anti-biofilm rates were as shown in Figure 4 .

[0095] From Figure 3 , 4 it can be seen that the anti-biofilm rate of Example 1 was the highest. In Example 2, the tail chain of the quaternary ammonium salt molecular monomer contained -O-, which increased the loading rate of metallic copper. In contrast, in Comparative Example 1, since 1-bromohexane did not contain -OH, the non-polar tail chain of the obtained (Cu@QAS@CM)3 structure did not have -OH at the end, resulting in a significantly reduced loading rate of metal ions and the lowest anti-biofilm rate. In Comparative Example 2, during the preparation of quaternary ammonium salt micelles, the addition concentration of the quaternary ammonium salt molecular monomer was 20 times the monomer critical micelle concentration. Although the loading rate of metal ions was the highest, the anti-biofilm rate was relatively low.

[0096] Comparative Example 3:

[0097] Compared with Example 1, only the amphiphilic quaternary ammonium salt molecular monomer, QAS, was prepared.

[0098] Comparative Example 4:

[0099] Compared with Example 1, only spherical quaternary ammonium salt micelles, QAS@CM, were prepared.

[0100] The obtained antibacterial agent in Example 1 was measured to study the film-removing ability (mass percentage of the biofilm), and the results are shown in Figure 5 . The antibacterial abilities of QAS in Comparative Example 3 and QAS@CM in Comparative Example 4 were also studied for comparison, and the results are shown in Figure 5 .

[0101] Method for studying the film-removing ability:

[0102] First, add 1 mL of SRB bacterial suspension into a 48-well plate and incubate at 37 °C for 7 days to form an SRB biofilm at the bottom of the plate. Then replace the old culture medium with the culture medium containing samples, including free QAS, QAS@CM, and Cu@QAS@CM (250 μM), and continue culturing for 24 h. Then remove the culture medium, add crystal violet solution (200 μL, 0.1 w / v%), and incubate at 37 °C. After culturing for 15 minutes, remove the crystal violet and add ethanol (200 μL, 95 v / v%) to dissolve the crystal violet. Finally, measure the optical density (550 nm) using a microplate reader. The calculation formula for the percentage of SRB biofilm biomass is as follows:

[0103] Percentage of SRB biofilm biomass = ODsamples / ODBlank × 100%.

[0104] It can be seen that Figure 5 under the same measurement conditions, after the antibacterial agent obtained in Example 1 acts on microorganisms, the mass percentage of the biofilm in the system is the lowest, at 22%. The mass percentage of the biofilm is the highest under the action of the molecular monomer in Comparative Example 3, reaching 57%. Under the action of spherical quaternary ammonium salt micelles, the mass percentage of the biofilm is in the middle, at 43%. It can be seen that although the spherical quaternary ammonium salt micelles have a certain film removal rate, the effect is not as good as that of the quaternary ammonium salt micelles doped with metal ions in Example 1. It can be seen that the antibacterial ability of the antibacterial agent is significantly improved under the synergistic effect of metal ions and quaternary ammonium salt micelles.

[0105] The quaternary ammonium salt micelle antibacterial agent doped with metal ions obtained in the present invention can efficiently penetrate the sulfate-reducing bacteria biofilm and accumulate at its bottom, and then release quaternary ammonium salt oligomers and metal ions in response to the microenvironment of the biofilm. The synergistic sterilization of quaternary ammonium salt oligomers and copper ions can not only efficiently remove the sulfate-reducing bacteria biofilm, but also significantly reduce the dosage of the antibacterial agent. Metal ions can affect the mass transfer of substrates required for the formation of sulfate-reducing bacteria biofilm and thus affect the formation of biofilm. The multi-drug combination strategy of quaternary ammonium salt oligomers and metal ions can reduce the risk of the generation of drug-resistant strains of sulfate-reducing bacteria. Therefore, it has broad application prospects in the control of oil and gas field bacteria.

[0106] The above specific embodiments further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A quaternary ammonium salt micelle antibacterial agent, characterized in that, It is obtained by introducing metal ion doping into spherical quaternary ammonium salt micelles, and the spherical quaternary ammonium salt micelles are formed by cross-linking amphiphilic quaternary ammonium salt molecular monomers; The metal ion is any one of divalent copper ions, divalent zinc ions, and monovalent silver ions.

2. The quaternary ammonium salt micelle antibacterial agent according to claim 1, characterized in that, The cationic structure of the amphiphilic quaternary ammonium salt molecular monomer is: Among them, R1, R2, R3, and R4 are non-polar tail chains, and at least one of R1, R2, R3, and R4 is a terminal structure containing cross-linking activity. The terminal with cross-linking activity can cross-link with the non-polar tail chains of other quaternary ammonium salt amphiphilic molecular monomers to form cross-linking bonds; The number of carbon atoms in any one of the non-polar tail chains of R1, R2, R3, and R4 is greater than or equal to 7 compared to the other three non-polar tail chains. The non-polar tail chain with the longest chain length contains any one of the structures -NH-, -O-, and -S-.

3. The quaternary ammonium salt micelle antibacterial agent according to claim 2, characterized in that, The cross-linking bond includes any one of disulfide bonds, thioether bonds, diselenide bonds, orthoester bonds, R-CH=N-NH2, and RRC=N-NH2 bonds.

4. The quaternary ammonium salt micelle antibacterial agent according to claim 2, characterized in that, The structure of the amphiphilic quaternary ammonium salt molecular monomer is any one of the following structures:

5. The quaternary ammonium salt micelle antibacterial agent according to claim 1, characterized in that, The source of the metal ion is any one of copper chloride, zinc sulfate, and silver nitrate.

6. A preparation method of the quaternary ammonium salt micelle antibacterial agent according to any one of claims 1 to 5, characterized in that, It includes the following steps: (1) Prepare amphiphilic quaternary ammonium salt molecular monomers; (2) Prepare uncrosslinked quaternary ammonium salt micelles, cross-link the quaternary ammonium salt micelles to obtain spherical quaternary ammonium salt micelles; (3) Introduce metal ions into the spherical crosslinked quaternary ammonium salt micelles to obtain a quaternary ammonium salt micelle antibacterial agent.

7. The preparation method of the quaternary ammonium salt micelle antibacterial agent according to claim 6, characterized in that, The preparation method of the uncrosslinked quaternary ammonium salt micelles in step (2) is: Dissolve the prepared amphiphilic quaternary ammonium salt molecular monomers in water, add dithiothreitol and a photoinitiator to prepare a mixed solution; Perform photo-crosslinking in a UV reactor; Remove unreacted monomers and impurities by dialysis to obtain crosslinked spherical quaternary ammonium salt micelles.

8. The preparation method of the quaternary ammonium salt micelle antibacterial agent according to claim 7, characterized in that, The photoinitiator is PI2959, and photo-crosslinking is performed in a UV reactor with a wavelength of 254 nm for 5 h; Preferably, the concentration of the added amphiphilic quaternary ammonium salt molecular monomer is 1 to 10 times the critical micelle concentration of the monomer.

9. The preparation method of the quaternary ammonium salt micelle antibacterial agent according to claim 6, characterized in that, The method of introducing metal ions into the spherical crosslinked quaternary ammonium salt micelles in step (3) is: Drop the metal ion solution into the spherical quaternary ammonium salt micelle solution; Slowly add the NaBH4 solution to the above solution until its color changes from colorless to light brown; Remove unreacted monomers and impurities by dialysis to obtain spherical quaternary ammonium salt micelles loaded with metal nanoparticles.

10. An application of the quaternary ammonium salt micelle antibacterial agent according to any one of claims 1 to 5 or the quaternary ammonium salt micelle antibacterial agent obtained by the method according to any one of claims 6 to 9, characterized in that, It is used to kill microorganisms that corrode mining equipment during oil and gas field exploitation.

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