Graphene Bioelectronics

Denmark

Preface

Ashutosh Tiwari , Editor

The legacy of graphene research at the interfaces of bioengineering and nanotechnology empowered bioelectronics innovations. Recently, graphene bioelectronics is a much broadly described arena in the cross-disciplinary field of biosensors. The leading scientists and graphene experts demonstrated the bioelectronics at nanoscale, and the bioengineering communities specified inputs for range of applications. This book is composed with expending field of graphene biomaterials, a wide span of biotechnological breakthrough, opportunities, possibilities, and compile challenges. It is the very first book on graphene bioelectronics: miniaturization of bioelectrode materials, bioelectrode interfaces, high-throughput biosensing platforms, and systemic approach for the development of electrochemical biosensors and bioelectronics for biomedical and energy applications. The adopted subject enables huge impact on other fundamental areas, such as advanced security, forensics, food, and the environmental monitoring. The chapters include the state of the art of two-dimensional (2D) bioelectronics and demand range of innovations in crosscutting disciplines starting from fabrication to applications.

The graphene and 2D-like nanomaterials permit exfoliation into atomically thick bioelectronic interfaces that percolate free bioelectronic movements into the 2D planes and regulate motion in the third plane with a nanometer thickness. Chapter 1 describes the various approaches to fabricate and characterize graphene and 2D-like nanomaterials, as well as demonstrate various electrochemical biosensors. The different biofunctionalization patterns of graphene and 2D-like nanomaterials are realized to enable high-performance enzyme-based, antibody-based, and DNA- or aptamer-based biosensors. Chapter 2 overviewed the synthesis, characterizations, and fundamental properties of vertical graphene-based electrochemical biosensors. The vertical orientation and open structures make it attractive to hold large amount of biofunctions for the bioelectronic submissions.

Chapter 3 discusses recent advancements in the metal alloy-graphene nanohybrid materials in biomolecule detection, including glucose, hydrogen peroxide, vanillin, methotrexate, dopamine (DA), chlorpyrifos, nifedipine, ascorbic acid (AA), nicotinamide adenine dinucleotide (NADH), DNA, RNA, and carcinoembryonic antigen. Typically, the metal alloy-graphene-based biosensors demonstrated excellent sensing performance, with good sensitivity, wide linear detection range, low detection limit, short time response, and long-term working and storage stability. Chapter 4 outlines the chemically modified graphene oxide with relatively enlarge surface plasmon resonance sensitivity and their applications in the clinical diagnostics such as protein-protein interactions for molecular detection of tumors. The bioelectronic sensors based on field-effect transistors (FETs) are widely reported due to their extremely high sensitivity, rapid response, and simple test procedure. The graphene-based FET biosensors are demonstrated for the detection of a range of biomolecules. Chapter 5 highlights graphene-based FET sensors for biomolecule detection including protein, DNA, living cell, and bacterium. The challenges and future prospective of graphene FET sensors in biomolecule sensing are also discussed.

Chapter 6 provides the recent developments of graphene-based electrochemical devices that have been applied in biomedical field, for the detection of a wide variety of analytes of interest ranging from small compounds to nucleic acids, antibodies, proteins, and bacteria. Focus interest on graphene-based lab-on-a-chip (LOC) devices capable to answer the important query introduced by the World Health Organization (WHO) toward the development of Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment free and Deliverable to end-users (ASSURED) devices and to replace traditional approaches in the biomedical self-diagnostic field. Chapter 7 emphasizes the emergent surface plasmon resonance (SPR) analysis of whole cells using graphene, electrochemically reduced graphene oxide (rGO), and graphene-oxide (GO)-coated plasmonic interfaces such as evaluation of adhesion strength of different Escherichia coli strains, for detection of Pseudomonas and on the use of whole cells, for example, Micrococcus lysodeikticus, as bioreporters. In this sequence, Chapter 8 discusses the recent trends in the development of label-free biosensors based on bioelectronic interfaces consisting of graphene decorated with DNA molecules, for example, conjugation of DNA onto graphene surface using covalent and noncovalent types of interaction toward the fabrication of biocompatible and conductive biosensing platforms with high specificity. The unique properties of the graphene/DNA biointerface can facilitate the selective detection of a wide range of molecular targets in the biomedical and environmental fields including nucleotides and metal ions. Likewise, Chapter 9 overviewed the various strategies and fabrication methods on the aptamer-functionalized graphene-based electrochemical analysis of tumor markers.

On the other hand, biofuel cells hold wide interests in the areas of bioelectronics, biomedicine, and applied bioelectrochemistry. The performance of biofuel cells is driven through creating biocompatible microenvironments for trapping biomolecules and enhancement of electron transfer kinetics with the new design, structural engineering, and smart architecture of nanostructured electrode materials. Chapter 10 offers an up-to-date overview of relevant advances in the research and development of enzymatic biofuel cells using graphene-supported functional composites as electrode materials, with the emphasis on the nanoscale engineering of electrode materials for enhancing the overall performance. The chapter focuses on several types of composite material systems, including metallic nanoparticle-decorated graphene materials, polymer-graphene, and metal hydroxide-graphene composites. Chapter 11 deals with different nanohybrids of graphene as anodes and cathodes improving the overall efficiency of microbial fuel cells (MFCs). Future perspectives that address some key challenges pertaining to the implication of graphene materials in MFCs are concluded at the end of chapter. In Chapter 12, recent advances in food analysis and quality monitoring using graphene-based nanomaterials, including graphene, GO, rGO, graphene quantum dots (GQDs), and their derivatives, are discussed. The chapter highlights current and future trends in graphene-based sensing devices used for food quality monitoring, miniaturization and development of portable devices, and smart food packaging technology to maintain the public health and reduce the waste of cost and food materials.

Moreover, graphene can be integrated into various flexible and stretchable electronic devices in a conventional, scalable fashion. The advantageous mechanical, electric, and optical properties of graphene and their integration with Internet-of-thing techniques could lead to a highly efficient, sophisticated, and cost-effective device for the real-time applications in electronics, energy-harvesting devices, sensors, and other systems. Recent research progress on graphene-based flexible and stretchable electronics and advantages of these next-generation device with their significant advancement from the past few years with a special emphasis on the advancement in wearable graphene-based bioelectronics as sensing and energy devices is discussed in Chapter 13. At first, the designing and fabrication steps used for making the devices are discussed. Afterward, the flexible, portable, stretchable, and wearable electronic devices enabled by graphene are summarized including chip-based or patch-based sensors, logic devices, and bioinspired or health-care devices. The chapter has been concluded by summarizing the challenges present in the field of graphene-based electronics and their potential solution, to improve their role in flexible and stretchable electronics. Chapter 14 discusses a wearable and flexible dry electrode design on graphene-based electrochemical nanomaterial substrate as a sensing platform. Graphene offers the possibility of continuous real-time measurements of wearable electrocardiography (ECG) monitoring for a miniaturized health-care application.

Thus, this book brings together innovative methodologies and strategies adopted in the research and developments of graphene bioelectronics. Well-known worldwide researchers deliberate subjects on (1) synthesis, characterizations, modeling, and properties; (2) state-of-the-art fabrication and design of bioelectrodes; and (3) innovative uses of graphene-based biointerfaces for range of bioelectronic applications. The book is written for readers from diverse backgrounds across chemistry, physics, materials science and engineering, nanoscience and nanotechnology, biotechnology, and biomedical engineering. It offers a comprehensive overview of cutting-edge research on graphene biosensors and bioelectronics. We acknowledge contributors and Mr. George Mishra for his hard work to produce this high-quality book.

Chapter 1

Graphene and 2D-Like Nanomaterials: Different Biofunctionalization Pathways for Electrochemical Biosensor Development

Riccarda Antiochia*; Cristina Tortolini*; Federico Tasca‡; Lo Gorton†; Paolo Bollella*    * Sapienza University of Rome, Rome, Italy

† Lund University, Lund, Sweden

‡ Universidad de Santiago de Chile, Santiago, Chile

Abstract

In the last decades, nanotechnology has played a key role in the electrochemical biosensor development based on the mediated and direct electrochemical communication between the biorecognition elements and the electrode surface. In particular, graphene and 2D-like nanomaterials (e.g., boron nitride nanosheets, graphitic carbon nitride (g-C3N4) nanosheets, and various transition metal dichalcogenides) have attracted an increasing interest due to their peculiar properties such as high specific surface area and the ease of biofunctionalization. Moreover, in the last few years, the successful integration of graphene and 2D-like nanomaterials with other nanomaterials such as metal nanoparticles, metal oxides, or quantum dots has dramatically increased the opportunities to develop novel electrochemical biosensors with highly enhanced performances, mainly due to the synergistic effects.

In this chapter, we would like to give the state of art of graphene and 2D-like nanomaterials employment for electrochemical biosensors development, by critically discussing the advantages and drawbacks. Successively, the discussion should be separately addressed to three different cases: (1) redox enzyme immobilization, (2) antibody immobilization, and (3) DNA/aptamer immobilization. Finally, we should critically define for which cases graphene and 2D-like nanomaterials are the most suitable electrochemical platform, within some conclusion remarks and future perspectives of both kinds of nanomaterials.

Keywords

Graphene; Graphene 2D-like nanomaterials (GLNs); Electrochemical biosensors; Biofunctionalization pathways

Chapter Outline

1 Introduction

1.1 Graphene and its Derivatives

1.2 Graphene 2D-Like Nanomaterials

1.3 Functional Comparison Between Graphene and GLNs

2 Graphene and GLNs for Electrochemical Biosensors Development

3 Graphene and GLNs Bio-Functionalization Pathway for Biosensors Development

4 Graphene and GLNs Based Redox Enzymatic Electrochemical Biosensors

4.1 Graphene-Based Redox Enzymatic Electrochemical Biosensors

4.2 GLNs-Based Redox Enzymatic Electrochemical Biosensors

5 Graphene and GLNs-Based Electrochemical Immunosensors

5.1 Graphene-Based Electrochemical Immunosensors

5.2 GLNs-Based Electrochemical Immunosensors

6 Graphene and GLNs-Based Electrochemical DNA/Aptasensors

6.1 Graphene-Based Electrochemical DNA/Aptasensors

6.2 GLNs-Based Electrochemical DNA/Aptasensors

7 Conclusions and Future Perspectives

Acknowledgments

References

Further Reading

Abbreviations

CPPU 1-(2-Chloropyridin-4-yl)-3-phenylurea

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

PBA-NHS 1-pyrenebutanoic acid succinimidyl ester

ATP adenosine triphospate

AFP alpha-fetoprotein

BN boron nitride

BSA bovine serum albumin

CA carbon aerogel

CILE carbon ionic liquid electrode

CNPs carbon nanoparticles

CEA carcinoembryonic antigen

CVD chemical vapor deposition

CR-GO chemically reduced graphene oxide

ChEt cholesterol esterase

ChOx cholesterol oxidase

nPd dendritic palladium nanoparticles

DET direct electron transfer

dsDNA double-strand DNA

ET electron transfer

ES electrophilic substitution

g-C3N4 graphitic carbon nitride

GC glassy carbon electrode

GOx glucose oxidase

AuNPs gold nanoparticles

Au-NH2 gold nanoparticles-NH2

GPH graphene

GLNs graphene 2D-like nanomaterials

GrONPs graphene oxide nanoparticles

GPHCS graphene-chitosan

HOPG highly oriented pyrolytic graphite

HRP horseradish peroxidase

ITO indium tin oxide

Fe3O4NPs iron(III) oxide nanoparticles

LDH l-lactate dehydrogenase

LOx l-lactate oxidase

MAA mercaptoacetic acid

MB methylene blue

MTMOS methyltrimethoxysilane

MoS2 molybdenum sulfide

MWCNTs multiwalled carbon nanotubes

NHS N-hydroxy succinimide

NuS nucleophilic substitution

PGE pencil graphite electrode

PrG photochemically reduced graphene

PANI polyaniline

PVP polyvinylpyrrolidone

PEG600 polyethyleneglycol

Ab1 primary antibody

rGO reduced graphene oxide

SPCE screen-printed carbon electrode

Ab2 secondary antibody

AgNSs silver nanospheres

ssDNA single-strand DNA

SDBS sodium dodecylbenzene sulfonate

SDS sodium dodecyl sulfate

TMOS tetramethoxysilane

Thi thionine

SnO2 tin dioxide

TiO2 titanium dioxide

TiO2-NPs titanium dioxide nanoparticles

TMDs transition metal dichalcogenides

TMOs transition metal oxides

WS2 tungsten disulfide

ZnO@Pd zinc oxide nanorods‑palladium nanoparticles

Acknowledgments

The authors would like to thank for financial funding the Swedish Research Council (Vetenskapsrådet project 2014-5908) and a scholarship of the Erasmus + Project Unipharma-Graduates, promoted by a Consortium of Italian Universities and coordinated by Sapienza Università di Roma. In particular, P. B. would like to acknowledge Patrizia Nadia Hanieh for carefully reading and continuously updating with her feedback. F.T. thanks the financial support of Fondecyt 11130167, and Dicyt 021342TG_DAS, and Proyectos Basales.

1 Introduction

In the last decades, graphene and its derivatives have become the most used 2D nanomaterials in science, medicine, and electronic engineering [1–3]. Graphene and its derivatives have been extensively used in electronic or optoelectronic devices in particular by considering chemical sensors and biosensors with electrochemical or optical transduction, due to their peculiar properties like fast electron transfer (ET), high thermal conductivity, high mechanical flexibility, optical transparency, and good biocompatibility [4,5]. Nevertheless, in last few years, many researchers have been investigating several new graphene 2D-like nanomaterials (GLNs) such as graphitic carbon nitride (g-C3N4), boron nitride (BN), transition metal dichalcogenides (TMDs), transition metal oxides (TMOs), and graphane in order to provide new engineered nanomaterials for the next-generation electronic devices [6]. All these nanomaterials represent a new source of 2D-layered nanomaterials considering both the structural similarities compared to graphene and its derivatives and some exotic electronic properties mainly related to their chemical structure [7,8].

In this book chapter, we would like to give a panoramic overview on the different biofunctionalization patterns of graphene and 2D-like nanomaterials in order to understand which are the best 2D nanomaterials to realize an enzyme-based, antibody-based, or DNA −/aptamer-based biosensor.

1.1 Graphene and its Derivatives

Graphene presents as single or double layer of carbon atoms linked by sp² bond in a honeycomb lattice [9], as reported in Fig. 1A. The different graphene layers are held together by weak van der Waals interactions along the distance of 3.35 Å [10,11]. However, graphene displays peculiar properties mainly due to the chemical structure where each carbon atoms is sp² covalently linked with other three carbon atoms in the 2D plane, so that one electron is freely moving in the third dimension generating high electronic conduction [12,13].

Fig. 1 (A) Structure of 2D graphene and its relation to other dimension carbon nanomaterials and (B) structure of graphene 2D-like nanomaterials. (A) Reproduced from Kulia et al. (2012) and (B) from J.C. Spear, B.W. Ewers, J.D. Batteas, 2D-nanomaterials for controlling friction and wear at interfaces. Nano Today 10 (2015) 301–314 with permission of Elsevier Science Ltd. (2012 and 2015).

During the last decade, several methods to synthesize graphene have been developed. There are basically two different classes of methods that can be resumed in top-down methods (e.g., graphite exfoliation through physical, chemical, or electrochemical procedures) and bottom-up methods (e.g., chemical vapor deposition CVD) [14–16]. The top-down methods are based on mechanical procedures that reduce interatomic van der Waals interactions between the graphene sheets, while the bottom-up methods consider mainly a chemical production with a low yield that does not allow to scale up the procedure to an industrial production [17,18]. The raw material mainly employed to synthesize graphene is graphite, which is naturally available with purity levels ranging between 80% and 98% due to the presence of nickel or iron as impurities, while considering the synthetic graphite is possible to achieve purity levels above 99% depending on the quality of carbon source and its thermal treatment [19].

Historically, graphene was synthesized the first time by using the mechanical exfoliation that allowed the fabrication of graphene sheets with minimal structural defects as detailed reported from Andre Geim and Konstantin Novoselov (both awarded with Nobel prize in Physics in 2010) using adhesive tape to separate graphene sheets from crystalline graphite [20]. Unfortunately, this method cannot be used for large-scale production, while it is useful to investigate physical properties and for some selected applications (e.g., field-effect transistor). The exfoliation can be also realized through the electrochemical oxidation of a carbon-based working electrode (e.g., highly oriented pyrolytic graphite HOPG) in a three-electrode electrochemical setup (working, reference, and auxiliary electrode usually platinum) [21]. The oxidation experiment is carried out in electrolyte diluted surfactant solution like sulfuric acid or poly(styrenesulfonate) in water-diluted sodium dodecyl sulfate (SDS), which acts during the intercalation process of negative charge from the electrolyte [22,23].

Among the graphene derivatives, we can mainly consider graphene oxide (GO) and reduced graphene oxide (rGO) [24,25]. However, also GO has been synthesized by exfoliation of graphite with strong oxidizing agents as fuming nitric acid, sulfuric acid (Hummer’s method), or a mixture of KMnO4 and NaNO3 in sulfuric acid as reported in the literature, while rGO was synthesized using reducing agents like hydrazine [26]. Although both GO and rGO have been discovered several years ago, their atomic structure has not been yet fully elucidated due to their amorphous character [27]. GO exhibits a layered structure similar to graphite, except for the higher oxygen content that is responsible for the higher interlayer distance and higher hydrophilic behavior [28]. Besides the epoxide groups (really reactive), GO presents several functional groups as carbonyl, hydroxyl groups, and phenols, which are useful in terms of biofunctionalization pathways with proteins, antibody, or aptamers/DNA in order to realize biosensors [29].

Besides the previously reported method, graphene can be synthesized by using thermal decomposition of SiC wafer under ultrahigh vacuum or chemical vapor deposition (CVD) growth on metal supports as ruthenium, nickel, or copper. Between these metals, ruthenium does not give a uniform layer thickness, while on copper, the graphene growth stops immediately after the first layer and nickel was found to be the best metal supports to produce high-quality graphene multiple layers [30]. According to Rafiee et al., nickel support is exposed to argon gas at roughly 1000°C with a production of methane, which is adsorbed on nickel support. The nickel‑carbon mixture is then cooled down in argon gas where carbon diffuses out from nickel layer to form graphene within the cooling process [31].

Finally, other methods have been suggested in order to simplify and scale up graphene and its derivative production like cutting and unraveling of multiwalled carbon nanotubes (MWCNTs) and unzipping carbon nanotubes with different methods (e.g., electrochemical, chemical, or physical) or epitaxial growth of graphene [32].

1.2 Graphene 2D-Like Nanomaterials

GLNs have attracted an increasing interest due to their particular chemical and physical properties (e.g., high electronic and thermal conductivity). In this class, there are several inorganic 2D-layered nanomaterials like BN, graphitic carbon nitride (g-C3N4), molybdenum disulfide (MoS2) [33,34], tungsten disulfide (WS2), TMDs [35], TMOs [36], graphane [37], and many others 2D nanomaterials [38,39]. However, some of GLNs present a lot of structural similarity with graphene, as shown in Fig. 1B, for example:

− BN (also called white graphene) is a promising 2D-layered nanomaterial that consists of alternating boron and nitrogen atoms in a hexagonal network [40].

− g-C3N4 shows up as multilayered GLN with interlayer weak van der Waals interactions, while carbon and nitrogen within the layer are covalently linked each other. g-C3N4 is based on two structural isomers, where the condensed tri-s-triazine is the most favorable in terms of energy.

− Graphane consists of carbon and hydrogen atoms in a 2D structure of covalently bonded hydrocarbon. It shows two different conformations: the chair structure with the hydrogen atoms alternating on both sides of the plane and a boat structure with the hydrogen atoms alternating in pairs.

However, the other GLNs present more a sandwich structure, as shown in Fig. 1B, like the following:

− TMDs, as for example MoS2 or WS2, exhibit a chemical formula MX2 where M are metal from groups IV, V, and VI, and X are chalcogenide atoms as S, Se, or Te with strong covalent interactions within the layer and weak interlayer van der Waals interactions. MoS2 presents a sandwich structure of Mo atoms between two layer of S atoms, covalently linked each other [41,42].

− TMOs displayed surprising superconductivity properties at the interface, while in the bulk part, they are well known to be insulating (e.g., LaAlO3 or SrTiO3) probably due to some complex electronic structure coulomb interactions at and between transition metal and oxygen ions [43].

Synthesis methods for inorganic 2D nanomaterials can be resumed as with graphene in two classes: (a) top-down methods (e.g., chemical, electrochemical, and physical exfoliation) and (b) bottom-up (e.g., CVD or thermal methods) [44].

In the last few years, GLNs have been synthesized mainly by using the mechanical exfoliation method [45], discovered by Geim and Novoselov in 2004 for graphene.

Among the top-down methods, mechanical exfoliation (scotch-tape method) has been used to produce high-quality BN monolayer or multiple layer as reported by Gorbachev et al. as proof of concept due to the low production yield [46]. Also, other inorganic nanomaterials have been synthesized with mechanical exfoliation as TMDs or TMOs as reported in the literature. However, there are other top-down methods consisting of ultrasonic radiation applied in certain solvents followed by exfoliation as shown by Coleman et al. [47] or the liquid-phase extraction that presents a high production yield from bulk to 2D-layered structure [48].

Moreover, within the bottom-up methods, CVD is the most used method to generate GLNs on large scale, by using silica nanoparticles as support or hard template to perform the deposition of a different layer, as reported in the literature [49,50].

1.3 Functional Comparison Between Graphene and GLNs

Recently, the success of 2D nanomaterials, as graphene and GLNs, has been connected to their particular physical and chemical properties, which are different in the bulk form. However, 2D nanomaterials display particular optical and electronic properties mainly related to the absence of interlayer intercalations, which are really important for the band structure determination. Thereafter, some differences in terms of mechanical properties are correlated to the particular geometry, quantum effects, and high surface-volume ratio [51,52].

Although graphene has been extensively investigated, most of the research efforts have been done to understand the electronic properties, probably derived either from its low dimensionality and the particular electronic band structure, where the electrons seem to lack their mass behaving like relativistic particles. Thus, regarding the microscopic structure, it has been proved that in the absence of defects, electrons may travel along few micrometers without any scattering phenomenon [53].

Finally, graphene displayed few proximity effects such as superconductivity and ferromagnetism mainly due to its 2D nature. All these important features make graphene very attractive for applications in electronics and sensing [54].

Contrary to what has been observed with graphene, for GLNs, physicochemical knowledge is still limited except for some TMDs and TMOs. Nevertheless, the potential properties of inorganic 2D nanomaterials, the ease of production, will be the drawn for future research either in terms of theoretical/physicochemical studies and practically as potential support in real devices’ developments (e.g., bioelectronics) [55].

2 Graphene and GLNs for Electrochemical Biosensors Development

NH2) that offer different biofunctionalization patterns through cross-linking, physical entrapment, or adsorption of biological interfaces onto the so-modified electrode surface [6,56,57].

As shown in Fig. 2, in the last decade, the number of papers reporting on the usage of graphene and its derivatives is exponentially increased up to 500 papers per year (e.g., 2015 and 2016; see Fig. 2), according to statistical data [58]. Nevertheless, the GLNs seem to be still in the early state concerning synthesis, chemical-physics characterization, and application in bioelectronics with < 100 papers in the last few years (Fig. 2), as reported on Web of Science [59–61].

Fig. 2 Statistical data for number of publication during the last 10 years about (A) graphene-based electrochemical biosensors and (B) GLN-based electrochemical biosensors with data collect from Web of Science.

In the enzymatic electrochemical biosensors based on direct electron transfer (DET), graphene and its derivatives provide the possibility to have an efficient wiring of several redox proteins, which can retain their intactness and activity [62,63]. Besides the biological stability aspects, graphene exhibits a high uniform distribution of electrochemical active sites compared with other different carbon structure like graphite, carbon nanotubes, or fullerenes, which may positively affect its electrochemical properties [64]. According to several electrochemical studies carried on graphene sheets, it is well known that the heterogeneous electron transfer from/to graphene sheet takes place on the edge of graphene sheet instead of the plane, where it is approximately zero [65]. It might contribute the presence of oxygen-containing groups on the edge of graphene sheet, which can speed up the electron transfer between redox protein and graphene-modified electrodes [66].

In the electrochemical immunosensors, graphene does not work out for the direct detection of antibody-antigen (Ab/Ag) interaction, which needs of an electrochemically active label (e.g., ferricyanide and hydrogen peroxide). In order to enhance the sensitivity concerning the detection of Ab/Ag interaction, there are two strategies to use graphene. In the first case, also so-called sandwich method, graphene can be used to wire the primary antibody, which interacts with the antigen followed by the secondary antibody labeled with a redox protein, usually horseradish peroxidase (HRP), which catalyzed the H2O2 reduction with an increased signal due to the presence of graphene [67]. Whereas in the second case, the graphene layer is placed close to the redox protein (labeling of secondary antibody), where it increases the amount of electrochemically active label produced from the redox enzyme, generating a larger signal [68].

In the electrochemical detection of DNA hybridization, graphene can contribute both through the direct detection of oxidative signal from DNA bases or by using an electroactive label [69]. The first platform presents the main advantage of being reagentless but as drawback lower sensitivity compares to the labeled method. In the first method, graphene plays a key role to increase the resolution of signal on the potential scale for different DNA bases like adenine (A), thymine (T), guanine (G), and cytosine (C). However, in the labeled mode, graphene might have the same role as in the electrochemical immunosensors [6].

3 Graphene and GLNs Bio-Functionalization Pathway for Biosensors Development

One very important aspect for the practical usage of graphene and GLNs in bioelectronics (e.g., electrochemical biosensors) is the biofunctionalization [70,71]. This section basically deals with different patterns of functionalization, covalent and noncovalent, for both graphene and GLNs.

Regarding graphene and mainly its derivatives (e.g., GO), the reaction is associated with a rehybridization of one or more sp² carbons into sp³ configuration with a simultaneous lack of electronic configuration [72]. There are four main routes achieving the covalent functionalization of graphene: nucleophilic substitution, electrophilic substitution, condensation, and addition [61,73,74], as shown in Fig. 3.

Fig. 3 Scheme of several functionalization pathways for graphene modification: (A) nucleophilic substitution, (B) electrophilic substitution, (C) condensation, (D) addition, and (E) noncovalent interaction. Reproduced from T. Kuila, S. Bose, A.K. Mishra, P. Khanra, N.H. Kim, J.H. Lee, Chemical functionalization of graphene and its applications, Progress in Materials Science 53 (2012) 1061–1105 by permission of Elsevier Science Ltd. (2012).

The nucleophilic substitution (NuS) occurs at the epoxy groups of GO. NuS is easily doable under mild reaction conditions (e.g., room temperature and water as solvent) [75,76]. Moreover, this method was considered a promising method for functionalization and biofunctionalization of graphene by using, for example, aliphatic or aromatic amines, amino acids, or amine-terminated biomolecules (e.g., enzymes, antibody, or DNA/aptamers) [77]. However, there are few drawbacks that may have to be taken into account during the reaction pattern: Long-chain amines need of high temperature and long-time reaction, and the products generally obtained from NuS pattern can be hydrophilic or organophilic lacking, in the last case, usability for biosensor development [78].

Afterward, another biofunctionalization pattern is the electrophilic substitution (ES) where hydrogen atoms displacement is promoted by an electrophile. One of the easiest way to realize an ES is the spontaneous grafting with aryl diazonium salts, while recently, another method has been developed through Friedel-Crafts acylation of GO [79].

Thus, hence, other two less useful methods of covalent modification are

− condensation reaction, which generally proceeds through the combination of two molecules to form one with a loss of entropy (e.g., diisocyanate, isocyanate, or with amine compounds by amides and carbamate ester bond, obviously in the presence of GO) [80];

addition reaction, which forms a larger organic molecule as, for example, the 1,3-dipolar cycloaddition of azomethine ylide on graphene surface (not so common) [81].

Besides the noncovalent biofunctionalization, we can consider the hydrophobic, van der Waals, and electrostatic interactions, which require the physical adsorption on graphene layer [82,83]. All these methods have been reported even for other nanomaterials [84–86].

On the other hand, functional groups can be added to graphene and to GLNs following the some of the previous principles reported for graphene. Sometimes, for GLNs, few modification pathways have been considered as doping procedures, which can alter both chemical and physical properties as, for example, with TMDs [87]. In fact, the substitution of V and Fe atoms for S in MoS2 can increase the magnetic moment as the doping with Au and Pd can induce it [88,89]. However, other studies have been carried on chemical functionalization of BN in order to tune the band gap [90]. This conception can be readapted also to graphene due to their high similarity on chemical structure [91].

Nevertheless, the knowledge on reaction patterns for graphene biofunctionalization has not been transferred yet to the new generation of GLNs, which surely will influence their diffusion in biosensors field as new low-cost nanomaterials with extraordinary electronic properties [92].

4 Graphene and GLNs Based Redox Enzymatic Electrochemical Biosensors

As reported above, graphene (GPH) has been extensively used as platform for electrochemical biosensor fabrication due to its peculiar features [93,94], which can be tuned through the combination with different nanomaterials such as ionic liquids, metal nanoparticles, and polymers to get graphene composites. The cooperation between the redox enzymes and the graphene composites improved the performances of the realized biosensors, especially the sensitivity and the selectivity, which plays a key role in real analytic applications [32,95]. So far, graphene has been combined with several redox enzymes as reported in literature. In this section, we focused mainly on two analytes for graphene, lactate, and cholesterol, which are of fundamental importance in clinical trials [96], while for GLNs, despite wider analyte range available, unfortunately, we found only few papers reporting on electrochemical biosensors. Nevertheless, the interest in the development of flexible biosensors as tools for health care and other possible applications has driven scientists to search for particular platform based on graphene and GLNs.

4.1 Graphene-Based Redox Enzymatic Electrochemical Biosensors

Nowadays, l-lactate is well known as biomarker to early assess the lactic acidosis, which is mostly related to the overproduction (mostly caused by circulatory, pulmonary, and hemoglobin transfer disorders) or underutilization of lactate (probably due to liver disease, inhibition of glucogenesis, pyruvate dehydrogenase deficiency, etc.) [97]. Lactic acidosis is also quite common in endurance-based activities such as the triathlon, cycling, or boxing as consequence of the incapability of aerobic metabolism to satisfy the energy body demand [98,99], whereas cholesterol is one of the most abundant steroids in the body, essential for maintaining a normal metabolism in humans. High cholesterol levels in the blood are related to several health diseases as diabetes, atherosclerosis, nephrotic syndrome, and liver disease [100].

For both analytes, the most useful enzymes to build electrochemical biosensors are oxidases (1) and dehydrogenases (2), which present different reaction pathways, as schematically reported below:

   (1)

   (2)

in order to get a current signal correlated with substrate concentration [101,102].

Briefly, we have reported few representative GPH-based electrochemical biosensors for lactate (Table 1) highlighting the different immobilization pattern herein used.

Table 1

Immobilization/Functionalization Method and Analytic Performances of Graphene-Based Enzymatic Electrochemical Biosensors for l-Lactate and Cholesterol

Abbreviations: 1-pyrenebutanoic acid succinimidyl ester (PBA-NHS), cholesterol esterase (ChEt), cholesterol oxidase (ChOx), dendritic palladium nanoparticles (nPd), glassy carbon electrode (GC), gold nanoparticles (AuNPs), graphene (GPH), graphene oxide nanoparticles (GrONPs), iron(III) oxide nanoparticles (Fe3O4NPs), l-lactate dehydrogenase (LDH), l-lactate oxidase (LOx), pencil graphite electrode (PGE), photochemically reduced graphene (PrG), polyaniline (PANI), polyvinylpyrrolidone (PVP), reduced graphene oxide (rGO), screen-printed carbon electrode (SPCE), sodium dodecyl benzene sulfonate (SDBS), titanium dioxide nanoparticles (TiO2-NPs), and zinc oxide nanorods‑palladium nanoparticles (ZnO@Pd).

a Functionalization methods are reported between the brackets.

Recently, a flexible biosensor based on GPH has been realized by transferring the nanomaterial on a flexible substrate as PET [103]. In this case, GPH sheets have been easily synthesized by CVD method and then transferred on PET, where two silver paste-based terminals were introduced as connectors and properly insulated by using epoxy glue to avoid any possible interference in the measurements. Graphene was further modified by incubating with a 5 mM 1-pyrenebutanoic acid succinimidyl ester (PBA-NHS) solution (2 h at room temperature), which is functionalized through van der Waals interactions between GPH and pyrene scaffold then able to covalently link the lactate dehydrogenase (LDH) due to butanoic acid succinimidyl ester. All measurements were carried out by applying a fixed potential of + 300 mV versus Ag | AgClsat and detecting a signal related to NADH reduction, indirectly connected with l-lactate reduction. The biosensor exhibited a wide linear range from 0.08 to 20 mM with a fast steady-state measuring time of 2 s, but the main advantage is represented by the possibility to run measurements under mechanical bending conditions.

An amperometric biosensor based on graphene composite including gold nanoparticles (AuNPs) and reduced graphene oxide (rGO) was developed for l-lactate detection [104]. rGO-AuNP suspension has been drop-cast on a pretreated screen-printed carbon electrode (SPCE), while LDH was immobilized by using sol-gel method. The enzyme solution was previously mixed with tetramethoxysilane (TMOS), methyltrimethoxysilane (MTMOS), polyethyleneglycol (PEG600), and an adequate volume of rGO-AuNPs suspension and then gently placed on the so-modified SPCE electrode. The so-prepared biosensor showed good analytic performances in terms of linear range (10–5 mM), selectivity, sensitivity (154 μA mM− 1 cm− 2) with a detection limit of 0.13 μM, and good operational stability. Finally, the device was tested on artificial serum samples concluding that compounds such as ascorbic acid, uric acid, and paracetamol do not interfere at all with the measurements.

COOH groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) [105]. Nevertheless, the biosensor showed a good sensitivity with a detection limit of 0.1 μM, fast response time (5 s), and smaller linear range (5–50 mM) compared with the previous paper probably due to the absence of metal nanoparticles, making possible to obtain similar analytic performances. Finally, the biosensor has been tested in real biological and food samples with analytic recoveries in the range 96%–98%.

Also, another lactate biosensor based on graphene composite has been developed by Casero et al. In this case, graphene was photochemically reduced and further combined titanium dioxide nanoparticles (TiO2-NPs) and then placed onto a glassy carbon electrode (GCE) [106]. The enzyme solution, lactate oxidase (LOx), was drop-cast on the so-modified surface. So far, the biosensor exhibited nice analytic performances, for example, low limit of detection (0.6 μM) and moderate linear range (2–400 μM) highlighting the effect of 2D and 3D nanomaterials combination on the current signal.

Herein, we analyzed an interesting dual biosensor based on graphene composite. In this work, Gu et al. synthesized a graphene composite based on the combination of zinc oxide nanorods‑palladium nanoparticles (ZnO@Pd) with graphene used as support, gaining sensitivity toward H2O2 due to the presence of ZnO@Pd but on the other hand also layer stability due to graphene [107]. In both cases, glucose oxidase (GOx) and LOx, we refer to first-generation biosensors because the signal is directly proportional to 2H+/2e− oxidation of H2O2, with good analytic performances as linear range (20–500 μM in both) and detection limits of 2.39 and 2.52 μM for glucose and l-lactate, respectively. The dual-enzyme oxidase biosensor based on graphene composite (GPH-ZnO@Pd) was introduced in an online microdialysis system, which made possible the continuous and simultaneous monitoring of glucose and l-lactate in the brain of freely moving