Fullerenes, Graphenes and Nanotubes: A

CHAPTER

Hybridized graphene nanomaterials for drug delivery, cyto-compatibility, and electrochemical biosensor application

10

Mohana Marimuthu1, Pramod K. Avti2, Velayutham Ravichandiran3 and Murugan Veerapandian4 1

Alagappa University, Karaikudi, Tamil Nadu, India 2Postgraduate Institute of Medical Education and Research, Chandigarh, Punjab, India 3National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India 4CSIR-Central Electrochemical Research Institute, Karaikudi, Tamil Nadu, India

CHAPTER OUTLINE 10.1 10.2 10.3 10.4

Introduction ...................................................................................................375 Physical and Chemical Properties of Graphene-Based Materials .......................377 Functionalized Graphene-Derivatives for Drug Delivery and Cyto-Compatibility ...379 Electrochemical Biosensors Based on Graphene Hybrid Nanomaterials .............383 10.4.1 Metabolic Biomarker Detection ....................................................385 10.4.2 Sensing of Infectious Agent.........................................................392 10.4.3 Sensing of Active Pharmaceuticals/Nutraceuticals.........................396 10.5 Summary and Future Perspective ....................................................................398 Acknowledgments ...................................................................................................399 References .............................................................................................................399 Further Reading ......................................................................................................411

10.1 INTRODUCTION Integration of novel nanostructures with improved properties for device development is an attractive prospect for materialists and engineers. Graphene derivatives

Volume VI: Carbon (Nanotube, Fullerene, Graphene) Nanomaterials.

Fullerenes, Graphenes and Nanotubes: A Pharmaceutical Approach. DOI: http://dx.doi.org/10.1016/B978-0-12-813691-1.00010-5 © 2018 Elsevier Inc. All rights reserved.

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376

CHAPTER 10 Hybridized graphene nanomaterials for drug delivery

are the novel two-dimensional (2D) nanomaterial which has recently attracted a great deal of consideration due to its variety of applications in biomedical and applied sciences (Dreyer et al., 2010; Georgakilas et al., 2012). Especially unique surface properties (oxygenated functional groups on its basal planes and edges), large surface area, layered structure, and easy exfoliation into monolayer in the aqueous phase, make graphene oxide (GO) a building block for fabricating versatile functional materials via covalent or noncovalent approaches (Dreyer et al., 2010). In recent years, there has been a rush of interest in functionalizing GO materials for diagnostics and pharmaceutical development (Veerapandian et al., 2012a). Surface modification through chemical reaction of active components on GO nanostructures influences the inherent sp2/sp3 carbon domains, which mediate the change of crystallite size, lattice orientation, and associated physicochemical properties (Krishnamoorthy et al., 2014). The different strategies employed to tune the physicochemical and biomedical functionality of graphene and its hybridized form are photoirradiation, elemental doping, and chemical anchoring of inorganic/organic materials (Matsumoto et al., 2010; Wang et al., 2012). Owing to their electrical and thermal conductivities, in addition to excellent barrier properties, graphene materials are generally projected as interconnectors for thermal dissipation in integrated circuits. Furthermore, graphene materials are studied to have various potential electronic applications, viz, touch screen, e-paper, foldable organic light-emitting diode (OLED), high-frequency transistor, logic transistor, and energy storage devices (Novoselov et al., 2012; Singh et al., 2011). The merits of integrating hybridized GO (i.e., metal or composite nanoparticles functionalized GO sheets) into thin solid films as an interface-layer for device fabrication over pristine carbon-based materials are: the synergistic effect of optical, thermal, quantum hall effect, electrochemical and mechanical properties (Singh et al., 2011). The physicochemical properties of hybridized GO materials render chemically versatile templates of high surface-to-volume ratio, which can be tuned to the needs of a variety of biological and biomedical applications, ranging from the detection of biomarkers to imaging and therapy, viz cancer and antimicrobials (Feng et al., 2013; Bitounis et al., 2013). Significant review articles have been recently devoted to the field of graphene and related 2D materials research. For instance, Singh et al. (2011) reviewed the synthetic strategies utilized in graphene derivatives, covering mechanical exfoliation, chemical vapor deposition (CVD) (including thermal CVD, plasma enhanced CVD and thermal decomposition on solid substrates like SiC), total organic synthesis of graphene-like polyacyclic hydrocarbons and the unzipping of carbon nanotubes. Furthermore, properties of chemically oxidized graphene derivatives such as oxygenated graphene (OG) and reduced graphene oxide (rGO) were also discussed in the literature (Singh et al., 2011). In another study, polymer nanocomposites with graphene-based fillers and their physicochemical properties were reviewed. Advancement in the field of gas and vapor sensors based on graphene and GO materials was available, which includes resistance-based gas/vapor

10.2 Physical and Chemical Properties of Graphene-Based Materials

sensors, field-effective transistor, surface acoustic waves, and microelectromechanical system (MEMS) (Basu and Bhattacharyya, 2012). Bitounis et al. (2013) have reviewed the prospects and challenges of graphene in biomedical applications with a focus on optical-based biomolecular sensing, bioimaging, photodynamic therapy, and antimicrobials. Furthermore, they have reviewed the recent research reports on tissue engineering, toxicity, and biocompatibility of graphene materials. Similarly, Sanchez et al. (2012) have reviewed the biological interactions of graphene family materials covering small molecules such as nucleic acids, lipids, proteins and biological degradation. In addition, the effect of deposition and clearance in the human respiratory system were discussed using mathematical models. According to that study, there are limited in vivo reports in the literature demonstrating the systemic biodistribution and biopersistence of graphene family nanomaterials. Furthermore, that report highlighted the need for extensive research in the evaluation of fundamental biological responses to graphene family nanomaterials, such as few layer graphene (FLG), GO and rGO. Due to its change of physical and chemical properties, such as lateral dimension and carbon-oxygen ratio, their individual toxicity also varies. Therefore, complete material characterization and mechanistic toxicity studies are preferred for optimized biological applications with minimal risks for environmental health and safety (Sanchez et al., 2012). Here, at first, the fundamental physical and chemical properties of graphene derivatives are briefly discussed. Secondly, the functionalization strategies demonstrated for the enhancement of inherent functionality of graphene-derivatives toward drug delivery and cell compatibility and proliferation are outlined. Later parts of the chapter are focused on the electrochemical biosensor studies of hybridized graphene materials, highlighting the sensor platform for the detection of metabolic biomarkers, infectious agents and active pharmaceuticals/nutraceuticals. Finally, we summarized the integration of feasibility of hybridized graphene materials in subdisciplines of nanotechnology and its advanced biomedical applications.

10.2 PHYSICAL AND CHEMICAL PROPERTIES OF GRAPHENE-BASED MATERIALS Graphene is a monolayer of sp2-hybridized carbon atoms assembled in a honeycomb 2D crystalline network with carboncarbon bond length of 0.142 nm (Slonczewski and Weiss, 1958; Singh et al., 2011). Due to its high Young’s modulus (1 Tpa) (Lee et al., 2008), high fracture strength, excellent electrical and thermal conductivity (5000 Wm21K21) (Balandin et al., 2008), fast mobility of charge carriers at room temperature (250,000 cm2/Vs) (Novoselov et al., 2005), large specific surface area, and biocompatibility, graphene possesses enormous

377

378


interests for interdisciplinary applications (Singh et al., 2011). Graphene is a fundamental building block for diverse materials with diverse geometries, which includes zero-dimensional fullerenes (wrapped spherical structures), onedimensional (1D) carbon nanotube such as structures and three-dimensional (3D) layered structures (graphite) (Geim and Novoselov, 2007). Graphene layers are composed of π-conjugated structure of six-atom rings, which can be considered as a planar aromatic macromolecule. GO is a chemical derivative of graphene having multiple negatively charged oxygenated functional groups on its basal plane and edges such as carboxylic acid, epoxy, carbonyl, and hydroxyl, which are useful in electrostatic interactions. With the advancement in the experimental characterization techniques, a range of structural models of GO have been presented in the literature. In 1939 the first model of GO was illustrated by Hofmann and Holst, which indicated that oxygen atoms were bound to carbon atoms of hexagonal layer by epoxide linkages with ideal formula C2O (Hofmann and Holst, 1939). Ruess (1946) proposed a model considering the hydrogen atoms of GO and denoted the sp3 hybridization form of basal plane structure. Scholz and Boehm (1969) proposed a structural model with completely removed epoxy and ether groups, substituting regular quinoidal species in a corrugated backbone. Nakajima and Matsuo (1994) represented a framework similar to poly(dicarbon monofluoride), (C2F)n by fluorination of GO through XRD pattern analysis, which forms a Stage 2 graphite intercalation compound. The fundamental structural features of GO proposed by Lerf et al., have been widely accepted. In the Lerf model, hydroxyl and epoxy groups are spread across the graphene planes, while carboxylic acid groups exist at edges, possibly in addition to keto groups. This model suggested a random distribution of aromatic and wrinkled regions on the basis of H NMR spectral studies (Lerf et al., 1998). Szabo et al. (2006) proposed a ribbon such as assembly of flat carbon hexagons connected by C 5 C double bonds. These individual models made a valuable contribution for the fundamental understanding of the chemical nature of GO by which various chemical modifications are feasible using organic moieties accordingly making GO organophilic. Unlike graphene, GO is insulating in nature with a highly disordered lattice assembly. However, the availability of electronegative oxygen functional groups with both sp2 and sp3 carbon domains make them interesting for scalable production of graphene by suitable reduction kinetics. By varying the oxidation level, i.e., carbon-oxygen ratio, the band gap of GO can be tuned for various optical applications (Jeong et al., 2009). Although GO is synthesized chemically from harsh oxidation of graphite, often by modified Hummers’ method, it still has sp2 carbon domains in its structure. The density-functional investigation on GO nanostructure show the existence of graphitic domains in the GO which create quantum confinement effects in GO (Saxena et al., 2010). Structural and functional properties of graphene can be modified by suitable reduction process of GO by optimizing the fractions of sp2 to sp3 clusters, which results in transition from insulator to semiconductor and to a metal-like

10.3 Functionalized Graphene-Derivatives

material (Shukla and Saxena, 2011). It has been studied that the optical properties of the graphene sheets can be tuned by removing oxygen functional groups on the surface of GO, which resulted into the formation of reduced GO with altered sp2 to sp3 carbon ratio (Krishnamoorthy et al., 2011). Different chemical routes for the reduction of GO have been studied; Agharkar et al. (2014) compiled all the important features of greener approach for the natural reduction of GO, which are based on the utilization of biomolecules, microorganisms and plant extract as reducing agents.

10.3 FUNCTIONALIZED GRAPHENE-DERIVATIVES FOR DRUG DELIVERY AND CYTO-COMPATIBILITY Graphene is highly hydrophobic and poorly soluble in aqueous solvents due to the sp2 hybridized carbon network which tends to agglomerate in solutions. Although oxidation of graphene improves the abundance of oxygen groups, both graphene and oxidized graphene exhibit poor solubility and stability in physiological saline, cell culture medium, and physiological conditions. For using the complete potential of graphene and its derivatives for efficient biomedical applications, various noncovalent and covalent strategies have been developed to improve its solubility and stability in biological environment. The noncovalent strategies involves the interaction of graphene’s hydrophobic surface with hydrophobic moieties/group adsorption through π-π stacking and electrostatic interactions (Hu et al., 2012, Liu et al., 2010, Yang et al., 2013), whereas the hydrophilic groups/chains impart aqueous solubility. This strategy retains the inherent electronic properties of the graphene for various pharmaceutical and biomedical applications. Conversely the covalent strategies involve the chemical bonding of surface groups on graphene after the harsh acid-based treatment strategy. These reactions introduce a variety of surface functional groups on graphene such as epoxide, hydroxide, and carboxylic acid groups. The harsh conditions might also introduce structural defects in graphene, resulting in altered graphene’s unique physicochemical properties. A classic example being the interaction of hydrophobic PPO segment of Pluronic F127 with graphene and the hydrophilic PEO moiety, which improves the aqueous dispersion and reduces agglomerationrelated toxicity. The covalent strategy involves the chemical reduction of graphene surface using bovine serum albumin, gelatin, cellulose, chitosan, dextran, and cyclodextrin (Justin and Chen, 2014, Guo et al., 2012; An et al., 2013; Kanakia et al., 2013; Chowdhury et al., 2013). Other strategies of chemical functionalization of graphene involve methods such as catechol chemistry, carbodiimide-assisted amidation, nucleophilic addition and 1,3-dipolar cycloaddition (Quintana et al., 2011; Hong et al., 2012; Zhang et al., 2013; Kim et al., 2013; Fan et al., 2013; Cao et al., 2013; Shen et al., 2010; Jung et al., 2010; Yang

379

380


et al., 2010; Nurunnabi et al., 2013; Cheng et al., 2013). Some of these approaches have shown the improved solubility and dispersion but also improve the efficient attachment of drug/ligand for delivery applications. It is also assumed that graphene has the ability to protect drugs from degradation, increase bioavailability, and allows targeted delivery (Aggarwal et al., 2009; Gao et al., 2013). This ability is due to graphene derivative easy uptake across the cellular membranes using energy-dependent clathrin-mediated endocytosis (Huang et al., 2012). Since 2008, graphene-based drug delivery systems have been developed to explore the potential delivery of low aqueous soluble pharmacological agents, such as amantadine, doxorubicin, methotrexate, paclitaxel, 5-fluorouracil and hypocrellin, to name few (Table 10.1). The enormous surface area of graphene (B2630 m2/g) has been utilized as an efficient platform for carrying pharmaceutical drugs with enhanced loading efficiency. It is shown that the drug doxorubicin mediates the π-π stacking with the graphene aromatic ring structures and achieves a very high loading efficiency of B200 wt% (Yang et al., 2008). The earliest graphene-based drug delivery system was reported by Hongjie Dai group, for SN38, a camptothecin analog and potent topoisomeraseI inhibitor, using PEGylated GO (Liu et al., 2008). The SN38 analog exhibited an IC50 efficacy of B6 nM (approximately .1000 fold compared to CPT-11) for HCT-116 human colon cancer cells. For tumor passive targeting, it is observed that nanoparticles of specific size loaded with drugs (1060 nm) are accumulated in the tumor tissues due to the enhanced permeability and retention (EPR) effect (Thakur and Karak, 2012; Zhu et al., 2010). The EPR effect is observed due to the tumor pathological characteristics, in which the leaky vascularization and the lack of lymphatic drainage leads to enlarged gap junctions between endothelial cells. Due to this leaky vasculature, the nanoparticles extravasate through the gap junctions and accumulate in the extravascular spaces of the tumor tissues. Studies by Liu’s group has shown that PEG-modified graphene (.65 nm) was taken up highly by the reticuloendothelial system, compared to small size PEGylated-graphene (2030 nm) (Yang et al., 2012a). A recent study has shown remarkable increase in the uptake of noncovalently conjugated graphene complexed PEG, compared to covalent linkage due to the compact nature of the complexation (Yang et al., 2012a). Drug delivery platforms based on 2D layered structures of GO-derivatives with modest surface functional groups are emerging as new competitive drug delivery systems with the potential to be explored for systemic, targeting, and local drug delivery platforms (Sanchez et al., 2012; Liu et al., 2013). An illustration depicting the structure of GO/graphene as nanotherapeutic carrier for different active pharmaceutical ingredients from small drug molecules, antibodies, nucleic acid/ genetic material, and proteins/peptides are presented in Fig. 10.1. In addition to drug delivery, reports have shown that the aromatic scaffold nature of graphene and GO have potential for enhancing cell behavior such as attachment, growth, proliferation and differentiation. This can promote the local

Table 10.1 Graphene-based Nanomaterials for Therapeutic Delivery Therapeutic Agents

Target Cell Lines

Amantadine

HeLa cell

Camptothecin

N2a cancer cells HepG2 and HeLa cell

Doxorubicin

MCF-7 MCF-7

LLC1 KB epidermal carcinoma cells PC-3

Glioma cells

HeLa and A549 cell

MDA-MB-231 cells CBRH7919 liver mouse cancer cell

Graphene-based Delivery System Cyclodextrin-GO nanohybrid Poly(2-(diethylamino) ethylmethacrylate)/GO Chitosan functionalized GO Folic acid conjugated GO Graphene-dextran nanohybrid; Gelatin functionalized graphene; Pluronic F127 coated GO Chitosan-modified rGO Hyaluronic acid-coated rGO Branched polyethylenimine and polyethylene glycol conjugated reduced GO Targeting peptide modified mesoporous silica-coated GO Polyvinylpyrrolidone (PVP) functionalized GO; Cyclodextrin-GO nanohybrid Poly(vinyl alcohol) coated GO Polyethyleneimine coated rGO

5-fluorouracil

CEM human lymphoblastic leukemia

Chitosan functionalized GO

Hypocrellin A; B

HeLa cell; A549, HeLa, SMMC-7721, SGC-7901 A549

GO

Methotrexate

MCF-7 Paclitaxel

L929 murine fibroblasts; SGC7901 human gastric cell lines A549; MCF-7; B6Melanoma A549

Ethylene oxide (covalent); Gelatin functionalized graphene GO Phosphorylcholine oligomer grafted perylene (Perylene-PCn) - GO

References Dong et al. (2013) Kavitha et al. (2013) Bao et al. (2011) Zhang et al. (2010) Jin et al. (2013); Liu et al. (2011); Hu et al. (2012) Wang et al. (2013a) Miao et al. (2013) Kim et al. (2013)

Wang et al. (2013b) Qin et al. (2013); Yang et al. (2012b)

Sahoo et al. (2011) Wei et al. (2012); Kim et al. (2011) Rana et al. (2011); Wang et al. (2013c) Zhou et al. (2011; 2012) Du et al. (2013); An et al. (2013) Wojtoniszak et al. (2013) Liu et al. (2016)

PEGylated GO

Xu et al. (2015a,b)

PLA-PEG-GO

Angelopoulou et al. (2015)

382


Antibodies

Small drug molecules O

OH

OH OH OH

DNA/RNA

OCH3 O

OH

H3C

O

OH

Genes

O HCI

NH2

Proteins/peptides

FIGURE 10.1 Illustration of application of graphene and GO as drug carrier for various biologically active therapeutic agents and biomolecule. Source: Adapted with permission from Liu, J., Cui, L., Losic, D., 2013. Graphene and graphene oxide as new nanocarriers for drug delivery applications. Acta Biomater. 9, 92439257, Copyright 2013, Elsevier Publications.

concentration of extracellular matrix (ECM) such as collagen, laminin, and fibronectin via noncovalent binding (Chen et al., 2008). Graphene and GO sheets are explored as biocompatible, transferable, and implantable platforms for stem cell culture. For instance, Lee et al. (2011) stated that noncovalent binding capability of graphene allows it to serve as a preconcentration platform for osteogenic inducers, which promote mesenchymal stem cell growth toward the osteogenic lineage. Similarly, Nayak et al. (2011) demonstrated that graphene acts as scaffold for proliferation of human mesenchymal stem cells (hMSCs) and specific differentiation into osteoblasts. An electronegative element, such as fluorine, was electrostatically functionalized on the surface of GO sheets (via C-F bond) and studied to have improved cyto-compatibility with MSCs (Wang et al., 2012). The polarization effect of the C-F bond was proposed to facilitate the cell alignment, cytoskeletal, and nuclear elongation through electrostatic induction at the interface of cell-fluorinated graphene. Likewise, Marimuthu et al. (2014) developed sodium (Na)-functionalized GO-coated titanium plates for improved corrosion resistance and cell viability. A solution-based blending approach was utilized for the Na1 functionalization. Owing to its known physiological functions, such as regulation of cellular osmotic balance through pumping small molecules, Na1

10.4 Electrochemical Biosensors Based on Graphene

modified on the functional groups of GO are hypothesized for augmenting the cellular growth of human dermal fibroblast cells. Song et al. (2015) studied that incorporation of GO sheets into the polycaprolactone (PCL) nanofibers improved the overall thermal and mechanical properties of the scaffold. Furthermore, the biocompatibility study with mouse marrow MSCs and low-differentiated rat pheochromocytoma (PC12-L) cells showed that moderate concentration of GO (0.3 and 0.5 wt%) significantly promoted the initial adhesion and spreading of those cell lines which are superior to those on pristine PCL scaffold. The cell morphologies were observed to be typical such as fibroblast- and neuron-like with obvious pseudopods and mature appearance, indicating GO nanofilled polymer composites have potential for tissue engineering application. Li et al. (2016) fabricated the GO/polyacrylamide (GO/PAM) composite hydrogels by in situ free radical polymerization and studied the attachment and proliferation of Schwann cells. The release of bio-factors by Schwann cells and adsorption matrix proteins were evaluated. This study observed that with increase of GO concentration, the surface porosity also largely varied. The hydrophobicity and mechanical features of hydrogel were improved with increased GO concentration. This experimental result shows a promising basis for further research on peripheral nerve regeneration especially utilizing hybridized GO composite. In other study, GO, hydroxyapatite nanoparticles and chitosan are self-assembled into a 3D hydrogel with the use of genipin as cross-linking agent. The developed hydrogel matrix is observed to have high mechanical strength, high fixing capacity of hydroxyapatite, and high porosity, suitable for bone-tissue engineering application (Yu et al., 2017). These studies are intended to highlight the utility of graphene/GO material as substrate for potential drug delivery and cyto-compatibility application, rather than comprehensive list on hybrids of graphene for pharmaceutical and cellbiological studies.

10.4 ELECTROCHEMICAL BIOSENSORS BASED ON GRAPHENE HYBRID NANOMATERIALS Functionalities of graphene/GO derivatives have been used in different types of biological sensors, which can be categorized based on the opto/electrochemical properties, such as fluorescence resonance energy transfer (FRET), laser desorption/ionization mass spectrometry (LDI-MS), surface-enhanced Raman spectroscopy, and electrochemistry (Lee et al., 2016) (Fig. 10.2, Table 10.2). Compared to conventional molecular assays, biosensors based on electrochemical approaches are widely explored for diagnostic device fabrication and field applications. Electrochemical biosensors are advantageous for the field and patient side use, due to its user-friendliness, portability, rapid response time, cost-efficiency, sensitivity, suitability for automation, miniaturization, and smart integration with other

383

m/z

N2 laser

Current

Graphene oxide -based biosensors Potential

Laser SERS

Graphene-based electrode

FIGURE 10.2 Graphene/GO-based biological sensors classified depending on the type of the detection signal. Source: Adapted with permission from Lee, J., Kim, J., Kim, S., Min, D., 2016. Biosensors based on graphene oxide and its biomedical application. Adv. Drug Deliv. Rev. 105, 275287, Copyright 2016, Elsevier Publications.

Table 10.2 Summary of Graphene Oxide-Based Biosensors Using Various Detection Techniques Detection Technique

Characteristics of Graphene Oxide

FRET

(1) Strong binding with biomolecules through pi-pi stacking and/or hydrogen bonding interactions, (2) fluorescence-quenching capability of nearby fluorescent dye (1) Strong absorbance at the excitation laser wavelength of 337 or 355 nm, (2) high affinity toward various amphiphilic biomolecules (electrostatic/ hydrophobic/pi-pi stacking interaction), (3) easy protonation of analytes by functional groups on GO (1) Outstanding electrocatalytic ability, (2) low charge-transfer resistance

LDI-MS

Electrochemistry

SERS

(1) Quenching the background fluorescence signal, (2) chemical enhancement in SERS induced by electron transfer

Purpose Biomolecule detection (Lu et al., 2009), signal amplification (Wang et al., 2015 ), enzyme assay (Song et al., 2013), cell/tissue imaging (Wang et al., 2014), highthroughput screening (Jang et al., 2013) Biomolecule detection (Huang et al., 2015), enzyme assay (Lee et al., 2010), cell/tissue imaging (Kim and Min, 2012), highthroughput screening (Liu et al., 2011)

Biomolecule detection (Veerapandian et al., 2012a,b; Srivastava et al., 2015), signal amplification (Wang et al., 2015), enzyme assay (Wu et al., 2012) Biomolecule detection (Lin et al., 2015), cell/tissue imaging (Liu et al., 2012)

Adapted and modified with permission from Lee, J., Kim, J., Kim, S., Min, D., 2016. Biosensors based on graphene oxide and its biomedical application. Adv. Drug Deliv. Rev. 105, 275287, Copyright 2016, Elsevier Publications.


gadgets (Vigneshvar et al., 2016). Direct electron transfer at the interface of electrode and recognition component (such as antibody, enzymes, proteins and nucleic acids/small oligonucleotides) is the fundamental process in electrochemical reaction. Evaluation of electrochemical behavior of graphene derivatives toward redox mediators requires fundamental understanding of electrochemical aspects of graphene (Pumera, 2009). Existence of oxygenated functional groups on the basal and edges of graphene surface are often tuned for enhancement of electron transfer rate. Graphene nanostructures have a low density of edge sites, compared to their abundant basal plane sites (Randviir and Banks, 2012; Brownson et al., 2012). Graphene nanosheets often tend to stack together due to the strong van der Waals force of interactions between the sheets. Stacked graphene sheets have reduced porosity, increase the diffusion resistance of active reactants/electrolyte species, which eventually reduce the number of exposed electroactive sites. Accordingly, researchers are attempting to carefully hybridize the graphene nanosheets to enable their use in high performance electrochemical biosensors and other practical applications.

10.4.1 METABOLIC BIOMARKER DETECTION Clinically important biomarkers have been identified for early diagnosis and management of different metabolic disorders, such as diabetes, galactosemia, hyperlipidemia, and hypercholesterolemia, to name few. Among the various metabolic diseases, diabetes mellitus is the most commonly studied clinical condition in larger populations, expressed with hyperglycemia associated to the insulin deficiency and reflected by blood/urine glucose concentration (Miyashita et al., 2009). A survey by International Diabetes Federation documented that 380 million people are expected to have hyperglycemia by 2025 and quoted that diabetes as a “global burden” (Veerapandian et al., 2012b). Although there are successful electrochemical assay kits available in the market for detecting blood/urine glucose level, there is still a growing interest in developing an advanced biosensor platform with a possibility of reusable self-monitoring device, with improved analytical performance and amicable integration on modern gadgets (Veerapandian et al., 2014a). For instance, Fig. 10.3 represents a functionalized GO (FGO)-based glucose sensor platform customized on a gold-printed circuit board (Au-PCB) chip, feasible for handheld diagnostics. Unlike conventional disintegrated three electrode system, this customized three-electrode on a single chip based on microelectromechanical system (MEMS) is comparatively versatile for field application. An FGO sensor platform on Au-PCB chip is comprised of metalloidpolymer nanoparticles (silver@silica-polyethylene glycol, Ag@SiO2-PEG) and enzyme glucose oxidase (GOx). Fig. 10.4A and B demonstrates the redox property of FGO sensor platform and its amperometric detection ability of different concentrations of glucose

385

386


Au-PCB electrode

Working electrode

(i) O2 plasma

GOx

(ii) FGO nanosheet Reference electrode

Au-PCB-FGO/GOx biosensor platform

Counter electrode

GO nanosheet

Ag@SiO2-PEG (MPHs)

FGO nanosheet

GOx enzyme

FIGURE 10.3 Illustration of fabricating functionalized graphene oxide (FGO) based glucose biosensor platform. Source: Copyright 2014, Elsevier Publications.

dissolved in N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid sodium salt (TES) buffer solution. As represented in Fig. 10.4A, curve b, the FGO electrode exhibited the well-resolved anodic peaks A1 at 10.11V and A2 at 10.54V, the former is ascribed to the monolayer oxide formation and the later is attributed to the oxidative reaction of specific Ag@SiO2-PEG functionalized on GO surface. The cathodic peak observed from FGO electrode at 10.11V, indicates the existence of overlap between the reduction reaction of Ag@SiO2-PEG and reduction of oxide layers from Au active site on the electrode surface. It was demonstrated that due to the functionalization of Ag@SiO2-PEG nanoparticles on the surface of GO nanosheets, the resulted FGO electrode exhibited an amplified peak current of 14.09 μA (anodic peak A2) during the oxidation reaction, supporting the better electron transfer process. Other types of hybrid graphene-based enzymatic biosensing of important biomarkers including glucose, cytochrome-C, nicotinamide adenine dinucleotide (NADH), hemoglobin (Hb), horseradish peroxide (HRP) and cholesterol has been reviewed in detail (Lawal, 2015). Table 10.3 represents the few of the enzymatic biosensor platforms derived from hybrids of graphene/GO with relevant relative standard deviation and limit of detection. Likewise, nonenzymatic graphene-based electrode materials are also demonstrated for sensing of biologically important molecules such as H2O2, ascorbic acid, uric acid and dopamine (Lawal, 2015).


(A)

(B) 4.50x10–6

A1

1.50x10–6

A1

0.00

3.0x10–8 2.5x10–8 2.0x10–8 1.5x10–8

–1.50x10–6

1.0x10–8

C1

–9

C1

–6

–3.00x10

–0.2

(C)

5.0x10

0.0 0.2 0.4 0.6 0.8 Applied potential (V) vs Au-PCB

0

(D)

2.5x10–8

1.2x10–7

2.0x10–8

1.0x10–7

–8

8.0x10–8

Current (A)

1.5x10

1.0x10–8

10 20 30 40 50 Glucose concentration (mM)

Au-PCB-FGO Accu-Chek

6.0x10–8 4.0x10–8 2.0x10–8

5.0x10–9

0.0 0.0 0

5 10 15 Number of days

20

25

60 300 280 260 240 220 200 180 160 140 120 100 80 60

Glucose (mg/dL)

Current (A)

R2 = 0.9981

3.5x10–8 Current (A)

Current (A)

3.00x10–6

–8

4.0x10

A2

(a) Bare-Au-PCB (b) Au-PCB-FGO

P1 P2 P3 P4 P5 P6 P7 P8 P9 Patients number

FIGURE 10.4 (A) Cyclic voltammogram of bare Au-PCB (curve a) and Au-PCB/FGO (curve b) in 100 mM TES buffer (pH 7.0). (B) Amperometric response of Au-PCB-FGO/GOx electrode against different glucose concentration samples in 100 mM TES buffer (pH 7.0). The applied potential was 0.7V. (C) Amperometric response of Au-PCB-FGO/GOx against 11.1 mM concentration of glucose sample in 100 mM TES buffer (pH 7.0) measured at different days of preparation. (D) Amperometric response of Au-PCB-FGO/GOx (square plot) and Accu-Chek against glucoseuria and hyperglycemic patient’s serum samples (circle plot), respectively. Source: Copyright 2014, Elsevier Publications.

Doping of electron-accepting nitrogen atoms is one of the hybridization procedures effectively implemented on the surface of graphene derivatives to vary the physicochemical properties (Wang et al., 2012; Van Khai et al., 2012; He et al., 2013). Introduction of nitrogen atoms delivers a relatively high positive charge density to adjacent atoms and creates an altered bonding configuration, within the carbon lattice, and exhibits high electrocatalytic activity (Van Khai et al., 2012; He et al., 2013). Kannan et al. (2013) developed the graphene-supported platinum nanoparticles (GN-PtNPs) and nitrogen-doped graphene-supported platinum nanoparticles (N-GN-PtNPs) by a simple chemical reduction method using ethylene glycol. Fig. 10.5A represents the possible interaction of nitrogen atoms on the

387

388


Table 10.3 Various Types of Enzymatic Electrodes Made From Graphene and GO. In all the Cases the Mode of Detection Is Change in Electrical Current Type of Biosensor

Sensor Materials

RSD %

Detection Limit

References

Glucose

a a

3 μM 6 0.5 μM 1 μM

Alwarappan et al. (2010) Baby et al. (2010)

4.21 a

1.0 3 10a6 M 0.1 mM

Chen et al. (2010) Huang et al. (2010a,b)

5.3 5.8 3.2

0.02 nM a 2a14 mM

Kang et al. (2009) Liu et al. (2010) Shan et al. (2009)

4.7 a 5.3 6.0 2.5 a a 4.3 3.2 4.2

180 μM a 0.7 mM 0.6 M 10 6 2 μM 0.376 mM 0.168 mM 2.0 μM 5 μM 5 μM

Shan et al. (2010a) Wang et al. (2009) Wang et al. (2011) Wu et al. (2009) Wu et al. (2010) Yang et al. (2010) Zeng et al. (2010a) Zhou et al. (2009) Zhou et al. (2010a) Shan et al. (2010b)

HRP

Graphene-PPy Metal decorated graphene Graphene/Nafion CVD grown graphene CS-GR GO PVDF-protected graphene CS-GR-AuNP GO Graphene-CdS CS-GR-AuNP Graphene CMG Graphene CR-GO Graphene IL functionalized graphene Graphene Fe3O4-graphene Graphene Graphene

3.5 1.6 a 4.48

Tang et al. (2009) He et al. (2011) Xu et al. (2010) Lu et al. (2010)

a 3.6 a

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NADH

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Zeng et al. (2010b) Zhou et al. (2010b) Zhou et al. (2011)

AuNP, gold nanoparticle; CdS, cadmium sulfide; CMG, chemically modified graphene; CR-GO, chemically reduced graphene oxide; CS, chitosan; CVD, chemical vapor deposition; Fe3O4, iron (II, III) oxide; GO, graphene oxide; GR, graphene; Hb, hemoglobin; HRP, horseradish peroxidase; IL, ionic liquid; PtNP, platinum nanoparticle; PPy, polypyrrole; PVDF, polyvinylidene fluoride; SDBS, sodium dodecyl benzene sulfonate. Copyright 2011, Elsevier Publications.


FIGURE 10.5 (A) Schematic of nitrogen-doped graphene-platinum nanoparticles (N-GN-PtNPs) depicting the nature of the bonding of nitrogen atoms in the graphene nanosheets. (B) Schematic representation of electrostatic, amino-Pt and thiol-Pt interactions between NGN-PtNPs and homocysteine molecules. Source: Adapted with permission from Kannan, P., Maiyalagan, T., Sahoo, N.G., Opallo, M., 2013. Nitrogen doped graphene nanosheet supported platinum nanoparticles as high performance electrochemical homocysteine biosensors. J. Mater. Chem. B 1, 46554666, Copyright 2013, Royal Society of Chemistry.

surface of a GN-PtNPs composite. The developed hybrid graphene sheetbased electrodes are explored as high performance nanocatalyst toward electrochemical oxidation of homocysteine. Presence of thiol (aSH) and amino (aNH3) group in the homocysteine molecule are studied to have better affinity to PtNPs in the GN composite. This occurs via PtaS and interparticle binding and electrostatic interactions between NH3 groups, which are expected to have three dimensional assembly on the substrate (Fig. 10.5B) (Kannan et al., 2013). Homocysteine is a biologically important amino acid, which does not directly exist in the diet, but is formed during the methionine metabolism. Normal blood plasma concentration of homocysteine is 5a16 μmol/L. Higher concentrations of homocysteine result in a clinical condition of hyperhomocysteinemia (#100 μmol/L) or homocystinuria (B500 μmol/L) (Medina et al., 2001). Physiological conditions shown that hyperhomocysteinemia is associated with folate and cobalamine deficiencies and might lead to early pregnancy loss, mental disorders, and tumors (Nelan et al., 2000). Furthermore, increased homocysteine concentration is also associated with an increased risk of coronary artery and cerebrovascular diseases, including atherosclerosis and thrombosis (Nelan et al., 2000). It is known that ascorbic acid is the coexistent component with homocysteine in biological fluids, but its concentration is relatively very much higher than homocysteine and is one of the main interferents for the determination of homocysteine. Therefore, simultaneous determination of homocysteine and ascorbic acid is vital to ensure the clinical safety of a

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person from the risk of above critical diseases. Kannan et al. (2013) have observed that a N-GN-PtNPs modified electrode show nearly three-fold enhancement in the oxidation current of homocysteine and ascorbic acid, and significantly reduced the oxidation overpotential of about B160 mV less than pristine GNPtNPs-modified electrode. This study further demonstrated that electrodes of N-GN-PtNPs exhibited higher electrocatalytic activity for homocysteine with an experimental detection limit of 200 pM. In addition, amperometric study of the nitrogen-doped graphene-supported platinum nanoparticle composites were studied to have selectivity for the electrochemical oxidation of homocysteine, even in the presence of ascorbic acid and other important interferents viz glucose, urea, uric acid, serotonin, and oxalate. Similar to nitrogen doping, photoirradiation on the surface of GO layers alters the inherent chemical state of the functional groups and its optical and electrochemical properties. Unlike chemical treatment, photoirradiation is recognized as a green method to optimize the functional groups of GO for device development. Veerapandian et al. (2015) have established an amino sugar molecule (glucosamine (GA))-anchored GO nanostructure and studied the effect of UV irradiation on the structural and opto-electrochemical properties of the GA-anchored GO nanosheets (GA-GO) toward an enzymatic biosensing of sialic acid. At first, the cyclic voltammetry of pristine GO or GA-GO and UV-irradiated GO or GA-GO electrode was studied using ruthenium(II) as model redox probe. This experiment revealed that UV-irradiated GA-GO electrode exhibited a better oxidation peak current (15.7 μA/cm2) for the redox probe ruthenium(II). The mechanism behind this enhanced electronic property is ascribed to the transducer material GA-GO, which has the configuration of C-N bond on the lattice, which provides the new sp3 and sp2 domains, lattice orientation, and relevant electroactive charge carriers. It is known that nitrogen has a superior electronegativity than carbon. Nitrogen species of GA bonded at the edges and basal planes of GO layers easily contribute to the electron transfer process through the carbon surface with high current density. This electron donation and back-donation process facilitates O2 dissociation on the neighboring carbon atoms (Veerapandian et al., 2015; Deng et al., 2011). Furthermore, UV irradiation-induced restoration of sp2 clusters via removal of O2 groups on the basal/edges of GO surface triggered the charge carriers for rapid electron transfer. Fig. 10.6 shows the plausible chemical structure of UV-irradiated GA-GO, indicating the chemical bonding between the NH2 group of GA and the functional groups GO (Veerapandian et al., 2015). The amperometric sensing ability of pristine GO or GA-GO and UV-irradiated GO or GA-GO has been studied against free sialic acid using enzyme, N-acetylneuraminic acid aldolase. This enzyme reversibly catalyzes the N-acetylneuraminic acid (NANA) 2 N-acetylmannosamine 1 pyruvate. Anionic monosaccharide sialic acid is the N-acetylated derivative of neuraminic acid. The normal concentration of total sialic acid in serum/plasma is 1.58a2.22 mmol/L, the free form of sialic acid is 0.5a3.0 μmol/L and the lipid-associated sialic acid


(A)

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FIGURE 10.6 Chemical structures of (A) GO (red colored oxygen functional groups are reactive to GA), (B) GA-bonded GO and (C) UV-irradiated GA-GO. Source: Adapted with permission from Veerapandian, M., Le´varay, N., Lee, M.H., Giasson, S., Zhu, X.X., 2015. Glucosamine-Anchored Graphene Oxide Nanosheets: Fabrication, Ultraviolet Irradiation, and Electrochemical Properties. ACS Appl. Mater. Interfaces 7, 14552 2 14556, Copyright 2013, American Chemical Society.

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is 1050 μmol/L (Sillanaukee et al., 1999). Owing to its essential physiological role in the expression of glycoconjugates and wide existence in biological fluids/ tissues, sialic acid is regarded as the vital biomarker for several types of clinical conditions including, cancer, cardiovascular disease and diabetes. Existence of NANA aldolase on the electrode surface readily oxidized free NANA (i.e., sialic acid) and hence supplies electrons to the electrode. This study observed that UVirradiated GA-GO/NANA aldolase electrode exhibits a better amperometric current against the different concentrations of NANA, which is superior to the other electrodes.

10.4.2 SENSING OF INFECTIOUS AGENT The food production and processing industry has the largest revenue worldwide. Certainly, securing the live-stock industry, such as cattle-farm, chicken-farm, dairy industries, food processing/packaging unit, and its associated environment from harmful infectious agents will preserve the individual and environment from serious outbreaks (Neethirajan, 2017). Therefore, development of smart sensing systems based on automatic or semi-automatic function suitable for various types of test samples ranges from liquid, semi-solid and solid, without complex sample pretreatment has a great demand with immediate application. In addition to food industries, personal health care diagnostic kits for rapid detection of infectious agents are highly demanded in small-to-large hospitals for early diagnosis of bacterial/viral diseases and management of hygienic environment. Bacterial/virus isolation culture are the conventional methods generally practiced for the detection of infectious agents. However, these methods are both time consuming and require intensive labor operation. Enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and reverse transcription PCR are also used, due to the superior sensitivity and less operational time, compared with conventional methods (Grabowska et al., 2014). However, limitations allied with these techniques are: necessity of expert personnel to isolate the genetic material and need of sophisticated instrumental facilities, which hampers the feasibility for small diagnostic laboratories and patient/bedside rapid monitoring (Rodovalho et al., 2015). Fabrication of user-friendly, rapid and efficient hand-held diagnosis system for on-site or patient-side monitoring is extremely valuable. Recent studies demonstrated that electrochemical biosensors, especially based on MEMS technology, offered better sensor platform potential for rapid diagnosis, without the need of complex sample procedure and perhaps suitable for on-site analysis (Veerapandian and Neethirajan, 2015; Rackus et al., 2015; Veerapandian et al., 2016a,b). Incorporation of electroactive metal-hybrid or carbon nanostructures as transducer element on the sensor device apparently enhances the total sensitivity of the device, with a flexibility of handling small volumes of test samples (Reverte et al., 2016). Devising a biocompatible transducer element with retained electrochemical property and chemical groups for immobilization of


bio-recognition moiety, such as antibodies, enzymes or small oligonucleotides for specific detection of particular target, with better response time, are key parameters for an efficient biosensor system (Veerapandian et al., 2015). Listeria monocytogenes (Lm), a gram-positive rod-shaped food-borne bacterium that often lead to a life threatening infection, listeriosis (Tully et al., 2008), particularly affecting pregnant women, newborns, aged people .65 and people with weakened immune systems. A study from Centers for Disease Control and Prevention (CDC), USA (June 2015) confirmed a multistate outbreak of listeriosis linked to food poisoning (CDC, 2015). Another report from the government of Canada projected that there may be B4 million cases of food borne illness every year. Common clinical symptoms resulted from food poisoning include fever, vomiting, headache, and diarrhea. In sever conditions, meningitis, septicemia and abortion can occur. Therefore, development of rapid and sensitive detection of pathogenic bacteria such as Lm in food processing unit is essential for public safety. Veerapandian et al. (2015) have established an electrochemical immunosensor platform for the detection of Lm. This immunosensor device composed of a hybrid nanoparticle (HNPs) made of Ag@[Ru(bpy)3]21/chitosan chemically modified on the surface of GO nanosheets. Abundant amino groups of chitosancoated Ag@[Ru(bpy)3]21 are studied to have significant chemical interaction with the oxygenated edges/basal planes of GO. These chemically interfaced HNPs-GO electrodes are experimented to have enhanced redox-property at the interface, suitable for sensor platform. A monoclonal antibody Lm was modified on the sensor element for specifically recognizing the target bacteria. A chronoamperometric technique was used to study the electrochemical immunosensing property of HNPs-GO/antiLm. Bacterial contaminations (artificially induced) in PBS and in milk samples were evaluated. The sensitivity of the HNPs-GO based immunosensor in PBS is calculated to be 1.82 3 10a5 A/101 Lm cells/mL and in milk is 5.61 3 10a6 A/101 Lm cells/mL. The detection limit was reported to be 2 cells/mL, which is better than the other electrode materials (TiO2 nanowire bundle microelectrode, planar Au, screen-printed Au and polypyrrole film) demonstrated for electrochemical immunoassay of Lm, reviewed elsewhere in the literature (Veerapandian and Neethirajan, 2015). Escherichia coli 0157:H7 is another food-borne bacterium also causing serious health concerns. A study by Pandey et al. (2017) reported that interfacing graphene with interdigitated microelectrodes of capacitors that were biofunctionalized with E. coli 0157:H7-specific antibodies are suitable for potential recognition of pathogenic bacteria detection. That group tested two distinct types of graphene nanosheets through interfacing with SiO2-substrates, such as defectfree monolayered-graphene and cost-efficient few-layered graphene nanoplates. The graphene interface enabled a high carrier mobility and biocompatibility with antibodies and bacteria. The limit of detection was found to be as low as 10100 cells/mL. The sensitivity of developed monolayered graphene and few-layered graphene biosensors was B4 pF and B1 pF in terms of change in biosensor

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signal per unit change in analyte (bacterial) concentration of 10a100 cells/mL, respectively. Likewise, hybrid GO derivatives are explored for other electrochemical immunosensor platform focused on targeting the specific infectious agents, such as virus antigen detection. For instance, methylene blue-electroadsorbed GO nanostructures are modified with monoclonal antibodies against hemagglutinin (HA) proteins of H5N1 and H1N1 (pathogenic viral strains) and proposed as dual immunosensor platform. Chitosan and protein-A molecules were incorporated as bio-functional layer at the interface of methylene blue-electroadsorbed GO sensor element and monoclonal antibodies, which are studied to have a synergistic effect in enriching the bio-activity of antibodies and resulted in immune complex formation. Fig. 10.7 illustrates the typical dual screen-printed electrode and the sequence of surface modification procedure implemented in the fabrication of immunosensor platform for two different avian influenza antigens. It has been observed that the fabricated dual immunosensor platform exhibited an amplified current sensitivity even at the picomolar concentration of antigens with a rapid analytical response time (,1 minutes). Infection caused by dengue virus (DENV) is other serious health issue which affects over 100 million people annually with a death rate of 2.5% (WHO report, 2015; CDC report, 2016). There are four serotypes of dengue virus, i.e., DENV-1, DENV-2, DENV-3 and DENV-4. According to the WHO, new tools for the diagnosis of diseases such as dengue should provide affordable, sensitive, specific, user-friendly, rapid, and robust measurements that can be obtained using point-ofcare and bedside approaches (Parolo and Merkoci, 2013). This will certainly help the clinician for proper medical care without having any fatalities. NS1 is a nonstructural dengue protein that is secreted from infected cells and has been used as an early surrogate biomarker for viremia and/or infected cell mass in patients. In addition, an NS1 antigen capture, ELISA has been developed which revealed that secreted NS1 is present in the sera of infected patients during the acute phase of disease. This suggests that it can be used as a diagnostic marker for dengue (Alcon et al., 2002). Furthermore, it has been reported that this test is able to detect all four dengue virus serotypes (Kumarasamy et al., 2007). Efforts have been taken for the development of immunosensing of DENV. For instance, poly (allylamine)-CNTs composites for detection of NS1 of DENV has been reported (Silva et al., 2015). In another recent study, a thin film of a copolymer matrixreinforced GO nanostructure has been spin-coated on Au-electrode and demonstrated for specific anchoring of DENV particles. An electrochemical impedance-based detection method was used to measure the change of charge transfer resistance (Rct), as a function of plaque-forming unit (pfu) concentration of DENV. Binding affinity of 4G2 antibody on four DENV serotypes compared. Here, DENV works as a component used to functionalize the GO-copolymer surface by triggering a self-assembly process that makes the polymer matrix sensitive and specific for re-localization of virus. The copolymer matrix used in this study is made from the monomers acrylamide, methacrylic acid and


CE RE GO WE2

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N CH3

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CH3 N CH3

(i) Chitosan (ii) Protein-A

(i) mAb (ii) BSA

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Antigen H5N1

H1N1

Antigen H1N1

Immunocomplex on electrode

FIGURE 10.7 Schematic representation of dual screen-printed electrode and sequence of surface modification in the preparation of immunosensor platform. WE, working electrode; CE, counter electrode; RE, reference electrode; mAb, monoclonal antibody; BSA bovine serum albumin. Source: Copyright 2016, Elsevier Publications.

N-vinylpyrrolidone, while N-N-(1,2-dihydroxyethylene) bisacrylamide and 2,20 azobis (isobutyronitrile) was used as the cross-linker and initiator, respectively. The linear dependence of Rct of an Au-electrode coated with GO-polymer matrix against virus concentrations was in the range 1 to 2 3 103 pfu/mL DENV with a detection limit of 0.12 pfu/mL (Navakul et al., 2016). In addition to immunodetection, the hybrid GO matrix is also explored for enzymatic biosensing of infectious agents, such as bacterial toxins, botulinum neurotoxin. Presently, there are seven serotypes (A to G) of botulinum toxins

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identified, in which botulinum neurotoxin A (BoNT/A) is the most frequent agent responsible for botulism events in human. Chan et al. (2015) fabricated an rGO/ Au electrode immobilized with SNAP-25-GFP peptide via pyrenbutyric acid as linker. Addition of enzyme BoNT serotype A light chain (BoNT-LcA) protease specifically cut the SNAP-25-GFP peptide at the cleavage sites from the electrode surface. This enzymatic activity of BoNT-LcA on the SNAP-26-GFP peptide at the sensor substrate resulted in the enhanced redox probe transfer rate, which is measured from the differential pulse voltammetry. The reported linear detection range is from 1 pg/mL to 1 ng/mL, with a detection limit around 8.6 pg/mL (Chan et al., 2015). These preliminary prototype sensor studies show the promising features of graphene-derivatives as sensor elements in electrode design potential for biodetection of infectious agents viz, bacteria, viral antigens, and bacterial toxins.

10.4.3 SENSING OF ACTIVE PHARMACEUTICALS/NUTRACEUTICALS Active pharmaceutical/nutraceutical components and semi-synthetic analogs inspired by natural product scaffold are significantly explored for effective therapeutics, especially antimicrobials and anticancer drugs (Harvey, 2008). Natural products (like phytoconstituents, marine compounds, and secondary metabolites from microbial sources) exhibit diverse and unique structural chemistry that are complementary to combinatorial libraries (Feher and Schmidt, 2003). However, technical limitations exist in the analysis of natural product chemistry, often hampering the pharmaceutical companies leading a compound into clinical trials (Li and Vederas, 2009). Modern research methods devised for natural product isolation, purification, and screening of structural properties aimed to revitalize the global perspective in natural product drug discovery. Various analytical techniques are utilized for the quantification of bioactive natural products, such as capillary electrophoresis (Desiderio et al., 2005), chemiluminescence (Yang et al., 2010), high-performance thin layer chromatography (HPTLC) (Attimarad et al., 2011), high performance liquid chromatography (HPLC) (Bittova et al., 2014) and LC-coupled mass spectrometry (MS) (Jeszka-Skowron and ZgolaGrzeskowiak, 2014). However, these analytical techniques are expensive, time consuming, and sophisticated. Electrochemical methods are comparatively advantageous, offering better sensitivity, simplicity, and low fabrication cost with an integrity for portable device, suitable for on-site use. Electrochemical methods recently received extensive interest for the analysis of various natural productderived constituents such as flavonoids. Flavonoids are an essential dietary component found in nutrimental plants with a broad range of physiological functionalities such as antioxidant, antiinflammatory, antitumor, antiallergic, antimicrobial/viral, and hemostatic (Guo and Wei, 2008; Park et al., 2002; Tao et al., 2007; Miao et al., 2014). Furthermore, flavonoids are redox active component suitable for studying electron transfer reaction at the interface (Mulazimoglu and Mulazimoglu, 2013). Fabrication of modified electrodes with improved current


sensitivity, stability and antifouling ability are the intrinsic parameters determining the better sensor platform (Hajian et al., 2014; Liu et al., 2017; Vilian et al., 2015). Owing to its synergistic electrocatalytic and mechanical strength, various nanomaterials are modified on the surface of graphene/GO nanosheets and formulated as a sensor platform for the quantification of flavonoids. For instance, fabricated the graphene-MnO2 nanocomposite modified carbon ionic liquid electrode and demonstrated the electrochemical detection of rutin (flavonoid glycoside compound), with a lowest detection limit of 2.73 nM/L. Yang et al. (2014) fabricated a flower-globular terbium hexacyanoferrate (TbHCF) particles-modified graphene (GR) on a carbon paste electrode (CPE) and studied the electrochemical behavior of rutin. Under studied experimental conditions, TbHCF/GR/CPE exhibited rutin with a broader detection range of 1.0 3 10a10 M/L a6.0 3 10a6 M/L with a lowest detectable concentration of 0.04 nM/L. This study also utilized the developed electrode to determine the concentration of rutin in pharmaceutical tablets. The same group has also formulated rGO-CPE with yttrium hexacyanoferrate NPs for nanomolar detection of detection (Yang et al., 2015). Likewise, poly (diallyldimethylammonium chloride)-functionalized graphene (Miao et al., 2014) was utilized for detection of rutin and quercetin in pharmaceutical and human plasma samples. Veerapandian et al. (2014b) developed Ag@SiO2/PEG hybrid nanoparticles (particle size B12.35 nm) modified GO sheets for direct electrochemical detection of quercetin (3,30 ,40 ,5,7-pentahydroxyflavone). In this study the functionalized GO (FGO) electrode observed to have an anodic peak potential at nearly 10.32V, which is similar to one of the inherent anodic peak potentials of quercetin (10.15, 10.30, 10.60 and 10.80V, ascribed to the OH groups of quercetin). Accordingly, the fundamental change in the electrochemical oxidation reaction at the FGO electrode interface was monitored, without requiring additional reagent. The strategy described herein showed a lowest detection limit of 3.57 nM quercetin (Veerapandian et al., 2014b). Baicalin is another flavonoids having antiinflammatory and other biological effects including antiallergy, free radical scavenging, and apoptosis in breast and prostatic cell lines. Sheng et al. (2017) demonstrated a simultaneous reduction of GO and Co21 to produce coamino-graphene using glycine as a reducing agent. An electrochemical sensor based on a co-amino-graphene nanocomposite is designed and demonstrated for the detection of baicalin. The observed linear range of detection is 1.0 3 10a8a8.0 3 10a7 M/L, with a lower detection limit of 5.0 3 10a9 m/L. Apart from the previously mentioned sensor design, other combinations of graphene hybrid electrodes are developed for flavonoid sensing, such as CeO2-poly(diallyldimethylammonium chloride)-graphene for eriocitrin (Wang et al., 2017), thioβ-cyclodextrin functionalized graphene/palladium NPs for rutin (Liu et al., 2017), and mesoporous NiCo2O4-decorated rGO electrodes for the detection of rutin and isoquercetin (Cui et al., 2017). In addition to natural products, other pharmaceutical or biochemical compounds are also quantified using electrochemical methods. For instance, Chen et al. (2016) formulated Au/ZnO hybrid nanocatalyst impregnated N-doped graphene electrode for simultaneous detection of ascorbic acid,

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acetaminophen, and dopamine. These studies provide insight on the synergistic electrochemical property of graphene hybrids and also enable the utility of electrochemical sensing approach for pharmaceutical analysis.

10.5 SUMMARY AND FUTURE PERSPECTIVE Integration of high performance materials in biomedical devices has gained important attention globally. Currently, nanotechnology driven products and their translational biomedical applications have shown promising utility in healthcare industries. For this purpose, several novel combinations of nanoscale materials such as carbon, metal, and polymer hybrid composites are progressively explored, with varied physicochemical features. Graphene and its derivatives are recently explored for several interdisciplinary applications, particularly after 2010, on the award of Nobel Prize in Physics to Andre Geim and Konstantin Novselov. To overcome the issues existing in large-scale synthesis of graphene derivatives and to enhance the physical/chemical and biological properties, several green chemistry or eco-friendly approaches were investigated, such as photoirradiation, utilization of biomolecular reduction, doping of bioactive moieties, and polymer hybridization, etc. Here, we attempted to review the recent reports on hybridized graphene nanomaterials particularly devoted for drug delivery, cyto-compatibility, and diagnostics, covering electrochemical biosensors. Considering the versatility of the 2D graphene and its derivatives, several active pharmaceutical ingredients are successfully functionalized and explored for various clinical applications, such as active carrier for transdermal drug delivery, antimicrobial, anticancer, photothermal therapy, cell-proliferation, wound healing and tissue engineering, to name few. In the case of electrochemical sensors, significant contributions are available utilizing hybrid graphene derivatives toward molecular diagnostics, Herein, focus was given to metabolic markers, infectious agents, and pharmaceuticals. Considering the optimistic possibilities of MEMS, especially microfluidicderived Lab-on-a-chip technology, bio-instrumentation engineering and its association with nanotechnology and amalgamation of high performance materials such as hybrid graphene derivatives have a lot of scope for real application. For instance, development of an implantable chip with simultaneous diagnosis and therapeutics, biosensor-integrated lab-chip for efficient bio-mimetic assay models, single chips for infectious agent detection and discrimination, nanobiosensors with multiplexed detection methods for biopharmaceuticals such as vaccines. Although some of these methods are already in exploration with hybrids of graphene nanomaterials, further optimization on real sample analysis with antiinterferent capability and reproducibility are essential needs which will indeed carry the translational scope.

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ACKNOWLEDGMENTS M. Veerapandian acknowledges the support of Department of Science and Technology, India for the DST-Inspire Faculty award (DST/INSPIRE/04/2015/002081).

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Further Reading

Zhou, K., Zhu, Y., Yang, X., Li, C., 2010a. Electrocatalytic oxidation of glucose by the glucose oxidase immobilized in graphene-Au-nafion biocomposite. Electroanalysis 22, 259264. Zhou, K., Zhu, Y., Yang, X., Luo, J., Li, C., Luan, S., 2010b. A novel hydrogen peroxide biosensor based on AugrapheneHRPchitosan biocomposites. Electrochim. Acta 55, 30553060. Zhou, K., Zhu, Y., Yang, X., Li, C., 2011. Preparation and Application of MediatorFree H2O2 Biosensors of Graphene-Fe3O4Composites. Electroanalysis 23, 862869. Zhou, L., Jiang, H.J., Wei, S.H., Ge, X.F., Zhou, J.H., Shen, J., 2012. High-efficiency loading of hypocrellin B on graphene oxide forphotodynamic therapy. Carbon 50, 55945604. Zhou, M., Zhai, Y., Dong, S., 2009. Electrochemical Sensing and Biosensing Platform Based on Chemically Reduced Graphene Oxide. Anal. Chem. 81, 56035613. Zhu, C.Z., Guo, S.J., Fang, Y.X., Dong, S.J., 2010. Reducing sugar: new functional molecules for the green synthesis of graphene nanosheets. ACS Nano 4, 24292437.

FURTHER READING Liu, Y., Yu, D., Zeng, C., Miao, Z., Dai, L., 2010. Biocompatible graphene oxidebased glucose biosensors. Langmuir 26, 61586160. Zhou, L., Wang, W., Tang, J., Zhou, J.H., Jiang, H.J., Shen, J., 2011. Graphene oxide noncovalent photosensitizer and its anticanceractivity in vitro. Chem.-Eur. J. 17, 1208412091.

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