Graphene quantum dots (GQDs) represent a class of zero-dimensional nanomaterials with exceptional optical and electronic properties, derived from their graphene-like structure and quantum confinement effects. Their synthesis can be broadly categorized into top-down and bottom-up approaches, each offering distinct advantages in terms of size control, surface chemistry, and scalability. The photoluminescence of GQDs is highly size-dependent, while surface functionalization further tailors their properties for specific applications, particularly in bioimaging. However, challenges related to toxicity and scalable production remain critical considerations for their widespread adoption.

Top-down synthesis methods involve the fragmentation of larger carbon-based precursors into nanoscale GQDs. One common approach is the oxidative cutting of carbon fibers or graphene sheets using strong acids or electrochemical exfoliation. For instance, carbon fibers treated with a mixture of sulfuric and nitric acids under sonication yield GQDs with sizes ranging from 2 to 10 nm, as confirmed by transmission electron microscopy (TEM). The acidic conditions introduce oxygen-containing functional groups such as carboxyl and hydroxyl groups on the edges of the GQDs, which influence their solubility and photoluminescence. Another top-down method involves hydrothermal or solvothermal cutting of graphene oxide (GO) sheets. Under high temperature and pressure, GO sheets break into smaller fragments, forming GQDs with lateral dimensions typically between 3 and 20 nm. The size distribution can be controlled by adjusting reaction parameters such as temperature, duration, and precursor concentration. A key advantage of top-down methods is the retention of the graphene lattice structure, which preserves the intrinsic electronic properties of GQDs. However, these methods often require harsh chemical treatments and suffer from broad size distributions, necessitating additional purification steps.

Bottom-up synthesis routes, on the other hand, construct GQDs from molecular precursors through controlled pyrolysis or carbonization. Citric acid pyrolysis is a widely studied method due to its simplicity and ability to produce uniform GQDs. When citric acid is heated to temperatures around 200°C, it undergoes dehydration and carbonization, forming GQDs with sizes typically between 2 and 7 nm. The process allows precise control over the size by varying the pyrolysis temperature and time. Another bottom-up approach involves the solution-phase synthesis of GQDs from polycyclic aromatic hydrocarbons (PAHs) or other small organic molecules. These precursors are subjected to controlled condensation and cyclodehydrogenation reactions, resulting in well-defined GQDs with tailored edge structures. Bottom-up methods generally offer better control over size and surface chemistry compared to top-down approaches, but they may introduce more defects in the graphene lattice, affecting electronic properties.

The photoluminescence (PL) of GQDs is a defining characteristic, exhibiting excitation-dependent emission due to quantum confinement and surface states. Smaller GQDs (below 5 nm) typically emit blue light, while larger dots (5–10 nm) show green to yellow emission. This size-dependent behavior arises from the quantum confinement effect, where the bandgap increases as the size decreases. Surface functional groups also play a critical role in PL properties. For example, GQDs with abundant carboxyl groups exhibit blue emission, while those with amine-functionalized surfaces may shift the emission toward longer wavelengths. The PL mechanism is further influenced by edge states and defect sites, which can introduce additional energy levels within the bandgap. Studies have shown that the quantum yield of GQDs can range from 10% to 80%, depending on the synthesis method and surface passivation.

Surface functionalization is essential for tuning the properties of GQDs for specific applications. Covalent modification with polyethylene glycol (PEG) improves biocompatibility and stability in physiological environments, making PEGylated GQDs suitable for bioimaging. Non-covalent functionalization with polymers or biomolecules can also enhance targeting capabilities. For instance, folic acid-conjugated GQDs have been used for selective imaging of cancer cells overexpressing folate receptors. The surface chemistry also affects the interaction of GQDs with biological systems, influencing their cellular uptake and distribution.

In bioimaging, GQDs serve as excellent fluorescent probes due to their high photostability, low toxicity, and tunable emission. Their small size enables efficient cellular uptake, and their surface can be modified for specific targeting. In vitro studies demonstrate that GQDs can label cells with high contrast and minimal background signal. In vivo imaging applications are also being explored, with GQDs showing promise in tracking tumor progression and monitoring drug delivery. However, the long-term biodistribution and clearance pathways of GQDs require further investigation to ensure safety.

Toxicity remains a critical concern for the biomedical application of GQDs. While in vitro studies often report low cytotoxicity at moderate concentrations, the effects of prolonged exposure or high doses are less understood. Surface chemistry plays a significant role in toxicity; for example, positively charged GQDs may exhibit higher cytotoxicity due to stronger interactions with cell membranes. In vivo studies have shown that GQDs can accumulate in organs such as the liver and spleen, raising concerns about potential long-term effects. Scalability is another challenge, as many synthesis methods are not easily translated to industrial-scale production. Bottom-up methods, while offering better control, often involve complex purification steps, whereas top-down methods may struggle with yield and reproducibility.

In summary, GQDs synthesized via top-down or bottom-up routes exhibit unique optical properties influenced by size and surface chemistry. Their applications in bioimaging are promising but require careful consideration of toxicity and scalability. Future research should focus on optimizing synthesis methods to improve yield and uniformity while ensuring biocompatibility for clinical translation. Addressing these challenges will be key to unlocking the full potential of GQDs in nanotechnology and medicine.