Exploring bimetallic Au–Ag core shell nanoparticles reduced using leaf extract of Ocimum tenuiflorum as a potential antibacterial and nanocatalytic agent

Biosynthesis of bimetallic Au–Ag core shell nanoparticle under ambient condition using Ocimum tenuiflorum (Krishna Tulsi) leaf extract is being reported for the first time. For the biosynthesized bimetallic core shell nanoparticles (OTAuAgNPs) visual and spectrophotometric analysis was performed. Development of a deep purple color suggested the creation of OTAuAgNPs. It was then validated by UV–Vis spectrophotometry within 4 h of incubation showing a surface plasmon resonance peak at 560 nm. FESEM-EDAX, FT-IR, particle analyzer and XRD were the analytical techniques employed to characterize the biosynthesized OTAuAgNPs. Using a known antibiotic as a control, OTAuAgNPs (10 mg/ml) had a substantial bacteriostatic effect against Gram + ve (Bacillus subtilis) and Gram −ve (E.coli, Pseudomonas aeruginosa) bacteria. The OTAuAgNPs (6.5 mg) showed good catalytic action for the adsorption of Coomassie brilliant blue R250 dye (CBB) (50 mg/L, 25 mL), confirmed by the reduction in absorbance maxima that is time dependent. A remarkable adsorption of CBB (94.79%) was attained within 120 min, and the results were best-suited PSO and Elovich models with adsorption kinetic constant of 5.96E−04 g/mg.min and 37.81 mg/g.min, respectively. Further the toxicity studies of brilliant blue R250 dye and metabolite of CBB (post nano-remediated dye solution) was carried out on Vigna radiata (green gram) germination and growth. OTAuAgNPs as nanocatalyst remediated water revealed that it was nontoxic. Thus an ecofriendly and cost-effective approach for bimetallic nanoparticle synthesis could be further explored for removal of CBB from wastewater effluents.


Introduction
The use of biological agents to synthesize bimetallic nanoparticles (NPs) has gained popularity due to its cost-effectiveness, environmental friendliness, and ease of scaling up without utilization of lot of energy and use of harmful chemicals (Khan et al. 2020;Padilla-Cruz et al. 2021). It also does not require cell culture care and can be stepped up for largescale synthesis (Gour et al. 2019). Plant extract-based NP biosynthesis is more stable and has a higher rate of synthesis than other biological processes. Researchers have recently begun concentrating on the production of bimetallic nanoparticles in order to investigate the potential of two metals for a variety of applications ). Furthermore, there are only a few recent studies on biological synthesis core and shell bimetallic nanoparticles, from aqueous plant extracts; to name a few Ag-Fe nanoparticles synthesized using Gardenia jasminoides leaf extract (Padilla-Cruz et al. 2021), silver-platinum nanoparticles biogenically synthesized using Schinusmolle L. leaf extract (Mares-Briones et al. 2019) and Au-Ag nanoparticles synthesized using weed Antigonon leptopus which is of no value (Ganaie et al. 2016), seed extract of Lawsonia inermis (Akilandaeaswari et al. 2021), clove bud extract (Sharma et al. 2020), trapa peel extract (Ahmad et al. 2019), and leaf extract of Stigmaphyllon ovatum (Elemike et al. 2019). Recently, bimetallic nanoparticles have been explored in diverse fields owing 1 3 to its extreme properties, to name a few, gold silver core shell nanoparticles synthesized by laser ablation technique have been explored for its electrical conductivity properties (Awwad et al. 2021), copper-silver nanoparticles for catalytic degradation of methylene blue (Al-Haddad et al. 2020), bacterial killing mediated by bimetallic gold-silver nanoparticles that disrupt the actin cytoskeleton, causing morphological alterations and death (Jena et al. 2020).
The dye industry's wastewater is the second most dangerous industrial effluent, with around 100 tonnes of dye released each year (Benkhaya et al. 2020). Because of their synthetic origins and intricate structures, dye discoloration is difficult (Mazzoli et al. 2021). Nanoparticles are increasingly being used to treat dye effluents due to their qualities such as greater nano-adsorptive capabilities, faster attainment of equilibrium, and large surface area (Homaeigohar et al. 2020). Textile effluents are known to have detrimental effects on plants by bioaccumulation/disrupting photosynthesis mechanism in aquatic flora (plants and algae) (Berradi et al. 2019). Adsorption, photocatalytic degradation, and adsorption plus degradation are all mechanisms for hazardous dye elimination by nanoparticles (Alkaykh et al. 2020). Since degradative products of dyes can sometimes be much more mutagenic and carcinogenic than the parent compounds, understanding the toxicity of nano-remediated water on plant growth is important (Lellis et al. 2019). Keeping this in view, the study was undertaken to see the effect of dye as well as nano-remediated dye solution on germination and growth of Vigna radiata.
The current study, thus, focuses on biological plantbased synthesis of bimetallic Au-Ag nanoparticles from Ocimum tenuiflorum (Krishna tulsi) leaf extract (OTAuAg-NPs). Holy basil, or O. tenuiflorum, is a plant native to the Indian subcontinent that is widely cultivated across the Southeast Asian tropics (Mousavi et al. 2018). Although it is known to have potential health benefits such as antimicrobial (Yamani et al. 2016), anti-diabetic (Mousaviwt et al. 2018, antioxidant, and protection of DNA from damage (Sarkar et al. 2014), there are no reports on biosynthesis of bimetallic gold-silver nanoparticles using this plant for the applications of environmental remediation. Synthesized OTAuAgNPs were characterized by UV-visible spectrophotometry, particle size and zeta sizer, Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) analysis. This is the first report on CBB adsorption by bimetallic Au-AgNPs produced from O. tenuiflorum, with focus on understanding the kinetics of adsorption and understanding the toxicity of the Dye as well as metabolite (nano-catalyst treated water) on Vigna radiata (green gram) germination and growth.

Plant material and chemicals
Fresh and firm O. tenuiflorum leaves were got from Nitte (13.1859° N, 74.9395° E) and Belman (13.1664° N, 74.8712° E) premises (Fig. 1a). AgNO 3 and HAuCl 4 required for nanoparticles synthesis was procured from Loba Chemie. Unless otherwise stated, all other compounds and reagents employed in the current study were of analytical grade. Coomassie Brilliant blue R250 analytical grade were bought from Hi-Media (Mumbai, India).

Ocimum tenuiflorum leaf extract preparation
Freshly plucked leaves of O. tenuiflorum (Krishna tulsi) got from the premises were washed thoroughly with tap water and distilled water. The finely chopped leaves suspended in distilled water at a ratio 1:10 were heated at 80 °C with occasional swirling for 30 min. Filtrate was then filtered and stored at refrigerator condition (4 °C) until further use.

Synthesis of OTAuAgNPs
OTAuAgNPs were synthesized by mixing O. tenuiflorum leaf extract with silver nitrate and gold chloride solution (1.0 mM) each such that 25 ml of the ready leaf extract was added to 115 ml of 1 mM aq. AgNO 3 and 110 ml of 1 mM HAuCl 4 . This blend was incubated at 27 ± 2 °C, 100 RPM agitation speed and observed for color change in the solution. The OTAuAgNP pellet was obtained by centrifuging the sample at 10,000 RPM for 30 min. The pellet was cleaned twice with sterile distilled water, dried in a desiccator, and reserved for future use.

Characterization of OTAuAgNPs
The colloidal solution of synthesized OTAuAgNPs was characterized by recording its absorbance between 200 and 670 nm using a UV-Vis spectrophotometer (Thermo-Merck, Germany). Dried OTAuAgNPs were subjected to gold sputtering and fixed on to aluminum stubs. In order to recognize the functional groups of O. tenuiflorum on OTAuAgNPs, FT-IR (PerkinElmer System 2000, England) was performed with potassium bromide between 4000 and 400 cm −1 . Their morphological characteristics and fundamental composition were determined by SEM-EDX (JEOLJSM-7800F) under different magnifications. X-ray diffraction (RigakuMini-flex600, Japan) is used for phase identification of crystalline OTAuAgNPs by finding out its diffraction angles. This method revealed the crystalline structure of nanoparticles.

Bactericidal activity of OTAuAgNPs
Strains and media Lyophilized P. aeruginosa, E. coli (Gram −ve) and B. subtilis (Gram + ve) were obtained from MTCC-Chandigarh. Bacterial growth initiation was done by adding a pinch of lyophilized bacteria in YPD medium and growing for 24 h.

Study of bactericidal activity of OTAuAgNPs
Bactericidal activity of OTAuAgNPs against Gram + ve (B. subtilis) and Gram −ve (E. coli, P. aeruginosa) was investigated. For antimicrobial assay, fresh microbial colonies (100 μl) were inoculated into 10 ml of nutrient broth medium. After 16-18 h of incubation the cfu/ml was calculated for 1:10 5 dilutions which corresponds to 1.7 × 10 9 , 9.3 × 10 8 and 6.7 × 10 8 cfu/ml for E. coli, B. subtilis, P. aeruginosa, respectively. OTAuAgNPs were reconstituted in DMSO at concentration 10 mg/ml. Bactericidal activity of OTAuAg-NPs against gram + ve and gram -ve microbes was carried out (Philip et al. 2011 and Ciprofloxacin (10 mg/ml) served as control. The inhibition zones were then measured as per standard protocol (Philip et al. 2011).

Adsorption of CBB by OTAuAgNPs
OTAuAgNPs (6.5 mg) were incubated with aqueous solution of CBB (50 mg/L, 25 mL) at 27 ± 2 °C, 100 RPM. Residual concentration of CBB was measured by recording absorbance of aqueous solution at 552 nm (λmax) every 30 min. % Adsorption and amount of adsorbed dye was calculated as per Eqs. 1 and 2, respectively. For accuracy, the same experiment procedures were repeated three times.
where Co and Ct are the initial and periodic CBB concentration, respectively.
where Qt = Dye adsorbed (mg/g), V is total volume (in liters) and W is OTAuAgNPs mass (g).

Adsorption kinetics
The adsorption kinetics of CBB by OTAuAgNPs were studied by fitting experimental values to pseudo first order (PFO), pseudo second order (PSO), Elovich and intraparticular diffusion kinetic equations. It is often noted that adsorption kinetics is a function of adsorbate concentration (Kamath Miyar et al. 2021). The following Eqs. 3 and 4 depict the PFO and PSO kinetics which were modeled to study the adsorption process of CBB by OTAuAgNPs.
where Qe and Qt = Adsorbed CBB ( mg/g) at equilibrium and time, respectively, and k ( min −1 ) is PFO rate constant where k 1 (g/mg.h) is the PSO rate constant. Elovich model describes the adsorption process as chemisorption and is denoted by the following Eq. (5) where α = preliminary adsorption rate (mg/gh) and β is a constant dependent on activation energy (g/mg).
Intraparticular diffusion model is essential in determination of rate limiting step in the adsorption process and is described by Eq. 6.
where kp is the intraparticular rate constant of diffusion (mg/ gh).

Toxicity studies of CBB and post nano-remediated water on Vigna radiata
CBB solution (50 mg/L) and post nano-remediated water (2.6 mg/L) were used for phytotoxicity study. The phytotoxicity study was carried out (at room temperature) in relation to Vigna radiata (6 seeds each). The seeds were soaked overnight in respective solutions, next day, the seeds were covered with moist cloth and kept in warm place (> 30 °C and < 37 °C) for germination. After soaking the seeds in distilled water, the control set was also kept in the same manner. Each treatment's percent germination, plumule (shoot) length, and radicle (root) length were measured every 24 h for about 5 days. For each batch, six seedlings were chosen to record morphological data such as shoot length and root length in cm scale. The tolerance index, percent germination, and percent phytotoxicity were also calculated Chou et al. (1978) proposed a formula for calculating the dye's and its metabolite's percent phytotoxicity

UV-Visible spectrophotometry
The synthesis of bimetall OTAuAgNPs nanoparticles was evidenced by a variation in the color of the solution from light yellow to give deep purple which is due to the reduction of precursor salts to zero-valent nanoparticles (Khan et al. 2020) (Fig. 1B). A single surface plasmon peak at 560 nm was confirmed by UV-Visible spectrophotometric reading which corresponds to gold shell (Loiseau et al. 2019). According to the literature, Au-Ag bimetallic core shell nanoparticles have a sole surface plasmon resonance peak that corresponds to the shell material, whereas Au-Ag alloy nanoparticles have two peaks (Borah et al. 2020). In %germination was calculated = Number of seeds germinated total number of seeds × 100 (8) %phytotoxicity = radicle length of control − radicle length of text seed radicle length of control × 100 the current study, single SPR was observed around 560 nm, corresponding to bimetallic core shell nature of OTAuAg-NPs (Fig. 1A).

FTIR spectroscopy
The FTIR peak at 3350 cm −1 vanished in the bimetallic core shell nanoparticles made from O. tenuiflorum extract, despite the fact that the peak could be found in the extract, indicating that phenolic groups are involved in precursor salt reduction to nanoparticles (Kulkarni et al. 2013). Similarly, the intensity of peak at 1647 cm −1 which was seen in extract was extinguished in nanoparticles confirming the involvement of amine groups as reducing agents (Gudimalla et al. 2021). The presence of proteins in the extract implicated in the creation of nanoparticles is confirmed by FTIR data, which showed increased intensity of the peak at 2360 cm −1 confirms the capping of bimetallic core shell nanoparticles with proteins (Nayak et al. 2020). FTIR analysis showed 3736 cm −1 as the maximum spike at which it matches to the frequency of OH-bonds stretching coming from carbohydrates or proteins in the sample, confirming that proteins were present in both the extract (Fig. 2) and the nanoparticles (Fig. 3) (Vincent et al. 2020).

SEM-EDX for analysis of morphology and composition of OTAuAgNPs
The metal content of OTAuAgNPs was analyzed by EDAX (Rajput et al. 2016). The mass % of gold in the nanoparticle was 57.35% and silver was 24.42%, trace amount of C (9%) and N (8%) and O (1%) was also present, which might have been from the plant phytochemicals capped around the nanoparticles (Fig. 4a). SEM-EDX on the other hand is a powerful tool for depiction of spatial resolution and composition of nanoparticles. On visualization of synthesized bimetallic OTAuAgNPs under SEM under various resolutions, a clear image of uniformly shaped spheres of sizes 20-30 nm (Fig. 4b). EDX analysis discovered the existence of characteristic sharp gold and silver peaks at around 2 keV and 3 keV confirming the formation of bimetallic OTAuAg-NPs (Fig. 4c). Figure 5 shows the size distribution by intensity of bimetallic nanoparticles with size of 202 nm and intensity of 100% having Z-avg of 179.9 nm and low polydispersity index (PDI) of 0.129 and negative Zeta potential value of 24.8 mV of OTAuAgNPs. This suggests that the molecules coated over OTAuAgNPs are primarily made up of negative charge groups and are also accountable for the nanoparticles' modest stability. The negative zeta potential values of Au-Ag bimetallic nanoparticles formed using Pulicaria undulata extract correspond to negatively charged plant phytochemicals capping the nanoparticles (Khan et al. 2020). Zeta potential values in the range of −9 to −19 mV were obtained, which are quite similar to the results described by (Khan et al. 2020).

XRD studies
The OTAuAgNPs XRD patterns were recorded (Fig. 6).  nanoparticles' unique peaks, in addition to the normal peaks. These peaks could be attributable to the extract's bioorganic components or proteins (Nayak et al. 2019).

Antibacterial activity of OTAuAgNPs
As demonstrated in Fig. 7, biologically produced OTAuAg-NPs had antibacterial action opposite Gram −ve (Pseudomonas aeruginosa and E.coli) and Gram + ve (Bacillus subtilis) bacteria. The zone of inhibition was defined as the bright zones surrounding the wells. The antibacterial activity of OTAuAgNPs against Gram + ve (B. subtilis) bacteria and was much higher than that of Gram −ve (P. aeruginosa and E.coli) bacteria possibly due to the negatively charged nanoparticle surface as confirmed by zeta analysis. It is a known fact that nanoparticles surface charge has a significant impact on its bactericidal activity against various test organisms (Abbaszadegan et al. 2015). The presence of carbohydrates and other functional groups of O. tenuiflorum on OTAuAgNPs as confirmed by FTIR were responsible for antimicrobial action against the tested organisms ).

Adsorption of CBB by OTAuAgNPs
The adsorptive ability of synthesized OTAuAgNPs was studied by addition of UL-NPs (6.5 mg) to an aqueous solution of CBB (50 mg/L). The adsorption increased rapidly till 2 h after which the process slowed down (Fig. 8). Maximum adsorption % of 94.79 ± 0.12% was achieved in 2 h of incubation while residual concentration of CBB in the solution was 2.605 ± 0.11 mg/L. For CBB, the highest adsorption capacity was found as 183 mg/g. Microwave synthesized hydroxyapatite nanoparticles had a highest adsorption capacity of 14.51 mg/g (Wang et al. 2019). The results reveal that OTAuAgNPs can be effectively used for remediation of CBB by dye adsorption mechanism.

Adsorption kinetics
Adsorption kinetic studies are an important in the design of adsorption processes because it helps to understand the adsorption mechanism and estimate diffusion. PFO, Elovich, PSO, and intraparticular models of diffusion were used to investigate the adsorption kinetics of CBB by OTAuAg-NPs (Fig. 8). PSO model well describes the adsorption of CBB by OTAuAgNPs (R 2 = 0.9839) and also predicted the equilibrium adsorption capacity (181 mg/L) close to the experimental value (183.3 mg/L). This shows that degree of CBB adsorption by OTAuAgNPs is proportionate to the CBB concentration and follows the PSO and Elovich   nanoparticles also fitted PSO and Elovich model which confirmed the chemisorption interaction among the dye and the nanoparticle surface (Dinh et al. 2021;Rahman et al. 2022). Congo red adsorption by green ZnO-CdWO 4 nanoparticles followed PSO kinetics and intraparticulate diffusion at initial dye concentration of 10 mg/L (Fatima et al. 2021). The rate constant of the process was observed as 5.96E −04 g/mg min. The intraparticulate diffusion plot revealed that CBB adsorption rate by OTAuAgNPs was initially controlled by intraparticulate diffusion (up to 45 min), which was evident from linear nature of the graph (up to 6.7 min) which indicates higher initial transport of dye molecule to the boundary layer of adsorbent. The nature of the curve after 6.7 min was not linear, which indicates multiple diffusion steps and pore diffusion were involved at later stages (Mittal et al. 2020;Rahman et al. 2021). This was indicated by the multilinear nature of the plot (Fig. 9d). Further, the kinetic parameters based on the studies of adsorption of CBB on OTAuAgNPs are shown in (Table 1). Table 2 shows the effect of brilliant blue (BB) and its metabolite (MBB) on seed germination, radicle and plumule length, and phytotoxicity on Vigna radiata. Plants treated with CBB had an average length of 7.75 cm, while those treated with its metabolite had an average length of 10.27 cm, which was higher than the control samples (plant treated with water). Apparently, it is evident from the previous scientific studies that gold nanoparticles enhance the growth of seed yield in Brassica juncea (Arora et al. 2012). The effect of CBB, MCBB and control (water) on germination and seedling of V. radiata is shown in Figs. 10 and 11, respectively. It is also evident from literature that colloidal solutions of metal nanoparticles have been used as a micronutrient fertilizer for cereals (Batsmanova et al. 2013).This might be the reason for better growth of plants which were treated with metabolite of CBB which was treated with OTAuAgNPs. Hence from this study it is evident that metabolite of CBB

Conclusion
In the design of processes utilizing innovative nanoadsorbents, determining adsorption kinetics and safety is critical. The current study reports for the very first time, the adsorption of CBB by bimetallic Au-Ag nanoparticles biologically produced using O. tenuiflorum leaf extract which is also a potent bactericidal agent. The synthesized OTAuAgNPs had an SPR of 560 nm, were capped by water soluble phytochemicals of O. tenuiflorum leaf and were crystalline in nature. They could effectively adsorb residual concentration of CBB (94.79 ± 0.12%) owing to their large surface area. Toxicity studies on seed germination, radicle length, plumule length, phytotoxicity on V. radiata revealed that the metabolite of the dye produced by catalytic reduction using OTAuAgNPs is less toxic and improved the growth of plants due to micronutrient effect of metal nanoparticle. Thus, employing bimetallic Au-Ag nanoparticles, the work proposes a new, environmentally acceptable, and seemingly safe strategy for treating the dye-polluted wastewaters with multiple benefits like nanoadsorption, antimicrobial effect and micronutrient, making it suitable for practical applications in the field. However, further studies on understanding the toxicity of OTAuAg-NPs on aquatic life need to addressed before actual use.
Authors contributions All authors have read and approved the final manuscript. SN performed the research and wrote the paper. CVR and SM helped in analysis of data, and mentored during paper writing.
Funding No funding have been received for this research work.
Availability of data and materials Not applicable, Supplementary material will be available on request.