Facile, One-pot Synthesis of S-doped Carbon Quantum Dots for Hydrogen Peroxide Detection

Facile, One-pot Synthesis of S-doped Carbon Quantum Dots for Hydrogen Peroxide Detection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Facile, One-pot Synthesis of S-doped Carbon Quantum Dots for Hydrogen Peroxide Detection Joyal Jain Palakulam, Jibin P O, Anupama Kuttappan, Tessy Paul, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2242696/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract ABSTRACT We have developed a green, facile and economical route to synthesise luminescent carbon quantum dots from pulp free juice of Citrus limon in ethylene glycol. The obtained graphitic carbon dots with an average size of 3 nm exhibits strong UV absorption. Dependence of excitation wavelength on blue emitting carbon dots is investigated. This spectral behaviour remains unchanged with sulphur doping in carbon dots. They show good biocompatibility while inhibiting the growth of DLA cancerous cells. Moreover optical properties of S-doped carbon quantum dots are utilised to detect hydrogen peroxide with a detection limit of 0.49 mM. GRAPHICAL ABSTRACT 1. INTRODUCTION Since their introduction in 2004, Carbon Quantum Dots (CDs) [1], tremendous scientific endeavors have been devoted to develop CDs based light emitting diodes [2], fluorescent sensors [3], drug delivery [4] and imaging [5]. Low cost, inexpensive precursors, eco-friendly nature, chemical inertness and low cytotoxicity makes these fluorescent carbonaceous materials an ideal alternative to many prominent semiconductor dots [6]. Many researchers achieved synthesis of fluorescent CDs from various chemicals like citric acid [7], urea [8] and phosphorus-rich phytic acid [9]. But the eco-friendly non-toxic starting materials was explored in a more meaningful manner on the synthesis of CDs from leaves of aloe [10], cardamom [11], P.amboinicus [12] etc fruits like papaya [13] lychee [14] etc, vegetables like potato [15], beetroot [16] cabbage [17]etc, and even from breads, biscuits jaggery etc. Easy availability and carbonization-friendly chemical composition of leaves, vegetables and fruits has made them the favourite source of carbon among the scientific community. Among natural sources, citrus fruits are reservoirs of carbon in the form of organic acids like citric acid and malic acid in their fleshy parts as well as oxalic acid and malonic acid in their peels [18]. CDs obtained by Federico Calì et al [19] from lemon waste were applied for a method of communication among biodevices. Lemon based CDs were prepared for Hg(II) [20], Fe 3+ ion [21] and riboflavin [22] detection and live cell imaging [23]applications. Hui Ding et. al. achieved synthesis of near-infrared emitting [24] and red emitting [25] CDs using lemon for bio-imaging applications. Moreover, CDs of much enhanced fluorescence properties, high quantum yield and tunable chemical properties can be obtained by doping heteroatoms, especially nitrogen (N) and sulphur (S) [26, 27, 28]. Even though these reports have sufficient data to establish the cyto-friendly nature of lemon prepared CDs, none of them proves to be therapeutic or shows anticancer activity. The significance of anti-cancer properties of CDs is widely discussed recently due to the influence of lifestyle, global warming and climatic conditions on human health. Tao Feng et. al [29] reacted with citric acid and diethylenetriamine in presence of nitrogen at 170°C to obtain carbon quantum dots that can act as pH responsive nano drug carriers with multicolour imaging abilities. Quinoline based carbon quantum dots synthesised by S. Karthik et. al [30] used a photo-response mechanism for designing an anticancer drug delivery system. The quenching of green luminescence in CDs prepared by Walaa E. Omer et. al [31] from ortho phenyl-enediamine(o-PD) was used to monitor changing concentration of cancer antigen-125 (CA-125) which provided an easy and cost-effective method for the early diagnosis of ovarian cancer. Toxic effects of mushroom based CDs on MDA-MB-231 breast cancer cells and HEK-293 cells from the kidney was explored by T. Boobalan et. al [32]. CDs prepared from four spices - black pepper, turmeric, red chilli and cinnamon by NagamalaiVasimalai et. al. showed an increase in death of cancerous human glioblastoma cells (LN-229) that affects the brain or spine [33]. Cancer cell line MCF-7, HepG-2, and A375 showed growth inhibition in presence of CDs obtained by Hua Yao et. al. from ginsenoside which is a natural glycoside extracted from plant genus Panax [34]. Moreover, considering the eco-friendly and biocompatibility, biomass derived CDs are to be explored more for their wide applicability. Among various biosensors, hydrogen peroxide sensors have always been utilised in chemical, pharmaceutical, biological, environmental and clinical processes. Zheng et al. has illustrated that peroxidase-like catalytic activity of CDs can be used for detection of hydrogen peroxide [35]. Fluorescence based H 2 O 2 detection method was proposed by J.Wei et al. which used gelatin-based CDs prepared by hydrothermal synthesis in presence of Fe 3+ ions [36]. Many carbon quantum dot based assays utilised MnO 2 nanosheets as a potential intermediate to accomplish sensing of various analytes. In vivo sensing of ascorbic acid was reached by WanyingZhai et. al. on suppressing fluorescence of 7-Hydroxycoumarin [37]. The same principle of fluorescence quenching by MnO 2 nanosheets was used by some researchers in H 2 O 2 sensing systems as well. A combination of first-order scattering, fluorescence, and second-order scattering was used by QianTang et. al. for dual-mode H 2 O 2 sensing systems in MnO 2 -blue fluorescent CD nanocomposite [38]. A sensing system for determining levels of H 2 O 2 in marine food samples was developed using CDs from citric acid and ethanediamine by Keke Ning et. al. [39]. Carbon quantum dots synthesised by Jiapei Gu et. al. from dried peony flowers also used fluorescence quenching mechanism by MnO 2 nanosheets for sensing of glucose in human serum samples by virtue of H 2 O 2 generation on enzymatic conversion of glucose by glucose oxidase [40]. Among them, most of them are based on aqueous or ethanolic solutions for their synthesis and easy solubility. Considering the long term stability of solutions free from aggregation induced fluorescence quenching, it is essential to explore H 2 O 2 sensors based on other solvents. Here, we report a simple route for synthesis of carbon quantum dots from lemon extract via heat treatment using ethylene glycol. Together with our effort for determining the cell viability and anticancer efficiency of CDs, we developed a turn-on fluorescence sensor for hydrogen peroxide detection. 2. EXPERIMENTAL SECTION 2.1. Materials Chemicals used including Ethylene Glycol (Merck, ≥ 99%), Potassium Permanganate (KMnO 4 ), MES buffer (2-( N -morpholino) ethanesulfonic acid, C 6 H 13 NO 4 S) and Thiourea (CH 4 N 2 S) were of analytical reagent grade. Ripe C. limon for carbon dot synthesis was purchased from the local market. All the solutions were prepared in ultra-pure water. Whatsman filter paper (~ 110 µm thickness) is used throughout for the filtering of solutions obtained. 2.2. Preparation of Carbon Quantum Dots The synthesis route of carbon dots is similar to the one reported in literature with slightly different experimental conditions [41]. CDs were formed by a simple one step heat treatment procedure of pulp free freshly squeezed lemon extract. 90 ml of filtered lemon juice was mixed with 60 ml of ethylene glycol and heated in a paraffin sealed beaker at 150°C for 24 hrs. Sulphur doped CDs (S-CDs) were synthesised by adding thiourea to the mixture before thermal treatment. Dark brown coloured carbon dot solutions obtained were filtered and used for further characterizations. 2.3. Preparation of MnO 2 nanosheets Manganese dioxide nanosheets were synthesised by reduction of potassium permanganate (KMnO 4 ) using 2-( N -morpholino) ethanesulfonic acid (MES) buffer [42]. 0.18 M KMnO 4 solution is added dropwise to equal volume of 0.11 M MES solution while stirring. After a few minutes, the solution starts to turn brown. When kept undisturbed after complete addition of KMnO 4 , a black-brown slurry is formed which is then filtered using filter paper. The filtrate is washed thoroughly with alcohol and ultra-pure water. The residue is kept at 80°C for about 16 hours to obtain MnO 2 nanosheets. 2.4. Detection of hydrogen peroxide In a typical process, MnO 2 nanosheet powder was weighed and sonicated with ethylene glycol for about 15 minutes to get 4, 8, 12, 16 and 20 millimolar concentrations. These were then added to the solution of S-CDs in ethylene glycol and stirred continuously for 30 minutes to obtain MnO 2 - modified CDs. The photoluminescence spectra on excitation at a wavelength of 370 nm was determined for each concentration. To the maximum concentration among them, various concentrations of freshly prepared hydrogen peroxide solution were added and thoroughly shaken. The spectra were recorded at the same excitation wavelength and slit width for emission measurements. 3. RESULTS AND DISCUSSIONS Transmission electron microscope (TEM) image (Fig. 1 .(a)) shows the crystalline nature and morphology of S-CDs. It can be seen that particles are uniformly formed and almost spherical in shape. The histogram plotted (inset of Fig. 1 .(a)) shows that particles have an average size of 3 nm. The existence of the crystalline graphite-like structure is confirmed in S-CDs from the HR-TEM image (Fig. 1 .(b)). The interplanar spacing of 0.253 nm in S-CDs is due to the (100) phase of hexagonal graphite. The Selected Area Electron Diffraction (SAED) pattern given in Fig. 2 indicates the poly-nanocrystalline nature of the S-CDs [43]. The interplanar distances calculated from the SAED pattern are found to be 0.20 nm and 0.24 nm which are associated with the (100) and (002) planes which illustrates effective formation of graphitic carbon structures. The XRD pattern of S-CDs shown in Fig. 3 . reveals that the sample has a broad diffraction peak around 2θ = 21 o which corresponds to the (002) plane of graphite ((JCPDS file number: 41-1487). This peak can be attributed to the turbostratic phase of carbon present in CDs [44]. The Energy-dispersive X-ray (EDX) spectroscopy images (Fig. 4 ) of CDs and S-CDs was analysed to determine elements present in carbon dots. Both CDs yielded a carbon-oxygen ratio of 3:2, indicating a fairly high formation of graphitic structures in them. The combined presence of 7 wt% nitrogen and sulphur in S-CDs confirms the S-doping in a larger ratio. From the Fourier transform infrared (FTIR) spectrum of the CDs (Fig. 5 ), the observed peak values are in good agreement with the reported data for carbon quantum dots. The characteristic bands observed at 3291 cm − 1 for both CDs and S-CDs can be attributed to stretching vibration of O-H groups during carbonization [45]. The dominant C = O stretching vibrations at 1737 cm − 1 is the evidence for the existence of more carboxylate groups on the surfaces of the CDs due to carbonization at high temperature [46]. A small band peaked at 1630 cm − 1 is due to C = C stretching mode [47]. A broad band peaked at 860 cm − 1 is due to C-H bending vibrations while bands in range 1000 cm − 1 to 1240 cm − 1 shows that the surface of the C-dots contains C-H and C-O-C bonds. The existence of the C-H bond [48] is indicated by the bands at 2940 cm − 1 and 2876 cm − 1 . The C-O-C stretching vibrations in CDs is due to contributions from compounds such as volatile acids, sugars and amino acids present in the lemon juice [45]. The absorption spectra (Fig. 6 ) of carbon quantum dots in ethylene glycol consists of a broad and strong absorption in the UV-Visible region of the spectrum. S-CDs shows strong absorption at 240 nm which is due to π – π* transition of sp 2 hybridised graphitic core. An absorption band between 250 nm and 300 nm wavelength present in both the CDs can be attributed to n – π* transition corresponding to edge states of the carbon dot core. These edge states are part of the interface between sp 2 hybridised and sp 3 hybridised carbon. This emissive centre is the reason for the bright blue luminescence of the carbon dots. The low energy absorption tail from 320–400 nm in CDs is due to the n – π* transition of the C = O bond. This is due to contributions from surface state transitions which are more prominent for CDs [49]. The emission spectra of both the quantum dots were recorded for varying excitation wavelengths. The CDs and S-CDs exhibit strong blue emission in the range of 440–470 nm upon UV excitations with maximum emission peaking at 448 nm wavelength. It can be observed that CDs (Fig. 7 ) and S-CDs (Fig. 8 ) showed a mixed nature of excitation dependence. From the normalised fluorescence spectra (inset of Fig. 7 and Fig. 8 ) the emission spectra of all excitations till 350 nm wavelength coincides for both QDs which suggests the excitation independent nature of fluorescence spectra of CDs. This behaviour changes to excitation dependent emission at higher excitation wavelengths with a red shift in emission wavelengths. Though the heterogeneous core-size distribution could be one of the factors responsible for red shift in PL as in many semiconductor nanodots, low synthesis temperature routes compared to other elemental quantum dots makes surface fluorescence emission a plausible explanation[50]. CDs and S-CDs were synthesised at 150 o C resulting in effective carbonization and pyrolysis which in turn lead to the presence of surface states along with molecular states in CDs. Surface of CDs is often composed of many small organic fluorescence moieties. They are attached with many functional groups like –OH,-COOH, -CO or even thiol groups. These groups are randomly linked to the dots with no well defined structural order and are often responsible for weak electronic transitions. The photoluminescence properties observed are attributed to the result of the synergistic contribution of these optically active centres in the core, molecular and surface states of the dots [51]. Thus CDs with excitation independent emission in aqueous medium changes into wavelength specific PL emitting CD in a less protic solvent like ethylene glycol. The mixed nature of wavelength dependence in our CDs can be intelligibly understood from the plot of emission peak wavelength for increasing excitation wavelength (Fig. 9 ). Both the CDs have almost constant PL peak wavelength till 350 nm excitations which are red shifted for higher excitations. The short term in vitro cytotoxicity studies were done on rat spleen cells (RSC) by comparing the cell toxicity with the addition of varying volumes of CDs using trypan dye. Dead cells take up the blue colour of the dye while live cells don’t acquire the colour. So, live cells are counted to evaluate % Cell viability. A similar trypan blue exclusion method was opted to find anticancer activity of dots towards Dalton’s lymphoma ascites(DLA) cells. In this case, dead cells are counted separately to evaluate the % Cell death. It can be observed that (Fig. 10 ) both CDs and S-CDs showed negligible cell killing effects on RSC and there is higher survival of these normal cells even at higher concentration. Though cell viability of normal cells remains almost constant even at all concentrations, it is evident that anticancer activity of both CDs increases with concentration. Compared to S-CDs, the undoped counterpart exhibits higher DLA tumour cell deaths at higher concentration. Absence of dopant chemicals and reduced surface functionalization in CDs also makes them more anti-cancer efficient compared to S-CDs. These dots are hence biocompatible with immense potential for application in the field of therapeutic treatment. To further investigate the H 2 O 2 sensing ability, emission spectra of S-CDs with various concentrations of MnO 2 are examined (Fig. 11 .a). It can be observed that emission intensity is quenched upon addition of MnO 2 . This can be well explained from the spectral overlap observed between UV-visible absorption spectrum of MnO 2 and emission spectrum of S-CDs (Fig. 11 .b). The broad absorption band ranging from 350 nm to 550 nm that overlaps with blue emissions of S-CDs makes MnO 2 nanosheets the perfect candidate for H 2 O 2 assay [52]. Ultrasound sonication of the MnO 2 nanosheet powder with S-CDs alters the surface features of dots. Nanosheets will mask the CDs and turn OFF its ability to emit radiation. These nanosheets act as an efficient quenching agent for luminescence by covering the surface of nanoparticles [42]. From Fig. 12 .a it can be inferred that fluorescence intensity of S-CDs are regained after addition of different concentrations of H 2 O 2 . The fluorescence regaining efficiency of MnO 2 modified S-CDs is linearly correlated with H 2 O 2 concentration from 0.06 mM to 1 mM (Fig. 12 .b). Thus the sensor exhibits a linear response to hydrogen peroxide concentration with a detection limit of 0.49 mM.On the addition of hydrogen peroxide solutions of various concentrations, the MnO 2 nanosheet modified dots will start deteriorating. Luminescence by CDs is gradually recovered on adding higher concentrations of H 2 O 2 solution. At a particularly higher concentration of H 2 O 2 the nanosheet will be completely decomposed and all CDs will show fluorescence. This recovery of luminescence can be attributed to reduction of MnO 2 to Mn 2+ mediated by H 2 O 2 . This reduction reaction can be represented as [52, 53, 54]. MnO 2 + H 2 O 2 + 2H + → Mn 2+ + 2H 2 O + O 2 Thus, by measuring the intensity of radiation emitted by MnO 2 modified carbon dots, we can even quantify the amount of H 2 O 2 added. The real images of fluorescence under 370 nm excitation wavelength, observed from MnO 2 modified carbon dots after addition of different concentrations of H 2 O 2 is shown in Fig. 13 . The biofriendly S-CDs can be effectively utilised for designing a fluorescent probe for sensitive determination of H 2 O 2 . 4. CONCLUSION In summary, carbon quantum dots were prepared via a facile heat treatment method using a natural green source in ethylene glycol. UV-visible absorption spectrum of CDs and sulphur doped CDs sheds light on core structure and surface states of CDs. Better understanding of photophysics and luminescence mechanisms of CDs are achieved from excitation dependent emission spectra of both CDs. Low cytotoxicity values and good optical properties made them a potential candidate for biosensing and bioimaging applications. A green eco-friendly method was proposed for detection of hydrogen peroxide using MnO 2 nanosheet - modified sulphur doped lemon CDs in ethylene glycol. The fluorescence regaining of S-CDs by H 2 O 2 opens up an economic and simple method to set up a biological assay. This methodology can be reciprocated in sensing biological enzymes which on catalysis produces hydrogen peroxide. Declarations Declarations Ethical Approval Not Applicable Competing interests All authors have no conflicts to disclose. Funding Not Applicable Authors' contributions All authors contributed to the study, conception and design. Material preparation, data collection and analysis were performed by Joyal Jain Palakulam and Jibin P O. Tessy Paul and Anupama K involved in interpretation and analysis of observations. The draft manuscript was prepared by Joyal Jain Palakulam. Experimental observations are analyzed in detail and final manuscript was prepared by Ann Mary K A. All authors read and approved the final manuscript. ACKNOWLEDGEMENT Authors are grateful to Centre for Nano and Soft Matter Sciences (CeNS), Bengaluru, Karnataka; Sophisticated Analytical Instrument Facilities (SAIF), Kottayam, Kerala and DST-FIST supported Central Instrumentation Facilities, St. Thomas College, Kerala for providing the characterization facilities. We also thank Amala Cancer Research Centre, Thrissur, Kerala for their assistance in cytotoxicity measurements. References 1. Xu, X.; Ray, R.; Gu, Y.; Ploehn, H. J.; Gearheart, L.; Raker, K.; Scrivens, W. A. Electrophoretic Analysis and Purification of Fluorescent Single-Walled Carbon Nanotube Fragments. J. Am. Chem. Soc. 2004, 126 (40), 12736–12737. https://doi.org/10.1021/ja040082h. 2. Zhang, X.; Zhang, Y.; Wang, Y.; Kalytchuk, S.; Kershaw, S. V.; Wang, Y.; Wang, P.; Zhang, T.; Zhao, Y.; Zhang, H.; Cui, T.; Wang, Y.; Zhao, J.; Yu, W. W.; Rogach, A. L. Color-Switchable Electroluminescence of Carbon Dot Light-Emitting Diodes. 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Jianfei Wei, Li Qiang, Jun Ren, Xiangling Ren, Fangqiong Tanga and Xianwei Meng; Fluorescence turn-off detection of hydrogen peroxide and glucose directly using carbon nanodots as probes; Anal. Methods, 2014,6, 1922–1927. https://doi.org/10.1039/C3AY41837E 37. WanyingZhai, Chunxia Wang, Ping Yu, Yuexiang Wang, Lanqun Mao; Single-Layer MnO2 Nanosheets Suppressed Fluorescence of 7-Hydroxycoumarin: Mechanistic Study and Application for Sensitive Sensing of Ascorbic Acid In Vivo; Anal. Chem. 2014, 86, 24, 12206–12213. https://doi.org/10.1021/ac503215z. 38. Qian Tang, Zhe Sun, Min Qing, Lei Wang, Yu Ling, Nian Bing Li, Hong Qun Luo; An optical sensing system with ratiometric and turn-off dual-mode of CDs@MnO2 nanosheets for the determination of H2O2 and glucose based on a combination of first-order scattering, fluorescence, and second-order scattering; Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 264, 2022, 120299. https://doi.org/10.1016/j.saa.2021.120299. 39. Keke Ning,GuoqiangXiang,CuicuiWang,FeihongHuang,JinzhiLiu,LanlanZhang,MinminYan,BingqianHu,Wen Lei; ‘Turn-on’ fluorescence sensing of hydrogen peroxide in marine food samples using a carbon dots–MnO2 probe; Luminescence. 2020;1–6. https://doi.org/10.1002/bio.3799 40. Jiapei Gu, Xiangqian Li, Zhan Zhou, Wanqiang Liu, Kai Li, Jinwei Gao, Ying Zhaob and Qianming Wang; 2D MnO2 nanosheets generated signal transduction with 0D carbon quantum dots: synthesis strategy, dual-mode behaviour and glucose detection; Nanoscale, 2019, 11, 13058. https://doi.org/10.1039/C9NR03583D 41. Dutta Choudhury, S.; Chethodil, J. M.; Gharat, P. M.; P. K., P.; Pal, H. PH-Elicited Luminescence Functionalities of Carbon Dots: Mechanistic Insights. J. Phys. Chem. Lett. 2017, 8 (7), 1389–1395. https://doi.org/10.1021/acs.jpclett.7b00153. 42. Renren Deng, XiaojiXie, Marc Vendrell, Young-Tae Chang and Xiaogang Liu; Intracellular Glutathione Detection Using MnO2-Nanosheet-Modified Upconversion Nanoparticles; J. Am. Chem.Soc. 2011, 133, 20168 − 20171. https://doi.org/10.1021/ja2100774. 43. Savita Chaudhary, Sandeep Kumar, Bhawandeep Kaur and S. K. Mehta; Potential prospects for carbon dots as a fluorescence sensing probe for metal ions; RSC Adv., 2016,6, 90526–90536. https://doi.org/10.1039/C6RA15691F 44. S. K. Bajpai, A. D’Souza, Basharat Suhail; Blue light-emitting carbon dots (CDs) from a milk protein and their interaction with Spinacia oleracea leaf cells; International Nano Letters (2019) 9:203–212 https://doi.org/10.1007/s40089-019-0271-9 45. Ajayeoba, Titilayo &Atanda, Olusegun & Adewale, Obadina& Bankole, Mobolaji &Brumbley, Steven; The potential of lemon juice-ogi steep liquor mixtures in the reduction of Listeria monocytogenes contamination of ready-to-eat vegetables; 2016, LWT - Food Science and Technology. http://dx.doi.org/10.1016/j.lwt.2016.08.022. 46. Pin-Che Hsu, Zih-Yu Shih, Chia-Hsin Lee and Huan-Tsung Chang; Synthesis and analytical applications of photoluminescent carbon nanodots; Green Chem., 2012,14, 917–920. https://doi.org/10.1039/C2GC16451E. 47. Poly(ethylene glycol)/carbon quantum dot composite solid films exhibiting intense and tunable blue–red emission; Y. Hao, et al.; Appl. Surf. Sci., 2014, http://dx.doi.org/10.1016/j.apsusc.2014.05.095. 48. Bui ThiHoan, Phuong Dinh Tam, Vuong-Hung Pham; Green Emission Carbon Quantum Dots from Lemon Juice for Selective Detection of Fe3 + Ions; VNU Journal of Science: Mathematics – Physics, Vol. 35, No. 1, 2019, 62–69. http://dx.doi.org/10.25073/2588-1124/vnumap.4317. 49. Reckmeier, C. J.; Wang, Y.; Zboril, R.; Rogach, A. L. Influence of Doping and Temperature on Solvatochromic Shifts in Optical Spectra of Carbon Dots. J. Phys. Chem. C 2016, 120 (19), 10591–10604. https://doi.org/10.1021/acs.jpcc.5b12294. 50. Sharma, A.; Gadly, T.; Gupta, A.; Ballal, A.; Ghosh, S. K.; Kumbhakar, M. Origin of Excitation Dependent Fluorescence in Carbon Nanodots. J. Phys. Chem. Lett. 2016, 7 (18), 3695–3702. https://doi.org/10.1021/acs.jpclett.6b01791. 51. Cao, L.; Meziani, M. J.; Sahu, S.; Sun, Y.-P. Photoluminescence Properties of Graphene versus Other Carbon Nanomaterials. Acc. Chem. Res. 2013, 46 (1), 171–180. https://doi.org/10.1021/ar300128j. 52. Hai-Bo Wang, Ying Chen, Na Li1 & Yan-Ming Liu; A fluorescent glucose bioassay based on the hydrogen peroxide-induced decomposition of a quencher system composed of MnO2 nanosheets and copper nanoclusters; Mikrochimica Acta; Heidelberg Vol. 184, Iss. 2, Feb 2017: 515–523. https://doi.org/10.1007/s00604-016-2045-7. 53. Juan Chen, Hongmin Meng, Yuan Tian, Ran Yang, Dan Du, Zhaohui Li, Lingbo Qu and Yuehe Lin; Recent advances in functionalized MnO2 nanosheets for biosensing and biomedicine applications; Nanoscale Horiz., 2019, 4, 321–338. https://doi.org/10.1039/C8NH00274F 54. Jing Yuan, Yao Cen, Xiang-Juan Kong, Shuang Wu, Chen-Liwei Liu, Ruqin Yu, and Xia Chu ; MnO2-Nanosheet-Modified UpconversionNanosystem for Sensitive Turn-On Fluorescence Detection of H2O2 and Glucose in Blood; ACS Appl. Mater. Interfaces 2015, 7, 19, 10548–10555. https://doi.org/10.1021/acsami.5b02188 Additional Declarations No competing interests reported. Supplementary Files SUPPLIMENTARYMATERIAL.docx SUPPLIMENTARYMATERIAL.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2242696","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":150920045,"identity":"f575c146-68a6-46dd-92d2-24c67629128b","order_by":0,"name":"Joyal Jain Palakulam","email":"","orcid":"","institution":"Department of Physics, St. Thomas College, Thrissur, Kerala, India-680001","correspondingAuthor":false,"prefix":"","firstName":"Joyal","middleName":"Jain","lastName":"Palakulam","suffix":""},{"id":150920046,"identity":"788007da-984a-40ef-87c2-16e05b7a8969","order_by":1,"name":"Jibin P O","email":"","orcid":"","institution":"Department of Physics, St. Thomas College, Thrissur, Kerala, India-680001","correspondingAuthor":false,"prefix":"","firstName":"Jibin","middleName":"P","lastName":"O","suffix":""},{"id":150920047,"identity":"3875928b-30b8-4cbd-a439-cfa4ed68fd7f","order_by":2,"name":"Anupama Kuttappan","email":"","orcid":"","institution":"Department of Physics, St. Thomas College, Thrissur, Kerala, India-680001","correspondingAuthor":false,"prefix":"","firstName":"Anupama","middleName":"","lastName":"Kuttappan","suffix":""},{"id":150920049,"identity":"daaad665-a471-4975-b334-00f46034ab3c","order_by":3,"name":"Tessy Paul","email":"","orcid":"","institution":"Department of Physics, St. Thomas College, Thrissur, Kerala, India-680001","correspondingAuthor":false,"prefix":"","firstName":"Tessy","middleName":"","lastName":"Paul","suffix":""},{"id":150920051,"identity":"32adad22-f903-401e-a608-3d65a5cfedd0","order_by":4,"name":"Ann Mary Kakkassery Aippunny","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBADAwb2BgYGxga4gAQRWngOMDYcJE2LRAKKFtyAf3bv0Q0/9xw25p/5/Pnjjztsovn7DzB++MFgkYdLi8Sdc2k3e54dNpO4nWPYcPBMWu6MGwnMkj0MEsU4rbmRY3aD58BhG4bbOUCHtR3ObbjBwCANNCsRlyPlgVpu/gFqkb95/CFYy/zzB5h/49NiANRyG2iLmcENBkOwlg0HEtjw2mII0iJzIN3Y8EyO4YyzbWm5G28ktln2GODWIgdy2JsD1obzjh9/8KGyzSZ33vnDh2/8qKjDqQUKmpE5oDRggF89ENQRVDEKRsEoGAUjGAAAWRNmjaPo5DkAAAAASUVORK5CYII=","orcid":"","institution":"Department of Physics, St. Thomas College, Thrissur, Kerala, India-680001","correspondingAuthor":true,"prefix":"","firstName":"Ann","middleName":"Mary Kakkassery","lastName":"Aippunny","suffix":""}],"badges":[],"createdAt":"2022-11-06 06:29:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2242696/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2242696/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30007468,"identity":"fc00ef3c-99c1-4bf0-a3ea-df452094c57c","added_by":"auto","created_at":"2022-12-07 12:14:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":259032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2242696/v1/5124fc10-b452-4ffd-9b3e-bdfd8898b70d.pdf"},{"id":28987174,"identity":"4be193ad-c261-492d-aa2d-e2057a1b6863","added_by":"auto","created_at":"2022-11-12 16:52:12","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":361349,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLIMENTARYMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-2242696/v1/106f5cb0da19558cca4b7e3f.docx"},{"id":28987838,"identity":"143fb03a-a77d-4e0d-a8f9-63956c11097a","added_by":"auto","created_at":"2022-11-12 17:00:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":361349,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLIMENTARYMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-2242696/v1/97ce288d9f939e8cda6cb6a3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Facile, One-pot Synthesis of S-doped Carbon Quantum Dots for Hydrogen Peroxide Detection","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eSince their introduction in 2004, Carbon Quantum Dots (CDs) [1], tremendous scientific endeavors have been devoted to develop CDs based light emitting diodes [2], fluorescent sensors [3], drug delivery [4] and imaging [5]. Low cost, inexpensive precursors, eco-friendly nature, chemical inertness and low cytotoxicity makes these fluorescent carbonaceous materials an ideal alternative to many prominent semiconductor dots [6]. Many researchers achieved synthesis of fluorescent CDs from various chemicals like citric acid [7], urea [8] and phosphorus-rich phytic acid [9]. But the eco-friendly non-toxic starting materials was explored in a more meaningful manner on the synthesis of CDs from leaves of aloe [10], cardamom [11],\u003cem\u003eP.amboinicus\u003c/em\u003e [12] etc fruits like papaya [13] lychee [14] etc, vegetables like potato [15], beetroot [16] cabbage [17]etc, and even from breads, biscuits jaggery etc. Easy availability and carbonization-friendly chemical composition of leaves, vegetables and fruits has made them the favourite source of carbon among the scientific community. Among natural sources, citrus fruits are reservoirs of carbon in the form of organic acids like citric acid and malic acid in their fleshy parts as well as oxalic acid and malonic acid in their peels [18]. CDs obtained by Federico Cal\u0026igrave; et al [19] from lemon waste were applied for a method of communication among biodevices. Lemon based CDs were prepared for Hg(II) [20], Fe\u003csup\u003e3+\u003c/sup\u003e ion [21] and riboflavin [22] detection and live cell imaging [23]applications. Hui Ding et. al. achieved synthesis of near-infrared emitting [24] and red emitting [25] CDs using lemon for bio-imaging applications. Moreover, CDs of much enhanced fluorescence properties, high quantum yield and tunable chemical properties can be obtained by doping heteroatoms, especially nitrogen (N) and sulphur (S) [26, 27, 28]. Even though these reports have sufficient data to establish the cyto-friendly nature of lemon prepared CDs, none of them proves to be therapeutic or shows anticancer activity.\u003c/p\u003e \u003cp\u003eThe significance of anti-cancer properties of CDs is widely discussed recently due to the influence of lifestyle, global warming and climatic conditions on human health. Tao Feng et. al [29] reacted with citric acid and diethylenetriamine in presence of nitrogen at 170\u0026deg;C to obtain carbon quantum dots that can act as pH responsive nano drug carriers with multicolour imaging abilities. Quinoline based carbon quantum dots synthesised by S. Karthik et. al [30] used a photo-response mechanism for designing an anticancer drug delivery system. The quenching of green luminescence in CDs prepared by Walaa E. Omer et. al [31] from ortho phenyl-enediamine(o-PD) was used to monitor changing concentration of cancer antigen-125 (CA-125) which provided an easy and cost-effective method for the early diagnosis of ovarian cancer. Toxic effects of mushroom based CDs on MDA-MB-231 breast cancer cells and HEK-293 cells from the kidney was explored by T. Boobalan et. al [32]. CDs prepared from four spices - black pepper, turmeric, red chilli and cinnamon by NagamalaiVasimalai et. al. showed an increase in death of cancerous human glioblastoma cells (LN-229) that affects the brain or spine [33]. Cancer cell line MCF-7, HepG-2, and A375 showed growth inhibition in presence of CDs obtained by Hua Yao et. al. from ginsenoside which is a natural glycoside extracted from plant genus \u003cem\u003ePanax\u003c/em\u003e [34]. Moreover, considering the eco-friendly and biocompatibility, biomass derived CDs are to be explored more for their wide applicability.\u003c/p\u003e \u003cp\u003eAmong various biosensors, hydrogen peroxide sensors have always been utilised in chemical, pharmaceutical, biological, environmental and clinical processes. Zheng et al. has illustrated that peroxidase-like catalytic activity of CDs can be used for detection of hydrogen peroxide [35]. Fluorescence based H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detection method was proposed by J.Wei et al. which used gelatin-based CDs prepared by hydrothermal synthesis in presence of Fe\u003csup\u003e3+\u003c/sup\u003e ions [36]. Many carbon quantum dot based assays utilised MnO\u003csub\u003e2\u003c/sub\u003e nanosheets as a potential intermediate to accomplish sensing of various analytes. In vivo sensing of ascorbic acid was reached by WanyingZhai et. al. on suppressing fluorescence of 7-Hydroxycoumarin [37]. The same principle of fluorescence quenching by MnO\u003csub\u003e2\u003c/sub\u003e nanosheets was used by some researchers in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensing systems as well. A combination of first-order scattering, fluorescence, and second-order scattering was used by QianTang et. al. for dual-mode H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensing systems in MnO\u003csub\u003e2\u003c/sub\u003e-blue fluorescent CD nanocomposite [38]. A sensing system for determining levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in marine food samples was developed using CDs from citric acid and ethanediamine by Keke Ning et. al. [39]. Carbon quantum dots synthesised by Jiapei Gu et. al. from dried peony flowers also used fluorescence quenching mechanism by MnO\u003csub\u003e2\u003c/sub\u003e nanosheets for sensing of glucose in human serum samples by virtue of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generation on enzymatic conversion of glucose by glucose oxidase [40]. Among them, most of them are based on aqueous or ethanolic solutions for their synthesis and easy solubility. Considering the long term stability of solutions free from aggregation induced fluorescence quenching, it is essential to explore H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensors based on other solvents. Here, we report a simple route for synthesis of carbon quantum dots from lemon extract via heat treatment using ethylene glycol. Together with our effort for determining the cell viability and anticancer efficiency of CDs, we developed a turn-on fluorescence sensor for hydrogen peroxide detection.\u003c/p\u003e"},{"header":"2. EXPERIMENTAL SECTION","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eChemicals used including Ethylene Glycol (Merck, \u0026ge; 99%), Potassium Permanganate (KMnO\u003csub\u003e4\u003c/sub\u003e), MES buffer (2-(\u003cem\u003eN\u003c/em\u003e-morpholino) ethanesulfonic acid, C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eNO\u003csub\u003e4\u003c/sub\u003eS) and Thiourea (CH\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eS) were of analytical reagent grade. Ripe \u003cem\u003eC. limon\u003c/em\u003e for carbon dot synthesis was purchased from the local market. All the solutions were prepared in ultra-pure water. Whatsman filter paper (~\u0026thinsp;110 \u0026micro;m thickness) is used throughout for the filtering of solutions obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of Carbon Quantum Dots\u003c/h2\u003e \u003cp\u003eThe synthesis route of carbon dots is similar to the one reported in literature with slightly different experimental conditions [41]. CDs were formed by a simple one step heat treatment procedure of pulp free freshly squeezed lemon extract. 90 ml of filtered lemon juice was mixed with 60 ml of ethylene glycol and heated in a paraffin sealed beaker at 150\u0026deg;C for 24 hrs. Sulphur doped CDs (S-CDs) were synthesised by adding thiourea to the mixture before thermal treatment. Dark brown coloured carbon dot solutions obtained were filtered and used for further characterizations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of MnO\u003csub\u003e2\u003c/sub\u003e nanosheets\u003c/h2\u003e \u003cp\u003eManganese dioxide nanosheets were synthesised by reduction of potassium permanganate (KMnO\u003csub\u003e4\u003c/sub\u003e) using 2-(\u003cem\u003eN\u003c/em\u003e-morpholino) ethanesulfonic acid (MES) buffer [42]. 0.18 M KMnO\u003csub\u003e4\u003c/sub\u003e solution is added dropwise to equal volume of 0.11 M MES solution while stirring. After a few minutes, the solution starts to turn brown. When kept undisturbed after complete addition of KMnO\u003csub\u003e4\u003c/sub\u003e, a black-brown slurry is formed which is then filtered using filter paper. The filtrate is washed thoroughly with alcohol and ultra-pure water. The residue is kept at 80\u0026deg;C for about 16 hours to obtain MnO\u003csub\u003e2\u003c/sub\u003e nanosheets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Detection of hydrogen peroxide\u003c/h2\u003e \u003cp\u003eIn a typical process, MnO\u003csub\u003e2\u003c/sub\u003e nanosheet powder was weighed and sonicated with ethylene glycol for about 15 minutes to get 4, 8, 12, 16 and 20 millimolar concentrations. These were then added to the solution of S-CDs in ethylene glycol and stirred continuously for 30 minutes to obtain MnO\u003csub\u003e2\u003c/sub\u003e - modified CDs. The photoluminescence spectra on excitation at a wavelength of 370 nm was determined for each concentration. To the maximum concentration among them, various concentrations of freshly prepared hydrogen peroxide solution were added and thoroughly shaken. The spectra were recorded at the same excitation wavelength and slit width for emission measurements.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSIONS","content":"\u003cp\u003e \u003c/p\u003e \u003cp\u003eTransmission electron microscope (TEM) image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.(a)) shows the crystalline nature and morphology of S-CDs. It can be seen that particles are uniformly formed and almost spherical in shape. The histogram plotted (inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.(a)) shows that particles have an average size of 3 nm. The existence of the crystalline graphite-like structure is confirmed in S-CDs from the HR-TEM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.(b)). The interplanar spacing of 0.253 nm in S-CDs is due to the (100) phase of hexagonal graphite.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Selected Area Electron Diffraction (SAED) pattern given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicates the poly-nanocrystalline nature of the S-CDs [43]. The interplanar distances calculated from the SAED pattern are found to be 0.20 nm and 0.24 nm which are associated with the (100) and (002) planes which illustrates effective formation of graphitic carbon structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe XRD pattern of S-CDs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. reveals that the sample has a broad diffraction peak around 2θ\u0026thinsp;=\u0026thinsp;21\u003csup\u003eo\u003c/sup\u003e which corresponds to the (002) plane of graphite ((JCPDS file number: 41-1487). This peak can be attributed to the turbostratic phase of carbon present in CDs [44].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Energy-dispersive X-ray (EDX) spectroscopy images (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) of CDs and S-CDs was analysed to determine elements present in carbon dots. Both CDs yielded a carbon-oxygen ratio of 3:2, indicating a fairly high formation of graphitic structures in them. The combined presence of 7 wt% nitrogen and sulphur in S-CDs confirms the S-doping in a larger ratio.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the Fourier transform infrared (FTIR) spectrum of the CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), the observed peak values are in good agreement with the reported data for carbon quantum dots. The characteristic bands observed at 3291 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for both CDs and S-CDs can be attributed to stretching vibration of O-H groups during carbonization [45]. The dominant C\u0026thinsp;=\u0026thinsp;O stretching vibrations at 1737 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the evidence for the existence of more carboxylate groups on the surfaces of the CDs due to carbonization at high temperature [46]. A small band peaked at 1630 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is due to C\u0026thinsp;=\u0026thinsp;C stretching mode [47]. A broad band peaked at 860 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is due to C-H bending vibrations while bands in range 1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1240 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shows that the surface of the C-dots contains C-H and C-O-C bonds. The existence of the C-H bond [48] is indicated by the bands at 2940 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2876 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The C-O-C stretching vibrations in CDs is due to contributions from compounds such as volatile acids, sugars and amino acids present in the lemon juice [45].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe absorption spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) of carbon quantum dots in ethylene glycol consists of a broad and strong absorption in the UV-Visible region of the spectrum. S-CDs shows strong absorption at 240 nm which is due to π \u0026ndash; π* transition of sp\u003csup\u003e2\u003c/sup\u003e hybridised graphitic core. An absorption band between 250 nm and 300 nm wavelength present in both the CDs can be attributed to n \u0026ndash; π* transition corresponding to edge states of the carbon dot core. These edge states are part of the interface between sp\u003csup\u003e2\u003c/sup\u003e hybridised and sp\u003csup\u003e3\u003c/sup\u003e hybridised carbon. This emissive centre is the reason for the bright blue luminescence of the carbon dots. The low energy absorption tail from 320\u0026ndash;400 nm in CDs is due to the n \u0026ndash; π* transition of the C\u0026thinsp;=\u0026thinsp;O bond. This is due to contributions from surface state transitions which are more prominent for CDs [49].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe emission spectra of both the quantum dots were recorded for varying excitation wavelengths. The CDs and S-CDs exhibit strong blue emission in the range of 440\u0026ndash;470 nm upon UV excitations with maximum emission peaking at 448 nm wavelength. It can be observed that CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and S-CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) showed a mixed nature of excitation dependence. From the normalised fluorescence spectra (inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) the emission spectra of all excitations till 350 nm wavelength coincides for both QDs which suggests the excitation independent nature of fluorescence spectra of CDs. This behaviour changes to excitation dependent emission at higher excitation wavelengths with a red shift in emission wavelengths. Though the heterogeneous core-size distribution could be one of the factors responsible for red shift in PL as in many semiconductor nanodots, low synthesis temperature routes compared to other elemental quantum dots makes surface fluorescence emission a plausible explanation[50].\u003c/p\u003e \u003cp\u003eCDs and S-CDs were synthesised at 150\u003csup\u003eo\u003c/sup\u003eC resulting in effective carbonization and pyrolysis which in turn lead to the presence of surface states along with molecular states in CDs. Surface of CDs is often composed of many small organic fluorescence moieties. They are attached with many functional groups like \u0026ndash;OH,-COOH, -CO or even thiol groups. These groups are randomly linked to the dots with no well defined structural order and are often responsible for weak electronic transitions. The photoluminescence properties observed are attributed to the result of the synergistic contribution of these optically active centres in the core, molecular and surface states of the dots [51]. Thus CDs with excitation independent emission in aqueous medium changes into wavelength specific PL emitting CD in a less protic solvent like ethylene glycol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mixed nature of wavelength dependence in our CDs can be intelligibly understood from the plot of emission peak wavelength for increasing excitation wavelength (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Both the CDs have almost constant PL peak wavelength till 350 nm excitations which are red shifted for higher excitations.\u003c/p\u003e \u003cp\u003eThe short term\u003cem\u003ein vitro\u003c/em\u003e cytotoxicity studies were done on rat spleen cells (RSC) by comparing the cell toxicity with the addition of varying volumes of CDs using trypan dye. Dead cells take up the blue colour of the dye while live cells don\u0026rsquo;t acquire the colour. So, live cells are counted to evaluate % Cell viability. A similar trypan blue exclusion method was opted to find anticancer activity of dots towards Dalton\u0026rsquo;s lymphoma ascites(DLA) cells. In this case, dead cells are counted separately to evaluate the % Cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt can be observed that (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) both CDs and S-CDs showed negligible cell killing effects on RSC and there is higher survival of these normal cells even at higher concentration. Though cell viability of normal cells remains almost constant even at all concentrations, it is evident that anticancer activity of both CDs increases with concentration. Compared to S-CDs, the undoped counterpart exhibits higher DLA tumour cell deaths at higher concentration. Absence of dopant chemicals and reduced surface functionalization in CDs also makes them more anti-cancer efficient compared to S-CDs. These dots are hence biocompatible with immense potential for application in the field of therapeutic treatment.\u003c/p\u003e \u003cp\u003eTo further investigate the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensing ability, emission spectra of S-CDs with various concentrations of MnO\u003csub\u003e2\u003c/sub\u003e are examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e.a). It can be observed that emission intensity is quenched upon addition of MnO\u003csub\u003e2\u003c/sub\u003e. This can be well explained from the spectral overlap observed between UV-visible absorption spectrum of MnO\u003csub\u003e2\u003c/sub\u003e and emission spectrum of S-CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e.b). The broad absorption band ranging from 350 nm to 550 nm that overlaps with blue emissions of S-CDs makes MnO\u003csub\u003e2\u003c/sub\u003e nanosheets the perfect candidate for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay [52]. Ultrasound sonication of the MnO\u003csub\u003e2\u003c/sub\u003e nanosheet powder with S-CDs alters the surface features of dots. Nanosheets will mask the CDs and turn OFF its ability to emit radiation. These nanosheets act as an efficient quenching agent for luminescence by covering the surface of nanoparticles [42].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e.a it can be inferred that fluorescence intensity of S-CDs are regained after addition of different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The fluorescence regaining efficiency of MnO\u003csub\u003e2\u003c/sub\u003e modified S-CDs is linearly correlated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration from 0.06 mM to 1 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e.b). Thus the sensor exhibits a linear response to hydrogen peroxide concentration with a detection limit of 0.49 mM.On the addition of hydrogen peroxide solutions of various concentrations, the MnO\u003csub\u003e2\u003c/sub\u003e nanosheet modified dots will start deteriorating. Luminescence by CDs is gradually recovered on adding higher concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution. At a particularly higher concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e the nanosheet will be completely decomposed and all CDs will show fluorescence. This recovery of luminescence can be attributed to reduction of MnO\u003csub\u003e2\u003c/sub\u003e to Mn\u003csup\u003e2+\u003c/sup\u003e mediated by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. This reduction reaction can be represented as [52, 53, 54].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMnO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e\u0026thinsp;\u003cb\u003e+\u0026thinsp;H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e+\u0026thinsp;2H\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e\u0026rarr; Mn\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e+ 2H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u0026thinsp;+\u0026thinsp;O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eThus, by measuring the intensity of radiation emitted by MnO\u003csub\u003e2\u003c/sub\u003e modified carbon dots, we can even quantify the amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e added. The real images of fluorescence under 370 nm excitation wavelength, observed from MnO\u003csub\u003e2\u003c/sub\u003e modified carbon dots after addition of different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e. The biofriendly S-CDs can be effectively utilised for designing a fluorescent probe for sensitive determination of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eIn summary, carbon quantum dots were prepared via a facile heat treatment method using a natural green source in ethylene glycol. UV-visible absorption spectrum of CDs and sulphur doped CDs sheds light on core structure and surface states of CDs. Better understanding of photophysics and luminescence mechanisms of CDs are achieved from excitation dependent emission spectra of both CDs. Low cytotoxicity values and good optical properties made them a potential candidate for biosensing and bioimaging applications. A green eco-friendly method was proposed for detection of hydrogen peroxide using MnO\u003csub\u003e2\u003c/sub\u003e nanosheet - modified sulphur doped lemon CDs in ethylene glycol. The fluorescence regaining of S-CDs by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eopens up an economic and simple method to set up a biological assay. This methodology can be reciprocated in sensing biological enzymes which on catalysis produces hydrogen peroxide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003eNot Applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eAll authors have no conflicts to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNot Applicable\u003c/p\u003e\u003ch2\u003eAuthors' contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study, conception and design. Material preparation, data collection and analysis were performed by Joyal Jain Palakulam and Jibin P O. Tessy Paul and Anupama K involved in interpretation and analysis of observations. The draft manuscript was prepared by Joyal Jain Palakulam. Experimental observations are analyzed in detail and final manuscript was prepared by Ann Mary K A. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eAuthors are grateful to Centre for Nano and Soft Matter Sciences (CeNS), Bengaluru, Karnataka; Sophisticated Analytical Instrument Facilities (SAIF), Kottayam, Kerala and DST-FIST supported Central Instrumentation Facilities, St. Thomas College, Kerala for providing the characterization facilities. We also thank Amala Cancer Research Centre, Thrissur, Kerala for their assistance in cytotoxicity measurements.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e1. Xu, X.; Ray, R.; Gu, Y.; Ploehn, H. J.; Gearheart, L.; Raker, K.; Scrivens, W. A. Electrophoretic Analysis and Purification of Fluorescent Single-Walled Carbon Nanotube Fragments. J. Am. Chem. 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Interfaces 2015, 7, 19, 10548\u0026ndash;10555. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1021/acsami.5b02188\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2242696/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2242696/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eABSTRACT\u003c/h2\u003e \u003cp\u003eWe have developed a green, facile and economical route to synthesise luminescent carbon quantum dots from pulp free juice of Citrus limon in ethylene glycol. The obtained graphitic carbon dots with an average size of 3 nm exhibits strong UV absorption. Dependence of excitation wavelength on blue emitting carbon dots is investigated. This spectral behaviour remains unchanged with sulphur doping in carbon dots. They show good biocompatibility while inhibiting the growth of DLA cancerous cells. Moreover optical properties of S-doped carbon quantum dots are utilised to detect hydrogen peroxide with a detection limit of 0.49 mM.\u003c/p\u003e\u003ch2\u003eGRAPHICAL ABSTRACT\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Facile, One-pot Synthesis of S-doped Carbon Quantum Dots for Hydrogen Peroxide Detection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-12 16:52:06","doi":"10.21203/rs.3.rs-2242696/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e6cf54d-4b0d-4322-86d9-8ebcca94d268","owner":[],"postedDate":"November 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-07T12:14:36+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-12 16:52:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2242696","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2242696","identity":"rs-2242696","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}