The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of Pseudomonas aeruginosa in microbial fuel cell-based biosensors

The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of Pseudomonas aeruginosa in microbial fuel cell-based biosensors | 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 Research Article The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of Pseudomonas aeruginosa in microbial fuel cell-based biosensors Wenguo Wu, Jia Lin, Dayun Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4239406/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Biofilm infections are resistant and seriously harmful to human health, real-time monitoring of the effects of anti-biofilm drugs on biofilms is critical for screening of new anti-biofilm drugs. Microbial fuel cell (MFC)-based biosensor with Pseudomonas aeruginosa as electricigens was constructed. Results The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of P. aeruginosa in MFC-based biosensor were investigated and compared with the anti-biofilm assessment results by crystal violet staining. The gold nanoparticles facilitated planktonic cells growth in anolyte and biofilm formation on the anode, while silver nanoparticles inhibited the growth of both planktonic cells and biofilm. The phenazine secreted in anolyte was decreased with the addition of gold nanoparticles but increased with the addition of silver nanoparticle. In comparison, the biofilm formed on the glass covers in microwell plates by crystal violet staining were inhibited by both of silver and gold nanoparticles. The growth restricted condition in MFC-biosensor and its discharging state resulted in the different response of cells to nanoparticles. Conclusions MFC-based biosensor as a potential method for the assessment of drug susceptibility, the actual cell metabolic activity and biofilm formation in it should be studied for the accurate comprehension of the interaction between drugs and electricigens. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Bacterial infection is a serious hazard to human health. The statistical research of NIH shows that more than 60% of infectious diseases are related to bacterial biofilm[ 1 ], such as periodontitis, diffuse bronchitis, pulmonary cystic fibrosis, valve endocarditis, staphylococcus aureus chronic osteomyelitis and so on[ 2 ]. In addition, implantation of biomaterials and medical devices often fail due to biofilm infection[ 3 ]. A variety of bacteria can form biofilms, such as Pseudomonas aeruginosa , Staphylococcus epidermidis , Streptococcus pneumonia , Klebsiella and so on[ 1 ]. Antibiotics are the main method of clinical treatment of bacterial biofilm infection. However, the resistance of bacteria to antibiotics is greatly enhanced as the biofilms are formed. It makes bacterial biofilm infection recur or even leads to the failure of medical device implantation, which greatly increases the difficulty and cost of treatment[ 4 ]. Screening of new anti-biofilm drugs with new inhibition pathways or targets becomes the hot issue[ 5 ]. The traditional plate colony count method is time-consuming, a high-throughput method based on 96 well plates is used to study the pathogenic biofilm drug resistance[ 6 ]. The process of transferring biofilm is easy to cause pollution and tedious[ 7 ]. The widely used method is to measure the biomass or cell activity of the biofilm by colorimetry and fluorescent probe staining, such as crystal violet staining, Alamar Blue assay and SYTO9 and PI staining[ 8 – 10 ]. Technologies are also established to evaluate the metabolic activity of the biofilm labeled with isotope or fluorescent substrates such as adenosine triphosphate (ATP) bioluminescence[ 11 ] and Raman spectromicroscopy[ 12 ]. However, none of these methods realize real-time monitoring of the interaction between pathogenic biofilms and drugs without labeling. Pseudomonas aeruginosa is an opportunistic pathogen easy to develop multidrug resistance and form biofilm attacking human health[ 13 ]. Various nanoparticles have been exploited as anti-biofilm drugs with new inhibition mechanism due to their intrinsic advantage of high surface-area-to-volume ratio[ 14 ]. Most of these researches were based on the traditional anti-biofilm assessment methods and the effects of nanoparticles on biofilms formation were also not monitored in real-time without labeling[ 15 – 18 ]. It is well known that the formation of biofilm goes through different stages, many important drugs that have a significant inhibitory effect on the process of biofilm formation may be missed. Real-time monitoring of the effects of anti-biofilm drugs on biofilms is essential to screen new anti-biofilm drugs. Microbial fuel cell (MFC) with the ability to directly convert biological signals into electricity signals can be used as biosensors in real-time monitoring of toxicity[ 19 ], BOD[ 20 ], DO[ 21 ], volatile fatty acid[ 22 ] and so on[ 19 ]. MFC-based biosensors for drug sensitivity test of Staphylococcus aureus and Escherichia coli to beta-lactam antibiotics[ 23 ], Shewanella loihica PV-4 to tobramycin and mixed-culture biofilm from wastewater to tobramycin[ 24 , 25 ]. P. aeruginosa is also a typical extracellular electrogenic strain, which can secrete a variety of phenazine compounds as electronic mediators to promote extracellular electron transfer between bacterial cells and electrodes in MFCs[ 26 ]. MFC-based biosensor with genetically modified P. aeruginosa as electricigen was developed to detect 3, 5-dichlorophenol in water[ 27 ]. The MFC-based biosensors were also suggested to have the potential in the assessment of the metabolic activity in label-free pathogenic biofilms[ 28 ]. However, most of these researches were focused on the relationship between drug concentrations and electrical signals of MFC-based biosensors, ignoring the actual cell growth and cell metabolism in biosensors. As we known, the environment of cell metabolism and biofilm formation in MFC-based biosensor and microwell plates by the classical crystal violet staining method was significantly different. In this paper, the effect of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) on the cell metabolic activity and biofilm formation of P. aeruginosa in MFC-based biosensors were studied and compared with the anti-biofilm assessment results by crystal violet staining. Results and discussion Particle size analysis of gold and silver nanoparticles The TEM diagram and particle size distribution of silver and gold nanoparticles were shown in Fig. 1 . Silver and gold nanoparticles are basically circular in shape and uniform in size. The average particle sizes of silver and gold nanoparticles are 25.70 ± 3.55 nm and 15.90 ± 1.4 nm, the particle sizes are normally distributed. Effect of silver and gold nanoparticles on the cell growth, cell activity and metabolic activity of P. aeruginosa in MFC-based biosensors The effect of silver and gold nanoparticles on the cell growth, cell activity and metabolic activity of P. aeruginosa in MFC-based biosensors were studied. The optical density of the planktonic cells in anodic chamber of MFC-based biosensors was monitored at different discharging time. The planktonic cells co-cultured with gold nanoparticles exhibited similar growth curves with blank control cells, the cell densities were greatly increased with discharging time and the optical density of cells at the concentration of 20 µg/mL gold nanoparticles reached the highest value. It indicated that gold nanoparticles facilitated the growth of planktonic cells in anolyte. Similar result was reported that gold nanoparticles even at concentrations of 150 µM promoted the growth of S. oneidensis in planktonic cells[ 32 ]. When silver nanoparticles were added into the biosensors, the growth curves of planktonic cells were increased in the early stage and then dramatically decreased to a low value in the later stage. These results suggested that the growth of planktonic cells in anolyte were inhibited by silver nanoparticles and the inhibition was decreased with the increase of silver nanoparticles concentration. Water soluble tetrazolium (WST) can be reduced by dehydrogenase to orange yellow water-soluble formaldehyde in the presence of electron coupling reagents and the amount of formaldehyde generated is proportional to that of living cells[ 33 ]. As shown in Fig. 3 , the activity of planktonic cells without the addition of nanoparticles and co-cultured with silver nanoparticles exhibited similar growth curve with low values. When gold nanoparticles were added into the biosensors, the cells activity was steadily increased to the maximum value and then dramatically dropped below zero after 96 h. The low cell activity was ascribe to the excessive consumption of nutrients in anolyte by the mass growth of planktonic cells. Phenazine is an extracellular electron transfer mediator secreted by P. aeruginosa cells that plays an important role in the electricity production efficiency of cells[ 26 ]. As shown in Fig. 4 , the concentration of phenazine secreted by P. aeruginosa cells without the addition of nanoparticles was increased in the early stage and then decreased in the later stage, while those secreted by cells co-cultured with silver nanoparticles were kept on growing until over the blank control value at the later stage and decreased with the increase of silver nanoparticles concentration. When gold nanoparticles were added into biosensors, the secretion of phenazine was increased in 48 h and then gradually decreased much lower than that of blank control cells. The cells co-cultured with 10 µg/mL gold nanoparticles produced the highest concentration of phenazine in 48 h. Effect of silver and gold nanoparticles on the biofilm formation of P. aeruginosa on the anodes of in MFC-based biosensors P. aeruginosa biofilms formed on the anodes of biosensors were further verified. As shown in Fig. 5 , the protein content of biofilm attached on the surface of carbon brush anode was significantly decreased with the addition of silver nanoparticles and did not showed obvious difference between different concentrations. However, that of biofilm co-cultured with gold nanoparticles was increased with the increase of gold nanoparticles concentration and an extremely high amount of protein content was harvested on the electrode at the concentration 40 µg/mL. These results suggested that gold nanoparticles promoted the formation of biofilms on anode while silver nanoparticles inhibited biofilm formation. The SEM results of biofilms attached on the anodes were in accordance with the determination of protein content results. As shown in Fig. 6 , the amount and morphology of biofilms formed on the anodes of biosensors were significantly different with that of biofilms formed on the cover glasses in microwell plates. Cells attached on the electrode surface with or without the presence of nanoparticles were all slim and elongate. The addition of nanoparticles only affected the number of attached cells on the electrode. A thin biofilm with several cells sparsely attached on the anode without the addition of nanoparticles. When silver nanoparticles were added into the biosensor, no biofilm was formed on the anode except single cell. However, the formation of biofilm on the anode was promoted by gold nanoparticles and increased with the increase of gold nanoparticles concentration, almost all the fibers of carbon brush anode were covered with dense biofilm packed with cells at the concentration of 40 µg/mL. The amount of gold nanoparticles with good biocompatibility adsorbed on the surface of carbon brush was increased with gold nanoparticles concentration, which played an important role in the promotion of biofilm formation on the anodes. Similar results were reported that more bacteria adhered to gold nanoparticles modified anodes in MFCs[ 34 – 36 ]. These results were accordance with the cell growth and cell activity results of planktonic cells. It could be concluded that gold nanoparticles facilitated both planktonic cells growth in anolyte and biofilm formation on the anode, while silver nanoparticles inhibited the growth of both planktonic cells and biofilm. Interestingly, the low concentrations of phenazine were detected in the biosensors with the thick biofilms on the anodes. It was reported that the phenazine secreted by cells could be adsorbed on the surface of biofilm and lead to the low concentration of phenazine in anolyte[ 37 , 38 ]. Effect of silver and gold nanoparticles on the biofilm formation of P. aeruginosa in microwell plates by crystal violet staining The effect of silver and gold nanoparticles on biofilm formation of P. aeruginosa was also studied by traditional crystal violet staining method in microwell plates. The crystal violet staining was an end-point method, therefore the biofilms co-cultured with different concentrations of silver and gold nanoparticles after 24 h were evaluated. As shown in Fig. 7 , the inhibition of silver nanoparticles on the growth of P. aeruginosa biofilm was increased with the increase of silver nanoparticles concentration. When the concentration of silver nanoparticles was 40 µg/mL, the inhibitory effect on the biofilm formation of P. aeruginosa was the most significant and the inhibition ratio was 72.7%. It was ascribed to the antibacterial effect of silver nanoparticles. With the increase of silver nanoparticles concentration, the number of cells decreased, leading to the reduction of biofilm formation. Compared with no obvious inhibition on biofilm at the concentration of 10 µg/mL silver nanoparticles, the biofilm formation was greatly inhibited by gold nanoparticles with an inhibition ratio of 21.4%. It was reported that metal-based nanoparticles with smaller particle size had stronger antibacterial effect[ 39 ]. The anti-biofilm effect of gold nanoparticles with smaller particle size was more obvious than that of silver nanoparticles at the low concentration. The antibacterial activity of gold nanoparticles was ascribed to the collapsed membrane potential and the inhibition of the subunit of ribosome from binding tRNA[ 30 ]. Then the biofilm inhibition was gradually decreased with the increase of gold nanoparticles concentration, the biofilm inhibition ratio of gold nanoparticles was only 14.3% at the concentration of 40 µg/mL. It could ascribe to the agglomeration effect of gold nanoparticles at high concentration blocking the entry of gold nanoparticles with small particle size into cells and leading to the low inhibition ratio of biofilm. The SEM images of P. aeruginosa biofilms on the cover glasses co-cultured with different concentrations of silver and gold nanoparticles in microwell plates after 24 h were shown in Fig. 8 . A thick biofilm with closely arranged cells in plump and short rod shape were formed on the surface of the cover glass without the addition of nanoparticles. With the increase of silver nanoparticles concentration, the number of cells in biofilm was decreased and the distance between them was increased, the shape of cells also became slim and elongate. It was most obvious at the concentration of 40 µg/mL silver nanoparticles. These results indicated the anti-biofilm activity of silver nanoparticles. Compared with the formed thick biofilm without the addition of nanoparticles, a loosed biofilm with widen cell spaces was formed at the concentration of 10 µg/mL gold nanoparticles. When the concentration of gold nanoparticles increased to 20 µg/mL and 40 µg/mL, the cells in biofilms seemed swollen and cells space recovered closely arrangement. It indicated that the low concentration of gold nanoparticles could inhibit biofilm formation and the inhibition was reduced with the increased concentration. It was ascribe to the agglomeration of gold nanoparticles into large particle size at high concentration which prevented them from entering cells and then reduced their antibacterial abilities. According to the quantitative results of crystal violet and SEM images, the inhibitory effects of silver nanoparticles and gold nanoparticles on P. aeruginosa biofilms at the concentration of 20 µg/mL was indistinguishable. The anti-biofilm effects of silver and gold nanoparticles at this concentration were further verified by the CLSM diagrams. As shown in Fig. 9 , the biofilm without the addition of nanoparticles was thick and covered a large area on the cover glass. After the addition of silver nanoparticles, the biofilm became thinner and the cell distance was increased. When the same concentration of gold nanoparticles was added, only part of area was covered with biofilm, no obvious biofilm formation was observed on the other area. These results verified that gold nanoparticles had stronger inhibition on P. aeruginosa biofilm than silver nanoparticles at the concentration of 20 µg/mL. The anti-biofilm results of silver and gold nanoparticles against P. aeruginosa cells in microwell plates and biosensor were compared. For the crystal violet staining results, the P. aeruginosa biofilms formed on glass covers were inhibited by both silver and gold nanoparticles after 24 h. The anti-biofilm performance of silver nanoparticles was a slightly weak than that of gold nanoparticles at low concentration, while it became the most obvious at high concentration. However, the anti-biofilm results were different in MFC-based biosensors. After 120 h of discharging time, the biofilm formed on the anodes were facilitated by gold nanoparticles and inhibited by silver nanoparticles and these phenomena were obvious even at low concentration. It seemed that the biofilm formation of P. aeruginosa cells in MFC-based biosensors was more sensitive to nanoparticles than that in microwell plates by crystal violet staining. The growth of Escherichia coli and Bacillus subtilis was significantly decreased at 20 ppm of Au(III) in LB liquid medium[ 40 ], while the G. sulfurreducens biofilm showed higher metabolic activity in MFC at this concentration[ 41 ]. The removal of 3-amino-5-methylisoxazole in MFC was faster than that in the open-circuit reactors. As we know, the biosensor should be in discharging state to realize real-time monitoring of the anti-biofilm effect of nanoparticles through the output of electrical signals. It suggested that the biosensor was in the state of current generation and under the growth restricted condition (anaerobic and electrolyte medium with glucose as substrate), which was different from the full cultured condition (aerobic and LB medium) in microwell plates by crystal violet staining. This different culture condition could change the oxidative stress[ 41 ] and ATP level of the MFC microbe[ 42 ] and leaded to the different response of cells to nanoparticles. When MFC-based biosensors were used for drug sensitivity test, not only the relationship between drug concentration and electrical signal, but also the actual cell growth, cell metabolism or biofilm formation in the biosensors should be systematically studied for the full comprehension of the interaction between drugs and electricigens Conclusions MFC-based biosensor has the potential in screening of new anti-biofilm drugs by real-time monitoring the effect of anti-biofilm drugs on biofilms through its discharging signals. In this paper, MFC-based biosensor with P. aeruginosa as electricigens was constructed. The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of P. aeruginosa in it were studied and compared with the anti-biofilm assessment results by crystal violet staining. The cell growth and cell activity of planktonic cells in anolyte were promoted by gold nanoparticles and inhibited by silver nanoparticles. The biofilm formed on the carbon brush anode was significantly inhibited at the low concentration of silver nanoparticles but greatly promoted by gold nanoparticles. The secreted phenazine could be absorbed on the surface of the thick biofilm and resulted in the low concentration of phenazine in anolyte. In comparison, the biofilm formed on the glass covers in microwell plates by crystal violet staining were inhibited by both of silver and gold nanoparticles. These results verified that the different culture condition between different anti-biofilm assessment methods had great impact on the evaluation results. MFC-based biosensor as a potential method for the assessment of drug susceptibility, it is very necessary to study the actual cell or biofilm growth and metabolic activity in it for the accurate comprehension of interaction between drugs and electricigens.. Methods Preparation of gold nanoparticles Gold nanoparticles were prepared by citrate reduction method[ 29 ]. 300 mL of 0.01% HAuCl 4 solution was heat to boiling and added with 10.5 mL of 1% C6H 5 Na 3 O 7 •2H 2 O aqueous solution under vigorously stir, reacted for 15 min until the solution color changing from clear and transparent to purple black and finally wine red. 1 mg/mL silver nanoparticles with a diameter of 25 nm were purchased from Shanghai Huzheng Nanotechnology Co., Ltd. Nanoparticles were dialyzed in deionized water for 48 h, sterilized them through a 0.22 mm filter (Millipore), and kept them at 4 ◦ C for use[ 30 ]. Characterization of silver and gold nanoparticles The transmission electron microscope (TEM, H-7650, HITACHI)) was used to observe the morphology of nanoparticles. The particle size distribution and average particle size were calculated by the software of Nano Measurer. Cell culture Pseudomonas aeruginosa strain (ATCC 9027) was cultivated in 10 mL Luria-Bertani (LB) broth (tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L) at 37 ◦ C, 150 rpm for 10 h. After centrifugation, cells were resuspended in anolyte (Na 2 HPO 4 6 g L − 1 , KH 2 PO 4 3 g/L, NH 4 Cl 1.0 g/L, NaCl 0.5 g/L, MgSO 4 0.2 g/L and CaCl 2 0.08 g/L) with glucose (10 g/L) and adjusted to the appropriate OD 600 value prior to being added into MFCs. Alternatively, the harvest cells were resuspended in fresh LB medium to adjust to the appropriate OD 600 value prior to being added into microwell plates. MFC-based biosensors operation A two chamber MFC (125 mL of each chamber) separated by a proton exchange membrane (PEM, Nafion 117, Dupont) was used in this work. Carbon brushes were used for the electrodes (both of the diameter and the length are 3 cm). The P. aeruginosa cells and silver or gold nanoparticles with a final concentration of 10 mg/mL, 20 mg/mL and 40 mg/mL were added into the anodic chamber filled with anolyte with glucose. The cathodic chamber was filled with 50 mM K 3 [Fe(CN) 6 ] in 0.1 M phosphate buffer solution. The MFC was connected with 1 KΩ external resistor and incubated at 37 ◦ C for power generation test. The output voltages of MFC-based biosensors were monitored continuously using an RBH8251 data acquisition system (Ruibohua, Beijing, China). Characterization of cell growth and cell activity in MFC-based biosensors 2.0 mL of anolyte was taken from anodic chamber at different discharging time. Cell density was measured at 600 nm with a UV-vis spectrophotometer (UV-1800, Mapada Instrument, Shanghai). Then the anolyte was centrifuged at 8000 rpm for 10 min. The supernatant was determined at 370 nm for the evaluation of phenazine concentration secreted by cells. The harvest cells were resuspended in 2 mL fresh LB medium and mixed well. 100 µL of cell suspension and 10 µL of WST-1 solution were added into a 96 well plate and mixed well. After 2 h incubation, the cell activity was detected at 450 nm by microplate reader (SpectraMax 250, Molecular Devices Corporation, USA). Protein content of biofilm on electrode surface The protein content was determined by Bradford spectrophotometer[ 31 ]. After power generation, 0.5 g carbon brush fiber of anode was cut off and soaked in 1 mol L − 1 NaOH solution for 1 h. 20 µL of sample solution and 200 µL of Coomassie Brilliant Blue G-250 were mixed and dyed for 2 min, and then measured the absorbance at 595 nm with a UV-vis spectrophotometer. The absorbance of the sample was compared with the standard curve to calculate its protein content. Morphology observation of the biofilm After power generation, the anode attached with biofilms was rinsed with PBS and immersed in 2.5% glutaraldehyde solution for 4 h. Then it was sequentially dehydrated with different concentration of ethanol (10%, 30%, 50%, 70%, 80%, 95% and 100%) for 10 min, followed by air-drying. The morphology of the biofilm on the anode was observed by Scanning electron microscopy (SEM, SU8010, HITACHI). The morphology of the biofilm on the cover glass in microwell plate was prepared as the same for SEM observation. Crystal violet staining The recovered P. aeruginosa cells were centrifuged and resuspended in fresh LB medium to adjust to the appropriate OD 600 value. 100 µL of cell suspension was co-cultured with 100 µL of fresh LB medium containing silver or gold nanoparticles with a final concentration of 10 mg/mL, 20 mg/mL and 40 mg/mL in microwell plate at 37 ◦ C for 24 h. The culture medium of each pore was removed, rinsed with PBS solution and dried. 250 µL of formaldehyde solution was added, fixed for 5 min and pipeted out. 250 µL of 0.1% crystal violet solution was added, dyed for 20 min and pipeted out. Then it was rinsed with PBS solution to wash the uncombined dye. 250 µL of 95% ethanol solution was added and retained for 5 min to elute crystal violet dye in cells. 150 µL of ethanol eluent for each well was transfer into a new 96 well plate to measure the absorbance at 595 nm with the microplate reader. Laser confocal scanning microscope (CLSM) The cell suspensions were co-cultured with silver or gold nanoparticles and round cover glasses at the bottom of each well at 37 ◦C for 24 h. Sucked out the culture medium, rinsed the well with PBS, dyed with SYTO9 fluorescent dye in dark for 30 min. Carefully taken out the cover glass and placed the attached bacteria face down to a glass slide with quenching agent in the center. The biofilm on the cover glass was observed by CLSM (TCS SP8, Leica, Germany). Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials Not applicable Competing interests The authors declare that they have no competing interests. Funding This work was supported by Natural Science Foundation of Fujian Province (Grant no. 2021J01313). Authors' contributions - provide individual author contribution Wenguo Wu participated in its design and coordination and helped to draft the manuscript. Jia Lin carried out all the experiments. Dayun Yang participated in its design and coordination and helped to revise the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. Authors' information Authors and Affiliations College of Chemical Engineering, Huaqiao University, Xiamen 361021, P.R. 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Chen M, Zhou XF, Liu X, Zeng RJ, Zhang F, Ye J, et al. Facilitated extracellular electron transfer of Geobacter sulfurreducens biofilm with in situ formed gold nanoparticles. Biosens Bioelectron. 2018;108:20–6. Wang L, Liu YL, Ma J, Zhao F. Rapid degradation of sulphamethoxazole and the further transformation of 3-amino-5-methylisoxazole in a microbial fuel cell. Water Res. 2016;88:322–8. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-4239406","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291965926,"identity":"462dd6ed-8ad9-40f0-bc0d-b267eacb0f69","order_by":0,"name":"Wenguo Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDADfgkwxUyCFskZJGsxuEGsFoMbOYaPC34dljO+3bxNgqHCOrGB/ewBQlqMjWf2HTY2u3OsTILhTHpiA09eAl4tZjdyt0nz9hxO3HYjx0yCse1wYoMEjwEhLdt/g7RsngHS8o84LduYeX4cTtwgAdLSQIQW+zPvP0vzNqQbS9xIK7ZIOJZu3MaTg1+LZHta4meeP9Zy/DOSN974UGMt289+Br8WMGBsA1MGDAlAko2wehD4A9UyCkbBKBgFowAbAABgRkWngDN07wAAAABJRU5ErkJggg==","orcid":"","institution":"Huaqiao University","correspondingAuthor":true,"prefix":"","firstName":"Wenguo","middleName":"","lastName":"Wu","suffix":""},{"id":291965927,"identity":"68b65551-6447-4510-9035-1ffed5a7cdcd","order_by":1,"name":"Jia Lin","email":"","orcid":"","institution":"Huaqiao University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Lin","suffix":""},{"id":291965928,"identity":"873ea0ec-6352-4b1b-a4c4-c82674777f63","order_by":2,"name":"Dayun Yang","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dayun","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-04-09 03:20:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4239406/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4239406/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54906727,"identity":"fa7b58c1-d098-4e43-b500-22eee5fe3740","added_by":"auto","created_at":"2024-04-18 11:50:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2037291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEM images and particle size analysis of silver (a, b) and gold nanoparticles (c, d).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/3d1fea55c82e11b786dce9cf.png"},{"id":54906731,"identity":"5df41dbd-738d-4c3c-b657-71c0e9deed58","added_by":"auto","created_at":"2024-04-18 11:50:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe optical density of the planktonic cells co-cultured with different concentrations of nanoparticles in anodic chamber of MFC-based biosensors.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/8ee9221dda9d6c1273000e3f.png"},{"id":54906733,"identity":"6bb4706a-3603-42a9-8e3e-9f09ef54810b","added_by":"auto","created_at":"2024-04-18 11:50:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111710,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cell activity of the planktonic cells co-cultured with different concentrations of nanoparticles in anodic chamber of MFC-based biosensors.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/4590c3d8a41e5b3a23be0329.png"},{"id":54906736,"identity":"1608832f-e9a4-4218-8bb2-3f3c617622c0","added_by":"auto","created_at":"2024-04-18 11:50:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe concentration of phenazine secreted by cells co-cultured with different concentrations of nanoparticles in anodic chamber of MFC-based biosensors.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/477a030e57478af062165b6c.png"},{"id":54906728,"identity":"be79ebda-ff81-42c0-adc8-2655af0f67ae","added_by":"auto","created_at":"2024-04-18 11:50:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":73156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe protein content of biofilms attached on carbon brush anodes with different concentrations of nanoparticles in anodic chamber of MFC-based biosensors.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/202c4947bf75a497b6119771.png"},{"id":54906730,"identity":"05c381ec-09cd-47cb-8041-d8c36f505fcb","added_by":"auto","created_at":"2024-04-18 11:50:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5054565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilms on the anodes of MFC-based biosensors added with different concentrations of silver and gold nanoparticles after 120 h.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/9929f4443036c72e90a93f8c.png"},{"id":54907236,"identity":"a9213848-658b-4b6c-86b9-8c3b235b0682","added_by":"auto","created_at":"2024-04-18 11:58:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":62799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of different concentrations of silver and gold nanoparticles on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm formation by crystal violet staining method.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/04b23603b65a3d6e65c61f76.png"},{"id":54906725,"identity":"6dc4fb80-0b48-46a0-a58b-fdd926025814","added_by":"auto","created_at":"2024-04-18 11:50:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5942401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilms on the cover glasses co-cultured with different concentrations of silver and gold nanoparticles for 24 h.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/a9a1430237330307bd43d782.png"},{"id":54906732,"identity":"e6329a36-d75b-4f30-9090-40edc15a8927","added_by":"auto","created_at":"2024-04-18 11:50:20","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1851965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLSM images of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilms on the cover glasses co-cultured with silver and gold nanoparticles at the concentrations of 20 µg/mL for 24 h.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/2143c34245c81a9f545df379.png"},{"id":57249849,"identity":"973a031c-b7e3-40f5-b323-bedd3af5fad2","added_by":"auto","created_at":"2024-05-28 06:57:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20189039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4239406/v1/535b2274-455c-4289-b8db-95f9e8221275.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of Pseudomonas aeruginosa in microbial fuel cell-based biosensors","fulltext":[{"header":"Background","content":"\u003cp\u003eBacterial infection is a serious hazard to human health. The statistical research of NIH shows that more than 60% of infectious diseases are related to bacterial biofilm[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], such as periodontitis, diffuse bronchitis, pulmonary cystic fibrosis, valve endocarditis, \u003cem\u003estaphylococcus aureus\u003c/em\u003e chronic osteomyelitis and so on[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition, implantation of biomaterials and medical devices often fail due to biofilm infection[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A variety of bacteria can form biofilms, such as \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, \u003cem\u003eStreptococcus pneumonia\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e and so on[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Antibiotics are the main method of clinical treatment of bacterial biofilm infection. However, the resistance of bacteria to antibiotics is greatly enhanced as the biofilms are formed. It makes bacterial biofilm infection recur or even leads to the failure of medical device implantation, which greatly increases the difficulty and cost of treatment[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Screening of new anti-biofilm drugs with new inhibition pathways or targets becomes the hot issue[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The traditional plate colony count method is time-consuming, a high-throughput method based on 96 well plates is used to study the pathogenic biofilm drug resistance[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The process of transferring biofilm is easy to cause pollution and tedious[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The widely used method is to measure the biomass or cell activity of the biofilm by colorimetry and fluorescent probe staining, such as crystal violet staining, Alamar Blue assay and SYTO9 and PI staining[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Technologies are also established to evaluate the metabolic activity of the biofilm labeled with isotope or fluorescent substrates such as adenosine triphosphate (ATP) bioluminescence[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and Raman spectromicroscopy[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, none of these methods realize real-time monitoring of the interaction between pathogenic biofilms and drugs without labeling.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is an opportunistic pathogen easy to develop multidrug resistance and form biofilm attacking human health[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Various nanoparticles have been exploited as anti-biofilm drugs with new inhibition mechanism due to their intrinsic advantage of high surface-area-to-volume ratio[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Most of these researches were based on the traditional anti-biofilm assessment methods and the effects of nanoparticles on biofilms formation were also not monitored in real-time without labeling[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is well known that the formation of biofilm goes through different stages, many important drugs that have a significant inhibitory effect on the process of biofilm formation may be missed. Real-time monitoring of the effects of anti-biofilm drugs on biofilms is essential to screen new anti-biofilm drugs.\u003c/p\u003e \u003cp\u003eMicrobial fuel cell (MFC) with the ability to directly convert biological signals into electricity signals can be used as biosensors in real-time monitoring of toxicity[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], BOD[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], DO[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], volatile fatty acid[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and so on[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. MFC-based biosensors for drug sensitivity test of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e to beta-lactam antibiotics[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003cem\u003eShewanella loihica\u003c/em\u003e PV-4 to tobramycin and mixed-culture biofilm from wastewater to tobramycin[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cem\u003eP. aeruginosa\u003c/em\u003e is also a typical extracellular electrogenic strain, which can secrete a variety of phenazine compounds as electronic mediators to promote extracellular electron transfer between bacterial cells and electrodes in MFCs[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. MFC-based biosensor with genetically modified \u003cem\u003eP. aeruginosa\u003c/em\u003e as electricigen was developed to detect 3, 5-dichlorophenol in water[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The MFC-based biosensors were also suggested to have the potential in the assessment of the metabolic activity in label-free pathogenic biofilms[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, most of these researches were focused on the relationship between drug concentrations and electrical signals of MFC-based biosensors, ignoring the actual cell growth and cell metabolism in biosensors. As we known, the environment of cell metabolism and biofilm formation in MFC-based biosensor and microwell plates by the classical crystal violet staining method was significantly different. In this paper, the effect of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) on the cell metabolic activity and biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e in MFC-based biosensors were studied and compared with the anti-biofilm assessment results by crystal violet staining.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticle size analysis of gold and silver nanoparticles\u003c/h2\u003e \u003cp\u003eThe TEM diagram and particle size distribution of silver and gold nanoparticles were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Silver and gold nanoparticles are basically circular in shape and uniform in size. The average particle sizes of silver and gold nanoparticles are 25.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55 nm and 15.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 nm, the particle sizes are normally distributed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of silver and gold nanoparticles on the cell growth, cell activity and metabolic activity of\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003ein MFC-based biosensors\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of silver and gold nanoparticles on the cell growth, cell activity and metabolic activity of \u003cem\u003eP. aeruginosa\u003c/em\u003e in MFC-based biosensors were studied. The optical density of the planktonic cells in anodic chamber of MFC-based biosensors was monitored at different discharging time. The planktonic cells co-cultured with gold nanoparticles exhibited similar growth curves with blank control cells, the cell densities were greatly increased with discharging time and the optical density of cells at the concentration of 20 \u0026micro;g/mL gold nanoparticles reached the highest value. It indicated that gold nanoparticles facilitated the growth of planktonic cells in anolyte. Similar result was reported that gold nanoparticles even at concentrations of 150 \u0026micro;M promoted the growth of \u003cem\u003eS. oneidensis\u003c/em\u003e in planktonic cells[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. When silver nanoparticles were added into the biosensors, the growth curves of planktonic cells were increased in the early stage and then dramatically decreased to a low value in the later stage. These results suggested that the growth of planktonic cells in anolyte were inhibited by silver nanoparticles and the inhibition was decreased with the increase of silver nanoparticles concentration.\u003c/p\u003e\u003cp\u003eWater soluble tetrazolium (WST) can be reduced by dehydrogenase to orange yellow water-soluble formaldehyde in the presence of electron coupling reagents and the amount of formaldehyde generated is proportional to that of living cells[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the activity of planktonic cells without the addition of nanoparticles and co-cultured with silver nanoparticles exhibited similar growth curve with low values. When gold nanoparticles were added into the biosensors, the cells activity was steadily increased to the maximum value and then dramatically dropped below zero after 96 h. The low cell activity was ascribe to the excessive consumption of nutrients in anolyte by the mass growth of planktonic cells.\u003c/p\u003e \u003cp\u003ePhenazine is an extracellular electron transfer mediator secreted by \u003cem\u003eP. aeruginosa\u003c/em\u003e cells that plays an important role in the electricity production efficiency of cells[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the concentration of phenazine secreted by \u003cem\u003eP. aeruginosa\u003c/em\u003e cells without the addition of nanoparticles was increased in the early stage and then decreased in the later stage, while those secreted by cells co-cultured with silver nanoparticles were kept on growing until over the blank control value at the later stage and decreased with the increase of silver nanoparticles concentration. When gold nanoparticles were added into biosensors, the secretion of phenazine was increased in 48 h and then gradually decreased much lower than that of blank control cells. The cells co-cultured with 10 \u0026micro;g/mL gold nanoparticles produced the highest concentration of phenazine in 48 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of silver and gold nanoparticles on the biofilm formation of\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eon the anodes of in MFC-based biosensors\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms formed on the anodes of biosensors were further verified. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the protein content of biofilm attached on the surface of carbon brush anode was significantly decreased with the addition of silver nanoparticles and did not showed obvious difference between different concentrations. However, that of biofilm co-cultured with gold nanoparticles was increased with the increase of gold nanoparticles concentration and an extremely high amount of protein content was harvested on the electrode at the concentration 40 \u0026micro;g/mL. These results suggested that gold nanoparticles promoted the formation of biofilms on anode while silver nanoparticles inhibited biofilm formation.\u003c/p\u003e\u003cp\u003eThe SEM results of biofilms attached on the anodes were in accordance with the determination of protein content results. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the amount and morphology of biofilms formed on the anodes of biosensors were significantly different with that of biofilms formed on the cover glasses in microwell plates. Cells attached on the electrode surface with or without the presence of nanoparticles were all slim and elongate. The addition of nanoparticles only affected the number of attached cells on the electrode. A thin biofilm with several cells sparsely attached on the anode without the addition of nanoparticles. When silver nanoparticles were added into the biosensor, no biofilm was formed on the anode except single cell. However, the formation of biofilm on the anode was promoted by gold nanoparticles and increased with the increase of gold nanoparticles concentration, almost all the fibers of carbon brush anode were covered with dense biofilm packed with cells at the concentration of 40 \u0026micro;g/mL. The amount of gold nanoparticles with good biocompatibility adsorbed on the surface of carbon brush was increased with gold nanoparticles concentration, which played an important role in the promotion of biofilm formation on the anodes. Similar results were reported that more bacteria adhered to gold nanoparticles modified anodes in MFCs[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These results were accordance with the cell growth and cell activity results of planktonic cells. It could be concluded that gold nanoparticles facilitated both planktonic cells growth in anolyte and biofilm formation on the anode, while silver nanoparticles inhibited the growth of both planktonic cells and biofilm. Interestingly, the low concentrations of phenazine were detected in the biosensors with the thick biofilms on the anodes. It was reported that the phenazine secreted by cells could be adsorbed on the surface of biofilm and lead to the low concentration of phenazine in anolyte[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of silver and gold nanoparticles on the biofilm formation of\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003ein microwell plates by crystal violet staining\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of silver and gold nanoparticles on biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e was also studied by traditional crystal violet staining method in microwell plates. The crystal violet staining was an end-point method, therefore the biofilms co-cultured with different concentrations of silver and gold nanoparticles after 24 h were evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the inhibition of silver nanoparticles on the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm was increased with the increase of silver nanoparticles concentration. When the concentration of silver nanoparticles was 40 \u0026micro;g/mL, the inhibitory effect on the biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e was the most significant and the inhibition ratio was 72.7%. It was ascribed to the antibacterial effect of silver nanoparticles. With the increase of silver nanoparticles concentration, the number of cells decreased, leading to the reduction of biofilm formation. Compared with no obvious inhibition on biofilm at the concentration of 10 \u0026micro;g/mL silver nanoparticles, the biofilm formation was greatly inhibited by gold nanoparticles with an inhibition ratio of 21.4%. It was reported that metal-based nanoparticles with smaller particle size had stronger antibacterial effect[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The anti-biofilm effect of gold nanoparticles with smaller particle size was more obvious than that of silver nanoparticles at the low concentration. The antibacterial activity of gold nanoparticles was ascribed to the collapsed membrane potential and the inhibition of the subunit of ribosome from binding tRNA[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Then the biofilm inhibition was gradually decreased with the increase of gold nanoparticles concentration, the biofilm inhibition ratio of gold nanoparticles was only 14.3% at the concentration of 40 \u0026micro;g/mL. It could ascribe to the agglomeration effect of gold nanoparticles at high concentration blocking the entry of gold nanoparticles with small particle size into cells and leading to the low inhibition ratio of biofilm.\u003c/p\u003e \u003cp\u003eThe SEM images of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms on the cover glasses co-cultured with different concentrations of silver and gold nanoparticles in microwell plates after 24 h were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. A thick biofilm with closely arranged cells in plump and short rod shape were formed on the surface of the cover glass without the addition of nanoparticles. With the increase of silver nanoparticles concentration, the number of cells in biofilm was decreased and the distance between them was increased, the shape of cells also became slim and elongate. It was most obvious at the concentration of 40 \u0026micro;g/mL silver nanoparticles. These results indicated the anti-biofilm activity of silver nanoparticles. Compared with the formed thick biofilm without the addition of nanoparticles, a loosed biofilm with widen cell spaces was formed at the concentration of 10 \u0026micro;g/mL gold nanoparticles. When the concentration of gold nanoparticles increased to 20 \u0026micro;g/mL and 40 \u0026micro;g/mL, the cells in biofilms seemed swollen and cells space recovered closely arrangement. It indicated that the low concentration of gold nanoparticles could inhibit biofilm formation and the inhibition was reduced with the increased concentration. It was ascribe to the agglomeration of gold nanoparticles into large particle size at high concentration which prevented them from entering cells and then reduced their antibacterial abilities.\u003c/p\u003e\u003cp\u003eAccording to the quantitative results of crystal violet and SEM images, the inhibitory effects of silver nanoparticles and gold nanoparticles on \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms at the concentration of 20 \u0026micro;g/mL was indistinguishable. The anti-biofilm effects of silver and gold nanoparticles at this concentration were further verified by the CLSM diagrams. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the biofilm without the addition of nanoparticles was thick and covered a large area on the cover glass. After the addition of silver nanoparticles, the biofilm became thinner and the cell distance was increased. When the same concentration of gold nanoparticles was added, only part of area was covered with biofilm, no obvious biofilm formation was observed on the other area. These results verified that gold nanoparticles had stronger inhibition on \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm than silver nanoparticles at the concentration of 20 \u0026micro;g/mL.\u003c/p\u003e\u003cp\u003eThe anti-biofilm results of silver and gold nanoparticles against \u003cem\u003eP. aeruginosa\u003c/em\u003e cells in microwell plates and biosensor were compared. For the crystal violet staining results, the \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms formed on glass covers were inhibited by both silver and gold nanoparticles after 24 h. The anti-biofilm performance of silver nanoparticles was a slightly weak than that of gold nanoparticles at low concentration, while it became the most obvious at high concentration. However, the anti-biofilm results were different in MFC-based biosensors. After 120 h of discharging time, the biofilm formed on the anodes were facilitated by gold nanoparticles and inhibited by silver nanoparticles and these phenomena were obvious even at low concentration. It seemed that the biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e cells in MFC-based biosensors was more sensitive to nanoparticles than that in microwell plates by crystal violet staining. The growth of \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e was significantly decreased at 20 ppm of Au(III) in LB liquid medium[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], while the \u003cem\u003eG. sulfurreducens\u003c/em\u003e biofilm showed higher metabolic activity in MFC at this concentration[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The removal of 3-amino-5-methylisoxazole in MFC was faster than that in the open-circuit reactors. As we know, the biosensor should be in discharging state to realize real-time monitoring of the anti-biofilm effect of nanoparticles through the output of electrical signals. It suggested that the biosensor was in the state of current generation and under the growth restricted condition (anaerobic and electrolyte medium with glucose as substrate), which was different from the full cultured condition (aerobic and LB medium) in microwell plates by crystal violet staining. This different culture condition could change the oxidative stress[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and ATP level of the MFC microbe[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and leaded to the different response of cells to nanoparticles. When MFC-based biosensors were used for drug sensitivity test, not only the relationship between drug concentration and electrical signal, but also the actual cell growth, cell metabolism or biofilm formation in the biosensors should be systematically studied for the full comprehension of the interaction between drugs and electricigens\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eMFC-based biosensor has the potential in screening of new anti-biofilm drugs by real-time monitoring the effect of anti-biofilm drugs on biofilms through its discharging signals. In this paper, MFC-based biosensor with \u003cem\u003eP. aeruginosa\u003c/em\u003e as electricigens was constructed. The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e in it were studied and compared with the anti-biofilm assessment results by crystal violet staining. The cell growth and cell activity of planktonic cells in anolyte were promoted by gold nanoparticles and inhibited by silver nanoparticles. The biofilm formed on the carbon brush anode was significantly inhibited at the low concentration of silver nanoparticles but greatly promoted by gold nanoparticles. The secreted phenazine could be absorbed on the surface of the thick biofilm and resulted in the low concentration of phenazine in anolyte. In comparison, the biofilm formed on the glass covers in microwell plates by crystal violet staining were inhibited by both of silver and gold nanoparticles. These results verified that the different culture condition between different anti-biofilm assessment methods had great impact on the evaluation results. MFC-based biosensor as a potential method for the assessment of drug susceptibility, it is very necessary to study the actual cell or biofilm growth and metabolic activity in it for the accurate comprehension of interaction between drugs and electricigens..\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of gold nanoparticles\u003c/h2\u003e \u003cp\u003eGold nanoparticles were prepared by citrate reduction method[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. 300 mL of 0.01% HAuCl\u003csub\u003e4\u003c/sub\u003e solution was heat to boiling and added with 10.5 mL of 1% C6H\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026bull;2H\u003csub\u003e2\u003c/sub\u003eO aqueous solution under vigorously stir, reacted for 15 min until the solution color changing from clear and transparent to purple black and finally wine red. 1 mg/mL silver nanoparticles with a diameter of 25 nm were purchased from Shanghai Huzheng Nanotechnology Co., Ltd. Nanoparticles were dialyzed in deionized water for 48 h, sterilized them through a 0.22 mm filter (Millipore), and kept them at 4 \u003csup\u003e◦\u003c/sup\u003eC for use[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of silver and gold nanoparticles\u003c/h2\u003e \u003cp\u003eThe transmission electron microscope (TEM, H-7650, HITACHI)) was used to observe the morphology of nanoparticles. The particle size distribution and average particle size were calculated by the software of Nano Measurer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain (ATCC 9027) was cultivated in 10 mL Luria-Bertani (LB) broth (tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L) at 37 \u003csup\u003e◦\u003c/sup\u003eC, 150 rpm for 10 h. After centrifugation, cells were resuspended in anolyte (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 6 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 3 g/L, NH\u003csub\u003e4\u003c/sub\u003eCl 1.0 g/L, NaCl 0.5 g/L, MgSO\u003csub\u003e4\u003c/sub\u003e 0.2 g/L and CaCl\u003csub\u003e2\u003c/sub\u003e 0.08 g/L) with glucose (10 g/L) and adjusted to the appropriate OD\u003csub\u003e600\u003c/sub\u003e value prior to being added into MFCs. Alternatively, the harvest cells were resuspended in fresh LB medium to adjust to the appropriate OD\u003csub\u003e600\u003c/sub\u003e value prior to being added into microwell plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMFC-based biosensors operation\u003c/h2\u003e \u003cp\u003eA two chamber MFC (125 mL of each chamber) separated by a proton exchange membrane (PEM, Nafion 117, Dupont) was used in this work. Carbon brushes were used for the electrodes (both of the diameter and the length are 3 cm). The \u003cem\u003eP. aeruginosa\u003c/em\u003e cells and silver or gold nanoparticles with a final concentration of 10 mg/mL, 20 mg/mL and 40 mg/mL were added into the anodic chamber filled with anolyte with glucose. The cathodic chamber was filled with 50 mM K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] in 0.1 M phosphate buffer solution. The MFC was connected with 1 KΩ external resistor and incubated at 37 \u003csup\u003e◦\u003c/sup\u003eC for power generation test. The output voltages of MFC-based biosensors were monitored continuously using an RBH8251 data acquisition system (Ruibohua, Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of cell growth and cell activity in MFC-based biosensors\u003c/h2\u003e \u003cp\u003e2.0 mL of anolyte was taken from anodic chamber at different discharging time. Cell density was measured at 600 nm with a UV-vis spectrophotometer (UV-1800, Mapada Instrument, Shanghai). Then the anolyte was centrifuged at 8000 rpm for 10 min. The supernatant was determined at 370 nm for the evaluation of phenazine concentration secreted by cells. The harvest cells were resuspended in 2 mL fresh LB medium and mixed well. 100 \u0026micro;L of cell suspension and 10 \u0026micro;L of WST-1 solution were added into a 96 well plate and mixed well. After 2 h incubation, the cell activity was detected at 450 nm by microplate reader (SpectraMax 250, Molecular Devices Corporation, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein content of biofilm on electrode surface\u003c/h2\u003e \u003cp\u003eThe protein content was determined by Bradford spectrophotometer[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. After power generation, 0.5 g carbon brush fiber of anode was cut off and soaked in 1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOH solution for 1 h. 20 \u0026micro;L of sample solution and 200 \u0026micro;L of Coomassie Brilliant Blue G-250 were mixed and dyed for 2 min, and then measured the absorbance at 595 nm with a UV-vis spectrophotometer. The absorbance of the sample was compared with the standard curve to calculate its protein content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMorphology observation of the biofilm\u003c/h2\u003e \u003cp\u003eAfter power generation, the anode attached with biofilms was rinsed with PBS and immersed in 2.5% glutaraldehyde solution for 4 h. Then it was sequentially dehydrated with different concentration of ethanol (10%, 30%, 50%, 70%, 80%, 95% and 100%) for 10 min, followed by air-drying. The morphology of the biofilm on the anode was observed by Scanning electron microscopy (SEM, SU8010, HITACHI). The morphology of the biofilm on the cover glass in microwell plate was prepared as the same for SEM observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCrystal violet staining\u003c/h2\u003e \u003cp\u003eThe recovered \u003cem\u003eP. aeruginosa\u003c/em\u003e cells were centrifuged and resuspended in fresh LB medium to adjust to the appropriate OD\u003csub\u003e600\u003c/sub\u003e value. 100 \u0026micro;L of cell suspension was co-cultured with 100 \u0026micro;L of fresh LB medium containing silver or gold nanoparticles with a final concentration of 10 mg/mL, 20 mg/mL and 40 mg/mL in microwell plate at 37 \u003csup\u003e◦\u003c/sup\u003eC for 24 h. The culture medium of each pore was removed, rinsed with PBS solution and dried. 250 \u0026micro;L of formaldehyde solution was added, fixed for 5 min and pipeted out. 250 \u0026micro;L of 0.1% crystal violet solution was added, dyed for 20 min and pipeted out. Then it was rinsed with PBS solution to wash the uncombined dye. 250 \u0026micro;L of 95% ethanol solution was added and retained for 5 min to elute crystal violet dye in cells. 150 \u0026micro;L of ethanol eluent for each well was transfer into a new 96 well plate to measure the absorbance at 595 nm with the microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLaser confocal scanning microscope (CLSM)\u003c/h2\u003e \u003cp\u003eThe cell suspensions were co-cultured with silver or gold nanoparticles and round cover glasses at the bottom of each well at 37 ◦C for 24 h. Sucked out the culture medium, rinsed the well with PBS, dyed with SYTO9 fluorescent dye in dark for 30 min. Carefully taken out the cover glass and placed the attached bacteria face down to a glass slide with quenching agent in the center. The biofilm on the cover glass was observed by CLSM (TCS SP8, Leica, Germany).\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of Fujian Province (Grant no. 2021J01313).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions - provide individual author contribution\u003c/p\u003e\n\u003cp\u003eWenguo Wu participated in its design and coordination and helped to draft the manuscript.\u0026nbsp;Jia Lin\u0026nbsp;carried out\u0026nbsp;all\u0026nbsp;the\u0026nbsp;experiments.\u0026nbsp;Dayun\u0026nbsp;Yang participated in its design and coordination and helped to\u0026nbsp;revise\u0026nbsp;the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; information\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eCollege of Chemical Engineering, Huaqiao University, Xiamen 361021, P.R. China\u003c/p\u003e\n\u003cp\u003eWenguo Wu, Jia Lin\u003c/p\u003e\n\u003cp\u003eFujian Key Laboratory of Translational Research in Cancer and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, 350108, PR China\u003c/p\u003e\n\u003cp\u003eDayun Yang\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLewis K. Riddle of biofilm resistance. Antimicrob Agents Ch. 2001;45(4):999\u0026ndash;1007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosterton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. 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Water Res. 2016;88:322\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4239406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4239406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBiofilm infections are resistant and seriously harmful to human health, real-time monitoring of the effects of anti-biofilm drugs on biofilms is critical for screening of new anti-biofilm drugs. Microbial fuel cell (MFC)-based biosensor with \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e as electricigens was constructed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e in MFC-based biosensor were investigated and compared with the anti-biofilm assessment results by crystal violet staining. The gold nanoparticles facilitated planktonic cells growth in anolyte and biofilm formation on the anode, while silver nanoparticles inhibited the growth of both planktonic cells and biofilm. The phenazine secreted in anolyte was decreased with the addition of gold nanoparticles but increased with the addition of silver nanoparticle. In comparison, the biofilm formed on the glass covers in microwell plates by crystal violet staining were inhibited by both of silver and gold nanoparticles. The growth restricted condition in MFC-biosensor and its discharging state resulted in the different response of cells to nanoparticles.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMFC-based biosensor as a potential method for the assessment of drug susceptibility, the actual cell metabolic activity and biofilm formation in it should be studied for the accurate comprehension of the interaction between drugs and electricigens.\u003c/p\u003e","manuscriptTitle":"The effects of silver and gold nanoparticles on the cell metabolic activity and biofilm formation of Pseudomonas aeruginosa in microbial fuel cell-based biosensors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-18 11:50:08","doi":"10.21203/rs.3.rs-4239406/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c465f124-5540-47b2-b7f5-33f3178259b7","owner":[],"postedDate":"April 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-28T06:48:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-18 11:50:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4239406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4239406","identity":"rs-4239406","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}