Ž .Bioelectrochemistry and Bioenergetics 45 1998 221–226

Direct electrochemistry of membrane-entrapped horseradish peroxidase.Part II: Amperometric detection of hydrogen peroxide

Tommaso Ferri a,), Alessandro Poscia a, Roberto Santucci b

a Dipartimento di Chimica, UniÕersita di Roma ‘La Sapienza’, P. le Aldo Moro 5, 00185 Roma, Italy`b Dipartimento di Medicina Sperimentale e Scienze Biochimiche, UniÕersita di Roma ‘Tor Vergata’, 00133 Roma, Italy`

Received 25 November 1997; revised 6 March 1998; accepted 9 March 1998

Abstract

Ž . Ž .Direct unmediated electrochemistry of horseradish peroxidase HRP immobilized within a polymeric film is investigated at apyrolytic graphite electrode by dc cyclic voltammetry, in the absence and in the presence of hydrogen peroxide. Under the latter

Ž .condition, a reduction wave centered at approx. y280 mV vs. SCE is observed, the intensity of which is strictly dependent on thehydrogen peroxide concentration. This permits a voltammetric investigation of the electrocatalytic reduction of hydrogen peroxide. Flowand flow–injection measurements carried out at constant potential under the same conditions, support voltammetric data. The suitabilityof the immobilized HRP-based electrodic system to monitor the presence of important analytes, such as glucose or choline, in solution, is

Ž .also discussed. To this issue, suitable amounts of HRP and glucose oxidase or, in turn, choline oxidase were simultaneously entrappedin the polymer. The results obtained are of potential value for basic and applied biochemistry and represent a first step for construction of

Ž .a mediator-free third-generation biosensor which may find application in the biosensoristic area. q 1998 Elsevier Science S.A. All rightsreserved.

Keywords: Protein entrapment; Cyclic voltammetry; Chemically modified electrode; Horseradish peroxidase; Biosensor

1. Introduction

Ž .Heterogeneous electron-transfer eT reactions of redoxproteins at the electrodes surface represent matter of inves-tigation because provide valuable information on the com-

Ž .plex mechanism s of biological eT and find potentialw xapplication in biotechnology 1–3 . It is known that direct

electrochemistry of soluble proteins is complicated byseveral factors, such as the strong adsorption of macro-

Žmolecules onto the electrode surface with consequent.structural alteration , the slow diffusion rate in solution,

the buried nature of the active site. Immobilization ofenzymes onto the electrode surfaces has become a well-established area of research since it avoids many of thecomplications linked to soluble systems; this emphasizesthe importance of engineering enzyme-modified electrodesand justifies the big development of immobilization tech-

Ž w x.niques in the recent years see, for example, Refs. 4,5 .

) Corresponding author: Fax: q 39-6-490631; e-m ail:ferri@axrma.uniroma1.it

Ž . ŽHorseradish peroxidase HRP is an enzyme FW ap-.prox. 44,000 which catalyzes the hydrogen peroxide-de-

pendent one-electron oxidation of a wide variety of sub-w xstrates 6 . The protein contains the heme as active site; in

Ž .the resting state, the heme-iron oxidation state is Fe III .The HRP catalytic mechanism is carried out through therapid reaction with hydrogen peroxide to give a two-equiv-alent oxidized form, called compound I, in which the

Ž Ž . .active site contains an oxyferryl center Fe IV s0 and aporphyrin p-cation radical; the rapid reaction of compound

Ž .I with the substrate then regenerates the Fe III -groundstate form via an intermediate called compound II. Inrecent years, the catalytic properties of HRP, in solutionŽ .in the presence of mediators or adsorbed onto an elec-trode surface, have been investigated at variety of elec-

w xtrodes, mostly by amperometric techniques 6–12 .Ž .Recently, we have shown that direct unmediated elec-

trochemistry of HRP entrapped within a polymeric film isŽ . Ž .achieved, with reference to the Fe III ´Fe II conver-

Ž . w xsion, at a pyrolytic graphite PG electrode 13 . Followingthe same line of research, we report here the direct electro-

0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved.Ž .PII S0302-4598 98 00102-0

( )T. Ferri et al.rBioelectrochemistry and Bioenergetics 45 1998 221–226222

chemistry of membrane-entrapped HRP in the presence ofhydrogen peroxide, a strong oxidant which converts the

Ž Ž ..protein from ground state Fe III to compound I. Theability of the electrodic system to work as an efficient

w xthird-generation biosensor 14 for detection of analytes insolution, is also reported. The purpose is to provide new

Ž . Žinformation relevant to: a the role of the solid matrix in.the form of a membrane in favoring direct heterogeneous

ŽeT of embedded HRP from the oxidized state as com-. Ž .pound I to the ground state, and b the potentialities of

Ž w x.the system to act as a mediator-free third-generation 14biosensor. Engineering electrodes with enzyme-entrappingsolid matrices able to enhance, once in contact with theelectrode surface, the electrochemical properties of a pro-tein is of potential value for basic and applied biochem-istry; coupled to a good efficiency, this approach providesundoubted advantages such as the possibility to operate inthe absence of mediators.

2. Materials and methods

2.1. Materials

Ž . Ž .HRP type VI-A glucoseoxidase GOD , cholineoxi-Ž . Ždase COD were purchased from Sigma St. Louis, MO,.USA and used as received. The polymeric film employed

Žfor protein entrapment, an anionic exchange resin poly-.styrene crosslinked with 1% divynil benzene bound to a

tributylmethyl phosphonium chloride polymer, was pur-Ž .chased from Fluka Chemical Buchs, Switzerland .

All the reagents used were of analytical grade.

2.2. Methods

2.2.1. Electrochemical measurements

2.2.1.1. Dc cyclic Õoltammetry measurements. Immobiliza-tion of the protein into the TBMPC membrane was achieved

w xas previously described 13 . Dc cyclic voltammogramsŽ .were run in a previously degassed 0.1 molrl phosphate

buffer, pH 7.0; during the experiment, the anaerobic envi-ronment was maintained by a gentle flow of high-purity

Žgrade N above the solution. A PG electrode Amel,2.Milan, Italy was the working electrode whose potential

was referred to a saturated calomel reference electrodeŽ .SCE and a Pt ring was the counter-electrode. An EG&G

ŽPAR 273A EG&G Princeton Applied Research, Prince-.ton, NJ potentiostatrgalvanostat controlled by EG&G

PAR 270 Research Electrochemical Software running on aŽ .80386 33 MHz IBM compatible computer was employed

for cyclic voltammetry measurements.

2.2.1.2. Amperometric measurements. Flow measurementsŽwere carried out by transferring the carrier solution 0.1

.molrl phosphate buffer, pH 7.0 to a Methrom 656 wall-jet

Ž .electrochemical cell Herisau, CH , cell volume-1 ml, byŽ .a Jasco PU-980 Tokyo, Japan HPLC pump at a 115

mlrmin flow rate. For flow–injection measurements, thecurrent response was monitored on addition of small vol-

Ž .umes 25 ml of a solution containing hydrogen peroxideto the carrier solution; the reagent was injected through aRheodyne model 7725i injection valve, connected to the

Ž .cell by a Teflon tube internal diameter 250 mm . Thesame procedure was used for detection of glucose andcholine in solution; in this case, modification of the elec-trode was achieved by entrapping suitable amounts of

Ž .glucose oxidase or choline oxidase and HRP within thesolid matrix; glucose and choline chloride were then in-jected, instead of hydrogen peroxide, into the carrier solu-

Ž .tion. An Amel 466 potentiostatrgalvanostat Milan, Italyequipped with an Amel 868 recorder was employed foramperometric measurements.

2.2.2. Absorbance measurementsAbsorbance measurements were carried out on a Uni-

Ž .cam UV2 Cambridge, UK spectrophotometer.

3. Results and discussion

3.1. Cyclic Õoltammetry measurements

At present, the electrochemical reduction of the oxi-dized states of HRP, i.e., compound I and compound II, tothe ground state of the protein is assumed to be too slow tobe revealed by voltammetry at the majority of electrode

w xsurfaces 3 . The process is mostly followed by ampero-metric measurements which are carried out in the presenceof hydrogen peroxide in excess. Amperometric data indi-cate that the heterogeneous electron transfer rate of theprocess increases at lower electrode potentials, in the

Ž . w xq200 mV to y200 mV vs. SCE range 9,15 . Noexplanation has however been furnished for this experi-mental evidence. Fig. 1 shows the dc cyclic voltammo-grams of TBMPC membrane-entrapped HRP at a PGelectrode in the absence and in the presence of hydrogen

w xperoxide. As previously reported 13 , the cathodic andanodic waves centered at approx. y460 mV and y300mV, respectively, are indicative for the one-electron reduc-

Ž Ž . Ž ..tion and successive re-oxidation Fe III ´Fe II of theheme-iron in the protein. Upon addition of hydrogen per-oxide, a cathodic wave centered at approx. y280 mV isalso observed, the intensity of which strictly depends onthe hydrogen peroxide concentration. Since at naked orprotein-free TBMPC-modified PG electrode addition ofthe reactant produces no electrochemical signal, we assignthis wave to the embedded protein. This result indicatesthat protein entrapment and the polymer charge enhanceheterogeneous electron-exchange at the electrode consider-ably, likely favoring a proper orientation of HRP macro-

( )T. Ferri et al.rBioelectrochemistry and Bioenergetics 45 1998 221–226 223

Fig. 1. Dc cyclic voltammograms of TBMPC-entrapped HRP at a PGŽ . Ž .electrode in the absence and in the presence of: PPP 0.3 mM,

Ž . Ž . Ž .- - - 0.48 mM, and -P-P-P 0.82 mM H O . The curve -PP-PP- refers2 2

to the cyclic voltammogram of 1.2 mM of H O observed at ‘bare’2 2

electrode. Inset: cyclic voltammograms of the protein subtracted of theone run in the absence of H O . Experimental conditions: 0.1 molrl2 2

phosphate buffer, pH 7.4. Scan rate: 200 mVrs.

molecules at reduced eT distance from the electrode sur-face, in the absence of adsorption phenomena.

The inset of Fig. 1 shows the dc cyclic voltammogramsof HRP as a function of hydrogen peroxide concentration,subtracted of the signals observed in the absence of reac-tant. A single cathodic wave is observed, the intensity ofwhich is strictly dependent on the reactant concentration.The hydrogen peroxide-dependent catalytic action of HRPcan be exemplified by the following scheme:Ž . w Ž .xa HRP Fe III qH O ´Compound IqH O2 2 2Ž .b Compound IqReductant´Compound IIŽ . w Ž .xc Compound IIqReductant´HRP Fe IIICompound I and compound II are two intermediates of

Ž Ž ..the protein, the former oxidation state V characterizedw Ž . xby oxyferryl iron Fe IV s0 and a porphyrin p-cationŽ Ž ..radical, the latter oxidation state IV achieved by the

one-electron reduction of compound I. The further reduc-tion of compound II returns HRP to the ground stateŽ Ž ..Fe III . At the electrode surface, the reduction reaction of

Ž .Compound I to HRP via compound II is usually detectedby amperometric techniques by setting the potential withina range ranging from 200 to y200 mV. The cyclic

Ž .voltammogram above reported see Fig. 1 provides directevidence for the reaction; further, it explains why, inamperometric measurements carried out at constant hydro-gen peroxide concentration, a current increase is observedas the set potential is decreased from 600 mV to y250

w xmV and then levels off 9 , simply because the reductioncompletes at y280 mV.

As above described, the cathodic wave centered atapprox. y280 mV is assigned to the reduction reaction:

step 1 step 2Compound I ™ Compound II ™ HRP Fe IIIŽ .

On the basis of the results obtained, the reaction isŽexpected to be characterized by 1 rapid thus, not de-

. Ž .tectable and 1 slower detectable step. The problem aboutwhich is the rapid and which the slow step of the reactioncan be solved by establishing which of the two intermedi-ates is stabilized under the conditions employed. A factorwhich strongly influences the stability of the two interme-diates is the H O rHRP molar ratio; from spectroscopic2 2

measurements, that will be described in more detail belowŽ .Section 3.2 , compound II is the intermediate showinghigher stability; thus, step 2 is the step detected by dccyclic voltammetry.

3.2. Absorbance measurements

In the HRP-catalyzed reduction reaction of hydrogenperoxide, the H O rHRP molar ratio influences the stabil-2 2

ity of the two intermediates compound I and compound IIsignificantly. The reaction was followed by absorbance

Ž .spectroscopy in the Soret 400–450 nm , a region directlyrelated to the active site microenvironment of the protein.In this wavelength range, compound I and compound IIshow single-banded spectra centered at two distinct wave-lengths, 403 nm and 415 nm, respectively. Fig. 2A showsthe absorbance spectrum of HRP after addition of a slight

Žexcess of hydrogen peroxide in solution 10:1 H O rHRP2 2.molar ratio . Initially, a spectrum centered at 403 nm,

typical of compound I, is observed; then, the maximumshifts of approx. 6 nm towards the red, while formation ofa shoulder centered at approx. 416 nm is observed. Thisindicates formation of a mixture of the two intermediates,the composition of which is time-dependent. By contrast,upon addition of a relatively large excess of hydrogen

Ž .peroxide 100:1 H O rHRP molar ratio a Soret spectrum2 2

centered at approx. 416 nm, typical of compound II, is

Fig. 2. Absorbance spectrum of HRP in the Soret region after addition ofŽ . Ž .H O . Mixing time; — — — after 15 min from mixing. 10:12 2

Ž . ŽH O rHRP molar ratio panel A ; 100:1 H O rHRP molar ratio panel2 2 2 2.B . HRP concentration: 7 mM, T s258C.

( )T. Ferri et al.rBioelectrochemistry and Bioenergetics 45 1998 221–226224

observed; the intensity of the absorbance band slightlyŽdecreases as a function of time at least, within the first 15

.min from mixing , but remains centered at approx. 416nm, as shown in Fig. 2B. The stabilization of compound II

Žinduced by the latter condition which is the one assumedto best resemble the conditions of cyclic voltammetrymeasurements, where the hydrogen peroxide concentration

.largely exceeds that of entrapped-HRP is supported fromthe changes observed in the color of the solution uponaddition of hydrogen peroxide: the brown color, typical ofHRP, rapidly changes to green, indicating formation ofcompound I, but reconverts to brown after a few seconds,

Ž .indicating a rapid reduction of compound I green toŽ .compound II brown . The stability of compound I ob-

served during the first minutes after addition of hydrogenŽperoxide i.e., the time needed to run cyclic voltammo-

.grams indicates that the voltammetric wave centered aty280 mV is linked to the reduction reaction of compound

Ž Ž ..II to HRP in the ground state Fe III . Further, the succes-sive reduction wave centered at y460 mV, related to the

Ž . Ž . w xreduction of HRP-Fe III to HRP-Fe II 13 , excludesformation of inactive forms of the protein, which are solely

Ž .observed at high 1 mM and higher hydrogen peroxidew xconcentration 16 .

3.3. Analysis of HRP electrocatalytic actiÕity

From dc cyclic voltammograms run in the presence ofhydrogen peroxide, we graphed the maximal peak responseof the HRP-modified electrode as a function of hydrogenperoxide concentration, to verify the behavior of the elec-trodic system towards the electrocatalytic reduction ofhydrogen peroxide basing on voltammetric data. The re-sults obtained are shown in Fig. 3. The two curves in thefigure refer to TBMPC membranes entrapping different

Fig. 3. Calibration curve for the H O reduction from measure at a PG2 2

electrode coated with TBMPC-membrane entrapping different amounts ofHRP. The plotted current intensities are obtained from cyclic voltammo-

Ž .grams subtracted of the buffer curve see inset of Fig. 1 . The curves areŽ . Ž .relative to an electrode with charge of 23.0 mC ` and 10.5 mC v .

Õs200 mVrs, other experimental conditions as in Fig. 1.

ŽFig. 4. Plot of peak current relative to the voltammetric cathodic wave. Ž .centered at y280 mV, see inset of Fig. 1 vs. glucose panel A and

Ž . Žcholine panel B concentration, at neutral pH 0.1 molrl phosphate. Ž .buffer . A di-enzymatic GOD- or COD-HRP -PG modified electrode

was employed for voltammetric measurements.

Žamounts of HRP. In one case that in which the HRP.concentration is higher , a linear response is observed up

to 0.8 mM H O ; for H O concentration G1.5 mM, the2 2 2 2

response decreases, in line with a progressive enzymeinactivation in the presence of high concentration of reac-

w xtant 16 . Comparison between the two curves illustratedsuggests that the working range of the electrode strictlydepends on the amount of HRP entrapped within themembrane.

Dc cyclic voltammograms of the electrodic system en-Ž .trapping the heme which is the protein active site instead

of HRP, were run in the presence of hydrogen peroxide forcomparative purposes; like HRP, the heme catalyzes the

w xreduction reaction of hydrogen peroxide efficiently 17 .Well-defined voltammograms, all showing the cathodicwave centered at y280 mV, just like HRP, were observedŽ .not shown . To quantitatively compare the protein andheme influence on the electrode response, the apparent

Žkinetic parameter K i.e., the apparent Michaelis–Mapp.Menten constant , which is strictly linked to the reaction

Žrate, was determined from the Lineweaver–Burk plot 1ripw x.vs. 1r H O . The values of K determined, equal to2 2 Mapp

5.5 mM for the HPR-modified electrode and 23.5 mM forthe heme-modified electrode, give clear evidence for thehigher sensitivity of the HRP-modified electrode.

In view of the important role played by multicomponentelectrodic systems for hydrogen peroxide detection in reac-tions where hydrogen peroxide is formed as product of acatalytic reaction, entrapment of HRP within the TBMPCmembrane was integrated with GOD and, in turn, COD.The issue is to gain a more detailed insight into thepotentialities of the HRP-modified electrodic system to beemployed as a biosensor.

The di-component electrodic system was investigatedby cyclic voltammetry, taking the wave centered at y280mV as reference. No signal was observed in the bufferŽwhich means that the cyclic voltammogram typical ofHRP in the absence of hydrogen peroxide was the only

.signal detected, see Fig. 1 , while a well-defined wave

( )T. Ferri et al.rBioelectrochemistry and Bioenergetics 45 1998 221–226 225

Fig. 5. Amperometric response of TBMPC-embedded HRP at a PGmodified electrode vs. flowing H O solutions. The buffer was 0.1 molrl2 2

phosphate buffer, pH 7.0.

centered at y280 mV was observed as glucose or cholinechloride were added to the buffer solution. As shown inFig. 4, analysis of data indicates for both systems a strictlinearity between the current generated and the substrate

Ž .concentration, up to 10 mM glucose Fig. 4A and 0.5 mMŽ .choline chloride Fig. 4B . At higher analytes concentra-

tion, the curve reaches a maximum indicating that theŽsystem is working under saturating conditions i.e., is

.limited by the oxidase activity .

3.4. Flow and flow–injection measurements

The electrocatalytic reduction of hydrogen peroxide wasfollowed also by flow measurements; amperometry is infact the technique most widely employed for this type ofinvestigation and may represent a valid tool to verify thevalidity of the electrodic system based on TBMPC-im-mobilized HRP. By amperometry, the reaction is followedthrough the current generated at a constant potential; in thepresent case, the potential was set at Esy280 mV

Žcorresponding to the peak potential of the voltammetricwave observed in the presence hydrogen peroxide, see Fig..1 .

At ‘bare’ and TBMPC membrane-modified electrode,no current is generated by hydrogen peroxide; by contrast,in the presence of entrapped HRP addition of hydrogenperoxide to the carrier solution generates a catalytic cur-rent, the intensity of which strictly depends on the hydro-gen peroxide concentration, as shown in Fig. 5.

The system rapidly responds to changes in the hydrogenperoxide concentration; further, the starting potential isalways restored at the end of each cycle.

The behavior of the di-component electrodic system forŽ .detecting bio-organic substrates as glucose and choline

was also investigated through flow–injection measure-ments. Fig. 6 shows the electrode response to replicate

Ž . Žinjections of choline chloride panel A or glucose panel.B at different concentrations.

The good results obtained evidence the interesting po-tentialities of the di-component electrodic systems to act asan efficient, unmediated amperometric electrode, whichmay represent a first step to build a third-generationbiosensor.

w xAs recently reported 13 , the electrodic system is effi-cient for weeks if kept, when not in use, in buffer at 58C.The high efficiency as multicomponent electroactive sys-

Žtem, of interest for performances where the hydrogen.peroxide is the final detectable product , renders thisŽbiosensor potentially utilizable in clinical for detection of

. Žglucose and cholesterol in blood and food for determina-.tion of glucose and glutamate areas. To this issue,

presently work is in progress in our laboratories.

4. Conclusions

In conclusion, the present paper demonstrates that HRPentrapped within a TBMPC polymeric film shows a well-

Ž .Fig. 6. Flow–injection response of bi-enzymatic systems. Response of COD-HRP system to different choline concentrations Panel A and of GOD-HRPto different glucose concentrations. Other experimental conditions as in Fig. 1. See text for details.

( )T. Ferri et al.rBioelectrochemistry and Bioenergetics 45 1998 221–226226

defined dc cyclic voltammetry in the absence as well as inthe presence of hydrogen peroxide. Under the latter condi-tions, a cathodic wave centered at y280 mV, is observed.From analysis of voltammetric and spectroscopic data, weassign this wave to the reduction reaction of compound II

w Ž .xto HRP in the ground state Fe III . This indicates that bysetting the potential at y280 mV, the HRP-catalyzedelectrochemical reduction of hydrogen peroxide can befollowed under optimal conditions, since high values ofcurrent are achieved in the absence of interferences due toundesired reactants. The modified electrodic system acts asan efficient voltammetric biosensor, since can rapidly de-tect the presence of analytes, such as glucose and choline,involved in reactions where hydrogen peroxide is pro-duced, and represents a valid example of mediator-free

Ž w x.amperometric third-generation 14 biosensor. If we thenconsider the relatively high stability shown by the elec-

Ž .trodic system it remains efficient for weeks , we concludethat HRP entrapment within a TBMPC membrane repre-sents a novel, efficient protein immobilization method forthe construction of biosensors with valuable potentialitiesin bioelectrochemical and biosensoristic application.

Acknowledgements

Work supported by grants from CNR and MURST.

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