Carbon dots (CDs)-based nanoparticles have been extensively explored for biological applications in sensing and bioimaging. However, the major translational barriers to CDs for imaging and sensing applications include synthetic strategies to obtain monodisperse CDs with tunable structural, electronic, and optical properties in order to achieve high-resolution deep-tissue imaging, intracellular detection, and sensing of metal ions with high sensitivity down to nanomolar levels. Herein, we report a novel strategy to synthesize and develop a multifunctional nitrogen-doped CDs probe of different sizes using a new combination of carbon and nitrogen sources. Our results show that the structural characteristics (i.e., the surface density of emissive traps and bandgaps levels) depend on the size of the CDs, which ultimately influences their optical properties. This work also demonstrates the development of a two-photon dual-emissive fluorescent multifunctional probes (3-FCDs) by conjugating fluorescein isothiocyanate on the surface of nitrogen-doped CDs. 3-FCDs show excellent near-infrared two-photon excitation ability, single-wavelength excitation, high photostability, biocompatibility, low cytotoxicity, and good cell permeability. Using two-photon fluorescence imaging, our multifunctional probe shows excellent deep-tissue high-resolution imaging capabilities with penetration depth up to 3000 and 280 μm in hydrogel scaffold and pigskin tissue, respectively. The designed probe exhibits ultrasensitivity and specificity toward Fe3+ ions with a remarkable detection limit of 2.21 nM using two-photon excitation. In addition, we also demonstrate the use of multifunctional CDs probe for ultrasensitive exogenous and real-time endogenous sensing of Fe3+ ions and imaging in live fibroblasts with rapid response times for intracellular ferric ion detection.

Bioprinting is a promising fabrication technique aimed at developing biologically functional, tissue-like constructs for various biomedical applications. Among the different bioprinting approaches, vat polymerization-based techniques offer the highest feature resolution compared to more commonly used extrusion-based methods and therefore have greater potential to be utilized for printing complex hierarchical tissue architectures. Although significant efforts have been directed toward harnessing digital light processing techniques for high-resolution bioprinting, the use of stereolithography (SLA) setups for producing distinct hydrogel filaments smaller than 20 μm has received less attention. Improving the bioprinting resolution is still a technical challenge that must consider both the practical limitations of the bioprinter apparatus and the formulation of the cytocompatible bioresin. In this study, we developed a novel bioresin compatible with SLA and capable of printing high-resolution features. This resin, composed of a biosynthetic polypeptide poly(L-glutamic acid) functionalized with tyramine moieties (PLGA-Tyr), was crosslinked using a visible-light photoinitiator system. Varying concentrations of PLGA-Tyr and the co-photoinitiator were evaluated for the hydrogel system's gelation ability, swelling characteristics, degradation profiles, mechanical properties, and cell viability post-encapsulation. This study introduces a custom-built, cost-effective, visible-light SLA bioprinting system named the "MicroNC". Using the newly developed visible-light bioresin, we demonstrated for the first time the ability to fabricate hydrogel scaffolds with well-resolved filaments (less than 8 μm in width) capable of supporting cell viability and proliferation and directing cellular morphology at the single-cell level for up to 14 days. Overall, these experiments have underscored the exciting potential of using the visible-light-photoinitiated PLGA-Tyr material system for developing physiologically relevant in vitro hydrogel scaffolds with feature resolutions comparable to the dimensions of individual human cells for a wide range of biomedical applications.