Graphene in Electrochemical Biosensors

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Hsing-Ying Lin*, Wen-Hao Chen†, Chen-Han Huang‡ Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States* Institute of Chemistry, Academia Sinica, Nankang, Taipei, Taiwan† Department of Biomedical Sciences and Engineering, National Central University, Taoyuan City, Taiwan‡

CHAPTER OUTLINE 1 Introduction ................................................................................................................................... 321 1.1 Voltammetric, Amperometric, Impedimetric, and Potentiometric Electrochemical Sensors ... 322 1.2 Small Molecule Biomarker Analysis ................................................................................. 323 1.3 Protein Analysis ............................................................................................................. 326 1.4 Nucleic Acid Analysis .................................................................................................... 328 1.5 Cell Analysis ................................................................................................................. 329 2 Conclusion .................................................................................................................................... 330 Acknowledgments ...............................................................................................................................330 References ......................................................................................................................................... 330

1 INTRODUCTION The development of high-performance, ultrasensitive, and miniaturized biosensors for precise and streamline monitoring of biomedical analytes in medical, environmental, food, and cosmetic applications has attracted lots of attention in the past decade (1–3). Within the biosensor fabrication process, several units are required. Among them, functional nanomaterials are abundantly applied due to their applicability to generate detectable signals from specific molecular recognition process to achieve the goal of tiny analyte detection. Until now, various detection forms have been developed, such as spectroscopic, chromatographic, colorimetric, fiber-optic, and electrochemical sensors (4–8). Researchers are considerably devoted to amalgamate a recognition element with an electronic unit to make advanced sensing devices. In comparison with other detection approaches, electrochemical sensors possess the merit of remarkable detection ability, robustness, user-friendliness, simplicity, low Biomedical Applications of Graphene and 2D Nanomaterials. https://doi.org/10.1016/B978-0-12-815889-0.00015-5 # 2019 Elsevier Inc. All rights reserved.

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detection limit, and fast analysis ability (9). As so, electrochemical sensors stand a foremost position among mostly available sensors and have achieved a commercial level. Enzyme, nucleic acids, antibodies, and oligonucleotides are widely used as the biorecognition elements in biosensors (10). Typical electrochemical biosensors convert the interaction between recognition events with the biomolecules of interest into an electric signal. To date, various electrochemical sensing formats have been adopted, for example, field-effect transistor (FET) (11), electrochemical impedance sensors (12), electrochemiluminescence sensors (13), amperometric sensors (14), and photoelectrochemical sensors (15). Advances in nanotechnology have facilitated the considerable progress of biosensor development. Nanomaterials and nanostructures have promoted the enhancement of both sensitivity and selectivity on biosensors. Most recently, graphene and its derivatives have been extensively applied in various sensing applications due to its several unique properties, for example, adjustable bandgap, strong photoluminescence emission, good biocompatibility, good optical transparency, ease of functionalization, high surface area, and high electronic and thermal conductivity (16). This two-dimensional (2D), sp2-bonded carbon nanomaterial can be synthesized and processed based on nonsolution and solution-route approaches. Generally, solution-based approaches are more suitable for biosensing applications than nonsolution-based ones, due to its compatibility with biochemical systems and inexpensive cost. Solution-based methods include chemical oxidation/reduction, functionalization of graphene oxide, liquid-phase mechanical exfoliation, electrolytic exfoliation, mechanical exfoliation with intercalation by small molecules, hydro-/solvothermal synthesis, and organic synthesis (17–19). Mechanical cleavage, chemical vapor deposition (CVD), epitaxial growth via ultrahigh vacuum graphitization, and other vapor-phase techniques are commonly studied nonsolution methods (20–22). So far, many researches demonstrate that exploiting functional graphene nanocomposites coupled with electrochemical analytic methods can effectively decrease the overpotential and enhance the current response, resulting the significant improved performance of electrochemical biosensors (23). Among these works, graphene-based electrochemical devices have been applied to analyze various biochemical analytes, including glucose (24), cysteine (25), proteins (26), biomarkers (Fig. 1) (27), and nucleic acids (28).

1.1 VOLTAMMETRIC, AMPEROMETRIC, IMPEDIMETRIC, AND POTENTIOMETRIC ELECTROCHEMICAL SENSORS Electrochemical reactions are electron charge transfer processes occurred at the electrode surface. These reactions usually involve electrolyte resistance, electroactive species adsorption, and mass transfer from bulk solution to the electrode surface. Each reaction can be represented by a simple electric circuit model consisting of resistance, capacitors, or constant phase elements combined in parallel or in series, such as Randles-Ershler electric equivalent circuit model (29, 30). Voltammetric and amperometric methods are the most prominent transducing techniques in sensor development since they can provide simple, high-sensitivity achievable, and cost-effective transducers (31). These two methodologies belong to potentiostatic technique where the electrode potential is controlled and applied to drive an electron transfer reaction, resulting in a measured current. Both are popularly used in electrochemical biosensors to detect significant biomarkers in clinical applications (32–34). Cyclic voltammetry has been adopted to improve the detection limit in quantitative measurements (35, 36). In cyclic voltammetry, usually, various excitation waveforms, for example, normal pulse voltammetry, square-wave voltammetry, and differential pulse voltammetry, are exploited to trigger the reaction (37–40). As to amperometry, a fixed potential is applied between a reference and a working electrode

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FIG. 1 Schematic representation of the electrochemical biosensing platform for vascular endothelial growth factor receptor 2 protein (27). Copyrights (2014) Springer Nature.

to cause the oxidation or reduction of electroactive species. The resultant current change is monitored over time (41, 42). The working electrode material strongly influences the performance of amperometric sensors. Thus, much effort has been devoted to electrode fabrication and maintenance (43, 44). Electrochemical impedimetry is a sensitive technique to investigate both bulk and interfacial electric properties of systems. This method can be applied to extract the electron transfer kinetics and diffusion characteristics related to reactions (45–47). It is usually employed to quantitatively monitor the parameter changes of electrochemical processes related to biorecognition events on the electrode surface, for example, enzyme catalysis reactions and specific binding reactions of proteins, antibodies, nucleic acids, or cells (48–50). Many immunosensors and aptasensors are based on impedimetric configuration. In impedimetry, once antibodies, antigens, or target sequences are bound and formed immunocomplexes or consequent conformational changes on the electrode surface, a surface impedance layer will form at the electrode, allowing direct detection of biomolecular recognition events without using enzyme labels (51–55). Potentiometry passively measures the potential difference between two electrodes. The reference electrode has a constant potential, while the other indicator electrode changes its potential in response to analyte concentration (56–58). Thus, it can be used to determine electrochemical properties relevant to the composition of sample.

1.2 SMALL MOLECULE BIOMARKER ANALYSIS Due to the fast electron transfer kinetics and superior electrocatalytic activity of graphene-based materials, a large number of works are dedicated to its application on developing novel electrochemical sensors for detecting several important small molecules, which are relevant to clinical diagnosis.

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Ascorbic acid, dopamine, uric acid, glucose, glutamate, β-nicotinamide adenine dinucleotide, hydrogen peroxide, and other small biomolecules are among the key small biomolecules affecting human health. Therefore, monitoring such kinds of small molecules plays a vital role in tracing the health status. Electrochemical identification of these three electroactive compounds, ascorbic acid, dopamine, and uric acid, is complicated and always a significant challenge in clear separation of their electrochemical signals since they have similar electrochemical properties (59). Dopamine is one of the most prominent catecholamines, functioning as a neurotransmitter in the central nervous system. The earlier study used graphite paste electrodes with chronoamperometry to selectively detect basal extracellular dopamine concentrations in the stratum brain tissue of rats (60). Thomas et al. applied biocompatible graphite oxide-modified carbon paste electrode to selectively and sensitively detect dopamine in the presence of large excess of ascorbic acid and uric acid at physiological pH, also in spiking human blood serum and cerebral fluid. This graphite oxide electrode could provide better resolution of anodic peaks in the differential pulse voltammogram (61). In addition to dopamine, melatonin and L-tryptophan also play vital roles and coexist in the extracellular fluid of central nervous system and serum (62). Melatonin is a hormone synthesized by the pineal parenchymal cells from serotonin through N-acetylation and O-methylation and then released into blood and cerebrospinal fluid. The dopamine deficiency and melatonin level variation have been presented to associate with Parkinson’s disease, one of the most common chronic and progressive neurodegenerative disorders (63, 64). Besides, abnormal metabolism of dopamine and melatonin is frequently observed in phenylketonuria, an inherited disorder of an amino acid phenylalanine buildup in the blood. These two small molecules are used as biomarkers to optimize treatment in phenylketonuria (65). L-Tryptophan is a precursor for serotonin, melatonin, and niacin. The decreased concentration of plasma tryptophan is correlated with major depressive disorder (66). Tadayon et al. adopted the carbon paste electrode together with synthesized nitrogen-doped graphene CuCo2O4 nanocomposites to directly determine the dopamine, melatonin, and tryptophan in human urine, serum, and pharmaceutical samples (Fig. 2). This strategy successfully advanced the oxidation peak currents and reduced the overpotential of these three analytes. The developed sensor achieved the simultaneous detection of target small molecules with linear ranges of 0.01–3.0 μM (67). In the human body, the balance of coexisting uric acid, ascorbic acid, dopamine, and nitrite (NO2
) is an important index for evaluation of physiological functions. Thus, sensitive biosensors for simultaneously detection of these four molecules with accurate concentration determination are highly desirable in analytic and diagnostic applications. NO2
is associated with NO, because NO is easily oxidized to NO2
in a few seconds (68, 69). NO is a neurotransmitter and also a neuromodulator in the central nervous systems. Simultaneous quantitative detection of these molecules in a mixture is always a challenge due to their close oxidation potentials. Hexadecyltrimethylammonium bromide-functionalized multiwalled carbon nanotubes (70), lanthanum-modified multiwalled carbon nanotubes (71), sol-gel-derived La(OH)3 nanorod-incorporated carbon nanotubes (72), and noncovalent iron(III)-porphyrin-functionalized multiwalled carbon nanotubes (73) are developed to modify glassy carbon electrodes for the simultaneous determination of these molecules. The effective and simple direct printing method to fabricate carbon electrodes is also developed. Graphene oxide nanoribbons and poly(3,4-ethylenedioxythiophene) derivatives deposited on electrodes are applied to successfully detect multiple species simultaneously (74). Large-area graphene is a semimetal with zero bandgap. Its valence and conduction bands are coneshaped and meet at the K points of Brillouin zone (75). Due to the lack of an electronic bandgap in graphene, its conductivity cannot be switched off electronically as in conventional semiconductor

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FIG. 2 Differential pulse voltammetry for different concentrations of dopamine (Dp), melatonin (Me), and L-tryptophan (Tp) in pH 3 Britton-Robinson (B-R) buffer solution. Concentrations of Dp, Me, and Tp from low to high: 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.7, 1, 1.5, 2, 2.3, 2.5, 2.7, and 3 μM (67). Copyrights (2015) the Royal Society of Chemistry.

materials. Chemical doping and surface modification with heteroatoms or nanoparticles on graphene are effective strategies for tailoring its surface chemistry and physical properties. They provide an efficient way to tune the bandgap structure and adjust Fermi level of graphene for various electric applications (76–78). Previous studies showed nitrogen is an excellent element for chemical doping. Its atomic size and five valence electrons can provide strong valence bonds with carbon. Owing to the doping of electron-rich nitrogen, the free charge carriers in the aromatic ring structure of graphene will increase, resulting in an enhanced conductivity and bandgap adjustment (79). Nitrogen-doped graphene has been applied in biosensors. Wang et al. prepared nitrogen-doped graphene by using nitrogen plasma treatment. The nitrogen percentage was controlled between 0.11% and 1.35% through tuning the exposure time. The produced nitrogen-doped graphene displayed a high electrocatalytic activity for reduction of hydrogen peroxide and fast direct electron transfer kinetics for glucose oxidase. Glucose oxidase is a homodimer containing two tightly bound flavin adenine dinucleotide cofactors. Redox peaks of glucose oxidase were hard to observe when using common electrode materials, for example, glassy carbon or graphite, because the direct two-proton and two-electron transfer reaction is not easily accessible at a general electrode surface. However, such kind of developed nitrogen-doped biosensor can provide a sensitive detection for glucose, and its detection limit can be as low as 0.01 mM in the presence of interferences (76). Lead (Pb) is a widely distributed toxic metal and is harmful to neurological, hematologic, gastrointestinal, cardiovascular, and renal systems. It can induce severe health issues, especially in children due to their immature nervous system. It has been reported that the Pb level of above 100 μg/L in blood leads to deleterious effects on children’s brain functions, including low intelligence and behavioral development problems (80). Due to the complicated blood matrix, it is challenging to develop a highly

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FIG. 3 Illustrative scheme of the preparation of graphene field-effect transistor (FET) (81). Copyrights (2016) Springer Nature.

sensitive detection technique with good selectivity, superior portability, and fast response. Wang et al. chose DNAzyme as the Pb sensing aptamer due to its high Pb2+ binding affinity and selectivity to develop a label-free and portable aptasensor based on graphene field-effect transistor (FET) for effective children blood Pb detection (Fig. 3) (81). They introduced a pyrene group on the 50 -end of enzyme strand to anchor this aptamer to the graphene surface through π-π stacking interactions. This strategy successfully avoids the nonspecific adsorption and denaturation of DNAzyme on the graphene surface. They replaced the cleavable site to uncleavable one of the DNAzyme, giving rise to significant signal changes with no need for the dissociation of cleaved substrate strand upon Pb2+ binding. This method improved the response speed of DNAzyme-based graphene sensor in Pb2+ detection. The detection limit is below 37.5 ng/L, which is three orders lower than the safe blood Pb level of 100 μg/L. This device also showed excellent selectivity over other metal cations, including Na+, K+, Mg2+, and Ca2+, suggesting the capability of working in a complex sample matrix. Such kinds of graphene FET aptasensors have significant potential applications of heavy metal ion health monitoring and disease diagnostics.

1.3 PROTEIN ANALYSIS Graphene is an ideal material to modify electrodes and biosensor platforms, especially in protein-based detection. Functionalized graphene is able to directly detect biomolecules by its own oxide components due to the synthesis composing lots of epoxide, hydroxyl, and carboxyl group on the edge and surface sites (82). Graphene oxide can provide an adsorption matrix for antibodies and proteins via oxygenmediated amide condensation reactions or electrostatic interactions (83, 84). The p53 protein is a well-known tumor suppressor and a transcription factor. It is found in 50% of human cancers. The p53 plays a crucial role in controlling cell growth and modulating DNA repair processes (85, 86). Thus,

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understanding p53 regulation remains an important goal to design broadly applicable anticancer strategies based on its pathways. The p53 can be stabilized and accumulated in nucleus of tumor cells as a result of p53 phosphorylation. In clinical research, the quantitative determination of phosphorylated p53 is of great significance for early diagnosis of cancers. Du et al. developed an ultrasensitive electrochemical immunosensor for phosphorylated p53 detection (87). They adopted graphene oxide, prepared through oxidizing graphite according to the modified Hummer’s method (88), as a nanocarrier for enzyme and antibody coimmobilization. Due to a large amount of enzymes loaded on the graphene oxide nanocarrier via amidization, the cascade electrocatalytic signal amplification resulting in greatly amplified sensitivity was achieved. The dynamic range of current response was proportional to the phospho-p53 concentration (0.02–2 nM) with a detection limit of 0.01 nM, which was 10-fold lower than that of the conventional immunosensor. Nerve growth factor (NGF) plays a trophic role during neuronal development, during neuronal differentiation, and in adulthood (89, 90). It maintains the phenotypic and functional characteristic of neurons and immune cells (91–93). The previous study showed that the severity of neurological impairment in patients that suffered from a cerebrovascular accident is strongly correlated with the physiological level of NGF (94). Li et al. demonstrated that the NGF administration significantly improved the microenvironment of grafted cells through the enhancement of dopaminergic content in regional brain tissue. Meanwhile, it further promoted the survival rate of transplanted cells in the host rat brain with Parkinson’s disease (95). The blood level of NGF is also significantly correlated with depressive disorder (96). Generally, the detection and quantification of NGF in body fluids are based on enzyme-linked immunosorbent assay (ELISA) techniques under immunologic reactions. Recently, Wei et al. applied the reduced graphene oxide-titanium nitride (TiN) nanocomposite on electrodes for sensitive electrochemical determination of NGF (97). The graphene oxide sheets provided good conductive supports for TiN nanoparticles, decreased the particle aggregation, and enhanced the electrochemical activity of particles through synergistic chemical coupling effects. This composite not only possessed long-term stability and reproducibility but also showed a high sensitivity with detection limit of 2.6 nM in electrocatalytic activity toward NGF oxidation. The linear dynamic range to NGF concentration is from 10 nM to 5 μM. The other similar material, amino group-functionalized reduced graphene oxide-supported Pd/Co nanoparticles, was employed as labels for the electrochemical detection of bladder cancer biomarker nuclear matrix protein-22 (NMP-22) in clinical urine samples (Fig. 4) (98). The immunosensor displayed a wide linear range of 0.01–20 ng/mL and a low detection limit of 0.33 pg/mL. Alpha-fetoprotein (AFP) is a glycoprotein produced by fetal liver and by a variety of tumors, including hepatocellular carcinoma, hepatoblastoma, and nonseminomatous germ-cell tumors of the ovary and testis (99). The elevated AFP concentration above the reference range was found in patient serum with viral hepatitis or cirrhosis (100, 101). The AFP blood test is especially important during pregnancy since abnormal levels may indicate certain problems with the fetus (102). Therefore, AFP has been considered as one of the important markers in clinical diagnostics. Wang et al. designed multifunctionalized graphene nanocomposites on electrochemical sensor to quantitatively detect AFP (103). The reduced graphene oxide was functionalized with ferric oxide and gold nanoparticles to further conjugate with specific antibodies and adsorb toluidine blue (TB). TB was used as an electron transfer mediator to provide measured signal. The redox of TB is promoted through ferric oxide nanoparticles. These fabricated electrodes can achieve effective and sensitive immunodetection of AFP with a linear analyzing range of 1.0  10
5 to 10.0 ng/mL and a low detection limit of 2.7 fg/mL. The other

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FIG. 4 Schematic representation of the preparation of the NH2-SAPO-34-Pd/Co-Ab2 (A) and fabrication process of modified immunosensor (B) (98). Copyrights (2016) Springer Nature.

similar label-free ultrasensitive impedimetric biosensor with lectin immobilization on graphene oxide for glycoprotein detection was also demonstrated and showed that utilizing the graphene-based interface can achieve the detection sensitivity down to an attomolar level (104).

1.4 NUCLEIC ACID ANALYSIS Nucleic acid sequencing is used to identify specific genes, full chromosomes, or even entire genomes. It helps to understand the nature of genetic information in a particular DNA segment. Many detection methodologies are developed to determine human genetic variations associated with diseases or pathological conditions. These approaches include northern blotting, polymerase chain reaction (PCR), and rolling cycle amplification (RCA). Though these methods can provide good sensitivity and selectivity in nucleic acid analysis, special equipments and reagents are required. In addition, sophisticated and time-consuming processes are involved, further increasing the experimental cost and complexity. Utilizing electrochemical DNA sensors allow the device miniaturization for low amount of sample volume analysis. Direct oxidation of DNA is the simplest approach to analyze nucleotide bases. Akhavan et al. deposited graphene oxide nanowalls with extremely sharp edges and vertical orientation on a graphite electrode (105). They applied differential pulse voltammetry and reduced graphene nanowalls to develop an ultrahigh-resolution electrochemical biosensor with single DNA resolution by directly screening the oxidation signals of individual nucleotide bases. It was also found that the electrochemical performances in stability, dynamic range of detection, and detection limit of reduced graphene nanowalls were better than those of reduced graphene nanosheet.

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Rasheed et al. developed a gold nanoparticle-incorporated and graphene-based electrochemical DNA sensor for breast cancer-related BRCA1 gene detection (106). The sensing assay was in the sandwich format, including capture DNA and reporter DNA. Capture DNA was functionalized on the graphene electrode, while reporter DNA was conjugated on gold nanoparticle surface. Once the target DNA was detected, the oxidation of gold nanoparticles can be observed through cyclic voltammetry and chronoamperometry. This kind of sensor can provide a high detection capability. It could detect one femtomolar BRCA1 gene. The other strategy is based on single-stranded reporter DNA fragments that were conjugated onto gold nanoparticles and then formed into larger particle clusters after hybridization with designed complementary DNA strands (107). After recognizing target DNA strands, these gold nanoparticle clusters were assembled on the graphene electrode surface and functionalized as signal amplification reporters, leading to significant enhancement in the electrochemical detection of BRCA1 gene. This method advanced the detection sensitivity with a detection limit down to 50 attomolar target DNA. Abnormal changes of nucleotide bases in gene can lead to the deficiency and mutation of immunity and also may induce various disorders, for example, cystic fibrosis, Parkinson’s disease, diabetes, Alzheimer’s disease, and cancers (108). The studies of single-nucleotide polymorphism can let us to further understand the underlying mechanism of single-base mismatch (109), which is helpful for early detection and treatment in clinical diagnosis. Sun et al. prepared copper-nitrogen-doped graphene nanocomposites as the electrocatalyst on electrodes to achieve the simultaneous determined bases of guanine (G), cytosine (C), adenine (A), and thymine (T) (110). Through the direct oxidation of nucleotide bases, considerable peak potential separations owing to the excellent electrocatalytic activity of doped graphene nanocomposites were observed. Simultaneous determination of four bases in a mixture was achieved without any separation pretreatment steps.

1.5 CELL ANALYSIS Since graphene is able to provide a biocompatible interface, it is an ideal material for good cell adhesion and further biochemical analysis. Guo et al. fabricated a smart biointerface of multilayered extracellular matrix protein nanostructures with an underlying graphene layer on the indium tin oxide (ITO) glass substrate (111). The graphene layer and ITO offered excellent electric conductivity for electric detection. Both layered artificial peroxidase (AP) and extracellular matrix protein enhanced the cell adhesion and cell growth capability. Results showed that including graphene can promote cell proliferation as compared with no graphene substrate. Breast cancer cells, MCF-7, were seeded on the substrate for the following in situ quantitative electrochemical detection of extracellular H2O2 released per cell. This study opened a window of developing smart electrochemical devices for in situ selective and quantitative molecular detection by cell-based assays. Due to the controllability and function tunability of graphene, the development of electrochemical sensing platform with controllable therapeutic drug release can be anticipated. Wu et al. developed an ultrasensitive and highly specific electrochemical and fluorescent immunosensing method for low-abundance circulating tumor cell detection (112). They used graphene to modify the sensor surface to accelerate the electron transfer and quantum dot-coated silica nanoparticle as tracing tags during detection. Both compose the double signal amplification strategy. Simultaneous measurements of two disease-specific biomarkers, epithelial cell adhesion molecule (EpCAM) and

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glypican 3 (GPC3), on liver cancer cell surface (Hep3B) were achieved by employing two kinds of nanoparticle tracers, anti-EpCAM-CdTe- and anti-GPC3-ZnSe-coated silica nanoparticles. Wu et al. developed a novel efficient electrochemical biosensing platform for the detection of extracellular oxygen released from human erythrocytes (113). They used hybrid material, laccase (Lac) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assembled graphene surface, as a good bioelectrocatalyst for the reduction of oxygen with inherent enzyme activity. ABTS was used as a redox mediator for the reduction of oxygen catalyzed by Lac. Though in comparison with the classical Clark electrode, this sensor still suffers a bit of slow response. This issue can be improved through modifying the size of electrode. The minimized electrode can be applied to measure the oxygen content release from single cell. The study established a general strategy in developing graphene-based hybrid materials via assembling enzymes and proteins on graphene. Moreover, it expanded graphene-based electrochemical sensor applications to in situ cellular analytic chemistry.

2 CONCLUSION Graphene-based materials have been widely applied in developing ultrasensitive and selective electrochemical biosensors. Lots of methodologies were developed and tested. The biosensing mechanisms are mainly relied on the charge transfer interactions with biomolecules and/or nanoparticlefunctionalized graphene electrodes. In this brief review, different approaches for the fabrication of graphene and the preparation of graphene-modified hybrid electrodes for electrochemical biosensing applications are introduced. Moreover, recent research advances on different graphene-based materials as an electrochemical platform for various biomolecules detection are discussed.

ACKNOWLEDGMENTS Authors thank to the funding supports: Ministry of Science and Technology, Taiwan, MOST-2112-M-008-001; 2018 Fund for Medical Discovery (FMD) Award, Massachusetts General Hospital.

REFERENCES [1] Yang Y, Asiri AM, Tang Z, Du D, Lin Y. Graphene Based Materials for Biomedical Applications. Mater Today 2013;16:365–73. [2] Reina G, Gonza´lez-Domı´nguez JM, Criado A, Va´zquez E, Bianco A, Prato M. Promises, Facts and Challenges for Graphene in Biomedical Applications. Chem Soc Rev 2017;46:4400–16. [3] Mehrotra P. Biosensors and Their Applications—A Review. J Oral Biol Craniofac Res 2016;6:153–9. € € [4] Apak R, Demirci C¸ekic¸ S, Uzer A, C¸elik SE, Bener M, Bekdeşer B, Can Z, Sag˘lam Ş, Onem AN, Erc¸ag˘ E. Novel Spectroscopic and Electrochemical Sensors and Nanoprobes for the Characterization of Food and Biological Antioxidants. Sensors 2018;18:186. [5] Han S-M, Kim Y-W, Kim Y-K, Chun J-H, Oh H-B, Paek S-H. Performance Characterization of TwoDimensional Paper Chromatography-Based Biosensors for Biodefense, Exemplified by Detection of Bacillus anthracis Spores. BioChip J 2018;12:59–68.

REFERENCES

331

[6] Zhai J, Yong D, Li J, Dong S. A Novel Colorimetric Biosensor for Monitoring and Detecting Acute Toxicity in Water. Analyst 2013;138:702–7. [7] Lin H-Y, Huang C-H, Cheng G-L, Chen N-K, Chui H-C. Tapered Optical Fiber Sensor Based on Localized Surface Plasmon Resonance. Opt Express 2012;20:21693–701. [8] Lin H-Y, Huang C-H, Park J, Pathania D, Castro CM, Fasano A, Weissleder R, Lee H. Integrated MagnetoChemical Sensor for On-Site Food Allergen Detection. ACS Nano 2017;11:10062–9. [9] El Harrad L, Bourais I, Mohammadi H, Amine A. Recent Advances in Electrochemical Biosensors Based on Enzyme Inhibition for Clinical and Pharmaceutical Applications. Sensors 2018;18:164. [10] Bhalla N, Jolly P, Formisano N, Estrela P. Introduction to Biosensors. Essays Biochem 2016;60:1–8. [11] Cheng S, Hideshima S, Kuroiwa S, Nakanishi T, Osaka T. Label-Free Detection of Tumor Markers Using Field Effect Transistor (FET)-Based Biosensors for Lung Cancer Diagnosis. Sensors Actuators B Chem 2015;212:329–34. [12] Huang M, Li H, He H, Zhang X, Wang S. An Electrochemical Impedance Sensor for Simple and Specific Recognition of G–G Mismatches in DNA. Anal Methods 2016;8:7413–9. [13] Wang Q, Chen M, Zhang H, Wen W, Zhang X, Wang S. Solid-State Electrochemiluminescence Sensor Based on RuSi Nanoparticles Combined With Molecularly Imprinted Polymer for the Determination of Ochratoxin A. Sensors Actuators B Chem 2016;222:264–9. [14] Seeber R, Pigani L, Terzi F, Zanardi C. Amperometric Sensing. A Melting Pot for Material, Electrochemical, and Analytical Sciences. Electrochim Acta 2015;179:350–63. [15] Zang Y, Lei J, Ju H. Principles and Applications of Photoelectrochemical Sensing Strategies Based on Biofunctionalized Nanostructures. Biosens Bioelectron 2017;96:8–16. [16] Liu B, Zhou K. Recent Progress on Graphene-Analogous 2D Nanomaterials: Properties, Modeling and Applications. Prog Mater Sci 2019;100:99–169. [17] Tang Z, Zhuang J, Wang X. Exfoliation of Graphene From Graphite and Their Self-Assembly at the Oil
Water Interface. Langmuir 2010;26:9045–9. [18] Ravula S, Baker SN, Kamath G, Baker GA. Ionic Liquid-Assisted Exfoliation and Dispersion: Stripping Graphene and Its Two-Dimensional Layered Inorganic Counterparts of Their Inhibitions. Nanoscale 2015;7:4338–53. [19] Yi M, Shen Z. A Review on Mechanical Exfoliation for the Scalable Production of Graphene. J Mater Chem A 2015;3:11700–15. [20] Bonaccorso F, Lombardo A, Hasan T, Sun Z, Colombo L, Ferrari AC. Production and Processing of Graphene and 2D Crystals. Mater Today 2012;15:564–89. [21] Bhuyan MSA, Uddin MN, Islam MM, Bipasha FA, Hossain SS. Synthesis of Graphene. Int Nano Lett 2016;6:65–83. [22] Abbas AN, Liu B, Narita A, D€ossel LF, Yang B, Zhang W, Tang J, Wang KL, R€ader HJ, Feng X, M€ ullen K, Zhou C. Vapor-Phase Transport Deposition, Characterization, and Applications of Large Nanographenes. J Am Chem Soc 2015;137:4453–9. [23] Vashist SK, Luong JHT. Recent Advances in Electrochemical Biosensing Schemes Using Graphene and Graphene-Based Nanocomposites. Carbon 2015;84:519–50. [24] Bharath G, Madhu R, Chen S-M, Veeramani V, Balamurugan A, Mangalaraj D, Viswanathan C, Ponpandian N. Enzymatic Electrochemical Glucose Biosensors by Mesoporous 1D Hydroxyapatite-on2D Reduced Graphene Oxide. J Mater Chem B 2015;3:1360–70. [25] Wang Y, Wang W, Li G, Liu Q, Wei T, Li B, Jiang C, Sun Y. Electrochemical Detection of L-Cysteine Using a Glassy Carbon Electrode Modified With a Two-Dimensional Composite Prepared From Platinum and Fe3O4 Nanoparticles on Reduced Graphene Oxide. Microchim Acta 2016;183:3221–8. [26] Jampasa S, Siangproh W, Laocharoensuk R, Vilaivan T, Chailapakul O. Electrochemical Detection of c-Reactive Protein Based on Anthraquinone-Labeled Antibody Using a Screen-Printed Graphene Electrode. Talanta 2018;183:311–9.

332

CHAPTER 15 GRAPHENE IN ELECTROCHEMICAL BIOSENSORS

[27] Wei T, Tu W, Zhao B, Lan Y, Bao J, Dai Z. Electrochemical Monitoring of an Important Biomarker and Target Protein: VEGFR2 in Cell Lysates. Sci Rep 2014;4:3982. [28] Muti M, Sharma S, Erdem A, Papakonstantinou P. Electrochemical Monitoring of Nucleic Acid Hybridization by Single-Use Graphene Oxide-Based Sensor. Electroanalysis 2011;23:272–9. [29] Randles JEB. Kinetics of Rapid Electrode Reactions. Discuss Faraday Soc 1947;1:11–9. [30] Ershler B. Investigation of Electrode Reactions by the Method of Charging-Curves and With the Aid of Alternating Currents. Discuss Faraday Soc 1947;1:269–77. [31] Bakker E, Qin Y. Electrochemical Sensors. Anal Chem 2006;78:3965–84. [32] Wei F, Patel P, Liao W, Chaudhry K, Zhang L, Arellano-Garcia M, Hu S, Elashoff D, Zhou H, Shukla S, Shah F, Ho C-M, Wong DT. Electrochemical Sensor for Multiplex Biomarkers Detection. Clin Cancer Res 2009;15:4446–52. [33] Ya´n˜ez-Seden˜o P, Campuzano S, Pingarro´n J. Multiplexed Electrochemical Immunosensors for Clinical Biomarkers. Sensors (Basel) 2017;17:965. [34] Martins GV, Tavares APM, Fortunato E, Sales MGF. Paper-Based Sensing Device for Electrochemical Detection of Oxidative Stress Biomarker 8-Hydroxy-20 -Deoxyguanosine (8-OHdG) in Point-of-Care. Sci Rep 2017;7:14558. [35] Elgrishi N, Rountree KJ, McCarthy BD, Rountree ES, Eisenhart TT, Dempsey JL. A Practical Beginner’s Guide to Cyclic Voltammetry. J Chem Educ 2018;95:197–206. [36] Weber SG, Long JT. Detection Limits and Selectivity in Electrochemical Detectors. Anal Chem 1988;60:903A–913A. [37] Brown AP, Anson FC. Cyclic and Differential Pulse Voltammetric Behavior of Reactants Confined to the Electrode Surface. Anal Chem 1977;49:1589–95. [38] Rifkin SC, Evans DH. Analytical Evaluation of Differential Pulse Voltammetry at Stationary Electrodes Using Computer-Based Instrumentation. Anal Chem 1976;48:2174–9. [39] Benedito da Silva O, Machado SAS. Evaluation of the Detection and Quantification Limits in Electroanalysis Using Two Popular Methods: Application in the Case Study of Paraquat Determination. Anal Methods 2012;4:2348–54. [40] Gerhardt GC, Cassidy RM, Baranski AS. Square-Wave Voltammetry Detection for Capillary Electrophoresis. Anal Chem 1998;70:2167–73. [41] Grieshaber D, MacKenzie R, V€or€os J, Reimhult E. Electrochemical Biosensors-Sensor Principles and Architectures. Sensors 2008;8:1400–58. [42] Helm I, Jalukse L, Leito I. Measurement Uncertainty Estimation in Amperometric Sensors: A Tutorial Review. Sensors 2010;10:4430. [43] Wang J. Real-Time Electrochemical Monitoring: Toward Green Analytical Chemistry. Acc Chem Res 2002;35:811–6. [44] Zhu C, Yang G, Li H, Du D, Lin Y. Electrochemical Sensors and Biosensors Based on Nanomaterials and Nanostructures. Anal Chem 2015;87:230–49. [45] Hayat A, Barthelmebs L, Marty J-L. Electrochemical Impedimetric Immunosensor for the Detection of Okadaic Acid in Mussel Sample. Sensors Actuators B Chem 2012;171, 172:810–5. [46] Bakas I, Hayat A, Piletsky S, Piletska E, Chehimi MM, Noguer T, Rouillon R. Electrochemical Impedimetric Sensor Based on Molecularly Imprinted Polymers/Sol–Gel Chemistry for Methidathion Organophosphorous Insecticide Recognition. Talanta 2014;130:294–8. [47] Pilehvar S, Dierckx T, Blust R, Breugelmans T, De Wael K. An Electrochemical Impedimetric Aptasensing Platform for Sensitive and Selective Detection of Small Molecules Such as Chloramphenicol. Sensors 2014;14:12059–69. [48] Chikkaveeraiah BV, Bhirde AA, Morgan NY, Eden HS, Chen X. Electrochemical Immunosensors for Detection of Cancer Protein Biomarkers. ACS Nano 2012;6:6546–61.

REFERENCES

333

[49] Bonanni A, del Valle M. Use of Nanomaterials for Impedimetric DNA Sensors: A Review. Anal Chim Acta 2010;678:7–17. [50] Wang X, Liu A, Xing Y, Duan H, Xu W, Zhou Q, Wu H, Chen C, Chen B. Three-Dimensional Graphene Biointerface With Extremely High Sensitivity to Single Cancer Cell Monitoring. Biosens Bioelectron 2018;105:22–8. [51] Fernandes FCB, Santos A, Martins DC, Go´es MS, Bueno PR. Comparing Label Free Electrochemical Impedimetric and Capacitive Biosensing Architectures. Biosens Bioelectron 2014;57:96–102. [52] Wu C-C, Huang W-C, Hu C-C. An Ultrasensitive Label-Free Electrochemical Impedimetric DNA Biosensing Chip Integrated With a DC-Biased AC Electroosmotic Vortex. Sensors Actuators B Chem 2015;209:61–8. [53] Zhang H, Chuai R, Li X, Zhang B. Design, Preparation and Performance Study of On-Chip Flow-Through Amperometric Sensors With an Integrated Ag/AgCl Reference Electrode. Micromachines (Basel) 2018;9:114. [54] O’Neill RD, Chang S-C, Lowry JP, McNeil CJ. Comparisons of Platinum, Gold, Palladium and Glassy Carbon as Electrode Materials in the Design of Biosensors for Glutamate. Biosens Bioelectron 2004;19:1521–8. [55] Morrin A, Killard AJ, Smyth MR. Electrochemical Characterization of Commercial and Home-Made ScreenPrinted Carbon Electrodes. Anal Lett 2003;36:2021–39. [56] Bakker E, Pretsch E. Potentiometric Sensors for Trace-Level Analysis. Trends Anal Chem 2005;24:199–207. [57] Grabowski LE, Goode SR. Determining a Solubility Product Constant by Potentiometric Titration To Increase Students’ Conceptual Understanding of Potentiometry and Titrations. J Chem Educ 2017;94:636–9. [58] Malon A, Vigassy T, Bakker E, Pretsch E. Potentiometry at Trace Levels in Confined Samples: Ion-Selective Electrodes With Subfemtomole Detection Limits. J Am Chem Soc 2006;128:8154–5. [59] Sun C-L, Lee H-H, Yang J-M, Wu C-C. The Simultaneous Electrochemical Detection of Ascorbic Acid, Dopamine, and Uric Acid Using Graphene/Size-Selected Pt Nanocomposites. Biosens Bioelectron 2011;26:3450–5. [60] Blaha CD. Evaluation of Stearate-Graphite Paste Electrodes for Chronic Measurement of Extracellular Dopamine Concentrations in the Mammalian Brain. Pharmacol Biochem Behav 1996;55:351–64. [61] Thomas T, Mascarenhas RJ, Nethravathi C, Rajamathi M, Kumara Swamy BE. Graphite Oxide Bulk Modified Carbon Paste Electrode for the Selective Detection of Dopamine: A Voltammetric Study. J Electroanal Chem 2011;659:113–9. [62] Hardeland R. Melatonin Metabolism in the Central Nervous System. Curr Neuropharmacol 2010;8:168–81. [63] Mack JM, Schamne MG, Sampaio TB, Pertile RAN, Fernandes PACM, Markus RP, Prediger RD. Melatoninergic System in Parkinson’s Disease: From Neuroprotection to the Management of Motor and Nonmotor Symptoms. Oxidative Med Cell Longev 2016;2016:3472032. [64] Videnovic A, Golombek D. Circadian Dysregulation in Parkinson’s Disease. Neurobiol Sleep Circadian Rhythms 2017;2:53–8. [65] Yano S, Moseley K, Azen C. Melatonin and Dopamine as Biomarkers to Optimize Treatment in Phenylketonuria: Effects of Tryptophan and Tyrosine Supplementation. J Pediatr 2014;165:184–189.e181. [66] Ogawa S, Fujii T, Koga N, Hori H, Teraishi T, Hattori K, Noda T, Higuchi T, Motohashi N, Kunugi H. Plasma L-Tryptophan Concentration in Major Depressive Disorder. J Clin Psychiatry 2014;75:906–15. [67] Tadayon F, Sepehri Z. A New Electrochemical Sensor Based on a Nitrogen-Doped Graphene/CuCo2O4 Nanocomposite for Simultaneous Determination of Dopamine, Melatonin and Tryptophan. RSC Adv 2015;5:65560–8. [68] Kelm M. Nitric Oxide Metabolism and Breakdown. Biochim Biophys Acta Bioenerg 1999;1411:273–89. [69] Ignarro LJ, Fukuto JM, Griscavage JM, Rogers NE, Byrns RE. Oxidation of Nitric Oxide in Aqueous Solution to Nitrite But Not Nitrate: Comparison With Enzymatically Formed Nitric Oxide From L-Arginine. Proc Natl Acad Sci USA 1993;90:8103–7.

334

CHAPTER 15 GRAPHENE IN ELECTROCHEMICAL BIOSENSORS

[70] Yang YJ, Li W. CTAB Functionalized Graphene Oxide/Multiwalled Carbon Nanotube Composite Modified Electrode for the Simultaneous Determination of Ascorbic Acid, Dopamine, Uric Acid and Nitrite. Biosens Bioelectron 2014;56:300–6. [71] Zhang W, Yuan R, Chai YQ, Zhang Y, Chen SH. A Simple Strategy Based on Lanthanum-Multiwalled Carbon Nanotube Nanocomposites for Simultaneous Determination of Ascorbic Acid, Dopamine, Uric Acid and Nitrite. Sensors Actuators B Chem 2012;166-167:601–7. [72] Zhang Y, Yuan R, Chai Y, Zhong X, Zhong H. Carbon Nanotubes Incorporated With Sol–Gel Derived La (OH)3 Nanorods as Platform to Simultaneously Determine Ascorbic Acid, Dopamine, Uric Acid and Nitrite. Colloids Surf B: Biointerfaces 2012;100:185–9. [73] Wang C, Yuan R, Chai Y, Chen S, Zhang Y, Hu F, Zhang M. Non-Covalent Iron(III)-Porphyrin Functionalized Multi-Walled Carbon Nanotubes for the Simultaneous Determination of Ascorbic Acid, Dopamine, Uric Acid and Nitrite. Electrochim Acta 2012;62:109–15. [74] Su C-H, Sun C-L, Liao Y-C. Printed Combinatorial Sensors for Simultaneous Detection of Ascorbic Acid, Uric Acid, Dopamine, and Nitrite. ACS Omega 2017;2:4245–52. [75] Schwierz F. Graphene Transistors. Nat Nanotechnol 2010;5:487. [76] Wang Y, Shao Y, Matson DW, Li J, Lin Y. Nitrogen-Doped Graphene and Its Application in Electrochemical Biosensing. ACS Nano 2010;4:1790–8. [77] Chang C-K, Kataria S, Kuo C-C, Ganguly A, Wang B-Y, Hwang J-Y, Huang K-J, Yang W-H, Wang S-B, Chuang C-H, Chen M, Huang C-I, Pong W-F, Song K-J, Chang S-J, Guo J-H, Tai Y, Tsujimoto M, Isoda S, Chen C-W, Chen L-C, Chen K-H. Band Gap Engineering of Chemical Vapor Deposited Graphene by In Situ BN Doping. ACS Nano 2013;7:1333–41. [78] Wu Y, Jiang W, Ren Y, Cai W, Lee WH, Li H, Piner RD, Pope CW, Hao Y, Ji H, Kang J, Ruoff RS. Tuning the Doping Type and Level of Graphene With Different Gold Configurations. Small 2012;8:3129–36. [79] Lv R, Terrones M. Towards New Graphene Materials: Doped Graphene Sheets and Nanoribbons. Mater Lett 2012;78:209–18. [80] Lanphear Bruce P, Hornung R, Khoury J, Yolton K, Baghurst P, Bellinger David C, Canfield Richard L, Dietrich Kim N, Bornschein R, Greene T, Rothenberg Stephen J, Needleman Herbert L, Schnaas L, Wasserman G, Graziano J, Roberts R. Low-Level Environmental Lead Exposure and Children’s Intellectual Function: An International Pooled Analysis. Environ Health Perspect 2005;113:894–9. [81] Wang C, Cui X, Li Y, Li H, Huang L, Bi J, Luo J, Ma LQ, Zhou W, Cao Y, Wang B, Miao F. A Label-Free and Portable Graphene FET Aptasensor for Children Blood Lead Detection. Sci Rep 2016;6:21711. [82] Georgakilas V, Otyepka M, Bourlinos AB, Chandra V, Kim N, Kemp KC, Hobza P, Zboril R, Kim KS. Functionalization of Graphene: Covalent and Non-Covalent Approaches, Derivatives and Applications. Chem Rev 2012;112:6156–214. [83] Cordeiro CA, de Vries MG, Cremers TIFH, Westerink BHC. The Role of Surface Availability in MembraneInduced Selectivity for Amperometric Enzyme-Based Biosensors. Sensors Actuators B Chem 2016;223:679–88. [84] Walcarius A, Minteer SD, Wang J, Lin Y, Merkoc¸i A. Nanomaterials for Bio-Functionalized Electrodes: Recent Trends. J Mater Chem B 2013;1:4878–908. [85] Toledo F, Wahl GM. Regulating the p 53 Pathway: In Vitro Hypotheses, In Vivo Veritas. Nat Rev Cancer 2006;6:909. [86] Evan GI, Vousden KH. Proliferation, Cell Cycle and Apoptosis in Cancer. Nature 2001;411:342. [87] Du D, Wang L, Shao Y, Wang J, Engelhard MH, Lin Y. Functionalized Graphene Oxide as a Nanocarrier in a Multienzyme Labeling Amplification Strategy for Ultrasensitive Electrochemical Immunoassay of Phosphorylated p53 (S392). Anal Chem 2011;83:746–52. [88] Shao Y, Wang J, Engelhard M, Wang C, Lin Y. Facile and Controllable Electrochemical Reduction of Graphene Oxide and Its Applications. J Mater Chem 2010;20:743–8. [89] Aloe L, Rocco ML, Bianchi P, Manni L. Nerve Growth Factor: From the Early Discoveries to the Potential Clinical Use. J Transl Med 2012;10:239.

REFERENCES

335

[90] Aloe L, Rocco ML, Balzamino BO, Micera A. Nerve Growth Factor: Role in Growth, Differentiation and Controlling Cancer Cell Development. J Exp Clin Cancer Res 2016;35:116. [91] Prencipe G, Minnone G, Strippoli R, De Pasquale L, Petrini S, Caiello I, Manni L, De Benedetti F, BracciLaudiero L. Nerve Growth Factor Downregulates Inflammatory Response in Human Monocytes Through TrkA. J Immunol 2014;192:3345–54. [92] Minnone G, De Benedetti F, Bracci-Laudiero L. NGF and Its Receptors in the Regulation of Inflammatory Response. Int J Mol Sci 2017;18:1028. [93] Garaci E, Caroleo MC, Aloe L, Aquaro S, Piacentini M, Costa N, Amendola A, Micera A, Calio` R, Perno C-F, Levi-Montalcini R. Nerve Growth Factor Is an Autocrine Factor Essential for the Survival of Macrophages Infected With HIV. Proc Natl Acad Sci 1999;96:14013–8. [94] Liu H-T, Liu A-B, Chancellor MB, Kuo H-C. Urinary Nerve Growth Factor Level Is Correlated With the Severity of Neurological Impairment in Patients With Cerebrovascular Accident. BJU Int 2009;104:1158–62. [95] Li M, Zhang S-Z, Guo Y-W, Cai Y-Q, Yan Z-J, Zou Z, Jiang X-D, Ke Y-Q, He X-Y, Jin Z-L, Lu G-H, Su DQ. Human Umbilical Vein-Derived Dopaminergic-Like Cell Transplantation With Nerve Growth Factor Ameliorates Motor Dysfunction in a Rat Model of Parkinson’s Disease. Neurochem Res 2010;35:1522–9. [96] Chen Y-W, Lin P-Y, Tu K-Y, Cheng Y-S, Wu C-K, Tseng P-T. Significantly Lower Nerve Growth Factor Levels in Patients With Major Depressive Disorder Than in Healthy Subjects: a Meta-Analysis and Systematic Review. Neuropsychiatr Dis Treat 2015;11:925–33. [97] Wei Z, Wang Y, Zhang J. Electrochemical Detection of NGF Using a Reduced Graphene Oxide-Titanium Nitride Nanocomposite. Sci Rep 2018;8:6929. [98] Wu D, Wang Y, Zhang Y, Ma H, Yan T, Du B, Wei Q. Sensitive Electrochemical Immunosensor for Detection of Nuclear Matrix Protein-22 Based on NH2-SAPO-34 Supported Pd/Co Nanoparticles. Sci Rep 2016;6:24551. [99] Sturgeon CM, Duffy MJ, Stenman U-H, Lilja H, Br€ unner N, Chan DW, Babaian R, Bast RC, Dowell B, Esteva FJ, Haglund C, Harbeck N, Hayes DF, Holten-Andersen M, Klee GG, Lamerz R, Looijenga LH, Molina R, Nielsen HJ, Rittenhouse H, Semjonow A, Shih I-M, Sibley P, S€ oletormos G, Stephan C, Sokoll L, Hoffman BR, Diamandis EP. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for Use of Tumor Markers in Testicular, Prostate, Colorectal, Breast, and Ovarian Cancers. Clin Chem 2008;54:e11–79. [100] Di Bisceglie AM, Sterling RK, Chung RT, Everhart JE, Dienstag JL, Bonkovsky HL, Wright EC, Everson GT, Lindsay KL, Lok ASF, Lee WM, Morgan TR, Ghany MG, Gretch DR, HALT-C Trial Group. Serum Alpha-Fetoprotein Levels in Patients With Advanced Hepatitis C: Results From the HALT-C Trial. J Hepatol 2005;43:434–41. [101] Sherman M. Current Status of Alpha-Fetoprotein Testing. Gastroenterol Hepatol 2011;7:113–4. [102] Sepp€al€a M, Ruoslahti E. Radioimmunoassay of Maternal Serum Alpha Fetoprotein During Pregnancy and Delivery. Am J Obstet Gynecol 1972;112:208–12. [103] Wang Y, Zhang Y, Wu D, Ma H, Pang X, Fan D, Wei Q, Du B. Ultrasensitive Label-Free Electrochemical Immunosensor Based on Multifunctionalized Graphene Nanocomposites for the Detection of Alpha Fetoprotein. Sci Rep 2017;7:42361. [104] 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;141:4278–82. [105] Akhavan O, Ghaderi E, Rahighi R. Toward Single-DNA Electrochemical Biosensing by Graphene Nanowalls. ACS Nano 2012;6:2904–16. [106] Rasheed PA, Sandhyarani N. Graphene-DNA Electrochemical Sensor for the Sensitive Detection of BRCA1 Gene. Sensors Actuators B Chem 2014;204:777–82.

336

CHAPTER 15 GRAPHENE IN ELECTROCHEMICAL BIOSENSORS

[107] Rasheed PA, Sandhyarani N. A Highly Sensitive DNA Sensor for Attomolar Detection of the BRCA1 Gene: Signal Amplification With Gold Nanoparticle Clusters. Analyst 2015;140:2713–8. [108] Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of Oxygen Radicals in DNA Damage and Cancer Incidence. Mol Cell Biochem 2004;266:37–56. [109] Huang Y, Yang HY, Ai Y. DNA Single-Base Mismatch Study Using Graphene Oxide Nanosheets-Based Fluorometric Biosensors. Anal Chem 2015;87:9132–6. [110] Sun S-W, Liu H-L, Zhou Y, Wang F-B, Xia X-H. Copper–Nitrogen-Doped Graphene Hybrid as an Electrochemical Sensing Platform for Distinguishing DNA Bases. Anal Chem 2017;89:10858–65. [111] Guo CX, Zheng XT, Lu ZS, Lou XW, Li CM. Biointerface by Cell Growth on Layered Graphene–Artificial Peroxidase–Protein Nanostructure for In Situ Quantitative Molecular Detection. Adv Mater 2010;22:5164–7. [112] Wu Y, Xue P, Kang Y, Hui KM. Highly Specific and Ultrasensitive Graphene-Enhanced Electrochemical Detection of Low-Abundance Tumor Cells Using Silica Nanoparticles Coated With Antibody-Conjugated Quantum Dots. Anal Chem 2013;85:3166–73. [113] Wu X, Hu Y, Jin J, Zhou N, Wu P, Zhang H, Cai C. Electrochemical Approach for Detection of Extracellular Oxygen Released From Erythrocytes Based on Graphene Film Integrated With Laccase and 2,2-Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid). Anal Chem 2010;82:3588–96.