Elsevier

Biosensors and Bioelectronics

Volume 26, Issue 12, 15 August 2011, Pages 4637-4648
Biosensors and Bioelectronics

Review
Recent advances in graphene-based biosensors

https://doi.org/10.1016/j.bios.2011.05.039Get rights and content

Abstract

A detailed overview towards the advancement of graphene based biosensors has been reviewed. The large surface area and excellent electrical conductivity of graphene allow it to act as an “electron wire” between the redox centers of an enzyme or protein and an electrode's surface. Rapid electron transfer facilitates accurate and selective detection of biomolecules. This review discusses the application of graphene for the detection of glucose, Cyt-c, NADH, Hb, cholesterol, AA, UA, DA, and H2O2. GO and RGO have been used for the fabrication of heavy metal ion sensors, gas sensors, and DNA sensors. Graphene based FETs have also been discussed in details. In all these cases, the biosensors performed well with low working potentials, high sensitivities, low detection limits, and long-term stabilities.

Highlights

► The sensing efficiency of graphene and CNT has been compared. ► Graphene based enzymatic and non-enzymatic electrodes can efficiently detect glucose, cytochrome-c, NADH, hemoglobin, HRP, and cholesterol, hydrogen peroxide, AA, UA, DA, respectively. ► The high sensitivity of graphene towards different gaseous molecules has led to its use in hydrogen, carbon monoxide, ammonia, chlorine, nitrogen dioxide, oxygen, and dinitrotoluene gas sensors.

Introduction

The historic development of enzyme electrodes by Leland C. Clark in 1962 opened a new area of research in medical science and technology (Clark and Lyons, 1962). Research on enzyme electrodes in various fields such as physics, chemistry, material science and biotechnology has resulted in more sophisticated and trustworthy biosensors. They are suitable for application in medicine, agriculture, biotechnology, as well as by the military and in bioterrorism detection and prevention (Wang, 2008, Chaubey and Malhotra, 2002, Yogeswaran and Chen, 2008). Recent work has examined ‘reagentless systems’, in which reagents are already immobilized and do not need to be added by the user (Schuhmann et al., 1993, Schmidt and Schuhmann, 1995, Senillou et al., 1999). Biosensors comprise a selective interface in close proximity to or integrated with a transducer, which relays information about interactions between the surface of the electrode and the analyte either directly or through a mediator (Fig. 1) (Yogeswaran and Chen, 2008). Biosensors can be categorized depending on the transducing mechanism: (i) resonant biosensors, (ii) optical-detection biosensors, (iii) thermal-detection biosensors, (iv) ion-sensitive FET biosensors, and (v) electrochemical biosensors (Chaubey and Malhotra, 2002). Electrochemical biosensors possess advantages over the others because their electrodes can sense materials present within the host without damaging the system (Chaubey and Malhotra, 2002). However, the sluggish ET of the biomolecules limits electrochemical efficiencies of the sensors (Kim et al., 2007, Huang et al., 2010a, Huang et al., 2010b, Shao et al., 2010, Wang, 2005). This is because of the structural features of the proteins and their unfavorable orientations at the surface of the electrodes. Generally electroactive prosthetic groups are embedded deep within the protein structure. To minimize the ET distance, nanomaterial “electron wires”, should be incorporated between the redox centers of the enzyme or protein and the electrode's surface (Shao et al., 2010).

Nanotechnology has allowed new applications of nanomaterials in electrochemical sensors and biosensors (Yogeswaran and Chen, 2008). Various nanomaterials, including metal NP, metal alloy NP, magnetic NP, nanowires, CNT, and CNF have been used as electrical connectors between the electrodes and the redox centers of the biomolecules (Shao et al., 2010, Kim et al., 2007, Huang et al., 2010a, Huang et al., 2010b, Sun et al., 2001, Shi and Ma, 2010, Haun et al., 2010, Yun et al., 2007). The use of metallic NP as biosensors is problematic because of their inconsistent signal amplification. The existence of CNT with metallic impurity is the main drawback while using in the modification of electrode (Pumera, 2009, Pumera, 2010). Such metallic impurities are electrochemically active and can dominate the electrochemistry of CNT. Impurities present at 50 ppm can be toxicological hazards as they can participate in redox reactions with the biomolecules (Pumera, 2009).

The discovery of graphene in 2004, added a new dimension to electrochemical biosensor research (Novoselov et al., 2004). The use of graphene can avoid the problems associated with metal alloy NP and CNT. The unique properties of graphene (fast electron transportation, high thermal conductivity, excellent mechanical flexibility and good biocompatibility) give it potential applicability in electrochemical biosensors (Pumera, 2009, Pumera et al., 2010, Allen et al., 2010, Brownson and Banks, 2010). Table 1 shows the comparative study of biosensing efficiency between CNT-based and graphene-based biosensors. Proper conjugation between biological molecules such as enzymes, ssDNA, RNA, Ab, receptors, and aptamers needs to be developed for graphene based electrochemical sensing electrodes. Appropriate functionalization of graphene and the immobilization of biomaterials on it are important, as functional groups can create defects on graphene surfaces (Shao et al., 2010).

There are several reported methods for the syntheses of graphene (Choi et al., 2010, Dreyer et al., 2010, Kuila et al., 2010, Park and Ruoff, 2009), including exfoliation and cleavage of natural graphite, CVD, PE-CVD, electric arc discharge, micromechanical exfoliation of graphite, epitaxial growth on electrically insulating surfaces, such as SiC, opening CNT and the solution-based reduction of GO (Choi et al., 2010, Kim et al., 2009, Kuila et al., 2010). Novoselov et al. (2004) discovered graphene sheets after the mechanical exfoliation of HOPG, a method now known as the scotch-tape method. Since their discovery, researchers have sought cost effective methods of producing large-area, single layers of graphene. CVD can produce large areas of single layer graphene (Reina et al., 2009). Li et al. (2009b) synthesized graphene by CVD using centimeter-scale copper substrates. Recently, Sun et al. (2010) synthesized graphene by CVD from solid PMMA deposited on a Cu substrate. Kim et al. (2009) synthesized graphene films on Ni substrates for stretchable transparent electrodes. Dong et al. (2009) prepared monolayers of graphene from natural flake graphite using 1,3,6,8-pyrenetetrasulfonic acid and D2O. Graphene can also be prepared on H2-etched surfaces of 6H-SiC when heated to 1250–1450 °C for a short time (1–20 min) (Wu et al., 2009b). Another mass production method involves the chemical or thermal reduction of GO (Schniepp et al., 2006) and to date is the most common and economical method. Graphene synthesized by chemical oxidation–reduction has been reported to possess many structural defects, which are advantageous for electrochemical applications (Shao et al., 2010).

DET is the fundamental process in electrochemical reactions. Study of electrochemical responses of graphene towards different redox systems requires knowledge of the electrochemical aspects of graphene (Pumera, 2009). It has been proposed that the presence of oxygen containing groups on graphene surface can enhance the ET rate. Heterogeneous charge transfer for the oxidation of endiol groups occurs through a proton-coupled ET mechanism (Pumera, 2009, Tang et al., 2009). Oxygen containing species take two protons from the endiol group and aid the oxidation reaction by reducing the overpotential voltage. Huang et al. (2011) measured EIS of GC and carboxyl-functionalized graphene electrodes. Yang et al. (2009) prepared graphene-based electrodes using vaseline as binder. CV found a pair of well-defined redox waves at the electrodes when the graphene content in the vaseline solution was 10.0 μg mL−1. This indicates that the redox couple at such electrodes is diffusion-controlled. The anti fouling ability and electrocatalytic behavior towards electrochemical oxidations of NADH and H2O2 at graphene-modified electrodes have also been evaluated (Lin et al., 2009). The proposed graphene-modified electrodes are expected to be useful in simple and effective electrochemical sensors and biofuel cells.

Biocompatibility of graphene or GO with microorganisms and living cells is very important. Extensive research efforts have been paid to study the biocompatibility of GO and graphene with the microorganisms (Akhavan and Ghaderi, 2009). It has been found that GO decreases cell adhesion when enters into cytoplasm and nucleus (Wang et al., 2011b). It also causes lung inflammation and kidney failure. However, the blood circulation time in GO deposited lungs are much better than other carbon based nanofiller (Zhang et al., 2011c). The growth of Gram-positive, Gram-negative, and Escherichia coli bacteria is affected significantly by the sharp edges of the reduced graphene nanowalls (Akhavan and Ghaderi, 2010, Hu et al., 2010). The ongoing research shows that the bacteria trapped within the aggregated graphene sheets are inactive without any chance for proliferation in a culture medium. But, after removing the aggregated graphene sheets from the surface of the bacteria by using sonication, they could be reactivated (Akhavan et al., 2011). Based on the cell culture experiments, Chen et al. (2008) showed that graphene paper may be biocompatible and therefore suitable for biomedical applications. Park et al. (2010) also proposed that graphene paper is noncytotoxic to three mammalian cell lines. Yang et al. (2011) showed that no obvious toxicity of pegylated graphene at the dose of 20 mg/kg to Balb/c female mice was observed in blood biochemistry, hematology, and histology analysis. However, the exact mechanism is still not clear. Therefore, to make it more biocompatible and to decrease or abolish the toxicity of GO is still a challengeable task for in vivo biomedical application.

This article reviews recent trends in the development of graphene-based electrochemical biosensors. Extensive research has been carried out on nanomaterial-based electrochemical biosensors; however, they suffer from limitations of sensitivity, electrochemical stability, production cost, biocompatibility and detection time. This work contains three main sections that examine graphene-based enzymatic, non-enzymatic and nano-electronic devices. The electrochemical detection of glucose, Cyt-c, NADH, Hb, HRP and cholesterol has been considered in the discussion of graphene-based enzyme electrodes. Non-enzymatic graphene-based electrodes for the detection of H2O2, AA, UA and DA are discussed in the second main section. Graphene-based nano-electronic devices for DNA sensing, heavy metal ion detection and gas sensing are reviewed in the last main section.

Section snippets

Graphene-based enzymatic electrodes

Direct electrochemistry of enzymes involves direct ET between the electrode and the active center of the enzymes without the participation of mediators or other reagents (Leger and Bertrand, 2008, Shao et al., 2010, Yao and Shiu, 2008). New mediator-free (or reagentless) biosensors, enzymatic bioreactors, and biomedical devices are sought that employ DET by immobilizing enzymes on conducting substrates. However, the redox centers of the biomolecules are usually embedded deep in their large

Graphene-based non-enzymatic electrodes

The preceding section discusses enzymatic biosensors. Strong performances have been reported with low detection limits and high accuracies. However, there are several disadvantages of enzyme-modified electrodes, such as their instability, the high cost of enzymes and the complexity of immobilization. Moreover, the activity of enzymes can be affected by temperature, pH, and toxic chemicals. To circumvent such problems, considerable attention has been paid to nonenzymatic electrodes (Li et al.,

Graphene-based nano-electronic devices

Interest in graphene arises from its potential applications in nano-electronic devices. Therefore, it has been used in the fabrication of nano-electronic devices for the detection of biomolecules, gas/water vapor and heavy metal ions. Table 4 shows different types of sensors made from graphene or GO for the detection of biomolecules, metal ions and gaseous molecules.

Conclusions

This review presents recent advances in the application of graphene for electrochemical biosensing. These graphene based biosensors and devices have exhibited good sensitivity and selectivity towards the detection of glucose, cholesterol, Hb, H2O2, small biomolecules, DNA, heavy metal ions and, poisonous gaseous molecules. Ionic liquid-modified graphene has potential applications in the routine clinical analysis of AA and DA. Nitrogen-doped graphene exhibited excellent electrocatalytic activity

Scope of future work

Different strategies have been explored for the construction of novel biosensors that employ graphene or graphene-like materials as sensing elements, yet there is still scope for further research. High surface area graphene prepared by CVD can be used to detect small biomolecules (DNA), gaseous elements and heavy metal ions. Heteroatom-doped graphene can be used to form novel biosensors. The electrochemical performances of IL attached to graphene can also be explored. Compared with CNTs,

Acknowledgements

This study was supported by the Human Resource Training Project for Regional Innovation, and the World Class University (WCU) program (R31-20029) through National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (MEST) of Korea.

References (144)

  • S. Andreescu et al.

    Anal. Biochem.

    (2008)
  • R. Arsat et al.

    Chem. Phys. Lett.

    (2009)
  • T.T. Baby et al.

    Sens. Actuators B: Chem.

    (2010)
  • Y. Bo et al.

    Electrochim. Acta

    (2011)
  • D.A.C. Brownson et al.

    Electrochem. Commun.

    (2011)
  • S. Chakraborty et al.

    Electrochem. Commun.

    (2007)
  • A. Chaubey et al.

    Biosens. Bioelectron.

    (2002)
  • Y. Ding et al.

    Biosens. Bioelectron.

    (2010)
  • Y. He et al.

    Electrochim. Acta

    (2011)
  • X. Kang et al.

    Biosens. Bioelectron.

    (2009)
  • F. Li et al.

    Talanta

    (2010)
  • J. Li et al.

    Anal. Chim. Acta

    (2009)
  • J. Li et al.

    Anal. Biochem.

    (2005)
  • L. Li et al.

    Talanta

    (2010)
  • W.J. Lin et al.

    Electrochem. Commun.

    (2009)
  • F. Liu et al.

    Biosens. Bioelectron.

    (2010)
  • Q. Lu et al.

    Talanta

    (2010)
  • R. Manjunatha et al.

    Sens. Actuators B: Chem.

    (2010)
  • M. Musameh et al.

    Electrochem. Commun.

    (2002)
  • Y. Ohno et al.

    Biosens. Bioelectron.

    (2010)
  • S. Park et al.

    Adv. Mater.

    (2010)
  • M. Pumera et al.

    Trac-Trend Anal. Chem.

    (2010)
  • W. Schuhmann et al.

    Synth. Met.

    (1993)
  • A. Senillou et al.

    Talanta

    (1999)
  • C. Shan et al.

    Biosens. Bioelectron.

    (2010)
  • C. Shan et al.

    Biosens. Bioelectron.

    (2010)
  • W. Shi et al.

    Biosens. Bioelectron.

    (2010)
  • W. Song et al.

    Biosens. Bioelectron.

    (2011)
  • L. Tan et al.

    Electrochem. Commun.

    (2010)
  • L. Valentini et al.

    Chem. Phys. Lett.

    (2004)
  • Y. Wan et al.

    Biosens. Bioelectron.

    (2011)
  • O. Akhavan et al.

    J. Phys. Chem. C

    (2009)
  • O. Akhavan et al.

    ACS Nano

    (2010)
  • O. Akhavan et al.

    J. Phys. Chem. B

    (2011)
  • M.J. Allen et al.

    Chem. Rev.

    (2010)
  • S. Alwarappan et al.

    J. Phys. Chem. C

    (2010)
  • Y. Bo et al.

    Analyst

    (2011)
  • D.A.C. Brownson et al.

    Analyst

    (2010)
  • H. Cai et al.

    Anal. Bioanal. Chem.

    (2003)
  • H. Chen et al.

    Adv. Mater.

    (2008)
  • X. Chen et al.

    Electroanalysis

    (2010)
  • W. Choi et al.

    Solid State Mater. Sci.

    (2010)
  • L.C. Clark et al.

    Ann. N. Y. Acad. Sci.

    (1962)
  • S.R. Dey et al.

    J. Phys. Chem. C

    (2010)
  • X. Dong et al.

    Nanoscale Res. Lett.

    (2011)
  • X. Dong et al.

    Phys. Rev. Lett.

    (2009)
  • R.D. Dreyer et al.

    Chem. Rev. Soc.

    (2010)
  • Z. Dursun et al.

    Electroanalysis

    (2010)
  • J.D. Fowler et al.

    ACS Nano

    (2009)
  • C.X. Guo et al.

    Adv. Mater.

    (2010)
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