Short Communication

Determination of Glucose at the Ru(NH3)63�-Based Paste EnzymeElectrodeLiang Chen and Waldemar Gorski*

Division of Earth and Physical Sciences, The University of Texas at San Antonio, San Antonio, TX 78249-0663e-mail: wgorski@utsa.edu.

Received: March 19, 2001Final version: May 1, 2001

AbstractThis paper describes a paste enzyme electrode based on a mediator-assisted electrical communication between redoxactive centers of glucose oxidase and graphite particles. The mediator is a solid inorganic redox salt composed of theRu(NH3)63� and Ru(CN)64� ions. The actual redox mediation is hypothesized to be accomplished by the paste-solubilized Ru(NH3)63�/Ru(NH3)62� couple that shuttles electrons between the enzyme and the graphite powder. Thepaste enzyme electrode operates at a potential of 0.0 V vs. Ag/AgCl/3M NaCl and displays superior analyticalproperties in terms of high operational stability, freedom from interferences, fast response time, extended long termstability, and ease of preparation.

Keywords: Amperometric glucose biosensor, Redox mediation, Paste electrode

Enzyme electrodes rely on electrical communication be-tween the active centers of redox enzymes and electrodematerial. A direct communication is usually hindered sinceenzymes active sites are typically located within an insulat-ing protein shell [1]. The indirect communication, on theother hand, can be accomplished by using redox mediators[2]. Several artificial mediators for coupling the enzymaticreactions to electrodes have been explored includingferrocenes [3, 4], osmium bipyridine complexes [5, 6],substituted bipyridine complexes of iron, ruthenium andosmium [7], cyano- and amino-complexes of ruthenium [8,9], octacyanotungstate(IV) and octacyanomolybdate(IV)[10], ferricyanide [11], viologens [12], quinones [13], tetra-thiafulvalene [14], diaminodurene [15], C60 [16], and violuricacid [17]. However, the selectivity of enzyme electrodesbased on mediated electron transfer is frequently compro-mised since the potential at which redox of the enzymeoccurs is primarily determined by the formal potential of themediator. Consequently, mediated enzyme electrodes oftenoperate at relatively high potentials where oxidation ofinterfering species takes place. In such a case, selectiveelectrode coatings have to be designed to alleviate theproblem of interferences [18]. In the present article, wedescribe an enzymeelectrode for glucose basedon the redoxmediator with a conveniently low formal potential. Theenzyme electrode functions at 0.0 V (vs. Ag/AgCl/3MNaCl), is free of typical interferences, and has operationalstability good enough for practical determinations ofglucose.Recently, we have initiated studies on a series of solid

redox salts basedon theRu(NH3)63� species asmediators forglucose oxidase [19]. Themediator used in the present workis a solid redox salt obtained bymixing aqueous solutions of

Ru(NH3)6Cl3 and K4Ru(CN)6 at pH� 3. The solid formedunder such conditions has a composition [Ru(NH3)63�]4[Ru(CN)64�]3 as indicated by the electrochemical andspectroscopic analyses. In order to shorten the notation,this solid will be referenced here as Ru-Ru.Figure 1 shows cyclic voltammograms for the homoge-

nous redox mediation of enzymatic reaction betweenglucose oxidase (GOx) and glucose by the Ru(NH3)63�

Fig. 1. Background corrected cyclic voltammograms recorded ata bare glassy carbon electrode in a solution containing 0.5 mMRu(NH3)63�, 40 �M glucose oxidase, and 50 mM glucose (1), andat a paste enzyme electrode in 20 mM glucose solution (2). Thepaste enzyme electrode contained 23 wt.% of Ru-Ru mediatorand was stable at E�� 0.25 V. Supporting electrolyte solution,0.05 M phosphate buffer at pH 7.40 deoxygenated with argon.Scan rate, 1 mV s�1.

78

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ions (curve 1), and by the Ru-Ru mediator immobilizedtogether with the enzyme in the paste electrode (curve 2).Both curves represent the net mediation current that wasobtained by subtracting corresponding voltammogramsrecorded in the presence and absence of glucose in thesolution. The voltammogram for the homogeneous system(curve 1) displays a current plateau at potentials morepositive than�0.20 V which indicates that the Ru(NH3)63�/Ru(NH3)62� couple (Ef

o��0.20 V) acts as an efficientredox mediator of the enzymatic reaction GOx(Ox)�glucose�GOx(Red)� gluconolactone. The kinetic analysisof the homogeneous system yielded the rate constant equalto (2.4� 0.2)� 103 L mol�1 s�1 for the mediation reactionGOx(Red)�Ru(NH3)63��GOx(Ox)�Ru(NH3)62� in a phos-phate buffer at pH 7.40.The voltammogram for the heterogeneous system (Figure

1, curve 2) displays a large current peak followed by thecurrent plateau, both in the same potential window as thatfor the homogeneous system. This implies that the Ru(NH3

)63�/Ru(NH3)62� couple, generated in the Ru-Ru mediatorpresent in the paste electrode, is responsible for themediation current recorded at the paste enzyme electrodein the presence of glucose. The existence of a current peakon curve 2 indicates that the rate of removal of Ru(NH3)62�

by electrolysis is not fully compensated by the rate of supplyof Ru(NH3)62� by the mediation reaction, which in turn canbe limited by the rate of the enzymatic reaction. The exactmechanism of the electrode process is not clear at present.Presumably, the Ru(NH3)63� species from the Ru-Ru solidsolubilize in the paste and shuttle electrons between theenzyme and the graphite powder. Mechanism notwithstan-ding, Figure 1 shows that the analytically useful level ofmediation current is observed at the paste enzyme electrodeeven on the plateau following the current peak.Figure 2 shows a set of calibration plots recorded at the

paste enzyme electrodes held at a constant potential of 0.0 Vin a stirred pH 7.40 phosphate buffer solution that wasspikedwith a glucose stock solution. It should be pointedoutthat no response to glucosewas observed at theRu-Rupasteelectrode prepared without the enzyme. The calibrationplots in Figure 2 illustrate two common trends. First, theanalytical signal in the form of meditation current increaseswith an increase in the content of theRu-Rumediator in thepaste enzyme electrode. Second, the signal of the pasteenzyme electrode containing a larger amount of the Ru-Rumediator is less sensitive to the changes in oxygen concen-tration in the sample. For example, the signal of theelectrode containing 23 wt.% Ru-Ru drops by only ca.20% when going from 5.0 mM glucose solution saturatedwith argon to the one saturated with air. In the case of theelectrode with 6 wt.% Ru-Ru the analogous change is ca.90%. In addition, the response of the electrode containing23 wt.% Ru-Ru is practically independent of oxygen atglucose concentrations above 10.0 mM. Consequently, thepaste electrode with 23 wt.% Ru-Ru was used in all thefollowing experiments.TheEadie-Hofstee plot I vs. I/Cglucose, based on calibration

points collected in deoxygenated solution (Figure 2,

curve 1), yielded a straight line in the entire concentrationrangewith a correlation coefficient of 0.994. The slope of theline gave the apparent Michaelis-Menten constant equal to32 mM, while the intercept produced the maximum currentdensity under conditions of enzyme saturation equal to0.20 mA cm�2. The relative standard deviation for the slopeand intercept was below 9%.The insert in Figure 2 shows that the determination of

glucose at thepaste enzymeelectrode is free of interferencesfrom the redox active species that are commonly present inphysiological samples of glucose. The response time,defined as the time to 90% of the full signal, was 10 s. Thedetection limit for glucose in deoxygenated solution was 8�10�5 M using the criterion of a signal of 3 times the peak-to-peak noise.The operational stability of the paste enzyme electrode

based on the Ru-Ru mediator was studied in a flow system.Figure 3 shows that the electrode response was stablewithin� 5% for at least 23 h under continuous polarizationand continuous flow of 10.0 mMglucose solution. The insertin Figure 3 illustrates the long-term stability of the elec-trode. The points are the average currents recorded at theelectrode exposed continuously to 10.0 mM glucose solu-tion in a flow system for at least 1 h on a given day. Theelectrode retained a constant response of 1.4� 0.1 (n� 10)�A for two weeks before it required a resurfacing to restoreits initial signal. The electrode was stored in pH 7.40phosphate buffer solution at room temperature when notin use.The reproducibility of the electrode preparation was

evaluated by assembling eleven paste electrodes, based on

Fig. 2. Calibration plots for glucose obtained with the pasteenzyme electrode in deoxygenated solution (1), and in air-saturated solution (2). The paste contained 23 (�) and 6 (�)wt.% of Ru-Ru mediator. Insert: An amperometric trace recordedwith the paste enzyme electrode (23 wt.% Ru-Ru) in air-saturatedsolution during the additions of 5.0 mM glucose (a), 0.10 mMascorbic acid (b), 0.10 mM uric acid (c), and 0.10 mM acetami-nophen (d). All measurements were performed in a stirred 0.05 Mphosphate buffer at pH 7.40 as a supporting electrolyte solution.Potential, 0.0 V.

79Determination of Glucose

Electroanalysis 2002, 14, No. 1

three different batches of paste with nominally the samecomposition, and measuring their response to 10.0 mMglucose in a stirred air-saturated solution. The resultingaverage signal was equal to 3.4� 0.3 (n� 11) �A whichillustrates a good reproducibility of electrode preparation.However, it should be pointed out that approximately one infive paste batches had to be discarded since it yieldedirreproducible electrodes due to, probably, inadequatemixing of all the components. The difference betweencurrents for 10 mM glucose recorded in a stirred solutionand those observed in a flow system can be ascribed to thevariations in the convective transport of glucose to theelectrode surface.In conclusion, the Ru-Ru mediator described here dis-

plays several advantageous features when compared withmany of the artificial mediators used previously in ampero-metric glucose biosensors. First, the mediator is able toefficiently couple the glucose oxidase/glucose system to anelectrode at a low applied potential. This translates into asensitive, fast, and interference free amperometric detectionof glucose at the Ru-Ru based paste electrode. Second, thepaste can be loaded with a substantial amount of solid Ru-Ru crystals without compromising its electronic conductiv-ity or damaging its texture. A high content of the Ru-Rumediator in the pasteminimizes the effects of oxygen on theresponse of the paste electrode to glucose. In addition, alarge reservoir of the Ru-Rumediator in the paste results ina paste electrode that has an operational stability adequatefor practical analysis of glucose samples. A third advantageof the present mediator is that it can be easily prepared bysimple precipitation from aqueous solutions. The design of apaste enzyme electrode based on the Ru-Ru mediator is

universal and can incorporate oxidoreductase enzymesother than glucose oxidase [20] to provide a host ofbiosensors for biologically and environmentally importantanalytes.

Experimental

Glucose oxidase fromAspergillus niger (EC 1.1.3.4, TypeX-S, 208,800units g�1) andpolyethylenimine [21] (Wt.Av.Mol.Wt. 750,000; 50 w/v%) were obtained from Sigma. Thegraphite powder, mineral oil, Ru(NH3)6Cl3, K4Ru(CN)6, �-�-glucose, �-ascorbic acid, uric acid, and acetaminophenwere purchased from Aldrich. Other chemicals, NaH2PO4 ¥H2O, NaCl, and NaOH were from Fisher.A redox mediator [Ru(NH3)63�]4[Ru(CN)64�]3 was pre-

cipitated by mixing equal volumes of 50 mM Ru(NH3)6Cl3and 50 mMK4Ru(CN)6 aqueous solutions. The purple solid(Ru-Ru) was stirred in mother liquor for 1 h, centrifugedand washed with deionized water 5 times, and dried at roomtemperature overnight before use.The pastes were prepared by hand mixing of 15 mg

glucose oxidase, 25 (or 5) mg solid Ru-Ru mediator, and5 mg polyethylenimine solution for 15 min. This mixturewas subsequently blended with 65 mg of carbon paste (50wt.% graphite powder � 50 wt.% mineral oil) for another30 minutes. Portions of the resulting paste were packed into3-mm diameter cavities of paste electrodes and polished ona weighing paper before use.Electroanalytical experiments were performed using the

CH Instruments Model 832 electrochemical detector in aconventional three-electrode system with a 3-mm diametercarbon paste or glassy carbon disk as the working electrode,a Ag/AgCl/3M NaCl (BAS) reference electrode, and aplatinum wire as a counter electrode. The infrared analysisand the energy-dispersiveX-raymicroanalysis of theRu-Rumediator were performed using the Nicolet Avatar 360 FT-IR spectrometer and Oxford Instruments INCA unit,respectively. All measurements were performed at roomtemperature (21� 1 �C).

Acknowledgement

This work was supported by the NIH/MBRS Grant GM08194.

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Fig. 3. An amperometric trace recorded at the paste electrode(23 wt.%) in a flow system. Arrows indicate the moment ofswitching from the glucose-free carrier solution to the solutioncontaining 10 mM glucose (a), and from 10 mM glucose solutionback to glucose-free solution (b). Insert: A long-term response ofthe paste enzyme electrode to 10 mM glucose in a flow system. Anarrow on day 15 illustrates a recovery of signal achieved byresurfacing the electrode with a 5-day old paste that was notexposed to any solutions. Carrier solution, 0.05 M phosphatebuffer at pH 7.40. Flow rate, 1.5 mL min�1. Potential, 0.0 V.

80 L. Chen, W. Gorski

Electroanalysis 2002, 14, No. 1

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