Effect of Codoping Zinc Oxide Nanoparticles with Sulfur and Nitrogen on Its Energy Bandgap, Antioxidant Properties, and Antibacterial Activity

Zinc oxide nanoparticles (ZnO-NPs) are used in various felds such as industrial, environmental remediation, catalytic, and antibacterial applications. However, their ability to absorb visible light is limited due to their high-energy bandgap and fast electron-hole recombination, which restricts their use. To enhance the efciency of ZnO-NPs in medical and other applications, surface functionality can be modifed through doping. Here, we investigated the efects of S and N doping on the energy bandgap of ZnO-NP and their antimicrobial and antioxidant activities. Te results showed that the optical bandgap energy of pure ZnO-NPs was 2.98eV while that of 6% N-ZnO, 4% S-ZnO, and S4-N6-ZnO was 2.78, 2.69, and 2.63eV, respectively. Te energy bandgap reduction is attributed to the changes in the electronic level of zinc oxide as the result of doping. Te crystal size of pure ZnO-NPs, 6% N-ZnO, 4% S-ZnO, and S4-N6-ZnO was 29.06, 27.05, 29.02, and 25.06nm, respectively, as calculated from XRD data using FWHM. Following the bandgap and particle size reduction, the antimicrobial activities of the dual-doped ZnO-NPs surpassed that of the pure ZnO-NPs. Moreover, dual doping improved the antioxidant activity of ZnO-NPs from 52.45% to 88.89% for the optimized concentration. Terefore, incorporating S and N as dual dopants can enhance the functionality and efciency of ZnO-NPs in various felds.


Introduction
Nanotechnology studies materials at the nanoscale of 1-100 nm.It is an interdisciplinary feld that has opened up new opportunities in material science and engineering [1][2][3].Metal oxide nanoparticles (MONPs) have several uses, including catalysis [4], optoelectronic materials [4], sensors [5], and environmental remediation [6].Due to their high surface area, nanoparticles are potentially valuable in biomedical applications since they can produce reactive oxygen species (ROS) more efectively.ROS production can be infuenced by exposure to ultraviolet (UV) light, disruption of intracellular metabolic activities, and antioxidant systems.As a result, oxidative stress can occur in cells, leading to damage to DNA, cell membranes, proteins, and even cell death [7][8][9][10].
To address these issues, researchers have explored various approaches to modify ZnO-NPs' properties.One such approach is doping, which involves the intentional introduction of impurities into a crystal lattice [6,9,[24][25][26].Dual doping is a promising technique that can enhance a limited portion of sunlight and improve the optical properties of ZnO-NPs.Doping can also minimize the energy bandgap, thereby enhancing ZnO's antioxidant and antimicrobial properties [14,[26][27][28].Te introduction of a limited amount of foreign materials, such as biomolecules and transition metals (Mn, Fe, Cr, and Cu) at the nanoscale, can increase and modify the surface area of ZnO-NPs, improving their functionality and efciency [5,6,9,29].Doping does not alter the precursor material but instead introduces new functionalities [9,18].Besides doping, the physical and chemical behavior of ZnO-NPs can be modifed by altering their shape using various methods and materials for synthesis [30].
ZnO nanocomposites are a better option than bare ZnO nanoparticles since they have improved properties [31,32].Nanocomposite materials have separate properties such as mechanical [33], electrical [34], optical [35], chemical [36], and catalytic properties [6,32].Nanoparticles have certain advantages over their counterparts, making them more favorable.Te Food and Drug Administration considers ZnO to be a safe material for biomedical applications, including drug delivery systems [37].ZnO nanoparticles have been used efectively in tissue engineering (TE) applications [32] with various shapes such as nanofower (NF) [13] and nanowires (NW) [34,38].In addition, several cell lines have shown better adhesion, growth, and diferentiation on these surfaces [39].
Tis study explores the impact of S and N codoping on the energy bandgap and antimicrobial activity of ZnO-NPs.Researchers have previously reported that the addition of sulfur to ZnO-NPs at varying temperatures reduces the bandgap energy from 3.34 to 3.22 eV [6,9,18].Similarly, Ag-N-codoped ZnO-NPs have been studied for their efects on the energy bandgap and photocatalytic degradation of organic pollutants by various authors [39][40][41].However, no prior research has investigated the impact of S-N-codoping on the energy band and its antimicrobial activity.
Te present study focuses on the dual nonmetal codoping of ZnO-NPs with S and N to minimize the energy bandgap and enhance their antimicrobial and antioxidant activity.Te dual-doped ZnO-NPs exhibit improved physicochemical properties, with their UV-vis absorbance wavelength shifting towards the red region, resulting in a reduced energy bandgap from 2.93 eV to 2.63 eV.Te dualdoped nanoparticles also demonstrate stronger antioxidant and antimicrobial activities than the ZnO-NPs.

Materials and Methods
ZnO nanoparticles (NPs) were prepared using the sol-gel method, which provides several benefts, such as repeatability, ease of synthesis, and regulation of composition.To create Sand N-codoped ZnO-NPs, the synthesized ZnO-NPs were mixed with appropriate amounts of urea and ZnS.Te main objective of this synthesis was to investigate the efect of doping on the physicochemical properties of ZnO-NPs.In this study, various types of laboratory equipment were used including beakers, measurement cylinders, magnetic stirrers, electronic balances, mufe furnaces, crystallizing dishes, burettes, funnels, flter paper, pH meters, spoons, sample bottles, and pipettes.In addition, advanced analytical instruments were used such as the directed ray radiometer (DRAWELL RADIometer-700), directed ray Cu radiometer (Cu Kα Radiometer (λ �1.54178 Å), direct ray visimeter (UV-Vis), and detailed ray spectrometer (Ray Spectrometer-200 PLUS-223E12).

Synthesis of ZnO-NPs.
Zinc oxide nanoparticles were synthesized using sol-gel techniques.Te frst step involved dissolving 0.4 M of zinc nitrate in 50 mL of distilled water.Next, 0.8 g of NaOH was dissolved in 100 mL of distilled water at room temperature to prepare 2 M sodium hydroxide.Continuous stirring of both solutions was carried out for 30 minutes using a magnetic stirrer.Te NaOH solution was then added slowly to the ZnO nitrate solution while stirring continuously, and the pH was adjusted to 10.After cooling for 2 hours, the solution was fltered using Whatman flter paper, washed 3 times with distilled water, and treated with ethanol.Te fltered sample was then dried in an oven at 60 °C and allowed to cool for 2 hours before being calcined at 500 °C in a mufe furnace.Te resulting white powders were confrmed to contain ZnO nanoparticles.

Preparation of N-Doped ZnO-NPs.
To select the optimized urea concentration, we dissolved 0.015%, 0.03%, 0.045%, and 0.06 g of urea in 25 mL of distilled water, at room temperature.We then added each concentration of urea to a previously prepared solution of hexahydrated zinc nitrate and stirred for 10 min to obtain a homogeneous solution.To prepare sodium hydroxide (2 M), we dissolved 0.8 g NaOH into 100 mL of distilled water.We then dropwise added the sodium hydroxide to the previously prepared zinc nitrate solution and urea solution, stirring until a precipitate formed.We controlled the pH to 10 and heated the mixture at 60 °C, stirring continuously for 2 hours.We kept the solution cool and fltered using Whatman flter paper, which was oven-dried at 60 °C and then calcinated in mufe furnaces at 500 °C for 2 hours.

Preparation of S-Doped ZnO NPs.
To prepare S-doped ZnO-NPs, frst varying amounts of ZnS salt (0.01, 0.02, 0.03, and 0.04 g) were measured and dissolved in 10 mL of distilled water.Next, 1 g of ZnO-NPs was mixed with the ZnS solution after being dissolved in 10 mL of ethanol.Te solution was stirred at room temperature for 3 hours.Te 2 Advances in Materials Science and Engineering S/ZnO mixture was fltered, and the residue was washed with distilled water until the pH of the removed water reached 7.
Te residue was air-dried overnight and then calcined at 500 °C for 2 hours.

Preparation of S-N-Co-Doped ZnO NPs. S-and N-doped
ZnO-NPs were prepared from zinc oxide nanoparticles by dissolving 1 g of ZnO in 10 mL of ethanol and mixing a selected amount (0.04 M) of urea solution and 0.04 M zinc sulfde solution.It was then stirred at room temperature for 3 hours, fltered, washed with distilled water until the pH of the removed water reached 7, left in air overnight, and calcined at 500 °C for 2 hours.

Antioxidant Activity Test.
As part of our research, we conducted a 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method to test the efectiveness of ZnO-NPs in scavenging free radicals.Te experiment involved observing how a methanol solution of DPPH changes when exposed to NPs.DPPH produces a purple or violet color in methanol, which turns yellow in the presence of antioxidants.We prepared a 0.1 mM DPPH methanol solution and mixed it with 1 mL of methanol solution containing pure ZnO NPS and S-and N-doped ZnO NPs at concentrations of 70, 90, 110, and 130 mg/mL.Te mixture was then vortexed and incubated in the dark at room temperature for 30 minutes.
We measured the absorbance of the mixture at 517 nm using a spectrophotometer to determine the percentage of DPPH radical scavenging activity.Ascorbic acid was used as a standard.

% DPPH radical scavenging activity �
where the absorbance of the control is A 0 and the absorbance of the sample is A 1 .
After plotting the percent of inhibition against concentration, the IC50 was derived from the graph.Te experiment was repeated three times at each concentration.

Antimicrobial Activity. Te Department of Biology at
Jimma University conducted bacterial studies using the agar disk difusion method.Tey tested the synthesized nanoparticles on bacterial strains such as Staphylococcus aureus, Salmonella typhimurium, Bacillus cereus, and Escherichia coli.Te nanoparticles were also tested for their antifungal activity against Candida albicans.For the test, cultures were used to inoculate the medium using test tubes with concentrations or densities approximately equal to 0.5 McFarland standard.Test bacteria and fungi were inoculated with freshly grown liquid cultures similar to 0.5 McFarland turbidity using Mueller-Hinton agar and potato dextrose agar, respectively.DMSO was used as the negative control, and gentamicin and Clo were used as positive controls for bacteria and fungi, respectively.Te test compound was dissolved in DMSO solutions containing 100 mg/ml.Te solution was then added to the culture wells and incubated at 37 °C for 24 hours.Te plate had an inhibition zone around a 6 mm diameter plain paper disc, and the antibacterial activity of the nanoparticles was detected by measuring the diameter of the inhibition zone formed around the sample.

Results and Discussion
UV-Vis spectroscopic measurements were performed to investigate the efect of doping on the absorption of ZnO-NPs.Here, diferent amounts of impurities were mixed with pure ZnO-NPs and their characteristic UV-Vis absorption was measured.Te introduction of N and S shifted the maximum absorption wavelength towards the red.Among all measurements, 6% N-ZnO, 4% S-ZnO, and S4-N6-ZnO showed the greatest change in UV-Vis absorption and was considered the optimal composition for this study.Figure 1 shows the UV-visible absorption spectra of 6% N-ZnO, 4% S-ZnO, and S4-N6-ZnO.Hybridization of Zn with the HOMO of the highest occupied molecular orbital resulted in the lowest unoccupied molecular orbital (LUMO).Te bandgap is due to the 2p orbital and leads to UV-induced absorption.It shows the absorption spectra of all freshly prepared puredoped and doubly-doped ZnO-NPs.Absorption values of ZnO are observed with other materials at wavelengths of 370 nm, 376 nm, 377 nm, and 379 nm.Tese apply to 4% S-ZnO, 6% N-ZnO, and S4-N6-ZnO, respectively.Tere are no other peaks.Only a redshift of ZnO to doped and doublydoped ZnO-NPs was observed.Tis indicates that the doping process did not change the crystal structure of ZnO-NPs.

Optical Bandgap Energy Analysis.
Te optical energy bandwidth (E g ) was calculated using the Tauc method [42,43].A Tauc curve can be plotted using the calculated UV-Vis absorption results using the following equation: where α stands for the absorption coefcient, h represents Planck's constant, v is the frequency of light, and E g is the optical band gap. Figure 2 illustrates a Tauc plot plotted between (a) 2 and hv using the equation above, where extrapolating the linear part of the plot gives the bandwidth value.Te calculated band values are 2.98 eV, 2.78 eV, 2.69 eV, and 2.63 eV for pure ZnO-NPs, 6% N-ZnO, 4% S-ZnO, and S4-N6-ZnO.As expected, there is a diference in the energy bands of doped and codoped compared to pure ZnO NPs.In particular, the energy bandgap of S4-N6-ZnO decreases compared to the energy bandgap of pure ZnO-NPs, 6% N-ZnO, and 4% S-ZnO.Tis may be due to changes in the electronic level of zinc oxide.More 2p nitrogen electrons compared to nitrogen-substituted oxygen induce a new energy level between the conduction and valence bands of ZnO, leading to a decrease in the bandgap of ZnO NPs.

Powder X-Ray Difraction (XRD) Analysis.
Te sample was produced using a Zn-(NO 3 ) 2 .6H 2 O precursor via a standard sol-gel process and calcined at 500 °C for 5 hours.Te product was characterized through Cu Kα radiation at Advances in Materials Science and Engineering 2θ of 10-80 °.Te XRD peaks are illustrated in Figure 3, demonstrating a hexagonal crystal structure (JCPDS).Te highest peaks were identifed at 2θ � 28.62 °, 31.86 °, 34.62 °, 36.28 °, 47.64 °, 56 °, 68.18 °, 78 °, and 78.15 °.Tese lines were identifed as the hexagonal phase of ZnO.Te particles display high crystallinity at 36.28 °for both single and double doping, as indicated by a sharp peak and high intensity.Te growth of larger crystals from smaller ones leads to a reduction in the number of smaller grains, whereas larger grains continue to grow.
In Figure 3, we can observe pure (doped), S-doped, and N-doped ZnO-NPs.Specifc peaks can be located at 20 (degree) � 28.62 °, 31.86 °, 34.62 °, 36.28 °, 47.64 °, 56.78 °, 63.15 °, and 68.18 °.Te difraction patterns of S-doped and N-doped ZnO-NP are very similar to those of pure ZnO-NP and are almost identical.Tis means that sulfur and nitrogen atoms were added to the ZnO crystals.Te absence of additional peaks indicates that the presence of sulfur and nitrogen compounds or complexes is excluded from the doped ZnO-NPs with respect to S and N.However, the additional peaks Te average size of crystallites of ZnO-NPs doped with S, N, and both S and N, as well as those that are pure, can be determined by using the full width at half maximum (FWHM) of the strongest peaks of their respective crystals.Te calculation involves using the Dybe-Scherrer equation, which is expressed as D � (0.9 λ)/βcosθ.Here, λ represents the X-ray wavelength, D is the average crystal size, θ is the Bragg difraction angle, and β is the FWHM in radians.It is observed that the size of crystallites decreases with increasing doping concentration, resulting in a decrease in the peak intensity.Te size of the synthesized NPs for the strongest peaks is recorded in Table 1 and is calculated based on XRD results using the Debye-Scherrer equation [6,9,43].Te crystallographic size in the decreasing order of the synthesized NPs is as follows: S-ZnO > ZnO NPs > S-N-ZnO > N-ZnO NPs.Te lower size of S-N-ZnO and N-ZnO-NPs is due to N doping, as N doping shows the lowest crystallite size (Table 1) [27].Te most notable trend as a function of particle size is that the surface area-to-volume ratio increases for smaller particles.Here, it is worth mentioning that the additional peaks introduced in the XRD patterns could be due to the possible introduction trace amounts of impurities during sample transportation from synthesis laboratory to the XRD characterizations.

FTIR-Analysis. Te transmission properties of bare
ZnO nanoparticles, doped ZnO nanoparticles, and codoped ZnO nanoparticles were measured by FTIR in the wave number range of 4000-400 cm −1 .Figure 4 shows the IR spectra of ZnO-NPs, 6% N-ZnO, 4% S-ZnO, and S4-N6-ZnO samples, highlighting the diferences in functional groups.Te IR spectrum reveals that the absorption band of zinc oxide in the Zn-O stretching mode is between 400 and 850 cm −1 , corresponding to the hexagonal crystal structure of ZnO [40,44].Te peak observed between 3300 cm −1 and 3500 cm −1 represents the O-H stretching mode of hydroxyl groups due to water adsorption on the surface, indicating the presence of impurities mainly near the ZnO surface.Furthermore, a strong absorption peak at 877 cm −1 was observed in all samples, which can be attributed to the substitution of H by O.A new peak was observed at 1410 cm −1 , which was not sharp due to the formation of the sample microstructure (Zn-S) during the doping process.Te peak at 1387 cm −1 corresponds to the Zn-N stretching vibration, and the peak at about 2426 cm −1 is due to the C=O stretching vibration.

Morphological Analysis.
To clarify the external structure of 6% N-ZnO, 4% S-ZnO, and S4-N6, SEM analysis was conducted.Te SEM images of pure ZnO-NPs, 6% N-ZnO, 4% S-ZnO, and S4-N6 are displayed in Figure 5. Te morphology of dual-doped ZnO-NPs (S4-N6) is signifcantly improved compared to single-doped and pristine ZnO-NPs.Te crystal size of pure ZnO-NPs, 6% N-ZnO, 4% S-ZnO, and S4-N6-ZnO are 29.06,22.44, 29.21, and 25.76 nm, respectively, as calculated from XRD data.Tere is good agreement between the calculated particle size from the XRD patterns and the morphology of the synthesized materials.It is worth mentioning that adding dopants to pure ZnO-NPs improved stability and morphology by modifying surface characteristics as revealed by the FTIR analysis.As a result of improvement in morphology, several physicochemical properties of the doped nanoparticle were enhanced compared to the pure one.
Te antimicrobial exercises of pure ZnO-NPs (undoped), N-doped ZnO-NPs, S-doped ZnO-NPs, and S and N codoped ZnO-NPs against Staphylococcus aureus, Salamonella typhi (−ve), Bacillus cereus, Escherichia coli (−ve), and Candida alibican were done utilizing the disk dissemination strategy and the chart for the zone of inhibition is demonstrated in Figure 6.Te antimicrobial action for pure ZnO-NPs (undoped), 6% N-doped ZnO-NPs, and 4% Sdoped ZnO-NPs were found to be compelling against all microscopic organisms' strains and organisms.In any case, the rate decay in S-N-ZnO NPs was found to signifcantly increment in bacterial action.Te antimicrobial impacts are in this way compared to engineered NPs, which were more dynamic for Gram-positive microscopic organisms.Te conceivable reason behind contrasts is (i) cell layer structure, (ii) physiology and metabolic exercises of the cell, and (iii)      Advances in Materials Science and Engineering degree of contact of Gram-positive and Gram-negative microscopic organisms.Te antibacterial movement of single-doped and dual-doped ZnO nanoparticles appeared that they were safer for Gram-negative microbes than that of Gram-positive microbes.For the most part, 6% N-doped ZnO NPs appeared way better antifungal movement when compared with undoped ZnO-NPs, as demonstrated by the inhibition zone in Table 2 and usually upheld in prior writing.Te improvement in antimicrobial activity is attributed to the improved stability and reduced particle size due to doping, which enables the particles to penetrate bacterial cell walls easily.Tis changes the structure of the cytoplasmic membrane, which in turn ruptures organelles and even leads to cell lysis.

Antioxidant Activity.
Te scavenging properties of diferent types of ZnO nanoparticles were evaluated using the DPPH method.Te types included pure (undoped) ZnO-NPs, 6% N-doped NPs, 4% S-doped NPs, and S4-N6 ZnO-NPs (Figure 7).Te results showed that all types of NPs had an enhanced radical scavenging activity (RSA) with     3. It is well-known that ZnO-NPs and 6% N-doped NPs have high antioxidant activity, whereas 4% S-doped and codoped NPs reach the break-even point when limited to 50 μg/mL.Te exchange of electron thickness from oxygen particles to nitrogen molecules in ZnO-NPs enables them to retain DPPH-free radicals containing odd electrons through n ⟶ π motion [25].Te increased in the antioxidant activities attributed to the productions species that scavenge the reactive oxygen species and mimic the antioxidant molecule due to their intrinsic physicochemical properties.

Conclusion
In this study, we examined the efects of sulfur and nitrogen doping, as well as codoping, on the properties of ZnO nanoparticles.Changes in the optical energy bandgap, antimicrobial activity, and antioxidant activity are examined in the study.For nitrogen and sulfur doping, the UV absorption spectra show a redshift from 370 to 377 nm and 378 nm, respectively, while for S-N-codoping, the shift is from 370 to 379 nm.When nitrogen doping is applied, the gap energy decreases from 2.97 to 2.78 eV, when sulfur doping is applied, and when S-N-co doping is applied, it decreases from 2.97 to 2.63 eV.In addition, XRD analysis showed that some additional peak patterns were observed with S-N-codoped nanoparticles, which are the result of the partial transformation of complex compounds S and N.At the same time, the hexagonal structure of ZnO-NPs remains unchanged.Te size of the NPs has been calculated using FWHM, and the size of N-S-dual-doped ZnO-NPs is smaller than that of single-doped and pure ZnO-NPs.ZnO-NPs with dual doping showed greater antimicrobial and antioxidant activity.Consequently, the dual-doped ZnO-NPs were much more efective than pure ZnO-NPs at inhibiting Gram-positive and Gram-negative bacteria species.Based on the results of this study, bandgap energy narrowing and surface-to-volume ratio increases can enhance the antimicrobial and antioxidant activity of ZnO-NPs.Te improvement in antimicrobial is attributed to the improved stability and reduced particle size due to doping, which enables the particles to penetrate bacterial cell walls easily.Tis changes the structure of the cytoplasmic membrane, which in turn ruptures organelles and even leads to cell lysis.Doping of ZnO-NPs increases their antioxidant properties (from 52.45% to 88.94%) as well.Tus, such doped nanoparticles are ideal candidates for targeted delivery in vivo, combating oxidative stress [44].

Figure 6 :
Figure 6: Picture showing the zone of inhibition of the pure and doped ZnO-NPs against some selected microorganisms using Genta and CLO as the positive control and DMSO as the negative control (a) S. aureus, (b) S. Typhi, (c) E. coli, (d) B. cerus, and (e) C. albican.