Sustainable approach to synthesis of carbon Dot/ silver nanoparticles for biological evaluation as antimicrobial agent

Green synthesis of metal nanoparticles is an attractive substitute for traditional methods using capping and reducing chemicals. In this study, silver nanoparticles (AgNPs) were synthesised using carbon dots (CDs) derived from bioresources as reducing, protecting, and stabilising agents in a single step using environmentally friendly and cost-effective synthetic methods. The optical and structural properties of prepared CD/AgNPs were explored using UV–vis (Ultraviolet-Visible Spectroscopy), Fluorescence spectroscopy, XRD (x-ray Diffraction), DLS (Dynamic Light Scattering), SEM-EDX (Scanning Electron Microscopy with Energy-Dispersive x-ray Spectroscopy) and TEM (Transmission Electron Microscopy). The synthesised CD/AgNPs are stable as zeta potential value is −14.7mV. From TEM the particle size exhibited as ∼12 nm. The prepared CD/AgNPs exhibited significant optical absorbance, good water dispersibility, stability and nano size. Also, CD/AgNPs revealed good biocidal effects against Gram-negative bacteria Escherichia coli (E. coli), Pseudomonas Aeruginosa (P. aeruginosa), Gram-positive Staphylococcus Aureus (S. aureus), Bacillus Cereus (B. cereus), and good anti-fungal activity against Aspergillus Niger (A. niger). The CD/AgNPs were further analyzed by live/dead assay. E. coli and A. niger with zone of inhibition around 3.1 and 40 mm, respectively when compared to ciprofloxacin (2.2 mm) and fluconazole (25 mm). The above investigation proved that the developed CD/AgNPs will be a new platform as an alternative to the traditional antibiotics for the generation of new kind of antibacterial materials and also provide the pathway for various metal/CD nanomaterials for diverse biomedical applications.


Introduction
Fluorescent carbon dots (CDs) have received a great interest from researchers all over the globe after the accidental discovery in 2004 while purifying single-walled carbon nanotubes [1].Mostly the fluorescent CDs are synthesized by various methods such as hydrothermal treatment, thermal decomposition, ultrasonic microwave irradiation, and chemical oxidation [2][3][4].The various methods for preparing CDs either require expensive equipment or the usage of substantial quantities of potentially hazardous chemicals.In this regard, it is crucial to establish easy and eco-friendly methods for making fluorescent CDs.Therefore, researchers have been working hard to develop new methodologies for synthesizing fluorescent CDs [5,6] using various natural resources including cranberry beans, crab shells, papaya leaves, coconut husk, green gram husks, pineapple peel, etc [7][8][9][10].Natural biomasses are inexpensive, abundant availability, easily accessible, nontoxic, eco-friendly, sustainable, and renewable, it has become an increasingly attractive energy source.Presently, natural waste resources are considered as noteworthy in the development of carbon nanomaterials and quantum dots due to their simple, economical, and eco-friendly synthetic strategy [11,12].But also, their water dispersibility, selfexisting surface functional groups, easy surface modification, excellent optical absorbance, fluorescent and electronic properties made them significant [7,13].These characteristics made them remarkable to develop fluorescent quantum, carbon dots, for various applications like bioimaging, drug delivery, catalysis, fluorescence detection [14][15][16].
In addition, new research shows that fluorescent CDs can also act as oxidizing and reducing agents due to their electron-donating/ accepting nature [17][18][19].This is because there are abundant oxygen rich functional groups (carbonyl, carboxyl, epoxy and hydroxyl,) on the CDs surface [20,21].In particular, fluorescent CDs were exploited as stabilizing, protecting, and reducing agent for the preparation of various metal nanoparticles including silver (Ag), gold (Au) nanoparticles (NPs) [18,22].In particular AgNPs have widely used in microbicidal applications against bacteria, fungus, and virus [23].AgNPs have better bactericidal, fungicidal ability than the other metal ions, antibiotics, quaternary ammonium salts, this may be due to the AgNPs can gently release Ag ions to inactivate bacteria, fungus [24,25].CDs play very important role in the reduction and stabilization of silver ions, in addition CDs can also influence in the increase of surface charge and hydrophilicity of AgNPs [26].Therefore, due to close proximity AgNPs could quench the fluorescent CDs which results in the enhancement of surface plasma energy transfer from CDs to silver nanoparticles [18].Though there are several reducing agents like poly (ethylene glycol), N, N-dimethylformamide (DMF), sodium borohydride (NaBH 4 ), ascorbate, sodium citrate, etc are used for the preparation of AgNPs.But this makes the process toxic and complex, So, to avoid this and it is also interesting to prepare CDs/AgNPs in economic and ecofriendly method.
In this study, fluorescent CDs was synthesized from natural waste resources such as citrus sinensis peel by sand-bath technique.The prepared CDs was explored as good stabilizing and reducing agent for synthesis of AgNPs by simple heating in the absence external reducing agents.The prepared, CDs/AgNPs was characterized by different analytical techniques to determine its physico-chemical properties.The prepared CD/AgNPs showed good water dispersibility, good optical absorbance, mono-dispersibility with nano-size.Further, we explored the CD/AgNPs to check the anti-bacterial activity against E. coli, S. aureus, P. aeruginosa, and B. cereus anti-fungal activity against A. niger which further supported by live/dead assay.

Experimental section 2.1. Materials
Silver nitrate (AgNO 3 ), ciprofloxacin, Mueller Hinton broth, potato dextrose agar, fluconazole was procured from by Sisco research laboratories, India.Live/dead bac light bacterial viability kit (L7012) purchased from Invitrogen, Korea.All the solvents and chemicals were utilized as received without further purification.Deionized water (DI) was used for the preparation of all aqueous solutions.

Sample pre-treatment
Citrus sinensis peels (from sweet oranges) were obtained from the local market and washed twice with tap water followed by DI water.Then the treated peels were dried in a hot air oven at 70 °C for two hours and grounded with mortar and pestle into a fine powder.

Synthesis of CDs
The CDs was synthesized following to previously reported sand bath method [27].Briefly, 2 g of citrus sinensis fruit peels fine powder was dispersed in 60 ml of DI water in a round-bottomed flask kept on a sand bath at 200 °C under a magnetic stirrer for 24 h.During the reaction the colour of solution slowly turns in to brown colour from orange colour.Then the reaction solution was undisturbed until it attains room temperature (RT).Later the reaction solution was subsequently centrifuged at 3000 RPM for 5 min.After that purified by syringe filtration (0.22 μM) to remove the larger particles.The CDs concentration is 50 μg/mL.The clear brown CDs solution obtained was refrigerated and used for further experiments.
2.4.Synthesis of CD/AgNPs 0.1mM AgNO 3 was prepared initially and added to CDs.For optimizing the suitable condition, several experiments were performed by varying the volume ratio of CDs and AgNPs (1:4, 4:1, 2:3 and 3:2 v/v).Each resultant solution was mounted on a magnetic stirrer for 90 min at 90 °C.Colour transformation from brown colour to black colour indicates the formation of CD/AgNPs due to reduction ( ) Ag Ag . + The synthesized CD/AgNPs were stored at 4 °C for further analysis.

Characterisation techniques
The Photoluminescence (PL) of CDs was observed using a handheld UV Lamp (Analytik Jena UVL-56, C.A, U.S. A. at 365nm).The UV-visible absorption spectra of CD and CD/AgNPs were recorded on UV-Visible Spectrophotometer (UV 1800, Shimadzu, Japan) using quartz cuvettes.The fluorescence spectra of CDs were obtained using quartz cuvettes on a Fluorescence Spectrophotometer (RF 6000, Shimadzu, Japan).Functional groups on the CD/AgNPs were analysed using a Fourier-transform infrared spectrometer (Bruker ATR-FTIR, U.S.A.).The morphology, size and shape of prepared CD/AgNPs were analysed with XRD (Model-D8 Advance Bruker AXS powder XRD) within of 10°-80°(2θ), SEM model of ZEIS Evo SEM, EDAX Oxford instruments, Transmission Electron Microscopy (JEOL 2100, 200 kV, TEM).A particle size analyzer, Nicomp 3000 (USA) was used to calculate the zeta potential.Image J software was used for analysing particle size of TEM image.Nikon Invitrogen fluorescence microscopy of Eclipse 80i, Japan to record fluorescent images.Triplicates of all the experiments were carried in the current study.

Antibacterial activity
Bacterial strains like E. coli and P. aeruginosa (Gram-negative) and S. aureus, and B. cereus (Gram-positive) was sub-cultured to calculate the antibacterial activity of CD/AgNPs by the agar-well diffusion method [28].The bacterial strains were cultured in sterilized Mueller Hinton broth and incubated for 16 h at 27 °C.The MHB medium containing bacterial inoculums were poured into sterile Petri dishes aseptically.Then wells (8 mm in diameter) were punched on Agar surface and CD/AgNPs at different concentrations of 25 μL, 50 μL, 75 μL, and 100 μL (1 mg ml −1 ) were added.Agar plates were incubated at 27 °C for 12 h (overnight) and the DIZ (diameter of the inhibition zone) surrounding each well was measured in mm to evaluate the antibacterial activity.Ciprofloxacin was used as a reference standard to compare the sensitivity of bacterial strains.Experiments were performed in triplicates.

Antifungal activity
Fungal strain A. niger was sub-cultured on a potato dextrose agar slant and incubated at 27 °C for 1-2 days to investigate the antifungal activity.At the same time, the agar-well diffusion method, was utilized to calculate the antifungal activity of CD/AgNPs.Potato dextrose agar plates were prepared by inoculating the inoculum by immersing a cotton swab which is sterile into the suspension and streaking the cotton swab overall on the surface of agar aseptically.Then wells (8 mm in diameter) were punched on agar surface and CD/AgNPs at different concentrations of 25 μL, 50 μL, 75 μL, and 100 μL (1 mg ml −1 ) were added.Agar plates were incubated at 27 °C for 4-7 days and the DIZ (diameter of the inhibition zone) surrounding each well was measured in mm to evaluate the antifungal activity.Fluconazole was used as a reference standard to compare the sensitivity of fungal strain.Experiments were performed in triplicates.
2.8.Live/dead assay CD/AgNPs were used to treat E. coli and B. cereus to determine cell viability by using live/dead assay.Bacterial cells were incubated for 60 min in the dark after treating live/dead cell staining solution (SYTO 9 for live cells and propidium iodide for dead cells).Both treated and untreated cells of E. coli and B. cereus were examined under fluorescence microscope.Nikon's live cell capture software was used to capture Images.

Statistical analysis
All the complete data shown in this research work is the mean ± SD of three independent experiments.Results taken from three-way analysis of variance (ANOVA) were considered statistically significant.The values were presented as the mean.

Synthesis and characterization of CD/AgNps
Fruit peels, such as those from citrus sinensis, represent a valuable renewable resource derived from natural waste.These peels contain polysaccharides, pectin, natural flavanone glycosides, and phenols, including phytochemicals like quercitrin, isoquercetin, methyl gallate, gallic acid, afzelin, and myricitrin [29].The presence of these phytochemicals suggests their potential role as capping and reducing agents in environmentally friendly synthesis approaches for AgNPs.In this study, CDs were prepared using a simple sand bath assisted method, utilizing the abundant bio-waste material of citrus sinensis fruit peels.Subsequently, the synthesized CDs were employed for the preparation of AgNPs (as depicted in figure 1).Notably, the CDs served as both reducing and stabilizing agents for AgNPs.The hydroxyl groups present on the CDs facilitated the interaction between silver ions in the silver nitrate solution and the oxygen atoms of the hydroxyl groups, contributing to the reduction process.Particularly at the 3:2 v/v (CDs: AgNO 3 ) UV-vis absorption spectra displayed a broad and intense absorption peak so the same ratio v/v (CD/AgNPs) is utilized for further characterizations and applications.
The optical properties of the CDs were analysed using UV-Visible and fluorescence spectroscopic techniques, along with a UV lamp.Under the UV lamp with a wavelength of 365 nm, the CDs exhibited blue fluorescence, whereas in visible light, they appeared as a reddish-brown colour.The UV-visible absorption spectrum of the CDs, depicted in (figure 2(a)), displayed an absorption peak at 276 nm, corresponding to the π-π * electronic transition of the C=C bonds present in the CDs.Fluorescence emission spectra was recorded at various excitation wavelengths ranging from 300 to 600 nm (figure 2(b)).As the excitation wavelength increased from 300 to 600 nm, the emission spectra exhibited a redshift, with the emission peak shifting from 500 to 680 nm.Notably, when excited at 450 nm, the CDs showed a maximum emission peak at 530 nm, indicating a dependence of fluorescence emission on the excitation wavelength.This characteristic emission spectra may suggest the presence of diverse emission sites on the surface of the CDs.Subsequently, the CDs were mixed with AgNO 3 at various volume ratios (1:4, 2:3, 3:2, and 4:1 v/v) to synthesized CD/AgNPs.The UV-vis absorption spectra (figure 2(c)) displayed a broad and intense absorption peak around 430-470 nm, indicative of the surface plasmon resonance (SPR) characteristic, confirming the successful formation of CD/AgNPs, particularly at the 3:2 v/v ratio.However, the fluorescence of the CD/AgNPs was observed to be quenched, as no fluorescence emission peak was detected under the excitation wavelength of 420 nm.This quenching behaviour is likely attributed to the interaction of silver ions and the functional groups present on the surface of CDs.
FT-IR analysis was employed to analyze the presence of CDs in the prepared CD/AgNPs.The FT-IR spectrum of the CD/AgNPs is depicted in figure 3(a).The spectrum exhibits a broad stretching vibrational peak at 3480 cm −1 , representing the presence of hydroxyl (-OH) functional groups on the surface of the CD/AgNPs.Additionally, a sharp and prominent peak at 1510 cm −1 corresponds to aromatic -C=C, another sharp peak at 1750 cm −1 corresponds to the vibration of the carbonyl (-C=O) functional group present in the amide bond of CDs of CD/AgNPs.Additionally, the crystalline nature of CD/AgNPs were characterized by XRD ranging from 10°to 80°.As depicted in figure 3(b), the formation of CDs was revealed by the XRD patterns of CDs shows two sharp intense peaks with the 2θ values of 28.6 and 40.7, which corresponds to (002) and (100) planes of graphene.Eventually for CD/AgNPs the slight shift of peak assigned to the plane (002) to 27.8, which may possibly be due to interaction between CD surface and silver nanoparticles.Additionally, significant sharp and broad peaks are evident at 38.2°, 44.4°, 64.5°, and 77.4°, corresponding to (111), ( 200), (220), and (311) reflections of silver nanoparticles (JCPDS No. 04-0783), respectively [30].Furthermore, the PXRD data reveals peaks at 27.8°, 32.3°, 54.9°, and 57.5°, which are characteristic of Ag2O nanoparticles (JCPDS No. 00-076-1393) [31].The lattice parameter was determined by measuring the highest intensity peak of the (111) reflection in the Ag nanoparticles using a formula .
The face centered cubic crystal structure of CD/AgNPs has unit cell edge value 'a' = 0.4078 nm correlating with the lattice parameter of silver nanoparticles, in reference with JCPDS file number 04-0783.The calculated crystalline size of the synthesised CD/AgNPs is found to be 10.6 nm which was estimated by using Scherrer's equation taking highest intense peak in to consideration [18].
Moreover, to measure the charge on the CD/AgNPs zeta potential was carried which in turns identifies the stability.The highest negative zeta potential value can create a repulsive force between CDs/AgNPs, which results in the stabilization of synthesised CDs/AgNPs.The zeta potential values of CDs and CDs/AgNPs

Antibacterial activity of CD/AgNPs
The antibacterial activity of the CD/AgNPs were evaluated against both Gram-negative (E.coli, P. aeruginosa) and Gram-positive (B.cereus, S. aureus) bacteria using the well diffusion method, as shown in figures 5(a)-(e).The anti-bacterial activity results are shown in the table 1.The anti-bacterial effectiveness of the CD/AgNPs increased linearly with incremental addition of the nanoparticles (25, 50, 75, and 100 μl), with the highest inhibition observed at 100 μl.Among all the bacterial strains tested, E. coli displayed the most extensive inhibition zone compared to B. cereus, S. aureus, and P. aeruginosa, even surpassing the reference antibiotic ciprofloxacin (1 mg ml −1 ), which also exhibited a clear inhibition zone.
The remarkable anti-bacterial properties of the green-synthesized CD/AgNPs can be attributed to their biocompatibility and morphological characteristics, allowing them to effectively combat a wide range of pathogenic bacteria.The antibacterial effect of the synthesized CD/AgNPs can be attributed to their smaller size and exceptionally high surface-to-volume ratio, which creates an ideal environment for interaction with bacterial cells.The mechanism behind the observed cell wall lysis in bacteria by the CD/AgNPs may be attributed to the loss of electrolytes, leading to bacterial cell death [30].Moreover, the release of silver ions from the CD/AgNPs enables interaction with intracellular components, such as proteins, which have penetrated the bacterial membrane effective [31,32].Gram-positive bacteria, with their firm plasma membrane, experience damage to the plasma membrane, while Gram-negative bacteria, with their flexible plasma membrane, are susceptible to the damage externally.However, the CD/AgNPs exhibited a comparable effect on both Grampositive and Gram-negative bacteria under similar conditions.

Antifungal activity of CD/AgNPs
The anti-fungal properties of the CD/AgNPs were examined against A. niger using the well diffusion method, as illustrated in figures 6(a)-(c).An increase in the inhibition zone was observed with incremental addition of CD/ AgNPs (25, 50, 75, and 100 μl), with the highest inhibition observed at 100 μl.The antifungal activity of CD/ AgNPs (100 μl) exhibited an inhibition region of approximately 40 mm, surpassing the performance of the reference antifungal agent fluconazole (1 mg ml −1 ), which showed an inhibition region of approximately 25 mm.The inhibition zones of the CD/AgNPs are presented in table 1.The observations indicates that the nanometer-sized CD/AgNPs are capable of easily penetrating the fungal cell membranes, leading to effective inhibition of fungal growth.Notably, smaller-sized CD/AgNPs particles demonstrated the highest growth inhibition activity.Therefore, the green synthesis of CD/AgNPs described in this research study provides an economical and environmental-friendly approach for various biomedical applications.

Live/dead assay
The effect of the CD/AgNPs on E. coli and B. cereus cells, specifically in terms of membrane damage, was examined using the live/dead assay.For differentiation purposes, the cells of B. cereus and E. coli were stained with SYTO 9 (yielding a green colour) and propidium iodide (yielding a red colour), respectively.The presence of green fluorescence in the cells indicates an intact membrane, indicating cell viability, while the presence of red fluorescence indicates a damaged cell membrane and cell death.As depicted in figure 7(a), the control E. coli cells displayed a distinctive intact morphology, accompanied by intense green fluorescence.However, upon treatment with CD/AgNPs at incremental additions (25, 50, 75, and 100 μl), there was a noticeable decrease in the number of cells exhibiting green fluorescence, concomitant with an increase in cells displaying red

Conclusion
In this work, CD/AgNPs was synthesized successfully by a simple and greener approach from CDs and AgNO 3 via simple reduction.The fluorescent CDs was synthesised from citrus sinensis fruits peels (sweet oranges) by the sand bath method.Later, the synthesised CDs, was utilised as reducing, protecting and stabilizing agents for the synthesis of CD/AgNPs.The AgNPs formation was confirmed by the appearance of strong absorption peak at 430 nm, which ascribed to SPR peak.The prepared CD/AgNPs was exhibited a particle size ∼12 nm (from TEM) with good water dispersibility and stability.Furthermore, the CD/AgNPs exhibited a good biocidal effect on microorganisms such as E. coli, B. cereus, P. aeruginosa, S. aureus, and A. niger.Therefore, the synthesized CD/ AgNPs will be considered as one of the potential materials antibacterial, antifungal, and other biomedical applications in future.

Figure 2 .
Figure 2. Spectral data of CDs & CD/AgNPs (a) UV Visible spectra of CDs.Insert: Optical image of CD under sunlight(left) and UV light (right) (b) Fluorescence emission spectra of CDs and CD/AgNPs.(c) UV-vis absorption spectra of CD with Varied Silver Nanoparticle Concentrations.

Figure 3 .
Figure 3. (a) (a) FT-IR spectra of CD/AgNPs (b) XRD patterns of the CDs and CD/AgNPs (c) Zeta potential of the CDs and CD/ AgNPs.

Figure 4 .
Figure 4. (a) SEM-EDX spectroscopy images of CD/AgNPs (b) TEM image of CD/AgNPs with a Insert of Size distribution of CD/ AgNPs.

Figure 5 .
Figure 5.The antibacterial & antifungal effect of CD/AgNPs (a) Optical image of E. coli (b) Optical image of S. aureus, (c) Optical image of P. aeruginosa, (d) Optical image of B. cereus and (e) Histogram of antibacterial activity.Standard deviation (SD) bars resulting from the mean of data from three individual experiments with * indicates P < 0.01 and ** indicates P < 0.001.

fluorescence (figures 7
(b)-(e)).Similarly, in figure 7(f), the control B. cereus cells exhibited an intact morphology, characterized by intense green fluorescence.However, upon treatment with CD/AgNPs at incremental additions (25, 50, 75, and 100 μl), a decrease in the number of green fluorescent cells was observed, while the number of cells displaying red fluorescence increased (figures 7(f)-(j)).These observations effectively confirm the significant bactericidal effect of the synthesized CD/AgNPs on both E. coli and B. cereus, indicating their excellent antimicrobial efficiency.

Table 1 .
Antibacterial and antifungal activity of CD/AgNPs.Ciprofloxacin for bacterial strains and fluconazole for fungal strain were used as controls.±SD values of this research work resulted from the mean of data from three individual experiments. a