Graphene and Its Derivatives as Biosensing Platform for Healthcare Applications

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Ramchander Chepyala*, Abu Zayed Md Badruddoza†, Mohammad Azad*,‡, Jason R. McCarthy§, Md Nurunnabi§ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States* Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, United States† Department of Chemical, Biological and Bioengineering, North Carolina A&T State University, Greensboro, NC, United States‡ Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States§

CHAPTER OUTLINE 1 Introduction ................................................................................................................................... 187 2 Epigenetics .................................................................................................................................... 188 2.1 Graphene-Based Biosensor for Detection of DNA Methylation ............................................ 190 2.2 Optical Sensors for miRNA Detection .............................................................................. 194 2.3 Electrochemical Sensors for miRNA Detection ................................................................. 196 3 Graphene Based Sensing Platforms for mRNA Detection .................................................................... 199 4 Graphene-Based Biosensors for Glycomics ....................................................................................... 204 5 Future Direction of Graphene Based Biosensing Platform .................................................................. 209 References ........................................................................................................................................ 212 Further Reading ................................................................................................................................. 215

1 INTRODUCTION Graphene is the two-dimensional form of carbon with planar sp2-hybridized bonding. Due to several of its unique physicochemical properties, which are highly tunable in nature, this material has received a tremendous attention in the scientific community due to a large number of potential applications in various fields. Most importantly, its giant carbon molecules and two-dimensional solid-state structure allow interaction of this material with other nanoscale objects or entities (e.g., biomolecules) with the minimal surface modification that makes this material a highly suitable candidate for sensing applications. Biomedical Applications of Graphene and 2D Nanomaterials. https://doi.org/10.1016/B978-0-12-815889-0.00009-X # 2019 Elsevier Inc. All rights reserved.

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Sensitive and highly specific sensing technology platforms for rapid detection of biomolecules at affordable price are highly desirable for various applications in healthcare, especially for disease diagnosis, prognosis, and treatment in resource-constrained and in resource-rich communities. Most importantly, the current methods applied for disease screening at molecular level (e.g., nucleic acids, DNA, glycoprotein, and glycan) require sophisticated instruments and trained man power and are far reach to the users. Therefore, there is a great need for developing sensitive, selective, effective, and rapid technology platforms for screening molecular level entities. The graphene-based nanostructures commonly used in sensing applications are graphene and derivatives of graphene, that is, graphene oxide (GO), reduced graphene oxide (rGO), graphene-based composite materials, and nanomaterials such as gold nanoparticles (AuNPs). This book chapter covers the latest developments in the use of graphene-based materials as sensing platforms for detection of various biomarkers exclusively for health-care applications. Specifically, the chapter will focus on the progress of research based on usage of various types of biomarkers, that is, genetic, epigenetic, proteomic, and glycomic as receptors or ligand molecules that are considered to decorate or modify the surfaces of graphene and its derivatives. The underlying molecular mechanisms and bioconjugation methodologies including various detection schemes for highly sensitive and selective detection of these biomarkers, along with the challenges and future perspectives in this rapidly developing field, are discussed here.

2 EPIGENETICS The knowledge of epigenetics provides the additional information of changes in chromosomal level that may cause irregular activity and expression of a normal gene. Understanding the changes in chromosomal level of a gene reveals the root cause of a disease and finding a convenient way of treatment. Some of the examples of changes that affect the activity of a gene without altering their original sequence are DNA methylation, chromatin remodeling, microRNA patterns, and modification of histone. The epigenetic changes usually sustain up to cell division level; however, sometimes, the changes are observed from generation to generation without altering the original DNA sequence of a specific organism. Many chronic diseases such as cancer and cardiovascular diseases are known as genetic and epigenetic diseases; therefore, the identification of a specific biomarker to detect and quantify the responsible epigenetics is considered as one of the key tools for detection of cancer at very early stage. For example, the risk of cancer of a specific environmental factor could be determined by understanding their effect on changes of epigenetics through DNA methylation. Hyper- and hypomethylation of cytosines in the promoter sequences of DNA are known to be the reason of many cancers. An instant example is that the prostate cancer patients are diagnosed as hypermethylation of CpG dinucleotides in the promoter sequences of the glutathione S-transferase Pi 1 (GSTP1). The changes in epigenetics could be classified into five different subcategories based on Fig. 1. These are as follows: 1. 2. 3. 4. 5.

Silencing of tumor suppressor gene by CpG island promoter methylation and histone deacetylation Global genomic hypomethylation Loss of imprinting event Epigenetic lack of the repression of intragenomic parasites The appearance of genetic defects in chromatin-related genes

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FIG. 1 DNA methylation at the center of the normal and malignant behavior of the cell. Reproduced from Esteller, M.; Herman, J. G. Cancer as an Epigenetic Disease: DNA Methylation and Chromatin Alterations in Human Tumours. J. Pathol. 2002, 196, pp. 1–7 with permission from Wiley.

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In this chapter, we describe the existing tools and technique on how to detect the DNA methylation, chromatin remodeling, and microRNA patterns and modification of histone for detecting cancer at earliest stage with a qualitative and quantitative manner.

2.1 GRAPHENE-BASED BIOSENSOR FOR DETECTION OF DNA METHYLATION Graphene derivatives have shown potential applications in biosensor and bioelectronics and for detection of DNA methylation. Li et al. (1) demonstrated an approach for detecting a specific site of DNA methylation and assay of methyltransferase activity with GO-modified electrode as shown in Fig. 2. In another work, Sarathy et al. (2) have summarized the ability of graphene nanopores for electronic recognition of DNA methylation especially for detection of methylated sites of a DNA. In this review,

Au GC Deposition

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S1: 5¢-SH-(CH2)6-CCT CTG TGC GCC GGT CTC TCC CAG GAC AGG CA-(CH2)6-NH2-3¢ S2: 5¢-TG CCT GTC CTG GGA GAG ACC GGC GCA CAG AGG-3¢ S3: 5¢-TG CCT GTC CTG GGA GAG ACT GGC GCA CAG AGG-3¢ FIG. 2 Schematic showing the gene-specific DNA methylation detection and MTase activity assay based on the electrochemical signal amplification of GO and restriction endonuclease. Reproduced from Li, W.; Wu, P.; Zhang, H.; Cai, C. Signal Amplification of Graphene Oxide Combining With Restriction Endonuclease for Site-Specific Determination of DNA Methylation and Assay of Methyltransferase Activity. Anal. Chem. 2012, 84 (17), 7583–7590 with permission from ACS.

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they have emphasized on the physical and chemical properties of graphene derivatives such as pore size, shape, position, and the presence of defects on a graphene and their effect on detection profile. The summary is having midgap (zero-energy) states in graphene construction that facilitates enhanced sensitivity for detecting methylated CpG sites and the presence of a large square deviation in a graphene further facilitating superior resolution in detection of multiple methylated sites of a DNA. In the recent years, Hoque et al. (3) proposed a simple and advanced method of detecting DNA methylation using base-dependent affinity interaction of DNA with graphene (Fig. 3). In this study, they have demonstrated that bisulfite-treated guanine-enriched methylated DNA leads to a stronger affinity to graphene-modified electrode compared with adenine-enriched unmethylated DNA. The guanine bases of DNA lead the DNA to get adsorbed with a stronger affinity. They were also able to quantify the level of methylation by monitoring the current (I) in differential pulse voltammetry.

FIG. 3 A schematic of a graphene-DNA adsorption-based methylation assay. The adsorption of single-stranded deoxyribonucleic acid (ssDNA) on graphene-modified screen-printed carbon electrode (g-SPCE) repulses [Fe(CN)6]3
molecules from accessing electrode surface, providing a significant DPV signal. Inset, typical differential pulse voltammetric signals showing the guanine-enriched methylated DNA that produces lower DPV currents in comparison with the adenine-enriched unmethylated DNA. Reproduced from Haque, M. H.; Gopalan, V.; Yadav, S.; Islam, M. N.; Eftekhari, E.; Li, Q.; Carrascosa, L. G.; Nguyen, N. T.; Lam, A. K.; Shiddiky, M. J. A. Detection of Regional DNA Methylation Using DNA-Graphene Affinity Interactions. Biosens. Bioelectron. 2017, 87, 615–621 with permission from Wiley.

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2.1.1 Graphene-based DNA biosensors Development of graphene-based DNA biosensors involves the interplay between the bulk and the interface that can be fine-tuned by optimization of various process protocols to enhance the electron charge transfer in order to result in a measurable signal. The fundamental forces that are responsible for the interaction between DNA bases and the graphene are van der Waals interactions (π–π interactions) and the solvation energy of the various solvent molecules used during functionalization (4). The interaction of DNAs with the surface of the graphene results in alteration of surface properties that further leads to the changes in the electrochemical signal generating from the surface. The generated electrochemical signals are highly sensitive to interactions at the surface; even the presence of a single nucleotide would lead to a significant change in the signal output. Hence, in the development of graphene-based sensors, this particular mechanism has been exploited for detecting DNAs with single-base specificity (5, 6). The GO is an oxidized form of graphene that retains its layered structure with a larger interlayer spacing (7), whereas rGO is prepared from the reduction of GO either by chemical, by electric, or by thermal treatments. The principle of DNA biosensors is based on the hybridization of probe and target forming DNA duplex on the electrode surface that results in a readable electrochemical signal (8). So far, several researchers have been using one or other forms of graphene or its derivatives to make highly sensitive DNA detection platforms; one such label-free DNA sensing assay platform was demonstrated by Hu et al. (9) for detecting HIV-1 pol gene on undecorated GO surface. In this work, these authors specifically designed the ssDNA to have a sequence for the probe and for immobilization onto the GO surface. When the probe hybridized with the target due to the π–π stacking, the interaction between the probe sequences and GO changed. Here, due to the changes in the charge and conformation of probe, it induced further changes in the interfacial properties of GO that was monitored and measured by electrochemical impedance spectroscopy (EIS). This simple label-free detection sensing platform using GO had the limit of detecting DNAs up to a concentration of 11 pM. Later on, by using a composite of molybdenum disulfide (MoS2), graphene, chitosan, and AuNPs, Cao (10) modified the glassy carbon electrode that was further functionalized with ssDNA to demonstrate an ultrasensitive electrochemical DNA biosensing. In this work, by interplay between the electric conductivity of the composite and signal amplification due to AuNPs, 2.2 fM of limit of detection (LOD) was achieved. This composite graphene-based DNA sensor was highly selective and capable of differentiating between single-base mismatch and three-base mismatch sequences of DNA. In the similar line, Hajihosseini et al. (11) developed a DNA biosensor for detecting Helicobacter pylori (H. pylori) by immobilizing the ssDNA on GO/AuNP-modified glassy carbon electrode (GO/AuNPs/ GCE) and carried out the hybridization reaction with the target DNA. These authors focused on signal amplification strategies in which by applying a redox indicator called oracet blue, the electrochemical signal of the DNA was amplified, and at the same time, the enhancement in the electric conductivity of the sensor surface was achieved due to embedded gold nanoparticles in graphene oxide (Go/AuNPs). All of these factors contributed to result in a picomolar (27.0 pM) level of detection for H. pylori. In another work carried out by Chen et al. (12), a label-free detection of DNA was demonstrated by growing large-area monolayer of graphene by chemical vapor deposition (CVD) followed by sensing of DNA by using graphene-based field-effect transistor (FET), which resulted in the LOD of 1 pM concentration. Several research groups focused on various strategies, materials, methods, etc. to achieve molecular level detection of DNA and nucleic acids. In the continued efforts, Dong et al. (13) were able

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FIG. 4 A schematic of graphene-based DNA biosensor with probe labeled with gold nanoparticles and modified electrode and showing carbon spheres functionalized with enzyme for signal tracer. Reproduced from Dong, H.; Zhu, Z.; Ju, H.; Yan, F. Triplex Signal Amplification for Electrochemical DNA Biosensing by Coupling Probe-Gold Nanoparticles-Graphene Modified Electrode With Enzyme Functionalized Carbon Sphere as Tracer. Biosens. Bioelectron. 2012, 33 (1), 228–232 with permission from Elsevier.

to attain the sensing of DNA down to attomolar concentration (5 aM) level. Here, they also focused on signal amplification strategies in which they developed a first of its kind of signal amplification strategy called a triplex signal amplification method. For this, they used electrochemically rGO-modified electrode and assembled DNA probe labeled with gold nanoparticles (ssDNA-AuNPs) with thiol group tagged (GT) DNA strand (d(GT)29SH). In the process, carbon spheres were functionalized with streptavidin (SA)-horseradish peroxidase (HRP) that worked as a signal tracer (Fig. 4). Recently, Benvidi et al. (14) demonstrated the lowest detection of DNA with concentrations down to zeptomolar level by optimizing the process protocols to make the graphene-based sensor. Here, the glassy carbon electrode was primarily modified with rGO for covalent immobilization of DNA probe that upon hybridization with target DNA followed by measuring the electrochemical signal resulted into 3.2 zM detection limit. They achieved this sensitivity by optimizing the process conditions through which the rGO resulted into a high-porosity material with large surface area and petallike graphene structures that were properly oriented to the substrate. The preferential vertical orientations of graphene nanoflakes with large lateral sizes contributed toward enhanced electrochemical activity of the sensor, leading to the high electron-transfer rates that in turn resulted in greater signal output. Most importantly, this is significant development in graphene-based DNA sensing technology where till date based on graphene and graphene-like 2-D materials, and this is the lowest LOD achieved for sensing DNA using electrochemical method.

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From the above discussion, it is evident that various strategies were followed in order to achieve highly sensitive graphene-based platforms for sensing DNA sequences. Majority of these methods would enable to detect the DNA sequences in the range of pM to fM in the controlled environment and needed sophisticated instrumentation and extensive process protocols to make these biosensors. In order to build effective graphene-based sensing platforms, we recommend three important aspects one need to focus while preparing these devices. Firstly, from fundamental point of view, efforts need to be made to deeply understand the interfacial effects, surface chemistry, reaction kinetics, and signal transduction mechanisms. Secondly, more efforts are needed to develop simple methods for synthesizing materials in which various materials including nanomaterials can be easily integrated to obtain the desirable properties. The third important aspect is to lay tremendous amount of efforts to demonstrate these sensing methods to be commercially viable by working on real samples and carrying out clinical trials followed by successful field trials. Further, easy to manufacture and deployment of these devices along with feasibility to operate in a simplest way possible in point of use would be highly desirable for effective health-care management.

2.1.2 Graphene based miRNA sensors In this section, we discuss about graphene-based sensing modules for miRNA detection. MicroRNAs (miRNAs) are a class of small nonprotein-coding single-stranded RNAs that regulate gene expression and several other biological processes such as cell development, differentiation, apoptosis, proliferation, and organ development (15, 16). Though these miRNAs are limited in number compared with the number of mRNAs and proteins present in most organisms, one miRNA regulates several of mRNAs that results in the substantial changes in gene expression. Therefore, usage of miRNA expressions used to have abundance of biological information, and the expression of these miRNAs shown to have strong correlation to various diseases (17). Hence, usage of miRNAs as potential biomarkers for disease diagnosis, screening, and prognosis and for therapeutic applications has tremendous attention in the research community (18). There is an abundance of research work carried out to detect these miRNAs by utilizing various nanomaterials such as nanoparticles, nanotubes, nanosilica, nanorods, and nanofibers. In these works, various novel strategies have been reported such as electrochemical detection (19), enzyme amplification (20), optical detection including fluorescence and colorimetric methods (21), surface plasmon resonance (SPR) (22), and other electrochemical methods. Here, we limit our discussion to usage of graphene and graphene-like derivative materials for sensing miRNAs.

2.2 OPTICAL SENSORS FOR miRNA DETECTION In the process of detecting miRNAs, Zhao et al. (23) developed a simple label-free colorimetric biosensor for sensing miRNA-21 based on GO and AuNP hybrids as shown in Fig. 5. Here, they used peptide nucleic acid (ssPNA) that upon absorption to graphene/AuNP hybrid surface caused peroxidase catalytic activity that in turn deactivated the π–π stacking interaction between ssPNA and graphene. This deactivation prevented the oxidation of tetramethylbenzidine (TMB) in the presence of hydrogen peroxide, which was released during the reaction. Once the miRNA-21 was added to the surface, the hybridization reaction between ssPNA and miRNA led to the release of PNA/DNA duplexes from the surface, resulting in catalytic activity of graphene-AuNPs forming blue color. Through this method, they demonstrated the LOD of 3.2 nM for miRNA-21. Though the LOD is in the nanomolar range, this work appears to be the first of its kind, where a simple colorimetric sensor was developed for visible

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FIG. 5 A schematic of the graphene/AuNP hybrid-based colorimetric sensor for detection of miRNA. Reproduced from Zhao, H.; Qu, Y.; Yuan, F.; Quan, X. A Visible and Label-Free Colorimetric Sensor for miRNA-21 Detection Based on Peroxidase-Like Activity of Graphene/Gold-Nanoparticle Hybrids. Anal. Methods 2016, 8 (9), 2005–2012 with permission from RSC.

detection of miRNAs on graphene-embedded nanomaterial platform. This platform seems to be very useful in point-of-care settings for binary detection of disease biomarkers. Over the years, several optical methods including fluorescence-based detection systems have been developed for sensing miRNAs by using GO as fluorescence quencher. One such label-free detection system was developed by Li et al. (24) by using magnetic silicon microsphere (MNP)-rGO sandwiched with DNA capture probes. By separating the sandwich nanostructures with a magnetic field, they achieved maximum fluorescence quenching. Though this process appears to be simple and does not require additional fluorescent labeling, increased signal-to-background ratio, the detection limit of 0.098 nM for miRNA-21 needs further efforts for using it for molecular level sensing of miRNAs. In order to improve the lower detection limits of graphene-based miRNA-sensing platforms, Treerattrakoon et al. (25) developed a multiplex circulating platform for detecting miRNA-44 and miRNA-29 using isothermal amplification along with fluorescence quenching of rGO. This sensitive graphenebased platform could detect circulating biomarkers down to 0.1 pM. Recently, Li et al. (26) used isothermal exponential amplification method by using SYBR Green I (SG) as the signal readout and GO as quencher (Fig. 6). This isothermal exponential amplification consisted of series of steps such as adsorption of hairpin probe (HP), the primer, and SG in the absence of target; unfolding of HP followed by polymerization; and strand displacement in the presence of target that further initiated target recycling process. As the enzyme recognized and cleaved the newly formed dsDNAs that further triggered sequence of reactions with the target and with HPs this led to circular exponential amplification, and upon intercalation of SG into dsDNAs, it resulted in enhanced fluorescence signal. This simplified label-free detection system yielded the miRNA detection limit of 3 fM that appears to be the lowest detection limit achieved so far using fluorescence-based detection method. In parallel, several research groups attempted various strategies and used graphene and its derivatives for detecting miRNAs by fluorescence detection methods. For example, Fan et al. (27) achieved lower LOD 0.18 nM for let-7a miRNA by introducing helicase into the reaction in which GO worked as a quencher. Further, Huang et al. (28) developed a nanophoton switch using quantum dots (QDs) and GO for multiplexing detection of miRNAs from tumor cells and demonstrated 1 pM detection limit.

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FIG. 6 A schematic of isothermal exponential amplification principle on GO platform for label-free detection of miRNA. Reproduced with permission from Li, W.; Hou, T.; Wu, M.; Li, F. Label-Free Fluorescence Strategy for Sensitive microRNA Detection Based on Isothermal Exponential Amplification and Graphene Oxide. Talanta 2016, 148, 116–121.

Recently, Laurenti et al. (29) developed an miRNA sensor based on graphene quantum dots (GQDs) and single-stranded DNA coupled with upconversion nanoparticles (ssDNA-UCNP@SiO2), which exhibited the lowest detection limit of 10 fM. However, from the above discussion and current research trend, it is evident that the fluorescence-based detection methods for sensing miRNAs on graphene platforms with several modified biological protocols demonstrated the lower LOD in the range of few nanomolars to femtomolars (30). And it appears that more research efforts are needed in this direction to detect less abundantly available, low stable miRNAs with minimal enriching and amplification protocols to concentrations down to single molecular level.

2.3 ELECTROCHEMICAL SENSORS FOR miRNA DETECTION As the need for highly sensitive, extremely specific sensors with very low LODs arises due to various disease conditions to be diagnosed at molecular level, development of electrochemical sensors has become a natural choice for addressing emerging challenges in healthcare. Electrochemical sensors are capable of detecting molecules with one-nucleotide difference, and its features such as simple processing steps, easy to use, miniaturization, and integrating into a point-of-care devices could make these electrochemical sensors to directly apply for applications in clinical diagnosis.

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FIG. 7 A schematic representing the process steps of CA-GO-PGE electrochemical sensor for miRNA detection. Reproduced with permission from Erdem, A.; Eksin, E.; Isin, D.; Polat, D. Graphene Oxide Modified Chemically Activated Graphite Electrodes for Detection of microRNA. Electroanalysis 2017, 29 (5), 1350–1358.

In an effort to develop simple electrochemical sensor by utilizing easily available pencil graphite electrodes (PGEs), Erdem et al. (31) demonstrated an miRNA detection on GO platform (Fig. 7). Here, they chemically activated the PGE surface by covalent agents (CA) such as mixture of N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form carboxyl functional groups on PGE surface. Later, they modified the surface of chemically activated PGEs with GO. The hybridization reaction of miRNA34a probe with specific target was carried out followed by measuring the signals with EIS. This simple, easy to handle, cost-effective, and disposable electrochemical sensor platform has yielded a lower LOD of 41.2 nM toward target miRNA34a.

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In another work, by using a novel electrochemical ELISA-like amplification strategy on rGO and carbon nanotube (CNT) composite, Tran et al. (32) developed an electrochemical biosensor for detecting miRNAs. Initially, the composite of rGO and multiwalled carbon nanotube (MWCNT) drop casted onto the screen-printed gold electrode where rGO mean to provide high surface area and CNTs to provide carboxylic functional groups for covalently binding to amino-modified DNA probes. After adding complementary miRNA followed by hybridization, the specific antibodies (anti-RNA-DNA antibody) were introduced that directly bind to RNA-DNA hybrids. Later on, a secondary antibody tagged to HRP further bonded to the primary antibody that had been modified for RNA-DNA. This process resulted in the generation of an electrochemical signal on the gold electrode surface that was measured with cyclic voltammetry. This novel ELISA-like enzyme amplification method performed on graphene- and carbon-nanotube-based platform detected miRNA down to 10 fM concentration. In the similar lines, by utilizing the gold electrodes along with HRP’s catalytic activity, other groups carried out further research to reduce the detection limits of the miRNAs with GQDs. For instance, Hu et al. (33) developed a highly sensitive and specific electrochemical sensor to detect miRNA155 from 1 fM to 100 pM with a lower LOD of 0.14 fM. These authors attributed the high performance of this sensor platform to the GQDs that provided high surface area for efficient reaction to occur on the surface. Further, an increase in the lower LOD for miRNAs was also demonstrated by Shuai et al. (34) on tungsten oxide-graphene composite platform by recycling target and enzyme signal amplification method (Fig. 8). The tungsten (WO3) and graphene composite were coupled with AuNPs on which immobilization of H1 probe was carried out followed by target hybridization. By adding another biotinylated hairpin DNA (H2), the target was released back to the solution that binds to opened HP in the subsequent steps. After adding streptavidin-conjugated alkaline phosphatase (SA-ALP), it initiated sequence of enzymatic reactions on the surface and generated an electrochemical signal, which was measured by EIS. This process turned out to be very sensitive due to enzyme and target recycling leading to detection of miRNA in the linear range from 0.1 fM to 100 pM with LOD of 0.05 fM toward target. Recently, Shuai et al. (35) developed an ultrasensitive miRNA electrochemical sensor on magnesium oxide (MgO)-GO-AuNP hybrid platform (schematic shown in Fig. 9). Similar to the work done by Shuai et al. (34), here, electrochemical-chemical-chemical (ECC) detection system was also used in which MgO nanoflower and AuNPs on GO electrode served as an efficient signal generators. This metal-oxide-based electrochemical sensor had the 50 aM as the lowest LOD for miRNA-21 with a dynamic range from 0.1 to 100 fM. The 50 aM of LOD toward an miRNA target appears to be the lowest limit reported in the literature till date. Apart from electrochemical detection systems, some research groups also developed other methods such as SPR method on GO and AuNP hybrids. In such reports, a LOD of 1 fM (36) and 0.1 fM (37) was demonstrated. In an another work (38), electrochemiluminescence detection method was used on boron-doped GQDs to detect oncogene miRNA-20a with a LOD 0.1 pM with a linear range of 0.1–1  104 pM. The above discussion indicates that graphene and its derivatives along with various nanomaterials as platform technologies for sensing miRNAs are becoming very promising to achieve concentration detection limits reaching down to attomolar level. However, much more efforts are required to improve miRNA profiling methods with significant reduction in the number of process steps along with developing rapid detection schemes. Since miRNAs are not available abundantly in organisms and they have low stability, rapid amplification strategies with multiplexing capabilities need to be developed. As stated in previous sections, these miRNA-sensing modules are required to be tested as well and demonstrated with real samples for efficient usage in disease management.

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Cycle

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FIG. 8 Schematic of electrochemical miRNA biosensor based on catalyzed hairpin assembly (CHA) target miRNA recycling and signal amplification of ALP and ECC redox cycling. Alkaline phosphatase (ALP), electrochemicalchemical-chemical (ECC), ascorbic acid 2-phosphate (AAP), ferrocene methanol (FcM), and tris(carboxyethyl) phosphine (TCEP). Reproduced with permission from Shuai, H.-L.; Huang, K.-J.; Xing, L.-L.; Chen, Y.-X. Ultrasensitive Electrochemical Sensing Platform for microRNA Based on Tungsten Oxide-Graphene Composites Coupling With Catalyzed Hairpin Assembly Target Recycling and Enzyme Signal Amplification. Biosens. Bioelectron. 2016, 86, 337–345.

3 GRAPHENE BASED SENSING PLATFORMS FOR mRNA DETECTION Ribonucleic acid (RNA) is one of the major biological macromolecules that is essential for organisms. In living organisms, DNA is considered to be a blueprint of the cell, whereas RNA is considered as the photocopy of the DNA. In the process of producing a certain type of protein, cell activates the particular protein’s gene present on DNA; this specific gene further produces multiple copies of the genetic code in the form messenger RNA (mRNA). The mRNAs convey the information from DNA to ribosomes that produce cell’s specific proteins. Since the mRNA contains the information about the genetic code and kind of protein to be produced for cell functioning, it has been shown that the levels of mRNA in living organism are a representation of physiological state and possibly a disease condition (39). Hence, sensing of intracellular mRNA levels helps in monitoring the progression of diseases and early

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I Na3C6H5O7×2H2O HAuCl4 GO

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FIG. 9 Schematic representation of the miRNA-21 sensor principle. Reproduced with permission from Shuai, H.-L.; Huang, K.-J.; Zhang, W.-J.; Cao, X.; Jia, M.-P. Sandwich-Type microRNA Biosensor Based on Magnesium Oxide Nanoflower and Graphene Oxide–Gold Nanoparticles Hybrids Coupling With Enzyme Signal Amplification. Sens. Actuators B 2017, 243, 403–411.

diagnosis of disorders (40, 41). It appears that few studies have been undertaken to detect these mRNAs for diagnosing various disease conditions. There are several conventional methods such as northern blotting, reverse-transcribed polymerase chain reaction (RT-PCR) and gene chip, rolling ring amplification technology, and rolling circle amplification (RCA) that have been used for mRNA assay in the laboratory settings. However, here, we limit our discussion to sensing of mRNAs using graphene and its derivative materials as platforms that have great potential to be translated to use in point-of-care settings. In the direction of detecting mRNAs, Li et al. (42) developed a novel GO-based sensor for detection of endonuclease activity of Ago2 protein. Here, for the first time, they developed an assay to monitor the RNA endonuclease activity of mammalian Ago2 protein. They functionalized the GO with probe labeled fluorophore carboxyfluorescein (P-FAM) that had two functional regions related to target mRNA sequence and longer DNA sequences. As the Ago2-miRNA complex was added onto P-FAM-coated GO, it cleaved the target mRNA at a single phosphodiester bond. Consequently, P-FAM underwent an irreversible stand scission at a specific scission site resulting in a release of oligonucleotide fragments followed by fluorescence (FL) intensity recovery. This cyclic activity (i.e., Ago2-miRNA complex release and initiation of reaction) of newly released entity can go on to further amplify the FL signal. The

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spectrofluorometer at 490 nm excitation wavelength was used to measure the FL intensity. This novel assay on GO platform had resulted in a lower detection limit of 1.7 nM for Ago2 endonuclease activity. Further, Ling Li et al. (43) reported a novel on microfluidic chip approach for capturing specific cancer cell and in situ detection of a tumor biomarker survivin mRNA (schematic is shown in Fig. 10). They used a composite of nanosized graphene oxide (NGO) and polyethylene glycol bis-amine (NGO-PEG) along with antisense oligonucleotide (F–S1) as a nanocomplex and signal tag to introduce into the captured cells. Once this nanocomplex entered into the cell, then S1 bonded to intracellular survivin mRNA followed by the release of F–S1 from NGO-PEG along with a fluorescence recovery. Through this antibodybased simple microfluidic chip system, they estimated the survivin mRNA in each PC-3 cell to be (4.8  1.8)  106 copies indicating a promising approach for early screening of malignancy.

step 1

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FIG. 10 Schematic of the microfluidic-based screening process for detecting survivin mRNA in captured cells. Microfluidic channel functionalized with anti-PSCA (step 1) and fluorescence-based assay for survivin mRNA (step 2). Anti-PSCA, prostate stem cell antigen (PSCA) monoclonal antibody; PC3-cells, prostate cancer cells; and LO2 cells, normal liver cells. Reproduced from Li, X.-L.; Shan, S.; Xiong, M.; Xia, X.-H.; Xu, J.; Chen, H.-Y. On-Chip Selective Capture of Cancer Cells and Ultrasensitive Fluorescence Detection of Survivin mRNA in a Single Living Cell. Lab Chip 2013, 13 (19), 3868–3875 with permission from RSC.

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FIG. 11 A schematic of GONS@SurMB sensing platform for mRNA detection. (A) MB OFF state, (B) GONS@SurMB OFF state, and (C) SurMB-mRNA ON state. Reproduced with permission from Stobiecka, M.; Dworakowska, B.; Jakiela, S.; Lukasiak, A.; Chalupa, A.; Zembrzycki, K. Sensing of Survivin mRNA in Malignant Astrocytes Using Graphene Oxide Nanocarrier-Supported Oligonucleotide Molecular Beacons. Sens. Actuators B 2016, 235, 136–145.

Further, studies were carried out by Stobiecka et al. (44) who reported the detection of survivin mRNA using GO nanocarrier-supported oligonucleotide (ONT) molecular beacons (MB), which could be used for screening for high-mortality cancers such as astrocytic cancer (Fig. 11). This research group utilized graphene oxide nanosheets (GONs) as theranostic nanocarriers (NCs) owing to several advantages over other metal or metal-oxide nanocarriers such as forming stable oligonucleotide complexes, effective transfection of cancer cells, and higher stability. Once they are inside the cell and most importantly, they release ONTs only by complimentary target mRNA or miRNA. With this platform and by using fluorescence detection method, they demonstrated a LOD for target DNA to be 24 nM, which opens up possibilities of detecting survivin mRNA in malignant cells in biopsies.

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FIG. 12 Schematic of the biomarker detection system using GO-UPNC platform in biopsy tissue samples. Reproduced with permission from Vilela, P.; El-Sagheer, A.; Millar, T. M.; Brown, T.; Muskens, O. L.; Kanaras, A. G. Graphene OxideUpconversion Nanoparticle Based Optical Sensors for Targeted Detection of mRNA Biomarkers Present in Alzheimer’s Disease and Prostate Cancer. ACS Sens. 2017, 2 (1), 52–56.

In another effort to detect the mRNAs with further lower detection limit, Vilela et al. (45) reported a novel method based on GO and upconversion nanoparticles (UCNPs) for sensing mRNAs relevant to Alzheimer’s disease (BACE-1) and prostate cancer (PCA3) in blood serum. As shown in Fig. 12, in a sequence of two steps, the UCNPs and GO were added to the whole cell lysate having either mRNA biomarker BACE-1 or PCA3. Once the hybridization of the probe and target completed, it results in reduced adsorption of UCNPs to the GO surface. This can be further detected by the fluorescence signature of the UCNPs by exciting these nanoparticles with 980 nm laser. They used lanthanide-doped nanoparticles as UCNPs due to their characteristic features such as to absorb low-energy photos and emit fluorescence at a shorter wavelength than the excitation along with other features as high stability and no blinking effects (or photobleaching). Here, GO aids as an energy transfer acceptor and efficient fluorescence quencher along with other characteristic features. They reported lower limits of detection for mRNAs down to femtomolar range (500 fM) by using a single-photon counting system. This simple, novel, highly sensitive method with lowest limits of detection appears to be one of the promising methods for detecting mRNAs for diagnosing diseases. Recently, Fan et al. (46) reported a sensitive detection scheme for mRNA using GON-based fluorescence method. Here, they used duplex-specific nuclease (DSN) that was specific to mRNA for amplifying the signals during the hybridization reaction. In the amplification process, the DSN cleaved the ssDNA present in the DNA/mRNA hybrid to produce small fragments that further initiated another hybridization. In this detection scheme, GO nanosheets provided maximum fluorescence quenching

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along with other characteristic benefits such as efficient signal carrier, protecting DNA probe from unspecific digestion. By maximizing the signal output in this mode, they could detect the mRNA down to 1.0 fM concentration that is significant in clinical diagnosis. From the above discussion, it is evident that the graphene-based materials combined with other signal amplification strategies are encouraging for clinical diagnosis of several diseases. However, it is evident that the research in the direction of detecting mRNAs has not progressed much compared with the sensing schemes developed for either DNAs or miRNAs. Since the degradation rate of mRNAs is much faster with shorter lifetime and the stability of mRNA appears to be a function of structural features of the gene than the function of genes (47), it limits the usage of mRNAs as biomarkers. Further, it has been shown that the origin of the tumor cell regulates the expression levels of mRNAs, and in some tumors, the abundancy of the mRNA was lower compared with normal cells (48, 49). Though mRNAs have some limitations as discussed above, they appear to be a promising biomarker for health-care applications since they contain the genetic information. Therefore, development of novel strategies for mRNA sensing using combination of nanomaterials, building efficient signal amplification methods, and most importantly tuning cellular origin-specific strategies with rapid processing protocols are highly desirable for using mRNAs as potential clinical markers for health-care applications.

4 GRAPHENE-BASED BIOSENSORS FOR GLYCOMICS Glycoproteins, which are one of the most important posttranslational modifications, play a critical role in a variety of biological activities, and they have been used as biomarkers for disease detection and as therapeutic targets for clinical diagnostics (50, 51). Enzymatic addition of a glycan (i.e., oligosaccharide chain covalently bound to proteins or lipids) in a process of glycosylation is a highly abundant form of co- and posttranslational modification of proteins. Glycans take active part in many physiological and pathological processes such as cell-cell adhesion, cellular differentiation, cell growth and cytokinesis, bacterial infection, viral invasion, and cancer metastasis. Better understanding of glycan involvement in pathological processes may be thus primarily important to cure diseases. As aberrant glycosylation can be the result of a disease, glycan profiling or changes can be used in early-stage diagnostics of numerous diseases, including various types of cancers with already approved biomarkers (50). Only recently, research focus has moved from the study of proteins and DNA (genomics and proteomics) to the study of glycans, creating a new field of science—glycomics. Glycans can be linear or branched with α- and/or β-linkages between neighboring carbohydrates and thus are considered to be a better biological information-coding/information-storing tool than the aforementioned proteins or DNA, because the number of possible unique sequences as compared with nucleic acids or proteins is staggering (Fig. 13) (51, 52). Even though the structural variability of glycans is huge, with an estimated number of unique glycan structures of 5000, carbohydrates as a building block of glycans have similar physicochemical properties, and this is why it is complicated to elucidate the exact glycan structure using instrumental techniques (nuclear magnetic resonance, mass spectrometry (MS), high-performance liquid chromatography, or electrophoresis). Recent progress in the field of glycomics was only possible due to advances in sample pretreatment and the use of sophisticated instrumentation. Even though such an approach can identify an exact glycan structure, there is a requirement for skilled operators and costly instrumentation. This is why alternative ways for the analysis of glycans emerged-using natural glycan-recognizing proteins, that is, lectins.

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FIG. 13 Graphic representation of complexity of glycans, showing variability of sugar building blocks, multiple branching, and attachment points. Reproduced with permission from Bertok, T.; Katrlik, J.; Gemeiner, P.; Tkac, J. Electrochemical Lectin Based Biosensors as a LabelFree Tool in Glycomics. Microchim. Acta 2013, 180 (1–2), 1–13.

Lectins are a class of nonimmune proteins (e.g., concanavalin A (ConA) and peanut agglutinin (PNA)) able to recognize free saccharides, mono- and oligosaccharides, or even whole cells specifically (53). Lectins have weak interaction with carbohydrates, in the form of glycoproteins, glycolipids, and glycans, in biological systems with dissociation constants (Kd) in the millimolar to micromolar range. These interactions are involved in various biological processes, including cell-cell communication, pathogen binding, tumor cell metastasis, and immune responses. It is important to understand and mimic carbohydrate and bacterial lectin interactions as the foundation of pathogen detection and prevention of bacterial infection. Therefore, lectin biosensors are very promising candidates for glycan analyses, and they can be used even in case when the target is unknown. Klukova et al. (51) have summarized in a review paper the most common lectins used in the preparation of lectin biosensors with specific binding and other characteristics. Recently, graphene materials have emerged as a prospective tool for fabrication of lectin-based biosensor allowing different ways of lectin immobilization. The smaller molecular size of lectins in comparison with antibodies allows the immobilization of lectins at higher density and a higher sensitivity/selectivity of assays compared with immunoassays. Moreover, prepared biosensor is able to recognize nanomolar to femtomolar concentration of analyte (52, 54).

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FRET 980 nm

980 nm +

547 nm

547 nm

UCP

(A)

conA

GO

Chitosan

Glucose

ConA

FBT Sugar

GO

(B) FIG. 14 (A) The upconversion phosphor-graphene oxide (UCP-GO) biosensing platform and the mechanism of glucose determination. (B) The principle of ConA detection with the graphene oxide/folate-biopterin (GO/FBT) hybrid probe. (A) Reproduced with permission from Zhang, C.; Yuan, Y.; Zhang, S.; Wang, Y.; Liu, Z. Biosensing Platform Based on Fluorescence Resonance Energy Transfer From Upconverting Nanocrystals to Graphene Oxide. Angew. Chem. Int. Ed. 2011, 50 (30), 6851–6854 and Wiley. (B) Reproduced with permission from Wang, L.; Pu, K. Y.; Li, J.; Qi, X.; Li, H.; Zhang, H.; Fan, C.; Liu, B. A Graphene-Conjugated Oligomer Hybrid Probe for Light-Up Sensing of Lectin and Escherichia coli. Adv. Mater. 2011, 23 (38), 4386–4391 and Wiley.

As shown in Fig. 14A, Zhang et al. (55) constructed a novel sensor for glucose determination based on fluorescence resonance energy transfer (FRET) from upconverting phosphors (UCPs) to GO. It is known that carboxyl, hydroxyl, and epoxy groups present on the surface of GO sheets make it more water-soluble and also enable covalent conjugation with various other molecules. Further, authors reported that the ConA and chitosan were covalently binded to UCPs and GO, respectively. It was anticipated that the known tight binding of ConA with chitosan to bring UCPs and GO into proximal distance leads to induce transfer of energy. Therefore, the expected FRET process could be inhibited because of competition between glucose and chitosan for ConA, and possibly, this phenomenon could be the driving principle for glucose sensing. When excited with the near-infrared light, the biosensor

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showed favorable analytic performance in a complex biological sample matrix. Unlike commonly used heterogeneous methods, such as enzyme-linked immunosorbent assay (ELISA), which needs multiple separating steps, the proposed UCP-GO sensor is capable of homogeneously detecting glucose in serum samples without background interference; therefore, the UCP-GO FRET system could be a favorable platform for biosensing. Moreover, Wang et al. (56) reported a GO-based bioassay using a conjugated oligomer as the watersoluble neutral probe for light-up detection of CoA and Escherichia coli (E. coli) (Fig. 14B). The E. coli is known as one of the most dangerous pathogens and can cause serious foodborne diseases; therefore, it requires quick and accurate detection. As E. coli is covered with lectin (the amino acid-sugar complex), the strong affinity between the carbohydrate groups and lectin is useful for E. coli detection. In this way, a conjugated oligomer called folate-biopterin transporter (FBT) was developed, which had a high density of α-mannose side chains and a relatively short backbone to increase water solubility. Although FBT binds specifically to ConA, it is not ideal for visual sensing due to the high background signals. Integration of GO can almost completely quench the fluorescence of FBT as a result of the π-π interaction between GO and FBT. This interaction can be inhibited by specific protein-carbohydrate interactions between FBT and ConA, and the quenched fluorescence of GO/FBT greatly recovers in the presence of ConA. As a result, this hybrid probe has the significant advantage of light-up visual discrimination of ConA from other proteins, which also allows quantification of ConA in the range 0–50 nM with an LOD of 0.5 nM. This specific protein-carbohydrate interaction induced a light-up response of GO/FBT probe that made it useful for distinguishing different E. coli strains. Similarly, Chen et al. (57) developed a novel biosensor to homogeneously detect ConA using pyrene-conjugated maltose assembled graphene based on FRET (Fig. 15A). In this study when the maltose-grafted-aminopyrene (Mal-Apy) was self-assembled on the surface of graphene by means of π–π stacking interaction, it was observed that the fluorescence quenching was adequate due to the graphene that acted as a nanoquencher of the pyrene rings because of FRET. Subsequently, this led to the competitive binding of ConA with glucose that further destroyed the π–π stacking interactions between the pyrene and graphene in the presence of ConA, leading to fluorescence recovery. Through this method, the authors could be able to demonstrate the selective sensing of ConA with a wide linear detection range of 2  10
2–1.0 μM and an LOD of 0.8 nM of ConA. In another study by Huang et al. (58), a sandwich-type SPR sensor based on GO covered by dextran was developed for sensitive detection of ConA (Fig. 15B). This particular sandwich-type SPR sensor platform was able to detect ConA with a detection limit of 0.39 μg mL
1 in the linear range of 1.0– 20 μg mL
1. In this study, the signal amplification was attributed to the specific binding between the ConA that was previously bound to GO/DexP/ConA sensing film and dextran-capped gold nanoparticles (Dex-AuNPs). Recently, Filip et al. (59) investigated the immobilization of ConA lectin on an electrochemically reduced graphene oxide (ErGO)/thionine (Thi) surface via glutaraldehyde (GA) cross-linking and utilized it for the impedimetric detection of the glycoprotein invertase (INV). An attachment of ConA/GA aggregates to the ErGO/Thi surface leads to a biosensor with a linear response in the concentration range of 10
14–10
8 mol for INV and a sensitivity of 6.1% of charge transfer resistance (RCT) change per decade of INV concentration (59). The detection of human acute lymphoblastic leukemia cells (CCRF-CEM) was demonstrated on glassy carbon electrode modified with a poly(amidoamine) dendrimer on a reduced graphene oxide (rGO-DEN) nanocomposite with an LOD of 10 cells/mL (60). The rGO-DEN offered a multivalent recognition interface for the immobilization of ConA that significantly enhanced the cell capture

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Mal-Apy (ii)

(i)

(A)

Con A

Graphene

(1) DexP

(1) PDDA

Con A

(2) Tween 20 SDS

(2) GO

Negatively charged goal chip Angle(m°)

500

Step 2

400 300 200 100

Dex-Au NPs

Step 1

0

Step 1

(B)

Step 2

0

500 1000 1500 2000 Time(s)

FIG. 15 (A) Self-assembled Mal-Apy and fluorescence recovery on the surface of graphene: (i) Mal-Apy was quenched when assembled to graphene due to π–π stacking interaction; (ii) the fluorescence recovers, while ConA combines with Mal-Apy causing the pyrene far away from the graphene surface. (B) Schematic illustration of the sandwich SPR sensor based on GO and Dex-AuNPs for detection of ConA. (A) Reproduced with permission from Chen, Q.; Wei, W.; Lin, J. M. Homogeneous Detection of Concanavalin A Using Pyrene-Conjugated Maltose Assembled Graphene Based on Fluorescence Resonance Energy Transfer. Biosens. Bioelectron. 2011, 26 (11), 4497–4502. (B) Reproduced with permission from Huang, C.-F.; Yao, G.-H.; Liang, R.-P.; Qiu, J.-D. Graphene Oxide and Dextran Capped Gold Nanoparticles Based Surface Plasmon Resonance Sensor for Sensitive Detection of Concanavalin A. Biosens. Bioelectron. 2013, 50, 305–310.

efficiency and improved the sensitivity of the cytosensing for cell surface glycan. After immobilization of ConA lectin and blocking with bovine serum albumin (BSA), cells were successfully bound to a surface, and the electrochemical signal was amplified by application of aptamer- and HRP-modified gold nanoparticles (HRP-aptamer-AuNPs) as nanoprobes in a sandwich format of analysis. In addition, at a concentration of 5.0  104 cells mL
1, this biosensor clearly distinguished the CCRF-CEM from five other types of cell lines used in the study (60). The same cell line was detected in a sandwich configuration with an LOD of 38 cells mL
1 using the biosensor having aptamer immobilized on a rGO-dendrimer-modified surface with electroluminescent detection (61). Moreover, Liu et al. (62) synthesized a new ternary composite biosensor (H-RGO-Au NRs) based on graphene, hemin, and gold nanorods, with high catalytic activity. This graphene-based ternary composite is attached to abundant positively charged Au NRs, which greatly improves the catalytic abilities of the graphene family of

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209

peroxidase mimetics. In addition, owing to the high electron-transfer rate of graphene and the synergistic interaction of three components, this biosensor realized sensitive detection of glycan expression on K562 cell surface with an extremely low detection limit of 10 cells (62). Furthermore, Li et al. (63) developed a glycan biosensor using anthraquinone-modified glycans attached to a graphene surface via π–π stacking interactions for the analysis of lectins and more importantly for detection of cancerous cells. In this work, the cyclic voltammetry was applied to monitor changes in the redox behavior of the quinone moiety as a redox probe upon interaction with an analyte. The binding specificity of six different lectins on glucose and galactose-modified graphene interfaces revealed that lectins could be detected down to nanomolar level on electrified anthraquinonyl glycoside surfaces (Fig. 16) (63). The hepatoma (liver cancer) cell line HepG2 could be detected down to a concentration of 5000 cells mL
1 with a narrow dynamic range. A comparative list of various graphene derivative-based biosensors for the analysis of lectin/glycan/cells is listed in Table 1.

5 FUTURE DIRECTION OF GRAPHENE BASED BIOSENSING PLATFORM The exploration of graphene and its derivatives for health-care applications has witnessed exciting advancement over the last few years, even though this research area is still in its infancy. The research on the interface of graphene and a variety of materials such as metals, metal oxides, polymers, QDs, and small molecules is emerging as one of the most dynamic areas in health-care research as it establishes excellent coupling with bioentities that forms the basis of developing efficient, long-lasting sensors for detection of a multitude of diseases, toxins, and biomarkers (64). The sensors developed based on graphene are faster in response, cheaper in cost, and smaller in size. However, still some challenges remain to be addressed for making efficient biosensors fabricated with the graphene and its derivative materials. First, graphene needs to be efficiently functionalized as the surface properties determine the subsequent performance of the sensors. The established chemical approaches for surface functionalization are still far from being applicable and reproducible. The reliable and simple methods have to be developed for functionalizing graphene in the future. Second, biosensors are a class of devices that are able to translate the biological information into other forms of information like electric or optical signals that can be readable with the help of various dedicated tools. The integration of the sensing bed with the signal translator and receiver is another factor determining the efficiency of the sensor. The translation must be fast, reliable, and readable. Third, detecting more than one target molecules is still a great challenge in the development of biosensors. The medical devices usually require multifunctionality with multiplexing facilities. Fourth, the sensors with a strong antiinterference ability are highly desirable. The real bioenvironment is much more complicated than the laboratory-built environment. In real settings, various biomolecules with similar properties often interfere with the detection of target molecules. To overcome this challenge, the efforts must be made on developing highly specific targeting agents, and these agents must be robust in various bioenvironments. In order to solve these challenges and to develop the robust, field-deployable sensors for solving real problems in the domain of diagnostics and healthcare, a holistic approach needs to be followed. In this approach, collective research and technology development efforts from various fields ranging from materials science to biology and from electronics to medical science (65) need to be directed toward building highly specific, robust addressing pressing needs of health-care needs of the society. For the development of graphene-based optical sensors, efforts need to be directed toward preparing soluble, well-defined (size, number of layers, and chemical functionalities) graphene or its derivatives

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1

1 0.8

0.6

I/I0

0.6

I/I0

GO-GA2

0.8

GO-GA1

0.4

0.4 0.2

0.2 0 0

5

10

15 C (10 –6

(A)

M)

20

25

–0.6

–0.5

–0.4

–0.3

0

E (V)

40

(B)

10

M)

20

25

–0.6

40

–0.5

–0.4

–0.3

–0.2

E (V)

Bare GO GO-GA2 + Con A + PNA

35 30

2

Zim (Ω cm )

30

5

15 C (10 –6

Bare GO GO-GA1 + PNA + Con A

35

Zim (Ω cm2)

0

–0.2

25 20 15

25 20 15

10

10

5

5

0

0 0

(C)

20

40

60

Zre (Ω cm2)

80

100

0

(D)

20

40

60

80

100

120

Zre (Ω cm2)

FIG. 16 Probing the specific sugar-lectin interactions on surface modified by (A) and (C) glucose and (B) and (D) galactose. The interaction between lectin and immobilized saccharide was detected by either (A) and (B) DPV or (C) and (D) EIS. Bare screen-printed electrode was modified by graphene oxide (GO), then by anthraquinonecontaining glucose (GO-GA1) or galactose (GO-GA2), and interaction between saccharide-containing surfaces was probed by two lectins: ConA, recognizing glucose, and peanut agglutinin, recognizing galactose. All experiments were performed in Tris-HCl (pH 7.0). Reproduced with permission from Li, Z.; Deng, S. S.; Zang, Y.; Gu, Z.; He, X. P.; Chen, G. R.; Chen, K.; James, T. D.; Li, J.; Long, Y. T. Capturing Intercellular Sugar-Mediated Ligand-Receptor Recognitions via a simple yet highly biospecific interfacial system. Sci. Rep. 2013, 3, 2293.

with improved optical properties and high quenching efficiency for better signal at lowest possible concentration. Further, given its structural features and exceptional physicochemical properties, design and construction of GO-based theranostic platform with multifunctionalities and multimodalities is a new direction to pursue. Therefore, joint efforts among different disciplines such as chemistry, biomedicine, materials sciences, and nanotechnology will be required to achieve this goal. In addition, development of suitable chemical synthesis and functionalization approaches for precise control over size, size distribution, morphology, structural defects, and oxygen-containing groups of GO is urgently needed, as this is closely correlated to the performance of the GO-based nanomaterials for biomedical applications and diagnostics (66).

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Table 1 Analytic Parameters of Graphene-Based Lectin/Glycan Biosensora

Method SPR EIS EIS FRET

Fluorescence FRET DPV DPV

Graphene-Modified Lectin/Glycan Biosensor GO/DexP/ConA/ Dex-Au ConA/GO/GCE GCE-ErGO-thionineConA/GA-CFB Pyrene-conjugated maltose assembled graphene GO/FBT hybrid probe GO-CS-ConA-UCP ConA-labeled H-RGO-Gold NRs ConA/rGO-DEN/ GCE

Analyte

LR

DL
1

References
1

ConA

1.0–20.0 μg mL

0.39 μg mL

(58)

Glycoprotein Glycoprotein invertase ConA

10 aM–100 pM 10
14–10
8 mol

1.0 aM –

(52) (59)

0.02–1.0 μM

0.8 nM

(57)

ConA

0 – 50 nM

0.05 nM

(56)

Glucose K562 cells

0.56 –2.0 μM 800– 50 cells mL
1

0.025 μM 10 cells ml
1

(55) (62)

CCRF-CEM cell (human acute lymphoblastic leukemia)

1  102– 5  104 cells mL
1

10 cells mL
1

(60)

a LR, linear range; DL, detection limit; EIS, electrochemical impedance spectroscopy; SPR, surface plasmon resonance; FRET, fluorescence resonance energy transfer; ECL, electrochemiluminescence; DPV, differential pulse voltammetry; UCP, upconverting phosphors; GCE, glass carbon electrode; CFB, carbon-free blocking solution; ConA, lectin concanavalin A; DexP, phenoxyderivatized dextran; FBT, folate-biopterin transporter.

Moreover, development of techniques to integrate GO into practical devices having high sensitivity, selectivity with acceptable reproducibility, reliability, and low cost remains a big challenge. It is known that graphene nanoribbons, which have the dimensional restriction of a graphene sheet of tens of nanometers, exhibit significantly different electric properties from large-sheet graphene. This is yet to be integrated in bio-FET (field-effect transistor) design and promises even higher sensitivity and selectivity for FET devices. It is also expected that zigzag and armchair graphene edges will exhibit different electrochemical properties, and although fabrication of purely zigzag or armchair edges is difficult, a breakthrough in this area is expected (67). Because the field is still young, it is expected to branch out into many applications to meet the needs of society in the areas of enhanced healthcare. Furthermore, high-surface-area graphene prepared by CVD can be used to detect small biomolecules (DNA), and heteroatom-doped graphene can be used to form novel biosensors. The electrochemical performances of ionic liquid attached to graphene can also be explored (68). As compared with CNTs, limited research has been carried out on graphene/conducting polymer nanocomposites and graphene/carbon paste electrode. Finally, the mass production of single-layer graphene is a major challenge that, if overcome, could benefit mankind. With continued research effort and interdisciplinary collaboration, it is expected that the use of graphene and its derivatives in biosensing will mature into a clinically useful field in the near future. The future of graphene in health-care applications looks brighter than ever, yet many hurdles remain to be conquered.

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REFERENCES [1] Li W, Wu P, Zhang H, Cai C. Signal Amplification of Graphene Oxide Combining With Restriction Endonuclease for Site-Specific Determination of DNA Methylation and Assay of Methyltransferase Activity. Anal Chem 2012;84(17):7583–90. [2] Sarathy A, Qiu H, Leburton JP. Graphene Nanopores for Electronic Recognition of DNA Methylation. J Phys Chem B 2017;121(15):3757–63. [3] Haque MH, Gopalan V, Yadav S, Islam MN, Eftekhari E, Li Q, Carrascosa LG, Nguyen NT, Lam AK, Shiddiky MJA. Detection of Regional DNA Methylation Using DNA-Graphene Affinity Interactions. Biosens Bioelectron 2017;87:615–21. [4] Varghese N, Mogera U, Govindaraj A, Das A, Maiti PK, Sood AK, Rao CNR. Binding of DNA Nucleobases and Nucleosides With Graphene. ChemPhysChem 2009;10(1):206–10. [5] Lv W, Guo M, Liang M-H, Jin F-M, Cui L, Zhi L, Yang Q-H. Graphene-DNA Hybrids: Self-Assembly and Electrochemical Detection Performance. J Mater Chem 2010;20(32):6668–73. [6] Tang X, Bansaruntip S, Nakayama N, Yenilmez E, Chang YI, Wang Q. Carbon Nanotube DNA Sensor and Sensing Mechanism. Nano Lett 2006;6(8):1632–6. [7] Li D, Muller MB, Gilje S, Kaner RB, Wallace GG. Processable Aqueous Dispersions of Graphene Nanosheets. Nat Nanotechnol 2008;3(2):101–5. [8] He P, Xu Y, Fang Y. Applications of Carbon Nanotubes in Electrochemical DNA Biosensors. Microchim Acta 2006;152:175–86. [9] Hu Y, Li F, Han D, Wu T, Zhang Q, Niu L, Bao Y. Simple and Label-Free Electrochemical Assay for SignalOn DNA Hybridization Directly at Undecorated Graphene Oxide. Anal Chim Acta 2012;753:82–9. [10] Cao X. Ultra-Sensitive Electrochemical DNA Biosensor Based on Signal Amplification Using Gold Nanoparticles Modified With Molybdenum Disulfide, graphene and horseradish peroxidase. Microchim Acta 2014;181(9–10):1133–41. [11] Hajihosseini S, Nasirizadeh N, Hejazi MS, Yaghmaei P. A Sensitive DNA Biosensor Fabricated From Gold Nanoparticles and Graphene Oxide on a Glassy Carbon Electrode. Mater Sci Eng C 2016;61:506–15. [12] Chen T-Y, Loan PTK, Hsu C-L, Lee Y-H, Tse-Wei Wang J, Wei K-H, Lin C-T, Li L-J. Label-Free Detection of DNA Hybridization Using Transistors Based on CVD Grown Graphene. Biosens Bioelectron 2013;41:103–9. [13] Dong H, Zhu Z, Ju H, Yan F. Triplex Signal Amplification for Electrochemical DNA Biosensing by Coupling Probe-Gold Nanoparticles-Graphene Modified Electrode With Enzyme Functionalized Carbon Sphere as Tracer. Biosens Bioelectron 2012;33(1):228–32. [14] Benvidi A, Rajabzadeh N, Mazloum-Ardakani M, Heidari MM, Mulchandani A. Simple and Label-Free Electrochemical Impedance Amelogenin Gene Hybridization Biosensing Based on Reduced Graphene Oxide. Biosens Bioelectron 2014;58:145–52. [15] Bartel DP. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 2004;116(2):281–97. [16] Pritchard CC, Cheng HH, Tewari M. MicroRNA Profiling: Approaches and Considerations. Nat Rev Genet 2012;13(5):358–69. [17] Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, et al. MicroRNA Expression Profiles Classify Human Cancers. Nature 2005;435(7043):834–8. [18] Guay C, Regazzi R. Circulating microRNAs as Novel Biomarkers for Diabetes Mellitus. Nat Rev Endocrinol 2013;9(9):513–21. [19] Miao X, Wang W, Kang T, Liu J, Shiu K-K, Leung C-H, Ma D-L. Ultrasensitive Electrochemical Detection of miRNA-21 by Using an Iridium(III) Complex as Catalyst. Biosens Bioelectron 2016;86:454–8. [20] Dong H, Meng X, Dai W, Cao Y, Lu H, Zhou S, Zhang X. Highly Sensitive and Selective microRNA Detection Based on DNA-Bio-Bar-Code and Enzyme-Assisted Strand Cycle Exponential Signal Amplification. Anal Chem 2015;87(8):4334–40.

REFERENCES

213

[21] Song C, Wang G-Y, Kong D-M. A Facile Fluorescence Method for Versatile Biomolecular Detection Based on Pristine α-Fe2O3 Nanoparticle-Induced Fluorescence Quenching. Biosens Bioelectron 2015;68:239–44. [22] Driskell JD, Seto AG, Jones LP, Jokela S, Dluhy RA, Zhao Y-P, Tripp RA. Rapid microRNA (miRNA) Detection and Classification via Surface-Enhanced Raman Spectroscopy (SERS). Biosens Bioelectron 2008;24(4):917–22. [23] Zhao H, Qu Y, Yuan F, Quan X. A Visible and Label-Free Colorimetric Sensor for miRNA-21 Detection Based on Peroxidase-Like Activity of Graphene/Gold-Nanoparticle Hybrids. Anal Methods 2016;8 (9):2005–12. [24] Li S, He K, Liao R, Chen C, Chen X, Cai C. An Interference-Free and Label-Free Sandwich-Type Magnetic Silicon Microsphere -rGO-Based Probe for Fluorescence Detection of microRNA. Talanta 2017;174:679–83. [25] Treerattrakoon K, Luksirikul P, Dharakul T, Japrung D. Isothermal Amplification and Graphene Based Detection of Circulating Multiplex miRNAs, In: IEEE 16th International Conference on Nanotechnology (IEEE-NANO). 2016; 2016. p. 347–50. [26] Li W, Hou T, Wu M, Li F. Label-Free Fluorescence Strategy for Sensitive microRNA Detection Based on Isothermal Exponential Amplification and Graphene Oxide. Talanta 2016;148:116–21. [27] Fan X, Qi Y, Shi Z, Lv Y, Guo Y. Molecular Mechanism of Helicase on Graphene-Based Hybridization Reaction Platform for microRNA Detection. RSC Adv 2017;7(58):36444–9. [28] Huang R, Liao Y, Zhou X, Fu Y, Xing D. Multiplexed Detection of microRNA Biomarkers From Tumor Cells and Tissues With a Homogeneous Nano-Photon Switch. Sensors Actuators B Chem 2017;247:505–13. [29] Laurenti M, Paez-Perez M, Algarra M, Alonso-Cristobal P, Lopez-Cabarcos E, Mendez-Gonzalez D, RubioRetama J. Enhancement of the Upconversion Emission by Visible-to-Near-Infrared Fluorescent Graphene Quantum Dots for miRNA Detection. ACS Appl Mater Interfaces 2016;8(20):12644–51. [30] Jamali AA, Pourhassan-Moghaddam M, Dolatabadi JEN, Omidi Y. Nanomaterials on the Road to microRNA Detection With Optical and Electrochemical Nanobiosensors. TrAC Trends Anal Chem 2014;55:24–42. [31] Erdem A, Eksin E, Isin D, Polat D. Graphene Oxide Modified Chemically Activated Graphite Electrodes for Detection of microRNA. Electroanalysis 2017;29(5):1350–8. [32] Tran H, V; Piro B, Reisberg S, Nguyen LH, Nguyen TD, Duc HT. Biosensors and Bioelectronics an electrochemical ELISA-Like Immunosensor for miRNAs Detection Based on Screen-Printed Gold Electrodes Modified With Reduced Graphene Oxide and Carbon Nanotubes. Biosens Bioelectron 2014;62:25–30. [33] Hu T, Zhang L, Wen W, Zhang X, Wang S. Enzyme Catalytic Amplification of miRNA-155 Detection With Graphene Quantum Dot-Based Electrochemical Biosensor. Biosens Bioelectron 2016;77:451–6. [34] Shuai H-L, Huang K-J, Xing L-L, Chen Y-X. Ultrasensitive Electrochemical Sensing Platform for microRNA Based on Tungsten Oxide-Graphene Composites Coupling With Catalyzed Hairpin Assembly Target Recycling and Enzyme Signal Amplification. Biosens Bioelectron 2016;86:337–45. [35] Shuai H-L, Huang K-J, Zhang W-J, Cao X, Jia M-P. Sandwich-Type microRNA Biosensor Based on Magnesium Oxide Nanoflower and Graphene Oxide–Gold Nanoparticles Hybrids Coupling With Enzyme Signal Amplification. Sensors Actuators B Chem 2017;243:403–11. [36] Wang Q, Li Q, Yang X, Wang K, Du S, Zhang H, Nie Y. Graphene Oxide–Gold Nanoparticles Hybrids-Based Surface Plasmon Resonance for Sensitive Detection of microRNA. Biosens Bioelectron 2016;77:1001–7. [37] Li Q, Wang Q, Yang X, Wang K, Zhang H, Nie W. High Sensitivity Surface Plasmon Resonance Biosensor for Detection of microRNA and Small Molecule Based on Graphene Oxide-Gold Nanoparticles Composites. Talanta 2017;174:521–6. [38] Zhang T, Zhao H, Fan G, Li Y, Li L, Quan X. Electrolytic Exfoliation Synthesis of Boron Doped Graphene Quantum Dots: A New Luminescent Material for Electrochemiluminescence Detection of Oncogene microRNA-20a. Electrochim Acta 2016;190:1150–8. [39] Panta P, Venna VR. Salivary RNA Signatures in Oral Cancer Detection. Anal Cell Pathol 2014;2014:1–7.

214

CHAPTER 9 GRAPHENE AND ITS DERIVATIVES AS BIOSENSING PLATFORM

[40] Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, Coller H, Loh ML, Downing JR, Caligiuri MA, et al. Molecular Classification of Cancer: Class Discovery and Class Prediction By Gene Expression Monitoring. Science 1999;286(5439):531–7. [41] Beer DG, Kardia SLR, Huang C-C, Giordano TJ, Levin AM, Misek DE, Lin L, Chen G, Gharib TG, Thomas DG, et al. Gene-Expression Profiles Predict Survival of Patients With Lung Adenocarcinoma. Nat Med 2002;8(8):816–24. [42] Li F, Chen M, Sun X, Wang X, Li P. A Novel Graphene Oxide-Based Fluorescence Assay for RNA Endonuclease Activity of Mammalian Argonaute2 Protein. Sensors Actuators B Chem 2013;182:156–60. [43] Li X-L, Shan S, Xiong M, Xia X-H, Xu J, Chen H-Y. On-Chip Selective Capture of Cancer Cells and Ultrasensitive Fluorescence Detection of Survivin mRNA in a Single Living Cell. Lab Chip 2013;13(19):3868–75. [44] Stobiecka M, Dworakowska B, Jakiela S, Lukasiak A, Chalupa A, Zembrzycki K. Sensing of Survivin mRNA in Malignant Astrocytes Using Graphene Oxide Nanocarrier-Supported Oligonucleotide Molecular Beacons. Sensors Actuators B Chem 2016;235:136–45. [45] Vilela P, El-Sagheer A, Millar TM, Brown T, Muskens OL, Kanaras AG. Graphene Oxide-Upconversion Nanoparticle Based Optical Sensors for Targeted Detection of mRNA Biomarkers Present in Alzheimer’s Disease and Prostate Cancer. ACS Sens 2017;2(1):52–6. [46] Fan J, Zhang X, Cheng Y, Xiao C, Wang W, Liu X, Tong C, Liu B. Increasing the Sensitivity and Selectivity of a GONS Quenched Probe for an mRNA Assay Assisted With Duplex Specific Nuclease. RSC Adv 2017;7 (57):35629–37. [47] Sharova LV, Sharov AA, Nedorezov T, Piao Y, Shaik N, Ko MSH. Database for mRNA Half-Life of 19 977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse Embryonic Stem Cells. DNA Res 2009;16(1):45–58. [48] Abbas T, Dutta A. p21 in Cancer: Intricate Networks and Multiple Activities. Nat Rev Cancer 2009;9 (6):400–14. [49] Nie F, Sun M, Yang J, Xie M, Xu T, Xia R, Liu Y, Liu X, Zhang E, Lu K, et al. Long Noncoding RNA ANRIL Promotes Non-Small Cell Lung Cancer Cell Proliferation and Inhibits Apoptosis by Silencing KLF2 and P21 Expression. Mol Cancer Ther 2015;14(1):268–77. [50] Palecˇek E, Tka´cˇ J, Bartosˇ´ık M, Berto´k T, Ostatna´ V, Palecˇek J. Electrochemistry of Nonconjugated Proteins and Glycoproteins. Toward Sensors for Biomedicine and Glycomics. Chem Rev 2015;115(5):2045–108. [51] Klukova´ L, Bertok T, Kasa´k P, Tkac J. Nanoscale-Controlled Architecture for the Development of Ultrasensitive Lectin Biosensors Applicable in Glycomics. Anal Methods 2014;6(14):4922–31. [52] Klukova L, Filip J, Belicky S, Vikartovska A, Tkac J. Graphene Oxide-Based Electrochemical Label-Free Detection of Glycoproteins Down to aM Level Using a Lectin Biosensor. Analyst 2016;141(14):4278–82. [53] Belicky S, Katrlik J, Tkac J. Glycan and Lectin Biosensors. Essays Biochem 2016;60(1):37–47. [54] Hushegyi A, Tkac J. Are Glycan Biosensors an Alternative to Glycan Microarrays? Anal Methods 2014;6 (17):6610. [55] Zhang C, Yuan Y, Zhang S, Wang Y, Liu Z. Biosensing Platform Based on Fluorescence Resonance Energy Transfer From Upconverting Nanocrystals to Graphene Oxide. Angew Chem Int Ed 2011;50 (30):6851–4. [56] Wang L, Pu KY, Li J, Qi X, Li H, Zhang H, Fan C, Liu B. A Graphene-Conjugated Oligomer Hybrid Probe for Light-Up Sensing of Lectin and Escherichia coli. Adv Mater 2011;23(38):4386–91. [57] Chen Q, Wei W, Lin JM. Homogeneous Detection of Concanavalin A Using Pyrene-Conjugated Maltose Assembled Graphene Based on Fluorescence Resonance Energy Transfer. Biosens Bioelectron 2011;26 (11):4497–502. [58] Huang C-F, Yao G-H, Liang R-P, Qiu J-D. Graphene Oxide and Dextran Capped Gold Nanoparticles Based Surface Plasmon Resonance Sensor for Sensitive Detection of Concanavalin A. Biosens Bioelectron 2013;50:305–10.

FURTHER READING

215

[59] Filip J, Zavahir S, Klukova L, Tkac J, Kasak P. Immobilization of Concanavalin A Lectin on a Reduced Graphene Oxide-Thionine Surface by Glutaraldehyde Crosslinking for the Construction of an Impedimetric Biosensor. J Electroanal Chem 2016;2017(794):156–63. [60] Chen X, Wang Y, Zhang Y, Chen Z, Liu Y, Li Z, Li J. Sensitive Electrochemical Aptamer Biosensor for Dynamic Cell Surface N-Glycan Evaluation Featuring Multivalent Recognition and Signal Amplification on a Dendrimer-Graphene Electrode Interface. Anal Chem 2014;86(9):4278–86. [61] Chen X, He Y, Zhang Y, Liu M, Liu Y, Li J. Ultrasensitive Detection of Cancer Cells and Glycan Expression Profiling Based on a Multivalent Recognition and Alkaline Phosphatase-Responsive Electrogenerated Chemiluminescence Biosensor. Nanoscale 2014;6(19):11196–203. [62] Liu J, Xin X, Zhou H, Zhang S. A Ternary Composite Based on Graphene, Hemin, and Gold Nanorods With High Catalytic Activity for the Detection of Cell-Surface Glycan Expression. Chem Eur J 2015;21 (5):1908–14. [63] Li Z, Deng SS, Zang Y, Gu Z, He XP, Chen GR, Chen K, James TD, Li J, Long YT. Capturing Intercellular Sugar-Mediated Ligand-Receptor Recognitions via a Simple Yet Highly Biospecific Interfacial System. Sci Rep 2013;3:2293. [64] Terse-Thakoor T, Badhulika S, Mulchandani A. Graphene Based Biosensors for Healthcare. J Mater Res 2017;32(15):2905–29. [65] Zhu Z. An Overview of Carbon Nanotubes and Graphene for Biosensing Applications. Nano-Micro Lett 2017;9(3):25. [66] Shen H, Zhang L, Liu M, Zhang Z. Biomedical Applications of Graphene. Theranostics 2012;2(3):283–94. [67] Pumera M. Graphene in Biosensing. Mater Today 2011;14(7–8):308–15. [68] Kuila T, Bose S, Khanra P, Mishra AK, Kim NH, Lee JH. Recent Advances in Graphene-Based Biosensors. Biosens Bioelectron 2011;26(12):4637–48.

FURTHER READING [69] Esteller M, Herman JG. Cancer as an Epigenetic Disease: DNA Methylation and Chromatin Alterations in Human Tumours. J Pathol 2002;196(1):1–7. [70] Bertok T, Katrlik J, Gemeiner P, Tkac J. Electrochemical Lectin Based Biosensors as a Label-Free Tool in Glycomics. Microchim Acta 2013;180(1–2):1–13. [71] Zhu X, Liu Y, Li P, Nie Z, Li J. Applications of Graphene and Its Derivatives in Intracellular Biosensing and Bioimaging. Analyst 2016;141(15):4541–53.