**3. Functionalization**

In spite of, the great potential related to pristine graphene, the fact that it possesses no band gap, it is practically chemically unreactive and it has a poor water dispersibility, have a severe effect limiting its applications in certain fields compared to other well‐established materials. Nevertheless, functionalization of graphene‐based materials has become one of the main alternatives to address these problems. Graphene derivatives, specifically GO and rGO, could be modified with a wide range of organic or inorganic molecules through chemical or physical functionalization [43]. The availability of different oxygen‐containing groups and the presence of sp<sup>2</sup> domains enable these materials to interact with a covalent, non‐covalent, and the combination of both interactions with other molecules. This produces hybrids or composite materials with a particular set of properties and potential applications [12], such as the increment in their dispersibility, processability, purification, device fabrication, biocompati‐ bility, band gap modification [44].

As Georgakilas emphasized [43, 45], the presence of several oxygen reactive sites in GO yields a higher covalent reactivity compared with rGO, and thus, GO is frequently chosen as a starting material where molecules will be attached to oxygen atoms. On the other hand, non‐covalent functionalization makes use of hydrophobic or π‐interactions on the surface or basal plane of the graphenic layer, giving rise to the preference of rGO as a starting material for this approach. Furthermore, the remnant oxygen moieties offer the possibility to form hydrogen bonding, electrostatic interactions, or the combination of all of them. Although these preferences are not a rule, it could be useful in some cases depending on the type of materials to immobilize [46].

#### **3.1. Covalent functionalization of GO**

**Reducing agents**

26610 Recent Advances in Graphene Research

Proteins

Hormones

Others Green reducing agents

**3. Functionalization**

bility, band gap modification [44].

*C/O ratio obtained by a*

of sp<sup>2</sup>

**C/O ratio**

*Shewanella* 3.1a Aerobic, 60 h [26] *E. coli* culture – 37°C, 48 h [26] *E. coli* biomass – 37°C, 72 h [26] Baker's yeast/NADPH 5.9a 35–40°C, 72 h [26] Wild carrots roots 11.9a 26°C, 72 h [26]

Bovine serum albumin/NaOH – 55–90°C, 3–24 h [26]

Melatonin/NH3 – 80°C, 3 h [26]

K2CO3 – 90°C, 2 h [41] H2‐rich water 6.26a 90°C, 3 h [42]

*Elemental analysis <sup>c</sup>*

In spite of, the great potential related to pristine graphene, the fact that it possesses no band gap, it is practically chemically unreactive and it has a poor water dispersibility, have a severe effect limiting its applications in certain fields compared to other well‐established materials. Nevertheless, functionalization of graphene‐based materials has become one of the main alternatives to address these problems. Graphene derivatives, specifically GO and rGO, could be modified with a wide range of organic or inorganic molecules through chemical or physical functionalization [43]. The availability of different oxygen‐containing groups and the presence

 domains enable these materials to interact with a covalent, non‐covalent, and the combination of both interactions with other molecules. This produces hybrids or composite materials with a particular set of properties and potential applications [12], such as the increment in their dispersibility, processability, purification, device fabrication, biocompati‐

As Georgakilas emphasized [43, 45], the presence of several oxygen reactive sites in GO yields a higher covalent reactivity compared with rGO, and thus, GO is frequently chosen as a starting material where molecules will be attached to oxygen atoms. On the other hand, non‐covalent functionalization makes use of hydrophobic or π‐interactions on the surface or basal plane of the graphenic layer, giving rise to the preference of rGO as a starting material for this approach. Furthermore, the remnant oxygen moieties offer the possibility to form hydrogen bonding,

*X‐ray Photoelectron spectroscopy <sup>b</sup>*

**Table 1.** Reducing agents for GO toward a green reduction.

**Conditions Refs.**

*Energy dispersive spectroscopy*

Covalent modification of GO yields chemical derivatives produced by the next routes, analogous to those obtained with other materials [47]:


#### *3.1.1. Chemical reactions by oxygen moiety*

Both, click chemistry and liker reaction are considered as a post‐functionalization of graphene oxide. Direct chemical attachment could be briefly explained in terms of the organic chemistry of the oxygen moieties in GO (**Figure 2**), according to Georgakilas [48].


After the functionalization, the remaining unreacted oxygen groups on GO could be removed with a post‐reduction chemical reaction to recover some of the graphenic character. This is a very common approach that exploits the dispersibility and chemical properties of GO, while the final material resembles in certain degree to graphene. In fact, in some cases, a partial reduction takes place through the functionalization reaction itself due to the presence of a reducing group (like amines) in the attached molecule or because of the conditions/catalyst employed. In either case, a covalently functionalized rGO is produced with improved properties such as conductivity, thermal stability, or mechanical properties, although the hydrophilic character is diminished [48].

**Figure 2.** Schematic representation of the covalent functionalization of GO through direct chemical attachment to its oxygen functionalities.

#### **3.2. Non‐covalent functionalization of rGO**

Unlike the strong covalent functionalization, non‐covalent functionalization is an attractive modification route because it offers the possibility to immobilize molecules on both sides of the graphenic basal plane without any further chemical modification of the carbon lattice, avoiding the generation of additional defects, and thus lowering the loss of desired properties, while new properties are introduced [43, 46, 51].

Non‐covalent functionalization consists of π‐stacking interactions, hydrophobic effects, van der Waals forces, electrostatic interactions, and hydrogen bonding, and in particular situations, even the geometry of the materials plays an important role [52]. In many cases, non‐covalent functionalization is accomplished through simple mixing of the materials in an adequate medium [48, 53, 54] or following an incubation protocol for specific biomolecules and cells [55]. As previously mentioned, even though rGO is frequently preferred for this approach, there are many works on the non‐covalent modification of GO, followed by a chemical reduction similar to the description above. These non‐covalent modifications are usually governed by electrostatic interactions, and thus they depend on the pH of the medium [56–60].

#### *3.2.1. Non‐covalent interactions*

employed. In either case, a covalently functionalized rGO is produced with improved properties such as conductivity, thermal stability, or mechanical properties, although the

**Figure 2.** Schematic representation of the covalent functionalization of GO through direct chemical attachment to its

Unlike the strong covalent functionalization, non‐covalent functionalization is an attractive modification route because it offers the possibility to immobilize molecules on both sides of the graphenic basal plane without any further chemical modification of the carbon lattice, avoiding the generation of additional defects, and thus lowering the loss of desired properties,

Non‐covalent functionalization consists of π‐stacking interactions, hydrophobic effects, van der Waals forces, electrostatic interactions, and hydrogen bonding, and in particular situations, even the geometry of the materials plays an important role [52]. In many cases, non‐covalent functionalization is accomplished through simple mixing of the materials in an adequate medium [48, 53, 54] or following an incubation protocol for specific biomolecules and cells [55].

hydrophilic character is diminished [48].

26812 Recent Advances in Graphene Research

oxygen functionalities.

**3.2. Non‐covalent functionalization of rGO**

while new properties are introduced [43, 46, 51].

π interactions occur at the aromatic domains on the surface of both GO and particularly on rGO. π‐stacking interactions are fundamental for the stabilization of aromatic systems as in the case of dyes, pyrene functionalized molecules, etc.; aromatic or π‐electron‐based polymers such as polystyrene, polypyrrol, polyaniline; and biomolecules such as nucleic acids, aromatic residues in polypeptides and some drugs. Such stability is a result of the strong π–π stacking interaction ruled by dispersion forces, not by electrostatic forces. There are other types of π complexes that can be formed with polar gases, cations, and anions. Hydrophobic and van der Waals interactions are exploited by molecules that lack of aromatics or charged groups, such as many polymers, surfactants, quantum dots, and predominantly hydrophobic polypeptides, among others [43, 46, 51].

Oxygen groups such as epoxy and hydroxyl on the surface, and carboxyl and carbonyl at the edges of the layers, allow the adsorption of polar and/or charged molecules through electro‐ static interactions and/or the formation of strong hydrogen bonding. While the surface charge of materials is a pH‐dependent property, hydrogen bonds is only presented with materials that possesses amine or hydroxyl moieties. Polycationic polymers, polysaccharides, proteins, and enzymes are some examples of molecules, which can present this type of interactions. Even when certain molecules present electrostatic interactions, it can occur that some biomo‐ lecules are pH independent, and thus, they are mainly adsorbed due to the hydrophobic or π‐ interactions [45, 52].

Thus, the combination of the previously discussed non‐covalent forces depends on the type of materials and the reaction conditions, and they are very important for the fabrication of new devices [46, 51]. Finally, a common approach consist on the synthesis of systems through the combination of covalent/non‐covalent interactions in multiple consecutive stages, yielding very complex hybrid materials or in a multilayer arranged, highly functional, selective, and efficient [46, 61].

Although functionalization of graphene is applicable to very diverse research fields, systems focus on biological applications which represent an attractive and rapidly growing area of research.
