A Comparative Study of Antibacterial Activity of ZnO and TiO 2 Nanoparticles Against Gram-Positive and Gram-Negative Bacteria

,


Introduction
In recent years, nanotechnology has experienced significant growth, with the development of nanomaterials being a crucial aspect of its advancement.Nanostructured materials, characterized by their unique properties and geometric dimensions at the nanoscale, have garnered substantial interest, particularly in biological and pharmaceutical applications [1].While antibiotics have traditionally been effective against specific disease-causing organisms, the emergence of nanomaterials has displayed their ability to eliminate a broader spectrum, targeting up to 650 cells [2,3].
Among nanomaterials, nano oxides, specifically zinc oxide (ZnO), have attracted considerable attention due to their antimicrobial properties and perceived safety for humans and animals [4].Nanoparticle laboratory experiments have demonstrated the rapid eradication of bacteria, viruses, and fungi upon contact.Understanding the impact of nanoparticles on bacteria is crucial, given their role at the lowest level of the ecosystem's food cycle [5].
In contrast to using organic and inorganic chemicals in the food sector as antibacterial agents, inorganic antibacterial materials like ZnO have gained prominence due to their stability under challenging processing conditions [6].Notably, nanoparticles formed of metal oxides, especially those smaller than 100nm, exhibit unique antimicrobial activities attributed to their distinctive properties, such as small particle size and large surface area [7].Research has shown that certain metal oxide nanoparticles, like ZnO, selectively harm bacteria without adverse effects on human cells [7].
The antibacterial mechanisms of ZnO nanoparticles are still under investigation, with proposed actions including damage to the cell membrane, the production of reactive oxygen species like hydrogen peroxide, and interactions with intracellular contents [8,9].Titanium dioxide (TiO 2 ), a photocatalyst known for its nontoxicity, photo-induced superhydrophobicity, and antifogging qualities, plays a significant role in environmental purification [10,11].Its applications range from self-cleaning 1233 and self-disinfecting surfaces in medical centers to water and air purification from germs and harmful chemical compounds [12,13].
Various morphologies of one-dimensional TiO 2 nanostructures, such as nanorods, nanowires, and nanotubes, have been successfully synthesized using different techniques [14,15].This study aims to investigate ZnO and TiO 2 nanoparticles' antibacterial effects against gram-positive and gram-negative bacteria across different concentrations.Exploring these nanomaterials' antibacterial properties holds promise for diverse applications, especially in fields requiring effective antimicrobial agents.

Preparation of ZnO NPs
The sol-gel method is employed to synthesize zinc oxide nanoparticles (ZnO NPs).Initially, materials were accurately weighed using a balance, and distilled water was measured with a graduated cylinder.Specifically, 2g of zinc acetate dehydrate was dissolved in 10ml of distilled water, and 7g of sodium hydroxide was added to the solution.The mixture was stirred continuously for approximately five minutes to ensure thorough blending.Upon achieving a homogeneous solution, the zinc acetate solution was stirred with a magnetic stirrer for five more minutes while the sodium hydroxide solution was carefully added drop by drop.As the reaction progressed, a white precipitate became evident.Subsequently, the solution underwent filtration, and the resulting substance was subjected to drying at 65 °C for one day.Post-drying, the substance was ground into a fine white powder.

Preparation of TiO 2 nanoparticles
In the synthesis of TiO 2 nanoparticles, the sol-gel method is employed.Specifically, 8 ml of titanium isopropoxide (TTIP), 140ml of ethanol, and 12ml of deionized water meticulously combined under constant stirring.The mixture was maintained at 80 °C for 5 hours, facilitated by a paraffin oil bath.Subsequently, the gel formed was dried in a hot air oven set at 70 °C.The next step involved calcination, in which the dried gel was heated at 500 °C for three hours.This critical step aimed to bring about the transformation of the gel into TiO 2 nanoparticles.The carefully controlled conditions during the synthesis process play a pivotal role in determining the characteristics and properties of the resulting nanoparticles, making them suitable for various applications in scientific research and technology.

Preparation of Bacterial Culture
Two types of bacteria (E.coli and Staph.aureus) were activated by culturing on nutrient agar at 37 °C for 24 hours.Then diluted by normal saline to obtain 107 CFU/ml in suspension (McFarland no.5).These were cultured with ZnO and TiO 2 in different concentrations (0.4, 0.8, and 1.6 mg/ml) and incubated in shaker incubation at 37 °C for 24 hours at 160rpm.Made serial dilutions three times, and 100 l of suspension, spread on a molar Hinton agar plate and incubated at 37 °C for 24 hours.The formula to calculate the bacteria's survival rate is as follows:

Fourier Transformation Infrared Results
The functional groups present in the synthesized titanium dioxide particles were characterized using FTIR analysis.The essential chemical bonds of ZnO powder are elucidated through the FTIR spectrum presented in Figure 1.The graph displays a prominent peak at 466.77cm -1 and a shoulder at 551.64 cm -1 , both indicative of Zn-O bonds [17].In Figure 2, the FTIR spectrum of Titanium dioxide particles reveals a broad band between 500 and 600 cm -1 , attributed to the vibrations of Ti-O bonds within the TiO 2 lattice.Additionally, peaks observed between 1620 and 1630 cm -1 and 3100 and 3600 cm-1 are attributed to the vibrations of hydroxyl groups (-OH) [18,19].
The produced particles' mean grain size (D) was determined using the Scherrer Equation 3 from the XRD line broadening measurement.The grain sizes of Titania and zinc oxide were 12.6nm and 18.6nm, respectively.
where: λ is the wavelength (Cu Kα), β is the full width at the half-maximum (FWHM) of the ZnO (101) line, and θ is the diffraction angle.

SEM Results
This study investigated the topography of powders derived from two materials, ZnO and TiO 2 , using scanning electron microscopy (SEM).The examination revealed that ZnO particles exhibited an average size of less than 1000nm, as illustrated in Figure 5, while titania particles displayed an average size of less than 500nm, as depicted in Figure 5.The SEM images indicated the presence of pores within the observed powders.Furthermore, the images highlighted the occurrence of agglomeration in both ZnO and TiO 2 particles.This agglomeration phenomenon is attributed to the tiny particles' high surface area, which encourages the particles to come together.The agglomeration phenomenon is evident in the SEM images, 1235 emphasizing the need for careful consideration of particle size and morphology in applications where dispersion and uniformity are crucial.

Antibacterial Test
In this research, the results compared the two types of Nanoparticles, ZnO, and TiO 2 , against two types of gram-positive bacteria (Staph.Aureus) and gram-negative bacteria (E.coli, Proteus) in different concentrations (0.4,0.8,1.6)mg/ml, and the results explain the ability of two types of Nanoparticles to effected on these types of bacteria in all concentration and different percentage by assessing the antibacterial activity of these materials.
The effect of TiO 2 Nanoparticles is less than the effect of ZnO Nanoparticles on all types of bacteria used in this research.The bacteriostatic of ZnO Nanoparticles in concentration (0.4, 0.8,1.6)mg/ml against (Staph.aureus) was (96, 98, 99)%, against (E.coli) was (97, 99,100)% and against (Proteus ) was (98, 99,100)% respectively, as shown in Table 1.The results of TiO 2 Nanoparticles as bacteriostatic were (60,70, and 90)% against (Staph.Aureus), (94.5, 96.3, and 97)% against (E.coli) and (97, 98, and 100)% respectively in the same concentration, as shown in Figures ( 6 and 7).Also, the results improved that bacteriostatic of both nanomaterials ( ZnO & TiO 2 ) raised with concentration increasing.The distinction between Gram-negative and Gram-positive bacteria's antibacterial activity is determined by a variety of variables, including the following: Differing outer layer or cell wall architecture [28].Gram-positive bacteria, like S. aureus, have thick cell walls that lack any periplasmic region and are mostly composed of peptidoglycans as well as other materials, such lipoteichoic acids (LTA).Gram-negative bacteria have relatively thin cell walls and periplasmic spaces between their inner and outer membranes [28]; (ii) the carotenoid pigments in S. aureus offer a better oxidant resistance [29] (iii) S. aureus can produce oxidative stress response gene products to combat oxidative stress conditions.In E. coli, comparable mechanisms are less effective [18].Also, additional elements, including how the Nps associate with the cell membrane, the capacity of the bacterial cell to dissolve ZnO, and cell permeability, must be considered [28].
The unique characteristic of nanoparticles' ability to effortlessly pass the cell membrane of bacteria with hole sizes smaller than their own is thought to be the mechanism [30].Additionally, the thiol (-SH) groups of the proteins found in the cell wall interact with the metal oxide nanoparticles' antibacterial activity, mostly visible on their surface.As a result of this interaction, cells become less permeable and lyse [31].
Cell death results from releasing genetic information, proteins, and minerals through damaged cell membranes.The protein leakage from bacterial pathogens was greater than that from fungal infections.These findings suggest that most of the cells exposed to nanoparticles were phantom cells that released intracellular content into the suspension of cells.The degree of harm to the cell membrane is determined by how long it is exposed to this stress on the cell wall.As a result, more lactate dehydrogenase enzymes were generated [32].

1238
This study looked at the antibacterial effects of various TiO 2 concentrations.One of the most significant agents responsible for nosocomial infections, E.coli, is also resistant to broad-spectrum antibiotics.Antibiotic resistance in bacteria has significantly grown due to excessive antibiotic use [33].
The rates of infectious disease mortality and morbidity can be decreased by the introduction of innovative antibacterial medications.Numerous bacterial strains have been demonstrated to be significantly inhibited by nanomaterials.Studies suggest that because metal oxides have a positive charge and microorganisms have a negative charge, there is an electromagnetic attraction between them that results in oxidation and finally results in the death of the microorganisms [34].Nanomaterials can also inactivate biological enzymes and DNA by collaborating with electron-donating groups like hydroxyls, indoles, thiols, polysaccharides, amino acids, and others.Bacterial cell walls develop pits, which increase permeability and cause cell death [35].
In the current work, we demonstrated that varying TiO 2 concentrations might stop an antibiotic-resistant strain of E.coli from growing.TiO 2 might be utilized as the appropriate disinfectant in hospital settings where resistant bacteria could spread readily and infect surgical wounds and burn victims.TiO 2 nanomaterials are used in the textile sector to create cotton textiles with an antibacterial effect [36].To reduce the risk of infection in patients, creating sutures or wound bands from such cotton materials could be conceivable.
As was seen for E.coli in the current study, strong binding of nanoparticles to the outer membrane of E.coli inhibits active transport, dehydrogenase, and periplasmic enzyme activity, which in turn inhibits RNA, DNA, and protein synthesis, ultimately leading to cell lysis [37].Such effective and quicker formulations may be useful in clinical settings where E.coli causes urinary tract infections (UTIs).Numerous bacterial strains have been demonstrated to be significantly inhibited by nanomaterials [38].To determine the optimal concentration that can have the most efficient antibacterial ability against the E. coli culture, several nanoscale TiO 2 and CdO concentrations were investigated throughout the current investigation.Our results are consistent with earlier research on the antibacterial properties of nanomaterials [38][39][40].Numerous studies have proposed potential pathways for nanomaterials' interaction with biological macromolecules.It is thought that metal oxides have a positive charge while microbes have a negative charge.The microbe and treated surface are attracted to one another in an "electromagnetic" manner.The moment the microorganism comes into touch, it oxidizes and promptly dies.The thiol groups (-SH) of the proteins on the bacterial cell surface are thought to interact with the ions produced by nanomaterials.These proteins protrude from the bacterial cell membrane, which allows nutrients to flow through the cell wall.Nanomaterials render proteins inactive, which reduces membrane permeability and ultimately causes cellular death [39] additionally, nanomaterials prevent the development of biofilms and bacterial adhesion.These proteins stick out from the bacterial cell membrane and allow nutrients to pass through the cell wall proteins rendered inactive by nanomaterials, which decreases membrane permeability and ultimately causes cellular death [39], nanomaterials prevent the development of biofilms and bacterial adhesion [40,41].A widely sought goal is an antimicrobial modification to stop the growth of harmful germs.A compact bio-film matrix is created because microbial cell development and colonization can shield the underlying microorganisms from antibiotics and the host's defensive systems.Serious illness may ensue from microbial invasion [42,43].These infections are also linked to material biofouling, food deterioration, and the development of food-borne illnesses [44].As a result, Much emphasis is being paid to creating antimicrobial goods and surfaces for application in the biomedical, food, personal hygiene, and health industries.The metal-based nanoparticles' broad-spectrum biocidal activity against various bacteria, fungi, and viruses [43].Nanomaterials are known to inactivate biological enzymes and DNA by coordinating with electron-donating groups such as thiols, carboxylates, amides, imidazole, indole, and hydroxyls.They puncture the bacterial cells' walls, increasing permeability and resulting in cell death [44].

Conclusion
Two distinct materials, ZnO and TiO 2 , were successfully synthesized and characterized using diagnostic tools such as XRD, FTIR, and SEM.The analyses confirmed the successful preparation of ZnO and TiO 2 in accordance with their respective phases.The determined granular sizes were 18.6nm for ZnO and 12.6nm for TiO 2 .The research demonstrates that both ZnO and TiO 2 are reliable materials with potential antibacterial applications.Notably, ZnO exhibited a superior antibacterial effect, displaying a more robust capability in eliminating Gram-positive and Gram-negative bacteria than TiO 2 .This highlights the differential antimicrobial properties of the two materials, with ZnO emerging as a safer and more effective option for combatting bacterial infections.These findings contribute valuable insights into the synthesis, characterization, and antibacterial efficacy of ZnO and TiO 2 nanoparticles, emphasizing their potential applications in various fields, including medicine and healthcare.

Figure 1 :Figure 2 :
Figure 1: FTIR spectra of Zinc oxide particles, illustrating a large peak and a shoulder, both consistent with Zn-O bonds

Figure 3 :Figure 4 :
Figure 3: XRD peaks of ZnO particles, the hexagonal ZnO particles' No more peaks seen, demonstrating this purity of the powder

Table 1 :
The antibacterial activity of TiO 2 &ZnO affected two types of bacteria with different percentages of kills