Synthesis and Functionalization of Graphene and Application in Electrochemical Biosensing

Nanotechnol Rev 2016; 5(4): 393–416

Review

Deepshikha Saini* Synthesis and functionalization of graphene and application in electrochemical biosensing

DOI 10.1515/ntrev-2015-0059 as “miracle material” for its outstanding characteris- Received October 13, 2015; accepted November 22, 2015; previously tics. Graphene is also the fundamental building block of published online February 4, 2016 carbon materials such as fullerenes, carbon nanotubes, and graphite [1, 2]. Its unique configuration and struc- Abstract: Graphene is a two-dimensional material with ture determine a number of fascinating properties such amazing characteristics, which grant it the title “wonder as a high planar surface area (2630 m2 g-1) [3], superior material”. It has grabbed appreciable attention due to its mechanical strength with a Young’s modulus of 1100 GPa exceptional electrical, optical, thermal, and mechani- [4], unparalleled thermal conductivity (5000 W m-1 K-1) [5], cal properties. Because of these interesting properties, remarkable high carrier mobility (10,000 cm2 V-1 s -1) [6], graphene has found its way into a wide variety of bio- high opacity (~97.7%), and the ability to quench fluores- sensing applications. It has been used as a transducer in cence [7]. Graphene is currently, without any doubt, the electrochemical biosensors, bio-field-effect transistors, most intensively studied material for a wide range of impedance biosensors, electrochemiluminescence, and applications that include electronics, energy, and sensing fluorescence biosensors. Functionalization of graphene outlets [8]. has further opened up novel fundamental and applied However, tuning physicochemical properties frontiers. The present article reviews recent works deal- becomes necessary [9] in many graphene applications ing with synthesis, functionalization of graphene, and because of intrinsic graphene properties like zero band its applications related to biosensors. Various synthesis gap, high sheet resistance, easy aggregation, and poor strategies, mechanism and process parameters, and types solubility, which are the big obstacles to the various of functionalization are discussed in view of biosensor applications [10, 11]. Functionalization is one of the effi- development. Some potential areas for biosensor-related cient ways to tailor the electrical properties of graphene. applications of functionalized graphene are highlighted, Furthermore, after modification, the as-synthesized including catalytic biosensors and bio affinity biosensors. hybrid materials could not only overcome the disadvan- Wherever applicable, the limitations of the present knowl- tages of intrinsic graphene but also be inculcated with edgebase and possible research directions have also been new desirable properties. Graphene is thought to become discussed. especially widespread in biosensors and diagnostics. The Keywords: electrochemical biosensors; functionalization; large surface area of graphene can enhance the surface graphene; synthesis. loading of desired biomolecules, and excellent conductivity and small band gap can be beneficial for conduct- ing electrons between biomolecules and the electrode 1 Introduction surface. Graphene also has significant potential for enabling the development of electrochemical biosensors Graphene is composed of single-atom thick sheets of sp2 based on direct electron transfer between the enzyme bonded carbon atoms that are arranged in a perfect two- and the electrode surface. Moreover, other applications dimensional (2D) honeycomb lattice. It is often dubbed including, but not limited to, synthesizing nanoelectronics [12], high-frequency electronics [13], energy storage and conversion devices [14], field-emission displays [15], and transparent conductors [16] are also proofs of their *Corresponding author: Deepshikha Saini, Beijing National Laboratory for Molecular Sciences, Laboratory of Molecular versatility. Nanostructure and Nanotechnology, Institute of Chemistry, In this comprehensive review, recent research efforts Beijing 100190, P. R. China, e-mail: [email protected] to the synthesis, functionalization of graphene, and its 394 D. Saini: Functionalized graphene-based biosensors application in the development of next-generation bio- Mechanical exfoliation sensors are addressed. This covers the latest develop- (research, prototyping) ments and importantly, offer insights on the underlying CVD detection mechanisms and on the unique advantages of (coating, bio, transparent) conductive layers, graphene in comparison with other materials. I hope that electronics, this article would inspire broader interests across various photonics) disciplines and stimulate more exciting developments in this still young, yet very promising, field of research. Quality

SiC (electronics, Molecular RF transistors) assembly 2 Synthesis of graphene (nanoelectronics)

As the first step to realize the applications, graphene must Liquid-phase exfoliation (coating, composites, be synthesized on a large scale with controlled size and inks, energy storage, morphology. Controllable and scalable production of gra- bio, transparent conductive layers) phene is of great importance for its applications in dif- Price (for mass production) ferent fields. Graphene has been synthesized in various Figure 1: Methods for the mass production of graphene. There ways and on different substrates. In the following, we are several choices depending on the particular application, each summarize the synthesis methods to obtain high-quality with differences in terms of size, quality (e.g. presence of defects graphene. The methods for graphene preparation can be and impurities), and price. Reprinted with permission from Ref. [8] divided into several groups: (Copyright 2012 Nature). (i) Mechanical exfoliation of graphene layers from highly oriented pyrolytic graphite [17–19] (i) Capability of automation and miniaturization (ii) Chemical vapor deposition with hydrocarbon decom- (ii) High electron transfer rate position on a metal [20–24] (iii) Biocompatibility (iii) Thermal decomposition of SiC (epitaxial growth) (iv) Easier operation [25–29] (v) Extraordinary carrier mobility and capacitance (iv) Electrical arc discharge method [30–33] (vi) Excellent transparency (v) Organic synthesis [34–37] (vii) Exceptionally large surface-to-volume ratio (vi) Chemical method using dispersion of graphite. (viii) Single atom thickness [38, 39] (ix) Lower cost (x) Robustness and flexibility The mainstream methods of graphene synthesis have (xi) Excellent conductivity been summarized in Figure 1. Table 1 summarizes the relative advantages and dis- These properties have also led to significant interest in advantages of the above synthesis methods in terms of developing graphene-based electrochemical biosensors the feasibility to scale-up the process for mass production, [48]. Graphene has been employed as electrode material materials and production costs, and the presence of defects. in various electrochemical biosensors for the detection of a range of analytes like glucose, glutamate, cholesterol, hemoglobin, and more [49]. Interestingly, the 2.1 Significance of graphene for electro- large surface area of and excellent conductivity leads chemical biosensing to the superior electrochemical biosensing performance of graphene over carbon nanotubes in terms of sensi- Graphene and sensors are a natural combination, as gra- tivity, signal-to-noise ratio, electron transfer kinetics, phene’s large surface-to-volume ratio, unique optical and stability [50, 51]. Additionally, graphene’s proper- properties, excellent electrical conductivity, high carrier ties can be tuned by amendable synthetic conditions, mobility and density, high thermal conductivity, and dimensions, number of layers, and doping components many other attributes can be greatly beneficial for sensor [52]. Therefore, graphene-based materials are playing, functions. The superior advantages offered by graphene and will continually play, a significant role in sensing for electrochemical biosensing are as follows applications. D. Saini: Functionalized graphene-based biosensors 395 [47] [45, 46] [43, 44] [32, 33] [28, 29] [18, 19] [40–42] [22–24] References

Nano electronics, Nano electronics, composites sensors, Novel composite composite Novel materials FET interconnects,NEMs FET interconnects,NEMs s composite Transparent electrodes, electrodes, Transparent fillers polymer sensors, Conductive inks and and inks Conductive fillers, polymer paints, sensors capacitors, super Graphene electronics Graphene Touch screens, smart smart screens, Touch LCDs, flexible windows, cells solar and OLEDS, Basic research purpose research Basic Applications

Delicate, time consuming Delicate, Non-uniform, impure Non-uniform, Time-consuming, moderate moderate Time-consuming, scalability Moderate quality, impure, impure, quality, Moderate yield low High defect density, low low density, High defect purity High process temperature temperature High process low (1500 ° C), high cost, discontinuous yields, High process temperature temperature High process ( > 1000 ° C), high cost, process transfer complex Delicate, low yields, uneven uneven yields, low Delicate, scalable not films, Disadvantages

High yield, cost effective cost yield, High High crystallinity, high purity, high purity, High crystallinity, production large-scale cost, low Low-cost, high quality, high high quality, Low-cost, yield Simplicity, high scalability, low low high scalability, Simplicity, cost High scalability, high yields, yields, high High scalability, processable easily cost, low Large-scale production, high production, Large-scale no defects quality, Scalable, high quality, uniform, uniform, high quality, Scalable, high compatibility Simplicity, high quality, less less high quality, Simplicity, defects Advantages

Few layer graphene layer Few Nanosheets (100s (100s Nanosheets > 10 μ m ) nm to of Nanoribbons (few (few Nanoribbons microns) Nanosheets (nm to (nm to Nanosheets μ m) a few Nanoflakes/powder Nanoflakes/powder μ m) a few (nm to Thin films ( > 50 μ m) Thin films Thin films ≤ 75 cm Thin films Flakes (5 to 100 μ m) (5 to Flakes Size

Carbon dioxide dioxide Carbon reduction Arc discharge of of discharge Arc graphite Carbon nanotubes nanotubes Carbon unzipping Liquid phase phase Liquid exfoliation Chemical reduction reduction Chemical oxide graphite of Epitaxial growth growth Epitaxial on SiC CVD (on Ni, Cu Co) CVD (on Ni, Cu Mechanical Mechanical cleavage Synthesis methods Synthesis

8. 7. 6. 5. 4. 3. 2. 1. Comparison of different graphene synthesis methods. synthesis graphene different of 1: Comparison Table No. Sr. 396 D. Saini: Functionalized graphene-based biosensors

3 Graphene functionalization the basal surface. Graphene can be covalently functionalized in two ways: To make full advantage of the superior properties of gra- (i) By reaction with unsaturated π-bonds of graphene phene, it is necessary to functionalize graphene to form [56] multifunctional hybrid materials [39, 53]. Surface func- (ii) Heteroatom doping [56] tionalization can transform pristine graphene into a chemically sensitive and soluble material, thus, enabling Covalent functionalization is associated with rehybridiza- its use in sensing technology [48, 54]. Graphene is more tion of one or more sp2 carbon atoms of the carbon network prone to effective, reproducible, and homogeneous func- into the sp3 configuration leading to the introduction of a tionalization compared to other carbon nanomaterials. scattering site in graphene. Further, covalent functionali- Various strategies have been used to functionalize gra- zation can play an important role for controlling the carrier phene. But mainly two methods are available to function- density and band gap engineering [57, 58]. If graphene is alize graphene as follows: covalently bonded to acceptor functional groups, this may (i) The covalent functionalization via the grafting of mol- give rise to p-type semiconductor properties. Conversely, ecules onto the sp2 carbon atoms of the π-conjugated if graphene is functionalized by donor functional groups, skeleton; the formation of n-type semiconductor is possible. (ii) The noncovalent functionalization based on the Being highly inert and thermally stable, gra- adsorption of polycyclic aromatic compounds or sur- phene requires high-energy processes to rehybridize its factants via π-stacking and hydrophobic interactions π-conjugated carbon network. The covalent function- on the carbon framework. alization of graphene can be achieved in four different ways: nucleophilic substitution, electrophilic addition, Figure 2 summarizes the most important methods for the condensation, and addition [59–69]. This results in the functionalization of graphene. formation of a large density of sp3 hybridized carbons in the graphene network [70], which disrupts the delocal- ized π cloud, converting graphene into an insulator. Dif- 3.1 Covalent functionalization of graphene ferent kinds of functional groups like amino, hydroxyl, sulfonate, or alkyl groups may be introduced onto gra- Covalent functionalization is the most studied form of graphene through covalent bonding [71, 72]. These groups phene functionalization for sensor applications [56]. The can further serve as chemical switches to insert functional structural alteration can take place at the edges and/or on molecules (proteins, carbohydrates, polymers) on the graphene surface [72]. Advantages of covalent functionalization: The formation of covalent carbon-carbon bonds involving the basal plane carbon atoms offers key advantages, such as: B 1. Greater stability of the hybrid material, 2. Controllability over the degree of functionalization, 3. Reproducibility. A C Disadvantages of covalent functionalization: Although covalent strategies can effectively, stably, and specifically install functionalities, they unavoidably causes 1. A loss of the free, sp2-associated π electron constitut- ing the π-cloud on graphene. D E 2. Causing severe decrease in carrier mobility 3. Leading to the introduction of a scattering site in Functionalization possibilities for graphene: (A) edge Figure 2: graphene functionalization, (B) basal-plane functionalization, (C) noncovalent adsorption on the basal plane, (D) asymmetric functionalization, 4. Altering the native electronic structure and physical 2 and (E) the self-assembly of graphene. Reprinted with permission properties of graphene by converting sp carbons to from Ref. [55] (Copyright 2014 J. Mater. Chem. A). sp3 ones. D. Saini: Functionalized graphene-based biosensors 397

3.2 Noncovalent functionalization of recognition element which is in direct spatial contact with graphene a transducer,” according to the IUPAC definition [109, 110]. Figure 3 schematically depicts the concept of a biosensor. Noncovalent functionalization is a simple and economic Biosensors can be classified by their biological recogni- approach to introduce specific functionalities to the gra- tion element or their transducer type. phene surface. Noncovalent linkages between graphene 1. Biological recognition element: Classified into five and other functional groups are mainly achieved through different major categories. These categories include π-π stacking, van der Waals, electrostatic forces, hydrophilic antibody/antigen, enzymes, nucleic acids/DNA/ and hydrophobic interactions. Noncovalent functionaliza- RNA, cellular structures/cells, and biomimetic. The tion strategies do not affect the transparency or conductiv- enzymes, antibodies, and nucleic acids are the main ity of the material. This, in turn, introduces scattering sites classes of bioreceptors, which are widely used in bio- by creation of high-density electron-hole puddles. Non sensor applications. covalent strategies also control design-based synthesis of 2. Transducers: The transducer plays an important role graphene derivatives. For instance, small- molecule aromat- in the detection process of a biosensor. In case of con- ics like quinones have been used to functionalize carbona- ducting polymer-based polymer biosensor, the con- ceous materials and were shown to effectively enhance the ductive polymer acts as a transducer that converts the performance of energy conversion and storage devices [73]. biological signal to an electrical signal. Various molecules can physically adsorb onto graphene materials without the need of any coupling Graphene, a single layer of sp2-bonded carbon atoms packed reagents. Noncovalent functionalization is primarily into a benzene ring structure, has been demonstrated as achieved by polymer wrapping, adsorption of surfactants an ideal electrochemical platform for the construction of such as sodium dodecyl sulfate, and hexadecyltrimethyl biosensors owing to its large specific surface area, high ammonium bromide and interaction with metal nano electric conductivity, good biocompatibility, thermal and particles (e.g. Au, Ag, Pt), porphyrins, or biomolecules chemical stability, and low-cost production [48, 112]. On the such as deoxyribonucleic acid (DNA) and peptides [74–77]. other hand, functionalization of graphene covalently and Advantages of noncovalent functionalization: noncovalently can effectively tune its electrical properties 1. Preserves the intrinsic properties of the graphene and enhance its electrocatalytic performances [113–115]. It 2. Does not destroy the original sp2-conjugated struc- is inferred that the electrochemically active sites generated ture of graphene, which further retains the high by functional groups triggers the adsorption and activation electroconductivity of analytes, anchoring of functional moieties, and accel- 3. Does not create sp3-hybridized carbons or defects. erating the charge transfer between electrode and analytes/electrolyte. This, in turn, would be advantages to the Disadvantages of noncovalent functionalization: enhanced electrochemical biosensing performances [116]. 1. Physical adsorption is nonspecific 2. There is no control over degree of functionalization 3. It changes the doping density, increases the density 5 Functionalized graphene-based of electron-hole puddles, and creates scattering sites. enzyme biosensors Different strategies for covalent and noncovalent func- Owing to their unique physical and chemical proper- tionalization of graphene are categorized in Table 2. ties, graphene-based materials have become important candidates for biosensing. The large surface area of graphene can enhance the surface loading of the desired bio molecules and direct electron transfer between enzymes 4 Current and emerging applications and electrodes. Thus, functionalized graphene could be of functionalized graphene in an excellent electrode material for enzyme biosensors. electrochemical biosensors 5.1 Glucose biosensor A biosensor is “a self-contained integrated device which is capable of providing specific quantitative or semi- The metabolic disorder of diabetes mellitus results quantitative analytical information using a biological in hyperglycemia. It is reflected by blood glucose 398 D. Saini: Functionalized graphene-based biosensors

Table 2: Different strategies for covalent and noncovalent functionalization of graphene.

Functionalization methods Procedure of Figure/illustration References functionalization

Covalent methods Addition of free [78–80] radicals to sp2 carbon atoms of graphene

Addition of [66, 81, 82] dienophiles to carbon-carbon bonds

Addition of [83–86] chromophores

Covalent linkage to [87–89] polymers

Covalent attachments [90–93] of hydrogen and halogens D. Saini: Functionalized graphene-based biosensors 399

Table 2: (continued)

Functionalization methods Procedure of Figure/illustration References functionalization

Noncovalent methods H-π Interaction [94–96]

Π-π Interaction [97–99]

Cation-π Interaction [100–102]

Anion-π Interaction [103–105]

Graphene-ligand [106–108] noncovalent interaction 400 D. Saini: Functionalized graphene-based biosensors

- e GOxFADHO+→GOxFAD +HO (2) Analytes ( 22) ( ) 22 Immobilized Cholesterol biomolecules Tr iglycerides +→ + (3) GOxF( AD) GlucoseGOx(FADH2 ) Gluconolactone Glucose Antibody Urea, etc. Functionalized graphene-based glucose biosensors Antigens Enzyme Biocompatible layer can be divided into the following categories: Target DNA Probe DNA Potential Response Organisms, Transducer bacteria (electrical, optical, physical) 5.1.1 Enzymatic functionalized graphene-based glucose biosensors Amplifier Signal Glucose oxidase (GOx) has been immobilized on graphene sheets for fabricating glucose biosensors via various meth- Microelectronics data processing odologies [112, 120, 121]. Shan et al. had designed CS-GR/ AuNP nanocomposite films for glucose sensing, which Figure 3: Schematic structure and operating principle of a biosensor. exhibited good amperometric responses to glucose with Reprinted with permission from Ref. [111] (Copyright 2011 Nature). linear ranges of 2–10 mm at -0.2 V and 2–14 mm at 0.5 V [122]. This was attributed to the synergy effect of graphene and the AuNP. G. Bharath et al. [123] have reported the direct concentration higher or lower than the normal range electrochemistry of glucose oxidase (GOx) on 1D hydroxya- of 80–120 mg dl-1. Persistent hyperglycemia can lead to patite (HAp) on reduced graphene oxide (RGO) nanocom- stroke, coronary heart disease, and circulation disorders posite-modified glassy carbon electrode (GCE) for glucose [117]. One of the main biomedical applications of gra- sensing (Figure 4). The electrocatalytic and electroanalytical phene in biosensors is to develop glucose concentration- applications of the proposed GOx/HAp/RGO-modified GCE measuring devices for patients suffering from diabetes. were studied by cyclic voltammetry (CV) and amperometry. Electrochemical detections of glucose have been well The reported biosensor exhibits a superior detection limit demonstrated using glucose oxidase (GOx) as the media- (0.03 mm) and higher sensitivity (16.9 mA mm-1 cm-2), respec- tor or recognition element [118, 119]. The redox reaction tively, with a wide linear range of 0.1–11.5 mm. mechanism is as follows: Liu et al. [124] fabricated a mono- and multilayered bio- In the absence of oxygen: sensor format with controlled biocatalytic activity. GOx was +++ - ↔ (1) first modified with pyrene functionalities in order to be self- GOxF( AD) 2H 2e GOxF( ADH2 ) assembled onto a graphene basal plane via noncovalent In the presence of oxygen: interactions. Using an alternate layer-upon-layer of

OH H O GOx (FAD) O 2 2 OH OH Glucose OH OH

OH O GOx (FADH ) OH O O2 O2 2 Gluconolactone OH OH e-

RGO

GCE Glucose oxidase (GOx)

HAp

Figure 4: Schematic illustration for the mechanism of glucose sensing based on an RGO/HAp/GOx nanocomposite. Reprinted with permission from Ref. [123] (Copyright 2015 J. Mater. Chem. B). D. Saini: Functionalized graphene-based biosensors 401 self-assembled graphene and pyrene-functionalized GOx, 5.1.2 Non-enzymatic functionalized graphene-based mono- and multilayered enzyme electrodes were readily glucose biosensors fabricated (Figure 5A). The biocatalytic activity of these enzyme electrodes increased with the number of graphene Enzymeless sensing of glucose is feasible using metal and GOx layers (Figure 5B). Such multilayered enzyme nanoparticles and nanowires even under neutral pH. In electrodes with controlled nanostructure exhibited reliable particular, a remarkable detection limit is obtained for application for the analysis of human serum samples with glucose (25 nm) using a 3D graphene foam modified with high sensitivity, good stability, and repeatability. Co3O4 nanowires [125]. Another non-enzymatic glucose The disadvantages of enzymatic electrochemical biosensor based on nickel-polyaniline-functionalized glucose sensors: reduced graphene oxide was fabricated by Bing Zhang The most common and serious problems associated et al. [126]. They had developed a new class of organic- with enzymatic electrochemical glucose sensors are as inorganic hybrid nanostructures, which was applied to follows: efficient non-enzymatic sensing of glucose (Figure 6). This (i) insufficient operational and storage stability biosensor format included a fast response (~2 s), high sen- (ii) lower reproducibility sitivity (6050 μA mm-1 cm-2), a linear range from 0.1 μm to (iii) influence of oxygen limitation. 1.0 mm, and a low detection limit (0.08 μm).

A O NH2 H N H H2N HN 2 EDC/NHS N OH O O + H H N NH2 N O 2 NH

H2N O O

H H2N HN N O H N O NH O

Graphene Graphene 1 Graphene 1 1

2 22

B 7 d 6 c 5

4 b

Current (µA) 2 a

-1 0.0 0.2 0.4 Potential (V)

Figure 5: (A) Schematic diagram of the modification of GOD with pyrene and the subsequent fabrication of mono- and multi-layered enzyme electrodes. (B) Cyclic voltammograms of the enzyme electrodes fabricated with mono- and multilayered graphene and pyrene functionalized GOD: (a) one layer, (b) two layers, (c) three layers, and (d) four layers. Data were recorded in 80 mm glucose solution in 0.1 M pH 7.4 phos- phate buffer at room temperature and potential scan rate of 10 mV s-1. Reprinted with permission from Ref. [124] (Copyright 2013 Analyst). 402 D. Saini: Functionalized graphene-based biosensors

Aniline

Glucose 2+ Polymerization Ni Glucolactone

rGO PANI-rGO Ni-PANI-rGO Ni(OH)2

A novel, stable, and sensitive non-enzymatic glucose biosensor for H2O2. The Pt/G hybrid provides an opera- sensor was developed by electrodepositing metallic Cu tive environment for maintaining the bioactivity of HRP. nanoparticles on graphene sheets [127]. The Cu-graphene It acts as a bridge for the transfer of electrons between the electrode sensor presented a wide linear range up to 4.5 mm active center of HRP and the electrode surface (Figure 8). glucose with a detection limit of 0.5 μm (signal/noise = 3) at Pt/G effectively reinforced the immobilization of HRP and a detection potential of 0.5 V in alkaline solution with a enhanced the utilization of HRP. The linear range for H2O2 very quick (2 s) response. Recently, highly sensitive hybrid was estimated to be from 3.0 × 10-6 mol l-1 to 5.2 × 10-3 mol l-1 structured non-enzymatic glucose sensors were fabricated. with a detection limit of 5.0 × 10-9 mol l-1. Platinum (Pt) nanoparticles were decorated on 3D gra- A sensitive and noble amperometric HRP biosensor phene oxide networks to increase the surface area. Large is fabricated via the deposition of gold nanoparticles surface area and high electrocatalytic activity resulted in (AuNPs) onto a 3D porous carbonized chicken eggshell high glucose sensitivity (137.4 μA mm-1 cm-2) and excellent membrane (CESM) [130]. Owing to the synergistic effects anti-interference characteristics [128]. of the unique porous carbon architecture and well-dis- tributed AuNPs, the enzyme-modified electrode shows an excellent electrochemical redox behavior (Figure 9). Fur-

5.2 Hydrogen peroxide (H2O2) biosensor thermore, the HRP-AuNPs-CESM/GCE electrode, as a bio-

sensor for H2O2 detection, has a good accuracy and high

H2O2 is also particularly important for the development of sensitivity with a linear range of 0.01–2.7 mm H2O2 and a biosensing systems. It is a substrate of peroxidases, enzy- detection limit of 3 μm H2O2. matic product of oxidases, and an important mediator in A facile one-pot hydrothermal approach was reported clinical as well as pharmaceutical analyses. The detection for cerium oxide-reduced graphene oxide CeO2-RGO nano- of H2O2 can be achieved using horseradish peroxidase composite formation (Figure 10), which is further assem- enzyme (HRP) immobilized onto graphene-based elec- bled with HRP for the detection of hydrogen peroxide trodes. The reaction mechanism of the catalytic process (H2O2) at trace levels [131]. This biosensor format exhibited can be summarized as follows: a wide linear range of H2O2 from 0.1 to 500 μM with a detection limit of 0.021 μm. +→ + HRPR( ed) HO22 HRPO( xH) 2O (4) In addition, many novel non-enzyme H2O2 bio sensors have been reported [132–134]. For instance, Dye HRPO( xH)+→ydroquinoneHRP(RedB)+ enzoquinone (5) et al. [132] developed a highly sensitive amperometric

biosensor based on the hybrid material, which is derived Benzoquinone++2e- 2H+ →Hydroquinone (6) from graphene and Pt nanoparticles for the detection of

H2O2 and cholesterol. This sensor shows high sensitiv-

Zonghua Wang et al. [129] fabricated platinum nano- ity and fast response toward H2O2 up to 12 mm with a particles/graphene nanohybrids (Pt/G) via in situ reduction detection limit of 0.5 nm (S/N = 3) in the absence of any 2- of PtCl6 in the presence of poly(diallyldimethylammonium redox mediator or enzyme. The combination of super- chloride)-modified graphene (Figure 7). These nanohy- conductive graphene sheets and catalytically active Pt brids were used as a matrix for the immobilization of HRP nanoparticles accelerated electron transfer for the oxi- to construct a HRP/Pt/G/glassy carbon electrode (GC) dation of H2O2. D. Saini: Functionalized graphene-based biosensors 403

PDDA

Graphene PDDA-Graphene

2- PtCI6

Microwave-assisted heating

Complex of PtCl 2- with Pt-Graphene 6 ammonium groups

Ammonium groups in PDDA

2- PtCI6 Pt nanoparticles

10 a GCE CESM 5 b c AuNPs HRP

f 0 Redox center of HRP e

i (µA) d -5

FeIV H O -10 2 2

- e- e - e H O -15 FeIII 2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 E (vs. SCE) Current signal

Figure 8: Cyclic voltammograms (CVs) of modified electrodes in Figure 9: Schematic illustration of the mechanism underlying the 0.1 mol l-1 pH ¼ 6.8 PBS, HRP/Pt/G/GC (A), Pt/G/GC (B), HRP/G/ detection of H2O2 using HRP-AuNPs- CESM-GCE electrochemical GC (C), G/GC (D), HRP/Pt/GC (E), and Pt/GC (F). Reprinted with biosensor. Reprinted with permission from Ref. [130] (Copyright permission from Ref. [129] (Copyright 2013 Anal. Methods). 2015 Plos One).

5.3 Cholesterol biosensor steroid hormones, bile acids, and vitamin D. The total blood cholesterol level should be < 200 mg/dl in the Cholesterol is an organic molecule. It is an essential struc- human body. However, undesired accumulation of cho- tural component of all animal cell membranes [135]. Cho- lesterol and its esters causes critical health problems lesterol also serves as a precursor for the biosynthesis of such as hypercholesterolemia, heart diseases, cerebral 404 D. Saini: Functionalized graphene-based biosensors

Ce3+ Adsorption Hydrothermal process GO CeO2/rGO

e HRP CeO2/rGO H2O2

Nafion - e + H2O

Figure 10: H2O2 detection scheme using HRP/CeO2-rGO modified glassy carbon electrode. Reprinted with permission from Ref. [131] (Copy- right 2015 RSC Adv.). thrombosis, and atherosclerosis. A smart, quick, accurate system (RGO-Fn Au NPs). The electrodes were fabricated determination of cholesterol in blood is an urgent need by the electrophoretic deposition technique. A syner- in clinical diagnosis. The following biochemical reaction gistically enhanced electrochemical sensing ability of occurs as a result of interaction of cholesterol oxidase 193.4 μA mm-1 cm-2 for free cholesterol detection was (ChOx) with cholesterol: achieved, which is much higher than that of the tradi- tional RGO system (Figure 12). Moreover, the RGO-Fn Au ++ChOx→ CholesterolO22Cholesterone HO2 NP platform promises a wider range of cholesterol detection (0.65–12.93 mm) with lower detection limit of 0.34 mm Dey and Raj [136] developed a highly sensitive amper- for free cholesterol. ometric cholesterol biosensor based on a hybrid material Deepshikha et al. have fabricated a novel nanobiocom- derived from PtNP and graphene for the detection of H2O2 posite bienzymatic amperometric cholesterol biosensor, (Figure 11). The cholesterol biosensor was developed by coupled with cholesterol oxidase (ChOx) and HRP, based on immobilizing cholesterol oxidase and cholesterol esterase the gold-nanoparticle decorated graphene-nanostructured on the surface of the GR/PtNP hybrid material. The sensitiv- polyaniline nanocomposite (NSPANI-AuNP-GR) film. The ity and detection limit of the electrode toward cholesterol nanocomposite film was electrochemically deposited onto ester were 2.07±0.1 μA/μm/cm2 and 0.2 μm, respectively. indium-tin-oxide (ITO) electrode from the NSPANI-AuNP- Shiju Abraham et al. [137] have reported the develop- GR nanodispersion [138]. The overall biochemical reaction ment of cost-effective bioelectrodes based on a reduced for ChOx-HRP/NSPANIAuNP- GR is shown in Figure 13. graphene oxide-functionalized gold nanoparticle hybrid The gold nanoparticle-decorated graphene- nanostructured polyaniline nanocomposite (NSPANI- AuNP-GR) offers an efficient electron transfer between Cholesteryl stearate underlining electrode and enzyme active center. The Pt bienzymatic nanocomposite bioelectrodes ChOx-HRP/ ChEt Pt NSPANI-AuNPGR/ITO have exhibited wider linearity (35 to 500 mg/dl), higher sensitivity (0.42 μA mm-1), low km Pt value of 0.01 mm, and higher accuracy for testing of blood RCOOH Cholesterol O Pt 2 serum samples than monoenzyme system. The novelty of the electrode lies on reusability, extended shelf life, and Pt accuracy of testing blood serum samples. ChOx H2O2

Cholesterol-4-ene-3-one 5.4 NADH biosensor Figure 11: Scheme illustrating the biosensing of cholesterol ester + with the GNS-nPt-based biosensor. Reprinted with permission from β-Nicotinamide adenine dinucleotide (NAD ) and Ref. [136] (Copyright 2010 J. Phys. Chem. C ). its reduced form (NADH) are a cofactor of many D. Saini: Functionalized graphene-based biosensors 405

OH N-H OH OH N-H N-H Pt electrode C = O N-H C = O C = O C = O C = O C = O H

ChOx ITO Immobilization B Via EDC-NHS Cholesterol coupling A C D Electrophoretic deposition RGO-Fn Au NPs set up

- N-H e N-H N-H e- C = O C = O C = O Electrochemical signal

Figure 12: Schematic representation of the cholesterol sensing process: (A) EPD set up for the fabrication of RGO and RGO-Fn Au NP thin films; (B) electrophoretically fabricated RGO-Fn Au NP thin film on an ITO substrate; (C) immobilization of ChOx on RGO-Fn Au NPs by EDC-NHS coupling; and (D) the immobilized RGO-Fn Au NP bioelectrode and the electrochemical reaction while adding cholesterol to the electrochemical cell containing the bioelectrode. Reprinted with permission from Ref. [137] (Copyright 2015 Anal. Methods).

acceptable analytical outcomes for the determination of Cholest-4-en-3-one + H O H O Cholesterol 2 2 2 NADH. Moreover, process is convenient, and low-cost Horseradish preparation is involved. Tang et al. adopted chemically peroxidase (HRP) reduced GO (CRGO), which showed a remarkable oxida- Polyaniline Cholesterol tion shift of about 30 mV compared to graphite and bare oxidase (ChOx) GC electrodes [143]. Graphene Gold nanoparticles ITO electrode 5.5 Dopamine biosensor Figure 13: Proposed biochemical reaction on the ChOx-HRP/ NSPANI-AuNP-GR. Reprinted from an Open Access Journal Ref. [138], Dopamine (DA), an important neurotransmitter, plays 2012. an important role in the central nervous, hormonal, renal, and cardiovascular systems. Its deficiency is correlated to Parkinson’s disease, which is a degenera- dehydrogenases. NAD+/NADH-dependent dehydroge- tive disorder of the central nervous system. The major nases have been attempted in the development of bio- symptoms of Parkinson’s disease result from the death sensors, biofuel cells, and bioelectronic devices. A large of dopamine-generating cells in midbrain. Therefore, overvoltage is necessary for anodic detection of NADH, a lot of research works aimed at developing sensitive, and electrode fouling is also encountered due to the for- rapid, and decentralized sensing devices for its detec- mation of reaction products. Apparently, graphene shows tion. With the excellent electroanalytical properties, promise in addressing these two drawbacks. Graphene graphene-based electrodes have demonstrated excellent functionalization with dyes like methylene green (MG) performances in detecting DA. Gregory Thien Soon How forms a stable complex via noncovalent bonding [139]. et al. [144] have reported a reduced graphene oxide/tita- The oxidation of NADH on MG-graphene electrode takes nium dioxide nanocomposite with highly exposed facets place at ~0.14 V, which is much lower than that of pristine (rGO/TiO2 {001}) for the detection of DA [144]. The modi- graphene [139] and CNTs [140, 141]. fied rGO/TiO2 {001} GCE shows enhanced electrochemi- Shan et al. [142] showed that graphene electrodes cal sensing toward DA under the interference effect. functionalized using ionic liquids could be used for both This method certainly opens up a new platform of facet NADH detection and the biosensing for ethanol. The manipulation studies for electrochemical applications ionic liquid modified-graphene-based sensor exhibited (Figure 14). 406 D. Saini: Functionalized graphene-based biosensors

A DAQ DA Current (µA )

GCE/rGO/TiO2 {001} Electrode Eapp=0.2 V vs. SCE Potential (V vs. SCE)

B 70 C 70 60 60

50 50 100 M µ y=0.376x+23.53 40 40 R2=0.971 1 µM 30 30 Current (µA) Current (µA) 20 20 y=0.99x+1.216 R2=0.994 10 10 0 0 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 02040 60 80 100 Potential (V vs. SCE) Concentration (µM)

Figure 14: (A) Mechanism for the electrocatalytic oxidation of dopamine at rGO/TiO2 {001} modified GCE. (B) DPV obtained for rGO/TiO2 {001} modified GCEs during the addition of 1, 2, 4, 8, 10, 15, 25, 30, 35, 40, 50, 60, 80, and 100 mm DA into the 0.1 M PBS (pH? 6.5). (C) Calibration plot observed for the oxidation peak current vs. concentration of DA. Reprinted with permission from Ref. [144] (Copyright 2014 Nature).

DAQ DA e- Cast on electrode

Mixed GCE GCE

Graphene/Fc-NH Graphene Fc-NH2 2 Nafion

DA coexists with endogenous electroactive species prevent the leakage of ferrocene (Figure 15). In the pres- such as ascorbic acid (AA) and uric acid (UA). They have ence of 1 mm AA and 0.1 mm UA, the electrode showed an overlapping voltammetric response, resulting in poor linear response in the range of 0.1 to 4 μm, and the detec- selectivity and sensitivity of DA. The presence of sp2 tion limit was 50 nm (S/N = 3). hybridized planes and various edge defects on the graphene surface might attribute to a better sensing performance toward DA [50] and can differentiate DA from AA 5.6 DNA biosensors and UA. Liu et al. [145] have used graphene/ferrocene derivative (graphene/Fc-NH2) nanocomposite to fabricate Electrochemical DNA sensors offer high sensitivity, high the Nafion/graphene/Fc-NH2-modified GCE for the detec- selectivity, and low cost for the detection of selected DNA tion of DA. The Fc-NH2 embedded on the graphene sig- sequences. These also detect mutated genes associated nificantly enhances the electrochemical response toward with human disease and provide a simple, accurate, relia- DA, charge-transport ability, stabilizes the graphene, and ble, and inexpensive platform of patient disease diagnosis D. Saini: Functionalized graphene-based biosensors 407

Modification of Target DNA bare DEP with CMG GCE materials SH 1) 3-/4- [Fe(CN)6] 2) Adenine o-PD [Fe(CN) ]3-/4- GCE GCE 6 H2O2

o-PD ox A

DPV Immobilization of anti-IgG blocking with BSA GCE C

AuNPs SWCNH Biotin FeTMPyP 3-/4- [Fe(CN)6] MB Streptavidin Carboxylic GO

Figure 16: Schematic illustration of graphene-supported ferric por- Incubation with IgG phyrin as a HRP mimicking trace label for electrochemical detection of DNA. Reprinted with permission from Ref. [155] (Copyright 2013 B Chem. Commun.).

Bare DEP [Fe(CN) ]3-/4- 6 Anti-IgG [146, 147]. Graphene, as a perfect 2D material with large surface area, can work as a carrier for DNA molecules GPO, GO, TR-GO, ER-GO IgG BSA loading either by (i) Direct absorption via π-π interactions [148, 149] Figure 18: An illustration of the protocol for IgG immunodetection based on the EIS method. (A) A bare DEP electrode modified with (ii) By other binding approaches via functional groups CMG material; (B) a CMG material modified DEP electrode after from modified graphene surface [150, 151]. the immobilization of anti-IgG and blocking with BSA; and (C) an anti-IgG and BSA-modified DEP electrode after the incubation with Furthermore, the excellent conductivity of graphene makes IgG. Reprinted with permission from Ref. [184] (Copyright 2012 it possible for fast electron transfer between DNA and Nanoscale).

OH HO HO OH O OH PG molecule NH Pyrene

O S NH O Glucose

Hg2+/DI solution Imine group DI water

Mercury ion

Reduced Alkyl chain graphene oxide

Figure 17: A proposed mechanism for an ultrasensitive and highly selective Hg2+ sensor based on the modified ERGO. Reprinted with permission from Ref. [160] (Copyright 2013 Chem. Commun.). 408 D. Saini: Functionalized graphene-based biosensors electrodes. Recently, the electrochemical detection for target by non-DNA-modified electrodes [153–155]. For example, DNA molecules has been reported [148–152]. For example, Quanbo Wang et al. [155] have designed a novel peroxidase Huang et al. [152] synthesized a tungsten sulfide-graphene mimic by loading ferric porphyrin and streptavidin onto gra-

(WS2-Gr) nanocomposite for DNA biosensor application. phene, which was used to recognize a biotinylated molecu- Graphene served as a 2D conductive support for a highly lar beacon (MB) for specific electrochemical detection of electrolytic accessible surface. However, the electronic con- DNA (Figure 16). The biosensor format had exhibited high ductivity of WS2 is much lower compared to graphene mate- specificity and excellent anti-interference ability. The bio- rials, thereby, limiting its application for sensitive detection. sensing method could discriminate target DNA from single- On the other hand, DNA detections can also be achieved base or three-base mismatched oligonucleotides.

Table 3: Characteristic features of functionalized graphene-based biosensors.

Sensor type Sensing material Detectable product Linearity range Detection References limit

Glucose N-doped graphene Glucose 0.1–1.1 mm 0.01 mm [186] RGO/hydroxyapatite Glucose 0.1–11.5 mm 0.03 mm [123] Graphene/polyaniline/Au Glucose 4.0–1.12 mm 0.6 mm [187] N-doped graphene/Cu Glucose 0.004–4.5 mm 1.3 mm [188] Carbon nitride dots-reduced graphene oxide Glucose 40 μm–20 mm 40 μm [189] Chitosan/AuNP/sulfonates poly(ether-ether- Glucose 0.5–22.2 mm 0.13 mm [190] ketone) functionalized ternary graphene Hydrogen Arginine-functionalized graphene Hydrogen Peroxide 5 nm–40 μm 1.1 nm [191]

Peroxide CeO2-RGO Hydrogen Peroxide 0.1–500 μm 0.021 μm [192] PtRu/3D graphene foam Hydrogen Peroxide 0.005–0.02 mm 0.04 μm [193] AuNP/stacked Graphene nanofibers Hydrogen Peroxide 0.08–250 μm 0.35 nm [194]

MnO2/graphene/CNT Hydrogen Peroxide 1–1030 μm 0.1 μm [195] PdNP-graphene nanosheets-Nafion Hydrogen Peroxide 0.1–1000 μm 0.05 μm [196] Cholesterol Pt-Pd-chitosan-graphene Cholesterol 2.2–5200 μm 0.75 μm [197] Graphene/polyvinylpyrrolidone/polyaniline Cholesterol 50 μm–10 mm 1 μm [198] PtNP-graphene Cholesterol Up to 12 mm 0.5 nm [136] Reduced graphene oxide-functionalized Cholesterol 0.65–12.93 mm 0.34 mm [199] AuNPs -8 -3 -9 NADH Graphene-TiO2 NADH 1 × 10 –2 × 10 m 3 × 10 m [200] Graphene-Au nanorods NADH 20–60 μm 6 μm [201] Electroreduced GO-polythionine NADH 0.01–3.9 mm 0.1 μm [202] Dopamine Trp-functionalized graphene Dopamine 0.5–110 μm 290 nm [203] N-doped graphene Dopamine 100–450 μm 0.93 μm [204] Sulfonated-graphene Dopamine 0.2–20 μm 40 nm [205] N-doped graphene/PEI Dopamine 1–130 μm 500 nm [206] DNA ssDNA/azophloxine/graphene nanosheets DNA 1 fm–0.1 pm 0.4 fm [207] ssDNA/AuNP/polyaniline/graphene sheets- DNA 10–1000 pm 2.11 pm [208] chitosan -12 -6 ssDNA/chitosan–Co3O4 nanorods–graphene DNA 1.0 × 10 –1.0 × 10 M 0.43 pm [209] ssDNA-MB/functionalized graphene DNA 100–350 nm 40 nm [148] Heavy Metal Graphene nanosheets/Au Pb2+ 0.4–20 nm 0.4 nm [166] Ions RGO/Au Pb2+ 50–1000 nm 10 nm [163] Graphene nanosheets/Au Cu2+ 1.5–20 nm 1.5 nm [166] 2+ SnO2/RGO Cu 0.2–0.6 μm 0.23 nm [170] 2+ SnO2/RGO Cd NA 0.10 nm [170] Polyvinylpyrrolidone protected graphene/Au Hg2+ 10–60 ppb 6 ppt [168] 2+ SnO2/RGO Hg 0.1–1.3 nm 0.28 nm [170] PG modified RGO Hg2+ 0.1 nm–4 nm. 0.1 nm [160] Immunosensor Chemically modified graphene (CMG) Immunoglobulin G 0.3 mg ml-1–7 mg ml-1 – [184] (IgG) RGO/thionine/gold Tumor markers 0.01 ng ml-1–300 ng ml-1 – [185] D. Saini: Functionalized graphene-based biosensors 409

5.7 Heavy metal ions biosensors fabrication of immunosensor (Figure 18). It was found that thermally reduced graphene oxide has provided the Heavy metal ions in potable water and food are major best performance with the linear detection range from health concerns. Their detection is critical to maintaining a 0.3 mg ml-1 to 7 mg ml-1. safe food supply [156, 157]. Therefore, the highly sensitive, Simultaneous detection of multiple tumor markers selective, and rapid detection of heavy metal ions has been was done by fabricating graphene nanocomposite immu- explored in depth using various analytical methodologies nosensor [185]. In this work, reduced graphene oxide/ [158–161]. Graphene- based sensors have been employed to thionine/gold nanoparticles nanocomposites were syn- detect and monitor the presence of heavy metal ions such as thesized and coated on ITO for the formation of an anti- Pb2+, Cd2+, Cu2+, and Hg2+ [160, 162–167]. For example, a gold body-antigen immunocomplex. Experimental results functionalized graphene electrode was produced via nonco- revealed that the multiplexed immunoassay enabled the valent interaction for the determination of Pb2+ and Cu2+ [168]. simultaneous determination of tumor markers with linear Chunmeng Yu et al. [160] have designed an ultrasensitive working ranges of 0.01–300 ng ml-1. and highly selective Hg2+ sensor through noncovalent modi- Characteristic features of various functionalized gra- fication of an electrochemically reduced GO (ERGO)-based phene-based biosensors are summarized in Table 3. diode with N-[(1-pyrenyl-sulfonamido)-heptyl]-gluconamide (PG) as the modifier. PG is comprised of a glucose residue and a pyrene residue. The glucose residue can work as multiple-receptor sites for Hg2+ in the medium. The large π system 6 Conclusion in the pyrene can be stably attached on the ERGO surface (Figure 17). The detection limit of this sensor for Hg2+ reaches Graphene, as an amazing and wonderful material among 0.1 nm, which is the best result of electronic sensors and is the nanocarbon family, has drawn considerable interest about 10 times as low as the previously reported results in many fields. Its unique structure contributes to its fas- [169]. Alkanethiol-modified graphene field effect transistors cinating chemical and physical properties, which lead to exhibited a sensitivity for Hg2+ detection at 10 ppm, which a broad range of applications in sensing. In this review, provides new opportunities for graphene-based electronics significant advances in functionalized graphene-based as heavy metal sensors. Sensors based on graphene have biosensors relative to their bulk counterparts have been also been reported for the detection of Cd2+, Cu2+, and Ag+ reported. The unique electronic structure, biocompat- [170–173]. ibility, and dimensional compatibility with biomolecules indicate that graphene can provide a new generation of smart transduction matrices for the control of a range of 5.8 Immunosensors biomolecular interactions, from the molecular to the cellular levels. However, graphene, as a newly discovered Electrochemical immunosensors are miniaturized ana- material, faces several challenges including improving lytical devices with higher sensitivity and selectivity. synthesis methods, extensive understanding of graphene Compared with the other methods like enzyme-linked surface, and extending the applications in various practi- immunosorbent assay (ELISA), immunosensors are cal fields. attractive because of their high sensitivity, low cost, There are currently two key challenges for the devel- short analytical time, simple instrumentation, and high opment of large-scale manufacturing of graphene-based specificity of immunological reactions [174]. Immunosen- products: sors are capable of detecting nucleic acids [175], viruses 1. Production of large quantities and high-quality uni- [176], antigens [177], and hormone [178] biomarkers. More form graphene recently, graphene has been utilized in a number of forms 2. Tailoring and tuning of graphene properties. for immunosensor applications [179–183]. Graphene is a material, which has immense potential for the fabrication The synthesis and cost of making graphene is relatively of immunosensors. A simple electrochemical impedimet- much cheaper in comparison to other carbon materials ric immunosensor for immunoglobulin G (IgG) based on such as CNTs. There have also been significant advances chemically modified graphene (CMG) surfaces was pro- in the preparation of graphene, their functionalization posed by A.H. Loo et al. [184]. The group had used graph- with the desired chemical entities, and the immobiliza- ite oxide, graphene oxide, thermally reduced graphene tion of biomolecules to the functionalized graphene sur- oxide, and electrochemically reduced graphene oxide for faces for biosensor applications. However, most of the 410 D. 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[205] Li SJ, He JZ, Zhang MJ, Zhang RX, Lv XL, Liand SH, Pang H, Electrochemical detection of dopamine using water-soluble Bionote sulfonated graphene. Electrochim. Acta 2013, 102, 58–65. [206] Li N, Zheng E, Chen X, Sun S, You C, Ruan Y, Weng X. Layer-by- Deepshikha Saini layer assembled multilayer films of nitrogen-doped graphene Beijing National Laboratory for Molecular and polyethylenimine for selective sensing of dopamine. Int. Sciences, Laboratory of Molecular J. Electrochem. Sci. 2013, 8, 6524–6534. Nanostructure and Nanotechnology, Institute [207] Guo Y, Guo Y, Dong C. Ultrasensitive and label-free electro- of Chemistry, Beijing 100190, P. R. China, chemical DNA biosensor based on water-soluble electroac- [email protected] tive dye azophloxine-functionalized graphene nanosheets. Electrochim. Acta. 2013, 113, 69–76. [208] Wang L, Hua E, Liang M, Ma C, Liu Z, Sheng S. Graphene sheets, polyaniline and AuNPs based DNA sensor for electrochemical determination of BCR/ABL fusion gene with Deepshikha Saini received her PhD degree (2012) from the functional hairpin probe. Biosens. Bioelectron. 2014, 51, Department of Chemistry, Amity University, Noida, India. Then, 201–207. she joined Peking University, Beijing, China, as a post-doctoral [209] Qi X, Gao H, Zhang Y, Wang X, Chen Y, Sun W. Electrochemi- fellow in 2012. At present, she is a Young International Scientist in

cal DNA biosensor with chitosan–Co3O4 nanorod–graphene Institute of Chemistry, Chinese Academy of Sciences, Beijing, China. composite for the sensitive detection of Staphylococcus Her research interests are focused on controlled modification of aureus nuc gene sequence. Bioelectrochemistry 2012, 88, graphene surfaces for photovoltaic and optoelectronic applications. 42–47. She has 8 years of research and 7 years of teaching experience. She received the best published paper award by KASCT, Springer (2012), and CAS young international scientist fellowship in 2014.