**4. Functionalization with biomolecules**

Even when graphene‐based materials have been studied intensively in different fields, their potential in biotechnology and biomedicine applications is still in development, but their research is growing quickly [46]. Graphenic surfaces are ideal to interact with several biomo‐ lecules as it has been seen with carbon nanotubes [47, 62]. The combination between graphene materials and biotechnology gives rise to new nano‐/bio‐interfaces [63] through different biofunctionalization process. Biofunctionalization is defined as the modification of a material by the attachment of biomolecules, ranging from organic groups to very large proteins or even cells. However, biofunctionalization can also be understood in terms of a temporal or perma‐ nent biological function as a result of the materials modification [43, 47, 64, 65]. This modifi‐ cation enhances graphene biocompatibility, solubility, immobilization of other molecules, and/ or molecular recognition [47, 63].

The different sort of biomolecules could be attached to graphene materials by means of the covalent and non‐covalent interactions previously discussed. Notwithstanding, a couple of descriptive examples include the incorporation of nucleic acids and aptamers through π interactions owing to the aromatic character of the nucleobases; the easy assembly of phos‐ pholipid chains onto GO and rGO layers through hydrophobic interactions; the immobiliza‐ tion of proteins and enzymes through a combination of hydrophobic and π interactions, and in some cases aided with the contribution of electrostatic forces, in accordance with the amino acid residue composition [46, 51].

Covalently, functionalized graphene biosystems are mainly produced by amidation or esterification reactions of the carboxyl groups, with the aid of coupling reagents or by means of a specific chemical reaction mechanism, although functionalization also includes the epoxy ring opening and hydroxyl modification [46].

Proteins and polysaccharides have the advantage of possessing a rich chemical structure and/ or a large amount of functional groups.

#### **4.1. Amine and other functional groups Interactions with graphenic surfaces**

Amine functional groups are nucleophiles that possess a basic nitrogen atom with a lone pair. Basically, amines can be classified in primary, secondary, and tertiary, although some cyclic/ aromatic amines could be present in certain molecules (tryptophan and histidine in proteins). Nevertheless, amines for chemical graphene modification are frequently found in their primary form, as a pendant or terminated group, whether they are intrinsically present or introduced by chemical modification (amination process).

Amines can react with carboxylic acids of graphene through a condensation reaction forming a stable amide bond. In addition, aminated compounds react through nucleophilic substitution to epoxide groups, yielding an amine addition and a hydroxyl with the ring opening. Chitosan is an excellent example for a macromolecule that can be amide bonded to GO. Also, the N‐ terminal or amino acid side chain from a protein is commonly reacted through an amide bonding, as well as the lysine residue, or through nucleophilic addition to epoxides. None‐ theless, if the nucleophile is an alcohol (forming an ester with carboxyl groups), a thiol or a carboxylate anion, the variety of reactions becomes larger, just as the non‐covalent interactions, as in the case of proteins, such as keratin (KE) among others.

#### **4.2. Keratin functionalization of GO and rGO**

lecules as it has been seen with carbon nanotubes [47, 62]. The combination between graphene materials and biotechnology gives rise to new nano‐/bio‐interfaces [63] through different biofunctionalization process. Biofunctionalization is defined as the modification of a material by the attachment of biomolecules, ranging from organic groups to very large proteins or even cells. However, biofunctionalization can also be understood in terms of a temporal or perma‐ nent biological function as a result of the materials modification [43, 47, 64, 65]. This modifi‐ cation enhances graphene biocompatibility, solubility, immobilization of other molecules, and/

The different sort of biomolecules could be attached to graphene materials by means of the covalent and non‐covalent interactions previously discussed. Notwithstanding, a couple of descriptive examples include the incorporation of nucleic acids and aptamers through π interactions owing to the aromatic character of the nucleobases; the easy assembly of phos‐ pholipid chains onto GO and rGO layers through hydrophobic interactions; the immobiliza‐ tion of proteins and enzymes through a combination of hydrophobic and π interactions, and in some cases aided with the contribution of electrostatic forces, in accordance with the amino

Covalently, functionalized graphene biosystems are mainly produced by amidation or esterification reactions of the carboxyl groups, with the aid of coupling reagents or by means of a specific chemical reaction mechanism, although functionalization also includes the epoxy

Proteins and polysaccharides have the advantage of possessing a rich chemical structure and/

Amine functional groups are nucleophiles that possess a basic nitrogen atom with a lone pair. Basically, amines can be classified in primary, secondary, and tertiary, although some cyclic/ aromatic amines could be present in certain molecules (tryptophan and histidine in proteins). Nevertheless, amines for chemical graphene modification are frequently found in their primary form, as a pendant or terminated group, whether they are intrinsically present or

Amines can react with carboxylic acids of graphene through a condensation reaction forming a stable amide bond. In addition, aminated compounds react through nucleophilic substitution to epoxide groups, yielding an amine addition and a hydroxyl with the ring opening. Chitosan is an excellent example for a macromolecule that can be amide bonded to GO. Also, the N‐ terminal or amino acid side chain from a protein is commonly reacted through an amide bonding, as well as the lysine residue, or through nucleophilic addition to epoxides. None‐ theless, if the nucleophile is an alcohol (forming an ester with carboxyl groups), a thiol or a carboxylate anion, the variety of reactions becomes larger, just as the non‐covalent interactions,

**4.1. Amine and other functional groups Interactions with graphenic surfaces**

or molecular recognition [47, 63].

27014 Recent Advances in Graphene Research

acid residue composition [46, 51].

ring opening and hydroxyl modification [46].

introduced by chemical modification (amination process).

as in the case of proteins, such as keratin (KE) among others.

or a large amount of functional groups.

Although there are several reports on the usage of proteins, particularly enzymes, for the functionalization of graphene‐based materials [46], few works have been done on the surface modification of graphene through the attachment of structural fibrous proteins,<sup>1</sup> and, to the best of our knowledge, only a couple of reports have used keratin [66, 67], specifically, chicken feather keratin, for this purpose.

Fibrous proteins give resistance and flexibility to several biological structures and they possess a high concentration of hydrophobic residues. Keratin is a distinctive fibrous protein compared to collagen, elastin, and myofibril proteins, due to its high degree of disulfide bonds due to the presence of cysteine in its sequence [68]. Keratins are chemically stable and durable against hard environmental conditions; thus, it is found in hair, wool, horns, claws, and feathers.

Chicken feather keratins are small polypeptide chains with a molecular mass of about 10 kDa, and a predominantly hydrophobic character, although it also has a high amount of serine and some other polar or charged groups. It possesses seven cysteine residues, but lacks of lysine, tryptophan, histidine, and methionine. Keratin is therefore adequate for covalent and non‐ covalent binding with graphene materials.

#### *4.2.1. Covalent attachment of keratin onto GO through a KMnO4 redox system*

Redox reaction systems with Mn(III) have been successfully used for the polymerization of vinyl monomers and the grafting of certain macromolecules [66, 69–73]. Mn(III) is obtained from the combination of KMnO4 in acid media with the aid of a reducing reagents, like malic acid, as an electron donor for the reduction of Mn(VII) into Mn(IV) as manganese dioxide. Mn(IV) reacts with the reducing agent to produce the highly reactive Mn(III) ions along with free radicals. This specie can generate active free radicals (primary radicals R) with the reducing reagents, or in the presence of a macromolecule such as a protein, polysaccharide, or graphenic material, yielding a macroradical. These macroradicals may also be formed by the direct attack of primary radicals [74–76].

Keratin and graphene oxide possess different X–H functional groups that may act as electron donors through the abstraction of a hydrogen, thus becoming a radical. In the case of KE, these groups are –SH from cysteine, –OH from serine, threonine, and tyrosine, –CONH2 from glycine and asparagine, and –COOH from glutamic and aspartic acids. In particular, KE also possesses some disulfide bonds (–S–S–) due to the oxidation of thiols. These groups could also be transformed into free radicals with the abstraction of a hydrogen from a neighbor carbon atom [68, 77, 78].

Keratin polypeptide chains are suitable to be grafted onto graphene oxide through a redox reaction system [67]. In this process, keratin needed to be dissolved and dialyzed in order to be chemically attached. Then, under mild conditions in aqueous media and in the presence of sulfuric acid, malic acid, and potassium permanganate, the functionalization was conducted

<sup>1</sup> We are not considering here the case of nanocomposites where graphene materials act as a reinforcement for the polymer matrix.

at two different conditions varying the amount of H2SO4/malic acid employed. Keratin polypeptide chains were successfully attached to graphene oxide as it is confirmed by the infrared spectroscopy in **Figure 3a**. The graph shows well‐defined peaks from keratin (num‐ bers in red) and graphene oxide (numbers in blue) characteristic vibrations in the covalent bonded materials, and apparently, there is an amide and/or ester bond formation during the reaction, where KE/GO‐1 seems to have a higher degree of polypeptide incorporation. In this case, graphene oxide was preferred over reduced GO, due to its higher degree of reactive oxygen sites thus giving a higher yield of chemical bonding.

**Figure 3.** (a) FTIR spectra of graphene oxide, keratin, and the functionalization between them at two conditions (KE/GO‐1 and KE/GO‐2) and TEM images of (b) GO, (c) KE/GO‐1, and (d) KE/GO‐2.

Typical TEM image of GO with its intrinsic foldings and wrinkles is shown in **Figure 3b**, where even though the sheets are very large (a couple of microns), they have a very thin structure. **Figure 3c** and **d** clearly shows that some morphologies are different than those of the carbon layer sheets, due to the presence of the keratin chains on the surface of the material. To further demonstrate the polypeptide immobilization onto the carbon nanostructure surface, a line‐ profile chemical composition analysis is also shown in **Figure 4**, confirming the presence of S and N.

**Figure 4.** STEM‐HAADF images with EDX line scan (red) and chemical profile (below) of (a) GO, (b) KE/GO‐1, and (c) KE/GO‐2.

#### *4.2.2. Non‐covalent attachment of KE onto rGO*

at two different conditions varying the amount of H2SO4/malic acid employed. Keratin polypeptide chains were successfully attached to graphene oxide as it is confirmed by the infrared spectroscopy in **Figure 3a**. The graph shows well‐defined peaks from keratin (num‐ bers in red) and graphene oxide (numbers in blue) characteristic vibrations in the covalent bonded materials, and apparently, there is an amide and/or ester bond formation during the reaction, where KE/GO‐1 seems to have a higher degree of polypeptide incorporation. In this case, graphene oxide was preferred over reduced GO, due to its higher degree of reactive

**Figure 3.** (a) FTIR spectra of graphene oxide, keratin, and the functionalization between them at two conditions

Typical TEM image of GO with its intrinsic foldings and wrinkles is shown in **Figure 3b**, where even though the sheets are very large (a couple of microns), they have a very thin structure.

(KE/GO‐1 and KE/GO‐2) and TEM images of (b) GO, (c) KE/GO‐1, and (d) KE/GO‐2.

oxygen sites thus giving a higher yield of chemical bonding.

27216 Recent Advances in Graphene Research

Reduced graphene oxide possesses a higher degree of sp<sup>2</sup> hybridization than its graphene oxide counterpart, thus these π‐electron domains may interact with hydrophobic or aromatic molecules as well as with aromatic pendant groups, for a non‐covalent modification. The oxygen remaining moieties are also useful for an electrostatic‐ or hydrogen‐bonding interac‐ tions. In the case of the previously described keratin, the mainly hydrophobic nature of the protein allows its immobilization or adsorption onto the surface of the rGO without the aid of any coupling reagent, while the charged and polar residues could also contribute for the protein tethering forming electrostatic functions depending on the mixing conditions.

Following the procedure for the solubilization of KE, a non‐covalent attachment of the protein had been made an aqueous media by simply mixing a KE/rGO mass ratio of 1:1 for 3 h [67]. From the keratin sequence, two amino acids may exhibit:


Arginine has a particular strong interaction with negatively charged groups like carboxylic acid, due to its positively charged structure. This residue could be attached to some remaining carboxyl groups in rGO, as it has certainly done in the case of GO [79]. In addition, it is possible that some of the polar residues of the KE will be oriented toward the aqueous media. The effectively non‐covalent functionalization of KE onto rGO is indicated by the FTIR spectra of the modified material compared with its precursors (**Figure 5a**).

**Figure 5.** (a) FTIR spectra of reduced graphene oxide, keratin, and the functionalization between them at a mass ratio of 1:1 (KE/rGO‐1:1). TEM images of (b) rGO, (c) KE/rGO‐1:1, (d) STEM‐HAADF images with EDX line scan, and (e) chemical profile of KE/rGO‐1:1.

In the same way as with keratin/graphene oxide, transmission electron microscopy and line scan chemical composition had been performed in order to further confirm the functionaliza‐ tion. As can be seen from **Figure 5b** and **c**, a surface roughness besides those intrinsically found in graphene is appreciated when keratin has been immobilized. Also, chemical compositions clearly show the presence of N and S on the surface of the graphenic layers (**Figure 5d** and **e**).

Based on these results, keratin has an affinity to interact with rGO in a strong manner, practically wrapping the carbon material, even when there is no covalent linkage and no coupling reagents present in the mixture. But there still more future research in order to manipulate the degree or sites of interactions between these two systems

#### **4.3. Chitosan functionalization of GO and rGO**

**i.** π-stacking: phenylalanine and tyrosine onto the graphene basal plane.

alanine, onto both, the basal, and the edges of the layers.

glutamic, and aspartic acids.

27418 Recent Advances in Graphene Research

chemical profile of KE/rGO‐1:1.

**iv.** Strong electrostatic/hydrogen bonding: arginine.

the modified material compared with its precursors (**Figure 5a**).

**ii.** Hydrophobic interaction: proline, glycine valine, cysteine, leucine, isoleucine and

**iii.** Electrostatic and/or hydrogen bonding: serine, glutamine, threonine, asparagine,

Arginine has a particular strong interaction with negatively charged groups like carboxylic acid, due to its positively charged structure. This residue could be attached to some remaining carboxyl groups in rGO, as it has certainly done in the case of GO [79]. In addition, it is possible that some of the polar residues of the KE will be oriented toward the aqueous media. The effectively non‐covalent functionalization of KE onto rGO is indicated by the FTIR spectra of

**Figure 5.** (a) FTIR spectra of reduced graphene oxide, keratin, and the functionalization between them at a mass ratio of 1:1 (KE/rGO‐1:1). TEM images of (b) rGO, (c) KE/rGO‐1:1, (d) STEM‐HAADF images with EDX line scan, and (e)

In the same way as with keratin/graphene oxide, transmission electron microscopy and line scan chemical composition had been performed in order to further confirm the functionaliza‐ tion. As can be seen from **Figure 5b** and **c**, a surface roughness besides those intrinsically found GO and rGO have been functionalized through covalent and non‐covalent interactions with several biopolymers; one of them with great relevance due to its potential applications such as tissue engineering, cell adhesion, and food delivery is chitosan (CS) [46]. CS exhibits an unusual combination of properties such as biological activities, mechanical, and physical properties that make it the most important derivative of chitin, the second most abundant natural polysaccharide [80]. CS is composed of β‐(1,4)‐2‐amino‐2‐deoxy‐D‐glucose, and it is the result of the deacetylation of chitin (β‐(1,4)‐2‐acetoamido‐2‐deoxy‐D‐glucose) [21] which

**Figure 6.** (a) Schematic illustration of covalent interaction between GO and CS trough amide bond formed between – COOH moieties of GO and –NH<sup>2</sup> groups, present in CS. (b) Fluorescence micrographs of immunocytochemistry of cell nuclei of pre‐osteoblasts after 2 day culture on GO (left) and GO–CS–HAP (right). The graphs shows high fluorescence intensity zones as pre‐osteoblasts growth, GO–CS–HAP system has notorious development in comparison to GO. (Adapted from [91] with permission of The Royal Society of Chemistry).

can be found in crustacean shells and shellfish wastes, both of them by‐products from marine bioprocessing plants and probably one of the most important sources of CS [81]. CS is a polycationic polymer with two amino and two hydroxyl groups and a carbohydrate backbone similar to cellulose as can be seen in **Figure 6a**. Molecular weight (MW) and degree of deacetylation depend on the alkaline reaction conditions yielding a random distribution of acetylated/deacetylated units coupled in its chains [80]. These two features are chiefly responsible of the physicochemical properties of CS, principally impacting on its biological characteristics [82]. The amino functional groups in CS provide a versatile behavior when CS is in aqueous solution. At low pH (<6), the amino groups are protonated, thus giving CS a polycationic behavior. On the other hand, when pH is higher than 6.5, functional groups in CS are deprotonated, losing its charge and reducing its hydrophilicity [83]. CS is a biocompatible, biodegradable, non‐toxic material approved by the FDA; additionally, amino and hydroxyl groups present in its structure offers reactive sites for functionalization; for these reasons, it has been considered together with GO and rGO as a promising material to synthesize new materials for different applications such as biomedical, pharmaceutical, gene therapy, and waste water treatment to mention but a few [80].

#### *4.3.1. Covalent functionalization of GO and rGO with CS*

The covalent functionalization of GO and rGO accordingly with reports is made through the interaction between the oxidized moieties on surface of 2D carbon nanomaterial (GO, rGO), and the functional groups present in CS molecule (amino). GO functionalized with CS have been used to developed novel materials via amidation [84], esterification [85], or nucleophilic addition [86, 87]. Esterification was reported by Xu et al., in order to obtain CS grafted onto GO. First GO was dissolved in dimethyl formamide (DMF) in sonication, followed by the addition of thionyl chloride (SOCl2) which reacts with –COOH groups to obtain acyl‐chloride functionalized GO (GO–COCl). Finally, –NH2 groups present in chitosan react with GO–COCl via esterification [85]. On the other hand, abundant epoxy groups in GO can react with – NH2 at elevated temperatures following the nucleophilic addition mechanism similar to cross‐ linking mechanism in epoxy resin during curing [86]. In spite of the successful grafting of CS onto GO via esterification and nucleophilic addition, the most common route to functionalize GO with CS is amidation, which is done by the interaction of –COOH and –NH2 groups present in GO and CS, respectively [84]. GO functionalized with CS (GO–CS) has been used as a scaffold in different fields due to its interesting properties.

Recently, GO–CS system was used as a platform for drug delivery and sensing devices [88– 94]. Depan et al. reported a biomimetic mineralization route of conjugated material made up of GO and CS for hydroxyapatite (HAP) biomineralization. HAP as a major component in bones with important features, like great biocompatibility and bioactivity, has been utilized as a part of new multicomponent system (GO–CS–HAP) for bone tissue engineering. The functionalization was completed via covalent modification between highly decorated GO with –COOH moieties and CS with amino groups. Fast Fourier transform infrared spectroscopy (FTIR) in earlier reports of Depan and coworkers showed the formation of amide bond. Prior to functionalization, the characteristic signals of functional groups in CS were observed in 1636 and 1597 cm-1 attributed to the stretching of C–O (in –NHCO amide group) and bending of N– H (in –NH2), respectively, whereas for GO grafted with CS, –NH2, signal was shifted to a lower value, while amide group (–NHCO) was shifted to a larger value, this point out that functional groups present in CS and GO interacted to form covalent bonds [95].

can be found in crustacean shells and shellfish wastes, both of them by‐products from marine bioprocessing plants and probably one of the most important sources of CS [81]. CS is a polycationic polymer with two amino and two hydroxyl groups and a carbohydrate backbone similar to cellulose as can be seen in **Figure 6a**. Molecular weight (MW) and degree of deacetylation depend on the alkaline reaction conditions yielding a random distribution of acetylated/deacetylated units coupled in its chains [80]. These two features are chiefly responsible of the physicochemical properties of CS, principally impacting on its biological characteristics [82]. The amino functional groups in CS provide a versatile behavior when CS is in aqueous solution. At low pH (<6), the amino groups are protonated, thus giving CS a polycationic behavior. On the other hand, when pH is higher than 6.5, functional groups in CS are deprotonated, losing its charge and reducing its hydrophilicity [83]. CS is a biocompatible, biodegradable, non‐toxic material approved by the FDA; additionally, amino and hydroxyl groups present in its structure offers reactive sites for functionalization; for these reasons, it has been considered together with GO and rGO as a promising material to synthesize new materials for different applications such as biomedical, pharmaceutical, gene therapy, and

The covalent functionalization of GO and rGO accordingly with reports is made through the interaction between the oxidized moieties on surface of 2D carbon nanomaterial (GO, rGO), and the functional groups present in CS molecule (amino). GO functionalized with CS have been used to developed novel materials via amidation [84], esterification [85], or nucleophilic addition [86, 87]. Esterification was reported by Xu et al., in order to obtain CS grafted onto GO. First GO was dissolved in dimethyl formamide (DMF) in sonication, followed by the addition of thionyl chloride (SOCl2) which reacts with –COOH groups to obtain acyl‐chloride functionalized GO (GO–COCl). Finally, –NH2 groups present in chitosan react with GO–COCl via esterification [85]. On the other hand, abundant epoxy groups in GO can react with – NH2 at elevated temperatures following the nucleophilic addition mechanism similar to cross‐ linking mechanism in epoxy resin during curing [86]. In spite of the successful grafting of CS onto GO via esterification and nucleophilic addition, the most common route to functionalize GO with CS is amidation, which is done by the interaction of –COOH and –NH2 groups present in GO and CS, respectively [84]. GO functionalized with CS (GO–CS) has been used as a

Recently, GO–CS system was used as a platform for drug delivery and sensing devices [88– 94]. Depan et al. reported a biomimetic mineralization route of conjugated material made up of GO and CS for hydroxyapatite (HAP) biomineralization. HAP as a major component in bones with important features, like great biocompatibility and bioactivity, has been utilized as a part of new multicomponent system (GO–CS–HAP) for bone tissue engineering. The functionalization was completed via covalent modification between highly decorated GO with –COOH moieties and CS with amino groups. Fast Fourier transform infrared spectroscopy (FTIR) in earlier reports of Depan and coworkers showed the formation of amide bond. Prior to functionalization, the characteristic signals of functional groups in CS were observed in 1636

waste water treatment to mention but a few [80].

27620 Recent Advances in Graphene Research

*4.3.1. Covalent functionalization of GO and rGO with CS*

scaffold in different fields due to its interesting properties.

On the other hand, HAP nucleation on GO–CS was corroborated by FTIR, X‐ray diffraction (XRD) and scanning electron microscopy (SEM). A fine dispersion of HAP was observed, and it was attributed to the electrostatic interactions with GO–CS system which promoted the HAP growth. The adhesion and proliferation of osteoblasts on CS–GO–HAP system was observed in order to evaluate their distribution and attachment, both features are necessary for tissue formation. Samples were analyzed after 1 and 24 h of cells incubation, fluorescence microscopy with DAPI showed a synergistic effect in mineralization of HAP in GO–CS–HAP system as can be seen in **Figure 6b**, attributed to interaction of CS and HAP. Another study about GO functionalized with CS was reported by Mohandes et al., using GO–CS–HAP nanocomposite as a scaffold to grow apatite (AP) due to its high porosity and interconnectivity, both features in nanocomposite are important for the cell attachment and new bone formation. Additionally, thermal stability of GO and GO–CS–HAP was evaluated by termogravimetric analysis which suggests that the loss of mass in GO of approximately 25 wt% is attributed to functional groups, while GO–CS–HAP shows stability at 310°C where the weight loss was important. New AP was formed in GO–CS–HAP nanocomposite after 14 days socked in simulated body fluid (SBF). Grain size of 20–15 nm was observed by scanning electron microscopy (SEM) [96].

Due to its haemostatic properties and safe excretion CS were used to build microneedle arrays as transdermal preloaded drug delivery nanocomposite. Fluorescein sodium (FS) was attached by non‐covalent interactions to CS–rGO at the same time reduction of GO was carried out. rGO–CS–FL array shows better mechanical properties and drug release in comparison to pristine CS. Nanocomposite with 2% of rGO achieved maximum release (91%) of available drug in 48 h, while CS–FL system only achieved around 33% according to the report [94]. Other approaches in drug delivery were reported by Rana et al. and Bao et al. The first one reported the use of ibuprofen (IBU) and 5‐fluoracil (5‐FU) drugs loaded in rGO–CS system via simple physisorption, and additionally, they evaluated the cytotoxicity and cell viability of GO–CS– IBU and GO–CS–5‐FU systems over CEM and MCF‐7 cancer cells [89]. The second one reported a novel nanocarrier of campthotecin (CPT), an inhibitor of topoisomerase I and anticancer drug. Cell viability of 80% was found in methylthiazoloterazolium (MTT) assay. Additionally, π–π stacking and hydrophobic interactions between GO–CS and CPT allow high load (20 wt %) of drug in nanocarrier [90].

Other approaches regarding removal of contaminants in water have been done. Removal of Cr(IV) from simulated wastewater with magnetic GO–CS ionic liquid (MCGO‐IL) was reported with a maximum of 143.35 mg/g of adsorption capacity (*Qmax*), this was described by the Langmuir isotherm. A removal mechanism was proposed. Briefly, first, the electrostatic attraction between Cr(IV) with –OH<sup>2</sup> + and –NH3 + takes place, second, the cooperation between ionic liquid, functional groups of GO–CS and Cr(IV) occurs and, third, the reduction of Cr(IV) to Cr(III) assisted by π electron of carboxylic six‐membered ring in MCGO‐IL is achieved [97]. On another report magnetic CS nanoparticles (MCGO) acting as magnetic bioadsorbent shown to have excellent properties. Magnetic biosorbents can be recovered, easily separated and CS adsorption is better in view of its high surface area along functional groups present [98]. According to Fuschine dye adsorption experiments, pH 5.5 is the optimal value to carry out the removal by MCGO. Moreover, the calculated and experimentally values of adsorption capacity were close, which confirms the pseudo‐second‐order kinetic model of adsorption proposed. Additionally, the studies over recycling of MCGO as adsorbent shows only slightly decay adsorption capacity of MCGO after the fifth cycle. These results suggested that this method can be used to yield graphene‐absorbents in commercial scale. In **Figure 7**, the synthesis of MCGO is briefly represented by a schematic diagram [99].

**Figure 7.** In the synthesis of MCGO, first magnetic particles are attached to CS and second trough amide bond forma‐ tion GO surface is functionalized. (Reproduced from [99] with permission of The Royal Society of Chemistry).

On the other hand, antifouling membranes were developed by depositing a thin film of GO– CS on polyamide surface to form brackish water thin‐film membrane for reverse osmosis (BWTFC‐RO). In this approach, the BWTFC‐RO with high content of GO shows better antifouling response because of the negative moieties in GO–CS increase hydrophilicity, which can reduced the interfacial energy among membrane surface and water and thus membrane fouling resistance was incremented. A water contact angle of 63.68° was measured for unmodified membrane, while membrane with GO–CS system has a contact angle of 19.13° [100]. In addition, in order to remove Uranium U(vi) released in wastewater, GO–CS systems grafted via covalent were done. Different probes of adsorption and desorption were made having *Qmax* = 225.78 mg/g at pH 4.0 and fitted to a Langmuir model. The removal of U(VI) can be attributed to interactions between amine groups and the metal; this was corroborated by FTIR where –NHCO– (at 1530 and 1630 cm-1) and –NH2 (at 1420 cm-1) peaks decreased when the U is adsorbed [101].

to have excellent properties. Magnetic biosorbents can be recovered, easily separated and CS adsorption is better in view of its high surface area along functional groups present [98]. According to Fuschine dye adsorption experiments, pH 5.5 is the optimal value to carry out the removal by MCGO. Moreover, the calculated and experimentally values of adsorption capacity were close, which confirms the pseudo‐second‐order kinetic model of adsorption proposed. Additionally, the studies over recycling of MCGO as adsorbent shows only slightly decay adsorption capacity of MCGO after the fifth cycle. These results suggested that this method can be used to yield graphene‐absorbents in commercial scale. In **Figure 7**, the

**Figure 7.** In the synthesis of MCGO, first magnetic particles are attached to CS and second trough amide bond forma‐ tion GO surface is functionalized. (Reproduced from [99] with permission of The Royal Society of Chemistry).

On the other hand, antifouling membranes were developed by depositing a thin film of GO– CS on polyamide surface to form brackish water thin‐film membrane for reverse osmosis (BWTFC‐RO). In this approach, the BWTFC‐RO with high content of GO shows better antifouling response because of the negative moieties in GO–CS increase hydrophilicity, which can reduced the interfacial energy among membrane surface and water and thus membrane fouling resistance was incremented. A water contact angle of 63.68° was measured for unmodified membrane, while membrane with GO–CS system has a contact angle of 19.13° [100]. In addition, in order to remove Uranium U(vi) released in wastewater, GO–CS systems grafted via covalent were done. Different probes of adsorption and desorption were made having *Qmax* = 225.78 mg/g at pH 4.0 and fitted to a Langmuir model. The removal of U(VI) can

synthesis of MCGO is briefly represented by a schematic diagram [99].

27822 Recent Advances in Graphene Research

Owing to the large surface‐to‐volume ratio and good electrochemical activity of GO and the compatibility of CS, GO–CS nanocomposite enhances transfer electron and DNA immobili‐ zation between electrode surface and DNA in electrochemical biosensor for typhoid diagnosis. This was reported using glutaraldehyde (GA) as a bridge between the GO–CS nanocomposite film on indium tin oxide (ITO) electrode and *Salmonella typhi,* specifically its 5'‐amine labeled single‐stranded DNA (ssDNA). GO–CS–ssDNA–ITO bioelectrode shows improvements in detection limits in comparison to other studies [88], and this is attributed to the excellent conductivity and small band gap from biomolecules; the bioelectrode shows the limits of 100 fM in buffer solution and shelf life of 15 days with a 100% recovery. Otherwise, *Candida rugose* lipase (CRL) immobilization was reported by magnetite particles (Fe3O4) assembled in GO–CS system. GO–CS–Fe3O4 nanocomposite was synthetized from ferric chloride hexahy‐ drate (FeCl3·6H2O) and 1,6‐hexadiamine by solvothermal reaction. The nanocomposite needs only one step to be synthetized which is important when large‐scale production can be possible. In this study, three strategies were used to immobilize CRL. Firstly, the electrostatic adsorption by GO–CS–Fe3O4, secondly, covalent bonding with GO–CS–Fe3O4–GA and thirdly metal‐chelate ligand anchorage on GO–CS–Fe3O4–IDA‐CU. The activity recovery was meas‐ ured for three systems and the adsorption results showed the best protein CRL immobilization for GO–CS–Fe3O4–IDA‐CU followed by GO–CS–Fe3O4–GA and GO–CS–Fe3O4 systems with 65.5, 60.2, and 57.2% of activity recovery, respectively. GO–CS–Fe3O4–IDA‐CU leads the activity recovery due to exposition of functional groups thus giving low diffuse resistance. On another approach, the biocompatibility of mouse mesenchymalstem C310T1/2 cells adhered to GO–CS covalent attached system was evaluated by DAPI fluorescence, tracing the nucleus of cells and low cytotoxicity was further reported. Additionally, cell viability after 24 h shown low death cell indications. This can be traduced in an acceptable biocompatibility of GO–CS system [93].

Another emergent field of study of GO–CS is catalysis. Green nanocomposite based on GO– CS have demonstrated high thermal stability (165°C) in differential scanning calorimetry (DSC) in comparison with pure CS (118–119°C), both were used as a support for novel catalyst. In this research, average size distribution of 50 nm was measured by atomic force microscopy (AFM) for GO–CS nanocomposite synthesized in free‐solvent conditions. Efficient synthesis of 2,4,5‐trisubstituied‐1H‐imidazoles were done, GO–CS in catalytic amount was combined with benzyl 2 or benzoin 1, benzaldehyde 3 and ammonium acetate at 120°C that shows great thermal stability of GO–CS system [102]. Another novel catalyst was developed for reduction of aromatic nitroarenes and azo dye degradation [103]. Silver (AgNPs) and gold (AuNPs) nanoparticles were attached onto GO–CS system in order to avoid aggregations of either Ag or Au nanoparticles which affect its catalytic activity. Both GO–CS‐AgNPs and GO–CS‐AuNPs catalysts were obtained in similar process using 0.1 M solution of silver nitrate (AgNO3) and tetrachloroauric (III) acid hydrate respectively in a solution of sodium borohydride (NaBH4). Particles of 20 nm for AgNPs and 5 nm for AuNPs were observed by high resolution trans‐ mission electron microscopy (HR‐TEM) that demonstrates homogeneous deposition of particles. The excellent catalytic activity and selective reduction of nitroarenes was mainly attributed to the high surface area of GO–CS system which improves adsorption of organic substrates, and to the presence of –NH2 and –OH groups in GO–CS system which might assist the stabilization and attraction of particles toward the catalytic sites, because of the combina‐ tion of hydrophobic–hydrophilic nature present in GO and CS.

#### *4.3.1.1. Covalent attachment of chitosan onto GO through a KMnO4 redox system*

Functionalization of GO with CS by redox system with KMnO4, H2SO4, and malic acid also has been reported [75]. In this approach, covalent attachment of CS onto GO was done varying temperature conditions, this shows influence over morphology and features of the final material produced. A better dispersion behavior was obtained as a result of chemical modifi‐ cation (grafting) originated from the interaction of GO and CS functional groups. As can be seen in **Figure 8a**, well‐exfoliated graphene oxide sheet alike a thin film and wrinkled surface is shown. Additionally, TEM images of CS grafted on GO exhibit the differences on morphol‐ ogy acquired in different conditions **Figure 8b**–**d**. At the lowest temperature, GO sheet is completely covered by CS. However, as the temperature is increase (75–80°C), CS only partially covers the GO sheets, and finally for the highest temperature, the GO–CS system shows great differences in its morphology, like a scrolled material. This can be attributable to the loss of free water in CS that increases as the temperature does, impacting on the amount of water and hydrogen bonds formed during grafting. Dispersion behavior of different GO–CS produced on water and hexane. A water stable dispersion even 24 h after the sonication process was

**Figure 8.** TEM images of (a) GO; (b) GO–CS obtained at 55–60°C; (c) GO–CS obtained at 75–80°C; (d) GO–CS obtained at 95–100°C; (e) FTIR spectra of (1) graphite; (2) graphite oxide; (3)GO; (4) GO–CS obtained at 55–60°C; (5) GO–CS ob‐ tained at 75–80°C; (6) GO–CS obtained at 95–100°C and (7) CS.

observed, unlike in hexane non‐polar solvent which shows poor dispersion and rapid precipitation of GO–CS. In addition, AFM measurements show a roughness between 3 and 6 nm for the GO–CS which is the result of bundles of CS chains grafted on GO that can be observed as dark dense zones in TEM images.

FTIR analysis was done in order to provide evidence about the chemical modification of graphite, and grafting of CS onto GO. The appearance of new signals respecting the interaction of moieties presents in both GO and CS can be clearly seen in the **Figure 8e**. Signal from new amides or carbamate esters formed during grafting, this band appears in 1535 cm-1 attributed to the combination of *ν*(C–N) and *δ*(CNH). Both, the band at 1156 cm-1 corresponding to *νa*(C– O–C) and signal at 899 cm-1 for C–O–C were detected, they are related with glycosidic linkage. The latter are typical in chitosan, whereas after grafting they are shifted to 881 cm-1 in GO–CS systems that show the modifications of C–O–C bond vibrations and the interaction of GO–CS hybrids. In addition, Raman spectroscopy and energy dispersive X‐ray spectroscopy (EDS) characterization techniques were used in order to corroborate the grafting of CS onto GO.

Finally based on previous works [99, 100] and evidence obtained in FTIR measurements, few reactions can be proposed for the novel bonds as a result of grafting of CS. As can be seen in Eqs. (3)–(5), first, linkage can be done by carbonyl moieties ((3) and (4)) and second with breaking of epoxy groups (5). Despite that other reactions can be carried out but evidence provided by FTIR points out just the aforementioned.

$$\frac{1}{2}GO-COOH + CS-NH\_2 \xrightarrow[H^+, \text{ nuclei ac.}]{KM nO\_4} \begin{array}{c} GO-COOH-CS + Subproduct \end{array} \tag{3}$$

$$\frac{1}{2}GO-COOH + CS-OH \xrightarrow[H^{+}]{KMnO\_{4}} \text{CaO}-COO-CS + Subproducts \tag{4}$$

Subproducts = HO, RH, ROH (R = Radical from malic acid)

mission electron microscopy (HR‐TEM) that demonstrates homogeneous deposition of particles. The excellent catalytic activity and selective reduction of nitroarenes was mainly attributed to the high surface area of GO–CS system which improves adsorption of organic substrates, and to the presence of –NH2 and –OH groups in GO–CS system which might assist the stabilization and attraction of particles toward the catalytic sites, because of the combina‐

Functionalization of GO with CS by redox system with KMnO4, H2SO4, and malic acid also has been reported [75]. In this approach, covalent attachment of CS onto GO was done varying temperature conditions, this shows influence over morphology and features of the final material produced. A better dispersion behavior was obtained as a result of chemical modifi‐ cation (grafting) originated from the interaction of GO and CS functional groups. As can be seen in **Figure 8a**, well‐exfoliated graphene oxide sheet alike a thin film and wrinkled surface is shown. Additionally, TEM images of CS grafted on GO exhibit the differences on morphol‐ ogy acquired in different conditions **Figure 8b**–**d**. At the lowest temperature, GO sheet is completely covered by CS. However, as the temperature is increase (75–80°C), CS only partially covers the GO sheets, and finally for the highest temperature, the GO–CS system shows great differences in its morphology, like a scrolled material. This can be attributable to the loss of free water in CS that increases as the temperature does, impacting on the amount of water and hydrogen bonds formed during grafting. Dispersion behavior of different GO–CS produced on water and hexane. A water stable dispersion even 24 h after the sonication process was

**Figure 8.** TEM images of (a) GO; (b) GO–CS obtained at 55–60°C; (c) GO–CS obtained at 75–80°C; (d) GO–CS obtained at 95–100°C; (e) FTIR spectra of (1) graphite; (2) graphite oxide; (3)GO; (4) GO–CS obtained at 55–60°C; (5) GO–CS ob‐

tained at 75–80°C; (6) GO–CS obtained at 95–100°C and (7) CS.

tion of hydrophobic–hydrophilic nature present in GO and CS.

28024 Recent Advances in Graphene Research

*4.3.1.1. Covalent attachment of chitosan onto GO through a KMnO4 redox system*

#### *4.3.2. Non‐covalent functionalization of GO and rGO with CS*

Non‐covalent functionalization of GO and rGO has been done through different ways such as hydrogen bonding, electrostatic interactions, Van der Waals forces, and ionic interactions. Electrostatic forces in functionalized GO have been reported in order to develop applications such as hemolytic activity and nanocomposites [104–107]. Li et al. reported removal of single or multi‐system ions based on Cu (II), Pb (II) and Cd (II). GO was functionalized with a sulfhydryl compound and CS resulting in CS–GO–SH adsorbent material, basically due to the affinity of CS, known as a cationic polymer with plenty –OH and –NH2 groups and negative charged in GO–SH complexes. In this work, ultrasonication was applied to yield GO–CS–SH nanocomposite; sulfhydryl groups were previously grafted on GO reacting in stirring with 4‐ aminothiophenol. Adsorption tests result in *Qmax* of 235, 226, and 117 mg/g for Cu (II), Pb (II), and Cd (II), respectively, from a starting concentration of 250 mg/L, and thus, a higher adsorption was obtained in comparison with other results reported with GO and CS as adsorbent. This suggests a synergistic effect originated by increasing the specific surface area and space between CS and GO–SH. Moreover, operational factors such as pH, adsorbent dosage, and temperature play important roles in results [107]. Other approach about electro‐ static interactions to functionalize GO was reported by Liao et al., they discussed the effect of GO size and coated GO over hemolytic activity. GO obtained in different exfoliation param‐ eters were tested by methylthiazolyldiphenyl‐tetrazolium bromide (MTT) and water‐soluble tetrazolium salt (ST‐8) to show blood compatibility. The functionalization of GO with CS reveals lower hemolytic activity in comparison of GO sheets; this can be attributable to disruption of red blood cells membrane (RBC) as a result of the strong electrostatic interaction between oxygenated functional groups of GO sheets with negative charge and positively charged phosphatidylcholine lipids of RBC outer membrane [104]. Unlike a traditional CS polymer with –NH2 groups which only become charged in acidic media, quaternized CS (QCS) is a polymer attached with quaternary ammonium groups which confers a cationic polyelec‐ trolyte permanently charged feature [108]. Consequently, cellulosic paper fibers can be cover with a positively charged coating made of QCS which attract the negatively charged GO sheets. So QCS can be used as a "glue" linking cellulose fibers to GO via electrostatic interactions. This was reported by Ling and co‐workers in order to fabricate composite paper in addition to load GO with AuNPs and its subsequent reduction by hydrogen iodide vapor. The final material shows an outstanding conductivity of 831 S/m [105]. Another interesting applications recently reported are Janus GO–CS membrane; in this work; non‐covalently assembly GO–CS mem‐ brane was used as a support of poly(styrene) (PS) and poly (N,N‐dimethyl methacrylate) (PDMAEMA) in upper and lower membrane surface; respectively. Photografting and photo‐ polymerization were used to yield Janus membranes a possible material for applications such as sensing or catalyst as a result of its versatility [109].

On the other hand, another route of non‐covalent functionalization of GO is through hydrogen bonding which has been reported to develop novel nanocomposites. Recently; a facile GO/CS conducting biocompatible hydrogel production method by extrusion printing was reported. CS and lactic acid (LA) as matrix and GO as reinforcement material were used, and L929 cells were cultured in diluted GO–CS–LA dispersions and were compared with GO–CS–LA nanocomposites.

Another via of non‐covalent functionalization of GO is the formation of hydrogen bond with CS which has been reported to develop novel nanocomposites. As a result of homogeneous distribution as well as excellent mechanical properties achieved in GO–CS nanocomposites originated from non‐covalent interactions, high‐performance nanocomposites have been prepared [110]. High mechanical properties exhibited in GO–CS nanocomposite can be used for drug delivery, GO–CS nanocomposites offer a platform to yield a tunable control through pH transdermal system for drug release [111]. Recently, another approach was done using CS and lactic acid (LA) as matrix and GO as reinforcement material by extrusion printing. Due to the relevance of the morphology, dimensions, shape, and biocompatibility in materials for tissue engineering, GO–CS was used to yield conducting biocompatible hydrogel. FTIR shows strong interaction by hydrogen bond between GO–CS–LA. Additionally L929 cells were cultured in composites; first diluted GO–CS–LA dispersions were used to note cell growth. Cell growth showed an increment of cell density 10–15 times (45 ± 4E4  cells cm-2 and 1.5% dead cells) in comparison to the original amount. No cell showed inclusion of dark material in either cytoplasm or any organelle; this was observed by bright field microscope. Second for GO–CS– LA films and scaffolds; no toxicity or changes on migration were observed in cells; moreover, mechanical properties were enhanced by the addition of GO having increments in tensile strength of 320% and 162% for dry and wet state for GO–CS–LA nanocomposite with only 3 wt% of GO added [112].

such as hemolytic activity and nanocomposites [104–107]. Li et al. reported removal of single or multi‐system ions based on Cu (II), Pb (II) and Cd (II). GO was functionalized with a sulfhydryl compound and CS resulting in CS–GO–SH adsorbent material, basically due to the affinity of CS, known as a cationic polymer with plenty –OH and –NH2 groups and negative charged in GO–SH complexes. In this work, ultrasonication was applied to yield GO–CS–SH nanocomposite; sulfhydryl groups were previously grafted on GO reacting in stirring with 4‐ aminothiophenol. Adsorption tests result in *Qmax* of 235, 226, and 117 mg/g for Cu (II), Pb (II), and Cd (II), respectively, from a starting concentration of 250 mg/L, and thus, a higher adsorption was obtained in comparison with other results reported with GO and CS as adsorbent. This suggests a synergistic effect originated by increasing the specific surface area and space between CS and GO–SH. Moreover, operational factors such as pH, adsorbent dosage, and temperature play important roles in results [107]. Other approach about electro‐ static interactions to functionalize GO was reported by Liao et al., they discussed the effect of GO size and coated GO over hemolytic activity. GO obtained in different exfoliation param‐ eters were tested by methylthiazolyldiphenyl‐tetrazolium bromide (MTT) and water‐soluble tetrazolium salt (ST‐8) to show blood compatibility. The functionalization of GO with CS reveals lower hemolytic activity in comparison of GO sheets; this can be attributable to disruption of red blood cells membrane (RBC) as a result of the strong electrostatic interaction between oxygenated functional groups of GO sheets with negative charge and positively charged phosphatidylcholine lipids of RBC outer membrane [104]. Unlike a traditional CS polymer with –NH2 groups which only become charged in acidic media, quaternized CS (QCS) is a polymer attached with quaternary ammonium groups which confers a cationic polyelec‐ trolyte permanently charged feature [108]. Consequently, cellulosic paper fibers can be cover with a positively charged coating made of QCS which attract the negatively charged GO sheets. So QCS can be used as a "glue" linking cellulose fibers to GO via electrostatic interactions. This was reported by Ling and co‐workers in order to fabricate composite paper in addition to load GO with AuNPs and its subsequent reduction by hydrogen iodide vapor. The final material shows an outstanding conductivity of 831 S/m [105]. Another interesting applications recently reported are Janus GO–CS membrane; in this work; non‐covalently assembly GO–CS mem‐ brane was used as a support of poly(styrene) (PS) and poly (N,N‐dimethyl methacrylate) (PDMAEMA) in upper and lower membrane surface; respectively. Photografting and photo‐ polymerization were used to yield Janus membranes a possible material for applications such

as sensing or catalyst as a result of its versatility [109].

nanocomposites.

28226 Recent Advances in Graphene Research

On the other hand, another route of non‐covalent functionalization of GO is through hydrogen bonding which has been reported to develop novel nanocomposites. Recently; a facile GO/CS conducting biocompatible hydrogel production method by extrusion printing was reported. CS and lactic acid (LA) as matrix and GO as reinforcement material were used, and L929 cells were cultured in diluted GO–CS–LA dispersions and were compared with GO–CS–LA

Another via of non‐covalent functionalization of GO is the formation of hydrogen bond with CS which has been reported to develop novel nanocomposites. As a result of homogeneous distribution as well as excellent mechanical properties achieved in GO–CS nanocomposites Due to the versatility and interesting capacity of self‐healing against damage, polymeric gels have attracted great attention. GO and CS hydrogels were prepared by non‐covalent interac‐ tions; in this regard, GO can be considered as a 2D cross‐linker as a result of functional groups present in both faces of GO sheets. Electrostatic and hydrogen bond are the predominant interaction in this kind of materials according with the report [113]. Water, CS, and GO are the molecules present in hydrogel, interacting mainly by electrostatic forces when mixed at room temperature. Particularly CS shows a compact state owing to hydrogen bond present in its structure, thus restricting the interaction with GO, this is diminished with the increase of temperature of hydrogel preparation according with the methodology. This kind of materials can be applied in fields such as biomaterials, absorbents, and so on. For example, recently heparin/GO–CS hybrid hydrogel (Hep‐GO–CS) were synthetized for bilirubin adsorption. Adsorption measurements in phosphate buffer solution (PBS) show for Hep‐GO–CS four times adsorption capacity than CS hydrogel; this can be attributable to GO added to hydrogel. Additionally, this material exhibits good hemocompatibility and low degree of hemolysis in the tests realized [114]. On the other hand, other applications for GO–CS system based on both electrostatic interactions and hydrogen bond have been reported in fields such as nanocom‐ posites for electrochemical biosensors [115], pathogen agent detection [116], membranes in microbial fuel cells [117], and cell growth [118]. Additionally, preparation of poly(vinyl alcohol) PVA–CS nanocomposite reinforced by rGO was reported; simultaneously, reduction and functionalization were done and synergistic effect by the interaction of rGO–CS–PVA was demonstrated with high‐performance nanocomposite obtained. CS acts as a bridge between rGO and PVA enhancing the quantity of formed hydrogen bonds, which improved mechanical properties taking for instance an increase of 40% in value of tensile strength in comparison of CS–PVA polymer [119].

As can be seen in **Figure 9**, multifunctional rGO magnetic nanosheet functionalized via non‐ covalent with chitosan (rGOMCS) was developed for *Pseudomonas aeruginosa* (BCRC 10303) and *Staphylococcus aureus* (BCRC 1045) detection in aqueous suspension and mouse blood. Owing to its inherent fluorescence properties and high surface area, rGO as a support to magnetic nanoparticles enhanced detection of bacteria in fluorescence spectroscopy and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). On one hand, fluorescence as bacteria detection method has been used widely, in spite of interference created by biomolecules in biological samples, furthermore external factors, among others make it a complicated task. On the other hand, great influence of CS and complementary effect of functional groups on graphene attached on rGOMCS was observed over bacteria in MALDI‐ MS. This allowed different non‐covalent interactions (hydrogen bond, hydrophobic and electrostatic interactions, acid‐base interactions, π–π interactions and polar functional groups interactions) together with pathogen agents reflecting high adsorption. Additionally, Abdel‐ hamid et al. [116] reported thermodynamic analysis which reveals better affinity of CS to BCRC 1045 as a result of increment on peptidoglycan content and consequently better sensitivity for this pathogen agent.

**Figure 9.** (a) Schematic diagram of the assembly of rGOMCS; (b) TEM images of rGO functionalized with magnetic particles and CS (c) Interaction between bacteria and rGOMCS. (Adapted from [116] with permission of The Royal So‐ ciety of Chemistry).

ζ potential measurements reveal the influence of ionic interactions between rGO–CS solutions, another via of non‐covalent functionalization of rGO. First, notable differences in the responses of GO–CS solutions were observed only changing the order of aggregation of the components. In the **Figure 10**, as can be seen when GO is added to CS solution the GO sheets are wrapped rapidly by ionic interactions with considerable amount of CS (ζ potential of +40 mV); however, if the GO is added greatly dispersed in solution to CS forms a "bridging" flocculation as a result of attachment of two or more GO sheets to polymer chain (ζ potential of +8 mV). Second, one can take advantage that after GO reduction by L‐AA in the presence of CS was done; the remaining functional groups on rGO and –NH2 groups in CS are still available for non‐covalent functionalization. This offers the possibility to control and change behavior of rGO–CS solutions inasmuch as pH is changed. ζ potential measurements show both behaviors on pH 6 (+33 mV) and at pH 7 (-9 mV) for a stable suspension and formation of agglomerates, respectively. It can be explained because at low pH, –NH2 groups are protonated creating a good environment for electrostatic repulsion between rGO sheets. In spite of that, if the pH is increased the intermolecular repulsion tend to decrease, originated by the deprotonation of NH2 groups of CS [60]. Ko et al., performed a nanocomposite for growth of *Escherichia coli* bacteria. They carried out the simultaneous reduction and non‐covalent functionalization of GO in acid media solutions, attributing to remaining protonated –NH3 <sup>+</sup> groups on CS the contributions for the well‐dispersed rGO–CS system. Moreover, enhanced antibacterial activity for *E. coli* was detected [120]. In **Table 2** are summarized some applications for both GO-CS and rGO-CS with covalent and non-covalent interactions.

and *Staphylococcus aureus* (BCRC 1045) detection in aqueous suspension and mouse blood. Owing to its inherent fluorescence properties and high surface area, rGO as a support to magnetic nanoparticles enhanced detection of bacteria in fluorescence spectroscopy and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). On one hand, fluorescence as bacteria detection method has been used widely, in spite of interference created by biomolecules in biological samples, furthermore external factors, among others make it a complicated task. On the other hand, great influence of CS and complementary effect of functional groups on graphene attached on rGOMCS was observed over bacteria in MALDI‐ MS. This allowed different non‐covalent interactions (hydrogen bond, hydrophobic and electrostatic interactions, acid‐base interactions, π–π interactions and polar functional groups interactions) together with pathogen agents reflecting high adsorption. Additionally, Abdel‐ hamid et al. [116] reported thermodynamic analysis which reveals better affinity of CS to BCRC 1045 as a result of increment on peptidoglycan content and consequently better sensitivity for

**Figure 9.** (a) Schematic diagram of the assembly of rGOMCS; (b) TEM images of rGO functionalized with magnetic particles and CS (c) Interaction between bacteria and rGOMCS. (Adapted from [116] with permission of The Royal So‐

ζ potential measurements reveal the influence of ionic interactions between rGO–CS solutions, another via of non‐covalent functionalization of rGO. First, notable differences in the responses of GO–CS solutions were observed only changing the order of aggregation of the components.

this pathogen agent.

28428 Recent Advances in Graphene Research

ciety of Chemistry).

**Figure 10.** (a) Schematic diagram of GO–CS suspensions behavior as a result of the variation in the order of the addi‐ tion of components. (b) Measurements taken by atomic force microscopy (AFM) of rGO extracted from rGO–CS sus‐ pension and deposited in cleaved mica. (Adapted with permission from [60]. Copyright 2016 American Chemical Society).



**Table 2.** Functionalization of GO and rGO by CS and its progress in different fields of application.
