INTRODUCTION Nanotechnology has immense potential to contribute for the development of various fields of sciences including medical, pharmaceutical and environmental clean-up (Kaur et al., 2021; Garg, 2021). Metal and metal oxide nanoparticles have been extensively used in all these scientific fields, but they have a number of disadvantages, including toxicity and non-biodegradability (Khayal et al., 2021). Carbon dots (CDs), the youngest member in carbon nanomaterial family were discovered by Xu et al. (2004) during the electrophoretic purification of single walled carbon nanotubes. These novel nanomaterials are considered as a viable substitute for metal-based nanoparticles due to their biocompatibility and feasibility (Wang et al., 2020). The synthesis methods of carbon dots can be divided into two main categories, top-down and bottom-up approaches (Wang and Hu 2014). Despite the fact that carbon dots can be synthesized from diverse biomasses, crop residues are considered as potential sources (Kang et al., 2020; Kurian and Paul 2021). Among the various crop residues, coconut wastes have been exploited for synthesis of carbon dots (Chunduri et al., 2016; Chauhan et al., 2020; Abinaya et al., 2021). Additionally, the use of coconut shell as a beginning precursor for the synthesis of CDs has been signified by the presence of cellulose, hemicelluloses, and lignin (Abinaya et al., 2021). The disposal issue of this coconut waste has been solved by converting bulk carbonaceous materials into effective CDs, which has antibacterial properties (Chauhan et al., 2020). These CDs are widely exploited for bioimaging and biosensing applications (Su et al., 2020) and research is being focussed to explore the possibilities of applying CDs as antibacterial agents (Yang et al., 2016; Lin et al., 2019). Therefore, the development of more potent antibacterial drugs for long-term usage is widely used to combat bacterial contamination. Earlier reports showed that the CDs has the ability to inhibit and suppress various microorganisms (Jijie et al., 2018). With this background, this study was designed to evolve a win-win strategy for effectively transforming coconut wastes into CDs and understand its potential as anti-bacterial agent. In this study, the antibacterial performance of CDs has been analysed using multi-assay approaches via Escherichia coli (E. coli), the most common indicator for faecal contamination in drinking water (Ishii and Sadowsky 2008). Hence, this current work has proposed a novel and green synthetic methodology for synthesising carbon dots using coconut waste. With more studies using in-vitro approaches for toxicological assessments, a clearer understanding of the toxicological behaviour of nanomaterials is essential. Among the different assays, the resazurin assay is simple, quick, versatile, economical (Pereira et al., 2020), and has a strong correlation with other methods used to measure cytotoxicity (Riss and Moravec 2004; Breznan et al., 2015). Other commonly used assays like agar dilution is a labor-intensive and time-consuming quantitative procedure that is frequently employed to determine MIC values (Elshikh et al., 2016). Additionally, other tests, such as disc diffusion method, which is a qualitative measure of antimicrobial activity for test materials, can only produce only zone of inhibition indicative results. Resazurin assay is therefore viewed as a direct indicator of bacterial metabolic activity that may be used to ascertain the minimum inhibitory concentration (MIC) of substances (Sarker et al., 2007). Secondly, in vitro cytotoxicity can be done by assessing the cell viability and among the several assays, the MTT assay is mostly recognised as a rapid, quantitative and colorimetric assay (Bahuguna et al., 2017). Accordingly, in this study, CDs were subjected to two assays namely, resazurin and MTT to observe its minimum inhibitory concentration and cytotoxicity on E. coli. MATERIALS AND METHODS Synthesis of carbon dots. The carbon dots were synthesized from coconut shell through muffle furnace mediated synthesis as outlined by Chauhan et al. 2020 and further subjected to sonication process. The obtained sediment after centrifugation was oven dried at 80C for 12 hours. The dried powder was ground using pestle and mortar and the resultant carbon dots were subjected for further characterization. The carbon dot suspension was homogenized by sonication prior to use in the following experiments. Characterization. High resolution transmission electron microscope (HR-TEM) is an imaging mode of the Transmission Electron Microscope used for higher magnification studies of nano-materials at the atomic scale. The size, morphology and uniformity of CDs were measured with the help of HR-TEM (JEOL, Japan) with 200 kV and the image was further developed by using “Image J” programme (Das et al., 2019) Microbial culture. Escherichia coli (E. coli) bacterial strain (MTCC 1652) was procured from Microbial Type Culture Collection, Chandigarh, India. Determination of Minimum Inhibitory Concentration (MIC) of CDs by Resazurin test Medium used for assay. Throughout the experiment, Muller Hinton medium (Himedia, India) was used as per the recommendation of NCCLS (National committee for clinical laboratory standard) for susceptibility testing. Preparation of resazurin solution. The resazurin solution was prepared by dissolving 0.4 % resazurin sodium (Himedia, India) in sterile distilled water. Further, the prepared dye solution was mixed well using a vortex mixer to ensure the dye was well-dissolved. MIC assessment. The minimum inhibitory concentration (MIC) assay was performed in a 96 well microplate using resazurin dye (Sarker et al., 2007). Initially, 100 μL of Mueller-Hinton broth was added to each well of a 96 well microplate. Subsequently, 100 μL of carbon dots was added by two-fold serial dilution 1000 ppm to 0.48 ppm concentration. Later, 10 μL of E. coli cell suspension was added to each well and then finally 10 μL of resazurin solution was added. The inoculated 96 well microplate was incubated at 37°C for 24h. Antibiotic ciproflaxin was used as positive control and was added to the Mueller-Hinton broth in serial dilution. Each test included a positive control (PC) antibiotic ciproflaxin and sterile control (BLK) (media alone). All tests were performed in triplicate and a negative control (NC) (without CDs) was added as an indicator to determine MIC (Chakansin et al., 2022). Assessing the cell viability through MTT assay. EZcountTM MTT cell assay kit was used to evaluate the cytotoxicity of CDs. The assay was carried out as per the manufacturer’s instructions with slight modifications. Briefly, E. coli cells at its early log phase (OD600 0.07) was seeded in 96-well plate, then exposed to different concentrations of CDs (1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.95, 0.97, 0.48 ppm) for 6 hours. The control group was kept as culture medium alone.10μl of MTT reagent (5 mg/ml concentration) was added and incubated for 3 hours and then 100μl of solubilizing agent was added. After 30 minutes, the optical density was measured at 570 nm by a spectrophotometer plate reader (BioTek, USA) RESULTS AND DISCUSSION HRTEM Characterization. In the present study, the coconut shell derived carbon dots were characterized using HR-TEM technique at 20 nm and 5nm scale magnification. At lower magnification scale (Fig. 1 a), HRTEM images of carbon dots derived from coconut shell were homogenous and spherical in shape, which is visible as dark spots. Surprisingly, the black dark spots were spotted as slanting lines at higher magnification scale, confirming the presence of carbon dots, and the average size of the carbon dots was observed as 7 nm (Fig. 1 b). The recorded size (less than 10 nm) of synthesized CDs in this study is similar to the reported size (3-5 nm) of carbon dots obtained from coconut shell (Chunduri et al., 2017). Determination of MIC of CDs by resazurin test. Carbon dots derived from coconut shell were tested for its cytotoxicity potential against E. coli. Although several cytotoxicity assays have been used to determine the cytotoxicity of nanoparticles, the resazurin dye-based method is one of the most effective and quick methods for visually determining the minimum inhibitory concentration of nanoparticles or any drugs of interest (Chakansin et al., 2022). Resazurin is a redox sensitive, non-fluorescent dye that is used to determine the cell viability. The non-fluorescent blue resazurin dye can be converted to the fluorescent pink resorufin by metabolically active cells. Non-living cells, on the other hand, do not reduce the resazurin, so the dye remains blue. As a result, the visible change in resazurin dye colour can indicate both viable and dead cells (Schmitt et al., 2013). In this work, the carbon dot at varying concentrations ranging from 1000 ppm to 0.48 ppm were tested against the E. coli (MTCC 1652) culture. The E. coli cell suspensions were seeded into the 96 well plate to carry out the MIC assay. After, 24 hours of the carbon dot and bacterial interaction, concentration dependent reduction in the dye intensity was observed. The carbon dots concentration, from 250 ppm to 0.48 ppm, did not significantly inhibit the E. coli cell growth and the resazurin blue dye was changed into pink color, indicating live cells (He et al., 2016), whereas at the higher concentrations of carbon dots at 1000 and 500 ppm, inhibition in the growth of E. coli cells was observed and that the dye retained its color. At end of 24 hours, the MIC of CDs against E. coli was observed as 500 ppm (Fig. 2). The positive control (Ciproflaxin) and negative control (E. coli culture + medium) were used as a reference point to measure the intensity of the dye's blue and pink colours. Our results corroborate with the previous studies which has reported that CDs are potent antibacterial agent. The MIC of CDs synthesized from glucose and polyethyleneimine (PEI) against E. coli was observed as 64 ppm (Dou et al., 2015). Recently, MIC of CDs synthesized from oyster mushroom through hydrothermal carbonization and tested with resazurin assay against pathogenic bacteria viz., S. aureus, K. pneumoniae, and P. aeruginosa was found to be 30 ppm for these three bacterial strains (Boobalanet al., 2020). In contradictory, Chauhan et al. (2020) reported that CDs derived from coconut waste (shell) through hydrothermal method and antibacterial test conducted through agar well diffusion method against E. coli showed no inhibition zone formation for any kind of bacterial species that were studied. Thus, this present study demonstrated that the coconut shell derived CDs had antibacterial property with MIC as 500 ppm against E. coli. The antibacterial effect of CDs might be due to the interaction of its surface functional groups with the cell membrane of the bacteria causing cell lysis. Dou et al. (2015) had reported that CDs were able to absorb onto the cytoplasmic membrane causing cell disruption of E. coli. Furthermore, Boobalan et al. (2021) had explained that antibacterial nature of CDs was due to its interaction with the cytoplasmic fluids inside the bacterial cell resulting in cell lysis including apoptosis. Assessing the cell viability through MTT assay. To evaluate the effect of synthesized CDs on the viability of E. coli, cell cultures were exposed to CDs at its exponential growth phase and the results of MTT assay showed a significant reduction in cell culture. Being one of the simplest cytotoxicity measurements, this assay utilizes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), a water-soluble yellow dye (Grela et al., 2018) that can be reduced to water-insoluble purple formazan crystals by the dehydrogenase enzyme of metabolicallyactive cells. The formazan crystals thus formed can be spectrophotometrically quantified by dissolution in a solvent, and the intensity is directly proportional to the number of metabolically active cells (Tunney et al., 2004). Cell cultures were exposed to CDs at its exponential growth phase and the results of MTT assay showed a significant reduction in cell viability. Looking closely at Fig. 3b, at the concentration of 1000 ppm, the cell viability was found to be 48.34 % and at 0.48 ppm it was 97.90 %. Our results demonstrated dose-dependent cytotoxicity of CDs with significant decrease in cell viability at higher concentration. These results are comparable with the findings of Alsadooni and Obada (2020) who performed MTT assay and found that graphene quantum dots derived from coconut husk had cytotoxicity effects against MCF 7 cell line recording lower cell viability (20 %) at 1000 ppm in comparison with higher cell viability (70 %) at 7.8 ppm. Recently, Chauhan et al. (2022) conducted MTT assay for coconut husk derived CDs against macrophage cell lines and established that 1 ppm of CDs was able to reduce the growth of tested cells by 32 %.