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Carbon dots have been gaining attention in the field of nanobiotechnology due to their superior photostability, high water solubility, ease of synthesis and surface functionalization, chemical inertness, low toxicity, and excellent biocompatibility. They also exhibit good two-photon absorption and unique tunable optical properties across a wide range of wavelengths, from ultraviolet to near infrared endowing them with potential for a variety of biological applications. Recently, there has been a growing interest in the synthesis and development of red-emissive two-photon carbon dots. Here we present recent progress in the design requirements for red-emissive two-photon carbon dots, and review current state-of-the-art systems, covering their applications in bioimaging, biosensing, and photothermal and photodynamic therapy. Carbon dots are suitable for a range of biological applications due to their unique physicochemical properties and biological behavior. This Review summarizes research related to the emerging field of red-emissive two-photon carbon dots for bioimaging, biosensing, and phototherapeutic applications.

In recent years, the development of carbon dot-based fluorescent nanoparticles for bioimaging applications has attracted the attention of scientific community. However, the functionality of the majority of these developed fluorescent systems is confined in to the blue-to-green region of the light spectrum, limiting their application as bioimaging agents. Here, we report the facile design and development of highly efficient two-photon excitable red-emissive carbon dots (D-CD) and demonstrate their high performance in bioimaging applications. Our comprehensive study on the effects of synthesis parameters on the photophysical and physicochemical properties of carbon dots demonstrated the importance of precursor selection and consideration of its molecular structure in developing red-emissive carbon dots. The developed D-CD is highly biocompatible and non-toxic, with remarkable photostability in cells under two-photon near-infrared excitation. The present study point to the great potential of D-CD as an efficient bioimaging agent for cellular biolabeling, long-term and real-time cellular imaging, and high-resolution deep-tissue imaging in complex biological systems.

Understanding the surface processes (deposition and surface diffusion) that occur at or close to the surface of growing nanoparticles is important for fabricating reproducibly stellated or branched gold nanoparticles with precise control over arm length and spatial orientation of arms around the core. By employing a simple seed-mediated strategy, we investigate the key synthetic variables for precise tuning of in situ surface processes (competition between the deposition and surface diffusion). These variables include the reduction rate of a reaction, the packing density of molecules/ions on the high surface energy facets, and temperature. As a result, the thermodynamically stabilized nanoparticles (cuboctahedron and truncated cube) and kinetic products (cube, concave cube, octapod, stellated octahedron, and rhombic dodecahedron) in different sizes with high quantitative shape yield (> 80%) can be obtained depending on the reduction rate of reaction and the packing density of molecules/ions. With computer simulation, we studied the stability of stellated (branched structure) and non-stellated (non-branched structure) gold nanoparticles at high temperature. We construct a morphology phase diagram by varying different synthetic parameters, illustrating the formation of both stellated and non-stellated gold nanoparticles in a range of reaction conditions. The stellated gold nanoparticles display shape-dependent optical properties and can be self-assembled into highly ordered superstructures to achieve an enhanced plasmonic response. Our strategy can be applied to other metal systems, allowing for the rational design of advanced new stellated metal nanoparticles with fascinating symmetry dependent plasmonic, catalytic, and electronic properties for technological applications.

Designing a two-photon ratiometric pH-sensitive nanoprobe for real time monitoring of intracellular pH in biological environment is of great importance for better understanding the pathogenesis of diseases and the design of intracellular drug delivery-based system. However, the development of such probe remains a challenge. Here, we report for the first time the design and development of the two-photon ratiometric carbon dot-based nanoprobe and demonstrate its capability to monitor and quantify the intracellular cytoplasmic pH value, in real time. The nanoprobe comprised of a fluorescent carbon dot functionalized with a pH-responsive fluorescein dye (FACD). With increasing pH, FACD exhibits a clear ratiometric change in the emission intensity ratio, with sensitivity across a wide pH range in both extracellular and intracellular compartments. FACD is non-toxic to adipose stem cells in cell imaging experimental conditions and exhibits remarkable thermal stability, photostability, and two-photon near-infrared excitation capability. Using real-time dual-channel two-photon confocal microscopy we demonstrate the great potential of FACD as an efficient nanoprobe with high accuracy for the intracellular sensing of pH in living adipose stem cells seeded on either cell-culture dishes or on a 3D printed bioactive scaffold.

Carbon based quantum dots find various applications such as imaging, diagnosis and therapeutics due to low toxicity, antibacterial activity, excellent water solubility, and good biocompatibility. However, the UV-blue emission of carbon based quantum dots is unsuited for applications in biomedicine and imaging as the short wavelength lights harm the living cells due to phototoxicity. In the present work, we have synthesised the long wavelength emitting heteroatoms (nitrogen (N) and sulfur (S)) co-doped graphene quantum dots (NS-GQD) via hydrothermal route. These NS-GQD have excitation-wavelength independent photoluminescence with high quantum yield in protonated and neutral forms. The non-covalent interaction of these NS-GQD when studied with a dye rhodamine B (RB) shows evidence of Forster resonance energy transfer (FRET). It has been shown here that the NS-GQD-RB composite exhibits prominent two-photon absorption at near infrared wavelength with a capability of deep-tissue imaging upto 175 µm.