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Traditional fluorophores and dyes used for imaging have certain limitations such as rapid photobleaching, multiple steps for synthesis, small Stokes shift, high production cost, narrow excitation spectra, short fluorescence lifetime and broad emission band [1, 2]. These drawbacks were overcome by advancements in nanotechnology and conventional semi-conductor quantum dots (QDs) were developed for use in photodetectors, cell imaging, solar cells and hydrogels [1, 2]. However, presence of heavy metals such as cadmium, lead etc. in conventional QDs lead to toxicity and hence, limits their use for biological applications. Owing to absence of heavy metals in carbon-based quantum dots (CQDs), these QDs emerged as alternative photoluminescent nanomaterials with a wide range of applications such as bioimaging, sensors, nanomedicine, electrocatalysis and light emitting diodes (LED) [1, 2]. CQDs exhibit excellent physicochemical attributes which include aqueous solubility, high crystallization, high photoluminescence, high electric conductivity, good catalytic activity, biocompatibility, low photobleaching, good dispersibility and ease of surface functionalization. Despite their photoactivity, there are reports that commercial CDs degrade under illumination resulting in photodegradation induced cytotoxicity, irrespective of the chemical composition . Under illumination, these photoactive CDs absorb UV-visible light due to π–π* transition of sp2 and generate electrons and holes, which drive various redox reactions and produce reactive oxygen species (ROS) . The generated ROS interacts with biomolecules (DNA, proteins and lipids) and induce a cascade of physiological and pathological effects, which damage the cells . Several types of CDs are known for producing ROS and therefore, function as potent antimicrobial agents . In the light of this background, it is evident that there is a need of biocompatible nano-probes with unique, tuneable optical and antioxidant properties for use in high-contrast fluorescence imaging, which can scavenge the ROS generated during imaging. Ascorbic acid (AA), also known as vitamin C is a natural antioxidant. AA also plays important role in physiological processes associated with aging, vascular injury, inflammatory damage and cancer . AA is the strongest ROS scavenger, well-known to protect cells from damage induced by free radicals. However, natural AA is unstable, therefore approaches for nano formulation have been used to enhance AA stability. AA is hydrophilic and non-toxic; therefore, its nano-formulation would yield nano-probes with excellent aqueous solubility, low toxicity, biocompatibility, and antioxidant activity, which can very well replace the metal-based semiconductor quantum dots used for bioimaging .
In this study, L-AA was used as carbon source for synthesis of CDs. AA-CDs were synthesized by one step-hydrothermal method at 180 ºC. Hydrothermal method is one of the best methods to synthesize fluorescent CQDs from various precursors. The as-synthesized AA-CDs showed bright blue fluorescence with a strong absorption peak at 340 nm (Fig. 1A), which corresponds to electronic transition from valence to the lowest empty bands (π → π* transition). The photoluminescence (PL) spectra (Fig. 1B) at different excitation wavelengths showed highest PL intensity at excitation wavelength 340 nm and emission wavelength 400 nm (the shift observed was 60 nm). The broad hump centered characteristic for amorphous carbon was obtained in AA-CDs at around 2𝜃=25º, which is evident in the X-ray diffraction (XRD) profile (Fig. 1C). EDX spectra revealed the elemental composition for AA-CDs as composed of carbon (46.38 wt.%) and oxygen (51.80 wt.%) (Fig.1D). We further evaluated the antioxidant activity of AA-CDs. For bioimaging applications, it is essential to determine the toxicity and cellular uptake of the nano-probe. AA-CDs were found to be non-toxic against HeLa cell lines. Microbial toxicity of AA-CDs was investigated against fungal pathogen (Candida albicans) using broth microdilution assay [9, 10, 11]. Our results demonstrated that AA-CDs were non-toxic, probably due to scavenging of ROS by AA-CDs. The cellular uptake of AA-CDs was determined by monitoring their internalization into the microbial cells as described previously . Best internalization results were obtained after incubation of AA-CDs with the fungal cells for 15 mins. Confocal microscopy images show internalized AA-CDs in the fungal cells which is visible as bright blue fluorescence inside the cells (Fig. 1E). We conclude that AA-CDs has promising anti-oxidant activity and may be used as eco-friendly, biocompatible, nano-probe for high contrast fluorescence imaging.
How to Cite
Ascorbic acid, Carbon-based quantum dots, Reactive Oxygen Species, ROS scavenger, Antioxidant, Bioimaging
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