Laboratory analysis of Au–Pd bimetallic nanoparticles synthesized with Citrus limon leaf extract and its efficacy on mosquito larvae and non-target organisms

The current study provides novel results on the synthesis of bimetallic nanoparticles (BNPs) of gold and palladium (Au–Pd) with an eco-friendly and non-toxic aqueous leaf extract of plant Citrus limon. The BNPs were characterized and toxicity bioassay was examined on the larvae of the pathogen vectors such as Anopheles stephensi and Aedes aegypti mosquitoes. The predation efficiency test was evaluated on the invertebrate non-target organisms such as natural predatory nymphs of dragonfly and damselfly. The results of material characterization using UV VIS spectroscopy confirmed the synthesis of Au–Pd BNPs with the appearance of the SPR bands. FT-IR spectroscopy indicates the presence of functional groups containing high amounts of nitro compounds and amines on the surface of BNPs. TEM result shows the presence of spherical polydisperse Au–Pd BNPs in the sample. The XRD pattern displayed the semi-crystalline nature and the changes in the hydrodynamic size and surface potential was determined for the sample at 0 h, 24 h, 48 h, and 72 h of synthesis through DLS and ZP analysis. Au–Pd BNPs Bioassay provided the effective lethal concentrations (LC50) against the I–IV instar larvae of An. stephensi and Ae. aegypti after 24 h, 48 h, and 72 h of exposure. The LC50 obtained from the larvicidal bioassay was used to test its effect on the predation efficiency of the selected nymphs which showed increased predation from 40 to 48 h of exposure as compared to the negative control. Hereby, we conclude that Au–Pd BNPs bioassay shows toxic mosquito larvicidal activity at the selected concentration with no lethal effect on the predation efficiency of the selected stage of the predatory non-target aquatic invertebrate insects.

The current COVID-19 pandemic has spread awareness about health and hygiene but it still can't prevent the transmission of vector-borne diseases. Like in COVID-19, the risky group comprises immuno-compromised individuals including pregnant women. The unavailability of potential vaccines against malaria, dengue virus, zika virus, and chikungunya virus, points on the importance of the vector control measures to reduce the epidemic transmission of diseases by the mosquito vectors such as Anopheles stephensi and Aedes aegypti 1,2 . The long term use of synthetic chemical insecticides have led to the micro-evolution of insecticide-resistant mosquitoes resulting in the constant occurrence of vector-borne disease cases despite the Integrated Vector Management (IVM) mediated controls in the endemic areas. This has led to the demand for vector control research in many sectors and also to formulate and evaluate the potential larvicidal formulations 3 . The research on the evaluation of the toxicity of different metal nanoparticles (NPs) for their potential use as larvicidal, pupicidal, and adulticidal agents in mosquito control have been well documented on monometallic NPs 4 . The increased one-pot synthesis of metal NPs using extracts of different biological origin like plant tissues, microbes, and fungi have provided an eco-friendly and cost-effective approach for the NPs synthesis [5][6][7] . The phytochemical extract of leaves of the plant Citrus limon is known to possess aromatic and medicinal importance with insect repellent properties. For instance, the aqueous leaf extract of Citrus limon is considered non-toxic and is also used in the preparation of green tea beverages due to its therapeutic activities. Its metabolomic profile shows the presence of active molecules of 26 different organic acids and their derivatives, 21 amino acids, and 13 sugars and sugar alcohols. Among Scientific Reports | (2020) 10:21610 | https://doi.org/10.1038/s41598-020-78662-y www.nature.com/scientificreports/ cles in the size range from ~ 1.5 to ~ 18.5 d. nm and the distribution curve shows the mean particle size (± SD) of ~ 9.63 d. nm (± 3.95 d. nm) (Fig. 2B). This data suggests the synthesis of the polydisperse spherical nanoparticles.
Energy dispersive x-ray (EDX) spectroscopy of SEM view. The EDX spectrum for Au-Pd BNPs (Fig. 3) was generated for the entire SEM view (Fig. 3A). The elemental composition confirmed to be pure with an absence of            Fig. 11A, a, c we can see that the dragonfly nymphs ( Fig. 11A, a) are voracious feeders on the larvae of An. stephensi (An.) in comparison with damselfly nymphs (Fig. 11A, c). Owing to the surface feeding behavior of An. stephensi larvae, the predation activity of damselfly nymphs (Fig. 11A, c) appeared to be slow as compared to all the other groups  The predation efficiency test at the selected test concentration of Au-Pd BNPs (6.67 mL/L) was done to evaluate predatory behavior of nymphs in the laboratory set up. The test concentration was selected from the observations made in mosquito larvae bioassay. In Fig. 11B, 100% predation at 48 h in all groups was observed as compared to the incomplete predation (88%) in (damselfly and An. stephensi) negative control at 48 h (Fig. 11A, c). Comparatively reduced predation in Au-Pd BNPs test (Fig. 11B, a, b, d) is observed for the test groups containing, Dragonfly nymph and An. stephensi, Dragonfly nymph and Ae. aegypti, and Damselfly nymphs, and Ae. aegypti at 24 h as compared to 100% predation at 24 h in the respective negative control (Fig. 11A, a, b, c). On the contrary, an increased (91%) predation at 24 h in Au-Pd BNPs test group of Damselfly nymphs and An. stephensi (Fig. 11B, c) was observed as compared to 60% predation after 24 h in the respective negative control (Fig. 11A, c).

Discussion
The characterization results of the unprocessed original samples of the prepared 10% aqueous leaf extract of Citrus limon and synthesized Au-Pd BNPs (2 mM) provided insight into its unique properties which can be studied for further alteration to improvise its use in other applications. In the current study, the absorbance of leaf extract samples in the UV region (Fig. 1I, A, a) shows a characteristic photons absorption spectrum facilitated by the bonds and rings of the biological macromolecules present in the selected leaf extract sample 8 . The localized surface plasmon resonance (SPR) band observed in the BNPs sample (Fig. 1I, B, b) is a result of the collective oscillations of the free electron in the conduction band of the nanoparticles 30 . The SPR band for Au NPs is usually observed in the wavelength range from 490 to 600 nm, while no SPR band for Pd NPs is observed due to the absence of free electrons (surface plasmons) in its outer shell 23,31 . The FT-IR analysis results for the current study suggest the presence of new surface functional groups in the Au-Pd BNPs sample (Fig. 1II, B) as compared to the leaf extract sample (Fig. 1II, A). The N-O stretching of nitro compounds and C-N stretching of amines is clearly observed in the transmittance spectra for the Au-Pd BNPs samples (Fig. 1II, B), which were absent in the leaf extract sample (Fig. 1II, A). The considerable utilization of the O-H groups (3600-2800 cm −1 ) and aromatic compounds (981-599 cm −1 ) of the leaf extract in the synthesis of Au-Pd BNPs (Fig. 1II, B), suggests the formation of the Au-Pd oxide BNPs 31 . This information can be utilized for further stabilization and surface modification of the Au-Pd BNPs samples though the use of surface compatible biogenic ligand molecules, polymers, or surfactants, for its use in biomedical applications. The resulting shape and size can be a combinatorial effect of the two metals and the eco-friendly approach of 10% aqueous leaf extract (Citrus limon) mediated synthesis. Metal combinations of Au-Pd BNPs, can also provide variable results in size and shape for each type of extract and method of synthesis used. The native activity of the extract used for the reduction and capping of the metal ions is also responsible for the stability of synthesized nanoparticles. Agglomeration and nanocluster formation can be a by-product of the ingredients present in the type of extract used 32,33 . The NPs often settle down to the bottom owing to their nucleation and growth and due to the interaction of the surface molecules on the nanoparticles which can be overcome with the use of polymeric stabilizers and sonication. The EDX analysis results showed a high concentration of oxygen in the composition of Au-Pd BNPs (Fig. 3). www.nature.com/scientificreports/ This indicates the synthesis of gold-palladium oxide BNPs which supports the results of FT-IR analysis (Fig. 1II,  B). The XRD diffractogram (Fig. 4) shows the semi-crystalline nature of the samples and the presence of high noise confirms the leaf extract based synthesis of Au-Pd BNPs 22 . The ZP (mV) deals with the surface charge of the particles, particularly with the electrostatic repulsive forces. However, insight on the stability of any nanoformulation can be assessed with both electrostatic repulsive forces and van der Waals attractive forces. For a solution having particles with weak van der Waals attractive forces, the colloid stability can be assessed by the mild electrostatic repulsive forces as reflected by the low ZP (± 10 to ± 15 mV). Therefore, a stable nanoparticles sample may have a low ZP since it only deals to provide the indicative information of the nature of surface charge i.e. positive or negative. The samples prepared in DI water often show 2-10 nm larger zeta size than the actual size in DLS results. The colored samples containing metal nanoparticles often show absorbance at the wavelength of laser (633 nm) which can lead to the estimation of the incorrect size of the particles. The metal nanoparticles having shapes other than spherical cannot be estimated by DLS. Hence, the determination of physicochemical properties of the metal nanoparticles using DLS and ZP results are often compared with the FT-IR spectroscopy and TEM results to draw conclusions on the surface functional groups and, shape and size of the nanoparticles, respectively. However, the visual results of TEM and intensity-based results of DLS are fundamentally different but the former provides an actual size and shape of the electron-dense metal nanoparticles, unlike the latter one which shows a comparatively bigger size based on the hydrodynamics of the particles present in the sample 34 .
The scattering plots of DLS are often inclined towards detecting data with combined high-intensity scattering, provides biased results. In polydisperse samples, the occurrence of masking of low-intensity scattering of the small-sized particles by high-intensity scattering of large-sized particles is evident 35 . The current DLS analysis of the diluted samples (1:10) of 2 mM Au-Pd BNPs (Figs. 5A, 6, 7, 8A), shows the hydrodynamic size of the particles, which includes both Au-Pd BNPs and the surface capping agents of the leaf extract. These biogenic surface functional groups (Fig. 1II, B) can aid nanoparticles to agglomerate in the Au-Pd BNPs sample over time.
This data clearly suggests that the rate of agglomeration in the Au-Pd BNPs solution can be due to the interaction between the denatured macromolecules of the leaf extract and the surface functional groups of the Au-Pd BNPs. The actual hydrodynamic size of the unaltered samples of synthesized Au-Pd BNPs shows the greater particle size (Z-Average) in comparison to the actual size of the metal nanoparticles which is enclosed in the leaf extract capping agents. The actual size of the metal nanoparticles can be determined by the ImageJ analysis of the obtained TEM micrograph 33 . In the present bioassay results, the selected concentration range was effectively toxic against the larvae of the selected mosquito larvae species and the data can be used for the selection of the lower concentrations for time-based efficacy for safe environmental applications. The positive control shows high larvicidal efficacy at the selected concentration. The absence of mortality after 72 h of exposure in the leaf extract and distilled water negative control shows, the non-toxic nature of aqueous leaf extract and survivability in the absence of food, respectively, for the selected mosquito larvae bioassay. The concentration was mentioned in mL/L to provide an accurate measurement of the test. From the obtained efficacy results we can see that the value of LC 50 concentration decreases with time for each stage (Fig. 10A, B). Hence, the time-dependent efficacy evaluation can also be adopted for the minimal LC 50 selection. The 100% mortality in Au-Pd BNPs bioassay was observed against I instar of An. stephensi mosquito larvae but not for I instar larvae of Ae. aegypti mosquito after 24 h. From this finding, we can say that the mode of action of BNPs concentrations has different effects on both species owing to their physical and biological complexity. The current study of the predation efficiency test shows that the initial predation in Au-Pd BNPs test (Fig. 11B, a-d) was slow at 24 h but increased after 24 h of Au-Pd BNPs exposure as compared to the negative control (Fig. 11A, a-d). This suggests the increase in predation due to the availability of slow-moving, inactive or morbid larvae as compared to normal quick-moving larvae.
No mortality in the predator (nymphs) group at the selected test concentration till 72 h was observed. The test nymphs were kept in the laboratory for 72 h after the test to observe the post-exposure effects on the predator which showed normal behavior and moulting. The absence of literature for the efficacy of Au-Pd BNPs bioassay against mosquito larvae and its effect on the predatory behavior of non-target aquatic invertebrate insects is due to the novelty of the current study. However, sufficient studies are available on the synthesis of Au-Pd BNPs from different biological extracts such as ascorbic acid and Cacumen platycladi leaf extract mediated Au-Pd BNPs (~ 40 nm) showed an efficient catalytic ability for the oxidation of benzyl alcohol to benzaldehyde 32 . The mono and bi-metallic nanoparticles of Au and Pd synthesized using the flower extract of Lantana camara plant showed enhanced catalytic activity of Au-Pd BNPs as compare to the individual Au NPs and Pd NPs for the reduction of hazardous borohydride dye 22 . The fenugreek plant polysaccharide mediated Au-Pd BNPs also showed heterogeneous catalytic hydrogenation of toxic 4-nitrophenol to the environmental friendly 4-aminophenol compounds 36 . Euphorbia condylocarpa root extract mediated Au-Pd BNPs can catalyze the ligand-free Suzuki and Heck coupling reactions in water with the unaltered catalytic activity even after several repeated cycles 37 . The novel biosynthesis of metal nanoparticles can also, be studied further by utilizing the purified active phytochemical compounds instead of using the whole crude leaf extract. This type of study can provide information on the effect of different types of active phytochemical compounds in the eco-friendly and non-toxic formation and stabilization of metal nanoparticles. This approach can enhance the surface functional properties of synthesized nanoparticles as it prevents the accumulation of the diverse organic ingredients responsible for nanoparticles agglomeration in the original solution. Based on the findings of current results, further research in the use of Au-Pd BNPs can be evaluated for environmental or biomedical applications. The present study on the eco-friendly synthesis of Au-Pd BNPs based mosquito larvicidal formulation shows the eco-toxicological findings of the metals nanoparticles alone. During the synthesis of the current Au-Pd BNPs, the structures of the active ingredients of non-toxic leaf extract were modified in the formation of the nanoparticles in the original solution as seen in FT-IR results (Fig. 1II,  A,B). Therefore, we suggest that an effective mosquito larvicidal formulation can be prepared by combining the plant-based active larvicidal ingredients with the synthesized nanoparticles. Further, the bioassay of such Scientific Reports | (2020) 10:21610 | https://doi.org/10.1038/s41598-020-78662-y www.nature.com/scientificreports/ formulation can be evaluated for the selection of an effective LC 50 . Lastly, we suggest further research in the scientific community to test the antibacterial activity of Au-Pd BNPs in the bacterial biofilms along with the anti-viral bioassays for its use in disinfectants.

Collection of Leaves, Mosquito larvae and Nymphs of Non-Target organisms. The fresh leaves
of Citrus limon (Linnaeus) Burm. f. were collected from the Botanical Garden of Dayalbagh Educational Institute (DEI), Agra, India (27°, 10′ N, 78° 05′ E). The larvae of Anopheles stephensi (Liston) and Aedes aegypti (Linnaeus) mosquitoes, and the nymphs of Dragonfly and Damselfly were also collected from the surrounding water bodies in DEI campus. The online identification keys from Walter Reed Biosystematics Unit (WRBU) were followed for mosquito species identification 38 . Pictorial keys from the Indian Biodiversity Portal were used to identify the native adult Dragonfly (Bradinopyga geminata (Rambur, 1842)) and Damselfly (Ischnura senegalensis (Rambur, 1842)) in the surrounding environment 39,40 .
Preparation of aqueous leaf extract of plant Citrus limon. The preparation of aqueous leaf extract and nanoparticles synthesis was performed using the one-pot synthesis method. The 10% aqueous leaf extract of plant Citrus limon was prepared with 10 gm of clean green finely chopped leaves into 100 mL of deionized water. The leaf broth was heated at 65-70 °C for 1 h. The filtered broth (10%) was used on the same day and stored at 4 °C for further use. Characterization. The prepared aqueous leaf extract (10%) and Au-Pd BNPs (2 mM), were characterized after 48 h ± 2 h of synthesis. The samples of both leaf extract and Au-Pd BNPs were diluted in DI water in 1:9 and 1:10 ratio, respectively, to be analyzed by UV-Visible (UV VIS) spectroscopy (Hitachi U-3900 spectrophotometer) and Fourier transform infrared (FT-IR) spectroscopy (Bruker TENSOR 37 FTIR). A blank containing DI water was set for the baseline correction for both techniques. The UV VIS spectroscopy was performed to obtain absorbance spectra and surface plasmon resonance (SPR) bands in the wavelength ranging from 300 to 700 nm, with 600 nm/min scan speed, 2 nm slit width, and 5.0 mm path length. The FT-IR data was used to analyze the modified functional groups present in Au-Pd BNPs samples as compared to the original functional groups in the leaf extract sample. The transmittance peaks were recorded in the mid-infrared region ranging from 599 to 3996 cm −1 in wavenumber. The pH of the synthesized 2 mM Au-Pd BNPs sample and 10% Citrus limon leaf extract was also measured at 29 °C. The undiluted sample of synthesized 2 mM Au-Pd BNPs was used for Transmission Electron Microscopy (Technai G 20 (FEI) TEM), Scanning Electron Microscopy (SEM -Zeiss EV040) with Energy Dispersive X-ray spectroscopy (PANalytical X'pert PRO), and X-ray Diffractometer (D8 ADVANCE Bruker) analysis. The TEM visualization of the dried Au-Pd BNPs sample over a formvar coated copper grids was done to generate the micrographs for the visual analysis of synthesized nanoparticles. The different locations of the same grid were observed to generate the micrographs of the test BNPs sample. In addition to the visual interpretation of the shape and size of the nanoparticles relative to the nanoscale, the selected TEM micrograph was also analyzed for the particle size distribution in diameter nm (d. nm). The particle area of the nanoparticles visible in the selected TEM micrograph was obtained using the ImageJ software and the diameter was calculated to generate the histogram displaying the size distribution bins. The distribution curve was generated from the histogram plot to obtain the value of the mean particle size ± standard deviation. The SEM-EDX analysis was used to assess the elemental composition of the dried Au-Pd BNPs sample over a carbon tape on the SEM stub. The crystallinity of the dried Au-Pd BNPs sample over the 1 cm x 1 cm glass slide was achieved by XRD analysis. The dynamic light scattering (DLS) and zeta potential (ZP) (Malvern Zetasizer Nano ZS90) was observed for the diluted Au-Pd BNPs (2 mM) samples in 1:10 ratio in the DI water, at 25 °C. The hydrodynamic size was measured through DLS (zeta size) and its surface electrical charge was analyzed through ZP at 0 h, 24  . The Au-Pd BNPs bioassay was performed on I, II, III, and IV instar of the larvae of selected mosquito species. The collected larvae were washed twice with distilled water and segregated according to the larval stages. The source water was incubated in a bio-incubator at 28 °C for the extraction of the newly hatched I instar. The selection of the test concentrations was done after the general laboratory survey of a wide range of test concentrations against the IV instar. The specific range of test concentrations was selected on the basis of 50% of observed larval mortality after 24 h. The test concentrations of 0.25, 0.5, 0.75, 1, and 2 mL of Au-Pd BNPs in 150 mL of distilled water containing 25 larvae were set up in three replicates for I, II, III, and IV instar. The selected test concentrations were equivalent to the strength of 1.67, 3.34, 5, 6.67, and 13.34 in mL/L of Au-Pd BNPs as such used in the data analysis for the bioassay. A triplicate of each control containing 0.2% Temephos (EC) for positive control and two types of negative controls containing 13.34 mL/L of 10% leaf extract of Citrus limon and another with plain distilled water were observed for mortality. The mortality was observed at 24 h, 48 h, and 72 h for Au-Pd BNPs bioassay and negative controls, and at 4 h, 8 h, and 24 h for positive control.

Synthesis of Au-Pd
Data analysis for Au-Pd bioassay. The dose-response relationships in the Au-Pd BNPs test concentrations were analyzed for the calculation of LC 50 through Probit Analysis (Finney 1952) 42 . The obtained mortality (%) data were subjected to corrected mortality (%) if 5-20% mortality in the negative control is observed (Abbott, 1925) 43 . The corrected mortality (%) was then transformed into respective probit values using the probit table (Finney 1948) 44 . The comparative regression graphs were plotted to generate the probit equations (ŷ-value) for the calculation of LC 50 or LC 99 values. The LC 50 values were also represented graphically to draw conclusive remarks on time and concentration based comparative data obtained for the I-IV larval instars of both Anopheles stephensi and Aedes aegypti mosquito species.
Predation efficiency test. The Predation Efficiency (P.E.) test was evaluated on the selected invertebrate predatory nymphs of dragonfly and damselfly which are generally found in the local water bodies along with the mosquito larvae 45 . The nymphs measuring ~ 1 inch in length were selected for the predation efficiency test 46 . The selected nymphs were fed on the III instar larval stage of each Anopheles stephensi and Aedes aegypti mosquito species in both test and negative control. The negative control containing 25, III instar larvae, and 1 nymph in plain distilled water was installed in triplicates. The test was evaluated for an effective lethal concentration as selected from the results of III instar larval mortality of each mosquito species in Au-Pd BNPs bioassay.