Graphene and its derivatives as biomedical materials: future prospects and challenges

Abstract

Graphene and its derivatives possess some intriguing properties, which generates tremendous interests in various fields, including biomedicine. The biomedical applications of graphene-based nanomaterials have attracted great interests over the last decade, and several groups have started working on this field around the globe. Because of the excellent biocompatibility, solubility and selectivity, graphene and its derivatives have shown great potential as biosensing and bio-imaging materials. Also, due to some unique physico-chemical properties of graphene and its derivatives, such as large surface area, high purity, good bio-functionalizability, easy solubility, high drug loading capacity, capability of easy cell membrane penetration, etc., graphene-based nanomaterials become promising candidates for bio-delivery carriers. Besides, graphene and its derivatives have also shown interesting applications in the fields of cell-culture, cell-growth and tissue engineering. In this article, a comprehensive review on the applications of graphene and its derivatives as biomedical materials has been presented. The unique properties of graphene and its derivatives (such as graphene oxide, reduced graphene oxide, graphane, graphone, graphyne, graphdiyne, fluorographene and their doped versions) have been discussed, followed by discussions on the recent efforts on the applications of graphene and its derivatives in biosensing, bio-imaging, drug delivery and therapy, cell culture, tissue engineering and cell growth. Also, the challenges involved in the use of graphene and its derivatives as biomedical materials are discussed briefly, followed by the future perspectives of the use of graphene-based nanomaterials in bio-applications. The review will provide an outlook to the applications of graphene and its derivatives, and may open up new horizons to inspire broader interests across various disciplines.

1. Introduction

The ‘wonder material’ called graphene is a two-dimensional (2D) sheet of sp²-hybridized carbon atoms packed into a honeycomb lattice (figure 1a). It shows tremendous potential in the fields of materials science, physics, chemistry, biology and others, ever since the few-layer graphene had been exfoliated from graphite [3–5]. Following the advent of other forms of carbon nanostructures such as fullerene (which is formed by wrapping up the 2D graphene layer into 0D molecule, figure 1b), carbon nanotube (formed by cylindrically rolling the 2D graphene layer into 1D nanotubes, figure 1c), graphite (2D graphene layers are stacked into 3D structure, figure 1d), the emergence of graphene has opened up several exciting new fields in science and technology [1,6], because of its many unique and fascinating properties. For example, charge carriers in graphene behave like massless relativistic particles or Dirac fermions due to the pinching off of the conduction and valance bands at the Brillouin zone corners (zero band gap), leading to a linear dispersion of the energy spectrum [7]. The metal-like behaviour can be tuned to semiconducting nature by creating graphene nanoribbons with controllable size. Graphene nanoribbons narrower than 10 nm revealed semiconducting properties due to quantum confinement effect, whereas for larger widths, the properties are largely dependent on the edge configurations [6]. Also, due to the delocalized out-of-plane π bonds arising from the sp² hybridization of carbon atoms, extraordinary electrical conductivity (mobility of charge carriers approx. 200 000 cm² V⁻¹ s⁻¹) is observed [8,9]. Graphene also showed the unusual half-integral quantum Hall effect for both electrons and holes at room temperature [10]. It also has some other unique properties like high mechanical strength (nearly 200 times stronger than steel with Young's modulus approx. 1100 Gpa), exceptionally thin nanostructure (almost 1 million times thinner than human hair), very high specific surface area (approx. 2630 m² g⁻¹), excellent thermal conductivity (approx. 5000 W m⁻¹ K⁻¹), high stretchability, full flexibility yet high impermeability, chemical inertness and intrinsic bio-compatibility [3–5].

Figure 1.

Schematic structures of (a) graphene layer (2D), (b) fullerene molecule (0D), (c) carbon nanotubes (1D) and (d) graphite (3D) (Reproduced with permission from [1] © 2009 American Physical Society). (e) Schematic of different derivatives formed from graphene (Reproduced with permission from [2] © 2014 Royal Society of Chemistry). (Online version in colour.)

Additionally, the recent advent of simple, cost-effective and scalable fabrication techniques for single-layer graphene made it one of the most attractive nanomaterials, which is having applications in a wide range of fields, including nanoelectronics, composite materials, energy technology, sensors and catalysis, among others [11–13]. Therefore, these unique properties, alongwith the biocompatibility of graphene and the realization of its biological and chemical functionalization processes [14–16] have attracted much attention in the scientific community for numerous potential applications in biotechnology, including biosensing, diagnostics, antibacterial/antiviral activity, anti-cancer activity, photothermal therapy (PTT), targeted drug delivery, electrical stimulation of cells and tissue engineering, among others [17–28].

2. Derivatives of graphene

Also, the recent advent of the other forms/derivatives of graphene (the so-called ‘cousins' of graphene) has attracted much attention for the potential applications in various fields including nano-biotechnology [2]. Various forms of graphene derivatives are schematically presented in figure 1e. Some of these include the following derivatives.

2.1. Graphene oxide

Graphene oxide (GO) has revealed some unique physico-chemical properties such as small size, large surface area, exceptional strength in 2D structure, interesting optical and electronic properties, among others [29,30]. As exfoliated graphene (from graphite) is hydrophobic in nature, it is not readily dispersible in water, and, hence, functionalization is a bit challenging, whereas GO, upon oxidation, becomes hydrophilic, and therefore becomes water soluble. Owing to this, GO attains excellent aqueous processability, amphiphilicity, surface functionalization capability, fluorescence quenching ability, etc., for promising biotechnological applications [31]. Additionally, nanocomposites of GO with polymers, gold and various magnetic nanoparticles have led to numerous biotechnological applications in phototherapy, bio-imaging, drug and gene delivery, biosensing, and antibacterial action, etc. Particularly, the π-π* transition leads to low-energetic electron movement, which favours biosensing and bioimaging capabilities of GO. In order to achieve good biomedical applications, the preparation and functionalization of GO should be properly done. Currently, GO is produced by Hummers methods (and its different variants) [32,33], which is basically the chemical treatment of graphite through oxidation, with subsequent dispersion and exfoliation in water or suitable organic solvents. Figure 2 (upper part) schematically represents the formation of GO from graphene (or graphite).

Figure 2.

Schematic of the formation of reduced graphene oxide (rGO) and graphene oxide (GO) from graphene (or graphite). Picture courtesy iInvestorIntel © 2017 InvestorIntel Corp. (Online version in colour.)

2.2. Reduced graphene oxide

Fabrication of graphene from GO has now become very important area of research, because it can be produced using inexpensive graphite as raw material by cost-effective chemical methods with a high yield. Also it is highly hydrophilic and can form stable aqueous colloids to facilitate the assembly of macroscopic structures by simple and cheap solution processes. Both of these attributes are very important for biomedical applications. The main process to convert GO to graphene is the reduction of GO via thermal, chemical and electrochemical treatments, which produces reduced graphene oxide (rGO), having much lesser number of oxygen functional groups than that of GO. Different reducing agents will lead to various carbon-to-oxygen ratio and chemical compositions in rGO. Even though it is not possible to fully reduce the GO structure to get pristine graphene, the cost-effectiveness and the controllability of the oxygen functional groups make rGO very attractive for biological applications [34]. Figure 2 (lower part) schematically represents the formation of rGO from GO.

2.3. Graphane

Graphane is the fully hydrogenated derivative of graphene, having CH composition. The addition of hydrogen to graphene causes the hybridization of carbon atoms to convert from the flat sp² to the tetrahedral sp³. This modification transforms the conducting graphene into the dielectric graphane, i.e. the zero bandgap of graphene is opened up to create a bandgap in graphane, thus boosting the prospects of graphane-based nanoelectronics and hydrogen-fuel technologies [35,36]. The binding energy (BE) calculations of graphane have revealed that the BE of graphane is higher than the other forms of hydrocarbon like benzene and acetylene [2], thus making graphane as the most stable extended 2D form of hydrocarbon with a stoichiometric formula unit of CH, leading to its potential applications in biotechnology. Reversible synthesis of graphane was done by exposing graphene to argon-diluted cold hydrogen plasma at low-pressure and temperature [37,38]. This process generally converts graphene into fully hydrogenated graphane via intermediate partially hydrogenated structures, which leads to the gradual changes of the electronic properties of the material. It is therefore expected that controlled incomplete hydrogenation of graphene may provide materials with the desired properties. Similarly, hydrogenation of predefined domains of a graphene sheet to form graphane could be the way to ‘cut out’ graphene fragments of desired shape, which can have potential applications in bio-delivery carriers. A schematic of the hydrogenation of graphene to graphane is shown in figure 3a.

Figure 3.

(a) Schematic of graphane from graphene sheet via hydrogenation (Picture courtesy Chemistry World (James Urquhart) © 2017 Chemistry World). (b) Geometry of graphone sheet (A) top view, (B) cell configuration, (C) side view (Reproduced with permission from [39] ©2015 Royal Society of Chemistry). (Online version in colour.)

2.4. Graphone

Semi-hydrogenated derivative of graphene is called graphone [40]. Although this material is yet to be realized through experimentations [41], but theoretical calculations revealed that unhydrogenated carbon atoms in graphone couple antiferromagnetically [42], and therefore, found potential applications in magnetism, spintronix, organic ferroelectrics, molecular packing [39,43–47], etc., some of which would be useful for biotechnology. Structurally, graphone is 50% hydrogenated graphene sheet having a stoichiometry C₂H with the hydrogen atoms bonded only on one side of the carbon sheet, resulting in a mixture of sp² and sp³ hybridized carbon atoms. Upon geometry relaxation, it was found that graphone has a somewhat zigzag shape as can be seen in figure 3b [39,45].

2.5. Fluorographene

Fluorinated graphene derivative is called fluorographene, which is a monolayer of graphite fluoride [2,48], and considered to be the thinnest insulator with a wide electronic band gap (figure 4a). It is also the counterpart of polytetrafluoroethylene (Teflon) as it is basically perfluorinated hydrocarbon [49,50]. Since its discovery, fluorographene has attracted remarkable attention from the scientific community due to its extraordinary physical and chemical properties. Numerous studies show that fluorographene is not chemically inert and undergoes various chemical reactions under ambient conditions. Owing to this unusually high reactivity, fluorographene provides an efficient strategy for synthesis of tailored graphene derivatives such as graphene acid, cyanographene and allyl-graphene with a high degree of functionalization [51,52]. Fluorographene was prepared by fluorination of graphene and mechanical or chemical exfoliation of graphite fluoride [49,50] (figure 4b). Owing to the interesting properties of fluorographene, it has found applications in wide ranging fields spanning sensing and bioimaging to separation, electronics and energy technologies [48].

Figure 4.

(a) Schematic structure of fluorographene (Picture courtesy Doc Brown's Chemistry website (www.docbrown.info). (b) Syntheses strategies of fluorographene via (A) exfoliation, (B) fluorination (the picture is adapted under the terms of the Creative Commons Attribution License (CC BY) from [48]). (Online version in colour.)

2.6. Graphyne and graphdiyne

Graphyne and graphdiyne are derivatives of graphene, which are one atom thick 2D sheets of sp and sp² bonded carbon atoms arranged in a crystal lattice, and having acetylenic linkages (sp hybridization) connecting the hexagons of graphene (benzene rings with sp² hybridization), as demon­strated in figure 5. The graphyne structure has one linear acetylenic chain connecting carbon hexagons, whereas graphdiyne consists of two acetylenic (di-acetylenic) chains in between carbon hexagons [53–55]. Both graphyne and graphdiyne are semiconductors with band gaps around 0.5–0.6 eV, having considerably small effective masses for carriers [56]. Graphyne generates great interest within the scientific community due to its unusual electronic structure, mechanical strength and optical properties, whereas graphdiyne shows very interesting electronic properties [2,57,58]. Both graphyne and graphdiyne were synthesized by organic syntheses procedures involving modern acetylene chemistry and new organometallic syn­thetic methodologies based on dehydrobenzo annulene framework [59]. The applications of graphyne and graphdiyne include nanoelectronics, desalinator, inorganic and organic sensors and other bio-related processes [2,57,58].

Figure 5.

Schematic structures of (a) graphene, (b) graphyne, (c) graphdiyne. Corresponding benzene rings connected with the acetylenic linkages are shown in the right. Red parallelograms are unit cells and blue squares are showing the acetylenic linkages (adapted with permission from [2] © 2014 Royal Society of Chemistry).

2.7. Doped graphene

Apart from the realization of various graphene derivatives, chemical doping of graphene becomes an interesting field because of the easy manipulation of the physico-chemical properties of graphene, which has allowed developing new materials with tailored properties for specific applications in diverse fields, such as energetics, sensors, photovoltaics, supercapacitors, superconductivity, nanoelectronics, magnetism, catalysis and biomedicine, among others [60]. Various non-metals (such as N, B, S, P, Se, O, Si, I, etc.) and metals (such as Mn, Fe, Co, Ni, Al, Ti, Pd, Ru, Rh, Pt, Au, Ag, etc.) have been introduced into graphene as dopants, which have shown quite interesting properties, including superconductivity, ferromagnetism, and enhanced chemical/electrochemical activity, for diverse applications [60–66]. Among these dopants, N and B have been used mostly, as these elements impart n- and p-type semiconductivity, respectively, in doped graphene to expand its potential application in semiconductor devices [61,62]. N and B co-doped graphene has also been explored to show interesting gas adsorption and catalytic properties [60]. N, P, F tri-doped graphene has exhibited excellent electrocatalytic activities for the oxygen reduction reaction, oxygen evolution reaction and hydrogen evolution reaction [63]. On the other hand, metal-doped graphene has shown excellent adsorption capability of toxic compounds for environmental remediation, electrocatalysis for renewable energy applications, electrochemical sensors and supercapacitive charge storage devices [65,66]. As far as biomedicinal applications of doped graphene are concerned, N-doped graphene has exhibited electrochemical biosensing properties, whereas B-doped graphene has been used as chemical sensors for biomolecules, support for cell culture and controlled drug release. Also B-doped graphene quantum dots (GQDs) have shown excellent DNA/RNA detection and other bio-imaging capabilities. Similarly, B and N co-doped graphene has exhibited potential candidature as nanoprobes for biosensing and bio-imaging applications [62].

Therefore, the advent of graphene and its derivatives and the realization of their biological and chemical functionalization processes [24,67] has made the graphene-based nanomaterials highly attractive for biomedical applications. Indeed, after the pioneering work on the use of GO as an efficient carrier for drug delivery [24], a new horizon has been opened up to explore the use of graphene-based nanomaterials for widespread biomedical applications. Although relatively new, this research area has become highly attractive and expanding rapidly with wide ranging applications, such as biological sensing and imaging, drug/gene delivery, antibacterial materials, biocompatible scaffold for cell culture, stem cell differentiation, mass spectrometry, etc. [67–72]. In fact, a recent study revealed that the biomedical applications of graphene and its derivatives supersede the non-medical applications. Figure 6a depicts that the biomedical applications of the graphene and its derivatives are almost 63% as against 37% for the non-medical applications [73]. The broad biomedical applications of graphene and its derivatives in some major fields are pictorially presented in figure 6b [71].

Figure 6.

(a) Comparative chart depicting biomedical applications of graphene and its derivatives against non-medical applications (adapted with permission from [73] © 2013 American Chemical Society). (b) Some major fields of biomedical applications of graphene and its derivatives (adapted with permission from [71] © 2013 American Chemical Society). (Online version in colour.)

3. Graphene and its derivatives as biosensing and bio-imaging materials

Owing to the unique chemical, electrochemical, optical, electrical and electronic properties of graphene and its derivatives, and the bio-functionalization of graphene-based nanomaterials with various biomolecules and cells along with their improved biocompatibility, solubility and selectivity, graphene and its derivatives reveal some interesting applications in optical/electrochemical sensors, bio-imaging, electronic devices, mass spectroscopy, etc. [74]. For example, a graphene-derivative/oligonucleotide nanocomplex is used as a platform for in situ sensing of biomolecules, DNA detection and analysis, in vivo imaging of biomolecules in living cells, protein detection, sensing of heavy metals, pathogens, etc. As far as the in situ sensing of biomolecules is concerned, graphene-field-effect-transistor-based biosensors have been extensively explored for the detection of nucleic acids, proteins, etc., and the growth factors have been successfully demonstrated by using appropriately functionalized graphene derivatives with nucleic acids, aptamers and carbohydrates for monitoring target-specific changes of electrical signal with high signal-to-noise ratio [19,28,71,75–78]. Figure 7a schematically illustrates the detection of vascular endothelial growth factor (VEGF) via nitrogen-doped graphene FET biosensor [76]. Similarly, figure 7b depicts a thermally reduced graphene oxide (TRGO)-based FET device, where anti-immunoglobulin G (anti-IgG) is anchored to the TRGO surface through Au NPs and performs as the specific recognition group for the IgG binding [77]. Therefore, graphene-based field effect transistors (FETs) become an attractive tool for electrical sensors for biomaterials and processes (like DNA hybridization, hormonal catecholamine molecules, protein-binding events, heavy metals, etc.). Regarding DNA detection and analysis, figure 7c schematically illustrates the mechanism of the interaction of fluorescent-tagged DNA with functionalized graphene, where both single-stranded (ss) DNA and double-stranded (ds) DNA are adsorbed onto a graphene surface (processes A and B, respectively). With ssDNA having stronger interaction than dsDNA, the fluorescence on the ssDNA darkens more, thus improving the biosensing capability. The adsorbed ssDNA can be detached from the graphene surface when a complimentary DNA nears the ssDNA (process C). Additionally, the adsorbed DNA onto the graphene is protected from being broken down by enzymes, thus improving the sensing efficiency [78]. Because of the aromatic character of graphene and its derivatives, and its ability to keep various ionic components onto its basal plane (which leads to the unique capacity to absorb bio-molecules), graphene nanomaterials could become excellent biosensors in near future.

Figure 7.

(a) Scheme of nitrogen-doped graphene FET biosensor for detection of VEGF (adapted with permission from [76] © 2012 American chemical Society). (b) Schematics of graphene nanomaterial-based FET for electrical detection of protein binding events (adapted with permission from [77] © 2010 John Wiley and Sons). (c) Schematic of the interaction of DNA with graphene for biosensing applications (adapted with permission from [78] © 2010 John Wiley and Sons) (Courtesy of Pacific Northwest National Laboratory). (Online version in colour.)

Regarding the bio-imaging applications, a nanohybrid of graphene derivatives and fluorescent molecules is used as an in vitro and in vivo fluorescent cellular imaging probe [79]. For example, in figure 8a, the in vitro cytotoxicity study of HeLa cells reveals that multi-functional graphene (MFG) can be used as a biocompatible imaging probe. Similarly, figure 8b shows the in vivo images of zebrafish treated with fluorescent-tagged MFG and the study reveals no significant abnormalities, nor does it affect the survival rate after microinjection of MFG. Confocal laser scanning microscopic images have revealed that MFG locates only in the cytoplasm region and exhibits excellent co-localization and bio-distribution from the head to the tail in zebrafish [79].

Figure 8.

(a) In vitro CSLM images of control (upper row) and treated (lower row, incubated with 20 mg ml⁻¹ of MFG for 24 h) HeLa cells. In all images, the nuclei of HeLa cells were stained with DAPI blue dye. (A) and (D) The nucleus images were recorded with a DAPI filter. (B) and (E) The green fluorescence from MFG was recorded with a FITC filter. (C) and (F) The image is an overlap of respective stained nucleus and the fluorescence from MFG. (b) In vivo images of the distribution of MFG inside a fully developed (72 hpf) zebrafish using CLSM (A) fluorescence image of MFG with a FITC filter, (B) DIC image and (C) overlay image (the images are adapted with permission from [79] © 2011 Elsevier Ltd).

Also, GQDs' photoluminescent properties have been explored for intracellular imaging without any surface processing or functionalization processes. For example, nitrogen-doped GQDs (N-GQD) are used to assess the prospects as bioimaging material, where N-GQDs were introduced into the HeLa cells to show their bioimaging capabilities using confocal microscopy. As shown in figure 9a, bright green luminescence is observed inside the cells, indicating that the N-GQDs have been internalized by the HeLa cells and are mainly localized in the cytoplasm region and could be used as efficient bioimaging probes [80]. Also a GO-magnetic nanoparticle composite is used for enhancing the magnetic resonance imaging (MRI) signal for in vivo and non-invasive cellular imaging [81,83]. Figure 9b depicts that aminodextran-coated Fe₃O₄ nanoparticles were conjugated with GO, followed by cellular uptake and subsequent MRI [81]. Additionally, radio-labelled GO (GO conjugated with some antibody) has been used for positron emission tomography (PET) imaging (a nuclear medical imaging technique) for cancer cells (figure 9c) [82].

Figure 9.

(a) Confocal fluorescence microphotograph of HeLa cells incubated with N-GQDs for 2 h (A). Bright-field microphotographs of cells (B). An overlay image (C) (adapted with permission from [80] © 2013 The Royal Society of Chemistry). (b) Schematic and pictorial representation of GO-magnetic nanoparticle composite for cellular MRI (adapted with permission from [81] © 2011 American Chemical Society). (c) In vivo PET/CT imaging of 64Cu-labelled GO conjugates in 4T1 murine breast tumour-bearing mice (adapted with permission from [82] © 2012 American Chemical Society).

Electrochemical sensing of bio-macromolecules, enzymes and small molecules (such as H₂O₂, β-nicotinamide adenine dinucleotide {NAD⁺}, dopamine, glucose, etc.) have also generated great attention, where a nanohybrid (consisting of graphene/GO/reduced GO-metallic/organometallic compound) is used as the electrochemical sensor [84–86]. For example, a direct electrochemical DNA sensor is constructed, based on gold nanoparticles/graphene film, and the synthetic sequence-specific DNA oligonucleotides is successfully detected via differential pulse voltammetric method and it has been observed that the established immobilization-free biosensor has the ability to discriminate single- or double-base mismatched DNA (figure 10a). The importance of this electrochemical approach is that the process is green and fast, and unlike chemical reduction it does not result in contamination of the reduced material. Also at highly negative potential, it can reduce the oxygen functionalities of the GO more efficiently [84].

Figure 10.

(a) Baseline-corrected DPVs of (A) G and A, (B) T, (C) C at GCE (B), CRGNO/GCE (C), ERGNO/GCE (O) and Au-NPs/ERGNO/GCE (—), respectively. (D) Baseline-corrected DPVs of a mixture of G, A, T and C at GCE (B), ERGNO/GCE (O) and Au-NPs/ERGNO/GCE (—), respectively (adapted with permission from [84] © 2010 The Royal Society of Chemistry). (b) Schematic of an array of graphene transistors on quartz and the detection of blood cells via conductance variations of graphene (adapted with permission from [87] © 2011 American Chemical Society). (Online version in colour.)

Additionally, integration of graphene transistor arrays into a microfluidic channel leads to the ‘flow-catch-release’ sensing of Malaria-infected red blood cells. Figure 10b shows the schematic diagram of an array of graphene transistors on quartz. The electrodes are protected by a SU-8 photoresist that conveniently acts as the side wall for the microfluidic channel through which cells flow. Malaria-infected red blood cells induce highly sensitive capacitively coupled changes in the conductivity of graphene. Therefore, by observing the distinct changes in the conductivity, the infected cells are detected [87]. Also, interfacing graphene and its derivatives with micro/nanoscale biomaterials/components leads to the fabrication of some bio-electronic devices for detection of DNAs, proteins and pH sensors, etc. Also graphene-based nanomaterials are used as a novel matrix for mass spectrometry assay of biological molecules. The ultra-high specific surface area of graphene with double-sided aromatic scaffold provides extremely large loading capacity in surface-enhanced laser desorption-ionization (SELDI) probe for DNA detection and analysis [77,88,89].

Although majority of the above-mentioned graphene-based biomedical applications involve pristine graphene, GO and rGO, some of the newly developed graphene derivatives are also reported to show interesting and novel applications in biomedicine. For example, graphdiyne showed promising amino acid detection capability and can potentially be used in biosensing applications. Quantum electronic transport properties of graphdiyne–amino acid systems are compared with the transport properties of pure graphdiyne, and it has been revealed that the amino acid molecules induce distinct changes in the electronic conductivity of graphdiyne [58]. Similarly, theoretical modelling depicted that pristine graphyne has excellent desalination properties. For example, comprehensive molecular dynamics simulations and first principles modelling have exhibited that graphyne can achieve 100% rejection of nearly all ions in seawater at an exceptionally high water permeability, about two orders of magnitude higher than those for commercial state-of-the-art reverse osmosis membranes at a salt rejection of approximately 98.5%. This complete ion rejection by graphyne, independent of the salt concentration and the operating pressure, is found to be originated from the significantly higher energy barriers for ions than for water [90]. Therefore, functionalized graphyne can be used for cyanobacterial cultures to generate large biomass for bio-desalination process [91]. On the other hand, graphone, the semi-hydrogenated graphene, where only one side of the C atoms is saturated with hydrogen, is a well-known magnetic semiconductor [40], whereas hydroxylized graphone, which has been obtained by replacing the H atoms on graphone with –OH, is multiferroic due to the coexistence of ferroelectricity and ferromagnetism. Therefore, graphone derivatives have promising applications in organic ferroelectrics [92], thereby opening up promising opportunities in the field of molecular ferroelectricity, which has interesting links with biological ferroelectricity [93]. Likewise, graphane, the fully hydrogenated graphene, has revealed improved biosensing properties over graphene and thus may be used in novel biosensing devices. For example, graphane has exhibited different electrochemical behaviour towards oxidation/reduction of important biomarkers, such as ascorbic acid, dopamine and uric acid when compared to ordinary graphene [94].

4. Graphene and its derivatives as bio-delivery carriers

Some of the unique properties of graphene and its derivatives, such as large surface area, chemical purity, easy bio-functionalization, availability of delocalized π-electrons for easy solubility and bondage of drug molecules, high drug loading capacity of the double-sided graphene sheet, its lipophilic nature to help cell membrane barrier penetration for in vivo drug delivery, etc., made graphene nanomaterials highly promising for bio-delivery carriers for nanomedicinal applications [95]. For example, figure 11 schematically depicts the manipulation of the hydrophilic–lipophilic properties of graphene (blue hexagonal planes) through chemical modification, which would allow interactions with biological membranes (purple-white double layer) for drug delivery into the interior of a cell (blue region) [96]. In particular, after the seminal work on the in vitro loading and release of anti-cancer drugs by GO [24], a series of works has been published on in vitro controlled drug loading and targeted delivery of drugs via graphene and its derivatives as bio-delivery carriers for cancer therapy as well as other non-cancer treatments [68–73,96–101]. For example, a superparamagnetic GO–Fe₃O₄ nanohybrid is used as the delivery carrier for targeting agents towards tumour cells, whereas the drug release is controlled by pH values. Figure 12a represents the schematics of the multi-functionalized GO-based anti-cancer drug delivery carrier with a dual-targeting function and pH-sensitive intelligently controlled release system. Firstly, a superparamagnetic GO–Fe₃O₄ nanohybrid is prepared by the chemical precipitation method, followed by conjugation with folic acid (FA) onto Fe₃O₄ nanoparticles via imide linkage with amino groups of 3-aminopropyl triethoxysilane modified GO–Fe₃O₄ nanohybrid. Doxorubicin hydrochloride (Dox) as an anti-tumour drug model is then loaded onto the surface of this multi-functionalized GO via π-π stacking. Furthermore, the release of Dox has exhibited pH dependence due to the carboxylic acid groups on GO [97]. Similarly, the biocompatibility and the drug release behaviour of chitosan-functionalized graphene oxide via the controlled release behaviour of two drug molecules, ibuprofen and 5-fluorouracil, has been successfully demonstrated (cf. figure 12b) [98]. Likewise, surface-functionalized exfoliated graphene is used as a nanocarrier to release tamoxifen citrate (a breast cancer drug) in a time-dependent manner to selectively enhance the death of transformed cancer cells compared to normal cells [101].

Figure 11.

Schematic of penetration of graphene inside a cell for drug delivery (adapted with permission from [96] © 2012 Macmillan Publishers Ltd). (Online version in colour.)

Figure 12.

(a) Schematic of the preparation of the multi-functionalized GO based anti-cancer drug-carrier with dual-targeting function and pH-sensitivity (adapted with permission from [97] © 2011 Then Royal Society of Chemistry). (b) In vitro cell viability assay. (A) Relative cell viability of CEM cancer cell line and (B) MCF-7 cancer cell line incubated with GO, FGOCs and drug-loaded FGOCs graphene sheets at different concentrations for 5 d (s.d. ± 3%) (adapted with permission from [98] © 2011 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). (Online version in colour.)

Apart from the in vitro delivery of small drug molecules, graphene and its derivatives are reported to show the capability of in vitro gene transfection, which is very important to treat various diseases caused by genetic disorders, including cystic fibrosis, Parkinson's disease and cancer. For example, plasmid DNA (pDNA), silencing RNA (siRNA), etc., are loaded in graphane derivatives for intracellular transfection into cancer cells with improved cell killing efficacy [102–107]. Figure 13a schematically illustrates the functionalization of GO by polyethylenimine (PEI), forming a positively charged GO-PEI complex, followed by loading of negatively charged pDNA on GO-PEI complexes via electrostatic interactions. Figure 13b shows the gene transfection efficiency of GO-PEI complexes on HeLa cells with enhanced green fluorescence protein (EGFP) [102]. Another interesting study in this direction has been the interaction of DNA nucleobases and nucleosides with graphene. The relative interaction energies of the nucleobases as well as nucleosides are found to approximately decrease in the order of guanine (G) > adenine (A) > cytosine (C) > thymine (T) in aqueous solutions [106]. In the field of gene therapy, the synthesis of efficient and safe gene vectors is still not satisfactory and, hence, poses a major challenge in the development of gene therapy [108]. Therefore, the use of graphene and its derivatives in the field of gene delivery can open up new possibilities to treat genetic disorders.

Figure 13.

(a) Schematic of the synthesis of GO-PEI-DNA complexes via a LBL assembly process. (b) Confocal fluorescence images of EGFP transfected HeLa cells using PEI-1.2 k (A), GO-PEI-1.2 k (B), PEI-10 k (C) and GO-PEI-10 k (D) at varying N/P ratios from 10 to 80. Scale bar ¼ 50 mm. Obvious toxicity was observed for HeLa cells after PEI-pDNA treatment at the N/P ratio of 80 (the figures are adapted with permission from [102] © 2011 The Royal Society of Chemistry).

Other important in vitro therapeutic applications of graphane nanomaterials are phototherapies, such as PTT and photodynamic therapy (PDT), which are used to control disease by specific light irradiation [109–116]. The advantage of phototherapy is that graphene nano-agents can specifically target and kill cancer cells which are exposed to the light, without significantly damaging the normal organs in dark, thus exhibiting remarkable advantages in terms of enhancing the cancer killing specificity and reducing the side effects [31]. In PTT, because of strong optical adsorption in the near-infrared (NIR) region of graphene, graphene-based nanomaterials are targeted to the cancer cells followed by NIR radiation to generate sufficient heat for cancer cell killing. Figure 14a schematically depicts that when rGO quantum dots (QDs) are irradiated, cell death is manifested and the QD fluorescence is quenched, which is also substantiated by the fluorescent imaging, which shows that, before irradiation, the cells (incubated with the FA-QD-US-rGO) are viable (green) and the internalized QDs are fluorescent (red), and after irradiation essentially all of the cells are killed and the fluorescence is absent [110].

Figure 14.

(a) Schematic of PTT. (A) Reduced GO (rGO) QDs are irradiated to kill cancer cells. Fluorescence images of cells before (B) and after (C) irradiation (adapted with permission from [110] © 2012 John Wiley and Sons). (b) Modified Jablonski diagram depicting the process of photodynamic therapy. IC, internal conversion; ISC, intersystem crossing; PS, photosensitizer; ¹PS*, Singlet excited photosensitizer (adapted from [111] © 2015 MDPI AG; the figure is from an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited). (c) Schemes of the experimental design in photothermally enhanced photodynamic therapy (adapted with permission from [115] © 2011 American Chemical Society).

On the other hand, in PDT, graphene-based nanomaterials are irradiated with a suitable wavelength of light to generate reactive oxygen species (or free radicals), which irreversibly damage/kill the cancer cells. Figure 14b represents the modified Jablonski diagram depicting the process of photodynamic therapy. When photosensitizers (PS) in cells are exposed to specific wavelengths of light, they are transformed from the singlet ground state (S₀) to an excited singlet state (S₁–S_n), which is followed by intersystem crossing to an excited triplet state (T₁). This can follow two separate pathways. Type I reaction occurs when the excited molecule reacts with organic substrates (R) and produce radical ions or radicals. Type II reaction takes place when the energy is transferred from the excited photosensitizer to molecular oxygen (³O₂) to form reactive oxygen intermediates. These intermediates react rapidly with their surroundings including cell wall, cell membrane, peptides and nucleic acids [111].

For enhancement of therapeutic efficacy, combinatorial therapy like PTT and chemotherapy have been used. In some cases, a combination of PTT and PDT showed much improved cell killing efficacy via improved delivery of PS through PTT, followed by generation of cytotoxic singlet oxygen through PDT effect of functionalized graphene derivatives (figure 14c). Some groups reported high therapeutic efficacy with graphene-photocatalyst nanohybrid via combinatorial PDT and photocatalytic therapy [114–116]. For example, a graphane oxide/TiO₂ hybrid (GOT) is prepared which has generated reactive oxygen species under visible light irradiation both in cell free conditions and in vitro. After the exposure of cells to GOT under irradiation, a marked decrease in mitochondrial membrane potential, cell viability, activities of superoxide dismutase, catalase and glutathione peroxidase, as well as increased malondialdehyde production are observed. Moreover, GOT has caused significant elevation in caspase-3 activity and has induced apoptotic death. The results have indicated that GOT has highly improved photodynamic anti-cancer activity without dark cytotoxicity, due to the combinatorial effect of PDT and photocatalytic therapy [116].

Although considerable progress has been made on the exploration of graphene and its derivatives as delivery carriers and therapeutic applications in vitro, in vivo testing needs considerable attention for real-time treatments of clinical cancer and other diseases. Only a handful of reports has been published in this direction. The first report [117] in this regard has presented the intravenous administration of bio-functionalized graphene derivatives (labelled with an NIR fluorescence dye, but not carrying any drug) for passive tumour targeting in mouse xenograft models and killing the cancer cells via PTT. Figure 15 shows the in vivo PTT study using intravenously injected nanographene sheet (NGS), functionalized with polyethylene glycol (NGS-PEG). All irradiated tumours on mice, injected with NGS have disappeared 1 day after laser irradiation, leaving the original tumour site with black scars, which fell off about one week after the treatment. No tumour regrowth is noted in this treated group over a course of 40 days [117]. After this report, only few groups have published on the in vivo test of graphene nanomaterials as delivery carries and therapeutic applications via PTT and PDT [22,118–120]. For example, a nanographene oxide (NGO)–hyaluronic acid (HA) conjugate (NGO–HA) is synthesized for photothermal ablation therapy of melanoma skin cancer using an NIR laser [121]. The effect of size and surface chemistry of graphene derivatives on the in vivo photothermal cancer treatment have also been studied. It has been observed that the nanostructured reduced graphene oxide (nRGO), having a non-covalent PEG coating, is able to effectively target the tumour via the enhanced permeation and retention effect with relatively low reticuloendothelial system retention. Owing to the high tumour passive uptake and strong NIR absorption, nRGO-PEG is observed to be an excellent photothermal agent that enables highly efficient in vivo tumour ablation by using an ultra-low laser power density, which is an order of magnitude lower than that usually applied for in vivo PTT treatment of cancer using many other nanomaterials [119].

Figure 15.

In vivo PTT study using intravenously injected NGS-PEG. (a) Tumour growth curves of different groups after treatment. The tumour volumes were normalized to their initial sizes. There were six mice in the untreated, 10 mice in the ‘laser only’, seven mice in the ‘NGS-PEG only’ and 10 mice in the ‘NGS-PEG+laser’ groups. While injection of NGS-PEG by itself or laser irradiation on uninjected mice did not affect tumour growth, tumours in the treated group were completely eliminated after NGS-PEG injection and the followed NIR laser irradiation. (b) Survival curves of mice bearing 4T1 tumour after various treatments indicated. NGS-PEG injected mice after PTT survived over 40 days without any single death. (c) Representative photos of tumours on mice after various treatments indicated. The laser irradiated tumour on NGS injected mouse was completely destructed. Error bars in (a) were based on standard deviations (adapted with permission from [117] © 2010 American Chemical Society).

Another very important area of research is the study of haemocompatibility and macrophage response of graphene and its derivatives [120]. This is because, most of the biomedical applications (such as drug/gene delivery, biosensing/imaging and therapeutic applications) of graphene-based nanomaterials require the intravenous injection of these nanomaterials, and, hence, evaluation of their haemocompatibility is highly important. For example, the macrophage response of pristine and functionalized graphene, in terms of the potential uptake by macrophages, effects on its metabolic activity, membrane integrity, induction of reactive oxygen stress, haemolysis, platelet activation, platelet aggregation, coagulation cascade, cytokine induction, immune cell activation and immune cell suppression, have been analysed in detail. The results have indicated that the toxicity effects of pristine graphene towards macrophage cells can be easily minimized by surface functionalization of the graphene-based nanomaterials with very good haemocompatibility [120].

5. Graphene and its derivatives as substrate materials for cell culture, tissue engineering and cell growth

Graphene-based nanomaterials have been used as scaffolds for cell culture and tissue engineering and have exhibited minimal harmful effect on the mammalian cells, very good adhesion properties, excellent gene transfection efficacy, high capability of the promotion of differentiation of stem cells into bone cells in a controlled manner for bone regeneration therapy, excellent boosting ability for the neurite sprouting and outgrowth for neural interfacing [122–134]. For example, it has been demonstrated that GO-coated substrates have significantly enhanced the differentiation of mouse embryonic stem (ES) cells to both primitive and definitive haematopoietic cells. Not only has GO no effect on the cell proliferation or survival of differentiated cells, it also enhances the transition of haemangioblasts to haemogenic endothelial cells, which is considered to be a key step during haematopoietic specification. Additionally, GO also improves human ES cell differentiation to blood cells. Figure 16a schematically represents the mechanism of action of GO to induce the transition of haemangioblast to haemogenic endothelium, thereby promoting the generation of haematopoietic stem and progenitor cells [126]. Similarly, it has been reported that neurite outgrowth in human neuroblastoma cells is significantly increased when seeded on graphene, compared with control glass substrate, both in neurite extension length and number, as shown in figure 16b [128,130]. Recently, three-dimensional printable graphene composite, consisting of graphene and a biocompatible elastomer, has revealed that it supports human mesenchymal stem cell adhesion, viability, proliferation and neurogenic differentiation with significant upregulation of glial and neuronal genes (in vitro) and has promising biocompatibility over the course of at least 30 days (in vivo) [135].

Figure 16.

(a) Schematic of mechanism of action of GO. GO induces the transition of haemangioblast to haemogenic endothelium thereby promotes the generation of HSPCs (adapted from [126] © 2017 Macmillan Publishers Limited; the figure is from an open access article distributed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited). (b) Images show SH-SY5Y neuroblastoma cells cultured on glass and graphene for 7 days without or with retinoic acid (RA). Neurite outgrowth was increased by RA. Graphene alone induced neurite outgrowth, which was further stimulated by RA (adapted with permission from [130] © 2015 Elsevier Ltd). (Online version in colour.)

Graphene and its derivatives as well as graphene-based nanocomposites have also revealed inhibition effect of bacterial growth on their surfaces due to the synergistic effect with other materials, and the oxidative stress, induced by membrane disruption. Also, the antibacterial activity is found to be controlled by the size of the graphene derivatives with minimal skin irritation [136–141]. For example, GO and rGO nanosheets revealed effective inhibition of the growth of E. coli bacteria while showing minimal cytotoxicity [136,137]. It has also been reported that GO-polymer composites have higher bacterial toxicity against pure GO-modified surfaces [138]. The antibacterial activity of GO–Ag composites showed remarkably enhanced antibacterial activity compared with the pure Ag nanoparticles [140].

It must be mentioned in this connection that the bactericidal effect of graphene and its derivatives is controversial [141–144]. For example, some reports showed that the antibacterial property of GO against E. coli is maximum as compared to other graphene derivatives like rGO, etc., whereas other reports have revealed faster bacterial growth, when the culture medium is supplemented with GO. This phenomenon is explained by the stimulation of bacterial proliferation via GO, which acts as a surface for cellular attachment and growth [142]. It has also been reported that the purification status of graphene and its derivatives has a significant effect on their antibacterial properties. For example, highly purified and thoroughly washed GO has shown minimal antibacterial properties against both Gram negative and positive bacteria. On the other hand, insufficiently purified GO has shown to behave as an antibacterial material, which is considered to be due to the pH of the GO and its contamination with low molecular weight contaminants [144]. Since graphene and its derivatives can interact with various cellular components like membranes, proteins, DNA, etc., and initiate a sequence of nanomaterial–bacterial interactions that rely on colloidal energies and active bio-physico-chemical interfaces, therefore the analyses of these interfaces is highly necessary for the development of anticipated bactericidal activity that depends on different physico-chemical features (such as shape, size, hydrophilicity, roughness, functionality, dispersibility, concentration, purity, etc.) of the graphene-based nanomaterials [142–144].

6. Challenges for the development of graphene and its derivatives as biomedical materials

Although there has been a surge of scientific activity in the field of graphene-based nanomaterials for bio-applications, the field is still in the nascent stage, and several key challenges are yet to be addressed before this field can be fully commercialized. As far as the major challenges in the field of graphene and its derivatives for biosensing and bio-imaging applications are concerned, the most important issue is the reproducibility and reliability of the materials and processes. Unless cost-effective and scalable processes can be developed with high reproducibility, alongwith the reliability of the materials' properties, the field cannot be moved to the next stage for practical applications. Secondly, for commercialization, the businesses are to be convinced about the practical advantages of the use of graphene and its derivatives over conventional bio-assay tools. Although a large volume of studies has been published in this area of research in recent times, and in many cases the detection sensitivities are found to be better than the conventional methods, yet, the batch-to-batch variations of the graphene-based nano-biosensors are far from satisfactory, and need more attention [72].

Similarly, as far as the applications of graphene and its derivatives in drug/gene delivery and therapy are concerned, the major challenges are the realization of sufficiently high drug loading capability for practical uses, development of suitable in vivo drug distribution and release profiles, finding proper chemical modification processes of graphene and its derivatives for cell-membrane-barrier penetration for drug delivery into the interior of a cell, proper understanding of in vitro and in vivo toxicity profiles, bio-distribution, bio-compatibility and bio-degradability of graphene and its derivatives. In particular, for toxicity profile of graphene and its derivatives, which is very important for therapeutic applications, although several groups have started to explore the long-term and short-term cytotoxicity effects of graphene-based nanomaterials in vitro [24,136] and in vivo [79,117,119,145–148], this field is very much open as the toxic effects of graphene is highly dependent on its particular form, such as the size, morphology, chemical structure, purity as well as the specific application in which it is used for. In particular, systematic studies on the effects of graphene and its derivatives on the immune systems, reproductive systems, nerve systems, etc., have not yet been investigated properly. Additionally, compared to the conventional bio-degradable organic macromolecules as drug delivery carriers, graphene and its derivatives, being an inorganic nanomaterial, hardly degrade into the biological system, and, hence, cannot be used simply as a delivery carrier, unless some novel means are discovered to make it bio-degradable. Therefore, unless the above challenges are addressed properly, the graphene nanomaterial-based drug delivery and therapeutic technology is not likely to hit the market in the near future, given the clinical and regulatory hurdles posed by the pharmacovigilance agencies.

As far as the major challenges for the use of graphene-based nanomaterials in the fields of cell culture, tissue engineering and antibacterial activities are concerned, the most important challenge is the scaling of the technology, for which proper understanding of the mechanism underlying stem cell differentiation, clear knowledge on the relative antibacterial activity of different graphene derivatives, proper understanding of the experimental conditions for bacterial activity, proper detection technique of the antibacterial activity to determine the cause and effect, etc., need to be developed properly. For example, graphene showed different stem cell differentiation against its derivatives, which is assumed to be due to the different interactions between the growth agents and graphene's (and its derivatives') surfaces. Unless the mechanism is properly understood, it will be difficult to scale the technology for commercialization. Similarly, there are controversies about the bacterial growth and inhibition on graphene. Some reports suggested enhancement of bacterial growth on graphene surfaces, rather than the inhibition [149], which clearly indicates the dependence of the antibacterial activity on the experimental conditions, which needs to be standardized for practical applications. Also the detection mechanism of the antibacterial activity of graphene-based nanomaterials should be done in the molecular level to understand the mechanism properly and monitor the antibacterial activity at the molecular level. Additionally, graphene-based nanomaterials sometimes generate oxidative debris, which may induce cytotoxicity. Therefore, the purity of graphene and its derivatives during bio-functionalization process should be considered properly.

7. Conclusion and future direction of graphene and its derivatives as biomedical materials

The use of graphene and its derivatives as biomedical materials has become highly interesting research field. Although the new and exciting properties of graphene-based nanomaterials lead to some interesting biomedical applications and dramatic progresses have been made in this direction in recent times, yet this area of research is still in its infant stage and needs proper future directions to convert it into a market-oriented research field.

One of the most important and essential future goal for the therapeutic application of graphene and its derivatives would be proper understanding of the toxicity profile of this kind of new nanomaterials in vitro and in vivo. Also, the toxicity effect of graphene and its derivatives can be exploited positively, by considering any particular toxicity profile (arose from a certain bio-activity of graphene nanomaterials), to use in therapeutic applications, like antibiotic and/or anti-cancer treatments.

Another interesting future application of graphene and its derivatives should be the development of combinatorial therapies with high yield. This may include two or more of the following therapies like PTT, PDT, photoacoustic, photocatalytic, RF ablation, etc., with the conventional therapeutic drugs, which would be extremely useful to overcome the multi-drug resistance problems, able to decrease the doses of drugs and reduce the side effects (which often pose hurdles in the current chemotherapies), thus improving the tumour treatment efficacy [150–152].

Gene transfection in vivo will be very important future biomedical application area of graphene-based nanomaterials [153]. Although there are several in vitro studies on the bio-functionalized graphene and its derivatives as gene transfection vectors, but in vivo gene delivery, using graphene-based nano-vector, has yet to be explored properly. This can be addressed by realizing better and smarter design of surface chemistry of graphene and its derivatives, for future treatment of genetic disorders.

Although the bulk of the graphene-based bio-research is aimed at novel bio-applications, very few attempts have been made to realize cost-effective, scalable and reproducible syntheses methods of stable and reliable graphene-based nanomaterials. Additionally, new bio-functionalization methods have to be realized to prevent the graphene nanomaterials from agglomeration during biomedical applications [154]. Therefore, future research should be aimed at the fundamental aspects of the properties and processes of graphene research.

In the field of bio-imaging, although GQDs have shown great potential for its low toxicity in vivo, the productivity is quite low. Therefore, future goals should be directed at investigating various techniques to increase the productivity of GQDs with high quantum yields [155–157].

As far as the biosensing capabilities of graphene and its derivatives are concerned, future research should be aimed at understanding the fundamental molecular mechanism of the biosensors and the physical/chemical parameters related to the performance of the sensors to improve the sensing efficiency for market applications [158].

Another novel future bio-application of graphene and its derivatives is the combined biomedical investigation and therapeutic application. This would include the in situ localization/detection via biosensing, followed by real-time monitoring/cell imaging and subsequent drug delivery for therapy.

Recently, graphene-based nanohybrid films, consisting of graphene nanomaterial/other carbon nanomaterials/metal nanoparticles, showed enhanced laser desorption/ionization (LDI) efficiency [159]. Therefore, graphene-based nanostructured films, as platforms for LDI-mass spectrometry (LDI-MS) analyses for phospholipase activity, small molecules and mouse brain tissue can have interesting future applications.

In the field of antibacterial activities of graphene and its derivatives, future research should be directed to the study of the effects of time, concentration, size, density, purity and surface structure of graphene nanomaterials, to understand the underlying mechanism from a molecular perspective, to develop efficient nanomaterial antibiotics [160].

In the field of tissue engineering, a multidisciplinary approach is needed, as this field uses expertise in medicine, biology and engineering to develop biomimetic tissue constructs for organ transplantation as well as for diagnostic and therapeutic research [131]. Also, graphene derivatives, supported by other bio-molecules, have shown the potential of generating mechanically strong, electro-conductive hydrogels, which can have important future applications in the field of cardiac tissue engineering and regeneration. Similarly, there is tremendous potential for graphene-based nanomaterials in the fields of neural, bone, cartilage, skeletal muscle, skin/adipose tissue engineering and regeneration [131].

Another highly interesting and novel application of graphene-based nanomaterials is DNA sequencing, which would lead to personalized medicine. Different approaches can be attempted to use graphene nanodevices for DNA sequencing, which would involve DNA passing through graphene nanopores, nanogaps and nanoribbons, and the physisorption of DNA on graphene nanostructures [161].

Besides experimental works, theoretical investigations of various aspects of graphene and its derivatives, including electronic structure, doping effects, optical/electrical/electrochemical/mechanical properties and dependence of these properties on the number of layers, surface structures, functionalization, etc., are very important to understand the properties of these new nanomaterials at the molecular level. This may lead to the rational designing of the biomedical devices combining with theoretical modelling with advanced experimental techniques to significantly shorten the cycle of graphene nanomaterial-based biomedical research.

Apart from graphene, GO and rGO (pristine and bio-functionalized), exploration of other ‘cousins’ of graphene, such as graphane, graphone, graphyne, graphdiyne, fluorographene and their doped versions, as biomedical materials can become an interesting future research field. Since these new nanomaterials are still in the nascent stage, and many of them are yet to be realized experimentally, therefore, the development of scalable syntheses procedures, investigation of the electrical, optical, mechanical, chemical, electrochemical, electronic and surface properties, proper understanding of the toxicity profile and bio-compatibility of these nanomaterials must be explored comprehensively [162–166].

Therefore, to bring the bio-applications of graphene and its derivatives from the research laboratory to the clinic, interdisciplinary approaches among physics, chemistry, biology, materials science and engineering are important. This is because, many of the unique advantages, fascinating properties and potential applications of graphene and its derivatives are yet to be explored completely, and several challenges need to be resolved, and for that, an effective multidisciplinary collaborative approach must be taken, which will accelerate the mechanistic understanding of graphene-based nanomaterials for bioapplications and make current efforts more routinely implemented in diverse applications.

Data accessibility

All the data are presented from the published literature with permission.

Competing interests

The author declares no conflict of interest.

Funding

We received no funding for this study.