Fluorescent Carbon Dots from Biomass Waste: Photoluminescent Behavior and Toxicity Profile Validated In Vitro and In...

Carbon dots (CDs) are emerging nanomaterials with tunable optical properties and promising scalability; however, their potential toxicity remains a key concern for biomedical and environmental applications. In this study, we investigated the in vitro and in vivo toxicity of CDs synthesized from spent coffee grounds (SCG-CD) via pyrolysis. Thermogravimetric analysis (TGA) was used to determine the optimal synthesis parameters, while fluorescence spectroscopy was used to confirm CD formation and excitation-dependent emission. The Design of Experiments (DoE) approach identified conditions yielding blue-emissive CDs with a maximum photoluminescence quantum yield of 6% (400 °C, 3 h, named S4_SCG-CD). Fluorescence analysis revealed pH-dependent emission enhancement under alkaline conditions. Structural characterization by Fourier-transform infrared spectroscopy and X-ray diffraction demonstrated surface functionalization and a semicrystalline framework. In vitro MTT assays indicated no cytotoxicity across tested concentrations, and in vivo assays in mice and zebrafish embryos revealed no acute toxicity. The S4_SCG-CD sample was classified as Category 5 (LD50 ≥ 5000 mg/kg) under the Globally Harmonized System and "practically nontoxic" (LC50 > 150 μg/mL) according to the Fish and Wildlife Service. These findings highlight spent coffee grounds as a sustainable precursor for generating biocompatible, low-toxicity materials with potential applications in bioimaging and eco-friendly nanotechnology.