Antibacterial Application of Copper Nanoparticles Biosynthesized by Water Caltrop Pod.

This study investigates the utilization of water caltrop pod, an abundant agricultural waste product, as a green extract for the optimized biosynthesis of copper nanoparticles (CuNPs). To comprehensively characterize the water caltrop pod and the biosynthesized CuNPs by water caltrop pod a sophisticated techniques were employed, including U.V-Vis spectrophotometry for probing their preliminary analysis of copper nanoparticles, FT-IR spectroscopy for elucidating the functional groups present in water caltrop pod, Scanning Electron Microscopy (SEM) for visualizing the morphology, Energy Dispersive X-ray (EDX) for elemental analysis while Energy-Dispersive X-ray (XRD) to determine crystalline structure of copper nanoparticles. Furthermore, the antibacterial application of these biogenic copper nanoparticles was explored. The antibacterial activity of copper nanoparticles (CuNPs) was investigated against both Gram-positive ( Staphylococcus aureus ) and Gram-negative ( Escherichia coli ) bacteria, revealing their efficacy in combating microbial growth employing a Well Diffusion method. Copper nanoparticles showed greater antibacterial treatment against Gram (+ve) bacteria i.e. Staphylococcus aureus as compared to Gram (-ve) bacteria i.e. Escherichia coli. This research paves the way for the sustainable production of bio-functional CuNPs from waste biomass, offering promising application in antibacterial therapies.

The green waste materials have an enriched composition like lignin and cellulose containing the carbohydrates, amino acids.Taking the advantage of this composition, an intention has been created to couple green waste material with the metal nanoparticles to biosynthesize metal nanoparticles of various shapes and sizes (Amrita et al., 2016).The green waste material extract (Kamalpreet et al., 2022) treating with metal-ions or metal-oxides was carried out.In the present work biosynthesis of copper nanoparticles (CuNPs) involving water caltrop pod (WCP) has been selected as it has gained considerable interest in recent years as a facile, effective, cheap (Kamalpreet et al., 2022) and eco-friendly method (Omnia and Deyab, 2023).Copper nanoparticles synthesis depends on several factors like extraction mode, biosynthesis parameters like temperature, pH, contact time and concentration of metal ions or metaloxides (Siyuan et al., 2023).Characterization of water caltrop pod and copper nanoparticles synthesized by water caltrop pod was done using various analytical instruments and copper nanoparticles utilized for antibacterial treatment against Gram (+ve) bacteria i.e.Staphylococcus aureusas as compared to Gram (-ve) bacteria i.e.Escherichia coli (Ahmed et al., 2024;Nervy et al., 2018).The objective of this work is to utilize green waste material like water caltrop pod to biosynthesize copper nanoparticles and its applications for treatment of pathogens such as Staphylococcus aureusas and Escherichia coli.

Material and Method
Water caltrop pod (WCP) Eleocharis-dulcis a Trapa nut is an aquatic annual herb that grows in ponds or marshes with its roots in the muddy soil at the bottom of water and with its upper leaves floating on the water surface.Water caltrop pods purchased from local market in Hyderabad, Sindh province, Pakistan.All the compounds or chemicals utilized throughout the practical work were chemically pure and analytically equivalent-grade.Water caltrop pod, CuSO4.5H2O,Deionized water, Filter papers, Conc.HCl, Conc.H2SO4, HNO3, 2-Propanol, NaOH, CH3COOH, CH3COONa, NH3 liquid, Sodium EDTA, Methanol, obtained from Sigma-Aldrich and Merck (Germany).
Water caltrop pods were separated from water caltrop and dried.To obtain water caltrop pod extract, a 20 g water caltrop pod was heated in 250 mL of deionized H2O for an hour at 90 0 C and centrifuged at 500 rpm for 2 hours.Filtered the water caltrop pod extract and utilized it to get biosynthesized copper nanoparticles (Khoiriah and Reza, 2018).The water caltrop pod extract treated with penta hydrated copper sulphate was carried out.Several factors like, concentration of metal precursor, temperature of solution, agitation-time, agitationspeed were reviewed (Siyuan et al., 2023).100 mL water caltrop pod extract equilibrated with 200 mL penta hydrated copper sulphate (0.1 M) in 1:2 at 80 0 C and centrifuged at 1000 rpm for 2 hours by using 79-1Magnetic Stirrer with hotplate Jiangsu Jinyi Instrument Technology Company Limited made in China used for biosynthesis of copper nanoparticles.The solution color switched from light blue to dark green indicated the biosynthesis of copper nanoparticles.A peak at 560 nm confirmed successful biosynthesized copper nanoparticles (Figure 1).Then dried the product, copper nanoparticles were put in the oven at 45 0 C for 4 h.
The antibacterial activity of these nanoparticles was evaluated by using Gram positive & Gram negative bacteria i.e.Staphylococcus aureus and Escherichia coli respectively employing a Well Diffusion Mode.Blood samples collected through swab consist of a sterile cotton tipped sticks and prepared media by adding copper nanoparticles.Blood samples then transferred to blood culture bottle.A slide test for bound coagulase and takes several hours to overnight to produce results.Applying MHA (Mueller Hinton Media), 1mg CuNPs + 1L H2O.Heated it then sterilized using steam sterilizer at 121 0 C for half an hour, nurtured at 37 0 C for 1 day then observed the inhibition zone (Ahmed et al., 2024;Nervy et al., 2018).
The mechanism of action of copper nanoparticles involve the release of metal ions, which can damage bacterial cell membrane and interfere with cellular processes, ultimately leading to inhibition of bacterial growt.  1 ).

Discussion
The biosynthesis of copper nanoparticles using water caltrop pod was studied for antibacterial application of copper nanoparticles against two bacterial strains Staphylococcus aureus and Escherichia coli as a positive and negative controls (Mehrab Pourmadadi et al., 2024).Biosynthesis of copper nanoparticles using water caltrop pod is an effective, cheap (Kamalpreet et al., 2022) and ecofriendly method (Omnia and Deyab, 2023).Copper nanoparticles synthesis depends on several factors like extraction mode, biosynthesis parameters like temperature, pH, contact time and concentration of metal ions or metal-oxides (Siyuan et al., 2023).
Characterization of water caltrop pod and copper nanoparticles biosynthesized by water caltrop pod was done using various analytical instruments (Siyuan et al., 2023).Preliminary identification of copper nanoparticles and water caltrop pod extract was done using U.V-Vis spectrometer.The CuNPs was formed in a brick brown color during the reaction time was investigated by UV-Vis spectrophotometer Perkins Elmer (Uberlingen, Germany) was used for the analysis of biosynthesized copper nanoparticles.A peak at 560 nm confirmed successful biosynthesized copper nanoparticles (Khoiriah and Reza, 2018).The FT-IR spectra of water caltrop pod (WCP) was attain to elaborate the functional groups present in water caltrop pod (WCP).The FT-IR spectra of water caltrop pod was evaluated in the range of 4000-625 cm -1 .The surface morphology of water caltrop pod (WCP) and CuNPs exhibit a micro-porosity at 1000x resolution while the images of water caltrop pod (WCP) and particle size of 10 micro-meter.Water caltrop pod (WCP) elemental composition was explored by EDX analysis.XRD measurement for CuNPs synthesized by water caltrop pod extract carried out in scattering range 2θ.The same analysis elaborated by Minakshi (Minakshi A Thakar et al., 2022) and indicated the CuNP's crystallinity & cubic geometry.
The antibacterial activity was evaluated by measuring the zone of inhibition surrounding the disc in mm and recorded the results.The antibacterial activity of these nanoparticles was evaluated by using Gram positive & Gram negative bacteria i.e.Staphylococcus aureus and Escherichia coli.Copper nanoparticles showed greater antibacterial treatment against Gram (+ve) bacteria i.e.Staphylococcus aureus as compared to Gram (-ve) bacteria i.e.Escherichia coli (Ahmed et al., 2024;Nervy et al., 2018).From inhibition zone results, biosynthesized copper nanoparticles from water caltrop pod showed higher inhibition activity against Staphylococcus aureua.A significant inhibition of zone was shown against grampositive bacteria.The zone of inhibition against Staphylococcu aureua was 15.6 mm and for Escherichia coli was 13.6 mm.The antibacterial effect is directly related to the nanoparticles size, minimum inhibitory concentration of nanoparticles, and oxidation degree of surface (Amrita Banerjee et al., 2016).The inhibition zone of bacteria decreases as the concentration of nanoparticles decreases.(Pratibha Kaushal et al., 2024).

Conclusion
Copper nanoparticles biosynthesized by water caltrop pod extract a natural waste material under optimized parameters.The biosynthesized copper nanoparticles were characterized by different analytical instrumental techniques such as FT-IR spectrum displayed a number of peaks appeared in the range 4000-625 cm -1 .The SEM images revealed the highly porous surface of copper nanoparticles biosynthesized by water caltrop pod.EDS and XRD depicted the confirmation of copper within the surface of material.Overall copper nanoparticles successfully utilized in antibacterial activity against Gram (+ve) bacteria i.e.Staphylococcus aureus by showing 15.6 mm zone of inhibition as compared to Gram (-ve) bacteria i.e.Escherichia coli having 13.6 mm zones of inhibition.Biosynthesized copper nanoparticles can be applied in number of commercial applications such as medicines, agriculture, optics and environmental remediation.

Figure 2 .
Figure 2. FT-IR spectra determines the molecular structure of water caltrop pod (WCP).Graph plotted Transmittance percentage (% T) vs wavelength ranges from 500 to 4000 per centimeter (cm -1 ).Peaks appeared were 3341.09,1594.86,1340.08,1129.96 and 945.23 cm -1 .Sem Analysis: The surface morphology of water caltrop pod and copper nanoparticles exhibit a micro-porosity at 100x resolution while the images of water caltrop pod have particle size of 10 micrometer.SEM analysis showed the images of water caltrop pod and copper nanoparticles biosynthesized by water caltrop pod. Figure 3. (A) and (B) SEM image of copper nanoparticles biosynthesized by water caltrop pod.

Figure 3 .
Figure 3. (A) and (B) scanning electron microscope (SEM) image of water caltrop pod and copper nanoparticles respectively.The surface morphology of water caltrop pod and copper nanoparticles was elaborated by Hitachi S-2300 SEM with 10 kV at x100 resolution and 100 micro meter (μm).The SEM was equipped with EDX, which elaborated the elemental composition of WCP and CuNPs surfaces.The samples were carbon -coated using Edwards-Scan coat.EDX analysis of CPP: Water caltrop pod elemental composition was explored by EDX analysis.The EDX analysis of water caltrop pod appeared in Figure 4. (A), elaborate the presence of different elements comprises maximum quantity of K. EDX spectrum of CuNPs biosynthesized by water caltrop pod shown in Figure 4. (B).

Figure. 6
Figure.6 (A) zone of inhibition in mili meter (mm) of Escherichia coli.The antibacterial activity of copper nanoparticles was evaluated by using Gram-negative Bacteria i.e.Escherichia-coli (E-coli) employing a Well Diffusion method.Applying MHA (Mueller Hinton Media) then observed the Inhibition zone.

Figure. 6
Figure.6 (B) zone of inhibition in mili meter (mm) of Staphylococcus aureus.The antibacterial activity of copper nanoparticles was evaluated by using Gram positive Bacteria i.e.Staphylococcus aureus employing a Well Diffusion method.Applying MHA (Mueller Hinton Media) then observed the Inhibition zone.
spectra of CPP: WCP infrared spectrum was attained to explore the presence of functional groups in water caltrop pod.

Table 1 .
Zone of inhibition in mili meter (mm) of Escherichia coli (E coli) and Staphylococcus aureus (S aureus) for copper nanoparticles (CuNPs).