## 3 Graphene and Graphene-Related Materials for Biological Applications
### From Material to Supramolecular Cluster: Graphene Quantum Dots  
Graphene and its related derivatives lack of some properties such as bandgap or optical properties. This reduces the possible applications, and an engineered material could offset these limitations. In recent times, graphene quantum dots (GQDs) have received great attention for their possible unique applications [167] as a solid alternative to classical graphene, GO, and rGO. Graphene quantum dots consist of graphene sheets, which could be single-sheet or multi-layered. They are nanoparticles with dimensions smaller than 100 nm, commonly under 20 nm, and their shapes are usually circular or elliptical. However, other shapes are produced with different synthetic routes [168]. The synthesis methods to produce GQDs are both top-down and bottom-up as summarized in Figure 5 by using different precursors and synthetic strategies. The most common approach is the top-down one that usually consists of physical processes that reduce the dimensions of carbon materials as carbon fibres [170] or coal [171], along with acid oxidation [172] and hydrothermal treatment [173]. Solvothermal route [174], microwave [175], and sonication [176] are also promising alternative approaches. A top-down physical process is the acid exfoliation, followed by the oxidation of the nano-obtained product. [PERSON] et al. [177] suggested an electrochemical synthesis route to produce a variety of GQDs, although the products differ in size, emission colours, and  
Figure 5: Scheme of graphene quantum dots (GQDs) top–down and bottom–up approaches as reported by [169] (under CC license). other intrinsic properties. Hydrothermal synthesis can be done just on precise precursors, such as GO, with a functionalization with oxidizing agents to introduce functional groups on the product surface [178]. The bottom-up method provides for the generation of aromatic molecules as precursors and can lead to high yield. [PERSON] et al. [179] developed a protocol to fabricate GQDs through direct pyrolysis on citric acid. The synthesis routes mentioned above lead to a differentiation of the GQDs produced and their different physicochemical properties which depend on procedure, precursors, defects, and functional groups [180; 181]. The GQDs' properties of interest are absorbance, photoluminescence (PL), electrochemical photoluminescence among the optical ones and of course their biocompatibility; all these properties make them great candidates for bioimaging and theragostic applications. The absorption characteristics of GQDs are shown in the short-wavelength region because of C=C bonds and around 270-390 nm they show peaks for the C=O bonds. The passivation of the surface with a functional group can alter the disposition of the peaks; it strongly depends on the type of dot. The principal and most fascinating property of the carbon dots is their photoluminescence. The unclear photoluminescence mechanism is one of the impediments to understand completely the physical mechanism behind these promising materials. The tuneable property of photoluminescence is size-dependent since it is related to quantum confinement. Moreover, other factors such as shape, defect, and doping also influence the effect. In detail, [PERSON] et al. [182] reported that PL is related to the quantum confinement effect of the electrons in sp\({}^{2}\) carbons [183]. Another structure-dependent characteristic of GQDs is the electron transfer [184]. Biocompatibility and almost absent toxicity make them eligible for in vivo applications. In vitro studies have been made by [PERSON] et al. [185], and they studied the GQDs' effects on the cellular life finding out how the nanomaterial did not affect either the viability or the reproduction capacity. In vivo studies have been made on mice by exposing them to different nanomaterials. The results showed no abnormalities in organs or habits and brought to awareness their excellent biocompatibility, raising interest in the possible bio-applications.