石墨烯为基础的生物传感器的研究进展

Biosensors and Bioelectronics 26 (2011) 4637–4648Contents lists available at ScienceDirectBiosensors andBioelectronicsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b i osReviewRecent advances in graphene-based biosensorsTapas Kuila a ,Saswata Bose a ,Partha Khanra a ,Ananta Kumar Mishra b ,Nam Hoon Kim c ,Joong Hee Lee a ,b ,c ,∗aDepartment of BIN Fusion Technology,Chonbuk National University,Jeonju,Jeonbuk 561-756,Republic of KoreabBIN Fusion Research Center,Department of Polymer &Nano Engineering,Chonbuk National University,Jeonju,Jeonbuk 561-756,Republic of Korea cDepartment of Hydrogen and Fuel Cell Engineering,Chonbuk National University,Jeonju,Jeonbuk 561-756,Republic of Koreaa r t i c l ei n f oArticle history:Received 16February 2011Received in revised form 25May 2011Accepted 25May 2011Available online 2 June 2011Keywords:GrapheneBiocompatibility Enzyme biosensor Graphene electrode DNA sensor Gas sensora b s t r a c tA 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 H 2O 2.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.© 2011 Elsevier B.V. All rights reserved.Contents 1.Introduction ..........................................................................................................................................46381.1.Synthesis of graphene ........................................................................................................................46381.2.Electrochemical aspects of graphene ........................................................................................................46391.3.Biocompatibility of GO and graphene ........................................................................................................46401.4.Scope of this review ..........................................................................................................................46402.Graphene-based enzymatic electrodes ..............................................................................................................46402.1.Glucose oxidase biosensor ...................................................................................................................46402.2.Cytochrome c biosensor ......................................................................................................................46412.3.NADH biosensor ..............................................................................................................................46412.4.Hemoglobin biosensor .......................................................................................................................46412.5.HRP biosensor ................................................................................................................................46412.6.Cholesterol biosensor (4642)Abbreviations:A,adenine;AA,ascorbic acid;Ab,antibody;AuNP,gold nanoparticle;BDD,boron doped diamond;CdS,cadmium sulfide;CEA,carcinoembryonic antigen;CEEs,chloroethylethyl sulfide;␤-CD,␤-cyclodextrin;C,cytosine;CMG,chemically modified graphene;CNT,carbon nanotube;CNF,carbon nanofiber;CRG,chemically reduced graphene;CR-GO,chemically reduced graphene oxide;CS,chitosan;CVD,chemical vapor deposition;CV,cyclic voltammetry;Cyt-c,cytochrome c;DA,dopamine;DET,direct electron transfer;DMMP,dimethylmethylphosphonate;DNA,deoxyribonucleic acid;DNT,dinitrotoluene;D 2O,heavy water;DPV,differential pulse voltammetry;EDTA,ethylenediamine triacetic acid;EG,epitaxial graphene;EIS,electrochemical impedance spectroscopy;ET,electron transfer;Fe 3O 4,iron (II,III)oxide;FET,field effect transistor;G,guanine;GC,glass carbon;GO,graphene oxide;GOx,glucose oxidase;GR,graphene;Hb,hemoglobin;HCN,hydrogen cyanide;HOPG,highly oriented pyrrolytic graphite;HRP,horseradish peroxide;IL,ionic liquid;H 2O 2,hydrogen peroxide;IgE,immunoglobulin;ITO,indium tin oxide;LiTaO 3,lithium tantalite;MB,molecular beacons;Mb,methylene blue;MG,methylene green;MGNF,multilayer graphene nanoflake;MnO 2,manganese dioxide;NADH,nicotinamide adenine dinucleotide;NP,nanoparticle;PANIw,polyaniline nanowire;PB,Prussian blue;PBS,phosphate buffer solution;PE-CVD,plasma-enhanced chemical vapor deposition;PMMA,poly(methyl methacrylate);ppb,parts per billion;ppm,parts per million;PSS,polystyrene sulfonate;PSSA-g-PPY,poly(styrenesulfonic acid-g-pyrrole);PtNP,platinum nanoparticles;PPy,polypyrrole;RGO,reduced GO;rGSFs,reduced graphene sheet film (rGSF);RNA,ribo nucleic acid;RSD,relative standard deviation;SDBS,sodium dodecyl benzene sulfonate;Sic,silicon carbide;SRB,sulfate-reducing bacteria;ssDNA,single strand deoxyribonucleic acid;S/N,signal to noise background ratio;T,thymine;UA,uric acid.∗Corresponding author at:Department of BIN Fusion Technology,Chonbuk National University,Jeonju,Jeonbuk 561-756,Republic of Korea.Tel.:+82632702342;fax:+82632702341.E-mail address:jhl@chonbuk.ac.kr (J.H.Lee).0956-5663/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2011.05.0394638T.Kuila et al./Biosensors and Bioelectronics26 (2011) 4637–46482.7.Catechol biosensor (4642)3.Graphene-based non-enzymatic electrodes (4642)3.1.Hydrogen peroxide (4643)3.2.Ascorbic acid,uric acid and dopamine (4643)4.Graphene-based nano-electronic devices (4643)4.1.DNA sensors (4644)4.2.Heavy metal ion detection (4645)4.3.Gas sensors (4645)4.4.Field effect transistor (4646)5.Conclusions (4646)6.Scope of future work (4647)Acknowledgements (4647)References...........................................................................................................................................4647Fig.1.Schematic presentation of a biosensor.1.IntroductionThe historic development of enzyme electrodes by Leland C. Clark in1962opened a new area of research in medical science and technology(Clark and Lyons,1962).Research on enzyme electrodes in variousfields such as physics,chemistry,material science and biotechnology has resulted in more sophisticated and trustworthy biosensors.They are suitable for application in medicine,agricul-ture,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 mech-anism:(i)resonant biosensors,(ii)optical-detection biosensors, (iii)thermal-detection biosensors,(iv)ion-sensitive FET biosen-sors,and(v)electrochemical biosensors(Chaubey and Malhotra, 2002).Electrochemical biosensors possess advantages over the oth-ers 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 electrochemi-cal efficiencies of the sensors(Kim et al.,2007;Huang et al.,2010a,b; 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 elec-trode’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 elec-trical connectors between the electrodes and the redox centers of the biomolecules(Shao et al.,2010;Kim et al.,2007;Huang et al., 2010a,b;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,2009,2010).Such metal-lic impurities are electrochemically active and can dominate the electrochemistry of CNT.Impurities present at50ppm can be tox-icological hazards as they can participate in redox reactions with the biomolecules(Pumera,2009).The discovery of graphene in2004,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).Table1shows the comparative study of biosensing efficiency between CNT-based and graphene-based biosensors.Proper conjugation between biolog-ical molecules such as enzymes,ssDNA,RNA,Ab,receptors,and aptamers needs to be developed for graphene based electrochem-ical sensing electrodes.Appropriate functionalization of graphene and the immobilization of biomaterials on it are important,as func-tional groups can create defects on graphene surfaces(Shao et al., 2010).1.1.Synthesis of grapheneThere 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 natu-ral graphite,CVD,PE-CVD,electric arc discharge,micromechanical exfoliation of graphite,epitaxial growth on electrically insulat-ing 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 haveT.Kuila et al./Biosensors and Bioelectronics26 (2011) 4637–46484639 Table1Comparative study of sensing efficiency between CNT and graphene-based biosensors/sensors.Sensor type Detected element Sensing materials RSD(%)a/response time b Detection limit ReferencesGlucose biosensor Glucose Graphene-PPy–3␮M±0.5␮M Alwarappan et al.(2010) Glucose CNT-PPy b15s0.2mM Wang and Musameh(2005) Glucose Graphene/Nafion a4.21 1.0×10−6M Chen et al.(2010)Glucose CNT/Nafion a2.8/b4s 3.0×10−9M Jeykumari andNarayanan(2008)CS–GR a5.3/0.02nM Kang et al.(2009) Glucose CS-CNT a5.00.05mM Zou et al.(2008)Glucose CS–GR-AuNP a6.00.6M Wu et al.(2009a)Glucose CS-CNT-AuNP a4.95/b<6s 3.0×10−6M Wang et al.(2009b) Cholesterol oxidase Cholesterol PtNP-Graphene–0.5nM Dey and Raj(2010) Cholesterol PtNP-CNT a2.6–4.1/b<20s–Shi and Peng(2005) NADH Graphene 3.5–Tang et al.(2009) NADH GO b<1s0.1␮m Zhang et al.(2011a)NADH CNT a2.55×10−3M Musameh et al.,2002NADH CNT b4s2␮m Chakraborty and Raj(2007)HRP H2O2Graphene a4.48 1.05×10−7M Lu et al.(2010b) H2O2AuNP-Graphene a3.6 1.0×10−6M Zhou et al.(2010b)H2O2CNT b<1s 1.0×10−7M Xu et al.(2005)H2O2CNT-Mb a4.5 1.0␮m Xu et al.(2003)Hb H2O2Graphene– 5.1×10−7M Xu et al.(2010) H2O2CNT a9.35 2.41␮m Ding et al.(2010)H2O2CNT-sodium alginate a2.3416.41␮m Zhao et al.(2009)Nitrite Hb-CS-DMF/GR a2.1 1.8×10−7M Liu et al.(2011a,b)Electrochemical sensor AA,UA,DA MGNF a0.17␮M–Shang et al.(2008) DA␤-CD/GR b30s0.2␮M Tan et al.(2010) DA CMG–0.01␮M Hou et al.(2010) AA,UA,DA PSS-CNT a4,2.9,2.5 5.0×10−7M,1.5×10−7M,5.0×10−7M Manjunatha et al.(2010)AA,UA,DA PtNP-CNT– 1.9×10−5M,3.2×10−8M,2.7×10−8M,Dursun and Gelmez(2010)DNA sensor DNA EG–1␮M Lim et al.(2010) DNA GO–200nM Liu et al.(2010a,b)DNA GR/PANIw a1.15 3.25×10−13M Bo et al.(2011b)DNA CNT-Mb–0.252nM Li et al.(2005)Phenolic pollutants CNT-DNA a3 1.6×10−5M Zheng et al.(2009)DNA CNT a11/b3s 1.0×10−10M Cai et al.(2003)Gas sensors NO2Thermally reduced GO–2ppm Lu et al.(2009b,c) NO2and NH3,DNT Hydrazine reduced GO–<5ppm(NO2and NH3),28ppb DNT Fowler et al.(2009)NO2and NH3CNT b<600s2ppm Kong et al.(2000)NO2CNT b<600s44ppb Li et al.(2003)NH3CNT b∼100s10ppm Suehiro et al.(2003)NO2CNT b∼600s5–100ppb Valentini et al.(2004)NH3,CO,ethanol Mechanically exfoliated graphene–1ppb Schedin et al.(2007)HCN CNT–4000Robinson et al.(2008)CEES0.5DMMP0.1DNT0.1HCN Reduced GO–70Robinson et al.(2008)CEES0.5DMMP5DNT0.1sought cost effective methods of producing large-area,single lay-ers 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 naturalflake graphite using1,3,6,8-pyrenetetrasulfonic acid and D2O.Graphene can also be prepared on H2-etched surfaces of6H-SiC when heated to1250–1450◦C for a short time(1–20min)(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 com-mon 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).1.2.Electrochemical aspects of grapheneDET 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 pres-ence 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)mea-sured EIS of GC and carboxyl-functionalized graphene electrodes. Yang et al.(2009)prepared graphene-based electrodes using vase-line as binder.CV found a pair of well-defined redox waves at the electrodes when the graphene content in the vaseline solu-tion was10.0␮g mL−1.This indicates that the redox couple at such4640T.Kuila et al./Biosensors and Bioelectronics26 (2011) 4637–4648Table2Various types of enzymatic electrodes made from graphene and GO.In all the cases the mode of detection is change in electrical current.Type of biosensor Sensor materials RSD(%)Detection limit ReferencesGlucose Graphene-PPy–3␮M±0.5␮M Alwarappan et al.(2010) Metal decorated graphene–1␮M Baby et al.(2010)Graphene/Nafion 4.21 1.0×10−6M Chen et al.(2010)CVD grown graphene–0.1mM Huang et al.(2010a,b)CS–GR 5.30.02nM Kang et al.(2009)GO 5.8–Liu et al.(2010b)PVDF-protected graphene 3.22–14mM Shan et al.(2009)CS–GR-AuNP 4.7180␮M Shan et al.(2010a)GO––Wang et al.(2009c)Graphene-CdS 5.30.7mM Wang et al.(2011a)CS–GR-AuNP 6.00.6M Wu et al.(2009a)Graphene 2.510±2␮M Wu et al.(2010b)CMG–0.376mM Yang et al.(2010)Graphene–0.168mM Zeng et al.(2010a)CR-GO 4.3 2.0␮M Zhou et al.(2009)Graphene 3.25␮M Zhou et al.(2010a) NADH IL functionalized graphene 4.25␮M Shan et al.(2010b)Graphene 3.5–Tang et al.(2009)Hb Fe3O4–graphene 1.60.5␮M He et al.(2011)Graphene– 5.1×10−7M Xu et al.(2010)HRP Graphene 4.48 1.05×10−7M Lu et al.(2010b)SDBS–graphene– 1.0×10−7M Zeng et al.(2010b)AuNP–graphene 3.6 1.0×10−6M Zhou et al.(2010b)CS–GR/Fe3O4/HRP– 6.0×10−7M Zhou et al.(2011) Cholesterol PtNP–graphene–0.5nM Dey and Raj(2010)electrodes is diffusion-controlled.The anti fouling ability and elec-trocatalytic behavior towards electrochemical oxidations of NADH and H2O2at graphene-modified electrodes have also been evalu-ated(Lin et al.,2009).The proposed graphene-modified electrodes are expected to be useful in simple and effective electrochemical sensors and biofuel cells.1.3.Biocompatibility of GO and grapheneBiocompatibility 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 with-out 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 pegy-lated graphene at the dose of20mg/kg to Balb/c female mice was observed in blood biochemistry,hematology,and histology analy-sis.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.1.4.Scope of this reviewThis 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 sensitiv-ity,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 dis-cussion 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.2.Graphene-based enzymatic electrodesDirect 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 biomedi-cal 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 three-dimensional structures.Recent research has shown that graphene can enhance DET between enzymes and electrodes.The use of metal NP with graphene has been reported to form exceptionally stable and cost-effective biosensors(Andreescu and Luck,2008;Xiao et al., 2003).Table2shows different types of enzymatic biosensor with their limit of detection and RSD.2.1.Glucose oxidase biosensorThe metabolic disorder of diabetes mellitus results in the defi-ciency of insulin and hyperglycemia and is reflected by blood glucose concentration higher or lower than the normal range of 80–120mg dL−1(4.4–6.6mM).The disease is a leading cause of death and disability(Wang,2008).Therefore,the diagnosis and management requires close monitoring of blood glucose levels. The application of graphene in highly sensitive and cost-effective biosensors can aid the aforementioned diseases(Wang et al.,T.Kuila et al./Biosensors and Bioelectronics26 (2011) 4637–464846412010b).Various groups have shown that graphene-based glucose biosensors exhibit good sensitivity,selectivity and reproducibility. The direct electrochemistry of GOx based on redox-active centers was confirmed by CV experiments in the potential range of0.8–0V to follow previously proposed reaction mechanisms(Yang et al., 2010):GO x(FAD)+2H++2e−↔GO x(FADH2)(In the absence of oxygen)(1) In the presence of oxygen,the reaction mechanism follows dif-ferent pathways:GO x(FADH2)+O2→GO x(FAD)+H2O2(2) GO x(FAD)+Glucose→GOx(FADH2)+Gluconolactone(3) Wang et al.(2009c)used electrochemically reduced single layers of graphene to detect glucose.The presence of CdS nanocrystal with graphene showed very low detection limit of0.7mM(Wang et al., 2011a).Shan et al.(2009)used a polyvinylpyrrolidone-protected graphene/polyethylenimine-functionalized IL/GOx electrochemi-cal biosensor to study the DET of GOx.CV measurements revealed characteristic reversible ET of the redox active center.Kang et al. (2009)fabricated CS–GR/GOx modified electrodes for direct elec-trochemical glucose sensing.The excellent performance of the biosensors was attributed to the large surface area to volume ratio and high conductivity of graphene and the good biocompatibility of chitosan with GOx.CS–GR/PtNP nanocompositefilm has also been used for the detection of glucose(Wu et al.,2009a,b).Introduc-tion of PtNP increased the sensor’s sensitivity,allowing a detection limit of0.6␮M glucose.The biosensor possessed good reproducibil-ity and long-term stability with negligible interference signals from AA and UA.Shan et al.(2010a)designed CS–GR/AuNP nanocompos-itefilms for glucose sensing,which exhibited good amperometric responses to glucose with a linear ranges of2–10mM(R=0.999)at −0.2V and2–14mM(R=0.999)at0.5V.This was attributed to the synergy effect of graphene and the AuNP.Zhou et al.(2010a)inves-tigated glucose sensitivity of Nafion-GR/AuNP biocompositefilms. This linear range of the sensor was15–5.8mM,with a detection limit of5mM(S/N=3).Zeng et al.(2010a)used organically modified graphene for enzyme-based glucose and maltose biosensing.The CV response of the modified electrode increased with increasing concentration of glucose in buffer solution.The detection limit and sensitivity were0.168mM(S/N=3)and0.261␮A mM−1cm−2,respectively. GO has also been used for the detection of glucose(Liu et al.,2010b; Song et al.,2010).Its biocompatibility with GOx led to a stable glu-cose biosensor with a sensitivity of8.045mA cm−2M−1.Yang et al. (2010)developed a glucose biosensor using CMG and IL.Chen et al. (2010)prepared Nafion-GR/GOxfilm-modified electrodes for the detection of glucose.The sensor showed good stability,with a RSD of4.21%(n=5).Huang et al.(2010a,b)used CVD-grown graphene to develop real-time glucose biosensors.Alwarappan et al.(2010) employed PPy-GR/GOx-based enzymatic biosensors for in vitro electrochemical glucose detection.Nafion-GR/PtNP or AuNP mod-ified electrodes exhibited best sensing performances,with a linear response up to30mM and an excellent detection limit of1␮M (Baby et al.,2010).Wang et al.(2010b)investigated the effect of nitrogen doping on the biosensing performances of graphene electrode.N-doped graphene exhibited excellent electrocatalytic activity for the reduction of H2O2,fast ET kinetics,high sensitivity and selectivity for glucose biosensing.2.2.Cytochrome c biosensorDET between Cyt-c and electrodes is receiving increasing research attention due to its biological functions(Xiang et al.,2008). However,bare GC does not exhibit a voltammetric response for Cyt-c detection due to slow ET kinetics at the electrode/electrolyte interface(Xiang et al.,2008).The development of carbon-based nanofiller has successfully overcome these problems.Wu et al.(2010a)tried to solve the same problem using CS–GR-modified electrodes to study the DET of Cyt-c.The formal potential of the CS–GR/Cyt-c modified GC electrode was estimated to be −0.28V.The ET rate constant of Cyt-c at the graphene electrode was estimated to be1.95s−1,which is much higher than that of the conventional electrodes.Therefore,Cyt-c on the surface of a CS–GR modified electrode maintains its bioactivity and shows an enzyme-like activity for the reduction of nitric oxide.2.3.NADH biosensorNADH is an important coenzyme that takes part in more than 300dehydrogenase enzymatic reactions(Liu et al.,2009).Elec-trocatalytic oxidation of NADH has been investigated as part of the development of dehydrogenase-based biodevices.However,its electrochemical oxidation at bare GC electrodes in neutral solu-tions occurs at a high overpotential(ca.0.5V)because of slow ET kinetics and electrode fouling(Raj and Ohsaka,2001).Therefore, the effective oxidation of NADH at low potentials would aid the development of NADH-based biodevices.Tang et al.(2009)studied the electrochemical behavior of NADH on rGSF.It showed increased ET kinetics and excellent electrocatalytic activity compared with bare GC electrodes.The peak potential of NADH oxidation shifted from0.75V on bare GC to0.42V on the rGSF/GC electrode.Liu et al.(2009)pre-pared a NADH sensor through noncovalent functionalization of graphene with water-soluble electroactive MG.The oxidation peak of NADH at a bare GC electrode occurs at+0.55V.However,this became+0.40V and+0.14V,respectively for electrodes of CRG and CRG functionalized with MG.Shan et al.(2010b)designed an IL-functionalized graphene biosensor for the electrochemical determination of NADH.The amperometric response at this elec-trode was more stable(95.4%and90%of initial activity remaining after10and30min,respectively,at1mM NADH solution)than that of the bare GC electrode(68%and46%).Moreover,the IL-CS–GR-modified electrode exhibited good linearity from0.25to2mM anda high sensitivity of37.43␮Am M−1cm−2.2.4.Hemoglobin biosensorHb is the most important part of blood and is responsible for transporting O2throughout the circulatory system.Change of Hb concentration in blood can cause several diseases and even death. Therefore,accurate determination of Hb content in blood is medi-cally very essential.Xu et al.(2010)used a CS–GR-modified electrode for the elec-troanalysis of Hb.The cyclic voltammogram of Hb at the CS–GR/GC electrode showed a well-resolved redox peak compared with a CS/GC electrode.The current response of Hb at the CS–GR/GC elec-trode increased linearly with scan rate from30to150mV s−1 (Fig.2),indicating a surface-controlled electrochemical process. He et al.(2011)studied the effect of magnetic NP on Hb detec-tion.The biosensor exhibited fast response time(<3s),broad linear response to H2O2in the range1.50–585␮M L−1and a rel-atively low detection limit of0.5␮M L−1(S/N=3).Wang et al. (2010a)designed CS–GR/Hb/GR/IL/GC electrode for the detection of nitromethane.The designed biosensor achieved a detection limit down to6.0×10−10M for quantitative nitromethane analysis.2.5.HRP biosensorAccurate determination of H2O2is useful in chemistry,biology, clinical control and environmental protection(Zhou et al.,2010b).。