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Sensors and Actuators B 202(2014)820–826

Contents lists available at ScienceDirect

Sensors and Actuators B:

Chemical

j 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 /s n

Prussian blue nanocubes modi?ed graphite electrodes for the

electrochemical detection of various analytes with high performance

Lifang Liu,Lei Shi,Zhenyu Chu,Jingmeng Peng,Wanqin Jin ?

State Key Laboratory of Materials-Oriented Chemical Engineering,College of Chemistry and Chemical Engineering,Nanjing Tech University,5Xinmofan Road,Nanjing 210009,PR China

a r t i c l e i n f o Article history:

Received 15April 2014

Received in revised form 3June 2014Accepted 8June 2014

Available online 14June 2014Keywords:

Electrochemical biosensor Prussian blue

Graphite electrode High sensitivity

a b s t r a c t

In this work,the morphology-controlled Prussian blue (PB)?lm modi?ed graphite electrodes were suc-cessfully fabricated by the self-assembly approach.PB ?lms with nanocubic structures were obtained on the surface of graphite electrode under an optimal synthesis parameter of 40assembly cycles and a temperature of 35?C.On account of the produced functional groups of –COOH on the graphite surface,the interaction of –NH 2in enzymes with the –COOH would enhance the stability of the fabricated biosensors.Based on the proposed PB modi?ed graphite electrodes,excellent biosensors have been constructed for detecting glucose,lactate and glutamate with corresponding oxidases.As a result,high sensitivities of 127,238and 12.5mA M ?1cm ?2were observed in the detection of glucose,lactate and glutamate respec-tively,as well as excellent selectivity and stability.It is believed that the proposed PB modi?ed graphite electrodes would be great promising in constructing more sensitive and selective biosensors.

1.Introduction

Prussian blue (PB),regarded as the “arti?cial peroxidase”[1,2],has been extensively used as an electron transfer mediator due to its excellent electrocatalysis toward the reduction of hydrogen peroxide (H 2O 2)[3,4],which is a side product generated from an enzymatic reaction [5,6].Therefore,the development of PB based biosensors has attracted enormous interest in many aspects,e.g.the food and fermentation industries,physiological parameters,environmental monitoring and clinical medicine [5,7,8].

PB ?lms with controlled morphologies,especially consisting of nanocubic PB structures,are preferred to construct promising PB based electrochemical biosensors with excellent sensitivity and selectivity,because more catalytic active sites exist in the edges of proposed PB nanocubes [5,9,10].Several techniques including the electrochemical deposition,chemical synthesis and self-assembly approach have been introduced in the preparation of PB ?lms [11,12].Considering the advantages of precise controlling on the thickness and morphology of PB ?lms,the self-assembly approach has become one of the most attractive techniques to fabricate the PB ?lms [12].In addition to the preparation methods,differ-ent substrates have also been proved to play a critical role in the

?Corresponding author.Tel.:+862583172266;fax:+862583172292.E-mail address:wqjin@https://www.wendangku.net/doc/7110482436.html, (W.Jin).formation of PB ?lms.The noble metal foils,e.g.gold and plat-inum,are frequently served as the substrates for the fabrication of PB ?lms because of their excellent conductivity and stabil-ity [13–16].Considering that there exist no chemical interactions between the PB molecules and these ?at substrates,additional link-ing agents,e.g.Poly(diallyldimethylammonium chloride)(PDDA)and chitosan,are needed to enhance the interaction.Nevertheless,the poor conductivities of these linking agents inversely hinder the performance of constructed PB based biosensors [17–19].Recently,graphite has been widely applied in various ?elds due to its good conductivity,excellent stability and easy chemical modi?cation [16,20,21].Especially,due to its rough and porous structure,the graphite electrode could be served as a fascinating and alterna-tive substrate for the growth of PB ?lms in the absence of linking agents,in which the enhanced interaction between the PB ?lms and graphite substrates would be realized through the capillarity effect [22,23].Moreover,the ability of easy chemical modi?cation of the graphite will improve its potential in the application of biosens-ing.Up to now,few reports were focused on the construction of PB ?lms based biosensors on the graphite substrates,especially with the preferred structure of nanocubes.

In this work,we successfully fabricated the PB ?lms on graphite electrodes without introducing any linking agents by the facile self-assembly approach.Through controlling the prepara-tion parameters,PB nanocubes with a diameter of around 200nm were formed on graphite substrate.With the pretreatment of HNO 3,

https://www.wendangku.net/doc/7110482436.html,/10.1016/j.snb.2014.06.022

L.Liu et al./Sensors and Actuators B202(2014)820–826821

functional groups of–COOH were produced on the graphite surface, which facilitated the process of enzyme immobilization through the chemical reaction with–NH2groups of enzymes and further enhanced the stability of the fabricated biosensors.Based on the proposed PB modi?ed graphite electrodes,various biosensors were constructed for the detection of glucose,lactate and glutamate respectively,which showed excellent sensitivity,wide linear range, as well as good selectivity and stability.

2.Experimental

2.1.Materials and reagents

K4[Fe(CN)6]·3H2O,FeCl3·6H2O,glucose oxidase(GOD)from Aspergillus niger(E.C.1.1.3.4,180200U g?1),lactate oxidase(LOD) from Pediococcus sp.(EC1.13.12.4,20U mg?1)and glutamic oxi-dase(GMOD)from Streptomyces sp.(EC1.4.3.11,5U mg?1)were purchased from Sigma–Aldrich.l-glutamic acid,monosodium salt monohydrate and sodium l-lactate were bought from Alfa-Aesar. Glutaraldehyde25%(v/v)was obtained from Shanghai Lingfeng Chemical Reagent Co.Ltd.(China).Hydrogen peroxide(H2O2,30%, w/v,solution),glucose,uric acid and ascorbic acid were received from Sinopharm Chemical Reagent Co.Ltd.(China).Other chemi-cals employed were all of analytical grade and triple distilled water was used throughout.

2.2.Pretreatment of the graphite electrodes

The graphite electrode(geometrical area of ca.0.32cm2)was ?rstly polished with the metallographic abrasive paper for remov-ing the oxidation layer on the surface.Then it was treated with the ultrasonication for15min to suf?ciently exfoliate the powder gen-erated in the polishing.Subsequently,the electrode was dipped into a concentrated nitric acid for12h to produce the functional groups of–COOH on the surface,followed by thorough washing with the distilled water.Finally,the freshly pretreated graphite electrode was dried with nitrogen gas and served for the growth of PB?lms.

2.3.Fabrication of the PB modi?ed graphite electrodes

Two precursor solutions were prepared for the self-assembly of PB?lms.Solution A:0.01M K4[Fe(CN)6]+0.1M KCl+0.1M HCl. Solution B:0.01M FeCl3+0.1M KCl+0.1M HCl.During a typi-cal self-assembly process,the pretreated graphite electrode was sequentially dipped into solution A for60s,then distilled water 30s for cleaning,solution B for60s,and again distilled water30s for cleaning.This procedure containing four steps was denoted as an assembly cycle.To control the assembly temperature,a constant temperature water-bathing was employed here.The pre-cursor solutions,as well as the washing water,were poured into the beakers?rstly and then slowly heated to the desired temperature in the water-bathing.The PB modi?ed graphite electrode was dried at room temperature and the modi?ed electrode was denominated as the PB/G electrode.

2.4.Enzyme immobilization

For the immobilization of enzymes,the enzyme solutions con-taining0.05%glutaraldehyde(v/v)were prepared?rstly,in which 10mg/ml GOD,1U/?l LOD and1U/?l GMOD were included respec-tively.Then the prepared PB/G electrodes were immersed into the enzyme solutions for3h.Finally,the enzyme immobilized PB/G electrode was stored at4?C when not in

use.Fig.1.(a)CVs of PB/G electrodes with different assembly cycles in0.05M PBS containing0.1M KCl,the inset showed the corresponding PB amount formed on electrode surface.Scan rate of CV was0.05V s?1.(b)Calibration curves for H2O2 detection performed at PB/G electrodes with different assembly cycles in0.05M PBS containing0.1M KCl,the inset was the dependence of sensitivities on the assembly cycles.

2.5.Characterizations of the graphite surface and fabricated PB

?lms

The morphology of the graphite surface and PB?lms was observed on?eld emission scanning electron microscope(FESEM) (Hitachi,ModelS-4800II,Japan).The spectroscopy of functional groups on the pretreated graphite electrode was investigated with Fourier-transform infrared(FT-IR)(Thermo Electron,Nicolet-8700, USA).

2.6.Electrochemical measurements

All of electrochemical measurements were carried out on CHI 660C electrochemical workstation(Shanghai Chenhua Instrument Co.,Ltd.,China).The three-electrode system used consisting of a platinum wire(Pt)counter electrode,a saturated silver–silver chloride(Ag/AgCl)reference electrode and the modi?ed graphite working electrode.All electrochemical measurements were imple-mented in0.05M phosphate buffered saline(PBS,pH 6.5) containing0.1M KCl at25?C.

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Fig.2.FESEM images of PB ?lms fabricated at various temperatures of (a)25?C,(b)30?C,(c)35?C and (d)40?C.

3.Results and discussion

3.1.Growth of PB ?lms on the graphite surface

In our previous work,it was found that the self-assembly technique was an effective approach to control the structures of fabricated PB ?lms,in which the thickness of the ?lms could be adjusted with different assembly cycles.Meanwhile,the temper-ature involved in the assembly process also played a critical role in the morphology of prepared PB ?lms [24].Therefore,these two essential parameters were explored in details here.

On the one hand,the PB ?lms fabricated with different assem-bly cycles were investigated by the cyclic voltammetry (CV)[4],as shown in Fig.1a.Once the graphite surface was modi?ed with PB ?lms,a couple of well-de?ned redox peaks appeared com-pared with that of the bare graphite,which was attributed to the transformation between PB and Prussian White (PW).The peak currents obviously increased with assembly cycles ranging from 10to 40,while with additional cycles up to 50,only a subtle current increase was obtained.Meanwhile,increased peak separation E was observed with more assembly cycles,indicating the electron transfer slowed down due to the increased thickness of PB ?lms.As the surface coverage of the PB was dependent on the assembly cycles,it could be evaluated with the following equation: =

Q where Q is the total charge of a single peak,n the average elec-tron transfer calculated by 57/ E ,F the Faraday constant and A is the electrode area [10].The inset in Fig.1a showed the obtained PB surface coverage as a function of the assembly cycles.The sur-face coverage was calculated as 0.486,0.944,1.571,3.370and 3.408nmol cm ?2with the assembly cycles of 10,20,30,40,and 50respectively.Based on the fabricated PB/G electrodes,the detec-tion of H 2O 2was performed.Fig.1b showed the calibration curves

obtained for amperomeric detection of H 2O 2based on PB ?lms with various assembly cycles.It was found that with the cycles increased from 10to 40,an enhanced sensitivity was observed (shown in inset Fig.1b).While with cycles of 50,the obtained sensitivity decreased obviously,due to the increased electron transfer resistance [20].The results con?rmed that less coverage of PB ?lms usually could not provide enough catalytic sites and lead to the generation of weak response signals,while a higher coverage resulted in an increased electron transfer resistance,which severely hindered the acquisition of response signals [25,26].Therefore,optimal assembly cycles of 40were selected,in which excellent signal response was observed.

On the other hand,the effect of temperature on the morphology of fabricated PB/G electrodes was inspected [27].As shown in Fig.2,the FESEM images showed the morphology changes of PB ?lms fab-ricated at various temperatures,which were assembled with cycles of 40.At a low temperature of 25?C,the PB ?lm consisting of a mass of analogous spherical particles was formed on the surface (Fig.2a),while with an increased temperature to 30?C,a compact PB ?lm was produced and a changing from the sphere to nanocube in particle shape was observed (Fig.2b).At a temperature of 35?C,an integrated PB ?lm with perfect nanocubes on the surface was successfully obtained,and these nanocubes possessed a diameter of around 200nm (Fig.2c).However,when the temperature was further increased to 40?C,large PB nanoparticles were produced and similar structure of nanocubes was disappeared (Fig.2d).The results demonstrated that the temperature played a critical role in the morphology of the fabricated PB ?lms,and the PB structure with nanocubes was tended to form at an optimal temperature of 35?C.

The PB/G electrodes fabricated at different temperatures showed distinct responses in H 2O 2detection.As shown in Fig.3,the sensitivities increased with the temperatures ranging from 25to 35?C,while a sharp decrease was observed on the PB/G fab-ricated at 40?C.The related sensitivities of ca.70,79,128and

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Fig.3.Calibration curves for H2O2detection in0.05M PBS containing0.1M KCl, which was performed on PB/G electrodes fabricated at different temperatures,the inset showed the dependence of sensitivities on the temperature.

66mA M?1cm?2were observed on PB/G electrodes prepared at temperatures of25,30,35and40?C respectively.As expected,a high electrocatalytic performance was obtained on the PB/G elec-trode fabricated at35?C,which was attributed to the integrated PB ?lm with cubic nanostructures[24].Therefore,an optimized condi-tion of assembly cycles of40at35?C was selected in the preparation of PB/G electrode.

Moreover,because no linking agents were used in the fabri-cation of PB/G electrode,the stability of the PB/G electrode was investigated here[4,28].The PB/G electrode prepared with assem-bly cycles of40and a temperature of35?C was consecutively scanned in the PBS and the corresponding signal responses were recorded in CVs.As shown in Fig.4a,it was found that a subtle decrease(ca.3.58%)in the peak current was observed even after the PB/G electrode was scanned after100cycles.The result showed an excellent stability was obtained on the PB/G electrode,due to the rough and porous surface of graphite shown in Fig.4b.The fab-ricated PB/G electrodes possessed a high electrocatalysis activity towards H2O2and an excellent stability,which would make them promising candidates in the construction of sensitive and stable biosensors.

3.2.Characterization of the enzyme modi?ed PB/G electrodes

FTIR was utilized to investigate the conformation variations of functional groups on the graphite[29,30].Fig.5displayed

the

Fig.5.FTIR spectra of crude and pretreated graphite electrodes.

FTIR spectra of crude graphite and pretreated graphite,which showed some distinct absorption bands due to produced groups on the pretreated graphite https://www.wendangku.net/doc/7110482436.html,pared with the crude graphite,the obviously exhibited band at around1650cm?1of pre-treated graphite corresponded to the stretching vibration of the –COO?,con?rming that abundant–COOH groups were formed.The present–COOH groups on the graphite could facilitate the covalent immobilization of enzyme and further enhance the stability of the fabricated biosensors.

The electrochemical behaviors of the PB/G electrodes before/after the immobilization of enzymes were investigated by CVs.In the case of the GOD,CVs of the PB/G and GOD/PB/G electrodes were tested,as shown in Fig.6.After the enzyme was immobilized on the PB/G electrode,it showed an obvious decline in peak current and a slight increase in E of0.071V compared to0.067V of the PB/G electrode.This may be ascribed to the poor conductivity of enzyme layer,which hindered the electron transfer towards the electrode surface and blocked the penetration of the counterion(K+)in the electrochemical reaction.In addition, the investigations on LOD and GMOD showed similar results,not shown here.

3.3.Performance of the enzyme immobilized PB/G electrodes

Different enzymes were immobilized on the PB/G electrodes, which were served for the detection of glucose,glutamate and lactate.The calibration curves of the fabricated biosensors for amperometric detection of glucose,glutamate and lactate

were

Fig.4.(a)Stability tests of the PB/G electrode after scanning in0.05M PBS containing0.1M KCl for100cycles.Scan rate of CV was0.05V s?1.(b)FESEM image of the bare graphite electrode.

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Table 1

Performance comparisons of our glucose,lactate and glutamate biosensors with those reported in the literatures.Analytes Functional electrodes

Potential (V vs.Ag/AgCl)Sensitivity (?A M ?1cm ?2)Linear range (mM)Reference Glucose

GA/PB/graphite ?0.051270.01–1.00This work GA/PB/Pt ?0.0580±40.01–1.00[11]GS/PB/Pt

?0.130.63NA

[31]Na?on/PB/graphite ?0.05 1.590.18–1.1[21]Cytc/GNPs/PAN/GCE

?0.263.10.01–3.2[32]BMIM-PF6-(SAMN@RITC-GO)/CPE ?0.145.850–1.5[33]CuNWs-MWCNs/GCE

?0.55 1.9950–3

[34]Glutamate

GA-GOD/PB/graphite ?0.052380.01–0.1This work GS-GMOD/PB/Pt

?0.112.36NA [31]PPD-BSA/PEA/PEI/PPD-BSA/PtD NA 71NA

[35]cMWCNT/AuNPs/CHIT 0.135155

0.005–0.5

[36]VACNT-NEA 0 2.2×10?30.01×10?3–20×10?3[37]CHIT-GDI 0.6100NA

[38]GA/SiO 2/PCB 0.696–1000.2–2.5[39]EDC/TGA

NA 20.750.0001–10[40]Lactate

GA-GOD/PB/graphite ?0.0512.50.01–0.1This work GS-LOD/PB/Pt

?0.1 4.5NA [31]Mucin/Albumin/Glu 0.6511.18NA [41]Hydrogel

0.4326NA

[42]Fe 3O 4/MWNTs/LDH/NAD +/GCE 07.670.005–0.5[43]LOD-FSM8.0/Naf/CoPC-SPCE 0.45 4.540.018–1.5[35]PB/gel membranes/alkoxysilane

0.18

0.001–10

[44]

Fig.6.CVs of PB/G and GOD/PB/G electrodes in 0.05M PBS containing 0.1M KCl.Scan rate of CV was 0.05V s ?1

Fig.7.Linear calibration curves for the current responses of prepared glucose,lac-tate and glutamate biosensors respectively.

shown in Fig.7,in which high sensitivities were calculated to be around 127,238and 12.5mA M ?1cm ?2respectively.In addition,wide linear ranges from 0.01to 1.2mM in glucose,0.01to 0.1mM in lactate and 0.01to 0.1mM in glutamate were obtained.The sen-sitivities of the prepared biosensors were signi?cantly higher than those reported in literatures,as listed in Table 1.Moreover,the response time was very short (within 3s)following the addition of analytes.

3.4.Anti-interference ability,reproducibility and stability of the enzyme immobilized PB/G electrodes

As the most common electrochemical interfering species,the ascorbic acid (AA)and uric acid (UA)were introduced to inves-tigate the anti-interference ability of enzyme immobilized PB/G electrodes.As shown in Fig.8,the current responses showed

Fig.8.The interference of AA and UA on the responses of the glucose,lactate and glutamate biosensors in 0.05M PBS containing 0.1M KCl.

L.Liu et al./Sensors and Actuators B202(2014)820–826825

obvious changes when0.1mM AA and0.1mM UA were added into the detection system,which were mainly attributed to the low operation potential of?0.05V(vs.Ag/AgCl)and the barrier effect of the enzyme layer.The results indicated that the proposed biosensors possessed good anti-interference ability.

Meanwhile,the reproducibility of the biosensors was evaluated with parallel experiments,in which?fteen electrodes were used in each experiment.The relative standard deviation(RSD)of4.1%,5.2% and5.6%in sensitivities were observed in the detection of glucose, lactate and glutamate respectively,indicating that the proposed enzyme biosensors possessed satisfactory reproducibility.

Moreover,the enzyme immobilized PB/G electrodes were?rstly stored in the refrigerator at4?C for3weeks and then exam-ined after adding the analytes.Based on the enhanced interactions between the graphite substrate and enzymes,the results of the experiments showed that the fabricated biosensors retained about 87%,82%and84%of their initial sensitivities in glucose,lactate and glutamate respectively,indicating an excellent stability of the fabricated biosensors.As comparisons,the crude graphite elec-trodes were also served in the construction of various biosensors. The results of stability tests exhibited that severe decreases in the responses were observed,in which only69%,65%and68%of their initial sensitivities in glucose,lactate and glutamate were obtained.

4.Conclusions

In this work,the PB?lm with nanocubic structures was suc-cessfully fabricated on graphite electrodes without introducing any linking agents by the self-assembly method.The PB/G elec-trode showed an excellent electrocatalysis and good stability.The additional functional groups of–COOH on the graphite surface facil-itated the process of enzyme immobilization and further enhanced the stability of the fabricated biosensors.Based on the proposed PB/G electrodes,various biosensors were constructed for the detec-tion of glucose,lactate and glutamate respectively,which showed excellent sensitivity,wide linear range response,good selectivity and stability.We hope that the proposed PB/G electrode would have a great promise in constructing more sensitive and selective biosensors.

Acknowledgements

This work was supported by the Innovative Research Team Program by the Ministry of Education of China(No.IRT13070), the Doctoral Fund of Ministry of Education of China(No. 20113221110001)and A Project Funded the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Biographies

Wanqin Jin is Professor of chemical engineering at Nanjing Tech University and currently researches on mixed-conducting membranes for oxygen separation and catalytic membrane reactors,organic/ceramic composite membranes for pervapor-ation,and biosensors.He was an Alexander von Humboldt Research Fellow(2001), and a visiting Professor at Arizona State University(2007)and Hiroshima University (2011,JSPS invitation fellowship).He has published over130internationally refer-eed journal papers and edited a book on materials-oriented chemical engineering. He serves as an editorial board member for several journals and is a council member of the Aseanian Membrane Society.

Lifang Liu received her B.Sc.degree in chemical engineering from the Department of Chemistry and Chemical Engineering,Qiqihar University,China in2010.Currently she is undertaking the master research at Nanjing Tech University.

Lei Shi received his B.Sc.degree in chemical engineering from the Department of Chemistry and Chemical Engineering,Nanjing Tech University,China in2010.He is currently working towards the Ph.D.degree at the same university.

Zhenyu Chu received his B.Sc.and Ph.D.degree in chemical engineering from the Department of Chemistry and Chemical Engineering,Nanjing Tech University,China in2008and2013.He studied in Institute of Technology Tallaght,Dublin,Ireland for 3months as a collaborative visitor in2011.He is now a lecturer at Nanjing Tech University.

Jingmeng Peng received her B.Sc.degree from the Department of Chemistry and Chemical Engineering,Changchun University of Science and Technology,China in 2012.She is undertaking the master research at Nanjing Tech University.