Air Gap Microcell for Scanning Electrochemical MicroscopicImaging of Carbon Dioxide Output. Model Calculation and GasPhase SECM Measurements for Estimation of Carbon DioxideProducing Activity of Microbial Sources

Andr�s Kiss,a Laszl� Kiss,b Barna Kov�cs,a, b G�za Nagy*a

a University of P�cs, Department of General and Physical Chemistry, P�cs, Ifjffls�g ffltja 6. H-7601, Hungaryb DDKKK Inc., 7632 P�cs, M�ra Ferenc Street 72/A, Hungary*e-mail: g-nagy@ttk.pte.hu

Received: March 29, 2011;&Accepted: July 5, 2011

AbstractMost of SECM studies have been carried out in liquid or gel phases. A Severinghaus type, CO2 detecting potentio-metric tip has been fabricated and used in gas phase SECM studies. The microcell tip is made without gas permea-ble membrane. Faster response, needed in SECM, was achieved by substituting the membrane with an air gap. Thecarbon dioxide concentration profile in the gas phase over a disc-shaped colony of yeast has been detected with thenew tip. Estimation of the carbon dioxide output of the fungi colony was attempted by fitting simulated curves tomeasured ones obtained with one dimensional (line) scans.

Keywords: Carbon dioxide, SECM, Air gap antimony electrode, Finite element method, Saccharomyces cervisiae

DOI: 10.1002/elan.201100180

1 Introduction

Scanning electrochemical microscopy (SECM) [1] intro-duced by Bard [2] and Engstrom [3] is a version of probemicroscopy. SECM is based on combined application of amicrosize electrochemical measuring tip, a high precision,three dimensional tip positioning device and appropriatecomputer programs for controlling tip movement, datacollecting and image forming steps.

Most often amperometric detection and ultramicrosizevoltammetric scanning tips are used in SECM. In am-perometric microscopy the tip–substrate distance throughmediator regeneration (positive feedback) or diffusionhindering (negative feed back) influences the current.Therefore the amperometric tip is considered active. Po-tentiometric SECM tips are passive in this respect. Theycan report only the local concentration of a species with-out giving information about tip–surface distance. Ion se-lective microsensors however, can detect ionic specieswith high selectivity, and they can be prepared with verysmall tip sizes. These are important advantages. Silver/silver-chloride microelectrodes were used for studyingchloride ion fluxes at polyaniline coated electrodes byDenault and co-workers [4]. Antimony microelectrodecould be used in both amperometric and potentiometricpH measuring modes by Bard�s group [5] and by others[6]. Different metal oxide, e.g., iridium oxide microelectr-odes [7], and conventional ion selective micropipette pHelectrodes [8] were also used in SECM for pH imaging.

A high electrode resistance can result in slow cell volt-age response in potentiometry. This limits the scanningrate in potentiometric SECM; high scanning rates resultsin distorted images. Different approaches [9,10] were re-ported for preparation of low resistance ion selectiveelectrodes for potentiometric SECM imaging. We suc-ceeded in making low resistance NH4

+ [11], K+ [12] andZn2+ [13] ion selective micropipettes for SECM applica-tions by employing a carbon fiber solid internal contact.

Working together with microbiologist colleagues, we re-alized that detecting certain gases released by microbialcolonies into the gas phase can have importance in differ-ent areas. Therefore developing special microtips, andmethods that could be used for high resolution SECMimaging of local concentrations in the gas phase over amicrobial colony was an obvious challenge.

Bard and co-workers [14] have already carried outSECM imaging in gas phase. They used a Clark type mi-crocell for studying oxygen flux through an array of 100mm diameter holes in a silicon wafer. SECM images wereobtained by measuring the reduction current of oxygenwhile scanning in the X–Y plane over the wafer surface.

Severinghaus [15] type potentiometric gas sensors candetect species involved in dissociation equilibrium in theelectrolyte film at the measuring electrode surface. Usual-ly a thin membrane permeable for the sample gas, buthaving ion transport blocking character separates thesample and the cell. The first Severinghaus electrodeswere built on pH sensitive glass electrodes and served for

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carbon dioxide measurements [16], however, NO2, HCN,SO2, NH3 etc. measuring Severinghaus electrodes havebeen also reported and used..

Carbon dioxide fluxes released by microorganisms intothe atmosphere can be used for quantification of theirmetabolic activity. Therefore in our studies the fabrica-tion of a carbon dioxide detecting microsize tip was pre-pared and its application of gas phase SECM imaging hasbeen attempted.

Microsize Severinghaus carbon dioxide measuring elec-trodes have been prepared by several groups before. E.g.ion selective liquid microelectrode with neutral ionophorwas used by Zhao and Cai [17]. They employed carbonicanhydrase enzyme for improving response time. Hansteinet al. [18] followed similar principle employing micropip-ette type pH electrode base sensing element and carbonicanhydrase enzyme additive. Tip diameter of their Severin-ghaus cell was as small as 2 mm. Metal oxide pH sensorused in potentiometric carbon dioxide cell can provideadvantageously lower resistance. Beyenal and co-workers[19] used iridium oxide micro pH electrode and carbondioxide permeable silicon rubber plug membrane prepar-ing Severinghaus sensor. It could be used detecting thecarbon dioxide concentration inside liquid phase ofStaphylococcus aureus biofilm.

For using as SECM tip in gas phase application fast re-sponding microsize gas sensor is needed. Our earlier ex-perience with air gap sensors [20] showed that substitut-ing the gas permeable membrane with air gap can dra-matically increase response time. Therefore air gap typeSeveringhaus electrode was fabricated using microsize an-timony base sensor. One dimensional SECM scanning ofthe tip in the gas phase across a disk shaped Saccharomy-ces cerevisiae colony was performed at different verticaldistances. The measured carbon dioxide concentration –distance plots were compared with calculated curves.

In this paper the fabrication of the carbon dioxide sens-ing tip, the results obtained in studying its properties andusing it in gas phase SECM over yeast colony is de-scribed.

The rate of carbon dioxide release could be estimatedfitting the calculated curves to experimental ones. Theobtained results were also used for estimation of the fluxof carbon dioxide following the method of Scott, Whiteand Bradley Phipps [21]. Details of sensor preparationprocedure, the measuring methods employed and resultsobtained are summarized in this work.

2 Experimental

2.1 Chemicals

AgNO3, glucose and glycerine were Reanal (Hungary)products, LiClO4 was purchased from Merck and NaCNfrom Chemapol. To enhance the formation of silver-ammin complexes in the electrolyzing solution, 25% NH3

was used (Interk�mia, Hungary). For making the microbi-al samples, dry agar-agar was dissolved (Szkarabeusz,

Hungary). To prepare the buffer solutions for the calibra-tions, 96 % acetic acid (Szkarabeusz) and Na-acetate(Reanal) were used.

2.2 Electrode Fabrication

Air gap Severinghaus electrode was used in these studies(see Figure 1). It was prepared as a thin glass capillarywith a pointy, pipette like end. In the lumen of the capil-lary a thin, glass coated antimony fiber was embedded inepoxy glue, while silver film deposited on the close to tippart of outer surface of the glass body served as quasi ref-erence. The pH sensing antimony indicating electrode ap-peared as a small metal disc on the circular endplate ofthe pipette side.

2.3 Preparation of the Antimony Fiber

Procedure used for preparing thin antimony fiber con-taining glass capillary used for electrode fabrication hasbeen described in details elsewhere [5]. Here a short sum-mary is given: Thick walled borosilicate glass capillaryhas been filled with melted antimony using suction. Aftercooling down the capillary was heated up and pulledusing standard manual glass blowing skill to obtain thincapillary. Under optical microscopic observation sectionswith continuous antimony fiber inside were selected andthese capillaries were further pulled using electric heatingcoil and small weight attached onto the capillary. In thisway, capillaries with submicron antimony fiber can beprepared easily. The Severinghaus microcells, however,were made with antimony fiber with diameter in therange of 20–50 mm.

2.4 Fabrication of the Cell

For preparing the Severinghaus microcell, an about30 mm long section of a thin capillary with about 10 mmouter diameter containing unbroken antimony fiberinside was selected. A copper contact wire was attachedon one end with silver epoxy glue. The antimony fibercontaining glass capillary was inserted all the way downinto the glass capillary serving as electrode body. The an-

Fig. 1. Schematic design of the Severinghaus cell used in the ex-periments, a) antimony fiber, b) epoxy glue, c) Ag film depositedon tip side of glass body.

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timony fiber containing capillary reached out to the circu-lar surface of the pointy end. The lumen of the cell bodywas filled up with epoxy glue. Silver coating, coveredwith a thin silver chloride layer was prepared on theouter surface of the conic part of the cell body to providea quasi reference electrode. The electric contact to thissmall cell was made by attaching with silver epoxy glue athin copper wire onto the silver film coated section of theglass surface. The tip was carefully polished with finesandpaper before using.

2.5 Preparation of the Silver Coating

As a first step, silver mirror coating was prepared by dip-ping the pipette tip into solution of basic diamino silvercomplex, and reducing the metal by glucose, according tothe Tollens reaction. In about half an hour the silvermirror deposition was completed. After this, the tip waswashed and the silver coating was electrochemicallythickened using controlled potential deposition techniquein a solution containing 3.4% silver nitrate, 3.3 % lithiumperchlorate and 5.3 % sodium cyanide. Silver wire servedfor counter electrode as well as one for reference. The ap-plied potential was �0.4 V vs. the reference. To make athin AgCl layer on the silver surface, the electrodes weredipped into 0.1 M FeCl3 solution for a few seconds. 0.1 MHCl solution was used to remove the ferrous traces fromthe surface. A little piece was cut from the tip with a scis-sor under microscope to expose a new, active antimonydisc.

2.6 Solutions for Silver Mirror Preparation

Solution A. Concentrated ammonia solution (25 %) wasadded drop wise to 60 cm3 0.1 M AgNO3 solution. In thebeginning silver oxide precipitate is forming. The additionwas followed as long as the precipitate completely dis-solved. After it 60 cm3 0.4 M NaOH solution was addedto the mixture. Brown precipitate was formed again, andit was dissolved after adding more ammonia solution toit.

Solution B. 1 g glucose, used here as reducing agentwas dissolved in 60 cm3 of water and 3 cm3 of ethanol wasadded to clean the glass surface.

80 %, and 20% volume fractions of solution A and Brespectively were mixed freshly to obtain silver mirrorpreparing solution.

2.7 Scanning Electrochemical Microscope, VoltageMeasurements

A homemade SECM apparatus was used in this work. Itsconstruction, working program, and functions have beenintroduced before [22]. The microscope employs New-port, Type M-MFN25PP linear motor based, three dimen-sion fine positioning unit. The smallest step of this is75 nm in all directions. A battery powered negative feedback voltage follower, based on operation amplifier (AD

515) connected to the cell, and a small digital voltmeter(Metex Instruments M-3640D 3 1/2 Digit DMM) wasused for cell voltage measurements and for feeding thevalues into the PC. The original scanning, data collectingand evaluating, programs of the microscope was used forSECM measurements, while standard potentiometricmeasuring techniques were applied for testing voltage –time, or voltage – concentration dependences of the elec-trode and the cell.

2.8 Microbial Procedures

Carbon dioxide gas emission of the Saccharomyces cerevi-siae (bakers yeast) colony was detected by scanning overit in gas phase with the Severinghaus microcell. Standardmicrobial methods were employed in growing and han-dling the yeast colony. The yeast strain used was obtainedfrom the collection of the Department of EnvironmentalMicrobiology of the University of P�cs. The yeast wasgrown on 2 % agarose gel cell culture media containing0.4 % meat broth extract, 0.4 % pepton, 1.0 % glucoseand 0.1 % yeast extract.

Before using the culture media was sterilized for 30 mi-nutes keeping the solution in high pressure and tempera-ture. The sterilized solution was poured into Petri dishand after gelling it was inoculated with a small needleplaced it into sterile hood. Only small spot or a short (1–2 cm long), thin (0.2 mm) line was formed with the yeastsuspension.

After inoculation the Petri dish was kept in laboratoryincubator at 30 8C and at 100% relative humidity for24 hours. It is known that in this condition the colony isin its active stage.

In order to make yeast colony appropriate for SECMstudies a small hole of about 1000 mm diameter was care-fully prepared with a microborer machine into a smallplexiglas sheet. This was filled with the cell culture mediacontaining agar solution cooled down to just slightlyabove gelling temperature. The plexiglas sheet was keptin humid atmosphere to avoid drying. Before SECM mea-surement a small droplet of 4% glucose solution wasadded onto the surface of the yeast colony.

3 Results and Discussion

The metal oxide pH electrodes, like antimony electrodesusually do not show theoretical function. In our studiesthe antimony electrode of the Severinghaus cell was cali-brated against a silver/silver chloride reference electrodeplaced in a separate half cell. Agar bridge was used forconnecting the two half cells. Calibration curve obtainedfor a cell with 50 mm antimony disc is shown in Figure 2.The slope of the pH function is DE/pH =54.38 mV/pHunit, that is acceptable.

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3.1 Calibrating the Gas Sensor

A small volume (8 cm3) vial with stopper was used forcalibrating the gas sensor. Two holes were made throughthe stopper. One of them served for the insertion of thegas sensor into the gas phase of the vial, while throughthe other solutions could be introduced (see Figure 3).Magnetic stirring was employed during measurements.Dipping the tip of the microcell into sodium hydrocar-bonate solution (1 mM, following original suggestion ofSeveringhaus [15]) containing 20 w% glycerin and 0.1 MLiCl to prevent drying as long as possible, a solution filmwas formed on the measuring surface. In order to avoidevaporation of the solvent from the film, the tip was im-mediately inserted into the lumen of the measuring vial.

2 mL 5 wt% sulfuric acid was added previously into thevial and the cell potential of the Severinghaus microcellwas continuously recorded. After achieving steady cellvoltage a small dose of sample solution containing calcu-lated amount of 0.1 M potassium hydrocarbonate solutionwas added through micropipette. As carbon dioxide wasliberated a quite fast potential change could be observed.After a relatively short time, the potential reading ach-ieved an other steady value. The gas phase carbon diox-ide concentration could be calculated knowing the hydro-carbonate amount added and the gas phase volume, ne-glecting the small amounts of carbon dioxide dissolved inthe acidic solution or escaped through the stopper. Thechange of cell voltage plotted against the decimal loga-rithm of gas phase carbon dioxide concentration wastaken as calibration curve of the Severinghaus sensor.SECM experiments were planned in atmospheric condi-tions over yeast culture. In this case the change of the at-mospheric cell voltage over the yeast colony is the signalindicating the microbial activity of the yeast culture.Therefore it was obvious to express the carbon dioxideconcentration in atmospheric carbon dioxide unit.Figure 4. shows the calibration curve of the Severinghauscell, Inset shows typical voltage – time curve obtaineddoing the calibration. Arrow indicates the time of potassi-um hydrocarbonate introduction.

3.2 SECM Studies

The Petri dish containing the agarose gel cell culturemedia with the small size yeast colony in it was placedand fixed with double sticky tape on the cell holder plateof the microscope. This plate moves in X and Y direction.The horizontal positioning of the gel surface was checkedor carefully adjusted. The Severinghaus microcell was at-

Fig. 2. Calibration curve of the antimony fiber pH electrode ob-tained with different buffer solutions. Outer diameter of the anti-mony containing glass capillary is about 50 mm.

Fig. 3. Microchamber used for gas phase calibration of carbondioxide measuring cell. (1) measuring tip, (2) and (3) contactleading to antimony and silver/silver chloride electrodes, (4) solu-tion input, (5) stopper with two holes, (6) syringe, (7) solution(aqueous sulfuric acid+potassium hydrocarbonate added later),(8) magnetic stirring bar, (9) glass microchamber.

Fig. 4. Gas phase calibration curve of the microcell. Inset: Typi-cal electrode potential – time curve obtained with gas phase Se-veringhaus microcell. Arrow indicates the time of additions ofhydrocarbonate solution doses to 2.0 mL 5%w sulfuric acid solu-tion.

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tached vertically on the tip holder of the microscope withthe measuring surface facing the gel. The tip was posi-tioned in vertical (Z) direction over the gel area that wasinvestigated. Usually during incubation the yeast culturegrowing arises from the level of the gel surface. Thereforein Z direction the tip was positioned about a few hundredmm over the gel surface. Optical microscope was used toobserve this tip – gel gap adjustment. One dimensional(line) scans passing over the center of the circular shapedcolony were carried out at different vertical distancesfrom the surface. The colony had about 1000 mm diame-ter. Total travel length was 6000 mm. 6 mm step size and25 mm/s scanning rate was adjusted. Using the voltage fol-lower and the potentiometric input of the electronic unitof the microscope 1000 voltage data were collected andaveraged at each step. The scans were made in two direc-tions (back and forth), and the data of one scan was uti-lized. About 480 s measuring time was needed for makinga one dimensional (two way) scan. In order to avoidevaporation of the measuring film of the tip near 100 %relative humidity was kept over the gel using a parafilmlayer on the Petri dish surface. (Of course there was anopening on the film allowing the tip move.)

Figure 5 shows one dimensional carbon dioxide concen-tration – distance curves obtained for scanning over thesource at different constant Z distances. These curveswere obtained transforming the recorded electrode poten-tial – horizontal distance curves to carbon dioxide con-centration – distance using the calibration curve shown inFigure 4.

Our experimental data could be used for a rough esti-mation of carbon dioxide outflow released by the colony.Two slightly differing approaches have been followed.One was the method of Scott et al. [21]. The other wayfor estimation was fitting our measured curves obtainedwith one dimensional scans to profiles obtained by modelcalculation.

3.3 Estimation by the Method of Scott, White andBradley Phipps

Following Scott�s work [21] we can write

rCðrÞ ¼ r0C0 ð1Þ

where C(r) is the concentration of the diffusing species atr radial distance from the surface of the hemisphericsource of radius r0. In this way by measuring r and C(r)we can get C0, if r0 can be estimated.

Saito [23] derived a rather complicated equation forthe concentration profile of a species diffusing from adisc shaped source of radius a. In order to take into ac-count the differences between the ideal hemisphericsource (ro) and our rather disc shaped one (a) we can usereff effective disc radius (reff =2a/p) [21]. Just consideringthe concentration of the species in z axial distance overthe center of the hemisphere at r radial distance theequation gets much simpler:

CðrÞ ¼ 2C0

parctg

az

� �ð2Þ

where C0 is the concentration at the source surface, a isthe radius of the source. So for rough estimation we canuse this reff value instead of r0 substituted in Eq. 1. Let bed the radial distance from the centre of the hemisphericsource. If r is the radial distance from the centre of thedisk shaped source, then d= r–reff. From Equation 1 wecan get:

1CðrÞ ¼

1C0

rreff� 1

� �¼ 1

C0

pr2a� 1

� �ð3Þ

Using the SECM we can find the centre of the diffu-sion profile over the centre of the disk source (locationwhere C(r) has maximal value at z distance from thesample sheet). Then z= r. After this, by moving stepwisethe measuring tip in z direction we can take C(r) concen-tration values at different r distances. Plotting 1/C(r)against r we can get C0 from the interception and eventhe source radius from the slope (1/C0reff).

Figure 6 shows the curve obtained plotting the 1/C(r)maximal carbon dioxide concentration values of Figure 5.against the distances. The source surface concentration C0

obtained from the intercept is 2.52 �10�4 M. The carbondioxide mass flow from the yeast containing disc shapedgel source is 7.93 nmol/s calculated from Equation 4 usingthe diffusion coefficient value DCO2 =0.16 cm2/s reportedfor air [24].

3.4 Flux Estimation with Finite Element Simulation

Carbon dioxide flux was estimated also by fitting simulat-ed curves to their corresponding measured ones. In thisway, nonradial diffusion could be considered, resulting ina more accurate flux estimation. Simulations were carried

Fig. 5. One dimensional carbon dioxide concentration – dis-tance scans obtained scanning over the center of the circularyeast colony at different vertical (Z directional) distances.

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out using the finite element method [25]. The experimen-tal system was modeled as a semicircle with a diameter of1 m to minimize wall-effect. Yeast colony was placed inthe middle of the base of the semicircle; the diameter ofthe colony was 1 mm, as in the measurements. Diffusioncoefficient of carbon dioxide was set to 0.16 cm2/s. Free-FEM+ + [26] was used to model the system, and towrite, and run the simulations as well. The diffusion fieldwas divided into 180430 triangles.

After calculating the equilibrium concentration valuesof carbon dioxide for each triangle, horizontal cuts weretaken at vertical distances of 100, 200, 300, 400, 500,1000 mm. Carbon dioxide flux through the surface of theyeast colony was adjusted to fit the peak of the simulatedcurves to the measured ones (Figure 7). Fitting was car-ried out at the horizontal distances mentioned above.

Based on the finite element simulation, the estimatedflux of carbon dioxide through the yeast colony surface is4.694�2.027 nmol/s, which is in fairly good agreementwith the flux value obtained with the method of Scott,White and Bradley Phipps (7.93 nmol/s). Table 1 showsthe flux values calculated by fitting curves recorded atdifferent heights. Asymmetry of the measured curves anddeviation from the calculated ones could be caused byseveral parameters like slow electrode response, relativelybig electrode size, asymmetric yeast cell distribution inthe source etc.

4 Conclusions

Miniaturized version of Severinghaus type carbon dioxidecell was prepared and was used as measuring tip in scan-ning electrochemical microscopy. While the conventionalSeveringhaus cells contain gas permeable membrane forproviding selectivity, the microcell presented here is pre-pared without membrane. It is an air gap type carbon di-

oxide sensor. This structure substantially decreases re-sponse time. Horizontal line scans were made in gasphase at different vertical distances over a small size, sur-face confined yeast colony without disturbing it. The col-lected data were used for preparing CO2 concentration –distance plots. Two methods were employed for estimat-ing the carbon dioxide flux from experimental yeastcolony. In practice of substrate generating – tip detecting(SG/TD) mode of SECM the delayed response, that isthe long response time can bring in distortions. The airgap construction with shorter response time can providemore realistic concentration profile with higher scanningrate than a slower membrane coated tip.

The preliminary results presented proves that potentio-metric SECM can be done scanning in the gas phase overa target without disturbing its conditions. Estimatingfluxes of gases from undisturbed surface confined micro-bial colonies can be a fruitful application of SECM in thefuture.

Fig. 6. Plot of inverse concentration vs. distance above thetarget at different vertical distances.

Fig. 7. Fitting simulated curves to the measured ones. One di-mensional [CO2] – distance plots over the center diameter of theyeast colony at different vertical (Z directional) distances (solidlines), and their simulation at the corresponding distances(dashed lines).

Table 1. Carbon dioxide flux through the yeast colony surfacecalculated from data collected by scans at carried out at differentZ distances.

Distance from the colony (mm) Estimated flux (nmol/s)

100 7.655200 6.343300 3.362400 5.157500 3.0191000 2.627Average 4.694Standard deviation 2.027

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Acknowledgements

The financial support of the Hungarian Czech BilateralResearch Funds is highly appreciated. The work was sup-ported by “Developing Competitiveness of Universitiesin the South Transdanubian Region” (SROP-4.2.1.B-10/2/KONV-2010-0002).

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