Journal of Industrial and Engineering Chemistry 16 (2010) 340–343

Amperometric study of hydrogen peroxide biosensor with butadiene rubberas immobilization matrix

Beom-Gyu Lee a, Keun-Bae Rhyu b, Kil-Joong Yoon b,*a Department of Chemistry, Chosun University, Kwangju 501-759, Republic of Koreab Division of Applied Sciences, Cheongju University, Cheongju 360-764, Republic of Korea

A R T I C L E I N F O

Article history:

Received 23 April 2009

Accepted 13 August 2009

Keywords:

Butadiene rubber

Biosensor

Hydrogen peroxide

Peroxidase

Enzyme electrode

A B S T R A C T

A carbon paste electrode bound by butadiene rubber has been newly constructed and its electrochemical

properties have been investigated to test the practicability of the enzyme electrode. The binder of carbon

powder was butadiene rubber dissolved in toluene and ground cabbage tissue was embedded in the

matrix as an enzyme source. The electrode, which showed a mechanical robustness after volatilization of

solvent, displayed good catalytic power (detection limit = 2.5 � 10�5 M, S/N = 2) and electrochemically

irreversible characteristics. Its symmetry factor and the exchange current density of the electrode used

were 0.23 and 1.71 � 10�3 A cm�2, respectively.

� 2010 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V.

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1. Introduction

It is essential to immobilize an enzyme on the surface of thebiosensor so that it can completely preserve its catalytic power andspecificity when one is trying to construct a biosensor and put it topractical use. This is due to an enzyme having the strikingcharacteristic of accelerating reactions by a factor of at least amillion, even in the case of lower temperature. A lot of methods havebeen introduced using mainly physical adsorption of an enzyme [1],a covalent bond with functional polymer [2], or polymer film forimmobilizing an enzyme [3]. Also, the peculiar method, in which thesource of enzyme is mixed with electrode material or doped into asol–gel matrix [4], has been developed intensively up to the present.All the methods mentioned above should go through very tediousphysical and chemical processes [5]. Those methodologies arelengthy processes, and therefore both costly and time-consumingfor fabrication. In order to cut down on this ineffectiveness,electrodes using mineral oil for the binder of carbon powder hadbeen designed and their electrochemical properties were studied inthis lab [6]. Even now, this is very useful in the study of enzymecharacteristics. However, since this cannot guarantee the mechani-cal robustness of the electrode, it is very far from being ready forpractical use. In order to solve this problem with mineral oil, wehave tried to find any binder which ensures the mechanical propertyof the enzyme electrode. We have confirmed that carbon paste

* Corresponding author. Tel.: +82 43 229 8540; fax: +82 43 229 8535.

E-mail address: kjyoon@cju.ac.kr (K.J. Yoon).

1226-086X/$ – see front matter � 2010 The Korean Society of Industrial and Engineer

doi:10.1016/j.jiec.2010.01.016

exerts such mechanical stability once the solvent has completelyescaped from the rubber solution after fabrication of electrode.These efforts led us to construct a lot of rubber electrodes usingvarious kinds of rubbers such as ethylene propylene dieneterpolymer [7], polybutadiene rubber [8], butyl rubber [9], andchloroprene rubber [10] as binders. Their usefulness was investi-gated and reported in journals several times. It is well known thatbutadiene rubber with sterical regularity is excellent in the aspect offlexibility and resistance to abrasion since it has no branch. Also, it isknown that butadiene rubber has excellent dynamic properties andis good for its filling ability and compatibility [11]. Therefore, weexpected that such characteristics might satisfy the mechanicalrobustness, which is a precondition for a practicable enzymeelectrode, and made the electrode using BDR as binder. On the otherhand, the peroxidase (Sigma, E.C.1.11.1.7) extracted from thehorseradish was used as an enzyme source in the previous work. It isknown that cabbage, being in the same family as cruciferae, containslots of peroxidase. The ground cabbage tissue as an enzyme sourcewas mixed directly with carbon paste in this system for economicreasons. In this study, the electrochemical properties of the enzymeelectrode made in this way was inspected, and the result is reportedin this paper.

2. Experiments

2.1. Materials

Butadiene rubber (abbr. BDR, Kumho petrochemical BR-01) wasused as a binder and the source of enzymes was the ground root

ing Chemistry. Published by Elsevier B.V. All rights reserved.

B.-G. Lee et al. / Journal of Industrial and Engineering Chemistry 16 (2010) 340–343 341

tissue of cabbage. Toluene and graphite powder were purchasedfrom Sigma–Aldrich (�99.9%) and from Fluka (�0.1 mm) respec-tively. Hydrogen peroxide (Junsei, EP, 35%) for substrate (abbr. S),NaCl (Shinyo pure Chem., �99.5%) for electrolyte, and ferrocene(Sigma) for the mediator to increase and stabilize the signal, wereused.

2.2. General procedures

After dissolving 0.09 g of ferrocene in 10 mL of CHCl3, 0.91 g ofthe graphite powder was added and then dried. By mixing 1.0 g ofthe produced graphite powder with the solution of BDR solution(5.0%) at a 1:1 ratio (wt/wt), carbon paste was made. 1 g of thispaste was completely mixed with 0.1 g of the ground cabbage root.The biosensor was constructed by packing this paste into a 6 mmi.d. and 1 mm depth polyethylene tube having ohmic contact. Itwas smoothed by friction on a spatula to make a flat workingsurface. The cyclic voltammograms were obtained in the states ofboth the unstirred and the stirred solution by the placement of theworking electrode. The current response to the step-excitation wasobtained as follows. When the decreasing tendency of thecondenser current keeps horizontal after applying the steppotential on the working electrode, substrate solution is addedin 10 mL of 0.1 M NaCl solution. Then the current differencebetween before and after adding the substrate was considered tobe the decomposition current of hydrogen peroxide. Ag/AgCl(BASMF2052) and Pt electrode (BAS MW 1032) were used for referenceand for auxiliary electrodes, respectively. The enzyme electrodewas connected to a BAS Model EPSILON (Bioanalytical System, Inc.,USA) to obtain cyclic voltammograms. The other amperometricmeasurements were performed with EG&G Model 362 potentio-stat (Princeton Applied Research, USA). Its output was recorded ona Kipp & Zonen x-t strip chart recorder (BD111, Holland).

3. Results and discussion

Fig. 1 shows two different linear sweep voltammograms (LSV).A is one obtained in 0.1 M NaCl electrolytic solution and B, in 0.1 MNaCl solution which contains 1.0 � 10�3 M hydrogen peroxide. Aconsists of two kinds of reduction currents which show differentincreasing tendencies with electrode potential. One is increasinglinearly below at�1.5 V and the other abruptly increasing above at�1.5 V. A glassy carbon electrode placed in 0.1 M NaCl electrolyticsolution did not show any current in this range of potential [10].

Fig. 1. The linear sweep voltammograms of the BDR-bound biosensor in the

unstirred 0.1 M NaCl electrolytic solution in the absence (A) and in the presence (B)

of 1.0 � 10�3 M H2O2. Initial potential: 0.0 V; scan rate: 0.1 V/s.

The standard reduction potential of ferricinium ion is +0.400 V. It isprobable that the tail of the reduction function of ferriciniumwould not have that much influence in the front part of theexperimental range. During the process of electrolysis at �1.5 V orhigher, one may observe air bubbles formed on the electrodesurface by the naked eye. The standard reduction potential ofwater is �0.828 V. However, expecting the overpotential to becaused by a rubber component, the current which is increasingsteeply above at �1.5 V may be considered to be the reductioncurrent of water. BDR is a mixture composed of various chemicalcomponents even though their electrochemical behavior has notbeen elucidated here yet. Current occurring below at �1.5 V inFig. 1 can be viewed as the reduction current of components ofBDR. In Fig. 1, A is deformed to B by adding substrate into theelectrolytic solution. Since this comes from the addition of H2O2

under the same conditions as A, it is clear that the reductioncurrent produced by the decomposition of substrate created thisdeformation. The current difference between A and B is plottedwith the applied electrode potential in Fig. 2. It is roughlysymmetric. On the assumption that the reduction function of H2O2

has Gaussian distribution, the pictorial representation of thesimulated result is a solid line. In the case of the completereversible system, irev

p depends on the scan rate but Ep isindependent of this. However, for the irreversible case, bothiirrev

p and Ep (peak potential) depend on v1=2. In other words, Ep

moves into the forward direction as v increases in the reductionreaction. The dependence of Ep on the scan rate in Fig. 3 tells usthat the system is irreversible. The reaction between ferrous ionand hydrogen peroxide occurring in the organism is as follows:

Fe2þ þH2O2 ! Fe3þ þOH� þOH�

and is one electron transfer reaction (n = 1) [12]. In the case of theirreversible system, kinetic parameters (i0, a, etc.) of the electrodereaction may be obtained in the Tafel area. The relation betweenoverpotential, E and ln{(il,c � i)/i} is linear and its slope andintercept are RT/anF and RT/anF(lni0/il,c) respectively.

Peak potential(Ep) representing the maximum current in Fig. 2is �1.047 V. The plot of E vs. ln{(il,c � i)/i}obtained by considering0.349 mA at�1.047 V as il,c is given in Fig. 4. The values of the slopeand intercept worked out from Fig. 4 are 0.112 and �0.596,respectively. Making efficient use of those values, the calculatedvalues of the symmetric factor (a) and exchange current density(i0) are 0.23 and 1.71 � 10�3 A cm�2, respectively. The value ofjEp � Ep/2j is 47.7/an (mV) when the system is irreversible.

Fig. 2. Current difference between A and B in Fig. 1. Solid line is a Gaussian

distribution (y = yo + (A/(w � sqrt(pi/2)))*exp(-2*((x-xc)/w)^2, yo: �0.884, xc:

�1047, w: 1635, A: 2527) after performing the fit.

Fig. 3. Dependence of the peak voltage on the scan rate in 2.0 � 10�2 M H2O2

solution.Fig. 5. Cyclic voltammograms in the stirred 0.1 M NaCl in the absence (A) and in the

presence (B) of substrate (1.0 � 10�3 M H2O2). Scan rate: 100 mV/s.

B.-G. Lee et al. / Journal of Industrial and Engineering Chemistry 16 (2010) 340–343342

Substituting the values obtained above for n and a, jEp � Ep/2jbecomes, 455 mV. This indicates that this system is an irreversibleone. Cyclic voltammograms of the enzyme electrode under stirringare given in Fig. 5. A represents the case for the electrolyticsolution without substrate and B represents that for theelectrolytic solution with addition of 0.1 M H2O2 100 mL to10 mL. Excepting the intensity of current, A and B are notconspicuously different from A in Fig. 1.

Plotting the current difference between A and B above at about�500 mV shows linearly increasing tendency with electrodepotential. This phenomenon is in contrast to that in Fig. 2, wherethe plot shows a peak current. There are various tendencies in thedependence of the reduction current of H2O2 on electrodepotential: e.c. linear [7,8], monotonous [13] increasing withelectrode potential or irrelevance to [14]. But the theoretical basisto explain the causes of the different tendencies has still not beenqualitatively established. Table 1 displays the changes (imax and t)of current with time when step potential is applied. As we can seein Fig. 2, this step potential is �1.0 V, which is sufficient todecompose H2O2. Regardless of stirring, Imax is smaller in thepresence of substrate. This says that the substrate has influence onthe migration of electrolytes and checks the rapidity with whichelectrolytes migrate as well. Also, one may observe that the timeconstant (t) increases with the addition of substrate. This gives theimpression that the decomposition current of the substrate makes

Fig. 4. Wave slope plot for the reduction of 1.0 � 10�3 M H2O2 in 0.1 M NaCl.

a contribution to the residual current. In the double reciprocal plotof the signal and the substrate concentration, if one obtains thelinear graphic, then it may be considered that the reaction is due tocatalysis by enzyme. The plot was a linear graphic with the equationthat y = A + Bx(A: 2.89� 106, B: 1.90� 103 mM/Na, R = 0.999).

This means that the peroxidase of cabbage tissue immobilizedby BDR discharges the role of catalyst normally on the electrodesurface and proves that BDR is a suitable binder for theimmobilization of peroxidase. Fig. 6 shows the typical current-time recording of the BDR-modified electrode upon addition ofH2O2 at a constant potential under stirring.

Immediately after applying an electrode potential in the absenceof substrate under constant convection, the abrupt shooting of thedouble layer charging current decreased rapidly with time andgradually approached its horizontal value in about 50 s. When H2O2

was added, the signal current rose steeply and then stabilized. Thesignal reached 90% of the maximum response in about 7�8 s. Thisspeedy appearance of the signal indicates that the root tissue ofcabbage has been successfully immobilized on the surface of thesensor and again BDR is a promising binder of carbon powder. Thedetection limit of the electrode used in Fig. 6 was 2.5� 10�5 M. Thisvalue is superior to that of Diaz et al. (6.7� 10�4 M) [15] but inferiorto that of Miao et al. (1.0� 10�6 M) [16]. If the factors which affectthe magnitude of signal, content of mediation and tissue, pH ofsolution, temperature, and the others are optimized properly, thenthe detection limit can be further improved even more. But J. Wanget al. [17] obtained 6.42� 10�8 M as detection limit using thechemiluminescence spectroscopic method and Tang et al. [18],3.89� 10�10 M based on fluorescence spectroscopic method. If onlythe detection limit is being considered, then voltammetry cannotcompete with the spectroscopic method. However, this electrodecan be used in practice and with repetition. In the event that morestudy for the improvement of the detection limit is carried outtogether, it is certain that the profits from the synthesis of the wholesituation in voltammetry using this biosensor will surpass thespectroscopic methods.

Table 1Double layer characteristics of the biosensor used in this work in 0.1 M NaCl. Step

potential: �0.80 V (vs. Ag/AgCl); [H2O2]: 5.0�10�4 M.

Substrate Technique

Unstirred Stirred

Without 7.73/4.46 7.79/4.37 imax (mA)/t (s)

With 7.69/4.50 7.70/4.39

Fig. 6. Amperogram obtained using BDR-bound biosensor in the stirred 10 mL of

0.1 M NaCl solution. 0.01 M H2O2 25 mL was added at �1.00 V (vs. Ag/AgCl).

B.-G. Lee et al. / Journal of Industrial and Engineering Chemistry 16 (2010) 340–343 343

4. Conclusion

The experimental results presented here prove that BDR is arecommendable binder for the development of carbon pasteelectrode that can be used repeatedly. This biosensor is currentlyinferior to the spectroscopic method in the aspect of detectionlimit. But the physical and chemical process of spectroscopicmethodologies are both time-consuming and costly. The presentstudy reports its attractive performance characteristics, such assimple construction, low cost, and suitability for mass production.If further study for the improvement of the detection limit is

carried out, then the advantages from this enzyme electrode maycompensate for the drawbacks which the voltammetries have incomparison with spectroscopic methods.

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