## 4 Synthesis of Graphene and Its Derivatives  
As the phenomenal electrochemical, physiochemical, and mechanical properties of graphene, GO, and rGO make it a superior material for electrochemical biosensors, the fabrication methods of the graphene which affect its characterization also influence the convenient advancement and performance of the electrochemical biosensors in terms of selectivity, sensitivity, and rapid cost-effective detection of the analyte [98; 99; 100]. Thus, the synthesis of graphene must take paramount considerations. On the one hand, it is vitally crucial to synthesize the controlled size (monolayer) and impurities-free graphene with restrained morphology to achieve structure-property compatibility. On the other hand, it is essential to design high yield, scalable, cost-effective industrial methods for their production [98; 101]. For a clear explanation, the synthesis of graphene, GO, and rGO can be categorized into two main methods known as bottom-up and top-down. Every method has its own strategy. While epitaxial growth [102] (using thermal chemical vapor deposition strategy) and unzipping carbon nanotubes [103] are considered as top-down methods, the isolation [102] and (mechanically or chemically exfoliations) and reduction strategies [104; 105; 106; 107; 108; 109] are considered as bottom-up methods as shown in Figure 8.  
Figure 8: Bottom-up and top-down approaches for graphene, GO, and rGO synthesis methods. The exfoliation method is originally based on isolation of a few layers from the bulk of graphene sheets by breaking the weak van dar Waals bonds and overcoming the attraction between adjacent layers. The number of mechanically and chemically approaches that depend on the exfoliation phenomena have exhibited promise for bulk and large-scale production [99; 110]. The scotch tape technique is employed to peel atomically thin layers from a slab of HOPG flakes using adhesive tape to get individual crystal planes. Micromechanical cleavage is then applied by rubbing large crystal flakes against each other [99; 111; 112]. This method can obtain pristine structured graphene crystals without repeating defects in the structure of 2D graphene layers [99; 110]. Although the mechanical exfoliation method provides a millimeter-sized graphene with electronic quality, their usage is restricted for biosensor applications because of the low yielding of mono-layer graphene sheets with a relatively high lateral dimension up to hundreds of micrometers besides the time consuming, making it only suitable for fundamental research and laboratories applications [98; 99; 101]. Liquid phase exfoliation (LPE) is another process of the same concept, it ordinarily includes dispersion of bulk graphite in an aqueous media, exfoliation, and purification. In principle through this concept, the presence of solvent applies external forces to overcome the interaction of van der Waal bonds as well as avoid substantial interruption of the plane of graphene. Thus, this method depends on the selection of optimum solvents that lower the interfacial tension between the solvent and graphene layer [113]. Recently, the usage of urea-based aqueous media shows greater capability superior to conventional toxic N-dimethylformamide (DMF)-based exfoliation [99; 101; 110]. Moreover, LPE can be improved by sonication, sheer mixing, or ball milling as well as by the addition of surfactants and intercalation with chemical species [101]. Sonication-assisted exfoliation via chemical wet dispersion involves the formation of shear forces and cavitation that act on the graphite bulk material and induce exfoliation followed by a solvent-graphene interaction that balance the interlayers attractive forces. However, this route involves the usage of high-boiling point solvents, e.g., N-methyl-pyrrolidone (NMP) and a long-time sonication, as well as, leads to the aggregation of graphene [113].