Preparation and Characterization of a Novel Rechargeable Antibacterial Polyurethane Sponge Foam Substrate Modified with Chlorinated N-Halamine for Efficient Disinfection

Preparation and Characterization of a Novel Rechargeable Antibacterial Polyurethane Sponge Foam Substrate Modified with Chlorinated N-Halamine for Efficient Disinfection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preparation and Characterization of a Novel Rechargeable Antibacterial Polyurethane Sponge Foam Substrate Modified with Chlorinated N-Halamine for Efficient Disinfection Ali Ashraf Derakhshan, Ali Akbar Zinatizadeh, Ali Rostami, Fariba Oulad, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6383007/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract This research offers a simple and convenient strategy for surface decoration of polyurethane sponge foam through the covalent attachment of 5,5-dimethylhydantoin as an N-halamine precursor using 1,6-hexamethylene diisocyanate as a crosslinking agent. After hypochlorite bleach treatment, the N-H bonds in the 5,5-dimethylhydantoin moieties on the polyurethane sponge foam surface change into N-Cl bonds. This modification enhances antibacterial performance against both Gram-positive and Gram-negative bacteria. The washing stability of the prepared Cl-DMH-PUSF substrates was studied after 20 rinsing cycles using the DPD tablet test. The results showed that no active chlorine was released from the Cl-DMH-PUSF substrate after multiple washing processes, implying the strong stability of the active chlorine on the N-halamine surface. To characterize the synthesized antiseptic samples, field emission scanning electron microscopy (FESEM) and energy dispersive X-ray analysis were applied. FESEM images revealed a collection of needle-shaped nanoparticles on the Cl-DMH-PUSF surface, which could act as a needle in contact with bacteria and contribute to pathogen death. Earth and environmental sciences/Environmental sciences Earth and environmental sciences/Environmental social sciences Physical sciences/Chemistry Physical sciences/Materials science Antibacterial N-Halamine Polyurethane Rechargeable Chlorination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The widespread contamination of water and the critical scarcity of freshwater sources are among the most formidable challenges faced by individuals and nations worldwide, primarily due to rapid urbanization and industrialization [ 1 , 2 ]. The failure to provide clean drinking water for all populations is considered one of the most significant shortcomings of the 20th century [ 3 ]. Increasing pollutant discharge into natural water resources has widened the gap between water availability and the growing demand for freshwater [ 4 , 5 ]. Despite environmental regulations, both groundwater aquifers and surface water sources remain contaminated with various inorganic and organic pollutants, including heavy metals, dyes, and phenolic compounds [ 6 , 7 ]. Beyond chemical contaminants, water sources are frequently inhabited by microorganisms and pathogens, leading to a range of acute and chronic illnesses in humans and other living organisms [ 8 , 9 ]. Contaminated drinking water has been linked to numerous health issues, including skin irritation, allergies, headaches, vomiting, and even cancer [ 10 , 11 ]. The urgent need for antibacterial and disinfectant compounds to combat pathogens is more critical than ever [ 12 ]. However, one of the primary concerns in this field is the increasing resistance of bacteria and pathogens to conventional disinfectants and antibacterial agents [ 13 ]. Nearly half of the population in developing countries suffers from waterborne diseases, with over 3 million people dying annually due to contaminated water and inadequate disinfection practices [ 14 ]. To address this issue, several antibacterial reagents have been developed, with biocidal polymers, such as N-halamine-based polymers, gaining attention in recent years [ 15 , 16 ]. Wang et al.[ 17 ] synthesized mesoporous composite-supported N-halamines (MCSNs) for water treatment, achieving 60% formaldehyde degradation within 10 minutes under alkaline conditions. MCSNs demonstrated strong antibacterial activity, reducing S. aureus and E. coli O157:H7 by over 7 logs within 1 minute, highlighting their potential for environmental remediation. In another related research, Zhu et al. [ 18 ] synthesized antibacterial N-halamine polymer materials based on protein gelatin sponges (GS-Cl) with oxidative chlorine as the active agent. The GS-Cl materials demonstrated high antibacterial efficiency against E. coli and S. aureus, with adjustable chlorine content and good biocompatibility, effectively promoting wound healing in bacterial infection models. Also, Wu et al. [ 19 ] developed a multifunctional antibacterial cotton fabric (QACs/Hals@cotton-Cl) using in-situ free radical copolymerization. The modified cotton exhibited strong antibacterial properties, eradicating S. aureus and E. coli within 10 minutes, promoting wound healing, and maintaining durability after multiple uses. The fabric showed excellent hydrophobicity and could be easily recharged with chlorine, indicating its potential for medical and hygiene applications. N-halamines are known for their long-term stability in both dry environments and aqueous solutions, effectively eliminating a wide range of microorganisms [ 20 , 21 ]. These compounds can rapidly kill infectious agents, including bacteria, viruses, fungi, and other microorganisms, through the action of halogen atoms within their structure that leading to the inactivation of the N-halamine molecule [ 22 ]. N-halamine compounds disinfect through two primary mechanisms. The first involves the direct transfer of an oxidized halogen (Cl⁺ or Br⁺) from the N-Cl or N-Br groups to the cell walls of microorganisms, causing oxidative damage and cell death. The second mechanism involves the breaking of N-Cl or N-Br bonds, releasing Cl⁺ or Br⁺ ions into water, which penetrate the microorganism and cause further oxidative damage [ 23 , 24 ]. According to the Kirby–Bauer test, both mechanisms are believed to occur simultaneously [ 25 , 26 ]. Traditional water treatment methods often rely on large volumes of chlorine or hypochlorite solutions, which have strong odors and corrosive properties. When these compounds interact with organic matter in water, they can produce carcinogenic by-products such as trihalomethanes [ 27 ]. One promising solution to mitigate these risks is the use of insoluble or immobilized disinfectants. Organic N-halamine compounds, in particular, offer a safer alternative, as they can perform effective disinfection without releasing harmful by-products [ 28 ]. These compounds are available in both solid and water-soluble forms and are known for being stable, odorless, non-corrosive, and non-toxic, making them ideal for disinfecting a wide range of pathogens [ 29 , 30 ]. In this study, organic N-halamine compounds were used to modify the surface of polyurethane sponge foam (PUSF) for efficient water disinfection. This approach presents a versatile strategy for surface functionalization by covalently attaching 5,5-dimethylhydantoin (DMH) using 1,6-hexamethylene diisocyanate as a crosslinking agent. Upon exposure to hypochlorite bleach, the N-H bonds in DMH moieties were converted into N-Cl bonds, resulting in durable oxidizing agents capable of eliminating bacteria. The synthesized antiseptic substrates were characterized using energy-dispersive X-ray analysis and field emission scanning electron microscopy. Antibacterial performance tests of the chlorinated DMH-PUSF substrates demonstrated excellent activity against both Gram-positive and Gram-negative bacteria. Additionally, the durability and rechargeability of the Cl-DMH-PUSF substrates were evaluated, with active chlorine content assessed through both quantitative and qualitative methods. The bonds of N-H in the DMH moieties be converted into bonds of N-Cl upon exposure to hypochlorite bleach treatment, which turns into durable oxidizing agents versus bacteria. The characterization of the prepared antiseptic substrates was evaluated by energy dispersive X-ray and field emission scanning electron microscopy. The results of the antibacterial performance evaluation of the chlorinated DMH-PUSF shown that these prepared substrates possessed great antibacterial activity against Gram-positive and negative bacteria. The durability and rechargeability of the Cl-DMH-PUSF substrates toward rinsing were studied. The active chlorine content of the disinfected samples was also measured by both quantitative and qualitative methods. 2. Experimental procedures 2.1. Materials 1,6-Hexamethylene diisocyanate (HDI), 5,5-dimethylhydantoin (DMH), and triethylamine (TEA) were obtained from Sigma–Aldrich. Toluene (Merck, p.a.) was used without further purification. Sodium hypochlorite solution (10% NaOCl), diethyl-p-phenylenediamine (DPD), potassium iodide, sodium thiosulfate standard solution, and starch powder were purchased from Yijishiye Company. The polyurethane sponge foam (PUSF) substrate was supplied by Dow Chemical Company. The bacterial strains used in this study, Escherichia coli and Staphylococcus aureus, were obtained from clinical isolates. 2.2. Preparation of the DMH grafted PUSF (DMH-PUSF) substrate The surface of the polyurethane sponge foam (PUSF) substrate was functionalized to impart disinfectant properties using 5,5-dimethylhydantoin (DMH) as a modifying agent and 1,6-hexamethylene diisocyanate (HDI) as a crosslinking agent. First, 5 grams of PUSF was cut into small pieces and placed under reflux in a balloon flask. Then, 40 ml of toluene, 4 ml of HDI, and 1 ml of triethylamine (TEA) catalyst were added. The mixture was refluxed under a nitrogen atmosphere at 60°C for 20 minutes. The polyurethane segments were then removed, washed with toluene, and dried. In the next step, the PUSF pieces were placed in a separate reflux balloon containing 100 ml of methanol and 10 g of DMH. The mixture was refluxed for 2 hours at 60°C. The resulting product was separated, washed with methanol, and dried [ 31 ]. 2.3. Preparation of the chlorinated DMH grafted PUSF (Cl- DMH-PUSF) substrate To activate the hydantoin-containing polyurethane surface and form N-Cl bonds within the hydantoin structure, the final product (DMH-PUSF) was immersed in a sodium hypochlorite solution (10% NaOCl) for 2 hours at room temperature and pH = 7. The activated DMH-PUSF was then washed with distilled water, heated at 45°C, and dried to remove excess chlorine [ 32 , 33 ]. Schematics of the performed reactions are shown in Fig. 1 . These reactions were conducted in three distinct ways, with variations in the amount of TEA catalyst, reaction time, and reflux temperature. 2.4. Characterization techniques Field emission scanning electron microscopy (FESEM, Hitachi S-4160, Japan) was used to characterize the morphological structure of the prepared antiseptic Cl-DMH-PUSF substrates. Energy-dispersive X-ray spectroscopy (EDX, LEO 1530) was employed to perform elemental analysis and detect the presence of chlorine (Cl), oxygen (O), nitrogen (N), and carbon (C) on the surface of the substrates. To evaluate the antibacterial performance and determine the percentage inhibition of bacterial growth, a microplate reader (ELISA) was used for optical density measurements of the prepared samples. 2.5. Measurement of active chlorine in disinfectant samples The active chlorine content in the N-halamine compounds of the disinfected samples was measured using both quantitative and qualitative methods. 2.5.1. The qualitative measurement of the active chlorine with DPD tablets The chemical method for measuring chlorine levels in water is more practical compared to electronic methods due to its simplicity, cost-effectiveness, and sufficient accuracy. This method allows for the easy determination of both free chlorine and total chlorine concentrations. In the chemical chlorine measurement test, diethyl-p-phenylenediamine (DPD) was used as a reagent. In the presence of chlorine, the DPD reagent changes color, and the chlorine concentration is determined based on the intensity of the resulting dye. Three tests were conducted to evaluate the release of active chlorine from the Cl-DMH-PUSF samples: In the first test, a small piece of the Cl-DMH-PUSF sample was immersed in 10 ml of distilled water for 2 hours. After the sponge sample was removed, the remaining water was tested with a DPD tablet to measure chlorine levels. In the second test, a DPD tablet was dissolved in 10 ml of distilled water, and a small piece of the Cl-DMH-PUSF sample was immersed in the solution to observe the chlorine release. In the third test, a DPD tablet was dissolved in 10 ml of distilled water, and a small piece of the Cl-DMH-PUSF sample, previously dried after undergoing 20 washing cycles, was immersed in the solution to assess the stability of active chlorine content. 2.5.2. The quantitative measurement of the active chlorine amount by iodometric titration Iodometric titration was used to measure the active chlorine content of the disinfectant solid samples. First, 1 g of potassium iodide was dissolved in 40 ml of distilled water, and the pH of the solution was adjusted to 4. Then, 0.05 g of the solid disinfectant sample was added to the solution and stirred continuously at room temperature for 1 hour. The iodine (I₂) produced in the reaction was titrated with a 0.01 mol/L standardized aqueous solution of sodium thiosulfate in the presence of a starch reagent to determine the active chlorine content of the Cl-DMH-PUSF substrate [ 34 ]. This protocol was also performed with the unchlorinated DMH-PUSF substrate as a control. The active chlorine content of the Cl-DMH-PUSF substrate was calculated using the following equation [ 34 ]: Cl % = \(\:\frac{35.5}{2}\times\:\frac{\left({V}_{Cl}-{V}_{0}\right)\:\times\:{\:10}^{-3}\:\times\:\:0.01}{{W}_{Cl}}\times\:100\) (1) Where V Cl is the consumed sodium thiosulfate volume (ml) in the Cl-DMH-PUSF titration. V 0 and W Cl are the consumed sodium thiosulfate volume (ml) in the unchlorinated DMH-PUSF titration and the Cl- DMH-PUSF weight (g), respectively. 2.6. Antibacterial functions evaluation To evaluate the antibacterial activity of the Cl-DMH-PUSF substrate, clinical isolates of Escherichia coli and Staphylococcus aureus were used as models for Gram-negative and Gram-positive bacteria, respectively. The bacterial isolates were cultured in a nutrient medium in a rotary incubator at 150 rpm and room temperature until they reached the mid-exponential growth phase. The bacterial cells were then harvested by centrifugation at 600 rpm for 10 minutes and washed three times with phosphate buffer solution [ 35 ]. The washed cells were re-suspended in buffer solution to a final concentration of approximately 10⁸ CFU/ml. A portion of the solid disinfectant sample was cut into smaller pieces, added to distilled water, and placed in an ultrasonic bath to achieve a homogeneous distribution. For the antibacterial test, 50 µl of the bacterial suspension was added to 500 µl of the disinfectant solution and mixed thoroughly. As a control, 50 µl of the bacterial suspension was added to 500 µl of nutrient culture medium without the disinfectant sample. The optical density (OD) of the solutions was measured at 630 nm using a microplate reader [ 36 ] before incubation (T₀). The culture media were then incubated at 37°C for 24 hours with constant shaking. After incubation, the OD of the solutions was measured again [ 37 ]. The percentage of antimicrobial activity was calculated using the following equation [ 38 ]: Percentage inhibition = \(\:[1-\frac{{\text{O}\text{D}}_{\:\text{t}\text{e}\text{s}\text{t}}}{{\:\text{O}\text{D}}_{\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}}]\text{*}100\) (2) 3. Results and discussion 3.1. Characterization of the disinfectant Cl-DMH-PUSF substrates Figure 2 shows the FE-SEM images of the disinfectant Cl-DMH-PUSF substrates at various magnifications. The images reveal both open and closed pores with a thin layer of polyurethane covering the surface. The main walls of the sponge foam are approximately 100 µm thick, while the thin film covering the pores is about 1–2 µm thick. At higher magnifications, smaller particles can be observed on the polyurethane surface, where needle-shaped nano-corals are visible. These nanoparticles are formed during the chlorination reaction of hydantoin compounds on the surface in the NaOCl solution. These nano-coral assemblies serve as the active disinfectant components on the sponge surface. Figure 3 shows higher magnification images of the N-halamine nano-corals on the sponge surface, which resemble marine corals in morphology. The needle-shaped nanoparticles are approximately 100 nm in length and 20 nm in thickness, with tips measuring about 19 nm in diameter. Also, the size of various microorganisms and pathogens is shown in Fig. 3 . Given that the size of bacteria ranges from 1 to a few micrometers and viruses range from 50 to 800 nm, these coral-shaped nano-needles can interact with bacteria or viruses, penetrating their cell bodies and releasing stabilized N-halamine chlorine, leading to pathogen death. The unique nano-coral morphology of these hydantoin compounds provides a high surface area for chlorine uptake, allowing them to store chlorine on the surface for extended periods. One significant advantage of these chlorinated N-halamine compounds is that they eliminate the need for high concentrations of free chlorine in water. Instead, the activated chlorine is stabilized within the solid substrate and is only released upon contact with bacteria. This reduces the risk of forming carcinogenic halo-methane compounds in water. The Cl-DMH-PUSF substrates retain sufficient stabilized chlorine for water disinfection, and when depleted, they can be easily recharged. Figure 4 presents the X-ray map analysis of the Cl-DMH-PUSF surface, showing the elemental distribution of key elements, including Cl, O, N, and C. The elemental distribution is represented by colored dots, where each color corresponds to a specific element on the surface of the substrate. The map was obtained from a Cl-DMH-PUSF sample after 20 washing cycles. As seen in the image, chlorine is uniformly and abundantly distributed across the polymer surface, confirming the stability of N-Cl compounds even after repeated washing. Additionally, the Cl-DMH-PUSF substrates appear yellow in color due to the presence of the chlorinated hydantoin layer, which is a direct result of the chlorination process. The yellow color forms as a visual indicator of the N-Cl bonds created during the reaction with NaOCl, demonstrating the successful formation of the active chlorine layer on the polyurethane sponge surface. Notably, the yellow color did not fade after multiple washing cycles, indicating the stability and durability of the Cl-DMH layer on the polyurethane sponge foam surface [ 9 ]. 3.2. The active chlorine content of antiseptic compounds In the qualitative method to detect the content of the active chlorine in the synthesized antiseptic product, the versatile DPD tablet test was applied. The DPD tablet test is a simple and economical approach to detect the presence of active chlorine in disinfectant materials or water. Since the use of rechargeable disinfection N-halamine systems requires determining the exact time of disinfectant substrate inactivation to reactivate the system with sodium hypochlorite, using the DPD test can be very effective and attractive. DPD tablets are commercially available in various packages and are very affordable. The compound Diethyl-p-PhenyleneDiamine (DPD), in contact with chlorine (N-Cl), quickly changes from a colorless state to a pinkish solution, with the intensity of the color change directly related to the concentration of active chlorine. In commercial samples of DPD tablets, a color change chart in terms of chlorine concentration (ppm) is provided with the product, allowing the operator to determine the approximate chlorine concentration by comparing the solution’s color with the chart. The DPD tests were performed on the disinfectant Cl-DMH-PUSF substrate before and after the washing process. In the first case, a small piece of the Cl-DMH-PUSF sample was immersed in 10 ml of distilled water for 2 hours. After removing the sponge sample, the remaining water was tested with a DPD tablet. No color change was observed upon dissolving the DPD tablet in this sample, indicating the absence of free active chlorine (Cl + ) in the water. Therefore, no active chlorine leaks from the solid polyurethane sample. In the second case, one DPD tablet was dissolved in 10 ml of distilled water, and a small piece of the Cl-DMH-PUSF sample was dipped into it. After about 30 seconds, the solution turned pink due to the contact of DPD molecules with the active surface of N-halamine in the sponge. Figure 5 shows the color change of the solutions in the presence of a polyurethane piece. By comparing the displayed color with the reference color chart, the amount of active chlorine was estimated to be about 0.5 ppm. To investigate the covalently bound stability of chlorine in the prepared Cl-DMH-PUSF substrate, the sample was rinsed 20 times, dried, and then tested with the DPD solution. Almost the same amount of discoloration was observed after 30 seconds (with the active chlorine content estimated to be about 0.5 ppm). According to the obtained active chlorine content, it was found that no active chlorine was released from the Cl-DMH-PUSF substrate after multiple washing processes. This test demonstrates the complete stability of the active chlorine on the N-halamine surface, which is not released by washing and only reacts to release Cl + upon contact with bacteria and organic molecules [ 39 ]. The accurate measurement of active chlorine content in synthesized disinfectant compounds is possible using iodometric titration. In this method, a potassium iodide solution of a certain concentration reacts with N-halamine active chlorine to form iodine (I 2 ), and the exact amount of active chlorine is determined by titration with a standard sodium thiosulfate solution [ 40 ]. The results of the active chlorine content in Cl-DMH-PUSF samples obtained from iodometric titration are presented in Table 1 . A non-functionalized PUSF sample exposed to chlorination by NaOCl solution, according to the previously mentioned methods, was prepared and examined as a blank sample [ 33 ]. The titration results showed that no active chlorine was present on the non-functionalized PUSF surface, indicating that the polyurethane sponge foam could not be chlorinated alone. The functionalized Cl-DMH-PUSF sample was titrated before and after 20 washing cycles (20w). The results indicated that multiple washes did not cause a significant reduction in chlorine content. The slight decrease in chlorine content after the 20 washing cycles may be attributed to the separation of thin and brittle polyurethane layers formed on the closed pores. During the washing process, some of these brittle films were lost, resulting in a slight reduction in active chlorine content. This issue can be addressed by using stronger foam filters to enhance durability. Table 1 Iodometric titration results in the synthesized solid samples Antiseptic sample Sample Weight (g) Sodium thiosulfate concentration (M) Sodium thiosulfate volume (ml) Weight percentage of active chlorine (w%) Cl-PUSF 0.5 0.1 ---- 0 Cl-DMH-PUSF 0.5 0.1 1.75 0.62 Cl-DMH-PUSF (20w) 0.5 0.1 1.5 0.53 Antiseptic sample Sample Weight (g) Remained active chlorine (w%) in chlorinated samples after washing cycles 0 5 10 15 20 Cl-PUSF 0.5 0 0 0 0 0 Cl-DMH-PUSF 0.5 0.62 0.60 0.58 0.55 0.53 Antiseptic sample Sample Weight (g) Percentage of active chlorine loading (w%) by rechlorination of samples after washing cycles 0 5 10 15 20 Cl-PUSF 0.5 0 0 0 0 0 Cl-DMH-PUSF 0.5 0.62 0.61 0.61 0.60 0.58 3.3. Antibacterial activity To investigate the antibacterial potential of the fabricated Cl-DMH-PUSF substrates, the antibacterial activity of these chlorinated N-halamine compounds was evaluated against the Gram-negative bacterium E. coli and the Gram-positive bacterium S. aureus. The obtained results are listed in Tables 2 and 3 , respectively. According to the results, the amount of antiseptic substance used affected the percentage of final antimicrobial activity. As observed, increasing the amount of the substance from 0.16 to 0.21 g caused an increase in antimicrobial activity from 46.43–48.87%. With higher concentrations of the synthesized substrates, the antimicrobial property increased significantly and could even reach 100%. This effect can be attributed to the presence of vacant spaces and porous walls in the polyurethane sponge foam, which create a large specific surface area for the binding of N-halamine compounds and act as barriers to the biocidal agent's attack [ 41 , 42 ]. To achieve the optimal amount of catalyst, the antimicrobial properties of the synthesized samples were evaluated with varying catalyst amounts. The results showed that the optimal catalyst content was 0.2 ml. The synthesized antiseptic substrate demonstrated almost identical antimicrobial activity against both Gram-positive and Gram-negative bacteria. This can be attributed to the high disinfection power of N-halamine compounds, which results in a broad-spectrum extinction of pathogenic agents [ 43 ]. Table 2 The results of the antibacterial test for prepared samples against Gram-negative bacteria of E-coli. Antiseptic sample Sample weight (g) Initial CFU Colony Forming Unit (CFU) Antibacterial activity (w%) Cl-DMH-PUSF (1 ml catalyst) 0.21 2.744 × 10 8 1.403 × 10 8 48.87 Cl-DMH-PUSF (0.2 ml catalyst) 0.16 2.744 × 10 8 1.470 × 10 8 46.43 Cl-DMH-PUSF (0.02 ml catalyst) 0.10 2.744 × 10 8 1.790 × 10 8 34.77 Table 3 The results of the antibacterial test for prepared samples against Gram-positive bacteria of S. aureus. Antiseptic sample Sample weight (g) Initial CFU Colony Forming Unit (CFU) Antibacterial activity (w%) Cl-DMH-PUSF (1 ml catalyst) 0.17 3.208 × 10 8 1.440 × 10 8 55.11 Cl-DMH-PUSF (0.2 ml catalyst) 0.17 3.208 × 10 8 1.456 × 10 8 54.61 Cl-DMH-PUSF (0.02 ml catalyst) 0.17 3.208 × 10 8 1.890 × 10 8 41.08 3.4. Antiseptic and rechargeability mechanism of the Cl-DMH-PUSF substrates In general, N-halamine compounds are divided into three categories: amine, amide, and imide compounds. The molecular structures of these compounds are shown in Fig. 6 . Amine compounds do not have any C = O electron-withdrawing groups, making their N-Cl bonds very stable and resistant to Cl + release [ 30 ]. On the other hand, imide compounds, due to the presence of two adjacent C = O groups near the nitrogen atom, have very weak N-Cl bonds, which are easily broken to release Cl + . Among these categories, amide N-halamine compounds exhibit moderate stability with only one adjacent C = O group. In these amide compounds, the stability of the N-Cl bond is sufficient to maintain its disinfectant properties over time while remaining reactive enough to release Cl + quickly in the presence of bacteria—a balance not achieved in amine or imide compounds [ 44 ]. Figure 6 illustrates the order of stability and the release rate of activated chlorine in these different compounds. Amide N-halamines, such as DMH, are more stable than imide compounds but react more effectively with bacteria. The synthesis of the disinfectant Cl-DMH-PUSF substrates is based on the use of HDI as an intermediate agent. This HDI compound forms a strong covalent bond with the polyurethane chain through one of its isocyanate groups, facilitated by the alkaline catalyst TEA. The other isocyanate group of HDI, which is highly reactive, binds to the DMH compound. The N-H bonds in the DMH moieties are converted into N-Cl bonds upon exposure to hypochlorite bleach treatment, resulting in durable oxidizing agents effective against bacteria. Figure 7 presents a schematic representation of the attachment of DMH molecules to the polyurethane foam surface and their antibacterial mechanism. According to the results obtained from Section 3.2 , it can be concluded that the disinfection mechanism of this system operates through direct contact with bacteria without releasing chlorine into the water. Therefore, to determine the exact time of inactivation of the disinfectant substrate in continuous disinfection systems, the user can simply remove a piece of the polyurethane sponge from the disinfection tank and test it with DPD tablets. If necessary, the disinfection tank can be recharged using a NaOCl solution. 4. Conclusion In this research, the N-halamine precursor of the 5,5-dimethylhydantoin was covalently grafted onto the surface of polyurethane sponge foam using 1,6-hexamethylene diisocyanate as a crosslinking agent. Upon hypochlorite bleach treatment, the N-H bonds in the 5,5-dimethylhydantoin moieties on the surface of the polyurethane sponge foam were converted into N-Cl bonds, providing rechargeable, durable, and powerful antimicrobial activity against both Gram-positive and Gram-negative bacteria. After multiple rinsing processes, the chemical method using Diethyl-p-PhenyleneDiamine (DPD) tablets was employed to evaluate the washing stability of the prepared Cl-DMH-PUSF substrates. The results showed that no active chlorine was released from the antiseptic Cl-DMH-PUSF substrate after repeated washing, indicating robust stability of the active chlorine on the N-halamine surface. The prepared antiseptic substrates were characterized through FESEM, EDX, DPD tests, and iodometric titration. Notably, the antimicrobial samples prepared in this work are rechargeable, and the inactivation time of the antiseptic substrates can be easily monitored using DPD tablets. If the samples become inactive, they can be recharged via additional chlorination treatments. The obtained results suggest that the prepared antiseptic Cl-DMH-PUSF substrates have significant potential for long-term antimicrobial applications. Declarations Author Contribution Ali Ashraf Derakhshan was the primary author of this study.Ali Akbar Zinatizadeh was the supervisor of this study.Ali Rostami wrote and edited the article and checked the data's validity. Fariba Oulad, Khosro Chehri, and Mozhgan Fathi Delphani also edited the article.All authors reviewed and approved the final manuscript. 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Dirty-water: estimated deaths from water-related diseases 2000–2020 (Citeseer, 2002). Hui, F. & Debiemme-Chouvy, C. Antimicrobial N-halamine polymers and coatings: a review of their synthesis, characterization, and applications. Biomacromolecules 14 (3), 585–601 (2013). Kocer, H. B. et al. A novel N-halamine acrylamide monomer and its copolymers for antimicrobial coatings. Reactive Funct. Polym. 71 (5), 561–568 (2011). Wang, Y. et al. Preparation of N-halamine-based mesoporous composites for decontamination of formaldehyde and simultaneous deactivation of bacteria. Colloids Surf., A . 700 , 134664 (2024). Zhu, J. et al. Preparation of N-Halamine Gelatin Sponge and Its Application in the Treatment of Skin Infection. Polymers 16 (18), 2579 (2024). Wu, K. et al. Fabrication of multifunctional cotton fabrics with quaternized N-halamine endowing the synergetic rechargeable antibacterial, wound healing and self-cleaning performances. Int. J. Biol. Macromol. 275 , 133493 (2024). Chang, J. et al. Alkyl substituted hydantoin-based n-halamine: preparation, characterization, and structure–antibacterial efficacy relationship. Ind. Eng. Chem. Res. 55 (35), 9344–9351 (2016). Lan, S. et al. Electrospun sesbania gum-based polymeric n-halamines for antibacterial applications. Polymers 11 (7), 1117 (2019). McCann, B. W. et al. Inter-and Intramolecular Mechanisms for Chlorine Rearrangements in Trimethyl-Substituted N-Chlorohydantoins. J. Phys. Chem. A . 116 (26), 7245–7252 (2012). Liang, J. et al. Polymerization of a hydantoinylsiloxane on particles of silicon dioxide to produce a biocidal sand. J. Appl. Polym. Sci. 97 (3), 1161–1166 (2005). Lin, J., Cammarata, V. & Worley, S. Infrared characterization of biocidal nylon. Polymer 42 (18), 7903–7906 (2001). Bauer, A. et al. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45 (4_ts), 493–496 (1966). Chen, Z., Luo, J. & Sun, Y. Biocidal efficacy, biofilm-controlling function, and controlled release effect of chloromelamine-based bioresponsive fibrous materials. Biomaterials 28 (9), 1597–1609 (2007). Black & Corporation, V. White's handbook of chlorination and alternative disinfectants (Wiley, 2011). Dong, A. et al. Chemical insights into antibacterial N-halamines. Chem. Rev. 117 (6), 4806–4862 (2017). Ahmed, A. E. S. I. et al. New approach to produce water free of bacteria, viruses, and halogens in a recyclable system. Appl. Environ. Microbiol. 77 (3), 847–853 (2011). Akdag, A. et al. The stabilities of N – Cl bonds in biocidal materials. J. Chem. Theory Comput. 2 (3), 879–884 (2006). Sun, X. et al. An N-halamine-based rechargeable antimicrobial and biofilm controlling polyurethane. Acta Biomater. 8 (4), 1498–1506 (2012). Cao, Z. & Sun, Y. Polymeric N-halamine latex emulsions for use in antimicrobial paints. ACS Appl. Mater. Interfaces . 1 (2), 494–504 (2009). Luo, J. & Sun, Y. Acyclic N-halamine‐based fibrous materials: Preparation, characterization, and biocidal functions. J. Polym. Sci., Part A: Polym. Chem. 44 (11), 3588–3600 (2006). Yao, J. & Sun, Y. Preparation and characterization of polymerizable hindered amine-based antimicrobial fibrous materials. Ind. Eng. Chem. Res. 47 (16), 5819–5824 (2008). Akujobi, C. & Njoku, H. Bioassay for the determination of microbial sensitivity to Nigerian honey. Global J. Pharmacol. 4 (1), 36–40 (2010). Kuda, T., Shimizu, K. & Yano, T. Comparison of rapid and simple colorimetric microplate assays as an index of bacterial count. Food Control . 15 (6), 421–425 (2004). Patton, T. et al. Use of a spectrophotometric bioassay for determination of microbial sensitivity to manuka honey. J. Microbiol. Methods . 64 (1), 84–95 (2006). Sarker, S. D., Nahar, L. & Kumarasamy, Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42 (4), 321–324 (2007). Cao, Z. et al. Rechargeable infection-responsive antifungal denture materials. J. Dent. Res. 89 (12), 1517–1521 (2010). Cao, Z. & Sun, Y. N-halamine‐based chitosan: Preparation, characterization, and antimicrobial function. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 85 (1): pp. 99–107. (2008). Denyer, S. P. Mechanisms of action of antibacterial biocides. Int. Biodeterior. Biodegrad. 36 (3–4), 227–245 (1995). Larson, M. A. & Mariñas, B. J. Inactivation of Bacillus subtilis spores with ozone and monochloramine. Water Res. 37 (4), 833–844 (2003). Liang, J. et al. N-halamine/quat siloxane copolymers for use in biocidal coatings. Biomaterials 27 (11), 2495–2501 (2006). Li, X. et al. Biocidal activity of n-halamine methylenebisacrylamide grafted cotton. J. Eng. Fibers Fabr. 10 (2), 155892501501000217 (2015). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Jun, 2025 Reviews received at journal 09 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 11 Apr, 2025 Reviewers invited by journal 11 Apr, 2025 Editor assigned by journal 11 Apr, 2025 Editor invited by journal 11 Apr, 2025 Submission checks completed at journal 11 Apr, 2025 First submitted to journal 05 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6383007","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":441822001,"identity":"18c9a1c2-6abf-4774-b814-24667bc80f6a","order_by":0,"name":"Ali Ashraf Derakhshan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYHACNgbGBijzA4jLTkA9D1wLGwMD4wwQzUyKFmYekBAhLfbsx689+LjDRk5+fvOzxza/tsnzMTMwfviYg8cWnpxyw5ln0owNjrGZG+f23TZsY2Zglpy5DZ/DctKkedsOJ25gYzCTzu25zQjUwsbMi08L/5s06b9ALfPb2L9JW/bctiesRSL9mDQjUEvDMR4zaYYftxMJa7nxhk2yF+yXnDLJ3obbyW3MjM14/cLen/5M4icoxJqPb5P48ee27fz25oMfPuLRArTHAMFmbAOTDfjUg+x5gMT5Q0DxKBgFo2AUjEgAAKYuTPeCedsIAAAAAElFTkSuQmCC","orcid":"","institution":"Razi University","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"Ashraf","lastName":"Derakhshan","suffix":""},{"id":441822002,"identity":"8a0572c2-2787-4050-b1a7-6318d4451c9a","order_by":1,"name":"Ali Akbar Zinatizadeh","email":"","orcid":"","institution":"Razi University","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Akbar","lastName":"Zinatizadeh","suffix":""},{"id":441822003,"identity":"51e7824b-c9ce-4427-8d9a-86487f1a6cf1","order_by":2,"name":"Ali Rostami","email":"","orcid":"","institution":"Razi University","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Rostami","suffix":""},{"id":441822004,"identity":"edaf6576-719b-482c-b221-ffb59d676cad","order_by":3,"name":"Fariba Oulad","email":"","orcid":"","institution":"Razi University","correspondingAuthor":false,"prefix":"","firstName":"Fariba","middleName":"","lastName":"Oulad","suffix":""},{"id":441822005,"identity":"42347723-0367-4527-820c-404688f880e6","order_by":4,"name":"Khosrow Chehri","email":"","orcid":"","institution":"Razi University","correspondingAuthor":false,"prefix":"","firstName":"Khosrow","middleName":"","lastName":"Chehri","suffix":""},{"id":441822006,"identity":"7ddd8721-700c-4924-bbe9-4c5d2b3068fc","order_by":5,"name":"Mozhgan Fatahi Dehpahni","email":"","orcid":"","institution":"Razi University","correspondingAuthor":false,"prefix":"","firstName":"Mozhgan","middleName":"Fatahi","lastName":"Dehpahni","suffix":""}],"badges":[],"createdAt":"2025-04-05 15:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6383007/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6383007/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16711-0","type":"published","date":"2025-08-20T16:29:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81147203,"identity":"cef96342-6fd0-4397-ad09-277da7c46b5d","added_by":"auto","created_at":"2025-04-22 18:25:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203130,"visible":true,"origin":"","legend":"\u003cp\u003eProduction steps of the disinfectant Cl-DMH-PUSF substrate.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/18ed55cb696ec1e21a65b3af.png"},{"id":81147207,"identity":"656f4968-0f22-4d8c-9ba1-b3e76ac765a0","added_by":"auto","created_at":"2025-04-22 18:25:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":842858,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of FE-SEM images for antiseptic Cl-DMH-PUSF substrates.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/a6fabd4ac4fa99087eca8f65.jpeg"},{"id":81148013,"identity":"44565a72-42eb-45f0-b0a2-5a7fbbb9e936","added_by":"auto","created_at":"2025-04-22 18:41:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":668712,"visible":true,"origin":"","legend":"\u003cp\u003eFE-SEM images of the N-halamine nanoparticles on the polyurethane surface in comparison with the size of various pathogens.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/9a7ec3622b79d1c33bc0e766.jpeg"},{"id":81147212,"identity":"a4a318b2-55ae-460c-a87e-f689c42448a7","added_by":"auto","created_at":"2025-04-22 18:25:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":821893,"visible":true,"origin":"","legend":"\u003cp\u003eThe result of the X-ray map analysis for the Cl-DMH-PUSF surface after 20-times washing processes.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/54dce3051cb666a9e2de50b0.jpeg"},{"id":81147205,"identity":"66257321-7359-4988-b131-4945aee5943c","added_by":"auto","created_at":"2025-04-22 18:25:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":171858,"visible":true,"origin":"","legend":"\u003cp\u003eActivated chlorine test using DPD tablets for the prepared antiseptic substrates.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/07201f55b54c13d5484154b4.jpeg"},{"id":81147206,"identity":"dddc81a4-cbd5-4139-bef0-775aa575582e","added_by":"auto","created_at":"2025-04-22 18:25:47","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":48092,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular structure and difference of stability properties of different amine, amide, and imide N-halamines compounds.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/e6f2edaeee9c1aaa00cfd23d.jpeg"},{"id":81147208,"identity":"6488d818-3a0c-4181-949d-c26e55a84d2a","added_by":"auto","created_at":"2025-04-22 18:25:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":588717,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the DMH molecule attaches to the surface of polyurethane foam and its antibacterial mechanism.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/a0ec56f705b5692f38afa0ea.png"},{"id":89847165,"identity":"9bf84141-d131-44c6-8d8c-65205714e109","added_by":"auto","created_at":"2025-08-25 16:41:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4273684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6383007/v1/c02bae42-deb7-4282-8c3e-d947dd7c31e3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation and Characterization of a Novel Rechargeable Antibacterial Polyurethane Sponge Foam Substrate Modified with Chlorinated N-Halamine for Efficient Disinfection","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe widespread contamination of water and the critical scarcity of freshwater sources are among the most formidable challenges faced by individuals and nations worldwide, primarily due to rapid urbanization and industrialization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The failure to provide clean drinking water for all populations is considered one of the most significant shortcomings of the 20th century [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Increasing pollutant discharge into natural water resources has widened the gap between water availability and the growing demand for freshwater [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite environmental regulations, both groundwater aquifers and surface water sources remain contaminated with various inorganic and organic pollutants, including heavy metals, dyes, and phenolic compounds [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Beyond chemical contaminants, water sources are frequently inhabited by microorganisms and pathogens, leading to a range of acute and chronic illnesses in humans and other living organisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Contaminated drinking water has been linked to numerous health issues, including skin irritation, allergies, headaches, vomiting, and even cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The urgent need for antibacterial and disinfectant compounds to combat pathogens is more critical than ever [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, one of the primary concerns in this field is the increasing resistance of bacteria and pathogens to conventional disinfectants and antibacterial agents [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nearly half of the population in developing countries suffers from waterborne diseases, with over 3\u0026nbsp;million people dying annually due to contaminated water and inadequate disinfection practices [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To address this issue, several antibacterial reagents have been developed, with biocidal polymers, such as N-halamine-based polymers, gaining attention in recent years [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Wang et al.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] synthesized mesoporous composite-supported N-halamines (MCSNs) for water treatment, achieving 60% formaldehyde degradation within 10 minutes under alkaline conditions. MCSNs demonstrated strong antibacterial activity, reducing \u003cem\u003eS.\u003c/em\u003e aureus and E. coli O157:H7 by over 7 logs within 1 minute, highlighting their potential for environmental remediation. In another related research, Zhu et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] synthesized antibacterial N-halamine polymer materials based on protein gelatin sponges (GS-Cl) with oxidative chlorine as the active agent. The GS-Cl materials demonstrated high antibacterial efficiency against E. coli and S. aureus, with adjustable chlorine content and good biocompatibility, effectively promoting wound healing in bacterial infection models. Also, Wu et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] developed a multifunctional antibacterial cotton fabric (QACs/Hals@cotton-Cl) using in-situ free radical copolymerization. The modified cotton exhibited strong antibacterial properties, eradicating \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e within 10 minutes, promoting wound healing, and maintaining durability after multiple uses. The fabric showed excellent hydrophobicity and could be easily recharged with chlorine, indicating its potential for medical and hygiene applications.\u003c/p\u003e \u003cp\u003eN-halamines are known for their long-term stability in both dry environments and aqueous solutions, effectively eliminating a wide range of microorganisms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These compounds can rapidly kill infectious agents, including bacteria, viruses, fungi, and other microorganisms, through the action of halogen atoms within their structure that leading to the inactivation of the N-halamine molecule [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. N-halamine compounds disinfect through two primary mechanisms. The first involves the direct transfer of an oxidized halogen (Cl⁺ or Br⁺) from the N-Cl or N-Br groups to the cell walls of microorganisms, causing oxidative damage and cell death. The second mechanism involves the breaking of N-Cl or N-Br bonds, releasing Cl⁺ or Br⁺ ions into water, which penetrate the microorganism and cause further oxidative damage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. According to the Kirby\u0026ndash;Bauer test, both mechanisms are believed to occur simultaneously [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Traditional water treatment methods often rely on large volumes of chlorine or hypochlorite solutions, which have strong odors and corrosive properties. When these compounds interact with organic matter in water, they can produce carcinogenic by-products such as trihalomethanes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. One promising solution to mitigate these risks is the use of insoluble or immobilized disinfectants. Organic N-halamine compounds, in particular, offer a safer alternative, as they can perform effective disinfection without releasing harmful by-products [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These compounds are available in both solid and water-soluble forms and are known for being stable, odorless, non-corrosive, and non-toxic, making them ideal for disinfecting a wide range of pathogens [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, organic N-halamine compounds were used to modify the surface of polyurethane sponge foam (PUSF) for efficient water disinfection. This approach presents a versatile strategy for surface functionalization by covalently attaching 5,5-dimethylhydantoin (DMH) using 1,6-hexamethylene diisocyanate as a crosslinking agent. Upon exposure to hypochlorite bleach, the N-H bonds in DMH moieties were converted into N-Cl bonds, resulting in durable oxidizing agents capable of eliminating bacteria. The synthesized antiseptic substrates were characterized using energy-dispersive X-ray analysis and field emission scanning electron microscopy. Antibacterial performance tests of the chlorinated DMH-PUSF substrates demonstrated excellent activity against both Gram-positive and Gram-negative bacteria. Additionally, the durability and rechargeability of the Cl-DMH-PUSF substrates were evaluated, with active chlorine content assessed through both quantitative and qualitative methods. The bonds of N-H in the DMH moieties be converted into bonds of N-Cl upon exposure to hypochlorite bleach treatment, which turns into durable oxidizing agents versus bacteria. The characterization of the prepared antiseptic substrates was evaluated by energy dispersive X-ray and field emission scanning electron microscopy. The results of the antibacterial performance evaluation of the chlorinated DMH-PUSF shown that these prepared substrates possessed great antibacterial activity against Gram-positive and negative bacteria. The durability and rechargeability of the Cl-DMH-PUSF substrates toward rinsing were studied. The active chlorine content of the disinfected samples was also measured by both quantitative and qualitative methods.\u003c/p\u003e"},{"header":"2. Experimental procedures","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003e1,6-Hexamethylene diisocyanate (HDI), 5,5-dimethylhydantoin (DMH), and triethylamine (TEA) were obtained from Sigma\u0026ndash;Aldrich. Toluene (Merck, p.a.) was used without further purification. Sodium hypochlorite solution (10% NaOCl), diethyl-p-phenylenediamine (DPD), potassium iodide, sodium thiosulfate standard solution, and starch powder were purchased from Yijishiye Company. The polyurethane sponge foam (PUSF) substrate was supplied by Dow Chemical Company. The bacterial strains used in this study, Escherichia coli and Staphylococcus aureus, were obtained from clinical isolates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of the DMH grafted PUSF (DMH-PUSF) substrate\u003c/h2\u003e \u003cp\u003eThe surface of the polyurethane sponge foam (PUSF) substrate was functionalized to impart disinfectant properties using 5,5-dimethylhydantoin (DMH) as a modifying agent and 1,6-hexamethylene diisocyanate (HDI) as a crosslinking agent. First, 5 grams of PUSF was cut into small pieces and placed under reflux in a balloon flask. Then, 40 ml of toluene, 4 ml of HDI, and 1 ml of triethylamine (TEA) catalyst were added. The mixture was refluxed under a nitrogen atmosphere at 60\u0026deg;C for 20 minutes. The polyurethane segments were then removed, washed with toluene, and dried. In the next step, the PUSF pieces were placed in a separate reflux balloon containing 100 ml of methanol and 10 g of DMH. The mixture was refluxed for 2 hours at 60\u0026deg;C. The resulting product was separated, washed with methanol, and dried [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of the chlorinated DMH grafted PUSF (Cl- DMH-PUSF) substrate\u003c/h2\u003e \u003cp\u003eTo activate the hydantoin-containing polyurethane surface and form N-Cl bonds within the hydantoin structure, the final product (DMH-PUSF) was immersed in a sodium hypochlorite solution (10% NaOCl) for 2 hours at room temperature and pH\u0026thinsp;=\u0026thinsp;7. The activated DMH-PUSF was then washed with distilled water, heated at 45\u0026deg;C, and dried to remove excess chlorine [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Schematics of the performed reactions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These reactions were conducted in three distinct ways, with variations in the amount of TEA catalyst, reaction time, and reflux temperature.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Characterization techniques\u003c/h2\u003e \u003cp\u003eField emission scanning electron microscopy (FESEM, Hitachi S-4160, Japan) was used to characterize the morphological structure of the prepared antiseptic Cl-DMH-PUSF substrates. Energy-dispersive X-ray spectroscopy (EDX, LEO 1530) was employed to perform elemental analysis and detect the presence of chlorine (Cl), oxygen (O), nitrogen (N), and carbon (C) on the surface of the substrates. To evaluate the antibacterial performance and determine the percentage inhibition of bacterial growth, a microplate reader (ELISA) was used for optical density measurements of the prepared samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Measurement of active chlorine in disinfectant samples\u003c/h2\u003e \u003cp\u003eThe active chlorine content in the N-halamine compounds of the disinfected samples was measured using both quantitative and qualitative methods.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. The qualitative measurement of the active chlorine with DPD tablets\u003c/h2\u003e \u003cp\u003eThe chemical method for measuring chlorine levels in water is more practical compared to electronic methods due to its simplicity, cost-effectiveness, and sufficient accuracy. This method allows for the easy determination of both free chlorine and total chlorine concentrations. In the chemical chlorine measurement test, diethyl-p-phenylenediamine (DPD) was used as a reagent. In the presence of chlorine, the DPD reagent changes color, and the chlorine concentration is determined based on the intensity of the resulting dye. Three tests were conducted to evaluate the release of active chlorine from the Cl-DMH-PUSF samples:\u003c/p\u003e \u003cp\u003eIn the first test, a small piece of the Cl-DMH-PUSF sample was immersed in 10 ml of distilled water for 2 hours. After the sponge sample was removed, the remaining water was tested with a DPD tablet to measure chlorine levels.\u003c/p\u003e \u003cp\u003eIn the second test, a DPD tablet was dissolved in 10 ml of distilled water, and a small piece of the Cl-DMH-PUSF sample was immersed in the solution to observe the chlorine release.\u003c/p\u003e \u003cp\u003eIn the third test, a DPD tablet was dissolved in 10 ml of distilled water, and a small piece of the Cl-DMH-PUSF sample, previously dried after undergoing 20 washing cycles, was immersed in the solution to assess the stability of active chlorine content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. The quantitative measurement of the active chlorine amount by iodometric titration\u003c/h2\u003e \u003cp\u003eIodometric titration was used to measure the active chlorine content of the disinfectant solid samples. First, 1 g of potassium iodide was dissolved in 40 ml of distilled water, and the pH of the solution was adjusted to 4. Then, 0.05 g of the solid disinfectant sample was added to the solution and stirred continuously at room temperature for 1 hour. The iodine (I₂) produced in the reaction was titrated with a 0.01 mol/L standardized aqueous solution of sodium thiosulfate in the presence of a starch reagent to determine the active chlorine content of the Cl-DMH-PUSF substrate [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This protocol was also performed with the unchlorinated DMH-PUSF substrate as a control. The active chlorine content of the Cl-DMH-PUSF substrate was calculated using the following equation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003eCl % = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{35.5}{2}\\times\\:\\frac{\\left({V}_{Cl}-{V}_{0}\\right)\\:\\times\\:{\\:10}^{-3}\\:\\times\\:\\:0.01}{{W}_{Cl}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e(1)\u003c/p\u003e \u003cp\u003eWhere V\u003csub\u003eCl\u003c/sub\u003e is the consumed sodium thiosulfate volume (ml) in the Cl-DMH-PUSF titration. V\u003csub\u003e0\u003c/sub\u003e and W\u003csub\u003eCl\u003c/sub\u003e are the consumed sodium thiosulfate volume (ml) in the unchlorinated DMH-PUSF titration and the Cl- DMH-PUSF weight (g), respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Antibacterial functions evaluation\u003c/h2\u003e \u003cp\u003eTo evaluate the antibacterial activity of the Cl-DMH-PUSF substrate, clinical isolates of Escherichia coli and Staphylococcus aureus were used as models for Gram-negative and Gram-positive bacteria, respectively. The bacterial isolates were cultured in a nutrient medium in a rotary incubator at 150 rpm and room temperature until they reached the mid-exponential growth phase. The bacterial cells were then harvested by centrifugation at 600 rpm for 10 minutes and washed three times with phosphate buffer solution [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The washed cells were re-suspended in buffer solution to a final concentration of approximately 10⁸ CFU/ml. A portion of the solid disinfectant sample was cut into smaller pieces, added to distilled water, and placed in an ultrasonic bath to achieve a homogeneous distribution. For the antibacterial test, 50 \u0026micro;l of the bacterial suspension was added to 500 \u0026micro;l of the disinfectant solution and mixed thoroughly. As a control, 50 \u0026micro;l of the bacterial suspension was added to 500 \u0026micro;l of nutrient culture medium without the disinfectant sample. The optical density (OD) of the solutions was measured at 630 nm using a microplate reader [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] before incubation (T₀). The culture media were then incubated at 37\u0026deg;C for 24 hours with constant shaking. After incubation, the OD of the solutions was measured again [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The percentage of antimicrobial activity was calculated using the following equation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003ePercentage inhibition = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:[1-\\frac{{\\text{O}\\text{D}}_{\\:\\text{t}\\text{e}\\text{s}\\text{t}}}{{\\:\\text{O}\\text{D}}_{\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}}]\\text{*}100\\)\u003c/span\u003e\u003c/span\u003e(2)\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of the disinfectant Cl-DMH-PUSF substrates\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the FE-SEM images of the disinfectant Cl-DMH-PUSF substrates at various magnifications. The images reveal both open and closed pores with a thin layer of polyurethane covering the surface. The main walls of the sponge foam are approximately 100 \u0026micro;m thick, while the thin film covering the pores is about 1\u0026ndash;2 \u0026micro;m thick. At higher magnifications, smaller particles can be observed on the polyurethane surface, where needle-shaped nano-corals are visible. These nanoparticles are formed during the chlorination reaction of hydantoin compounds on the surface in the NaOCl solution. These nano-coral assemblies serve as the active disinfectant components on the sponge surface. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows higher magnification images of the N-halamine nano-corals on the sponge surface, which resemble marine corals in morphology. The needle-shaped nanoparticles are approximately 100 nm in length and 20 nm in thickness, with tips measuring about 19 nm in diameter. Also, the size of various microorganisms and pathogens is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Given that the size of bacteria ranges from 1 to a few micrometers and viruses range from 50 to 800 nm, these coral-shaped nano-needles can interact with bacteria or viruses, penetrating their cell bodies and releasing stabilized N-halamine chlorine, leading to pathogen death. The unique nano-coral morphology of these hydantoin compounds provides a high surface area for chlorine uptake, allowing them to store chlorine on the surface for extended periods. One significant advantage of these chlorinated N-halamine compounds is that they eliminate the need for high concentrations of free chlorine in water. Instead, the activated chlorine is stabilized within the solid substrate and is only released upon contact with bacteria. This reduces the risk of forming carcinogenic halo-methane compounds in water. The Cl-DMH-PUSF substrates retain sufficient stabilized chlorine for water disinfection, and when depleted, they can be easily recharged. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the X-ray map analysis of the Cl-DMH-PUSF surface, showing the elemental distribution of key elements, including Cl, O, N, and C. The elemental distribution is represented by colored dots, where each color corresponds to a specific element on the surface of the substrate. The map was obtained from a Cl-DMH-PUSF sample after 20 washing cycles. As seen in the image, chlorine is uniformly and abundantly distributed across the polymer surface, confirming the stability of N-Cl compounds even after repeated washing. Additionally, the Cl-DMH-PUSF substrates appear yellow in color due to the presence of the chlorinated hydantoin layer, which is a direct result of the chlorination process. The yellow color forms as a visual indicator of the N-Cl bonds created during the reaction with NaOCl, demonstrating the successful formation of the active chlorine layer on the polyurethane sponge surface. Notably, the yellow color did not fade after multiple washing cycles, indicating the stability and durability of the Cl-DMH layer on the polyurethane sponge foam surface [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The active chlorine content of antiseptic compounds\u003c/h2\u003e \u003cp\u003eIn the qualitative method to detect the content of the active chlorine in the synthesized antiseptic product, the versatile DPD tablet test was applied. The DPD tablet test is a simple and economical approach to detect the presence of active chlorine in disinfectant materials or water. Since the use of rechargeable disinfection N-halamine systems requires determining the exact time of disinfectant substrate inactivation to reactivate the system with sodium hypochlorite, using the DPD test can be very effective and attractive. DPD tablets are commercially available in various packages and are very affordable. The compound Diethyl-p-PhenyleneDiamine (DPD), in contact with chlorine (N-Cl), quickly changes from a colorless state to a pinkish solution, with the intensity of the color change directly related to the concentration of active chlorine. In commercial samples of DPD tablets, a color change chart in terms of chlorine concentration (ppm) is provided with the product, allowing the operator to determine the approximate chlorine concentration by comparing the solution\u0026rsquo;s color with the chart. The DPD tests were performed on the disinfectant Cl-DMH-PUSF substrate before and after the washing process.\u003c/p\u003e \u003cp\u003eIn the first case, a small piece of the Cl-DMH-PUSF sample was immersed in 10 ml of distilled water for 2 hours. After removing the sponge sample, the remaining water was tested with a DPD tablet. No color change was observed upon dissolving the DPD tablet in this sample, indicating the absence of free active chlorine (Cl\u003csup\u003e+\u003c/sup\u003e) in the water. Therefore, no active chlorine leaks from the solid polyurethane sample.\u003c/p\u003e \u003cp\u003eIn the second case, one DPD tablet was dissolved in 10 ml of distilled water, and a small piece of the Cl-DMH-PUSF sample was dipped into it. After about 30 seconds, the solution turned pink due to the contact of DPD molecules with the active surface of N-halamine in the sponge. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the color change of the solutions in the presence of a polyurethane piece. By comparing the displayed color with the reference color chart, the amount of active chlorine was estimated to be about 0.5 ppm.\u003c/p\u003e \u003cp\u003eTo investigate the covalently bound stability of chlorine in the prepared Cl-DMH-PUSF substrate, the sample was rinsed 20 times, dried, and then tested with the DPD solution. Almost the same amount of discoloration was observed after 30 seconds (with the active chlorine content estimated to be about 0.5 ppm). According to the obtained active chlorine content, it was found that no active chlorine was released from the Cl-DMH-PUSF substrate after multiple washing processes. This test demonstrates the complete stability of the active chlorine on the N-halamine surface, which is not released by washing and only reacts to release Cl\u003csup\u003e+\u003c/sup\u003e upon contact with bacteria and organic molecules [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe accurate measurement of active chlorine content in synthesized disinfectant compounds is possible using iodometric titration. In this method, a potassium iodide solution of a certain concentration reacts with N-halamine active chlorine to form iodine (I\u003csub\u003e2\u003c/sub\u003e), and the exact amount of active chlorine is determined by titration with a standard sodium thiosulfate solution [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The results of the active chlorine content in Cl-DMH-PUSF samples obtained from iodometric titration are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A non-functionalized PUSF sample exposed to chlorination by NaOCl solution, according to the previously mentioned methods, was prepared and examined as a blank sample [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The titration results showed that no active chlorine was present on the non-functionalized PUSF surface, indicating that the polyurethane sponge foam could not be chlorinated alone. The functionalized Cl-DMH-PUSF sample was titrated before and after 20 washing cycles (20w). The results indicated that multiple washes did not cause a significant reduction in chlorine content. The slight decrease in chlorine content after the 20 washing cycles may be attributed to the separation of thin and brittle polyurethane layers formed on the closed pores. During the washing process, some of these brittle films were lost, resulting in a slight reduction in active chlorine content. This issue can be addressed by using stronger foam filters to enhance durability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIodometric titration results in the synthesized solid samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntiseptic sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample Weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSodium thiosulfate concentration (M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSodium thiosulfate volume (ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWeight percentage of active chlorine (w%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-PUSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF (20w)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntiseptic sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample Weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eRemained active chlorine (w%) in chlorinated samples after washing cycles\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-PUSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntiseptic sample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample Weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003ePercentage of active chlorine loading (w%) by rechlorination of samples after washing cycles\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-PUSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antibacterial activity\u003c/h2\u003e \u003cp\u003eTo investigate the antibacterial potential of the fabricated Cl-DMH-PUSF substrates, the antibacterial activity of these chlorinated N-halamine compounds was evaluated against the Gram-negative bacterium E. coli and the Gram-positive bacterium S. aureus. The obtained results are listed in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, respectively. According to the results, the amount of antiseptic substance used affected the percentage of final antimicrobial activity. As observed, increasing the amount of the substance from 0.16 to 0.21 g caused an increase in antimicrobial activity from 46.43\u0026ndash;48.87%. With higher concentrations of the synthesized substrates, the antimicrobial property increased significantly and could even reach 100%. This effect can be attributed to the presence of vacant spaces and porous walls in the polyurethane sponge foam, which create a large specific surface area for the binding of N-halamine compounds and act as barriers to the biocidal agent's attack [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo achieve the optimal amount of catalyst, the antimicrobial properties of the synthesized samples were evaluated with varying catalyst amounts. The results showed that the optimal catalyst content was 0.2 ml. The synthesized antiseptic substrate demonstrated almost identical antimicrobial activity against both Gram-positive and Gram-negative bacteria. This can be attributed to the high disinfection power of N-halamine compounds, which results in a broad-spectrum extinction of pathogenic agents [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of the antibacterial test for prepared samples against Gram-negative bacteria of E-coli.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntiseptic sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInitial CFU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColony Forming Unit (CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAntibacterial activity (w%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF (1 ml catalyst)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e2.744 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e1.403 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF (0.2 ml catalyst)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e2.744 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e1.470 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF (0.02 ml catalyst)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e2.744 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e1.790 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of the antibacterial test for prepared samples against Gram-positive bacteria of S. aureus.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntiseptic sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInitial CFU\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColony Forming Unit (CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAntibacterial activity (w%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF (1 ml catalyst)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e3.208 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e1.440 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e55.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF (0.2 ml catalyst)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e3.208 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e1.456 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl-DMH-PUSF (0.02 ml catalyst)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e3.208 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e \u003cp\u003e1.890 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e41.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Antiseptic and rechargeability mechanism of the Cl-DMH-PUSF substrates\u003c/h2\u003e \u003cp\u003eIn general, N-halamine compounds are divided into three categories: amine, amide, and imide compounds. The molecular structures of these compounds are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Amine compounds do not have any C\u0026thinsp;=\u0026thinsp;O electron-withdrawing groups, making their N-Cl bonds very stable and resistant to Cl\u003csup\u003e+\u003c/sup\u003e release [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. On the other hand, imide compounds, due to the presence of two adjacent C\u0026thinsp;=\u0026thinsp;O groups near the nitrogen atom, have very weak N-Cl bonds, which are easily broken to release Cl\u003csup\u003e+\u003c/sup\u003e. Among these categories, amide N-halamine compounds exhibit moderate stability with only one adjacent C\u0026thinsp;=\u0026thinsp;O group. In these amide compounds, the stability of the N-Cl bond is sufficient to maintain its disinfectant properties over time while remaining reactive enough to release Cl\u003csup\u003e+\u003c/sup\u003e quickly in the presence of bacteria\u0026mdash;a balance not achieved in amine or imide compounds [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates the order of stability and the release rate of activated chlorine in these different compounds. Amide N-halamines, such as DMH, are more stable than imide compounds but react more effectively with bacteria. The synthesis of the disinfectant Cl-DMH-PUSF substrates is based on the use of HDI as an intermediate agent. This HDI compound forms a strong covalent bond with the polyurethane chain through one of its isocyanate groups, facilitated by the alkaline catalyst TEA. The other isocyanate group of HDI, which is highly reactive, binds to the DMH compound. The N-H bonds in the DMH moieties are converted into N-Cl bonds upon exposure to hypochlorite bleach treatment, resulting in durable oxidizing agents effective against bacteria. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e presents a schematic representation of the attachment of DMH molecules to the polyurethane foam surface and their antibacterial mechanism.\u003c/p\u003e \u003cp\u003eAccording to the results obtained from Section \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e, it can be concluded that the disinfection mechanism of this system operates through direct contact with bacteria without releasing chlorine into the water. Therefore, to determine the exact time of inactivation of the disinfectant substrate in continuous disinfection systems, the user can simply remove a piece of the polyurethane sponge from the disinfection tank and test it with DPD tablets. If necessary, the disinfection tank can be recharged using a NaOCl solution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this research, the N-halamine precursor of the 5,5-dimethylhydantoin was covalently grafted onto the surface of polyurethane sponge foam using 1,6-hexamethylene diisocyanate as a crosslinking agent. Upon hypochlorite bleach treatment, the N-H bonds in the 5,5-dimethylhydantoin moieties on the surface of the polyurethane sponge foam were converted into N-Cl bonds, providing rechargeable, durable, and powerful antimicrobial activity against both Gram-positive and Gram-negative bacteria. After multiple rinsing processes, the chemical method using Diethyl-p-PhenyleneDiamine (DPD) tablets was employed to evaluate the washing stability of the prepared Cl-DMH-PUSF substrates. The results showed that no active chlorine was released from the antiseptic Cl-DMH-PUSF substrate after repeated washing, indicating robust stability of the active chlorine on the N-halamine surface. The prepared antiseptic substrates were characterized through FESEM, EDX, DPD tests, and iodometric titration. Notably, the antimicrobial samples prepared in this work are rechargeable, and the inactivation time of the antiseptic substrates can be easily monitored using DPD tablets. If the samples become inactive, they can be recharged via additional chlorination treatments. The obtained results suggest that the prepared antiseptic Cl-DMH-PUSF substrates have significant potential for long-term antimicrobial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAli Ashraf Derakhshan was the primary author of this study.Ali Akbar Zinatizadeh was the supervisor of this study.Ali Rostami wrote and edited the article and checked the data's validity. Fariba Oulad, Khosro Chehri, and Mozhgan Fathi Delphani also edited the article.All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOulad, F. et al. 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Fibers Fabr.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (2), 155892501501000217 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antibacterial, N-Halamine, Polyurethane, Rechargeable, Chlorination","lastPublishedDoi":"10.21203/rs.3.rs-6383007/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6383007/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis research offers a simple and convenient strategy for surface decoration of polyurethane sponge foam through the covalent attachment of 5,5-dimethylhydantoin as an N-halamine precursor using 1,6-hexamethylene diisocyanate as a crosslinking agent. After hypochlorite bleach treatment, the N-H bonds in the 5,5-dimethylhydantoin moieties on the polyurethane sponge foam surface change into N-Cl bonds. This modification enhances antibacterial performance against both Gram-positive and Gram-negative bacteria. The washing stability of the prepared Cl-DMH-PUSF substrates was studied after 20 rinsing cycles using the DPD tablet test. The results showed that no active chlorine was released from the Cl-DMH-PUSF substrate after multiple washing processes, implying the strong stability of the active chlorine on the N-halamine surface. To characterize the synthesized antiseptic samples, field emission scanning electron microscopy (FESEM) and energy dispersive X-ray analysis were applied. FESEM images revealed a collection of needle-shaped nanoparticles on the Cl-DMH-PUSF surface, which could act as a needle in contact with bacteria and contribute to pathogen death.\u003c/p\u003e","manuscriptTitle":"Preparation and Characterization of a Novel Rechargeable Antibacterial Polyurethane Sponge Foam Substrate Modified with Chlorinated N-Halamine for Efficient Disinfection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 18:25:42","doi":"10.21203/rs.3.rs-6383007/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-09T19:08:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-09T17:11:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9803040619564592958169977392180063601","date":"2025-06-02T23:17:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T03:31:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324063279730303022330677124819569195873","date":"2025-04-11T08:40:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-11T07:09:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-11T07:08:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-11T06:52:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-11T06:51:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-05T15:43:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b96fc5d9-bcf8-4bb0-8898-c14b19cc03dd","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47047152,"name":"Earth and environmental sciences/Environmental sciences"},{"id":47047153,"name":"Earth and environmental sciences/Environmental social sciences"},{"id":47047154,"name":"Physical sciences/Chemistry"},{"id":47047155,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2025-08-25T16:32:34+00:00","versionOfRecord":{"articleIdentity":"rs-6383007","link":"https://doi.org/10.1038/s41598-025-16711-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-20 16:29:05","publishedOnDateReadable":"August 20th, 2025"},"versionCreatedAt":"2025-04-22 18:25:42","video":"","vorDoi":"10.1038/s41598-025-16711-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16711-0","workflowStages":[]},"version":"v1","identity":"rs-6383007","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6383007","identity":"rs-6383007","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}