Notes Bull.Korean Chem. Soc. 2009, Vol. 30, No. 10 2457
Electrochemical Investigation of Carbon Paste Biosensor Bound with Natural Rubber Solution
Beom-GyuLee, Keun-Bae Rhyu「andKil-Joong Yoon'，*
Department of Chemistry, Chosun University, Kwangju501-759,Korea
‘Division ofApplied Sciences, Cheongju University, Cheongju360-764,Korea. *E-mail: kjyoon@cju.ac.kr Received March 10, 2009, Accepted August 24, 2009
KeyWords: Biosensor,Peroxide,Peroxidase,Carbon paste electrode,Naturalrubber
Therate of reactionscatalyzed using enzymes is more than a milliontimes higher compared to cases withoutcatalysis.
Therehavebeen a lot of effortstomake andput touse a bio sensor usingthis catalytic specificity of enzymes.Here,itis very important to immobilize anenzyme to keep itsspecificity on thesurface of the biosensor. In order to do this, many methods have been developed such as using the physical adsorption property of the enzyme,1 the covalent bonding ability of the enzyme with a functionalpolymer,2 the polymer film3 and the immobilization of the enzyme in a mixture of electrode ma terial, especially, sol-gel, withthe enzyme source.4,5 Inthis lab, variousgraphite electrodes using mineraloilas a binder have been made and their electrochemical characteristics have been studied.6 This methodachievesvery high efficiency in theaspects of cost and time for makingthebiosensor com pared withthepreviouslymentionedmethodssince it doesnot require tedious physical and/or chemical procedures. This method is still used efficiently in the field of research about characteristics of enzymes.So that the binder may be of much use in actual life, the mechanical stability of the electrode should be guaranteed.However wecannotexpect such stability frommineral oil. We had been tryingtofind a binderof graphite to ensure the mechanical property in order to obviate this inadequacy of mineral oil. Theinquiry revealed a surprising fact that the rubber liquefied in toluene shows mechanical stabilityifthesolventis volatilized duringthe finalstage of makingthe electrode. Asa result, electrodes using various kinds of rubberssuch as ethylenepropylene diene terpolymer,7 polybutadienerubber,8 butylrubber9andchloroprenerubber10 have beenmade and theirapplicability has beenstudiedand reported in related research. Research activities revolving around thisareincreasingatthistime.
The naturalrubber molecules consist virtually entirely of cis-1,4-polyisoprene which has no evidence for any trans material or for any 1,2- or 3,4-isoprene polymer, in contrast to the synthetic polyisoprenes. It is known that natural rubber has abetter shelf life than synthetics and has a highwet gel strengthevenatlowtemperatures.11 Italso attainsthe excellent characteristics of membrane strength and elasticity, which cannot be expected from synthetics. Expecting that those characteristics shouldsatisfythe pre-requisite conditions for application,wehave constructed an enzymeelectrode for the determination of hydrogen peroxide. In this context, wereport its details here.
Experiments
Reagents and Measurements. The ground root tissue of cabbagewas used as a source of enzymes and natural rubber (NR, RSS#1,Mooney viscosity; 61, density;0.92) was a pro duct of Malaysia. Toluene and graphitepowder werepurchased from Sigma-Aldrich (> 99.9%) and from Fluka (< 0.1 mm) respectively.Hydrogen peroxide (Junsei, EP,35%) forsubstrate (abbr. S), NaCl(Shinyopure Chem. >99.5%) for electrolyte and ferrocene (Sigma)for the mediator toincrease and stabilize thesignal were used. Ag/AgCl(BASMF2052) and Ptelectrode (BAS MW 1032) were used for the referenceand for auxiliary electrodes, respectively. The enzymeelectrode was connected to aBAS ModelEPSILON (Bioanalytical System, Inc., U.S. A.) to obtaincyclic voltammograms. The other amperometric mea
surementswere performedwithEG&G Model 362 potentio stat (Princeton Applied Resaerch, U. S. A.). Its output was recorded on aKipp & Zonen x-tstrip chart recorder(BD111, Holland).Origin7wasutilized for allthecalculations.
Constuctionof EnzymeElectrode. After dissolving0.09 g of ferrocene in 10 mLofCHCls, 0.91 g ofthegraphitepowder wasadded and then dried.By mixing 1.0 g of theproduced graphite powder with thesolution of naturalrubber (1.5%) at a 1 : 1 ratio (wt/wt), carbon paste wasmade. 1 gof thispaste wascompletelymixed with 0.1 g of theground cabbage root.
The biosensor wasdesigned bypacking the paste into a 6mm i. d. and 2mm depthpolyethylene tube having ohmiccontact.
Itwassmoothed by frictionon a spatulato make a flat wor
king surface.Thecyclic voltammograms were obtainedinthe states of boththe unstirred and stirredsolution by the place
ment of the working electrode. Amperometric current was obtained as follows. When the decreasing tendency of the charging current keeps horizontal after applying the step potential onthe working electrode,substratesolution is added in 10 mL 0.1 M NaCl solution. Then the current difference between before and after addingthe solution wasconsidered tobethedecomposition current of hydrogen peroxide.
Results and Discussion
Figure 1 shows the comparison of the cyclic voltammo- grams before(a) and after(b) adding substrate H2O2 in the system. It showsthe increase of the reduction current accor
ding to theelectrode potential increases in anegativedirection
2458 Bull.KoreanChem. Soc. 2009, Vol. 30, No. 10 Notes in the electrolytic solution when it does not contain the
substrate (a-1). Inthecase of aglassycarbonelectrode,this system does not yieldanyreductioncurrentinthis potential range.10 However,wemay take various electrochemical reac
tions intoour consideration since the enzymeelectrode which is used in thisworkcontainsmediator,ferrocene,electrolytic solvent, andrubber. Firstof all, with ferroceneas a mediator, the standard reduction potential of the ferricinium ion was 0.400 V.It seems that theinfluence of thereduction function of the ferricinium ion is not great in the experimental rangeof potential. Also, we note that the current increases rapidly above -1.5 V. The standard reduction electrode of water is -0.828 V. However,wemay expect that the enzyme electrode will undergo large overvoltage compared with the Pt elec-
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Figune 1. Comparison of cyclic voltammogram without (a) and with (b) adding substrate (0.1 M H2O2 100 pL) in an unstirred 10.0 mL of 0.1 M NaCl solution. 1 and 2 are the direction of the forward and the reverse scan respectively. Initial potential: 0.0 V, switching potential:
-1.8 V, scan rate: 100 mV/sec.
trode even though wecannot explainthe precise cause. The reason is conjectured as follows. Since we can see the air bubbleswith thenakedeye duringelectrolysis at above-1.5 V, it would be no problem to consider the highcurrent at above -1.5 V asthecurrent of reduction of water.Rubber is a complex mixture with various components. Therefore, the linearly increasing currentwith electrode potential can be attributed to electrochemical reactionsamong thecomponents ofrubber. It can be seen that a-1 is deformed to b-1 after adding thesubstrate, H2O2, in theelectrolytic solution. Since thischangecomes fromthe addition of H2O2 while maintain
ing the sameconditions, one may consider itto be the con
tribution of thereductioncurrent of H2O2.Figure 2 shows the variation in the differencebetween a-1 andb-1. The graphic of current difference is approximately symmetric and the normalized result is represented in a solid line. The peak potential (Ep), which represents the maximum current, is -1.029 V. Inthecasewherethe system is completely rever
sible, Ep has no correlation with the scan speed and irpev increases depending onthe scan speed. However, whenthe system is irreversible, iiprrev is relevanttov1/2 but the peakpoten- tial(Ep), which is the functionofpotentialscan speed, changes to the negative(-) direction as v increases for the reduction reaction.Figure3 is the plot of dependenceofEp accordingto changes in scanspeed and showsclearlythetendency of the previously mentioned irreversible system. Theelectron transfer reaction of ferrous ion and hydrogenperoxide, which happens in bio-organisms, is Fe2+ + H2O2 — Fe3+ + OH- + OH* and is oneelectrontransfer reaction (n = 1).12-14 In theirreversible system, one may obtain thekineticparameters (i。，a, etc.) in theTafel area wherethe overvoltage is relativelyhigh. In other words, the relation between the overvoltage E and ln(i1,c -i)/i}
is linear and theslope and intercept areRT/anFand (RT/anF) ln(i0/i1,c), respectively. The plot of E vs. ln((i1,c - i)/i} is in Figure4 for thecase of 0.427mA obtained at -1.029V as i1,c. Here 0.125 and-0.528are obtained as thevalues of slope(RT/
anF) and intercept (RT/anF)ln(ic/i1,c) respectively. Fromthese wecan get 0.21 and 5.62 x 10-3Acm-2 as thevalues of a and i0, respectively.
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Figure 2. Calculated current difference between b-1 and a-1 with applied electrode potential in Figure 1. Solid line: y = y0 + (A/
(w*sqrt(pi/2)))*exp(-2*((x -xc)/w)A2), y0: -0.347, xc: -1029, w:
1267, A: 1230.
0 -200 -400 -600 -800 -1000 -1200 -1400 -1600-1800
Potential (mV)
Figune 3. Variation of peak potential with scan speed in 2.0 乂 10-2 M H2O2 solution.
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Notes Bull.Korean Chem. Soc. 2009, Vol. 30, No. 10 2459 In theirreversiblesystem, |Ep 一 Ep/2| =47.7/an (mV).Substi
tuting the obtainedvalues for n and a, then |Ep - Ep/2| = 239 (mV). Sincethisis smaller than the551 (mV) obtained from Figure 2, wecanhypothesizethat this system is irreversible.
Figure 5 shows twocyclicvoltammograms of enzyme elec trodes in thestirredsolution. Theadvantage of thishydrody namic amperometry is that condenser current doesn't enter into the measurementsbecause the rate ofmasstransfer is much larger than that ofdiffusion. So that therelative contribution of the effect of mass transfer to electron transfer kinetic is smaller.Line aisfor the case without substrate and b isfor the additionof 0.1M H2O2 100卩L in 10mL ofelectrolytic solution.
When compared with ain Figure 1, aand b show little di fference exceptfor the magnitude of current. Plotting the difference between aand b withtheelectrodepotential,gives a good practical linearity. In contrast withthepeak currentin the static solution (Figure 2, b-1), it is shown to increase linearly.In this lab, we haveobserved these tendencies of the current difference with applied potential using variousanimal and plant tissues. There havebeen many different cases of
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-2-10 12
In (ii -i)/i
linear increasing,6"7,8monotonousincreasing,11 andindepen
dence of theelectrode potential.15 However,there is no theo
retic basis toexplain thecause of these changes yet.Table 1 provides the results ofobservations of the changesforcharging currentwiththelapse time atsteppotential in the static and stirred solutions. The steppotential is- 0.5 V, which isthe possible potential for thecompletedecomposition of H2O2 as Figure 1 shows. However, maximumcurrent (imax) and time constant (t) are identical in not being connected with the existenceofsubstratein the static solution.Here the samemaxi
mum currentswithout regardto the presence of substrateindi cates that thereduction of thesubstratemakes no contribution tothe signalcurrent because of the slowmasstransfer in the start of step potential. But twithstirring is higher than twithout stirring with no regardtothe existence ofsubstrate. This means thatreductioncurrent has been added in the long time span of potential step. Those phenomena reflect the fact that the diffusion of substrateattheactive site of theenzymeis relati
vely slow. Ontheotherhand,onemayobserve that maximum current instatic solutions is higherthan thatof stirredones and the time constant is smaller. This canbe attributedto con
vection by thestirring bar disturbingthe Levicheffect.Figure 6 shows the typical time-current recording at - 0.4 V (vs.
Ag/AgCl)in thestirred solution. Within 10 seconds,onemay observe that the current is saturated after the addition of substrate.Thisshows that theenzyme of cabbageroottissue bound by natural rubber is effectivelyembedded on theelec-
Table 1. i-t behavior resulting from potential step. Step potential:
-0.5 V. Conc. of H2O2： 10 乂 10-3 M.
Substrate Technique
unstirred stirred without
with
3.52 / 0.17 3.52 / 0.17
3.24 / 0.21 3.23 / 0.21
imax (mA) / t (sec)
2.00
-0.75 0.0 1.31
0.63
-0.06
Figure 4. Linear plot of E vs. ln{(ii -i)/i}.
-437.5 -875.0 -1312.5 서750.0
Poteritial (mV)
Fiure 5. Cyclic voltammetric response of the bioelectrode in the absense(a) and presense(b) of substrate (1.0 乂 10-3 M H2O2) in a
stirred 100 mL of 0.1 M NaCl solution. Scan speed: 100 mV/sec. Figure 6. Typical current-time response of the bioelectrode. 0.1 M H2O2 50 卩L was added at 0.4 V (vs. Ag/AgCl).
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2460 Bull.KoreanChem. Soc. 2009, Vol. 30, No. 10 Notes trode surface. Therefore,this fact implicitly tellsus that natural
rubbercan be utilized as a binder. If a double reciprocal plot of thesignal andof the substrate concentration showslinearity, theelectrodereaction isthroughcatalysis of theenzyme.This plotsetsup a linear equation, Y =2.69+ 5.13X(X : 1/[S]( x 103, 1/mM),Y : 1/i(x 106,1/nA), R = 0.999}. From this, one mayconclude that the peroxide of cabbage root tissue, which is embedded by natural rubber, playsthe role of catalyst on the electrode surface normally. Also, this proves thatnatural rubber canbe used as a good binder oncemore. The mostinfluential factor for thesignal of a biosensor is the enzyme immobilizing method.The immobilizing methodusing carbon paste,which hastheadvantages of easy production and high possibility of applicability,does not show a gooddetection limit compared with other methods. The detection limit obtained fromthe electrodeused in Figure 6is 2.5 x 10-4M. This value can be comparable withthat (6.7 x 10-4 M) ofA. N. Diazet al.16 who madeHRP electrode with thesol-gel method, butis inferiorto that (1.0 x 10-6 M) of M. Y. Miao etal)1 ThepH of the solution, the existence and concentration of the mediator, and the content of the enzyme canhave additional effects. If those can beoptimized,it is clear that the detection limitwillbemuch improved from the present state.Usingthe chemiluminescence method,J.Wang et al.18 obtained 6.42 x 10-8 M as its detection limit and B.Tang19 achieved3.89 x 10-10 Musing thefluori- metric method. If weconsider only the detection limit, the potentiostatic method cannotcompete withthespectroscopic method.However, itis possible to put this electrode to use and touse it permanently. If moreresearch for theimprovement of thedetection limit is achieved together,the profits from the advantage of thismethodmayfar surpass the costs that the higher detection limitincurs compared to the spectroscopic method.
Con이usion
Ithasbeendemonstrated in this study thatnatural rubber immobilizestheperoxidase contained in cabbage root tissue to maintainits catalyticpower. Also, thisproves that natural rubber would be a recommendable binder for applying an enzymeelectrodeusing carbon paste.Itoffersthepossibility of thesuccessful development of a carbon paste biosensor that canbe usedpermanently. In spite of thefeasibility ofcon
struction and the possibility ofapplication, the sensor made bythemethod of carbon paste isinferior tothespectroscopic methods in the aspect ofdetectionlimit. If studies for the improvement of the detection limitwere carried outside by side,then the advantage of a biosensor bound with natural rubber may coverthe cost of the disadvantage in detection ability.
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