Fluorescent enhancement of CNDs from Cinnamon bark with L-Arginine doping for Yeast cell Imaging

Fluorescent enhancement of CNDs from Cinnamon bark with L-Arginine doping for Yeast cell Imaging | 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 Fluorescent enhancement of CNDs from Cinnamon bark with L-Arginine doping for Yeast cell Imaging Urvi M. Lad, Dhruti J. Dave, Bhumi N. Desai, Devesh H. Suthar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4142590/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract In this study, we present an economical and efficient synthesis method for carbon nanodots (CNDs) derived from cinnamon bark wood powder, supplemented with L-arginine doping at varying ratios. Extensive structural and optical characterization was conducted through techniques such as FTIR, XRD, HRTEM, DLS, UV-Vis, and PL spectra, providing a comprehensive understanding of their properties. Quantum yields (QY) were quantified for all three samples, contributing to the assessment of their fluorescence efficiency. The synthesized CNDs were successfully applied for bioimaging of yeast cells, employing fluorescence microscopy to visualize their interaction. Remarkably, L-arginine-doped CNDs exhibited enhanced fluorescence, particularly at a higher doping ratio (1:0.50), showcasing the influence of the dopant. The non-toxic nature of these CNDs was rigorously investigated, confirming their biocompatibility. This work not only contributes to the synthesis and characterization of CNDs but also highlights their potential for diverse applications, emphasizing their structural, optical, and biological attributes. The findings underscore the versatility of CNDs derived from cinnamon bark wood powder and their potential in advancing biotechnological and imaging applications. Carbon nanodots Hydrothermal L-Arginine Photoluminescence Bioimaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Nanomaterials are a family of materials with distinct qualities and promising properties, with applications like bioimaging to drug delivery in medical, biosensing in technology, and energy conversion and storage in environmental sciences[ 1 ]. Some of its key characteristics are its nanostructured scale (> 100 nm in size) and essential optical, electrical, and conductive capabilities such as sensing, photocatalysis, electrocatalysis, and so on. The merging of nanotechnology and several areas of research has increased scientists’ interest in developing superior nanoscale materials. The use of photoluminescent carbon-based nanoparticles has increased substantially in past 10 years[ 2 ]. These particles are mostly amorphous nanocarbon variants with a high heteroatom concentration[ 3 – 6 ]. Various carbon nanomaterials, including individual carbon nanotubes, graphene nanosheets, carbon nanodots (CNDs), nanodiamonds, carbon nanoribbons/nanorods, and fluorescent polymer dots, are garnering attention due to their wide-ranging applications, spanning from high-performance electrochemistry to biomaterials[ 7 , 8 ]. CNDs are nanoparticles characterized by their zero-dimensional nature, presenting as amorphous quasispherical particles with cores ranging from amorphous to nanocrystalline. These cores predominantly consist of graphitic or turbostratic carbon-based material and typically measure less than 10 nm. They exhibit a hybridized carbon structure comprising both sp 3 and sp 2 carbon bonding. CNDs derived from natural biomass hold greater appeal compared to chemically synthesized counterparts due to their customizable photoluminescence (PL) features, diverse emission colors, water solubility, outstanding long-term photostability (lack of photobleaching), cost-effectiveness, simplicity of preparation, and amenability to functionalization [ 9 – 12 ]. There are two types of manufacturing techniques used: TOP-DOWN and BOTTOM-UP, with different thermal and chemical strategies. Some of them are extremely cost-effective, environmentally friendly, as well as simple methods of synthesising CNDs from different sources of carbon. The hydrothermal method is a widely used thermal method with no loss of material and a closed, pressurised reaction performed at higher temperatures without exposing any environmental hazards. CNDs with properties like size and PL can be procured by employing various synthetic techniques [ 13 , 14 ]. The photoluminescence (PL) exhibited by CNDs possessing particle dimensions below 10 nm is attributed to quantum confinement effects and the composition of their surface functional groups. These factors contribute to alterations in energy band gaps, leading to modifiable emission characteristics. Catalysis, drug carrier, imaging, sensing, solar-energy conversion, and lightning, are just a few of the areas that show tremendous promise[ 15 ], and much more research is underway like optical sensors[ 16 ] and information encryption[ 17 ]. In biomedicine, CNDs have been used as antibacterial and anticancer agents, as well as for wound healing and bioimaging[ 4 , 14 , 18 , 19 ]. CNDs made of carbon materials have a lower toxicity toward the environment and living organisms than metal nanoparticles (NPs)[ 20 ]. Major role of water solubility performed by CNDs has been proven by Hydrophilic CNDs. For example, hydrophilic CNDs linked to drugs serve as effective carriers due to their excellent dispersion in water, facilitated by their ability to engage in multiple hydrogen-bonding interactions. As demonstrated by Wang et al., they synthesized CNDs using commercial beer and then attached them to doxorubicin hydrochloride, thereby creating a promising anticancer treatment approach[ 21 ]. Quang et al. synthesized carbon nanodots (CNDs) from waste wine cork via the hydrothermal method, yielding CDs with an average diameter of ~ 6.2 ± 2.7 nm. The characterized optical properties revealed excitation-dependent photoluminescence associated with surface functional groups, achieving a quantum yield of 1.54%. Successfully applied in bioimaging, these CDs showcase promise for fluorescence imaging applications [ 22 ]. Nevertheless, the quantum yield (QY) of the produced CNDs remains relatively low, limiting their potential applications. Therefore, it remains essential and imperative to synthesize CNDs with high fluorescence. Heteroatom doping represents an effective strategy for enhancing the surface activity and electrical properties of CNDs, thereby boosting their QY and fluorescence characteristics [ 23 – 25 ]. To facilitate doping, various heteroatom sources can be employed, such as urea, thiourea, and nitrogen-rich compounds like amino acids, including Alanie, Glutamine, Tyrosine, and Arginine[ 23 ]. In our study, we utilized L-Arginine as the nitrogen source, known for being non-toxic and abundant in nitrogen. This choice of nitrogen source not only enhanced the fluorescence but also had a significant impact on the quantum yield of the produced carbon nanodots (CNDs). Specifically, CNDs derived from cinnamon bark wood powder were doped with L-Arginine amino acid to augment their fluorescence. The surge in popularity of medicinal herbs aligns with the integration of innovative materials like cinnamon bark powder and carbon nanodots (CNDs). Historically, these herbs have served as effective home remedies for various ailments such as coughs, digestive issues, and skin allergies. Cinnamon, renowned for its high antioxidant content and more than 30 compounds combating oxidative stress, seamlessly fits into this traditional healing practice[ 26 ]. When considering the use of these materials together, both possessing medical applications, a notable potential outcome emerges. Utilizing a medically oriented carbon source for synthesizing CNDs, cinnamon bark powder proves particularly advantageous in this regard. In this study, we employed a green synthesis approach for the preparation of highly fluorescent nitrogen-doped carbon nanodots (CNDs). Cinnamon bark served as the carbon source in a hydrothermal reaction, and L-Arginine was used for doping, enhancing both fluorescence and quantum yield (QY). The structural and optical properties of the synthesized CNDs and nitrogen-doped CNDs were comprehensively analysed using various techniques. UV-Vis spectroscopy provided insights into the absorption characteristics, while fluorescence spectroscopy shed light on the emission properties. Transmission electron microscopy (TEM) allowed for a detailed examination of the particle morphology. Additional structural analysis was carried out using Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD). Furthermore, excitation and emission spectra were investigated to identify the optimal excitation wavelength for fluorescence imaging. The quantum yield was calculated to quantify the efficiency of fluorescence. To validate the practical application, we conducted fluorescence microscopy on yeast cells treated with both CNDs and nitrogen-doped CNDs. This analysis confirmed the successful synthesis and fluorescence enhancement of the carbon nanodots, providing visual evidence of their effectiveness in biological settings. 2. Materials and Method Cinnamon bark was procured from a local grocery store in Valsad, Gujarat. L-arginine (reagent grade) purchased from Sigma-aldrich lab and production materials. Solvents such as dimethyl sulphide (DMS), dimethyl sulfoxide (DMSO), and methanol were employed to assess the photoluminescence (PL) properties of CNDs. Solutions of 98% H 2 SO 4 and NaOH were prepared to achieve various pH levels. All the materials were used as purchased without further purification. The study involved employing Yeast Extract Peptone Dextrose (YEPD) petri plates for bioimaging analysis. 2.1. Preparation of Cinnamon bark wood powder derived CNDs In the pursuit of highly efficient carbon nanodots (CNDs), a hydrothermal synthesis method was applied to cinnamon bark wood powder (scheme S1). The process involved finely grinding dried cinnamon bark into a powder, followed by the ultrasonication of 5 g of this powder with 70 ml of distilled water. The homogenized mixture underwent a hydrothermal reaction at 180 ℃ for 5 hours in a Teflon-lined stainless-steel autoclave, with subsequent natural cooling. Filtration removed unreacted material, and centrifugation at 12000 rpm for 5–6 minutes separated a brown-colored CNDs solution. Dialysis against a 3.5 kDa cellulose membrane in water for 24 hours further purified the CNDs, resulting in nano-sized fluorescent particles. The yellow-colored CNDs solution obtained was stored at 4 ℃ for subsequent characterization and application, showcasing the efficacy of this hydrothermal synthesis approach using cinnamon bark wood powder. 2.2. Preparation of L-arginine doped CNDs For the preparation of L-arginine-doped carbon nanodots (CNDs) from cinnamon bark wood powder, the synthesis process remained consistent, introducing L-arginine during the ultrasonication step. Two different ratios, 1:0.25 and 1:0.5 (cinnamon bark powder to L-arginine), were employed, resulting in two distinct samples. The addition of the amino acid L-arginine during ultrasonication enhanced the properties of the CNDs derived from cinnamon bark wood powder, providing two variations of doped CNDs for subsequent analysis and application studies. 2.3. Labelling and imaging of Yeast cells with CNDs Yeast cells were cultured overnight in 100 ml Erlenmeyer flasks, each containing 20 ml of YEPD broth. The flasks were placed in a rotary shaker incubator at 28 ℃ and 200 rpm for continuous agitation. After the overnight growth period, the broth was centrifuged at 10,000 rpm for 5 min, and the resulting cell pellet was washed with PBS buffer and resuspended in 4 ml of PBS buffer. For each labeling assay, a 0.01 g/ml solution of CNDs and doped-CNDs in deionized water was mixed with 400 µl of yeast cells (at a concentration of 108 cfu/ml). This mixture was then incubated at 37 ℃ for 1 h with gentle shaking. After the incubation, non-internalized CNDs in the supernatant were removed by centrifugation at 10,000 rpm for 5 min. The yeast cells labeled with CNDs and doped-CNDs were washed twice and suspended again in PBS buffer. Subsequently, the labeled yeast cells were imaged using a fluorescence microscope, specifically the Nikon Eclipse Ti2E microscope, equipped with a 60x objective lens. 2.4. Characterization The optical spectrum of CNDs obtained from cinnamon bark wood powder and L-arginine-doped CNDs was analyzed using a UV-1800 Shimadzu Spectrophotometer. Fluorescence properties were investigated with an RF-6000 Spectro Fluorometer (Shimadzu Corp). Structural characterization involved powder X-ray diffraction analysis performed on a Rigaku Smart Lab SE, 3 kW XRD instrument. High-resolution transmission electron microscopy (HR-TEM) images were captured using a Jeol/JEM 2100/200 kV microscope at SAIF-COCHIN. FTIR spectra of the synthesized materials in the range of 4000 to 400 cm − 1 were recorded using a Shimadzu FTIR-8400 S model, employing KBr pellets for analysis. 2.5. QY measurement The quantum yield (QY) of the synthesized CNDs and doped-CNDs was assessed using a reference solution containing 0.1 molar H2SO4 and quinine sulfate solution, which has a known quantum yield efficiency of 0.54 [ 27 ]. QY determination was performed with excitation at a wavelength of 365 nm, utilizing the equation provided below: Q CNDs = Q R ∙ I CNDs /I R ∙ A R /A CNDs ∙ η 2 CNDs /η 2 R Here, the symbols represent the following parameters: Q signifies the fluorescence quantum yield, I denote the fluorescence intensity, η represents the refractive index of the solvent in the experiments, A stands for absorbance, R corresponds to the reference solution, and CNDs refers to the carbon nanodots. 3. Results and Discussion 3.1. HR-TEM analysis The morphological features of the synthesized CNDs are depicted in Fig. 1 , showcasing the presence of nanodots with varying sizes. The transmission electron microscopy (TEM) image provides valuable insights into the spherical structure of these particles (Fig. 1 (b) and (c)), offering a detailed visualization crucial for understanding their geometry. Using ImageJ software, the average particle size was quantitatively determined as 6.5 ± 0.5 nm (Fig. 1 (d)), providing a reliable assessment considering the observed size variations in the micrograph. The TEM image not only confirms the presence of spherical CNDs but also highlights their polydispersity, indicating a range of sizes. The quantitative measurement with ImageJ supports this observation, presenting a mean size with an associated standard deviation to account for the size distribution. In summary, the provided TEM image offers insights into the spherical morphology of the synthesized CNDs, while quantitative analysis with ImageJ establishes an average size of 6.5 ± 0.5 nm, emphasizing the diverse sizes within the nanodot population. In addition to HR-TEM analysis, Dynamic Light Scattering (DLS) measurements were conducted to estimate particle size. The DLS measurements on CNDs derived from Cinnamon bark wood powder at 180℃ by Hydrothermal reaction in green solvent reveal a predominant broad distribution peak within the range of 1–10 nm (Fig. S1 ), indicating that a significant portion of the freshly prepared CNDs possesses particle sizes within this specific range. 3.2. FT-IR spectroscopy The FT-IR spectra of Cinnamon bark wood powder and L-arginine (Fig. S2, S3) exhibit significant variations in peaks when compared to the synthesized carbon nanodots (CNDs), as depicted in Fig. 2 here. The O-H stretching band in CNDs is noted at 3397 cm − 1 , which broadens to 3426 cm − 1 with the addition of L-arginine due to combined O-H and N-H stretching. Following the doping of L-arginine, the peak at 1670 cm − 1 becomes sharper, corresponding to the amide I band. Additionally, new peaks emerge around 1078 cm − 1 and 1028 cm − 1 after the introduction of the doping agent. These changes in the spectral peaks signify the successful formation of CNDs and the incorporation of L-arginine in varying amounts. The Fig. 2 illustrates the distinctive infrared absorption patterns, highlighting the impact of L-arginine doping in varying ratios on the spectral characteristics of the CNDs. 3.3. XRD patterns The predominant amorphous nature of the CNDs is evident in the broad hump centered around 2θ = 20.7° in the XRD profile, as depicted in Fig. 3 . This characteristic hump is commonly observed in XRD patterns of amorphous carbon [ 28 ]. Additionally, the absence of lattice fringes in the HR-TEM image further approves the amorphous nature of the material. The broadness of the peak in the XRD pattern, specifically observed at 2θ = 20.7°, is attributed to the compact size of the CNDs, providing compelling evidence for their amorphous nature. This amorphous characteristic can be ascribed to the arrangement of C-C, C = O, and C = C bonds within the structural composition of the CNDs. 3.4. UV-Vis spectroscopy The UV–Vis absorption spectra of CNDs derived from cinnamon and L-arginine (LA) doping exhibit typical characteristics observed in CNDs (Fig. 4 ). These spectra display strong absorption in the UV region, with the absorbance gradually decreasing into the visible range.[ 29 ]. The π-π* transition manifests as a peak at 270 nm, and the electronic transition of the n-π* star transition is noticeable at 324 nm[ 30 ]. In the absence of doping, carbon nanodots (CNDs) show lower absorbance. However, upon introducing LA doping, the absorbance increases, and this enhancement varies with the doping ratio, ranging from 0.25 to 0.50. This modulation in absorbance highlights the impact of LA doping on the electronic transitions within the CNDs, with the degree of enhancement correlating with the varying doping ratios. 3.5. Fluorescence spectroscopy Fluorescence spectra were obtained using diluted samples of synthesized carbon nanodots (CNDs) in distilled water. Figure 5 illustrates the emission spectra of all three CND types when excited at 365 nm, demonstrating that an increase in the doping ratio enhances fluorescence. Consistent parameters, such as excitation wavelength, bandwidth, and room temperature, were maintained for all measurements. Systematically varying the excitation wavelength from 280 nm to 420 nm with 10 nm increments revealed excitation-dependent fluorescence (Fig. 6 )[ 31 ]. CNDs derived from cinnamon bark wood powder without doping exhibited weak fluorescence emission, with the maximum emission observed at 310 nm. After doping with two varying ratios, the emission spectra of CNDs with a 0.25 ratio (1:0.25) showed a 30 nm shift from undoped CNDs, and with an increased LA ratio (1:0.50), the fluorescence intensity became stronger (Fig. 6 (c)). The corresponding normalized fluorescence spectra (lower panel) illustrate the shift in emission with varying excitation wavelengths (Fig. 6 (e-g)). The QY value were obtained from the above-mentioned equation for Cinn CNDs, LA-doped CNDs (1: 0.25), and LA-doped CNDs (1: 0.50) are, 5.1, 10.5 and 14.2, respectively. 4. Fluorescence Microscopy Fluorescence microscopy was employed to visualize yeast cells labelled with CNDs. In Fig. 7 , the top panel displays bright-field images, while the bottom panel shows fluorescent images of CNDs-labelled yeast cells. Notably, untreated cells with CNDs showed no fluorescence, and cinnamon-derived CNDs-treated yeast cells exhibited weak or barely noticeable fluorescence. With increasing doping, the fluorescence became more intense. Remarkably, yeast cells treated with L-arginine-doped CNDs (1:0.50) exhibited robust fluorescence. Under illumination within the 320–365 nm wavelength range, CNDs-labelled yeast cells demonstrated uniform and intense fluorescence emission at 461 nm. Cells labelled with CNDs from L-arginine-doped (1:0.50) CNDs synthesis displayed significantly enhanced blue fluorescence compared to those from cinnamon-derived CNDs and L-arginine-doped (1:0.25) CNDs synthesis. This observation highlights the efficacy of a higher ratio of L-arginine doping into CNDs derived from cinnamon bark wood powder. The exceptional properties of CNDs, including their non-toxic nature, biocompatibility, strong photostability, and tunable photoluminescence, underscore their versatile utility in various biotechnological and imaging applications. 5. Conclusion This study has successfully synthesized and characterized carbon nanodots (CNDs) derived from cinnamon bark wood powder and L-arginine-doped CNDs at varying ratios. Comprehensive structural and optical analyses, including FTIR, XRD, HRTEM, DLS, UV-Vis, and PL spectra, provided a thorough understanding of their properties. The quantum yield (QY) values, essential for assessing fluorescence efficiency, were determined, revealing an increase with the L-arginine doping ratio. Specifically, QY values for Cinn CNDs, LA-doped CNDs (1:0.25), and LA-doped CNDs (1:0.50) were found to be 5.1, 10.5, and 14.2, respectively. The higher QY values at increased doping ratios underscore the effectiveness of L-arginine in enhancing the fluorescence of CNDs. The application of these synthesized CNDs in bioimaging of yeast cells demonstrated their efficacy, particularly the LA-doped CNDs (1:0.50), exhibiting robust fluorescence. The investigation into their non-toxic nature further enhances their potential for biological applications. In conclusion, this work establishes the utility of CNDs derived from cinnamon bark wood powder, emphasizing their versatility and potential advancements in biotechnological and imaging applications. Declarations Acknowledgement We express our gratitude to the Government of Gujarat for their financial support through the SHODH-ScHeme of Developing High-Quality Research program. Special thanks to the Sophisticated Test and Instrumentation Centre, Cochin University of Science and Technology, Cochin, India, for facilitating HRTEM analysis. We acknowledge the Department of Applied Chemistry, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Vadodara, India, for providing the fluorescence spectrophotometer facility under the DST PURSE program (SR/PURSE Phase 2/28 (C)) of the Government of India. Additionally, we thank the Department of Applied Physics, The Maharaja Sayajirao University of Baroda, Vadodara, India, for granting access to the XRD facility under the DST-FIST program (No. SR/FST/PS-II/ 2017/20). Author contributions All authors contributed to the study’s conception and design. The roles of the authors are as follows: Urvi M. Lad: Investigation (lead), Material preparation, Methodology, Writing the Original draft (equal), Visualization Dhruti J. Dave: Data collection (supporting), Analysis (supporting) Bhumi N. Desai: Data collection (supporting), Analysis (supporting) Devesh H. Suthar: Resources (supporting), Data collection (supporting), Analysis (supporting) Chetan K. Modi: Conceptualization (lead), Writing — Review & Editing (lead), Project Administration and Supervision (lead) Ethical Approval This declaration is “not applicable”. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. 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Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Apr, 2024 Reviews received at journal 24 Apr, 2024 Reviews received at journal 19 Apr, 2024 Reviews received at journal 18 Apr, 2024 Reviewers agreed at journal 17 Apr, 2024 Reviewers agreed at journal 17 Apr, 2024 Reviewers agreed at journal 12 Apr, 2024 Reviewers agreed at journal 12 Apr, 2024 Reviews received at journal 25 Mar, 2024 Reviewers agreed at journal 24 Mar, 2024 Reviewers agreed at journal 24 Mar, 2024 Reviewers invited by journal 24 Mar, 2024 Editor assigned by journal 22 Mar, 2024 Submission checks completed at journal 22 Mar, 2024 First submitted to journal 21 Mar, 2024 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. 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Lad","email":"","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":false,"prefix":"","firstName":"Urvi","middleName":"M.","lastName":"Lad","suffix":""},{"id":283692200,"identity":"b80447dd-46b3-4644-9323-569c2645ae90","order_by":1,"name":"Dhruti J. Dave","email":"","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":false,"prefix":"","firstName":"Dhruti","middleName":"J.","lastName":"Dave","suffix":""},{"id":283692201,"identity":"f47a6cc0-ea9a-4190-888a-74246be965f0","order_by":2,"name":"Bhumi N. Desai","email":"","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":false,"prefix":"","firstName":"Bhumi","middleName":"N.","lastName":"Desai","suffix":""},{"id":283692203,"identity":"ec57775a-9dde-4246-94a3-2b5656546be0","order_by":3,"name":"Devesh H. Suthar","email":"","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":false,"prefix":"","firstName":"Devesh","middleName":"H.","lastName":"Suthar","suffix":""},{"id":283692205,"identity":"dbf1b69e-1c14-453e-b695-16121c0c3380","order_by":4,"name":"Chetan K. Modi","email":"data:image/png;base64,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","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":true,"prefix":"","firstName":"Chetan","middleName":"K.","lastName":"Modi","suffix":""}],"badges":[],"createdAt":"2024-03-21 10:05:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4142590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4142590/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53662750,"identity":"00678456-73ad-486b-8a2a-00bfbed4908a","added_by":"auto","created_at":"2024-03-28 16:22:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":578668,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-resolution TEM images (a-c) depicting the synthesized CNDs and (d) histogram illustrating the particle size distribution.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/cf9b94c0e74e964cecb6b4b2.jpg"},{"id":53662758,"identity":"a31501e5-4b7d-4b5f-8157-4f2ff6e19e56","added_by":"auto","created_at":"2024-03-28 16:22:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":495090,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra of the as-synthesized carbon nanodots (CNDs) from cinnamon bark wood powder (black), L-arginine doped (1:0.25) CNDs (red), and L-arginine doped (1:0.50) CNDs (blue).\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/447256325effd73c05a72b42.jpg"},{"id":53662751,"identity":"3f8c3275-095f-41f0-8fa7-179f349233e7","added_by":"auto","created_at":"2024-03-28 16:22:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":488442,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of synthesized CNDs from cinnamon bark wood \u0026nbsp;powder.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/981bfae2afcbbc2cccd68461.jpg"},{"id":53662755,"identity":"790f3ac6-108b-4536-a62a-e8a34ad89a7c","added_by":"auto","created_at":"2024-03-28 16:22:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1595076,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis absorption spectra of as-synthesized CNDs and doped – CNDs.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/71e72a7f26b6a37770a9b039.jpg"},{"id":53662753,"identity":"3742d73f-f71c-4398-ad4e-9631c0b3ca17","added_by":"auto","created_at":"2024-03-28 16:22:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":507586,"visible":true,"origin":"","legend":"\u003cp\u003ePhotoluminescence emission spectra of CNDs and effect of doping on emission spectra.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/db80dca3ee0ee0ab3d7cddc5.jpg"},{"id":53662754,"identity":"1e8ff4cf-984e-4386-aed9-16cb2b72831d","added_by":"auto","created_at":"2024-03-28 16:22:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":272530,"visible":true,"origin":"","legend":"\u003cp\u003ePhotoluminescence spectra of (a) cinnamon bark wood powder derived CNDs, (b) Cinn + LA (0.25) CNDs, (c) Cinn + LA (0.50) CNDs , and (e-g) corresponding normalized fluorescence spectra (lower panel).\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/e9ec1e0a04e4faaf3ea9aefa.jpg"},{"id":53662760,"identity":"740ccbc8-3b84-494c-830d-60361c4c1ca8","added_by":"auto","created_at":"2024-03-28 16:22:46","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":172028,"visible":true,"origin":"","legend":"\u003cp\u003eThe images showcase yeast cells labelled with various carbon nanodots (CNDs), presenting both bright-field (upper panel) and fluorescence (lower panel) perspectives.\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/ed7d0415c94a376bc34bd937.jpg"},{"id":53662849,"identity":"159f6f07-44ae-4dae-a3bd-5e0a02a87fd9","added_by":"auto","created_at":"2024-03-28 16:22:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1142916,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/0c2759bc-14db-4bb5-9e71-7e7e7ab04121.pdf"},{"id":53662752,"identity":"4c82e47e-5cf0-48a1-b993-ccbfb27ebdda","added_by":"auto","created_at":"2024-03-28 16:22:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1749679,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4142590/v1/156a8ddf21410f2616ef2a51.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fluorescent enhancement of CNDs from Cinnamon bark with L-Arginine doping for Yeast cell Imaging","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanomaterials are a family of materials with distinct qualities and promising properties, with applications like bioimaging to drug delivery in medical, biosensing in technology, and energy conversion and storage in environmental sciences[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Some of its key characteristics are its nanostructured scale (\u0026gt;\u0026thinsp;100 nm in size) and essential optical, electrical, and conductive capabilities such as sensing, photocatalysis, electrocatalysis, and so on. The merging of nanotechnology and several areas of research has increased scientists\u0026rsquo; interest in developing superior nanoscale materials. The use of photoluminescent carbon-based nanoparticles has increased substantially in past 10 years[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These particles are mostly amorphous nanocarbon variants with a high heteroatom concentration[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Various carbon nanomaterials, including individual carbon nanotubes, graphene nanosheets, carbon nanodots (CNDs), nanodiamonds, carbon nanoribbons/nanorods, and fluorescent polymer dots, are garnering attention due to their wide-ranging applications, spanning from high-performance electrochemistry to biomaterials[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. CNDs are nanoparticles characterized by their zero-dimensional nature, presenting as amorphous quasispherical particles with cores ranging from amorphous to nanocrystalline. These cores predominantly consist of graphitic or turbostratic carbon-based material and typically measure less than 10 nm. They exhibit a hybridized carbon structure comprising both \u003cem\u003esp\u003c/em\u003e\u003csup\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003esp\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e carbon bonding. CNDs derived from natural biomass hold greater appeal compared to chemically synthesized counterparts due to their customizable photoluminescence (PL) features, diverse emission colors, water solubility, outstanding long-term photostability (lack of photobleaching), cost-effectiveness, simplicity of preparation, and amenability to functionalization [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. There are two types of manufacturing techniques used: TOP-DOWN and BOTTOM-UP, with different thermal and chemical strategies. Some of them are extremely cost-effective, environmentally friendly, as well as simple methods of synthesising CNDs from different sources of carbon. The hydrothermal method is a widely used thermal method with no loss of material and a closed, pressurised reaction performed at higher temperatures without exposing any environmental hazards. CNDs with properties like size and PL can be procured by employing various synthetic techniques [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The photoluminescence (PL) exhibited by CNDs possessing particle dimensions below 10 nm is attributed to quantum confinement effects and the composition of their surface functional groups. These factors contribute to alterations in energy band gaps, leading to modifiable emission characteristics. Catalysis, drug carrier, imaging, sensing, solar-energy conversion, and lightning, are just a few of the areas that show tremendous promise[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and much more research is underway like optical sensors[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and information encryption[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In biomedicine, CNDs have been used as antibacterial and anticancer agents, as well as for wound healing and bioimaging[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. CNDs made of carbon materials have a lower toxicity toward the environment and living organisms than metal nanoparticles (NPs)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Major role of water solubility performed by CNDs has been proven by Hydrophilic CNDs.\u003c/p\u003e \u003cp\u003eFor example, hydrophilic CNDs linked to drugs serve as effective carriers due to their excellent dispersion in water, facilitated by their ability to engage in multiple hydrogen-bonding interactions. As demonstrated by Wang et al., they synthesized CNDs using commercial beer and then attached them to doxorubicin hydrochloride, thereby creating a promising anticancer treatment approach[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Quang et al. synthesized carbon nanodots (CNDs) from waste wine cork via the hydrothermal method, yielding CDs with an average diameter of ~\u0026thinsp;6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 nm. The characterized optical properties revealed excitation-dependent photoluminescence associated with surface functional groups, achieving a quantum yield of 1.54%. Successfully applied in bioimaging, these CDs showcase promise for fluorescence imaging applications [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Nevertheless, the quantum yield (QY) of the produced CNDs remains relatively low, limiting their potential applications. Therefore, it remains essential and imperative to synthesize CNDs with high fluorescence. Heteroatom doping represents an effective strategy for enhancing the surface activity and electrical properties of CNDs, thereby boosting their QY and fluorescence characteristics\u003c/p\u003e \u003cp\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To facilitate doping, various heteroatom sources can be employed, such as urea, thiourea, and nitrogen-rich compounds like amino acids, including Alanie, Glutamine, Tyrosine, and Arginine[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In our study, we utilized L-Arginine as the nitrogen source, known for being non-toxic and abundant in nitrogen. This choice of nitrogen source not only enhanced the fluorescence but also had a significant impact on the quantum yield of the produced carbon nanodots (CNDs). Specifically, CNDs derived from cinnamon bark wood powder were doped with L-Arginine amino acid to augment their fluorescence.\u003c/p\u003e \u003cp\u003eThe surge in popularity of medicinal herbs aligns with the integration of innovative materials like cinnamon bark powder and carbon nanodots (CNDs). Historically, these herbs have served as effective home remedies for various ailments such as coughs, digestive issues, and skin allergies. Cinnamon, renowned for its high antioxidant content and more than 30 compounds combating oxidative stress, seamlessly fits into this traditional healing practice[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. When considering the use of these materials together, both possessing medical applications, a notable potential outcome emerges. Utilizing a medically oriented carbon source for synthesizing CNDs, cinnamon bark powder proves particularly advantageous in this regard.\u003c/p\u003e \u003cp\u003eIn this study, we employed a green synthesis approach for the preparation of highly fluorescent nitrogen-doped carbon nanodots (CNDs). Cinnamon bark served as the carbon source in a hydrothermal reaction, and L-Arginine was used for doping, enhancing both fluorescence and quantum yield (QY). The structural and optical properties of the synthesized CNDs and nitrogen-doped CNDs were comprehensively analysed using various techniques. UV-Vis spectroscopy provided insights into the absorption characteristics, while fluorescence spectroscopy shed light on the emission properties. Transmission electron microscopy (TEM) allowed for a detailed examination of the particle morphology. Additional structural analysis was carried out using Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD). Furthermore, excitation and emission spectra were investigated to identify the optimal excitation wavelength for fluorescence imaging. The quantum yield was calculated to quantify the efficiency of fluorescence. To validate the practical application, we conducted fluorescence microscopy on yeast cells treated with both CNDs and nitrogen-doped CNDs. This analysis confirmed the successful synthesis and fluorescence enhancement of the carbon nanodots, providing visual evidence of their effectiveness in biological settings.\u003c/p\u003e"},{"header":"2. Materials and Method","content":"\u003cp\u003eCinnamon bark was procured from a local grocery store in Valsad, Gujarat. L-arginine (reagent grade) purchased from Sigma-aldrich lab and production materials. Solvents such as dimethyl sulphide (DMS), dimethyl sulfoxide (DMSO), and methanol were employed to assess the photoluminescence (PL) properties of CNDs. Solutions of 98% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and NaOH were prepared to achieve various pH levels. All the materials were used as purchased without further purification. The study involved employing Yeast Extract Peptone Dextrose (YEPD) petri plates for bioimaging analysis.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of Cinnamon bark wood powder derived CNDs\u003c/h2\u003e \u003cp\u003eIn the pursuit of highly efficient carbon nanodots (CNDs), a hydrothermal synthesis method was applied to cinnamon bark wood powder (scheme S1). The process involved finely grinding dried cinnamon bark into a powder, followed by the ultrasonication of 5 g of this powder with 70 ml of distilled water. The homogenized mixture underwent a hydrothermal reaction at 180 ℃ for 5 hours in a Teflon-lined stainless-steel autoclave, with subsequent natural cooling. Filtration removed unreacted material, and centrifugation at 12000 rpm for 5\u0026ndash;6 minutes separated a brown-colored CNDs solution. Dialysis against a 3.5 kDa cellulose membrane in water for 24 hours further purified the CNDs, resulting in nano-sized fluorescent particles. The yellow-colored CNDs solution obtained was stored at 4 ℃ for subsequent characterization and application, showcasing the efficacy of this hydrothermal synthesis approach using cinnamon bark wood powder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of L-arginine doped CNDs\u003c/h2\u003e \u003cp\u003eFor the preparation of L-arginine-doped carbon nanodots (CNDs) from cinnamon bark wood powder, the synthesis process remained consistent, introducing L-arginine during the ultrasonication step. Two different ratios, 1:0.25 and 1:0.5 (cinnamon bark powder to L-arginine), were employed, resulting in two distinct samples. The addition of the amino acid L-arginine during ultrasonication enhanced the properties of the CNDs derived from cinnamon bark wood powder, providing two variations of doped CNDs for subsequent analysis and application studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Labelling and imaging of Yeast cells with CNDs\u003c/h2\u003e \u003cp\u003eYeast cells were cultured overnight in 100 ml Erlenmeyer flasks, each containing 20 ml of YEPD broth. The flasks were placed in a rotary shaker incubator at 28 ℃ and 200 rpm for continuous agitation. After the overnight growth period, the broth was centrifuged at 10,000 rpm for 5 min, and the resulting cell pellet was washed with PBS buffer and resuspended in 4 ml of PBS buffer. For each labeling assay, a 0.01 g/ml solution of CNDs and doped-CNDs in deionized water was mixed with 400 \u0026micro;l of yeast cells (at a concentration of 108 cfu/ml). This mixture was then incubated at 37 ℃ for 1 h with gentle shaking. After the incubation, non-internalized CNDs in the supernatant were removed by centrifugation at 10,000 rpm for 5 min. The yeast cells labeled with CNDs and doped-CNDs were washed twice and suspended again in PBS buffer. Subsequently, the labeled yeast cells were imaged using a fluorescence microscope, specifically the Nikon Eclipse Ti2E microscope, equipped with a 60x objective lens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Characterization\u003c/h2\u003e \u003cp\u003eThe optical spectrum of CNDs obtained from cinnamon bark wood powder and L-arginine-doped CNDs was analyzed using a UV-1800 Shimadzu Spectrophotometer. Fluorescence properties were investigated with an RF-6000 Spectro Fluorometer (Shimadzu Corp). Structural characterization involved powder X-ray diffraction analysis performed on a Rigaku Smart Lab SE, 3 kW XRD instrument. High-resolution transmission electron microscopy (HR-TEM) images were captured using a Jeol/JEM 2100/200 kV microscope at SAIF-COCHIN. FTIR spectra of the synthesized materials in the range of 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were recorded using a Shimadzu FTIR-8400 S model, employing KBr pellets for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. QY measurement\u003c/h2\u003e \u003cp\u003eThe quantum yield (QY) of the synthesized CNDs and doped-CNDs was assessed using a reference solution containing 0.1 molar H2SO4 and quinine sulfate solution, which has a known quantum yield efficiency of 0.54 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. QY determination was performed with excitation at a wavelength of 365 nm, utilizing the equation provided below:\u003c/p\u003e \u003cp\u003eQ\u003csub\u003eCNDs\u003c/sub\u003e = Q\u003csub\u003eR\u003c/sub\u003e ∙ I\u003csub\u003eCNDs\u003c/sub\u003e/I\u003csub\u003eR\u003c/sub\u003e ∙ A\u003csub\u003eR\u003c/sub\u003e/A\u003csub\u003eCNDs\u003c/sub\u003e ∙ η\u003csup\u003e2\u003c/sup\u003e\u003csub\u003eCNDs\u003c/sub\u003e/η\u003csup\u003e2\u003c/sup\u003e\u003csub\u003eR\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eHere, the symbols represent the following parameters: Q signifies the fluorescence quantum yield, I denote the fluorescence intensity, η represents the refractive index of the solvent in the experiments, A stands for absorbance, R corresponds to the reference solution, and CNDs refers to the carbon nanodots.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. HR-TEM analysis\u003c/h2\u003e \u003cp\u003eThe morphological features of the synthesized CNDs are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, showcasing the presence of nanodots with varying sizes. The transmission electron microscopy (TEM) image provides valuable insights into the spherical structure of these particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b) and (c)), offering a detailed visualization crucial for understanding their geometry. Using ImageJ software, the average particle size was quantitatively determined as 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d)), providing a reliable assessment considering the observed size variations in the micrograph. The TEM image not only confirms the presence of spherical CNDs but also highlights their polydispersity, indicating a range of sizes. The quantitative measurement with ImageJ supports this observation, presenting a mean size with an associated standard deviation to account for the size distribution.\u003c/p\u003e \u003cp\u003eIn summary, the provided TEM image offers insights into the spherical morphology of the synthesized CNDs, while quantitative analysis with ImageJ establishes an average size of 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nm, emphasizing the diverse sizes within the nanodot population. In addition to HR-TEM analysis, Dynamic Light Scattering (DLS) measurements were conducted to estimate particle size. The DLS measurements on CNDs derived from Cinnamon bark wood powder at 180℃ by Hydrothermal reaction in green solvent reveal a predominant broad distribution peak within the range of 1\u0026ndash;10 nm (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating that a significant portion of the freshly prepared CNDs possesses particle sizes within this specific range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. FT-IR spectroscopy\u003c/h2\u003e \u003cp\u003eThe FT-IR spectra of Cinnamon bark wood powder and L-arginine (Fig. S2, S3) exhibit significant variations in peaks when compared to the synthesized carbon nanodots (CNDs), as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e here. The O-H stretching band in CNDs is noted at 3397 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which broadens to 3426 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with the addition of L-arginine due to combined O-H and N-H stretching. Following the doping of L-arginine, the peak at 1670 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e becomes sharper, corresponding to the amide I band. Additionally, new peaks emerge around 1078 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1028 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after the introduction of the doping agent. These changes in the spectral peaks signify the successful formation of CNDs and the incorporation of L-arginine in varying amounts. The Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the distinctive infrared absorption patterns, highlighting the impact of L-arginine doping in varying ratios on the spectral characteristics of the CNDs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. XRD patterns\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe predominant amorphous nature of the CNDs is evident in the broad hump centered around 2θ\u0026thinsp;=\u0026thinsp;20.7\u0026deg; in the XRD profile, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. This characteristic hump is commonly observed in XRD patterns of amorphous carbon [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additionally, the absence of lattice fringes in the HR-TEM image further approves the amorphous nature of the material. The broadness of the peak in the XRD pattern, specifically observed at 2θ\u0026thinsp;=\u0026thinsp;20.7\u0026deg;, is attributed to the compact size of the CNDs, providing compelling evidence for their amorphous nature. This amorphous characteristic can be ascribed to the arrangement of C-C, C\u0026thinsp;=\u0026thinsp;O, and C\u0026thinsp;=\u0026thinsp;C bonds within the structural composition of the CNDs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4. UV-Vis spectroscopy\u003c/h2\u003e \u003cp\u003eThe UV\u0026ndash;Vis absorption spectra of CNDs derived from cinnamon and L-arginine (LA) doping exhibit typical characteristics observed in CNDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These spectra display strong absorption in the UV region, with the absorbance gradually decreasing into the visible range.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The π-π* transition manifests as a peak at 270 nm, and the electronic transition of the n-π* star transition is noticeable at 324 nm[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the absence of doping, carbon nanodots (CNDs) show lower absorbance. However, upon introducing LA doping, the absorbance increases, and this enhancement varies with the doping ratio, ranging from 0.25 to 0.50. This modulation in absorbance highlights the impact of LA doping on the electronic transitions within the CNDs, with the degree of enhancement correlating with the varying doping ratios.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Fluorescence spectroscopy\u003c/h2\u003e \u003cp\u003eFluorescence spectra were obtained using diluted samples of synthesized carbon nanodots (CNDs) in distilled water. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the emission spectra of all three CND types when excited at 365 nm, demonstrating that an increase in the doping ratio enhances fluorescence. Consistent parameters, such as excitation wavelength, bandwidth, and room temperature, were maintained for all measurements. Systematically varying the excitation wavelength from 280 nm to 420 nm with 10 nm increments revealed excitation-dependent fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. CNDs derived from cinnamon bark wood powder without doping exhibited weak fluorescence emission, with the maximum emission observed at 310 nm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter doping with two varying ratios, the emission spectra of CNDs with a 0.25 ratio (1:0.25) showed a 30 nm shift from undoped CNDs, and with an increased LA ratio (1:0.50), the fluorescence intensity became stronger (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(c)). The corresponding normalized fluorescence spectra (lower panel) illustrate the shift in emission with varying excitation wavelengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(e-g)). The QY value were obtained from the above-mentioned equation for Cinn CNDs, LA-doped CNDs (1: 0.25), and LA-doped CNDs (1: 0.50) are, 5.1, 10.5 and 14.2, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Fluorescence Microscopy","content":"\u003cp\u003e \u003c/p\u003e \u003cp\u003eFluorescence microscopy was employed to visualize yeast cells labelled with CNDs. In Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the top panel displays bright-field images, while the bottom panel shows fluorescent images of CNDs-labelled yeast cells. Notably, untreated cells with CNDs showed no fluorescence, and cinnamon-derived CNDs-treated yeast cells exhibited weak or barely noticeable fluorescence. With increasing doping, the fluorescence became more intense. Remarkably, yeast cells treated with L-arginine-doped CNDs (1:0.50) exhibited robust fluorescence. Under illumination within the 320\u0026ndash;365 nm wavelength range, CNDs-labelled yeast cells demonstrated uniform and intense fluorescence emission at 461 nm. Cells labelled with CNDs from L-arginine-doped (1:0.50) CNDs synthesis displayed significantly enhanced blue fluorescence compared to those from cinnamon-derived CNDs and L-arginine-doped (1:0.25) CNDs synthesis. This observation highlights the efficacy of a higher ratio of L-arginine doping into CNDs derived from cinnamon bark wood powder. The exceptional properties of CNDs, including their non-toxic nature, biocompatibility, strong photostability, and tunable photoluminescence, underscore their versatile utility in various biotechnological and imaging applications.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study has successfully synthesized and characterized carbon nanodots (CNDs) derived from cinnamon bark wood powder and L-arginine-doped CNDs at varying ratios. Comprehensive structural and optical analyses, including FTIR, XRD, HRTEM, DLS, UV-Vis, and PL spectra, provided a thorough understanding of their properties. The quantum yield (QY) values, essential for assessing fluorescence efficiency, were determined, revealing an increase with the L-arginine doping ratio. Specifically, QY values for Cinn CNDs, LA-doped CNDs (1:0.25), and LA-doped CNDs (1:0.50) were found to be 5.1, 10.5, and 14.2, respectively. The higher QY values at increased doping ratios underscore the effectiveness of L-arginine in enhancing the fluorescence of CNDs. The application of these synthesized CNDs in bioimaging of yeast cells demonstrated their efficacy, particularly the LA-doped CNDs (1:0.50), exhibiting robust fluorescence. The investigation into their non-toxic nature further enhances their potential for biological applications. In conclusion, this work establishes the utility of CNDs derived from cinnamon bark wood powder, emphasizing their versatility and potential advancements in biotechnological and imaging applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our gratitude to the Government of Gujarat for their financial support through the SHODH-ScHeme of Developing High-Quality Research program. Special thanks to the Sophisticated Test and Instrumentation Centre, Cochin University of Science and Technology, Cochin, India, for facilitating HRTEM analysis. We acknowledge the Department of Applied Chemistry, Faculty of Technology \u0026amp; Engineering, The Maharaja Sayajirao University of Baroda, Vadodara, India, for providing the fluorescence spectrophotometer facility under the DST PURSE program (SR/PURSE Phase 2/28 (C)) of the Government of India. Additionally, we thank the Department of Applied Physics, The Maharaja Sayajirao University of Baroda, Vadodara, India, for granting access to the XRD facility under the DST-FIST program (No. SR/FST/PS-II/ 2017/20).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study\u0026rsquo;s conception and design. The roles of the authors are as follows:\u003c/p\u003e\n\u003cp\u003eUrvi M. Lad: Investigation (lead), Material preparation, Methodology, Writing the Original draft (equal), Visualization\u003c/p\u003e\n\u003cp\u003eDhruti J. Dave: Data collection (supporting), Analysis (supporting)\u003c/p\u003e\n\u003cp\u003eBhumi N. Desai: Data collection (supporting), Analysis (supporting)\u003c/p\u003e\n\u003cp\u003eDevesh H. Suthar: Resources (supporting), Data collection (supporting), Analysis (supporting)\u003c/p\u003e\n\u003cp\u003eChetan K. Modi: Conceptualization (lead), Writing \u0026mdash; Review \u0026amp; Editing (lead), Project Administration and Supervision (lead)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis declaration is \u0026ldquo;not applicable\u0026rdquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included in the main article as well as Supplementary material in the online version.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHu Q, Gong X, Liu L, Choi MMF (2017) Characterization and Analytical Separation of Fluorescent Carbon Nanodots. 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Nanoscale 12:591\u0026ndash;601. https://doi.org/10.1039/C9NR08461D\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carbon nanodots, Hydrothermal, L-Arginine, Photoluminescence, Bioimaging","lastPublishedDoi":"10.21203/rs.3.rs-4142590/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4142590/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we present an economical and efficient synthesis method for carbon nanodots (CNDs) derived from cinnamon bark wood powder, supplemented with L-arginine doping at varying ratios. Extensive structural and optical characterization was conducted through techniques such as FTIR, XRD, HRTEM, DLS, UV-Vis, and PL spectra, providing a comprehensive understanding of their properties. Quantum yields (QY) were quantified for all three samples, contributing to the assessment of their fluorescence efficiency. The synthesized CNDs were successfully applied for bioimaging of yeast cells, employing fluorescence microscopy to visualize their interaction. Remarkably, L-arginine-doped CNDs exhibited enhanced fluorescence, particularly at a higher doping ratio (1:0.50), showcasing the influence of the dopant. The non-toxic nature of these CNDs was rigorously investigated, confirming their biocompatibility. This work not only contributes to the synthesis and characterization of CNDs but also highlights their potential for diverse applications, emphasizing their structural, optical, and biological attributes. The findings underscore the versatility of CNDs derived from cinnamon bark wood powder and their potential in advancing biotechnological and imaging applications.\u003c/p\u003e","manuscriptTitle":"Fluorescent enhancement of CNDs from Cinnamon bark with L-Arginine doping for Yeast cell Imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 16:22:34","doi":"10.21203/rs.3.rs-4142590/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-26T11:48:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-24T06:45:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-19T17:07:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-18T12:50:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7ae395e8-e9c4-4495-aa5a-77cd0f54edc0","date":"2024-04-17T19:43:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3233b714-3b5b-472b-bd0a-6a0c728257da","date":"2024-04-17T18:12:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2260e286-dd57-4f8a-a615-ef872525f7ca","date":"2024-04-12T14:27:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15b2bf4e-98e2-41c6-8d60-9da5669f7f79","date":"2024-04-12T12:17:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-25T15:39:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f1d25cc7-2e38-49a3-b6c6-01420c5300a5","date":"2024-03-24T18:13:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1eb44375-c10c-4826-a5c5-03f6623725de","date":"2024-03-24T17:55:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-24T17:35:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-22T06:44:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-22T06:44:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2024-03-21T10:02:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d59375a6-8f94-4e06-966b-a6a838c92043","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-06T16:50:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-28 16:22:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4142590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4142590","identity":"rs-4142590","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}