Design and Synthesis of Polyacrylamide Quaternary Ammonium Salt with Antibacterial Activity

Propenamine quaternary ammonium salt (NNDBAB) containing unsaturated bonds was synthesized from propenamine and bromobutane by alkylation reaction. Using NNDBAB as a monomer, PNNDBAB was further self-polymerized by free radical polymerization to produce propenamine quaternary ammonium salt (PNNDBAB). The structure of NNDBAB was characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. The antibacterial properties of PNNDBAB were characterized by an antibacterial zone test, minimum inhibitory concentration, and smear plate counting. The results showed that PNNDBAB had good bacteriostatic activity, the diameter of the bacteriostatic zone was 27 mm, the minimum bacteriostatic concentration was 0.5 mg/L, and the bacteriostatic rate of 1wt% was 71.0%. The bacteriostatic effect was significant. The antibacterial effect of PNNDBAB is long-lasting. When added to the emulsion, the antibacterial effect of PNNDBAB can be sustained within 15 days.


Introduction
The bacteriostatic agent is a substance or product with antibacterial and bactericidal properties, mainly a metal bacteriostatic agent, quaternary ammonium salt bacteriostatic agent, acrylamide bacteriostatic agent, polymer bacteriostatic agent, etc [1] .Compared with ordinary physical and chemical disinfection methods, bacteriostatic material has the advantages [2] of long-term effectiveness, safety, and economy.Nowadays, antibacterial materials have been widely [3][4] used in coatings, household appliances, automotive decoration, and public facilities, and the development of low-cost, high-antibacterial, longterm antibacterial, large-scale production of antibacterial materials has become a trend [5][6] .Quaternary ammonium salt bacteriostatic agent is an organic compound containing an N + structure prepared by chemical reaction.N + adsorbs negatively charged bacteria through electrostatic force, hydrogen bond force, and hydrophobic binding between protein molecules, and gathers on the cell wall, resulting in chamber resistance effect, bacterial growth, and death [7] .Quaternary ammonium salt bacteriostatic agent has become the most widely used bacteriostatic agent [8][9] in the world due to its characteristics of high efficiency, low toxicity, easy usage, and unsusceptibility to pH change.However, quaternary ammonium salt has small molecular weight, instability, low charge density, and weak [10] bacteriostatic performance and chemical stability.It can introduce carbon-carbon double bonds and synthesize polyquaternary ammonium salt macromolecules through free radical polymerization by using unsaturated groups, to enhance its antimicrobial persistence and stability.
Because of the amino group in the structure, acrylamine is easy to alkalize with halogens to form a quaternary ammonium salt structure, and its double bond can be used in polymerization [11][12][13] .Therefore, in this study, from the perspective of the development of quaternary ammonium salt polymer antibacterial agents, the first alkylation reaction of propylene amine and bromobutane containing a halogen to produce quaternary ammonium salt with unsaturated bond, and then further polymerization to synthesize polyquaternary ammonium salt.Through this study, the research results of quaternary ammonium salt bacteriostatic agents can be enriched and provide a scientific and reasonable theoretical basis for the preparation of long-term bacteriostatic materials.

Materials
Acrylamine and bromobutane were purchased from Shanghai Maclin Biochemical Industrial Park.Ammonium persulfate was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Company.Calcium carbonate (K 2 CO 3 ) was purchased from Tianjin Beichen Founder Reagent Factory.Diluted hydrochloric acid, Tetrahydrofuran (THF), Dichloromethane (CH 2 Cl), Nutritious broth, and Nutrient AGAR were purchased from Sigma Chemical Co.The E. coli from the Biochemistry Laboratory of Qilu Institute of Technology.All chemicals used were of analytical grade.

Methods
We put up the three-mouth flask on the heating set, add acetone, propylene amine, and potassium carbonate, stir to dissolve it, slowly add excess bromobutane, react at a certain temperature for a certain time, and recrystallize the product after the reaction.We add an appropriate amount of deionized water as a solvent, add the monomer generated by the reaction of the previous step, take ammonium persulfate as the initiator, and adjust the speed after the reaction at a certain temperature for 5 hours.After the reaction to tetrahydrofuran as a precipitating agent, the polymer is prepared.We rinse with anhydrous ethanol three to four times to obtain a polymer with less impurity and higher purity.The reaction equation for this step is shown in Figure 1.

Bacteriostatic test
Each petri dish was placed with a filter paper soaked in saline as a control, and then 1-2 filter paper pieces containing other reagents were placed at a certain distance.Finally, the samples were cultured in a constant temperature incubator at 37℃ for 24 h, the diameter of the antibacterial zone was observed and measured, and the average value was calculated [14] .The sample concentrations in tubes 1 to 15 were 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0, and 0 mg/L, and then 0.1 mL bacterial suspension was added to tubes 1 to 14 and cultured at 37℃ for 24 h to observe the turbidity of each tube.The concentration of the sample in the test tube that began to muddy with the culture solution was the lowest inhibitory concentration [15] .Three parallel experiments were set up for this experiment.We take a certain amount of normal saline in a conical bottle and add 1 g, 2 g, 3 g, 4 g, and 5 g NNDBAB, respectively, so that the total mass of the solution is 100 g.We configured NNDBAB solution with the concentration of 0.00wt%, 1.00wt%, 2.00wt%, 3.00wt%, 4.00wt% and 5.00wt%; Normal saline was used as blank control, and 0.2 µL Escherichia coli solution was taken by pipetting gun and cultured at constant temperature for 24 h in blank normal saline and normal saline with different concentrations of NNDBAB and PNNDBAB.0.1 µL bacterial solution was taken, and the antibacterial rate [16] of NNDBAB and PNNDBAB at each concentration was measured by three parallel experiments using the dilution-coated plate method.Figure 2 shows the NMR spectrum of NNDBAB.It can be seen that the peaks at δ=5.62 are the two hydrogen proton signal peaks on the molecule methyl-CH 2 -, and the chemical shift of the hydrogen proton is shifted to the lower field due to the shielding effect of N + .The information obtained from the infrared spectra of the compound is in agreement with the results obtained from the nuclear magnetic resonance spectra.

Bacteriostatic performance characterization 3.2.1 Bacteriostatic zone experiment.
The comparison of the experimental results of the antibacterial zone between PNNDBAB and NNDBAB is shown in Figure 3. Third filter paper produces an obvious antibacterial zone with a diameter of 27 mm, which is larger than that of No.1, indicating that PNNDBAB has a significant antibacterial effect, and its antibacterial effect is stronger than that of monomer NNDBAB.

Dilution coating plate method.
Figure 5 shows the bacterial-inhibiting pictures of the blank control group, NNDBAB, and PNNDBAB respectively.It can be seen from the figure that NNDBAB and PNNDBAB all showed good bacterial-inhibiting performance.Compared with the control group, the plate colony number with 1wt%NNDBAB and PNNDBAB added significantly decreased.The calculated bacteriostatic rates were 69.2% and 71.0%, respectively.The data showed that HMMBA selfpolymerization not only did not affect the activity of the functional groups of capsaicin but could even slightly improve the bacteriostatic ability of HMMBA.To test the antibacterial persistence of PNNDBAB, it was physically blended with ordinary emulsion, the addition amount was 2.00wt%, and the antibacterial activity of the three emulsions was observed by comparing the blank emulsion and the emulsion with 2.00wt%NNDBAB.The emulsion was placed in the air for 3 days, 6 days, 9 days, 12 days, and 15 days, and the results are shown in Figure 6.The antibacterial rate of the emulsion supplemented with PNNDBAB changed from 91.72% to 90.89%, basically maintaining the same, while the antibacterial rate of the emulsion supplemented with NNDBAB decreased significantly, indicating that the antibacterial rate of PNNDBAB was long-lasting, and the antibacterial persistence increased after the polymerization of NNDBAB into macromolecules.The result is shown in Figure 6.

Conclusions
The inhibitory zone diameter of PNNDBAB was 27 mm, the minimum inhibitory concentration of PNNDBAB was 0.5 mg/L, and the inhibitory rate of 1wt% was 71.0%.The antibacterial effect of PNNDBAB is long-lasting, and after adding the emulsion, the antibacterial effect of PNNDBAB can last for 15 days.When the dosage of 2wt% was added, the antibacterial rate changed from 91.72% to 90.89%, and the antibacterial rate remained unchanged.Given these results, PNNDBAB is expected to be widely used in health care and daily life as a bacteriostatic additive.
Figure 4 were analyzed: the liquid in test tube No.15 was clarified and no bacteria grew; In test tube No.14, the liquid was cloudy and the bacteria grew well; The liquid medium in test tubes No.1-13 became cloudy from the test tube No.10, and the concentration of quaternary ammonium salt in the test tube No.10 was 0.5 mg/L, that is, AMCE-2023 Journal of Physics: Conference Series 2713 (2024) 012027 IOP Publishing doi:10.1088/1742-6596/2713/1/0120274 the MIC of the synthesized quaternary ammonium salt was 0.5 mg/L.

Figure 4 .
Figure 4. Comparison of the test results of the minimum bacteriostatic concentration of quaternary ammonium salt.

Figure 6 .
Figure 6.Antibacterial rate of NNDDAB and PNNDBAB emulsion added for different time pers.