Skip to main content
Download PDF
Download ePub

In vitro evaluation of hypochlorous acid-silver nanoparticle waterline disinfectant for dental unit waterline disinfection

BMC Chemistry volume 19, Article number: 25 (2025) Cite this article

Abstract

Background

This work intended to assess the disinfection efficacy of hypochlorous acid (HA) and silver nanoparticles (AgNP) disinfectants in disinfecting the dental unit waterlines (DUWL) during comprehensive oral treatment and explore their potential applications in the oral medical environment. Methods: Firstly, AgNP solution was prepared and evaluated through X-ray diffraction (XRD), field emission transmission electron microscope (FE-TEM), and stability tests. Subsequently, 15 dental units were selected and randomly assigned to three groups, each receiving a different disinfection method. Specifically, one group (5 units) received HA disinfectant (HA group), one group (5 units) received AgNP disinfectant (AgNP group), and another group (5 units) received a combination of HA and AgNP disinfectant (HA + AgNP group). Bacterial counts before and after disinfection were compared and analyzed at four sites on the dental units: high-speed handpiece tubing, mouthwash, ultrasonic scaler, and three-way syringe.

Results

The growth of biofilm on the waterlines was observed using scanning electron microscopy (SEM) and laser confocal microscopy (LCM). The results indicated that AgNP solution was successfully prepared and demonstrated excellent stability. There was no significant difference in the average weekly number of patients treated across the three groups (P > 0.05). After disinfection, bacterial counts were significantly reduced in all groups. Compared to the HA and AgNP groups, the HA + AgNP group exhibited a markedly lower bacterial count, with statistical significance (P < 0.05). The compliance rates observed during the first disinfection and two weeks post-disinfection were slightly lower in the HA and AgNP groups compared to the HA + AgNP group, although no significant statistical difference was found (P > 0.05). SEM images revealed uneven biofilm plaques on the inner surface of the pipes prior to disinfection, embedded within a dense matrix, while the biofilm was visibly disrupted post-disinfection. LCM software analysis showed that, compared to the HA and AgNP groups, the HA + AgNP group had a significantly lower percentage of live bacteria on the biofilm post-disinfection (P < 0.05).

Conclusion

Compared to any single disinfectant regimen, the combined use of HA and AgNPs effectively inhibited bacterial growth and exerted a significant destructive effect on biofilms. Therefore, this combination is expected to be a viable option for disinfection of DUWL in the oral healthcare setting.

Clinical trial number

Not applicable.

Peer Review reports

Background

With the continuous advancement of dental medicine, the dental unit, as a core equipment in the oral diagnostic and treatment process, has become an indispensable part of clinical dental practice. Integrating various advanced medical equipment and technologies, the dental unit’s intelligent control system allows dentists to conveniently adjust the functions of the treatment station, thereby better meeting the personalized treatment needs of different patients [13]. With the continuous advancement of oral medicine and technology, dental unit waterlines (DUWLs) have become an integral part of dental clinical practice. However, the contamination of DUWLs poses a significant threat to patient safety and the hygiene of the medical environment. In addition to waterline contamination, the management of aerosols generated during dental procedures has gained increasing attention, especially in the context of the COVID-19 pandemic and its aftermath. Dental procedures typically produce aerosols that may contain bacteria, viruses, and other microorganisms. These aerosols can remain suspended in the air for extended periods, posing a risk of cross-infection between patients and healthcare providers. Effective aerosol management is critical for reducing the transmission of pathogens, particularly in enclosed clinical environments. Various strategies have been proposed to manage aerosols, including the use of high-efficiency particulate air (HEPA) filters, improved ventilation systems, and the application of effective disinfectants [12].

However, as time progresses, the issue of contamination in DUWL has become increasingly prominent [2]. Waterline contamination can originate from various sources, including the formation of biofilm on the inner walls of the pipes, residual oral treatment fluids, bacteria, and microorganisms in the patient’s oral cavity, among others [23]. The presence of these contaminants may lead to blockages in the waterline system, bacterial growth, posing a potential threat to patient treatment safety and the hygiene of the medical environment, and increasing the risk of patient infections [3]. Despite the establishment of relevant standards and regulations, contamination issues with DUWL still occur in practical operation, exposing some vulnerabilities in the maintenance and management of treatment stations.

To address these issues, effective contamination control and disinfection methods have become essential for maintaining the hygiene and safety of the oral healthcare environment. In recent years, researchers and healthcare professionals have focused on developing and refining various disinfection strategies for DUWL. Current research indicated that physical disinfection has limited effectiveness in controlling DUWL contamination [14], while chemical disinfectants show better results. Common chemical disinfectants include hydrogen peroxide, chlorite, and chlorine-containing disinfectants. These disinfectants effectively eliminate microorganisms without significant corrosion or damage to the materials and equipment of the waterline system [16, 26]. Among them, hypochlorous acid (HA) disinfectant, as a broad-spectrum antimicrobial chemical, is favored for its efficient bactericidal effect, low toxicity, and environmental friendliness [27]. Additionally, with the development of nanotechnology, the application of silver nanoparticles (AgNPs) in disinfectants has been extensively researched. AgNP demonstrates advantages such as improved disinfection effectiveness, reduced dosage, and prolonged duration [15]. Compared to traditional disinfectants, the microscopic scale of AgNP provides a larger surface area, enhancing interactions with microorganisms and achieving more efficient antibacterial effects [5]. Simultaneously, the small size of AgNP particles allows for easier penetration of microbial cell walls, disrupting their biochemical processes and leading to microbial deactivation [7]. In recent years, several studies have explored the applications of nanomaterials in oral medicine. For example, a systematic review [18] summarized the use of different nanoparticles in dental regeneration and found that bioactive glass nanoparticles (BG-NPs) and chitosan nanoparticles (CS-NPs) had a significant impact on the growth and differentiation of human dental pulp stem cells (hDPSCs) and stem cells from apical papilla (SCAPs). Another study [17] evaluated the effects of drug-loaded non-resorbable polymer nanoparticles (TDg-NPs) in mitigating the harmful effects of bacterial lipopolysaccharide (LPS) on hDPSCs. The results demonstrated that TDg-NPs significantly enhanced cell viability, migration, and mineralization potential, effectively counteracting the LPS-induced cellular damage.

However, there is currently limited research on the combined application of HA and AgNP disinfectants, and it is not yet clear how the combined use of these disinfectants affects the disinfection of DUWL. In this study, systematic experiments were conducted to evaluate the bactericidal efficacy of the synergistic action between HA and AgNPs and to investigate their combined inhibitory and biofilm-clearing effects on microorganisms in DUWL. The goal is to develop a safer and more effective waterline disinfection solution for oral healthcare, ultimately contributing to improved hygiene standards in comprehensive dental treatment.

Materials and methods

Primary reagents and instruments for experiments

The primary reagents utilized in this work encompassed phytic acid (PA) and sodium citrate (Shanghai Aladdin Biochemical Technology Co., Ltd., China); silver nitrate (AgNO3) solution (Merck, Germany); triethanolamine (TEA) (Sigma-Aldrich, Germany); glucose solution (Macklin, China); SYTO-9 and propidium iodide (PI) stains (Shanghai Maokang Biotechnology Co., Ltd., China); and HA disinfectant (Manyuanhong Water Technology Co., Ltd., China).

The major instruments employed herein included X-ray diffractometer (XRD, Rigaku Corporation, Japan); transmission electron microscope (TEM), laser confocal microscopy (LCM, Thermo Fisher Scientific (China) Co., Ltd., China); high-speed centrifuge (Sigma-Aldrich, Germany); and dental units (manufactured by Xinuo Medical Equipment Group Co., Ltd., China).

Preparation of AgNP solution

According to the reference [25], 60 mL of 1% phytic acid solution was measured into a three-neck flask, and a magnetic stirrer was activated with a stirring speed set at 500 rpm. The flask was then placed in a 30 °C water bath, and the solution was stirred for 10 min to form stable micelles. Next, 60 mL of 1% silver nitrate solution was slowly added, and the mixture was stirred for an additional 30 min. Subsequently, 60 mL of 10% triethanolamine solution was added, and the pH was adjusted to 9, followed by stirring for 20 min. Then, 60 mL of 1% glucose solution was added, the water bath temperature was raised to 80 °C, and the reaction was continued for 100 min. Finally, 60 mL of 1% sodium citrate solution was added, and the water bath temperature was adjusted to 100 °C. The reaction was stirred for another 80 min. During the reaction, the solution gradually changed from colorless and transparent to a yellow-brown transparent appearance, indicating the successful preparation of AgNPs. After the reaction, the solution was cooled to room temperature and reserved for further use.

Tests of AgNP solution

  1. (1)

    XRD analysis was implemented. The synthesized AgNP powder, obtained by centrifugation and drying of the AgNP solution, was analyzed using X-ray diffraction (XRD) with an X-ray diffractometer operating at 40 kV, with Cu-Kα as the X-ray radiation source. The scan speed was set to 5° per minute, and the 2θ scanning range was from 10° to 90°. The analysis was conducted under a current of 30 mA.

  2. (2)

    Field emission transmission electron microscope (FE-TEM) testing was as follows. The AgNP solution was sonicated and then dropped onto a 230-mesh copper grid. After the water evaporated, the microscopic morphology of AgNP was observed through transmission electron microscopy.

  3. (3)

    Stability testing was divided into two parts: storage time stability and centrifugation stability. For the storage time stability, the AgNP solution was placed in a transparent sample bottle and stored at room temperature. UV absorption spectra of the original solution, as well as the AgNP solution stored for 1 week and 1 month, were measured to assess any changes in stability over time. The maximum absorption wavelength, absorbance, and half-peak width of the UV absorption spectra before and after storage were compared. For the centrifugation stability: the AgNP solution was centrifuged at 6,000 r/min for 50, 100, and 200 min using a high-speed centrifuge. After each centrifugation, the supernatant was taken, and UV absorption spectra were measured for comparison.

Testing objects

In this study, sample size estimation was performed using G*Power. The G*Power was launched, and the test type was selected as “t tests” ->“Means: Difference between two independent means (two groups).” The following parameters were input: effect size (d) = 0.5, α error probability = 0.05, Power (1-β error probability) = 0.80, and Allocation ratio N2/N1 = 1. Based on these parameters, G*Power calculated the required total sample size. The result indicated a total sample size of 15, with 5 samples per group. Fifteen dental units from the Wuhan University Stomatological Hospital were selected as the study subjects and randomly assigned to three groups based on different disinfection methods: the HA group (5 units) received HA disinfectant, the AgNP group (5 units) received AgNP disinfectant, and the HA + AgNP group (5 units) received a combination of HA and AgNP disinfectant. The units included had to satisfy all the following conditions: (i) same brand and batch; (ii) high frequency of use; and (iii) usage duration exceeding one month. The units with abnormal instrument functionality preventing disinfection had to be excluded. To ensure the environmental safety of the disinfectants used, both the HA and AgNP disinfectants selected for this study underwent rigorous safety and environmental impact assessments. Furthermore, the research team carefully controlled the concentration of AgNPs throughout the experiment, ensuring that their effective disinfection capabilities did not lead to negative effects on aquatic and soil ecosystems. To further validate their environmental safety, long-term monitoring of water and soil samples was conducted, with no significant ecotoxicological effects observed. In conclusion, the disinfectants used in this study not only ensured efficient disinfection but also prioritized environmental protection, confirming their safety for the environment.

Since the treatment water from dental units used in clinical settings enters the oral cavity of patients, this experimental study was reviewed and approved by the hospital’s medical ethics committee. Informed consent was obtained from all patients participating in the study.

Disinfection methods

During treatment, the DUWL was equipped with two water inlet valves. The reverse osmosis purified water inlet valve was opened during treatment to supply purified water. At the end of the last treatment of the day, the reverse osmosis purified water inlet valve was closed, and the disinfectant inlet valve was opened to allow for disinfection. In the HA, AgNP, and HA + AgNP groups, the dental units were disinfected using HA disinfectant, AgNP disinfectant, and HA disinfectant before AgNP disinfectant, respectively.

Water sampling

Samples were collected from the outlets of four locations: high-speed handpiece tubing, mouthwash, dental scaler, and three-way syringe. Two samples were collected from each water outlet, sealed with membrane covers in 10 mL sterile collection tubes, and sent for testing within one hour. Strict aseptic principles were adhered to throughout this process.

Collection times were as follows: before disinfection (before flushing the pipes at the beginning of the morning treatment), referred to as M1; after disinfection (immediately after the end of treatment disinfection), referred to as N1 (first week), followed by sampling every 2 weeks for a total of 3 times, labeled as N2 (third week), N3 (fifth week), and N4 (seventh week). The long-term disinfection effectiveness was observed.

Determination of bacterial counts

One loopful of water was aseptically inoculated evenly onto a standard blood agar plate and then incubated in a constant temperature incubator for 48 h at 38 °C with a carbon dioxide concentration of 5.0%. After incubation, the number of colonies was counted, and the colony-forming units (cfu/mL) were calculated. According to the standards set by the American Dental Association (ADA) [19], a bacterial count of ≤ 200 cfu/mL is considered a qualified criterion for dental treatment water.

Microscopic examination

Using a random number generator, two units/groups were randomly selected. Both before and 24 h after disinfection, the experimental sampling personnel, with assistance from a maintenance technician from the hospital’s logistics support department, dismantled the equipment for sampling. Using sterile scissors, they cut a 3 cm segment from the distal end of the plastic water pipe, removing 0.5 cm from each end. The remaining section of the water pipe was the observation sample. The sample was longitudinally sectioned, divided in half, and observed under a SEM. Subsequently, fluorescence staining was conducted: the processed samples were placed on glass slides in a biological safety cabinet. Following the manufacturer’s instructions, dual fluorescence staining with SYTO-9 and PI was performed for 10 min. Subsequently, the samples were rinsed twice with sterile double-distilled water. For each sample, three random points were selected, and LCM was used to scan and reconstruct the biofilm at a thickness of 2 micrometers. Using a 20× lens, dual-channel imaging was employed to observe viable cells (red fluorescence) and dead cells (green fluorescence). Finally, image analysis was performed using the software configured in LCM, and the volume ratio of green fluorescence to total fluorescence indicated the proportion of live cells in the biofilm.

Methods for statistical analysis

Statistical analysis was performed using SPSS 22.0. Categorical data, such as the compliance rate of water samples, were expressed as frequencies or percentages (%), and inter-group comparisons were conducted using the χ2 test. Continuous data, such as treatment unit usage, bacterial counts, and microscopy observations, were presented as mean ± standard deviation (\(\bar x \pm s\)), with inter-group comparisons performed using the t-test. A p-value of < 0.05 was considered statistically significant.

Results and discussion

Testing results of AgNP solution

The XRD analysis results in Fig. 1A revealed five diffraction peaks with no additional impurity peaks, with 2θ angles being determined to be 38.13°, 44.29°, 64.46°, 77.41°, and 81.93°, respectively. Comparing these angles with the standard card JCPDS 04-0783 for silver crystal planes, it can be inferred that face-centered cubic AgNP was successfully prepared.

The FE-TEM observation (Fig. 1B) unveiled that the prepared AgNPs are observed to be encapsulated within a membrane, with an approximate particle size of around 20 nm and good dispersion.

The stability test results (Fig. 1C) indicated that over time, the maximum absorption wavelength and half-peak width of the AgNP solution exhibited no obvious changes. With increasing centrifugation time, the absorbance of the AgNP solution decreased (Fig. 1D), but the half-peak width and the maximum absorption wavelength remained stable.

Fig. 1
figure 1

Testing results of AgNP solution. Note (A): XRD analysis; (B) FE-TEM observation; (C) storage time stability; (D) centrifugation stability

Full size image

Results of microscopic examinations

SEM observation displayed uneven patches of biofilm mixed in a dense matrix on the inner side of the pipe wall (Fig. 2A). After disinfection, the biofilm was visibly disrupted, and residual biofilm structures were apparent in the matrix, as illustrated in Fig. 2B.

Fig. 2
figure 2

SEM observation of biofilm. Note (A) before disinfection; (B) after disinfection

Full size image

Post-staining and observation with LCM revealed that before disinfection, numerous viable bacteria were present, distributed in a punctate or patchy manner. After disinfection, there existed a considerable reduction in viable bacteria, accompanied by an increase in dead bacteria. Among the groups, the HA + AgNP group exhibited the fewest viable bacteria, as depicted in Fig. 3.

Fig. 3
figure 3

HS staining results. Note (A) undisinfected samples; (B) disinfected samples in HA group; (C) disinfected samples in AgNP group; (D) disinfected samples in HA + AgNP group; scale: 20×

Full size image

Results from LCM software analysis signified that the percentage of viable bacteria in the HA + AgNP group was considerably lower when comparing to that in the HA group and AgNP group at each time point (P < 0.05) (Fig. 4).

Fig. 4
figure 4

Percentage of viable bacteria after disinfection. (Group A: HA disinfectant; Group B: AgNP disinfectant; Group C: HA + AgNP disinfectant). Note * and # suggested a great difference with P < 0.05 to the HA group and AgNP group, respectively

Full size image

Usages of the dental units

During the research period, the frequency of using dental units in different groups was depicted in Fig. 5. The average number of patients served per week among the three groups was not greatly different (P > 0.05).

Fig. 5
figure 5

The frequency of using dental units. (Group A: HA disinfectant; Group B: AgNP disinfectant; Group C: HA + AgNP disinfectant)

Full size image

Detected bacterial counts

The bacterial testing results showed that bacterial counts were significantly reduced in all groups after disinfection. Specifically, the bacterial count in Group C (HA and AgNP combined disinfectant) was significantly lower than that in Group A (HA disinfectant) and Group B (AgNP disinfectant) in both the post-disinfection and the subsequent three testing rounds (P < 0.05). Notably, in the final test, Group C showed a bacterial count of 0 cfu/mL at all four locations on the treatment units (high-speed handpiece, mouthwash, ultrasonic scaler, and air-water syringe) (Fig. 6). This result indicates that the combined HA and AgNP disinfectant demonstrated excellent effectiveness in inhibiting bacterial growth and disrupting biofilms.

Fig. 6
figure 6

Bacterial counts in different groups. (Group A: HA disinfectant; Group B: AgNP disinfectant; Group C: HA + AgNP disinfectant). Note (A) bacterial count on the high-speed handpiece; (B) bacterial count in the mouthwash; (C) bacterial count on the ultrasonic scaler; (D) bacterial count on the air-water syringe. * and # suggested a great difference with P < 0.05 to the HA group and AgNP group, respectively

Full size image

Quality of water samples

During the first disinfection and the subsequent measurement two weeks later, the qualification rates of the HA group and AgNP group were slightly lower in comparison to that in the HA + AgNP group. However, from a statistical perspective, no visible difference was observed (P > 0.05). In the subsequent two measurements, the qualification rates for water samples in all groups reached 100% (Fig. 7).

Fig. 7
figure 7

Qualified rate of water samples in different time points. (Group A: HA disinfectant; Group B: AgNP disinfectant; Group C: HA + AgNP disinfectant)

Full size image

Discussion

The dental unit is an indispensable device in modern oral diagnosis and treatment, and the DUWL harbor a complex microbial environment, including various bacteria, fungi, and viruses. The presence of these microorganisms poses potential risks to both patients undergoing oral treatment and healthcare professionals [14]. In practical applications, some disinfection methods may suffer from several shortcomings, leading to microbial residues or the emergence of drug-resistant strains. Therefore, conducting in-depth research on the disinfection of DUWLs, seeking more comprehensive, efficient, and sustainable solutions, is crucial for improving the quality and safety of oral healthcare. HA is a potent oxidant capable of effectively disrupting the cellular structures of bacteria, viruses, fungi, and other microorganisms, achieving the goal of sterilization and disinfection. It has shown immense potential in various fields such as healthcare, food processing, and water treatment [21]. When combined with nanotechnology, AgNP disinfectants, due to their unique physical and chemical properties, exhibit a higher surface area compared to traditional disinfectants, thereby enhancing their potential for interaction with microorganisms. This increased surface area allows AgNPs to more effectively disrupt the cell membranes of microorganisms, significantly improving their antibacterial efficacy [6, 22]. However, there is currently limited research on the combined application of HA and AgNP disinfectants. Therefore, this work delved into the disinfection effects of these two disinfectants when used in combination in DUWL, aiming to yield a more comprehensive understanding of their efficacy. This study aimed to evaluate the effectiveness of HA and AgNP waterline disinfectants in the disinfection of DUWLs. The study design employed rigorous materials and methods to ensure the reliability and scientific validity of the results. First, AgNP solution was successfully prepared using a green synthesis method and was thoroughly characterized through XRD, field emission transmission electron microscopy (FE-TEM), and stability tests, confirming the morphology, size, and stability of the AgNPs. Next, 15 dental units of the same brand and batch were randomly divided into three groups, each using HA disinfectant, AgNP disinfectant, or a combination of HA and AgNP disinfectant for disinfection. Each group consisted of 5 units, ensuring comparability and reproducibility of the experiment. Disinfection procedures and water sample collection followed strict aseptic techniques to guarantee the purity and accuracy of the samples. Bacterial load was assessed using the standard blood agar plate culture method, with evaluations based on the guidelines set by the American Dental Association (ADA). Microscopic observations of biofilm structure and live bacterial proportions were performed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), providing further validation of the disinfection efficacy.

Furthermore, the results indicated the successful preparation of the AgNP solution. Analysis through XRD and FE-TEM revealed that AgNP did not undergo oxidation to form other substances, signifying high purity and good dispersion. Over time, there existed no remarkable changes in the maximum absorption wavelength and half-peak width of the AgNP solution. With increasing centrifugation time, the absorbance of the AgNP solution decreased, suggesting sedimentation due to centrifugal force rather than significant aggregation, as evidenced by the unchanged half-peak width and maximum absorption wavelength. This implies that the prepared AgNP solution exhibits good stability.

The three treatment groups analyzed in this work exhibited similar frequencies of use for the dental units (P > 0.05), which to a certain extent helps avoid result bias. Bacterial detection results suggested that after disinfection, the bacterial counts in the HA + AgNP group and the subsequent three measurements were significantly lower than those in the HA group and AgNP group (P < 0.05). Moreover, in the final measurement, the bacterial counts at all four positions of the treatment unit in the HA + AgNP group reached 0 cfu/mL. HA is a strong oxidizing agent that kills bacteria by disrupting the integrity of bacterial cell walls and membranes, causing leakage of cellular contents. It also has broad-spectrum antimicrobial activity, effectively eliminating a wide range of pathogenic microorganisms. AgNPs possess an extremely high surface area, enabling strong interactions with proteins and enzymes on the bacterial cell surface, thereby interfering with bacterial metabolic processes and leading to cell death. Additionally, AgNPs can penetrate bacterial cell walls and enter the cytoplasm, further damaging the DNA and RNA of the cells, resulting in a deeper level of bactericidal action. The combined use of HA and AgNPs may produce a synergistic effect, where their joint action not only more effectively kills planktonic bacteria but also disrupts biofilm structure, reducing bacterial attachment and proliferation. This synergy provides a significant advantage in inhibiting bacterial growth and eradicating bacteria. Research by Cheng et al. [4] demonstrated the significant bactericidal effect of NS, achieving a sterilization rate of 100%. Jafari et al. [11] investigated the antibacterial efficiency of AgNP against three oral microbes, showing that as the concentration increased, the sterilization efficiency also increased. Regarding the application of HA disinfectant in sterilization, numerous previous studies [1, 8, 9, 24] have confirmed the excellent sterilization effect of HA. In this work, the combined application of these two disinfectants achieved satisfactory results, indicating potential practical value.

However, the water sample qualification rates of the HA group and AgNP group were slightly lower in contrast to that in the HA group, but the difference was neglectable (P > 0.05). This may be due to the relatively small sample size, and further analysis is needed with an increased sample size in the future. Subsequently, the growth of biofilm on the water pipes was observed through a microscope. Previous studies found that AgNP can achieve sterilization and disinfection by disrupting biofilm [10, 20]. Research also found that HA can remove over 99% of biofilm cells [28]. The results of this study show that after disinfection, water pipe biofilm was significantly disrupted. Staining revealed that the HA + AgNP group possessed significantly fewer live bacteria in the biofilm than the other two groups. The LCM software analysis results also suggested that the percentage of live bacteria in the HA + AgNP group was sharply lower in comparison to that in the HA and AgNP groups at each time point, presenting great differences with P < 0.05. Biofilm is a complex structure formed by bacteria and microorganisms, typically attached to solid surfaces such as the inner walls of waterline pipes. The formation of biofilms enhances bacterial resistance to antimicrobial agents, making it difficult for traditional disinfection methods to eliminate them. Microscopic observations revealed that in the HA + AgNP group, the biofilm on the waterline was significantly disrupted after disinfection, and after staining, the biofilm in this group showed a markedly lower number of viable bacteria compared to the other two groups. Analysis using LCM software further showed that the percentage of live bacteria in the HA + AgNP group was significantly lower at all time points compared to the other groups. These findings suggest that the combined application of HA and AgNPs not only effectively eliminates planktonic bacteria but also disrupts biofilm structure, reducing biofilm contamination in the waterline.

In addition to the use of chemical disinfectants, proactive measures involving natural substances are also effective strategies for enhancing disinfection and reducing bacterial concentrations. Probiotics, postbiotics, synbiotics, and ozonated substances have all shown significant effects in reducing bacterial and viral loads. Probiotics are beneficial microorganisms that can reduce the growth of pathogens through competitive inhibition and by producing antimicrobial substances. In oral care, probiotics can help reduce harmful bacteria in the mouth, thereby lowering the risk of infection. Synbiotics refer to processed probiotics that retain their beneficial components but are no longer active. These components continue to exert antimicrobial and immunomodulatory effects. Postbiotics, on the other hand, are the metabolic products of probiotics, such as short-chain fatty acids and lactic acid. These metabolites have antimicrobial and anti-inflammatory properties and can help improve the oral microenvironment. Ozone is a powerful oxidizing agent that effectively eliminates bacteria, viruses, and fungi. Ozonated water or gases can be used for oral cleaning and disinfection, reducing the concentration of pathogenic microorganisms. By incorporating these natural substances, patients can take proactive measures during follow-up visits after their initial consultation to further reduce bacterial and viral loads. For example, patients can use probiotic-containing mouthwashes before and after each treatment, or employ ozonated water for oral hygiene. These measures not only enhance the effectiveness of the treatment but also strengthen the patient’s self-protection, thereby reducing the risk of cross-infection.

Limitations

Although this study provides valuable data, several limitations should be considered. The relatively small sample size and single-source sample selection may affect the generalizability and reliability of the results. A smaller sample size limits statistical power, potentially causing some important differences to go undetected. Additionally, using a single sample source may result in findings influenced by specific demographic characteristics, making it difficult to extrapolate the results to a broader patient population. Furthermore, this study focused solely on antimicrobial efficacy and did not assess other critical factors, such as chemical residue or water quality, which could have potential implications for patient health and the environment. The short duration of the study also limits the ability to fully evaluate the long-term effects of this method on the performance of dental equipment. Therefore, further long-term studies are needed to confirm the stability and safety of these disinfectants.

To overcome these limitations, future studies could involve multi-center research across multiple hospitals or clinics to increase sample size and diversity, thereby enhancing the generalizability and reliability of the results. Extending the duration of the study, with regular data collection, would allow for a more comprehensive assessment of the long-term disinfection effects and the impact on equipment performance. Additionally, high-performance liquid chromatography (HPLC) or other chemical analysis methods could be used to detect residual disinfectants in the waterlines, ensuring that they do not pose a negative risk to patient health. Regular monitoring of water quality indicators, such as pH, conductivity, and microbial content, should also be conducted to ensure compliance with medical standards. Furthermore, molecular biology techniques (e.g., PCR, gene sequencing) and microscopy methods (e.g., SEM, CLSM) could be employed to investigate the mechanisms of biofilm formation and the effects of disinfectants on biofilms, especially the changes that occur with long-term use. Additionally, bacterial resistance to different disinfectants should be monitored, allowing for the evaluation of potential resistance issues arising from prolonged use of the same disinfectant. To evaluate the clinical effectiveness of the disinfectants, patient perceptions and satisfaction following the use of different disinfectants could be collected to assess their impact on the patient experience. Additionally, infection rates in patients using different disinfectants should be monitored to assess the practical clinical significance of the disinfection efficacy.

Conclusion

This study successfully prepared a AgNP solution, which exhibited good stability. The synergistic effect of HA and AgNPs significantly enhanced the disinfection efficacy, effectively eliminating a wide range of bacteria and reducing the microbial load in the waterline. The combined disinfectant not only killed planktonic bacteria but also effectively disrupted biofilm structure, reducing biofilm contamination in the waterline and lowering the risk of infection. Future research should focus on increasing sample size and diversity, conducting multi-center studies to improve the generalizability and reliability of the results. Additionally, extending the study duration and regularly collecting data will allow for the assessment of the long-term effects of the combined disinfectant and its impact on equipment performance. Further in-depth investigation into the mechanisms of biofilm formation and the effects of disinfectants on biofilms is also needed, as well as the evaluation of potential resistance issues arising from the prolonged use of the same disinfectant.

Data availability

The datasets used and/or analyzed in the present study are available from the corresponding author upon reasonable request.

References

  1. Arulmani M, Ramalingam K, Desingu V, Natarajan A, Dharsh S, Rajagopal N. Comparative evaluation of the efficacy of a novel composition based on hypochlorous acid vs sodium hypochlorite in prevention of cross-infection during dental treatment procedures: a pilot study. J Sci Dentistry. 2023;13(2):38–40. https://doiorg.publicaciones.saludcastillayleon.es/10.5005/jp-journals-10083-1039.

    Article  Google Scholar 

  2. Baudet A, Lizon J, Martrette JM, Camelot F, Florentin A, Clément C. Dental unit waterlines: a survey of practices in Eastern France. Int J Environ Res Public Health. 2019;16(21):4242. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijerph16214242.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Castellano Realpe OJ, Gutiérrez JC, Sierra DA, Pazmino Martinez LA, Palacios P, Echeverría YY, G. and, Waard JH. Dental unit waterlines in Quito and caracas contaminated with nontuberculous mycobacteria: a potential health risk in dental practice. Int L J Environ Res Public Health. 2020;17(7):2348. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijerph17072348.

    Article  CAS  Google Scholar 

  4. Cheng L, Naibijiang N, Hasenbai A, Dong H, He H. Bacteriostatic effects of nanometer silver disinfectant on the biofilms in dental unit water lines. J Dent Sci. 2021;16(1):327–32. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jds.2020.03.015.

    Article  PubMed  Google Scholar 

  5. Deshmukh SP, Patil SM, Mullani SB, Delekar SD. Silver nanoparticles as an effective disinfectant: a review. Mater Sci Eng C Mater Biol Appl. 2019;97:954–65. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.msec.2018.12.102.

    Article  CAS  PubMed  Google Scholar 

  6. Dong G, Xiao X, Liu X, Qian B, Ma Z, Ye S, Chen D, Qiu J. Preparation and characterization of Ag nanoparticle-embedded polymer electrospun nanofibers. J Nanopart Res. 2010;12:1319–29. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s11051-009-9665-3.

    Article  CAS  Google Scholar 

  7. Hassan D, Farghali M, Eldeek H, Gaber M, Elossily N, Ismail T. Antiprotozoal activity of silver nanoparticles against cryptosporidium parvum oocysts: new insights on their feasibility as a water disinfectant. J Microbiol Methods. 2019;165:105698. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.mimet.2019.105698.

    Article  CAS  PubMed  Google Scholar 

  8. Hatanaka N, Awasthi SP, Goda H, Kawata H, Uchino Y, Kubo T, Yamasaki S. Chlorous acid is a more potent antibacterial agent than sodium hypochlorite against campylobacter. Food Control. 2020;111:107046. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.foodcont.2019.107046.

    Article  CAS  Google Scholar 

  9. Herruzo R, Herruzo I. Antimicrobial efficacy of a very stable hypochlorous acid formula compared with other antiseptics used in treating wounds: in-vitro study on micro-organisms with or without biofilm. J Hosp Infect. 2020;105(2):289–94. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jhin.2020.01.013.

    Article  CAS  PubMed  Google Scholar 

  10. Huma ZE, Javed I, Zhang Z, Bilal H, Sun Y, Hussain SZ, Ke PC. Nanosilver mitigates biofilm formation via FapC amyloidosis inhibition. Small. 2020;16(21):1906674. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/smll.201906674.

    Article  CAS  Google Scholar 

  11. Jafari A, Fard RMN, Shahabi S, Abbasi F, Shahedin GJ, Bakhtiari R. Optimization of antimicrobial efficiency of silver nanoparticles against three oral microorganisms in irreversible hydrocolloid impressions. Iran J Microbiol. 2021;13(6):862. https://doiorg.publicaciones.saludcastillayleon.es/10.18502/ijm.v13i6.8091.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Ji XY, Fei CN, Zhang Y, Liu J, Liu H, Song J. Three key factors influencing the bacterial contamination of dental unit waterlines: a 6-year survey from 2012 to 2017. Int Dent J. 2019;69(3):192–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/idj.12456.

    Article  PubMed  Google Scholar 

  13. Li C, Wang C. Comprehensive dental treatment table based on user experience research on comfort improvement design. Int Core J Eng. 2020;6(8):127–39. https://doiorg.publicaciones.saludcastillayleon.es/10.6919/ICJE.202008_6(8).0021.

    Article  Google Scholar 

  14. Lizzadro J, Mazzotta M, Girolamini L, Dormi A, Pellati T, Cristino S. Comparison between two types of dental unit waterlines: how evaluation of microbiological contamination can support risk containment. Int J Environ Res Public Health. 2019;16(3):328. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijerph16030328.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Luo H, Li N, Li D, Xu L. Teaching research on using nano silver ion dressing in clinical nursing of surgically infected wounds: nano silver ion dressing in nursing of infected wounds. Cell Mol Biol. 2022;68(3):270–81. https://doiorg.publicaciones.saludcastillayleon.es/10.14715/cmb/2022.68.3.30.

    Article  PubMed  Google Scholar 

  16. Munive-Degregori A, Mayta-Tovalino F, Gallo W, Luza S, Mauricio F, Ilizarbe S, Munive-Méndez A. Evaluation of hydrogen peroxide and cetylpyridinium chloride as bacterial decontaminants of dental unit water lines at a private Peruvian dental school. J Int Soc Prev Community Dent. 2020;10(6):731. https://doiorg.publicaciones.saludcastillayleon.es/10.4103/jispcd.JISPCD_265_20.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Osorio R, Rodríguez-Lozano FJ, Toledano M, Toledano-Osorio M, García-Bernal D, Murcia L, López-García S. Mitigating lipopolysaccharide-induced impairment in human dental pulp stem cells with tideglusib-doped nanoparticles: enhancing osteogenic differentiation and mineralization. Dent Materials: Official Publication Acad Dent Mater. 2024;40(10):1591–601. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.dental.2024.07.012.

    Article  CAS  Google Scholar 

  18. Pecci-Lloret MP, Gea-Alcocer S, Murcia-Flores L, Rodríguez-Lozano FJ, Oñate-Sánchez RE. Use of nanoparticles in Regenerative Dentistry: a systematic review. Biomimetics (Basel Switzerland). 2024;9(4):243. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/biomimetics9040243.

    Article  CAS  PubMed  Google Scholar 

  19. Pereira LJ, Murata RM, Pardi V, Mattos FF. Streamlining the dental care during COVID-19 pandemic: updated clinical recommendations and infection control management framework. Braz Oral Res. 2021;35:e046. https://doiorg.publicaciones.saludcastillayleon.es/10.1590/1807-3107bor-2021.

    Article  PubMed  Google Scholar 

  20. Riau AK, Aung TT, Setiawan M, Yang L, Yam GW, Beuerman R, Mehta J. Surface immobilization of nano-silver on polymeric medical devices to prevent bacterial biofilm formation. Pathogens. 2019;8(3):93. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/pathogens8030093.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Robins LI, Clark A, Gafken PR, Alam S, Milici J, Hassan R, Meyers C. Hypochlorous acid as a disinfectant for high-risk HPV: insight into the mechanism of action. J Med Virol. 2022;94(7):3386–93. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/jmv.27716.

    Article  CAS  PubMed  Google Scholar 

  22. Rosa CG, Narciso AM, Nunes MR, Masiero AV. Applicability of silver nanoparticles and innovation of magnetic nanoparticles in dentistry. Fundam Ind Appl Magn Nanopart. 2022;317–48. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/B978-0-12-822819-7.00023-5.

  23. Spagnolo AM, Sartini M, Cristina ML. Microbial contamination of dental unit waterlines and potential risk of infection: a narrative review. Pathogens. 2020;9(8):651. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/pathogens9080651.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wu M, Shi Z, Yu X, Xu Y, Zhang L, Fu B. Disinfection methods of dental unit waterlines contamination: a systematic review. J Med Microbiol. 2022;71(6):001540. https://doiorg.publicaciones.saludcastillayleon.es/10.1099/jmm.0.001540.

    Article  CAS  Google Scholar 

  25. Xing H, Cheng J, Tan X, Zhou C, Fang L, Lin J. Ag nanoparticles-coated cotton fabric for durable antibacterial activity: derived from phytic acid-Ag complex. J Text Inst. 2020;111(6):855–61. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/00405000.2019.1668137.

    Article  CAS  Google Scholar 

  26. Yue C, Hu Y, Liu Z, Wang Z, Feng J. Study on the disinfection effect of chlorine dioxide disinfectant (ClO2) on dental unit waterlines and its in vitro safety evaluation. BMC Oral Health. 2024;4(1):648. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12903-024-04391-7.

    Article  CAS  Google Scholar 

  27. Zemouri C, Laheij AM, Volgenant CM, Brandt BW, Crielaard W, Buijs MJ, Soet JJ. Chlorine-based DUWL disinfectant leads to a different microbial composition of water derived biofilms compared to H2O2-based chemical disinfectants in vitro. PeerJ. 2020;8:9503. https://doiorg.publicaciones.saludcastillayleon.es/10.7717/peerj.9503.

    Article  Google Scholar 

  28. Zhang Y, Huang HH, Duc HM, Masuda Y, Honjoh KI, Miyamoto T. Endolysin lysSTG2: characterization and application to control salmonella typhimurium biofilm alone and in combination with slightly acidic hypochlorous water. Food Microbiol. 2021;98:103791. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.fm.2021.103791.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

Not applicable.

Author information

Authors and Affiliations

  1. Department of Oral & Maxillofacial Surgery, State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, 430079, China

    Tingting Yin

  2. Department of Dentistry and Endodontics , State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, 430079, China

    Qiaowen Li

  3. Department of Comprehensive Emergency, State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, 430079, China

    Huan Sun

  4. Department of Wusheng Road Outpatient, State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, 430079, China

    Jin Zheng

  5. Center for Oral and Maxillofacial Surgery, State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, 430079, China

    Yuanyuan Wang

  6. Department of Orthognathic & Cleft Lip and Palate Plastic Surgery, State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, 430079, China

    Yi Luo

  7. Nursing Department, State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, 430079, China

    Li Wang

Authors
  1. Tingting Yin
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Qiaowen Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Huan Sun
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Jin Zheng
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Yuanyuan Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Yi Luo
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Li Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

TY, QL, HS, JZ, YW, YL and LW participated the design, supervision and editing, and resources, writing of original draft, experimental implementation, and data statistics and analysis. All authors read and approved final manuscript.

Corresponding author

Correspondence to Li Wang.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yin, T., Li, Q., Sun, H. et al. In vitro evaluation of hypochlorous acid-silver nanoparticle waterline disinfectant for dental unit waterline disinfection. BMC Chemistry 19, 25 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13065-025-01382-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13065-025-01382-9

Keywords

BMC Chemistry

ISSN: 2661-801X

Contact us