黃海平　徐　亮　岳亞鋒　姜立萍

(1江西理工大學冶金與化學工程學院，贛州341000)

(2生命分析化學國家重點實驗室，南京大學化學化工學院，南京210093)

基于氧化鉺-石墨烯氧化物復合納米材料的葡萄糖氧化酶直接電化學性能及對葡萄糖的檢測

黃海平1,2徐亮1岳亞鋒1姜立萍＊,2

(1江西理工大學冶金與化學工程學院，贛州341000)

(2生命分析化學國家重點實驗室，南京大學化學化工學院，南京210093)

將稀土納米材料Er2O3用于構建葡萄糖生物傳感器。Er2O3和氧化石墨烯形成復合基底，將葡萄糖氧化酶(GOD)固載在玻碳電極表面。首先利用SEM和XRD技術對所制備的Er2O3和氧化石墨烯納米材料進行表征。利用EIS和CV對整個生物傳感器制備過程進行表征。Er2O3的存在能有效地保持GOD的生物活性并加速其與電極之間的電子傳遞。由于Er2O3和氧化石墨烯之間的協同效應，使得制備的傳感器在CV圖中呈現一對明顯的氧化還原峰，證實GOD和電極之間的直接電子傳遞性能。當用于對葡萄糖的電催化氧化時，傳感器的CV響應隨著葡萄糖濃度的增加而變弱。在葡萄糖濃度為1～10 mmol·L-1范圍內，CV響應值與葡萄糖濃度成線性關系。此外，傳感器具有好的穩(wěn)定性和重現性。

氧化鉺；石墨烯氧化物；葡萄糖氧化酶；生物傳感器

0　Introduction

Nowadays,the rare-earth based nanomaterials have attracted much interest due to their narrow emission bands,noncytotoxicity and excellent biocompatibility for construction of various desired biosensors. As back in 2002,Prof.Chen[1]first prepared the lanthanum hexacyanoferrate(LaHCF)modified platinum electrode by cyclic voltammetric(CV)technique.The modified electrode showed considerable electrochemical behavior and might be used for fabrication of electrochemical sensor.In 2006,Ru′s group[2]developed CeO2/Chitosan(CHIT)composite matrix for the immobilization of single-stranded DNA(ssDNA)probe and the fabrication of DNA biosensor.Combining the advantages of good biocompatibility and electronic conductivity,the prepared matrix exhibited the enhanced loading of ssDNA probe on the electrode surface.In order to improve the loading amount,the sensitivity and the selectivity,carbon nanotubes and other nanomaterials were employed to form the nanocomposite with CeO2[3].Under the synergistic effect of this nanocomposite matrix,the immobilization of the DNA probes was greatly enhanced and the sensitivity of the detection of target DNA was markedly improved.What′s more,some rare earth elements could also promote the conformational change of DNA secondary structure.For example,in the presence of Tb3+,the conformation of single stranded G-rich DNA probe could be changed to form the compact quadruplex[4].With their excellent biocompatibilities, rare earth elementscould enhance the catalytic activities of bio-enzymes.Many enzymes such as horseradish peroxidase(HRP)[5],glucose oxidase(GOD)[6],and cholesterol oxidase(ChOx)[7]were reported to be immobilized onto the rare earth based nanomaterials for the construction of biosensor.To take ChOx as an example,ChOx could be immobilized onto the sol-gel derived nano-structured cerium oxide with ITO as glass substrate.The result proved that the sol-gel NSCeO2film could provide better configuration for immobilization of ChOx.Another rare earth oxide of Tm2O3was employed for the immobilization of GOD[6]. The direct electron transfer(DET)with an apparent heterogeneous electron transfer rate constant was achieved on the GOD immobilized Nafion-Tm2O3film. Other elements like Y[8],Sm[9-10]were reported for the fabrication of biosensor for the detection of serum uric acid,oxidized low density lipoprotein,etc.The above successful reports for the achievement of enzymes DET and the electrochemical detection of small biomolecule proved a new promising strategy for the fabrication of electrochemical biosensors based on the rare earth oxide nanomaterials.Many other elements among the rare earth are still remain to be explored and studied.For this purpose,herein we synthesized Er2O3nanomaterial and studied its electrochemical property.

As a novel two-dimensional monolayer nanomaterial,graphene[11]exhibited excellent thermal,electronic,and mechanical properties,such as high surface area,unique transport performance[12],excellent electrical conductivity,ultra-strong mechanical properties and high stability[13].In recent years,graphene oxide (GO)based materials were widely used for electrochemical biosensor applications[14-16].

In this paper,in order to further expand the electrochemical study of rare earth elements,the Er2O3nanomaterial was firstly synthesized via hydrothermal homogeneous method and then characterized.Glucose oxidase(GOD),as an ideal model enzyme,was employed for use in the next bioelectrochemistry. Graphene oxide was used to form the composite with the prepared Er2O3nanomaterial so as to achieve better electrochemical performance.

1　Experimental

1.1Reagents and apparatus

Chitosan(low molecular weight),GOD from Aspergillus niger(E.C.1.1.3.4,Type X-S,100～250 kU·g-1) and D-(+)-glucose were purchased from Sigma-Aldrich and used without further purification.Bulk Er2O3was obtained from Tongji Institute of Trace Element (Beijing,China).All other chemicals were of analytical grade and used without further purification. Millipore ultrapure water(Resistivity≥18.2 MΩ)wasused throughout the experiment.Phosphate buffer solution(PBS)(pH 7.0,0.1 mol·L-1)was employed as a supporting electrolyte by mixing the stock solutions of NaH2PO4and Na2HPO4.

X-ray diffraction(XRD)patterns were recorded on an X-ray diffractometer(PANalytical X′Pert Pro) with Cu Kα radiation(λ=0.154 18 nm)for crystal phase identification.The XRD was operated at 40 kV accelerating voltage and 40 mA tube current,with the degree range of 5°～80°.Scanning electron microscope (SEM)was taken using a Philips XL30 microscope, using an accelerating voltage of 200 kV.The electrochemical impedance spectroscopy(EIS)analyses were performed on an Autolab PGSTAT12(Ecochemie,BV, The Netherlands)with the frequency range of 0.1～1.0×105Hz.A CHI 660D Electrochemical Workstation(Shanghai CH Instruments Co.,China)was used for the cyclic voltammograms(CVs).The electrochemical system consisted of a modified glassy carbon electrode(GCE)as working electrode,a platinum wire auxiliary electrode and a saturated calomel electrode (SCE)as reference electrode.

1.2Synthesis of nanomaterials

GO was synthesized according to the modified Hummers method[17-18].The last suspension was centrifuged under 3 000 r·min-1.The supernatant was collected and put in the refrigerator for further use.Er2O3nanomaterial was prepared according to the reported previously method with some modifications[6].In briefly,0.1 mmol bulk Er2O3was firstly dissolved in hot concentrated HCl(36%).After that,the pH value was adjusted by 0.2 mol·L-1NaOH solution to about 10.The solution was then poured into the Teflon-lined autoclaves(100 mL)and held at 150℃for 12 h. After cooled to room temperature naturally,the precipitates were collected by centrifugation and then calcined at 400℃for 2 h.

1.3Fabrication of biosensor

First of all,the glassy carbon electrode(GCE,4 mm in diameter)was carefully polished to a mirror by 1.0,0.3 and 0.05 μm alumina powder.After ultrasonically cleaned in ethanol and water respectively,GCE was then dried by flowing N2before it was used.1 mg prepared Er2O3nanomaterial was dispersed in 1 mL H2O by ultrasonicating to form a stable suspension. Then a homogeneous solution,which finally contained about 6 mg·mL-1GOD,0.3 mg·mL-1Er2O3nanomaterial,0.6 mg·mL-1GO was formed by thoroughly mixing the Er2O3suspension,GO suspension with GOD solution(20 mg·mL-1)at 1∶1∶1 ratio(V/V).A volume of 10 μL of the resulting solution was dropped onto the pretreated GCE.The electrode was left in desiccator to dry at 4℃.At last,5 μL of 5 mg·mL-1chitosan solution was dripped onto the GOD/GO/ Er2O3/GCE for sealing.The GOD/GCE,GOD/Er2O3/ GCE,GOD/GO/GCE were fabricated through a similar procedure with pure water as the substitute.

2　Results and discussion

2.1Characterization of nanomaterials

The morphology of the synthesized Er2O3nanomaterial was characterized by SEM,which was shown in Fig.1A.It could be seen from Fig.1A that the Er2O3were relatively uniform in square-shaped size with about 500 nm in length.Fig.1B was the XRD pattern of the as-synthesized GO.As indicated in the pattern,a well-defined d001peak of 2θ=10.3° confirmed that the GO formed a well-ordered layered structure[19].The surface of the prepared biosensor was shown in Fig.1C,which contained ternary nanocomposites of Er2O3nanomaterial,GO and GOD.As could be seen from Fig.1C,Er2O3nanomaterials and GODs were dispersed on the surface of GO.

2.2Electrochemical characterization of the biosensor

As an effective tool to inspect the estates of the electrode surface,EIS is widely used to understand the chemical transformations and processes associated with the conductive electrode surface[20].The electron transfer resistance of the electrochemical reaction,Ret, reveals the electron transfer kinetics of the redox electrochemical probe at the electrode interface. Another electrochemical technique of cyclic voltammogram(CV)is also considered as a powerful method to monitor the electron transfer behaviour between the solution species and the electrode.Herein,the EISand CV were used to examine the modified electrode after each self-assembly step,which were shown in Fig.2.

Fig.1(A)SEM of the prepared Er2O3Nanomaterial;(B)XRD pattern of the prepared graphene oxide; (C)SEM of the ternary composites containing Er2O3nanomaterial,GO and GOD

Fig.2　EIS(A)and CV(B)of the electrode at different stages in 0.1 mol·L-1KCl+2 mmol·L-1[Fe(CN6)]3-/[Fe(CN6)]4-

As presented by the EIS spectrum in Fig.2A,the GO modified electrode(curve a in Fig.2A)showed relative small electron-transfer resistance(Ret)as compared to the Er2O3modified electrode(curve b in Fig. 2A),which suggested that the GO owned better electronic conductivity than Er2O3.After the GOD was mixed with Er2O3and coated on the bare electrode (curve d in Fig.2A),the resistance increased dramatically.This phenomenon could be attributed to the hindrance effect of electron-transfer kinetics between the redox probe and electrode surface[21].The similar result was also observed when GOD was mixed with GO(curve c in Fig.2A).Accordingly,the ternary nanocomposites GOD/GO/Er2O3modified electrode exhibited a moderate Ret(curve e in Fig.2A),which wassmaller than the GOD/Er2O3and larger than the GOD/ GO modified electrode.

Fig.2B showed the CVs of the redox probe [Fe(CN6)]3-/[Fe(CN6)]4-on the modified electrode at different stages.As could be seen in Fig.2B,stepwise modifications on the GCE were accompanied by the changes in the amperometric response of the redox probe.On the GO modified electrode,a pair of welldefined redox peaks was observed(curve a in Fig.2B), showing the excellent electron-transfer kinetics of [Fe(CN6)]3-/[Fe(CN6)]4-,so as the Er2O3modified GCE (curve b in Fig.2B).After the GOD was mixed with GO,the amperometric response decreased and the peak-to-peak separation enlarged(curve c in Fig.2B), due to the fact that the bulky GOD molecules blocked the electron exchange.The CV response was further decreased after GOD was mixed with Er2O3(curve d in Fig.2B).The shape of the redox peaks become better than Er2O3/GOD when GO was added to form the ternary nanocomposites(curve e in Fig.2B).From the above results,it was obvious that the CV changes were consistent with the EIS changes.

2.3Direct electrochemistry of GOD/GO/Er2O3/ GCE

For the purpose of investigating the direct electrochemical property of the modified electrodes, the cyclic voltammograms(CV)of the modified electrodes at different steps were detected.Fig.3A was the CV of GOD/GCE,GOD/Er2O3/GCE,GOD/GO/GCE and GOD/GO/Er2O3/GCE in PBS solution(0.1 mol·L-1, pH 7.0)at the scan rate of 100 mV·s-1.

As could be seen from the results,when the GOD was directly dropped onto the GCE surface(curve a in Fig.3A),a very small redox wave was observed.This wave could be attributed to the characteristic of a reversible electron transfer process between the redox active center(flavin-adenine dinucleotide,FAD)in GOD and the electrode[22-23].The CV curve of the GOD/ GO/GCE(curve c in Fig.3A)showed more distinguished redox waves with larger peak current,which was attributed to the excellent biocompatibility and electronical conductivity of GO.A comparison with the CV curves of the GOD/Er2O3/GCE(curve b in Fig. 3A)and GOD/GO/GCE presented similar redox waves, which proved that the prepared Er2O3nanomaterial could also provide a friendly microenvironment to maintain the bioactivity of GOD and the electron transfer between the modified electrode and GOD. Furthermore,after GOD was mixed with GO and Er2O3to form the ternary nanocomposites,the GOD/GO/ Er2O3/GCE(curve d in Fig.3A)displayed a pair of more distinct and better-defined redox peaks, indicating the faster DET rate between the redoxactive site of GOD and GCE.The synergistic effect of the GO/Er2O3nanocomposite was considered to effectively accelerate electrical transfer between redox-active center of GOD and electrode surface, leading to the increased peak current.Combining theSEM picture and CV curves,it could be deduced that GO not only promoted the electron transfer rate,but also provided the necessary supported matrix to form the ternary nanocomposites.

Fig.3(A)CV of the modified electrodes at different steps in PBS solution(0.1 mol·L-1,pH 7.0)at the scan rate of 100 mV·s-1; (B)CVs of GOD/GO/Er2O3/GCE measured in PBS solution(0.1 mol·L-1,pH 7.0)at the different scan rates

For the purpose of further understanding the property of electron transfer between the GOD and the electrode,the cyclic voltammograms of the GOD/GO/ Er2O3/GCE at various scan rates were investigated, which were displayed in Fig.3B.It could be obviously seen from Fig.3B that the redox peak currents increased with the increase of the scan rates in the range of 10～200 mV·s-1,coupled with slightly enlarged peak-to-peak separation.Inset in Fig.3B was the calibration plot of the peak current vs the scan rate. The redox peak currents linearly increased with the increase of the scan rates,confirming that this redox reaction of GOD was a surface-controlled electrochemical process,not a diffusion-controlled process[24-25].

2.4Detection of glucose

The amperometric response of the prepared biosensor to the target was investigated in various concentrations of glucose.As was proved,via the enzyme catalyzed reaction,the D-(+)-glucose could result in the reductive form of GOD(GOD-FADH2) according to the following chemical equations[26-27]:

GOD-FAD+2e+2H+?GOD-FADH2(1) Glucose+GOD-FAD→gluconolactone+GOD-FADH2(2) Therefore,when glucose was added,the electrocatalytic reaction(Eq.1)would be restrained by the enzyme catalyzed reaction(Eq.2).This directly induced the decrease of the GOD-FAD concentration, followed by the decrease of the reduction current.

Fig.4A was the typical cyclic voltammograms of the prepared biosensor in blank 0.1 mol·L-1PBS solution with the different concentrations of glucose from 0 to 10.0 mmol·L-1.With more glucose added to the PBS solution,the reduction current decreased,i.e., the higher glucose concentration caused the decrease of the reduction current.Fig.4B is the CV response calibration curve of the prepared biosensor against the concentrations of glucose.The calibration curve corresponding to the CV response is linear against the concentrations of glucose ranging from 1 to 10 mmol· L-1with the detection limit of 0.3 mmol·L-1(S/N=3).The regression equation is i(μA)=-12.94+0.85c(mmol·L-1) with the correlation coefficient(R)of 0.998.

The stability of the biosensor was investigated in 0.1 mol·L-1PBS.The relative standard deviations(RSD) were 4.1%for 10 successive assays in the presence of 1.0 mmol·L-1glucose,indicating that the enzyme electrode was stable in buffer solution.The fabrication reproducibility for four electrodes gave a RSD of 6.5% for CV determination at 1.0 mmol·L-1glucose.After storing at 4℃in the refrigerator for 10 days,the response to 1.0 mmol·L-1glucose retained 95.8%of its initial current,demonstrating good long-term stability.It can be attributed to the biocompatibility ofthe GO/Er2O3nanocomposite,which can provide an excellent microenvironment for GOD to retain its bioactivity

Fig.4(A)CVs of GOD/GO/Er2O3/GCE in PBS solution(0.1 mol·L-1,pH 7.0)with different glucose concentration; (B)Calibration plot of response current vs glucose concentration

3　Conclusions

In summary,the rare earth oxide of Er2O3was employed to form the nanocompositewith GO.SEM and XRD were used to characterize the prepared nanomaterials.The EIS and CV were used to check the electrochemical behaviors of different modified electrode.The results proved that the nanomaterials owned good electronical conductivity.The direct electrochemical properties of GOD/GO/Er2O3/GCE suggested that the Er2O3/GO supported matrix could effectively immobilize GOD onto the GCE,while still maintaining the excellent bioactivity.Detecting performance of the prepared biosensor towards the electrocatalytic oxidation to glucose revealed a wide linear range,good stability and reproducibility.On the basis of the above electrochemical measurements,the Er2O3nanomaterial,which owns good electronical conductivity and biocompatibility,exhibits great potential applications in the field of electrochemical biosensor.This provides the possibility of novel nanomaterials for the construction of electrochemical biosensor.Further work is still in progress to explore new rare earth elements nanomaterials for the electrochemical and biological applications.

References:

[1]Liu S Q,Chen H Y.J.Electroanal.Chem.,2002,528(1/2): 190-195

[2]Feng K J,Yang Y H,Wang Z J,et al.Talanta,2006,70(3): 561-565

[3]Zhang W,Yang T,Zhuang X M,et al.Biosens.Bioelectron., 2009,24(8):2417-2422

[4]Zhang J,Chen J H,Chen R C,et al.Biosens.Bioelectron., 2009,25(2):378-382

[5]Xiao X L,Luan Q F,Yao X,et al.Biosens.Bioelectron.,2009, 24(8):2447-2451

[6]Li Y,Gao Y F,Zhou Y,et al.J.Electroanal.Chem.,2010, 642(1):1-5

[7]Ansari A A,Kaushik A,Solanki P R,et al.Electrochem. Commun.,2008,10(9):1246-1249

[8]Kodaira C A,Lourenco A V S,Felinto M C F C.et al.J. Lumin.,2011,131(4):727-731

[9]Chinnu M K,Anan K V,Kumar R M,et al.Mater.Lett., 2013,113:170-173

[10]Wu M H,Lin T W,Huang M D,et al.Sens.Actuators B, 2010,146(1):342-348

[11]Novoselov K S,Geim A K,Morozov S V,et al.Science,2004, 306(5696):666-669

[12]Du X,Skachko L,Barker A,et al.Nat.Nanotechnol.,2008, 3:491-495

[13]Balandin A A,Ghosh S,Bao W,et al.Nano Lett.,2008,8 (3):902-907

[14]Wang W X,Ge L,Sun X M,et al.Mater.Inter.,2015,7(51): 28566-28575

[15]Zhang R Z,Sun C L,Lu Y J,et al.Anal.Chem.,2015,87 (24):12262-12269

[16]Liu J Y,Wang X H,Wang T S,et al.ACS Appl.Mater. Interface,2014,6(22):19997-20002

[17]Hummers W,Offeman R.J.Am.Chem.Soc.,1958,80(6): 1339-1339

[18]Wang Z J,Zhou X Z,Zhang J,et al.J.Phys.Chem.C, 2005,113(32):14071-14075

[19]Lambert T N,Chave C A,Sanch B H,et al.J.Phys.Chem. C,2009,113(46):19812-19823

[20]Bard A J,Faulkner L R.Electrochemical Methods:Fundamentals and Applications.New York:Wiley,1980.

[21]Deng S Y,Jian G Q,Lei J P,et al.Biosens.Bioelectron., 2009,25(2):373-377

[22]Zhang Y J,Shen Y F,Han D X,et al.Biosens.Bioelectron., 2007,23(3):438-443

[23]Zhou Y,Yang H,Chen H Y.Talanta,2008,76(2):419-423

[24]Ivanova E V,Magner E.Electrochem.Commun.,2005,7(4): 323-327

[25]Deng C Y,Chen J H,Chen X L,et al.Biosens.Bioelectron., 2008,23(8):1272-1277

[26]Yang L Q,Ren X L,Tang F Q,et al.Biosens.Bioelectron., 2009,25(4):889-895

[27]Liu S Q,Ju H X.Biosens.Bioelectron.,2003,19(3):177-183

Er2O3-Graphene Oxide Nanocomposite Supported Glucose Oxidase: Direct Electrochemistry and Biosensing to Glucose

HUANG Hai-Ping1,2XU Liang1YUE Ya-Feng1JIANG Li-Ping＊,2
(1School of Metallurgy and Chemical Engineering,Jiangxi University of Science and Technology,Ganzhou,Jiangxi 341000,China)
(2State Key Laboratory of Analytical Chemistry for Life Sciences,School of Chemistry& Chemical Engineering,Nanjing University,Nanjing 210093,China)

A new rare earth oxide of Er2O3was employed for the construction of glucose biosensor.Er2O3was mixed with graphene oxide(GO)to form the supported matrix for immobilization of glucose oxidase(GOD)onto the glassy carbon electrode(GCE).The nanomaterials of Er2O3and GO were firstly synthesized and characterized by SEM,XRD.The fabrication process for the biosensor was monitored by electrochemical impedance spectroscopy (EIS)and cyclic voltammetry(CV).The presence of Er2O3could effectively maintain the bioactivity of GOD and enhance the electron transfer rate.The prepared biosensor showed a pair of distinct and well-defined redox peaks,indicating the fast direct electron transfer(DET)rate between the redox-active site of GOD and GCE,which could be attributed to the synergistic effect of the GO/Er2O3nanocomposite.When employed to the electrocatalytic detection of glucose,the CV response of the prepared biosensor decreased against the concentrations of glucose. The calibration curve corresponding to the CV response was linear against the concentrations of glucose ranging from 1 to 10 mmol·L-1.Moreover,the biosensor showed good stability and reproducibility.

Er2O3;graphene oxide;glucose oxidase;biosensor

TB333

A

1001-4861(2016)11-2034-07

10.11862/CJIC.2016.268

2016-05-28。收修改稿日期：2016-09-30。

國家自然科學基金(No.21465013，21475057，21005034)、中國博士后科學基金(No.2014M551550)、江西省自然科學基金(No.20114BAB213014，GJJ13433)和江西理工大學清江青年英才支持計劃資助項目。

＊通信聯系人。E-mail：jianglp@nju.edu.cn