Fluorescence analysis of antibacterial activity of ZnO/TiO 2 electrospun nanofibers: A molecular approach to reveals the insights of physiochemical interactions of materials with bacteria

Fluorescence analysis of antibacterial activity of ZnO/TiO 2 electrospun nanofibers: A molecular approach to reveals the insights of physiochemical interactions of materials with bacteria | 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 Fluorescence analysis of antibacterial activity of ZnO/TiO 2 electrospun nanofibers: A molecular approach to reveals the insights of physiochemical interactions of materials with bacteria Ali Hassan, Shahzad Anwar, Rashad Rashid, Rafaqat Ali Khan, Saba Ibrahim, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5222566/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Dec, 2024 Read the published version in Journal of Fluorescence → Version 1 posted 9 You are reading this latest preprint version Abstract Fluorescence spectroscopy employed to compute the antibacterial potential of pure ZnO and Titania (TiO 2 ) loaded ZnO (TiO 2 : 2%, 4%, 6%, and 8%) electrospun nanofibers. The study of electrospun nanofibers followed by their structural, morphological and antibacterial properties has been revealed through fluorescence spectroscopy. X-ray diffraction (XRD) analysis of nanofibers calcinated at 600°C revealed the presence of polycrystalline wurtzite hexagonal crystallographic planes of ZnO with preferred orientation along (101) direction. Scanning electron microscopy (SEM) confirmed that calcination of electrospun nanofibers resulted in smooth and pure ZnO nanofibers due to ethanol evaporation and polyvinylpyrrolidone (PVP) decomposition. Two bacterial strains Escherichia coli and Pseudomonas aeruginosa were used for fluorescence spectroscopy-based evaluation of antibacterial activity of ZnO and TiO 2 -ZnO nanofibers. Agar well technique was employed to investigate the antibacterial activity and functioning mechanism of nanofibers against Escherichia coli and Pseudomonas aeruginosa . The consistent zones of inhibition have been observed for pure ZnO and Titania loaded ZnO nanofibers. Fluorescence spectroscopy revealed the insights of bacterial killing with nanofibers. The mechanistic study of interaction between nanofibers and bacterial cells leads to cell membrane breakdown and confirmed with SEM imaged micrographs. Furthermore, the nanofibers calcinated at 600°C efficiently ruptured the bacteria resulting in higher antibacterial phenomenon as compare to the other nanofiber structures. TiO2/ZnO Electrospinning Nanofibers Fluorescence spectroscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Study of antibacterial activity of metal oxide has got immense attraction because of increasing bacterial infection in the human civilization [ 1 ]. Zinc oxide is one of the most widely used antibacterial metal oxide nanomaterial due to its favorable chemical and physical properties [ 2 – 4 ]. Since ZnO belongs to "Generally Recognized as A Safe (GRAS)" category, therefore it has a great deal of recognition in the field of nanomedicine, food packaging, plasters, painting, cosmetics and sunscreen for UV protection [ 5 – 8 ]. ZnO is suitable for a wide range of biological uses, including the treatment of diabetes, cancer, wound healing, infection, and inflammation because it is less expensive and safer than other metal oxides like SnO 2 and CuO [ 9 – 11 ]. Moreover, ZnO nanoparticles have shown antibacterial action against both Gram-positive and Gram-negative oral bacteria where Gram-negative bacteria are more resistant against ZnO nanoparticles [ 12 ]. Zinc oxide has the ability to combat microorganisms originated viral disease like H1N1, SARS-CoV-2, COVID-19 pandemic, therefore it is very important and essential to investigate possible antiviral properties of Zinc related compounds [ 13 – 15 ]. Nanofibers are preferred for antibacterial applications because of their large surface area and compact size which provide better particle penetration into bacteria and increased adsorption of the packages on the cell membrane. Among several synthesis techniques of nanofibers, electrospinning is one of the straightforward and low-cost technique to synthesize high quality long and continuous fibers of different materials including polymers and metal oxide [ 2 , 3 , 15 , 16 ]. Numerous parameters of the nanostructures such as morphological (particle size, shape), crystalline structure and concentration of inclusion affect the antibacterial activity of ZnO. Embedding a suitable material in metal oxide and formation of composites is a promising approach to enhancing the antimicrobial properties of ZnO for food packaging industries [ 17 ]. It is reported that antibacterial activity index of ZnO increases with the inclusion of TiO 2 atoms in ZnO matrix [ 18 ]. Antiviral mechanism of Zinc is commonly described by one of the following models: inhibition of virus replication, reduction of angiotensin-converting enzyme 2 (ACE2) activity, conditional up regulation of IFN-α (interferon α), inhibition of nuclear factor-kappa B (NF-κB) with an immunologic role, serving as a cofactor for multiple viral proteins, blocking the entry of the virus from the cell membrane, and reduction of the risk of bacterial coinfection by strengthening direct antibacterial effects against Streptococcus pneumonia . But more comprehensive experimental data and clinical research are required in this field [ 14 , 19 ]. ZnO nanoparticles have efficiently been used against a broad spectrum of bacteria and the photocatalytic and antibacterial capabilities of ZnO are mostly determined by its shape [ 20 ]. Their ability to induce the production of reactive oxygen species (ROS), interfere with the function of proteins and DNA, and break bacterial cell membranes is how they exhibit their antimicrobial action [ 21 , 22 ]. In this study, ZnO and TiO 2 -loaded ZnO nanofibers have been synthesized by using electrospinning techniques followed by their morphological and structural characterizations by SEM and XRD techniques. The antibacterial activity of the synthesized Nanofibers against multi drug resistant bacteria were investigated by employing a noninvasive, quick and efficient fluorescence spectroscopy technique. Mechanistic study of interaction between nanofibers and bacterial cells is explained with the help of SEM. These outcome can be employed for the development of versatile material for antiviral, antibacterial, and self-cleaning application. Materials and Methods Materials Zinc Acetate 99.8% pure (Sigma Aldrich), Sigma–Aldrich Corp.), polyvinylpyrrolidone (PVP, Mw = 1 360 000, Sigma–Aldrich Corp.), Titanium Isopropoxide (TIP Mw 284.26, Sigma Aldrich), ethanol (anhydrous 99.5%, Sigma–Aldrich Corp.), Sodium Hydroxide (NaOH) Sigma–Aldrich Corp.), and deionized water were used, without any further purification. NUTRIENT AGAR (CM0003) made in OXOID LTD and Nutrient broth (ENUB205000) MADE IN BIO LAB ZRT hungry are used for cell culture and cultivation of micro-organisms. Methods Synthesis of ZnO and ZnO/TiO 2 Nanofibers Electrospinning technique is used to produce the nanofibers of the composite materials. To execute the electrospinning process, the homogeneous solution of zinc oxide (ZnO) and TiO 2 loaded ZnO was created by using Sol-Gel process. Initially 1.7 g polyvinylpyrrolidone (PVP) were added in 10 ml deionized (DI) water and the mixture was stirred at room temperature for 30 minutes. Then 0.5 g of precursor zinc acetate dehydrate was added in PVP/DI solution and agitated for a further 30 minutes at room temperature. To speed up the synthesis process, 0.5 g of sodium hydroxide (NaOH) was further added in the mixture and stirred again for 30 minutes. Finally, 1 ml ethanol was added in the mixture while stirring continuously at room temperature. Titanium isopropoxide (TIP) of different concentration 2%, 4%, 6%, and 8% is used as source to create multiple TiO 2 -ZnO solutions. Additionally, to regulate precursor solubility and enhance the quality of the resultant nanofibers, ethanol is used as a co-solvent in the synthesis process. Now the prepared solutions of ZnO and ZnO/TiO 2 are ready to be used for synthesis of nanofibers with electrospinning technique. In the electrospinning experiment, an injector (syringe) equipped with 25-gauge metallic needle was filled with the solution of ZnO and ZnO/TiO 2 . To supply electric potential to the solution the tip of the needle was wired to a 50 kV high-voltage DC power source. The feeding rate of the solution is controlled and kept at 1ml/hour with a microcontroller. To collect the nanofibers, a metallic collector is used which is made of a copper plate and encased in a circle of aluminium foil. The spacing between the collector and syringe tip was fixed at 12 cm for all the experiment. The applied voltage ~ 20 kV was also kept constant for all the experiments. After the electrospinning process, the ZnO synthesis fibers are then post annealed at varying temperature from 400–600 o C by using a box furnace to eliminate polymer content and to enhance structure quality. All the samples of ZnO/TiO 2 composite electrospun nanofibers were calcinated at 600°C for two hours at 5°C/min heating rate. Structural and Morphological Study of ZnO and ZnO/TiO NFs The crystal structure of the prepared ZnO and ZnO/TiO 2 NFs was examined by employing ARL EQUINOX 3000 X-RAY Diffractometer (Thermo SCIENTIFIC) equipped with Cu-K α radiation source (λ = 1.540 Å). The XRD measurement of all the nanofibers is conducted with fixed detector arm over a 2θ angular range of 10–90 degree. The morphology of synthesized nanofibers was examined by using scanning electron microscopy (SEM) TESCAN (MAIA3 Triglav™) system operated at an accelerating voltage of 20 kV. Elemental composition of nanofibers was determined by using energy dispersive x-ray (EDX) equipped with the SEM system. Fluorescence Analysis of ZnO and ZnO/TiO NFs Fluoroax-4 Spectrofluorometer (HORIBA Scientific) was used to measure fluorescence spectra of ZnO NFs and TiO 2 loaded ZnO aqueous solution. Spectra were captured using a 1 ml path quartz cell in the 385–600 nm wavelength range. Using equally wide excitation and emission slits (5 nm), the fluorescence spectra was recorded at an excitation wavelength of 527 nm. Antibacterial Activity of ZnO and ZnO/TiO 2 Nanofibers The agar diffusion method for anti-bacterial activity of ZnO and TiO 2 loaded ZnO was employed. The prepared dilutions of NFs and pored 50µl using micropipettes into wells on petri plates with two bacterial strains E. coli and Pseudomonas aeruginosa. Then these petri plates were placed into incubator at room temperature for 12 hours. A digital vernier caliper was used to measure the zones of inhibition in millimeters. To get mean values with standard deviations, each experiment was run in triplicate for ZnO and TiO 2 loaded ZnO with different concentrations of TiO 2 (2%, 4%, 6%, and 8%). Mechanistic study of bacterial interaction with NFs The prepared suspensions of ZnO nanofibers synthesized at 500°C and 600°C, as well as Ti loaded ZnO with varying TiO 2 concentrations of 2%, 4%, 6%, and 8%. These suspensions were inserted into the petri dishes of diameter 95mm using the agar diffusion method. Subsequently, bacterial cultures (10µm) of both E. coli and Pseudomonas aeruginosa were spread onto the agar surface. The petri dishes were then placed in an incubator at 37°C overnight to allow for bacterial-nanofiber interactions. Following the overnight incubation, samples were collected from regions exhibiting pure bacterial colonies and regions displaying zones of inhibition (ZOI) where bacterial growth was inhibited due to interaction with the nanofibers. Two samples of each bacterial strain were prepared for scanning electron microscopical (SEM) analysis. Result and Discussion Structural Analysis X-ray diffraction (XRD) is an effective tool to find out the crystalline nature of nanostructured materials. To investigate the crystal structure of pure ZnO nanofibers and the impact of TiO 2 loading on ZnO crystal structure, and XRD analysis in 2θ geometry ranging from 10° to 90°. Figures 1 (a) and (c) showed XRD patterns of pure ZnO and TiO 2 loaded ZnO nanofibers. The patterns in Fig. 1 (a) exhibited distinct crystallographic peaks corresponding to (100), (002), and (101) planes clearly indicating the presence of ZnO nanofibers having wurtzite hexagonal structure (JCPDS card 36–1451) [ 23 – 25 ]. Figure 1 (c) clearly showed that diffraction peaks along (002) plane start appearing when the loading concentration of TiO 2 increased up to 6% in the composite. This diffraction peak belongs to anatase phase of TiO 2 . Figure 1 (b) depicted that crystallite sizes of the ZnO NFs increased from 1.46 nm to 22. 42 nm with increasing calcination temperature from 400°C to 600°C [ 26 ]. Figure 1 (d) showed that the diffraction peak intensity of the TiO 2 loaded samples decreased with increasing TiO 2 content resulting in the crystallinity reduction of the ZnO/TiO 2 NFs. The grain size of the ZnO and TiO 2 loaded ZnO nanofibers were calculated from the Scherrer formula \(\:D=K\lambda\:⁄\beta\:cos\theta\:\) . Here, D, λ, θ and β represented the average crystallite size, wavelength of x-ray, Bragg’s angle and full width at half maximum (FWHM) of the diffraction peak respectively. The calculated grain size with increasing calcination temperature and TiO 2 loading. The decrease in grain size with increasing TiO 2 loading is observed because of increasing density of nucleation centers in the host nanofibers [ 27 ]. Morphological and Elemental Analysis of ZnO and ZnO/TiO 2 NFs The surface morphology of the grown NFs is observed by scanning electron microscopy (SEM) images and the results are shown in Fig. 2 (a & b). Figure 2 (a) confirmed the growth of high quality smooth and lengthy ZnO nanofibers at 600°C calcination temperature. The effect of calcination temperature (from 400–600°C) on morphology of NFs is depicted in Figure S1 (a-d) of supporting information. At low temperature PVP polymer is not decomposed completely but at 600°C it was completely evaporated which resulted in smooth and pure ZnO NFs [ 28 , 29 ]. SEM image of TiO 2 /ZnO nanofibers with 8 wt% TiO 2 is shown in Fig. 2 (b) which also revealed the growth of smooth and lengthy NFs of TiO 2 loaded ZnO. The SEM images of TiO 2 loaded ZnO with varying TiO 2 is shown in figure S2 (a-d) of supporting information. The energy dispersive x-ray spectroscopy (EDX) is employed to measure the elemental composition of pure and TiO 2 loaded ZnO NFs calcinated at 600°C. The EDX spectra shown in Fig. 2 (c, d) clearly depicted the formation of ZnO and existence of titanium in the ZnO/TiO 2 when TiO 2 is loaded up to 8 wt%. Growth Inhibition Study of ZnO and ZnO/TiO 2 Nanofibers Against E. coli and Pseudomonas aeruginosa The effects of ZnO (calcinated at 500°C, 600°C) and TiO 2 loaded ZnO NFs with different concentrations of TiO 2 (2%, 4%, 6%, and 8%) on the growth inhibition of E. coli and Pseudomonas aeruginosa bacterial strains were investigated. Particle growth inhibition at various doses was evaluated as part of the study. In Fig. 3 (b) and (c) the average size of growth inhibition zones for ZnO 500°C and ZnO 600°C increased from, 0 mm to 11.97 mm in comparison to the TiO 2 loaded ZnO, indicating the particle's activity against the E. coli . Figure 3 (a) and (d) the average size of growth inhibition zones for ZnO 500°C and ZnO 600°C increased from,0 mm to 11.62 mm in comparison to the TiO 2 -loaded ZnO, indicating the particle's activity against the Pseudomonas aeruginosa illustrates the dose-dependent behavior of ZnO 500°C, ZnO 600°C, and TiO 2 loaded ZnO with Titanium at varying concentrations of 2%, 4%, 6%, and 8% in the E.coli and Pseudomonas aeruginosa strain. Fluorescence based antibacterial activity of ZnO and Ti-loaded ZnO Nanofibers Fluorescence-based investigation to assess the antibacterial activity of ZnO NFs and Titanium (Ti)-loaded Zinc Oxide (ZnO) Nanofibers against both E. coli and Pseudomonas aeruginosa bacteria was evaluated. By employing fluorescence spectroscopy. The results demonstrated a remarkable antibacterial effect of the Ti-loaded ZnO nanofibers against both bacterial strains. Upon exposure to the nanofibers, observed a significant reduction in bacterial viability, as evidenced by a notable decrease in fluorescence intensity associated with bacterial cells. Moreover, fluorescence imaging revealed morphological alterations in bacterial cells, including membrane damage and cellular fragmentation, further indicating the bactericidal activity of the Ti-loaded ZnO nanofibers. Overall fluorescence-based antibacterial activity study highlights the efficacy of ZnO and Ti-loaded ZnO nanofibers as potent antibacterial agents against both E. coli and Pseudomonas aeruginosa bacteria. The direct visualization provided by fluorescence microscopy offers valuable insights into the mechanisms underlying the antibacterial action of these nanofibers, paving the way for their potential applications in combating bacterial infections and advancing the development of novel antibacterial materials. Figure 4 (a), (b), (c), and (d) The interaction between the E.coli and Pseudomonas aeruginosa bacteria cells and the nanofibers caused a sudden rise in fluorescence intensity when the cells were exposed to ZnO at 500°C and 600°C nanofiber nanofibers [ 30 , 31 ]. Figure 5 (a), (b), (c), and (d) the interaction between the E.coli bacterial cells and the nanofibers caused a sudden rise in fluorescence intensity when the cells were exposed to 2%, 4%, 6% and 8% Ti loaded ZnO nanofibers. Figure 6 (a), (b), (c), and (d) The interaction between the Pseudomonas aeruginosa bacteria cells and the nanofibers from 0 min to 60 min caused a sudden rise in fluorescence intensity when the cells were exposed to 2%, 4%, 6% and 8% Ti loaded ZnO nanofibers [ 30 ]. .Reactive oxygen species (ROS) were created as a result of this interaction and were able to cause oxidative stress in the bacterial cells by producing ROS on the nanofiber surface. As a result of the oxidative stress, the bacteria eventually die as a result of many biological reactions, including damage to membranes, proteins, and DNA. As a result of bacterial cell death and decreased metabolic activity, the fluorescence intensity at 527nm decreased with time [ 32 ]. Interaction study of ZnO and ZnO/TiO 2 NFs with E. coli and Pseudomonas The SEM of untreated E. coli and Pseudomonas aeruginosa bacteria is shown in Fig. 7 (a), demonstrating the bacterium's flawless rod-shaped morphology. As bacteria are treated with Ti loaded ZnO with 4%, 6%, and 8% Ti concentration nanofibers, the cellular membrane becomes damaged, which causes the cells to shrink, as Fig. 7 (b), (c) for both bacteria demonstrate. As observed in Fig. 7 (d) and for both bacteria, further treatment of bacteria with nanofibers causes membrane disarray and intracellular material leakage, which ultimately ends in cell death. [ 32 , 33 ]. Figure 9 illustrates Mechanistic study of ZnO NFs and Ti loaded ZnO NFs against multidrug resistant bacterial strains. Conclusion This study highlights the potent antibacterial and Fluorescence properties of both Zinc Oxide (ZnO) and Titanium (Ti)-loaded ZnO nanofibers against E. coli and Pseudomonas aeruginosa . Furthermore, calcination at temperatures ranging from 400°C to 600°C resulted in surface diameter reduction attributed to various thermal processes. Scanning Electron Microscopy (SEM) provided valuable insights into the morphological changes induced by Ti loading, leading to smoother nanofiber surfaces. Antibacterial efficacy against E. coli and Pseudomonas was robustly demonstrated through the Well Plate method, with consistent zones of inhibition observed for pure ZnO nanofibers synthesized at 500°C and 600°C, and Ti-loaded ZnO nanofibers at various Ti concentrations, The results of the agar disc diffusion technique demonstrate exceptional antibacterial activity, with the lowest inhibition zone measuring 0 mm and the greatest inhibition zone measuring 11.97 mm. Fluorescence spectroscopy confirmed the ZnO NF and Ti loaded ZnO NF antibacterial activity against E. coli and Pseudomonas aeruginosa . Fluorescence spectra confirmed the antibacterial activity of nanofibers. SEM study further elucidated the interaction between nanofibers and bacterial cells, revealing significant cell membrane disruption and subsequent leakage of cellular contents, ultimately resulting in bacterial cell death. These findings highlight the promising potential of ZnO and Ti-loaded ZnO nanofibers as effective antibacterial agents, offering novel paths for combating bacterial infections. Declarations Source of Funding There was no external funding involved during this research. Data Availability The data has not been used in this research. Ethical Approval No ethical approval is required because there was no human and animal subject involved in study Conflict of Interest All authors have no conflict of interest. Author Contribution Ali Hassan completed the experiment. Shahzad Anwar: Writing, review & editing, Writing, original draft, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization. Rafaqat Ali: Writing – review & editing, Validation, Methodology, Data curation. Rashad Rashid : Writing – review & editing, Methodology. Saba and Bisma: Review the results and findings. Atta Ullah and Muhammad Raffi Contributed in data analysis and Methodology Acknowledgement The authors are grateful for MS Fatima Batool from the National Institutes of Lasers and Optronics who has helped in the completion of this research project. References Kadiyala, U., N.A. Kotov, and J.S. VanEpps, Antibacterial Metal Oxide Nanoparticles: Challenges in Interpreting the Literature. Curr Pharm Des, 2018. 24 (8): p. 896-903. Thangavel, K., et al., Structural, morphological and antibacterial properties of ZnO nanofibers fabricated by electrospinning technique. 2020. 33 : p. 2160-2166. Thakur, S., et al., Fabrication and characterization of electrospun ZnO nanofibers; antimicrobial assessment. 2020. 264 : p. 127279. Babayevska, N., et al., Fabrication of gelatin-zno nanofibers for antibacterial applications. 2020. 14 (1): p. 103. Mishra, P.K., et al., Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications. 2017. 22 (12): p. 1825-1834. 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Cite Share Download PDF Status: Published Journal Publication published 30 Dec, 2024 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 19 Nov, 2024 Reviews received at journal 07 Nov, 2024 Reviewers agreed at journal 30 Oct, 2024 Reviewers agreed at journal 22 Oct, 2024 Reviewers agreed at journal 22 Oct, 2024 Reviewers invited by journal 22 Oct, 2024 Editor assigned by journal 21 Oct, 2024 Submission checks completed at journal 21 Oct, 2024 First submitted to journal 08 Oct, 2024 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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Lahore","correspondingAuthor":false,"prefix":"","firstName":"Bisma","middleName":"","lastName":"Khanam","suffix":""},{"id":380020699,"identity":"ddde2100-6616-480c-8eab-2b3835425cdb","order_by":6,"name":"Attaullah Shah","email":"","orcid":"","institution":"Pakistan Institute of Engineering and Applied Sciences","correspondingAuthor":false,"prefix":"","firstName":"Attaullah","middleName":"","lastName":"Shah","suffix":""},{"id":380020700,"identity":"4a03a655-4c75-4918-b008-d9b6e5b6c754","order_by":7,"name":"Muhammad Raffi","email":"","orcid":"","institution":"Pakistan Institute of Engineering and Applied Sciences","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Raffi","suffix":""}],"badges":[],"createdAt":"2024-10-08 06:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5222566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5222566/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-024-04085-x","type":"published","date":"2024-12-30T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70548920,"identity":"3b6aa53c-9c66-483e-8a88-43278292062a","added_by":"auto","created_at":"2024-12-04 09:44:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":770663,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e XRD graph of pure ZnO NFs annealed at 400°C, 500°C, and 600°C \u003cstrong\u003e(b)\u003c/strong\u003e Grain size variation of pure ZnO NFs with annealing\u003cstrong\u003e(c) \u003c/strong\u003eXRD graph for TiO\u003csub\u003e2\u003c/sub\u003e-ZnO NFs with 2%, 4%, 6% and 8% TiO\u003csub\u003e2\u003c/sub\u003e concentration \u003cstrong\u003e(d) \u003c/strong\u003eGrain size variation with TiO\u003csub\u003e2\u003c/sub\u003e concentration in TiO\u003csub\u003e2\u003c/sub\u003e-ZnO NFs.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/2b275eac92f8400f579046be.png"},{"id":70548926,"identity":"f00a6b10-35ab-4deb-b1b2-c6592b4866af","added_by":"auto","created_at":"2024-12-04 09:44:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1029302,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image of \u003cstrong\u003e(a) \u003c/strong\u003ePure ZnO NF calcinated at 600 \u003csup\u003eo\u003c/sup\u003eC\u003cstrong\u003e (b) \u003c/strong\u003eZnO/TiO\u003csub\u003e2 \u003c/sub\u003enanofibers with 8% TiO\u003csub\u003e2\u003c/sub\u003e. EDX spectra of \u003cstrong\u003e(c)\u003c/strong\u003e Pure and \u003cstrong\u003e(d)\u003c/strong\u003e ZnO/TiO\u003csub\u003e2 \u003c/sub\u003enanofibers with 8% TiO\u003csub\u003e2.\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/8174591d9f023f8bf3eef7ce.png"},{"id":70548924,"identity":"15840d15-3596-42f6-851f-7820852b4de3","added_by":"auto","created_at":"2024-12-04 09:44:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1212639,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition zone of ZnO and Ti-loaded ZnO NFs treatment with \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e bacterial strains. Standard deviation was calculated against each value (±SD).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/2fb09126b6b9a23a65b7cec2.png"},{"id":70548925,"identity":"cc3b3be9-75a6-4019-966c-d91bcabad504","added_by":"auto","created_at":"2024-12-04 09:44:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":459958,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra illustrate interaction of (a) ZnO NF at 500°C \u003cem\u003eE.coli\u003c/em\u003e (b) ZnO NF at 500°C (c) ZnO NF at 600°C \u003cem\u003eE.coli\u003c/em\u003e (d) ZnO NF at 600°C Nanofibers with \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/801d7e8cc40978b33347c8bf.png"},{"id":70548928,"identity":"4146f25b-cd46-4807-83f3-150a3d54e78b","added_by":"auto","created_at":"2024-12-04 09:44:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":794883,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra illustrate interaction of (a) 2% TiZnO NF (b) 4% TiZnO (c) 6% TiZnO (d) 8% TiZnO Nanofibers with \u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/6e09edd7506a4f9b7a10df18.png"},{"id":70548929,"identity":"642583ae-ead0-40f3-8dae-f35d68c389f8","added_by":"auto","created_at":"2024-12-04 09:45:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":446299,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra illustrate the interaction of (a) 2% TiZnO NF (b) 4% TiZnO NF (c) 6% TiZnO NF (d) 8% TiZnO Nanofibers with \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/36b3aa0abeaa30cba2beb3df.png"},{"id":70549063,"identity":"9610ae29-b159-45a0-9a4d-202e62fa8366","added_by":"auto","created_at":"2024-12-04 09:52:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":904036,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of (a) control untreated \u003cem\u003eE. coli and Pseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e bacteria and (b), (c), and (d) treated bacterial strain using the prepared NFs of ZnO at 500°C and ZnO at 600°C showing the leakage of intracellular materials leading to the cell death.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/ad348707d0fa1089c6bab25a.png"},{"id":70548923,"identity":"8d08b4a3-a335-4fb3-bba0-bb17096e0319","added_by":"auto","created_at":"2024-12-04 09:44:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":793129,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of (a) control untreated \u003cem\u003eE. coli and Pseudomonas aeruginosa\u003c/em\u003e bacteria and (b), (c), and (d) treated bacterial strain using the prepared NFs of Ti loaded ZnO with 2%, 4%, 6%, and 8% Ti concentration showed the leakage of intracellular materials leading to the cell death.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/ba125dc1f4b2993be2c68553.png"},{"id":70548922,"identity":"f96cf73d-514f-402e-8eab-c1f4152ee90d","added_by":"auto","created_at":"2024-12-04 09:44:52","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":195714,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic study of ZnO NFs and Ti loaded ZnO NFs against multidrug \u0026nbsp;resistant bacterial strains (\u003cem\u003eE. coli\u003c/em\u003eand \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/7041d0145d47c55b51a55ef4.png"},{"id":73093509,"identity":"7cad5e62-4968-45de-bf08-4970e4cbf10b","added_by":"auto","created_at":"2025-01-06 16:21:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7205305,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5222566/v1/437aa10d-6589-491d-ad3f-387193b44c26.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fluorescence analysis of antibacterial activity of ZnO/TiO 2 electrospun nanofibers: A molecular approach to reveals the insights of physiochemical interactions of materials with bacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStudy of antibacterial activity of metal oxide has got immense attraction because of increasing bacterial infection in the human civilization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Zinc oxide is one of the most widely used antibacterial metal oxide nanomaterial due to its favorable chemical and physical properties [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Since ZnO belongs to \"Generally Recognized as A Safe (GRAS)\" category, therefore it has a great deal of recognition in the field of nanomedicine, food packaging, plasters, painting, cosmetics and sunscreen for UV protection [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. ZnO is suitable for a wide range of biological uses, including the treatment of diabetes, cancer, wound healing, infection, and inflammation because it is less expensive and safer than other metal oxides like SnO\u003csub\u003e2\u003c/sub\u003e and CuO [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, ZnO nanoparticles have shown antibacterial action against both Gram-positive and Gram-negative oral bacteria where Gram-negative bacteria are more resistant against ZnO nanoparticles [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Zinc oxide has the ability to combat microorganisms originated viral disease like H1N1, SARS-CoV-2, COVID-19 pandemic, therefore it is very important and essential to investigate possible antiviral properties of Zinc related compounds [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nanofibers are preferred for antibacterial applications because of their large surface area and compact size which provide better particle penetration into bacteria and increased adsorption of the packages on the cell membrane. Among several synthesis techniques of nanofibers, electrospinning is one of the straightforward and low-cost technique to synthesize high quality long and continuous fibers of different materials including polymers and metal oxide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous parameters of the nanostructures such as morphological (particle size, shape), crystalline structure and concentration of inclusion affect the antibacterial activity of ZnO. Embedding a suitable material in metal oxide and formation of composites is a promising approach to enhancing the antimicrobial properties of ZnO for food packaging industries [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is reported that antibacterial activity index of ZnO increases with the inclusion of TiO\u003csub\u003e2\u003c/sub\u003e atoms in ZnO matrix [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntiviral mechanism of Zinc is commonly described by one of the following models: inhibition of virus replication, reduction of angiotensin-converting enzyme 2 (ACE2) activity, conditional up regulation of IFN-α (interferon α), inhibition of nuclear factor-kappa B (NF-κB) with an immunologic role, serving as a cofactor for multiple viral proteins, blocking the entry of the virus from the cell membrane, and reduction of the risk of bacterial coinfection by strengthening direct antibacterial effects against \u003cem\u003eStreptococcus pneumonia\u003c/em\u003e. But more comprehensive experimental data and clinical research are required in this field [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. ZnO nanoparticles have efficiently been used against a broad spectrum of bacteria and the photocatalytic and antibacterial capabilities of ZnO are mostly determined by its shape [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Their ability to induce the production of reactive oxygen species (ROS), interfere with the function of proteins and DNA, and break bacterial cell membranes is how they exhibit their antimicrobial action [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, ZnO and TiO\u003csub\u003e2\u003c/sub\u003e-loaded ZnO nanofibers have been synthesized by using electrospinning techniques followed by their morphological and structural characterizations by SEM and XRD techniques. The antibacterial activity of the synthesized Nanofibers against multi drug resistant bacteria were investigated by employing a noninvasive, quick and efficient fluorescence spectroscopy technique. Mechanistic study of interaction between nanofibers and bacterial cells is explained with the help of SEM. These outcome can be employed for the development of versatile material for antiviral, antibacterial, and self-cleaning application.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eZinc Acetate 99.8% pure (Sigma Aldrich), Sigma\u0026ndash;Aldrich Corp.), polyvinylpyrrolidone (PVP, Mw\u0026thinsp;=\u0026thinsp;1 360 000, Sigma\u0026ndash;Aldrich Corp.), Titanium Isopropoxide (TIP Mw 284.26, Sigma Aldrich), ethanol (anhydrous 99.5%, Sigma\u0026ndash;Aldrich Corp.), Sodium Hydroxide (NaOH) Sigma\u0026ndash;Aldrich Corp.), and deionized water were used, without any further purification. NUTRIENT AGAR (CM0003) made in OXOID LTD and Nutrient broth (ENUB205000) MADE IN BIO LAB ZRT hungry are used for cell culture and cultivation of micro-organisms.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of ZnO and ZnO/TiO\u003csub\u003e2\u003c/sub\u003e Nanofibers\u003c/h2\u003e \u003cp\u003eElectrospinning technique is used to produce the nanofibers of the composite materials. To execute the electrospinning process, the homogeneous solution of zinc oxide (ZnO) and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO was created by using Sol-Gel process. Initially 1.7 g polyvinylpyrrolidone (PVP) were added in 10 ml deionized (DI) water and the mixture was stirred at room temperature for 30 minutes. Then 0.5 g of precursor zinc acetate dehydrate was added in PVP/DI solution and agitated for a further 30 minutes at room temperature. To speed up the synthesis process, 0.5 g of sodium hydroxide (NaOH) was further added in the mixture and stirred again for 30 minutes. Finally, 1 ml ethanol was added in the mixture while stirring continuously at room temperature. Titanium isopropoxide (TIP) of different concentration 2%, 4%, 6%, and 8% is used as source to create multiple TiO\u003csub\u003e2\u003c/sub\u003e-ZnO solutions. Additionally, to regulate precursor solubility and enhance the quality of the resultant nanofibers, ethanol is used as a co-solvent in the synthesis process. Now the prepared solutions of ZnO and ZnO/TiO\u003csub\u003e2\u003c/sub\u003e are ready to be used for synthesis of nanofibers with electrospinning technique.\u003c/p\u003e \u003cp\u003eIn the electrospinning experiment, an injector (syringe) equipped with 25-gauge metallic needle was filled with the solution of ZnO and ZnO/TiO\u003csub\u003e2\u003c/sub\u003e. To supply electric potential to the solution the tip of the needle was wired to a 50 kV high-voltage DC power source. The feeding rate of the solution is controlled and kept at 1ml/hour with a microcontroller. To collect the nanofibers, a metallic collector is used which is made of a copper plate and encased in a circle of aluminium foil. The spacing between the collector and syringe tip was fixed at 12 cm for all the experiment. The applied voltage\u0026thinsp;~\u0026thinsp;20 kV was also kept constant for all the experiments. After the electrospinning process, the ZnO synthesis fibers are then post annealed at varying temperature from 400\u0026ndash;600 \u003csup\u003eo\u003c/sup\u003eC by using a box furnace to eliminate polymer content and to enhance structure quality. All the samples of ZnO/TiO\u003csub\u003e2\u003c/sub\u003e composite electrospun nanofibers were calcinated at 600\u0026deg;C for two hours at 5\u0026deg;C/min heating rate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStructural and Morphological Study of ZnO and ZnO/TiO NFs\u003c/h3\u003e\n\u003cp\u003eThe crystal structure of the prepared ZnO and ZnO/TiO\u003csub\u003e2\u003c/sub\u003e NFs was examined by employing ARL EQUINOX 3000 X-RAY Diffractometer (Thermo SCIENTIFIC) equipped with Cu-K\u003csub\u003eα\u003c/sub\u003e radiation source (λ\u0026thinsp;=\u0026thinsp;1.540 \u0026Aring;). The XRD measurement of all the nanofibers is conducted with fixed detector arm over a 2θ angular range of 10\u0026ndash;90 degree. The morphology of synthesized nanofibers was examined by using scanning electron microscopy (SEM) TESCAN (MAIA3 Triglav\u0026trade;) system operated at an accelerating voltage of 20 kV. Elemental composition of nanofibers was determined by using energy dispersive x-ray (EDX) equipped with the SEM system.\u003c/p\u003e\n\u003ch3\u003eFluorescence Analysis of ZnO and ZnO/TiO NFs\u003c/h3\u003e\n\u003cp\u003eFluoroax-4 Spectrofluorometer (HORIBA Scientific) was used to measure fluorescence spectra of ZnO NFs and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO aqueous solution. Spectra were captured using a 1 ml path quartz cell in the 385\u0026ndash;600 nm wavelength range. Using equally wide excitation and emission slits (5 nm), the fluorescence spectra was recorded at an excitation wavelength of 527 nm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Activity of ZnO and ZnO/TiO\u003csub\u003e2\u003c/sub\u003e Nanofibers\u003c/h2\u003e \u003cp\u003eThe agar diffusion method for anti-bacterial activity of ZnO and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO was employed. The prepared dilutions of NFs and pored 50\u0026micro;l using micropipettes into wells on petri plates with two bacterial strains \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa.\u003c/em\u003e Then these petri plates were placed into incubator at room temperature for 12 hours. A digital vernier caliper was used to measure the zones of inhibition in millimeters. To get mean values with standard deviations, each experiment was run in triplicate for ZnO and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO with different concentrations of TiO\u003csub\u003e2\u003c/sub\u003e (2%, 4%, 6%, and 8%).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMechanistic study of bacterial interaction with NFs\u003c/h3\u003e\n\u003cp\u003eThe prepared suspensions of ZnO nanofibers synthesized at 500\u0026deg;C and 600\u0026deg;C, as well as Ti loaded ZnO with varying TiO\u003csub\u003e2\u003c/sub\u003e concentrations of 2%, 4%, 6%, and 8%. These suspensions were inserted into the petri dishes of diameter 95mm using the agar diffusion method. Subsequently, bacterial cultures (10\u0026micro;m) of both \u003cem\u003eE. coli and Pseudomonas aeruginosa\u003c/em\u003e were spread onto the agar surface. The petri dishes were then placed in an incubator at 37\u0026deg;C overnight to allow for bacterial-nanofiber interactions. Following the overnight incubation, samples were collected from regions exhibiting pure bacterial colonies and regions displaying zones of inhibition (ZOI) where bacterial growth was inhibited due to interaction with the nanofibers. Two samples of each bacterial strain were prepared for scanning electron microscopical (SEM) analysis.\u003c/p\u003e"},{"header":"Result and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStructural Analysis\u003c/h2\u003e \u003cp\u003eX-ray diffraction (XRD) is an effective tool to find out the crystalline nature of nanostructured materials. To investigate the crystal structure of pure ZnO nanofibers and the impact of TiO\u003csub\u003e2\u003c/sub\u003e loading on ZnO crystal structure, and XRD analysis in 2θ geometry ranging from 10\u0026deg; to 90\u0026deg;.\u003c/p\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a) and (c) showed XRD patterns of pure ZnO and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO nanofibers. The patterns in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a) exhibited distinct crystallographic peaks corresponding to (100), (002), and (101) planes clearly indicating the presence of ZnO nanofibers having wurtzite hexagonal structure (JCPDS card 36\u0026ndash;1451) [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (c) clearly showed that diffraction peaks along (002) plane start appearing when the loading concentration of TiO\u003csub\u003e2\u003c/sub\u003e increased up to 6% in the composite. This diffraction peak belongs to anatase phase of TiO\u003csub\u003e2\u003c/sub\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b) depicted that crystallite sizes of the ZnO NFs increased from 1.46 nm to 22. 42 nm with increasing calcination temperature from 400\u0026deg;C to 600\u0026deg;C [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (d) showed that the diffraction peak intensity of the TiO\u003csub\u003e2\u003c/sub\u003e loaded samples decreased with increasing TiO\u003csub\u003e2\u003c/sub\u003e content resulting in the crystallinity reduction of the ZnO/TiO\u003csub\u003e2\u003c/sub\u003e NFs. The grain size of the ZnO and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO nanofibers were calculated from the Scherrer formula \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:D=K\\lambda\\:\u0026frasl;\\beta\\:cos\\theta\\:\\)\u003c/span\u003e\u003c/span\u003e. Here, D, λ, θ and β represented the average crystallite size, wavelength of x-ray, Bragg\u0026rsquo;s angle and full width at half maximum (FWHM) of the diffraction peak respectively. The calculated grain size with increasing calcination temperature and TiO\u003csub\u003e2\u003c/sub\u003e loading. The decrease in grain size with increasing TiO\u003csub\u003e2\u003c/sub\u003e loading is observed because of increasing density of nucleation centers in the host nanofibers [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMorphological and Elemental Analysis of ZnO and ZnO/TiO\u003csub\u003e2\u003c/sub\u003e NFs\u003c/h2\u003e \u003cp\u003eThe surface morphology of the grown NFs is observed by scanning electron microscopy (SEM) images and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a \u0026amp; b). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a) confirmed the growth of high quality smooth and lengthy ZnO nanofibers at 600\u0026deg;C calcination temperature. The effect of calcination temperature (from 400\u0026ndash;600\u0026deg;C) on morphology of NFs is depicted in Figure S1 (a-d) of supporting information. At low temperature PVP polymer is not decomposed completely but at 600\u0026deg;C it was completely evaporated which resulted in smooth and pure ZnO NFs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. SEM image of TiO\u003csub\u003e2\u003c/sub\u003e/ZnO nanofibers with 8 wt% TiO\u003csub\u003e2\u003c/sub\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b) which also revealed the growth of smooth and lengthy NFs of TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO. The SEM images of TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO with varying TiO\u003csub\u003e2\u003c/sub\u003e is shown in figure S2 (a-d) of supporting information.\u003c/p\u003e \u003cp\u003eThe energy dispersive x-ray spectroscopy (EDX) is employed to measure the elemental composition of pure and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO NFs calcinated at 600\u0026deg;C. The EDX spectra shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c, d) clearly depicted the formation of ZnO and existence of titanium in the ZnO/TiO\u003csub\u003e2\u003c/sub\u003e when TiO\u003csub\u003e2\u003c/sub\u003e is loaded up to 8 wt%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth Inhibition Study of ZnO and ZnO/TiO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eNanofibers Against\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effects of ZnO (calcinated at 500\u0026deg;C, 600\u0026deg;C) and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO NFs with different concentrations of TiO\u003csub\u003e2\u003c/sub\u003e (2%, 4%, 6%, and 8%) on the growth inhibition of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e bacterial strains were investigated. Particle growth inhibition at various doses was evaluated as part of the study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (b) and (c) the average size of growth inhibition zones for ZnO 500\u0026deg;C and ZnO 600\u0026deg;C increased from, 0 mm to 11.97 mm in comparison to the TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO, indicating the particle's activity against the \u003cem\u003eE. coli\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (a) and (d) the average size of growth inhibition zones for ZnO 500\u0026deg;C and ZnO 600\u0026deg;C increased from,0 mm to 11.62 mm in comparison to the TiO\u003csub\u003e2\u003c/sub\u003e-loaded ZnO, indicating the particle's activity against the \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e illustrates the dose-dependent behavior of ZnO 500\u0026deg;C, ZnO 600\u0026deg;C, and TiO\u003csub\u003e2\u003c/sub\u003e loaded ZnO with Titanium at varying concentrations of 2%, 4%, 6%, and 8% in the \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence based antibacterial activity of ZnO and Ti-loaded ZnO Nanofibers\u003c/h2\u003e \u003cp\u003eFluorescence-based investigation to assess the antibacterial activity of ZnO NFs and Titanium (Ti)-loaded Zinc Oxide (ZnO) Nanofibers against both \u003cem\u003eE. coli and Pseudomonas aeruginosa\u003c/em\u003e bacteria was evaluated. By employing fluorescence spectroscopy. The results demonstrated a remarkable antibacterial effect of the Ti-loaded ZnO nanofibers against both bacterial strains. Upon exposure to the nanofibers, observed a significant reduction in bacterial viability, as evidenced by a notable decrease in fluorescence intensity associated with bacterial cells. Moreover, fluorescence imaging revealed morphological alterations in bacterial cells, including membrane damage and cellular fragmentation, further indicating the bactericidal activity of the Ti-loaded ZnO nanofibers.\u003c/p\u003e \u003cp\u003eOverall fluorescence-based antibacterial activity study highlights the efficacy of ZnO and Ti-loaded ZnO nanofibers as potent antibacterial agents against both E. \u003cem\u003ecoli and Pseudomonas aeruginosa\u003c/em\u003e bacteria. The direct visualization provided by fluorescence microscopy offers valuable insights into the mechanisms underlying the antibacterial action of these nanofibers, paving the way for their potential applications in combating bacterial infections and advancing the development of novel antibacterial materials.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a), (b), (c), and (d) The interaction between the \u003cem\u003eE.coli and Pseudomonas aeruginosa\u003c/em\u003e bacteria cells and the nanofibers caused a sudden rise in fluorescence intensity when the cells were exposed to ZnO at 500\u0026deg;C and 600\u0026deg;C nanofiber nanofibers [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (a), (b), (c), and (d) the interaction between the \u003cem\u003eE.coli\u003c/em\u003e bacterial cells and the nanofibers caused a sudden rise in fluorescence intensity when the cells were exposed to 2%, 4%, 6% and 8% Ti loaded ZnO nanofibers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (a), (b), (c), and (d) The interaction between the \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e bacteria cells and the nanofibers from 0 min to 60 min caused a sudden rise in fluorescence intensity when the cells were exposed to 2%, 4%, 6% and 8% Ti loaded ZnO nanofibers [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. .Reactive oxygen species (ROS) were created as a result of this interaction and were able to cause oxidative stress in the bacterial cells by producing ROS on the nanofiber surface. As a result of the oxidative stress, the bacteria eventually die as a result of many biological reactions, including damage to membranes, proteins, and DNA. As a result of bacterial cell death and decreased metabolic activity, the fluorescence intensity at 527nm decreased with time [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eInteraction study of ZnO and ZnO/TiO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eNFs with\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe SEM of untreated \u003cem\u003eE. coli and Pseudomonas aeruginosa\u003c/em\u003e bacteria is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(a), demonstrating the bacterium's flawless rod-shaped morphology.\u003c/p\u003e \u003cp\u003eAs bacteria are treated with Ti loaded ZnO with 4%, 6%, and 8% Ti concentration nanofibers, the cellular membrane becomes damaged, which causes the cells to shrink, as Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b), (c) for both bacteria demonstrate. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(d) and for both bacteria, further treatment of bacteria with nanofibers causes membrane disarray and intracellular material leakage, which ultimately ends in cell death. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates Mechanistic study of ZnO NFs and Ti loaded ZnO NFs against multidrug resistant bacterial strains.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the potent antibacterial and Fluorescence properties of both Zinc Oxide (ZnO) and Titanium (Ti)-loaded ZnO nanofibers against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Furthermore, calcination at temperatures ranging from 400\u0026deg;C to 600\u0026deg;C resulted in surface diameter reduction attributed to various thermal processes. Scanning Electron Microscopy (SEM) provided valuable insights into the morphological changes induced by Ti loading, leading to smoother nanofiber surfaces. Antibacterial efficacy against \u003cem\u003eE. coli\u003c/em\u003e and Pseudomonas was robustly demonstrated through the Well Plate method, with consistent zones of inhibition observed for pure ZnO nanofibers synthesized at 500\u0026deg;C and 600\u0026deg;C, and Ti-loaded ZnO nanofibers at various Ti concentrations, The results of the agar disc diffusion technique demonstrate exceptional antibacterial activity, with the lowest inhibition zone measuring 0 mm and the greatest inhibition zone measuring 11.97 mm. Fluorescence spectroscopy confirmed the ZnO NF and Ti loaded ZnO NF antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Fluorescence spectra confirmed the antibacterial activity of nanofibers. SEM study further elucidated the interaction between nanofibers and bacterial cells, revealing significant cell membrane disruption and subsequent leakage of cellular contents, ultimately resulting in bacterial cell death. These findings highlight the promising potential of ZnO and Ti-loaded ZnO nanofibers as effective antibacterial agents, offering novel paths for combating bacterial infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSource of Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no external funding involved during this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data has not been used in this research.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical Approval\u003c/h2\u003e \u003cp\u003eNo ethical approval is required because there was no human and animal subject involved in study\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of Interest\u003c/strong\u003e \u003cp\u003eAll authors have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAli Hassan completed the experiment. Shahzad Anwar: Writing, review \u0026amp; editing, Writing, original draft, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization. Rafaqat Ali: Writing \u0026ndash; review \u0026amp; editing, Validation, Methodology, Data curation. Rashad Rashid : Writing \u0026ndash; review \u0026amp; editing, Methodology. Saba and Bisma: Review the results and findings. Atta Ullah and Muhammad Raffi Contributed in data analysis and Methodology\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful for MS Fatima Batool from the National Institutes of Lasers and Optronics who has helped in the completion of this research project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKadiyala, U., N.A. Kotov, and J.S. 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Gopinath, \u003cem\u003eEfficient adsorption and antibacterial properties of electrospun CuO-ZnO composite nanofibers for water remediation.\u003c/em\u003e 2017. \u003cstrong\u003e321\u003c/strong\u003e: p. 611-621.\u003c/li\u003e\n\u003cli\u003eAbebe, B., et al., \u003cem\u003eA review on enhancing the antibacterial activity of ZnO: Mechanisms and microscopic investigation.\u003c/em\u003e 2020. \u003cstrong\u003e15\u003c/strong\u003e: p. 1-19.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TiO2/ZnO, Electrospinning, Nanofibers, Fluorescence spectroscopy","lastPublishedDoi":"10.21203/rs.3.rs-5222566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5222566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFluorescence spectroscopy employed to compute the antibacterial potential of pure ZnO and Titania (TiO\u003csub\u003e2\u003c/sub\u003e) loaded ZnO (TiO\u003csub\u003e2\u003c/sub\u003e: 2%, 4%, 6%, and 8%) electrospun nanofibers. The study of electrospun nanofibers followed by their structural, morphological and antibacterial properties has been revealed through fluorescence spectroscopy. X-ray diffraction (XRD) analysis of nanofibers calcinated at 600\u0026deg;C revealed the presence of polycrystalline wurtzite hexagonal crystallographic planes of ZnO with preferred orientation along (101) direction. Scanning electron microscopy (SEM) confirmed that calcination of electrospun nanofibers resulted in smooth and pure ZnO nanofibers due to ethanol evaporation and polyvinylpyrrolidone (PVP) decomposition. Two bacterial strains \u003cem\u003eEscherichia coli and Pseudomonas aeruginosa\u003c/em\u003e were used for fluorescence spectroscopy-based evaluation of antibacterial activity of ZnO and TiO\u003csub\u003e2\u003c/sub\u003e-ZnO nanofibers. Agar well technique was employed to investigate the antibacterial activity and functioning mechanism of nanofibers against \u003cem\u003eEscherichia coli and Pseudomonas aeruginosa\u003c/em\u003e. The consistent zones of inhibition have been observed for pure ZnO and Titania loaded ZnO nanofibers. Fluorescence spectroscopy revealed the insights of bacterial killing with nanofibers. The mechanistic study of interaction between nanofibers and bacterial cells leads to cell membrane breakdown and confirmed with SEM imaged micrographs. Furthermore, the nanofibers calcinated at 600\u0026deg;C efficiently ruptured the bacteria resulting in higher antibacterial phenomenon as compare to the other nanofiber structures.\u003c/p\u003e","manuscriptTitle":"Fluorescence analysis of antibacterial activity of ZnO/TiO 2 electrospun nanofibers: A molecular approach to reveals the insights of physiochemical interactions of materials with bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-04 09:44:47","doi":"10.21203/rs.3.rs-5222566/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-19T13:26:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T14:16:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32484104794407822224267296579301734969","date":"2024-10-30T13:26:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41543552280541599199092665454766488699","date":"2024-10-23T03:14:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265212179765787051666405865890376027895","date":"2024-10-22T20:28:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-22T18:50:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-21T14:39:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-21T14:36:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2024-10-08T06:37:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9f42b290-7cb2-470e-a593-6181fdee7342","owner":[],"postedDate":"December 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-06T16:05:21+00:00","versionOfRecord":{"articleIdentity":"rs-5222566","link":"https://doi.org/10.1007/s10895-024-04085-x","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2024-12-30 15:57:26","publishedOnDateReadable":"December 30th, 2024"},"versionCreatedAt":"2024-12-04 09:44:47","video":"","vorDoi":"10.1007/s10895-024-04085-x","vorDoiUrl":"https://doi.org/10.1007/s10895-024-04085-x","workflowStages":[]},"version":"v1","identity":"rs-5222566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5222566","identity":"rs-5222566","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}