A Bienzyme Carbon Paste Electrode for the Sensitive Detectionof NADPH and the Measurement of Glucose-6-phosphateDehydrogenaseTina Huang,�� Axel Warsinke,� Olga V. Koroljova-Skorobogat'ko,��� Alexander Makower,� Theodore Kuwana,*��

and Frieder W. Scheller�

� Department of Analytical Biochemistry, Institute of Biochemistry and Molecular Physiology, Potsdam University, c=o Biotechnology Park,

D-14943 Luckenwalde, Germany�� Department of Chemistry, The University of Kansas, Lawrence, KS 66045, USA���A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow 117071, Russia

Received: October 29, 1998

Final version: December 10, 1998

Abstract

A novel bienzyme-based biosensor was constructed for the sensitive detection of NADPH. The sensing system consists of two enzymes, p-hydroxybenzoate hydroxylase (HBH) and laccase, both immobilized inside a carbon paste electrode. The detection scheme for NADPH isas followed: ®rst, the HBH converts p-hydroxybenzoate (pHB) to 3,4-dihydroxybenzoate (3,4-DHBred) in the presence of NADPH. Thenthe 3,4-DHBred is further oxidized by laccase to its corresponding o-quinone form (3,4-DHBox) and reduced back to 3,4 DHBred at thesurface of the electrode (at Eappl�ÿ50 mV vs. Ag=AgCl). The recycling of the 3,4-DHBred between the laccase and the electrode results inan ampli®ed current signal that is proportional to NADPH concentration. The low applied potential is a desirable feature for minimizinginterferants in biological applications. This bienzyme system showed an increased sensitivity for NADPH by a factor of ca. 18 whencompared with the single enzyme electrode (HBH only, 3,4-DHB oxidation at Eappl� � 350 mV vs. Ag=AgCl). A linear NADPH cali-bration plot was obtained from 5±30mM, with a detection limit of 1 mM. The pH optimum of this sensor was around 6. The bienzymesensor showed consistent response over 7 hours in pH 6.2 phosphate buffer at room temperature. Both enzymes remained stable inside thecarbon paste for 2 months when stored dry at 4 �C. The utility of this NADPH-detecting biosensor was demonstrated by measuring theactivity of glucose-6-phosphate dehydrogenase in test blood samples. The results obtained with the sensor setup were compared with thoseobtained with the spectrophotometric assay. Good correlation was found between the two methods with a correlation coef®cient of 0.98.

Keywords: NADPH, Bienzyme CPE containing p-hydroxybenzoate hydroxylase and laccase, Glucose-6-phosphate dehydrogenase, Blood samples

1. Introduction

We are pleased to dedicate this article in celebration of Dr.Ralph Adams 75th birthday. The bienzyme sensor reportedherein is made possible by the use of the carbon paste electrode

(CPE), which was invented by Ralph in 1958 [1]. One of us (TK)recalls the excitement of transferring what was to be a `̀ drop-ping'' CPE for anodic oxidations, in analogy to the dropping Hgelectrode for cathodic reductions, to a pool con®guration thatworked. Since the time of his invention, his research activity hasspanned from studying the electrochemistry of organic com-pounds to investigating neurotransmitter in brain tissues. Hiscontribution in the area of electrochemistry and neurochemistryis truly extraordinary. His ®rst book, Electrochemistry at Solid

Electrodes [2], is a timeless piece of work which containsinvaluable practical information. The latest monograph, Voltam-

metric Methods in Brain System [3], edited by him, providesinsightful views on current experimental approaches in neuro-chemistry. Even though his recent research project does notinvolve carbon paste electrode, this unique electrode has foundits way back into many researchers laboratory in the past decade,especially in the area of biosensor research.

The unique property of CPE makes it easy to add modi®erssuch as organic compounds, polymeric materials, enzymes, andcell tissues to the electrode surface. In the case of enzyme basedsensors, the use of organic binder (such as paraf®n oil or Nujol)can also provide an environment that is favorable in keepingenzymatic activities [4, 5] Several reviews published in recent

years has compiled the research progress involving carbon pasteelectrodes [6±8].

The sensitive detection of NAD(P)H is important in thedetermination of many analytes. For example, in clinical analysis,the quantitation of analytes (such as ammonia, lactate, andpyruvate) and the measurement of enzymatic activities (i.e.aspartate aminotransferase, lactate dehydrogenase, creatinekinase) are based on monitoring the formation or disappearanceof NAD(P)H spectrophotometrically [9, 10]. The detection ofNAD(P)H is especially pertinent to the development of bio-sensors because NAD(P)H participates in the enzymatic catalysisof the dehydrogenases (which is the largest group of oxidor-eductases with more than 250 NAD+-dependent and about 150NADP�-dependent enzymes). Therefore, the development ofNAD(P)H-detecting sensors allows the incorporation of thedehydrogenase enzymes, in which a large variety of sensors canbe designed for a variety of bio-substances. The recent progressin dehydrogenase-based biosensors is detailed in several reviews[11, 12]. Various measuring methods are currently used for thedetection of NAD(P)H and the most common approach for theelectrooxidation of NAD(P)H is with the use of mediators [12].The oxidation of the coenzyme can also be achieved by ways ofenzymatic conversion. Several diaphorase- and NADH oxidase-based systems have been reported for the oxidation NAD(P)H[13±18]. This approach presents a more natural and ef®cient wayof recycling the NAD(P)+=NAD(P)H redox couple than withmediators, because the formation of NAD(P)+ is guaranteed bythe enzymatic catalysis [12].

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295

In a previous work, we demonstrated the sensitive detection ofNADH using a bienzyme (salicylate hydroxylase and tyrosinase)recycling system inside a carbon paste electrode (CPE) [19]. Thissystem was immobilized together with phenylalanine dehy-drogenase for the determination of L-phenylalanine in serum andblood samples. In this article, we present results for a newbienzyme sensor, which consists of p-hydroxybenzoate hydro-xylase (HBH) and laccase, for the sensitive detection of NADPHand its application in measuring the activity of glucose-6-phos-phate dehydrogenase (G6PDH) in blood samples.

G6PDH is a common clinical analyte, and the quantitation ofG6PDH activity is important in the diagnosis of chronic hemo-lytic anemia or episodic hemolytic anemia caused by drugs orinfections [9]. This condition is mainly genetic and is prevalentamong certain populations in the world. For example, the highestgene frequency has been found in Kurdish Jews (with a genefrequency of 0.7) [20]. G6PDH de®ciencies are also quite com-mon (ca. 10%) among Africans, African-Americans, and certainpopulations of Mediterranean countries and southeast Asia.Although the condition is mild among African-American chil-dren, G6PDH de®ciencies can be severe and life-threatening withoxidative stress (causing acute hemolysis), especially with viralinfections and fava bean ingestion [9]. In some populations (i.e.Thailand), this condition can lead to severe hyperbilirubinemia innewborns [21, 22].

The current method for determination of G6PDH activity isbased on spectrophotometric assays which monitor the change inabsorbance with the formation of NADPH at 340 nm. Variousother methods based on potentiometry [23], amperometry [24],and electrochemiluminecence [25] have been reported for themeasurement of G6PDH activity. Although these methodsshowed good sensitivity, some of them still require highlysophisticated devices, extensive sample preparation and lengthyincubation periods [25]. In the case of amperometric detection[24], the electrode suffered from interferences due to the positiveapplied potential (�500 mV vs. Ag=AgCl). In this article, weshow that the bienzyme CPE method requires no sample pre-treatment, has a fast analysis time, and is not prone to inter-ferences because of the low applied potential (ÿ50 mV vs.Ag=AgCl).

The measuring principle of this bienzyme system is illustratedin Figure 1A. HBH is a hydroxylating ¯avin enzyme whichconverts p-hydroxybenzoate (pHB) to 3,4-dihydroxybenzoate(3,4-DHBred) in the presence of NADPH. This enzyme waschosen due to its high speci®city towards NADPH [26]. Laccaseis a copper-containing dioxygen oxidoreductase that couples theoxidation of phenolic compounds to the 4 electron reduction of

dioxygen to water [27]. The enzymatic reactions for HBH andlaccase are shown in Reactions 1 and 2.

The current signal comes from the 3,4-DHBox being reducedback to 3,4-DHBred at the surface of the electrode (see Reaction3).

The recycling of 3,4-DHBred and 3,4-DHBox between laccaseand the electrode results in an ampli®ed signal which increasesthe sensitivity of the overall system. Figure 1B shows how adehydrogenase enzyme can be incorporated into a biosensorsetup. In this study, we show a different application of theNADPH detecting sensor, the analyte of interest is the enzymeitself instead of the substrate. The measurement of G6PDHactivity is accomplished via the conversion of NADP� toNADPH by G6PDH in the presence of glucose-6-phosphate(G6P).

2. Experimental

2.1. Reagents and Materials

p-Hydroxybenzoate hydroxylase (HBH, from Pseudomonas

sp., EC 1.14.13.2) was obtained from Toyobo Enzymes (Tokyo,Japan). Laccase (from Coriolus hirsutus, EC 1.10.13.2) wasisolated and puri®ed in F. Scheller's laboratory using the pro-cedures described by Koroljova-Skorobogat'ko et al. [27]p-Hydroxybenzoate (p-HB,), 3,4-dihydroxy-benzoate (3,4-DHB), glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49, from Leuconostoc mesenteroides, Recombinant ex-pressed in E. Coli), and G6PDH controls (lyophilized G6PDH ina human red cell hemolysate base) were purchased from Sigma(St. Louis, MO). NADPH and NADP� were from Gerbu (Gai-berg, Germany). Potassium dihydrogen phosphate, di-sodiumhydrogen phosphate, graphite powder MO, Millipore) was usedfor solution preparation.

2.2. Instrumentation

All electrochemical experiments were performed on a CypressSystems (Lawrence, KS) 1090 computer-controlled potentiostat.Data were recorded and processed with a Pentium-100 personalcomputer. A three electrode con®guration was used, with theenzyme CPE as the working (1.2 mm diameter), a platinumauxiliary and a Ag=AgCl reference (DriRef-2, WPI, Inc., Sar-atosa, FL). All experiments were conducted in a homemade cell(cell volume 1 mL) equipped with a magnetic stirrer. Constantpotential experiments were performed using the chrono-amperometry mode of the CS-1090 system.

Fig. 1. A) Measuring principle of the HBH=laccase bienzyme CPE. B)Detectionscheme for measurement of G6PDH activity.

p-hydroxybenzoate + NADPH + O2

p-hydroxybenzoateHydroxylase

3,4-DHBred + NADP+ + 2 (1)

3,4-DHBred + O2Laccase

3,4-DHBox + 2 (2)H2O

H2O

3,4-DHBox

CPE, Eappl = −50 mV3,4-DHBred (3)

296 T. Huang et al.

Electroanalysis 1999, 11, No. 5

2.3. Preparation of Enzyme-Modi®ed Carbon Paste

Electrodes

The preparation procedures and the construction of enzyme-modi®ed CPEs were as previously described [19], except for theenzymes used.

2.4. NADPH Measurement Using the HBH- and

HBH-Laccase Modi®ed CPE

10 mM stock solutions of pHB and NADPH were made bydissolving the compound in pH 7 phosphate buffer. Currentresponse for NADPH was obtained using the following proce-dure: 1) ®ll the measuring cell with pH 6.2 phosphate buffer(0.066 M), and after a baseline current is established (usuallyafter 1 min), then 2) add 20mL of the pHB stock solution, and 3)add stepwise aliquots of NADPH stock solution to the measuringcell. The addition of NADPH solution resulted in increase in thecurrent which reaches a steady state current after 30 s. The cur-rent signal (Di) was determined by subtracting the baseline fromthe steady state current. A calibration plot for NADPH wasobtained by plotting Di vs. the various concentrations. Currentresponse was monitored at Eappl of ÿ50 mV (reduction of the 3,4-DHBox) and �350 mV (oxidation of 3,4-DHBred) for the HBH-laccase bienzyme CPE and the HBH-only CPE, respectively.

2.5. Determination of G6PDH Activity Using the

HBH-Laccase Bienzyme CPE

For determination of G6PDH activity in buffer, the G6PDHstock solution was made by dissolving 1.2 mg enzyme(190 U=mg) in 100mL HEPES buffer (25 mM, pH 7). The stocksolution was divided into smaller portions and stored in thefreezer. On the day of use, the stock solution was diluted further(1 : 100). The activity of G6PDH was measured by adding dif-ferent aliquots of the diluted G6PDH solution to the measuringcell (which contains 200mM pHB, 800mM G6P, and 400mMNADP�). Upon the addition of the enzyme solution, an increasein current was observed. The slope of the response curve (currentvs. time) was used to plot the graph of current response (innA=min) vs. G6PDH activity.

For determination of G6PDH activity in blood, the samplesused were the G6PDH controls from Sigma. A sample of 10 mLvolume was added to the measuring cell containing pH 6.2phosphate buffer. After the baseline current stabilized (after 1±2minutes), aliquots of the pHB, G6P, and NADP� stock solutionwere added to the measuring cell. The ®nal volume was 1 mL,the concentration of the substrates were the same as those mea-sured in buffer. With the addition of NADP�, a current increasewas observed and recorded for 5 minutes. G6PDH activities ofthe blood samples were calculated based on the rate of NADPHformation (recorded as nA=min) and the current signal obtainedwith 10 mM NADPH (mM=nA).

2.6. Determination of G6PDH Activity by

Spectrophotometric Method

The procedure for G6PDH measurement was a modi®cation ofthe method by Sigma Diagnostics (procedure No. 345-UV).Phosphate buffer pH 6.2 containing all the substrates as descri-

bed in the preceding section was used instead of the reagent andsubstrate solution supplied by Sigma. The total volume was2 mL. The formation of NADPH was monitored at l � 340 nmfor 5 minutes using a Shimadzu UV-vis (Model 160A) spectro-photometer.

2.7. Warning

The reconstituted G6PDH control solution should be treated inexactly the same manner as a test blood sample. The potential ofcontracting hepatitis and=or HIV from blood samples poses aserious risk. Latex gloves and protective clothing were wornwhen conducting experiments with blood samples. All pipettetips, centrifuge tubes, gloves, and used blood samples werecollected in a biohazard container and disposed of properly. Themeasuring cell and laboratory bench tops were washed andscrubbed with a disinfectant spray.

3. Results and Discussion

3.1. Characterization of the HBH and HBH-Laccase

CPEs

The HBH CPE was characterized by cyclic voltammetry (CV)to determine whether the enzyme remained active after theimmobilization process. For CV obtained in 1 mM pHB and1 mM NADPH (Fig. 2, CV B), the peak potentials were ca.325 mV and 225 mV (vs. Ag=AgCl) for the oxidative andreductive peaks, respectively. These values correspond to CVobtained in 1 mM 3,4-DHBred (Fig. 2, CV C). The CVs indicatethat the enzyme HBH is active inside the CP and is capable ofconverting pHB to 3,4-DHBred in the presence of NADPH. Theworking potential was set at ÿ50 mV to insure that the 3,4-DHBox is quantitatively reduced back to 3,4 DHBred. The activityof laccase after immobilization was checked with a bienzymeelectrode (at Eappl�ÿ50 mV) by adding 3,4-DHBred to the

Fig. 2. CVs obtained using the HBH single enzyme CPE. CV A)obtained in 1 mM pHB, CV B) (dotted line) in 1 mM pHB and 1 mMNADPH and CV C) in 1 mM 3,4-DHB (all in pH 7.0 phosphate bufferand at a scan rate of 50 mV=s).

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Electroanalysis 1999, 11, No. 5

measuring cell. The resulting cathodic current veri®ed that thelaccase was effective in converting the 3,4-DHBred to 3,4-DHBox.

The HBH and laccase have different pH optima. In solution,the working optimum for HBH is in the pH range of 7.5±8.5 [28],whereas for laccase it is 4.5 [27]. The bienzyme CPE was studiedin phosphate buffer with pH values ranging from 5 to 7.5 and theresponse of the sensor to 50 mM NADPH (with 500mM pHB insolution) was recorded (Fig. 3). The optimum pH range for thesensor was between 5.5 and 6.2, and pH 6.2 was chosen as theworking pH for the HBH-laccase system.

The current vs. time curves obtained with the bienzyme (HBHand laccase) and single enzyme (HBH only) are shown in Figure4, curves A and B, respectively. Figure 5 is the plot of theNADPH calibration plots for the bienzyme (curve A) and singleenzyme (curve B) CPEs. As previously noted in the experimentalsection, the current signal for the HBH-single enzyme CPEcomes from the direct oxidation of 3,4-DHBred atEappl� � 350 mV. The oxidative current is plotted in the samedirection as the reductive current (obtained by the bienzymeCPE) in order to compare the current level of the two systems.The linear range for NADPH is ca. 5±30mM for the bienzymeCPE and 50±500mM for the single enzyme CPE. The detectionlimit of the bienzyme CPE is 1 mM vs. 20mM for the singleenzyme CPE. The sensitivity for NADPH in curve A (Fig. 5) isabout 18 times greater than that of curve B. This is partly due tothe recycling of the products and the signal ampli®cation in thebienzyme system as opposed to that of the single enzyme system.

Possible interferants such as ascorbic acid and acetaminophenwere tested using the bienzyme CPE. With the addition of100mM ascorbic acid to the measuring cell, current response ofthe bienzyme sensor to 10 mM 3,4-DHB and 10 mM NADPH wasdecreased by 8±10%. This decrease is most likely caused by thechemical reaction of ascorbic acid with the quinoid forms of 3,4-DHB [29]. A reductive current was observed with the addition of100mM acetaminophen (the current level is similar to thoseresulted from 10±20 mM NADPH). This may be due to the factthat laccase can also catalyze acetaminophen to its correspondingquinones or to radicals at the working potential. Even thoughacetaminophen can act as a competitive substrate, the slope

(nA=min) of the current vs. time curve for acetaminophen (usinga plain CPE and with laccase in solution) is about 4 times lesscompared to 3,4-DHB. The decrease in slope observed foracetaminophen may be due to 1) the conversion of acet-aminophen by laccase is slower compared to 3,4-DHB or 2) thequinoid forms of acetaminophen is less electroactive than that of3,4-DHBox, or 3) the enzymatic product of acetaminophen isundergoing other side reactions. The maximum physiologicalconcentration for acetaminophen is about 100 mM. In practicalapplications, the blood samples are diluted so that interference

Fig. 3. The effect of pH on the current signal of the bienzyme CPE.Response to 50mM NADPH in the presence of 200 mM pHB.

Fig. 4. Current vs. time curves for NADPH, obtained with the bienzyme(HBH and laccase, curve A) and single enzyme (HBH only, curve B)CPEs. In curve A, the concentration of NADPH used is as indicated. Incurve B, the arrows indicate the addition of 50mM NADPH. Both curveswere obtained in pH 6.2 phosphate buffer and in the presence of 200 mMpHB. The applied potentials were ÿ50 mV and �350 mV (vs.Ag=AgCl)for the bienzyme and single enzyme CPE, respectively.

Fig. 5. Change in current vs. NADPH concentration. Curve A and Bwere obtained using the bienzyme (HBH and laccase) and single enzyme(HBH only) CPEs, respectively.

298 T. Huang et al.

Electroanalysis 1999, 11, No. 5

from these sources are decreased further. Therefore, the catalyticoxidation of acetaminophen and ascorbic acid does not pose aproblem for the overall analytical utility of the sensor.

It has been proposed that NAD(P)H can react with quinones toform NAD(P)+ and the reduced form of the quinones [30±32].Researchers have utilized this reaction and use the coenzyme(mostly NADH) to recycle the quinones back to their reducedform [33,34]. Others have used quinoid compounds as a mediatorto catalyze the oxidation of NAD(P)H [35±39]. In our case, thisreaction may decrease the response of the bienzyme sensorbecause the NADPH would be competing with the electrode forthe conversion of 3,4-DHBox to 3,4-DHBred. This possiblereaction of NADPH with 3,4-DHBox was investigated by using alaccase CPE and recording the current signal for 10mM 3,4-DHBred in buffer and in the presence of 150mM and 1 mMNADPH. The recycling of 3,4 DHB by laccase and the electrodewas not affected by 150mM NADPH, whereas a 23% currentdecrease was observed in 1 mM NADPH. This shows that thereaction of NADPH with 3,4-DHBox does occur, but only affectthe sensor response when the NADPH concentration is in the mMrange (which is far beyond the measuring range of our sensor).

The storage stability of the HBH-laccase inside the carbonpaste is shown in Figure 6. The current responses of variousHBH-laccase CPEs to 20mM NADPH (in 200mM pHB) areplotted vs. time. All the enzyme CPEs were made from the samebatch of HBH-laccase CP. From Figure 6, one can see that theactivity of the enzymes remain essentially unchanged even aftertwo months, when the enzyme is immobilized inside the CP andstored dry at 4 �C. The solid line indicates the average value(417� 37 nA) among the 6 HBH-laccase CPEs. The workingreproducibility of the HBH-laccase CPE is consistent in phos-phate buffer pH 6.2 and at room temperature. For this particularelectrode, the average current response to 10 mM NADPH was271� 21 nA for twelve measurements made over a period of 7hours. Table 1 summarizes the current values for 10 mM NADPHobtained with 11 bienzyme CPEs from three different batches ofenzyme-modi®ed carbon paste. The coef®cient of variationamong the three different batches of carbon paste is ca. 15% andthe variation among the individual electrodes within a particularbatch is 10±15%.

The sensitivity of the HBH-laccase bienzyme CPE (ca.2900 mA Mÿ1 cmÿ2, calculated based on the average valuesshown in Table 1) is higher than other NADPH sensors reportedin the literature (9.8 mA Mÿ1 cmÿ2 for a poly(o-phenylenedia-mine) modi®ed CPE [40], and 15.8 mA Mÿ1 cmÿ2 for a ¯avinreductase=pyrrole modi®ed glass carbon electrode [41]). Thereproducibility and stability of the HBH-laccase bienzyme CPEare comparable to the other NAD(P)H sensors surveyed in lit-erature [11, 12]. The linear range for NADPH is smaller (oneorder of magnitude) compare to the other sensors (2±4 orders ofmagnitude), but this behavior is typical of a recycling systemwhere the analytical range is less in order to achieve highersensitivity.

3.2. Determination of G6PDH Activity

The activity of G6PDH determined in buffer is shown inFigure 7, where the current response (in nA=min) is plotted vs.activity (U=mL). A linear response was observed from0.02 U=mL to 0.34 U=mL, with a detection limit of 0.001 U=mL.This range is suf®cient for determination of G6PDH activity inblood samples (diluted 1 : 100). The interpretative referencerange for G6PDH is 8.34� 1.59 U=g Hb [9] or 0.98� 0.9 U=mL(calculated based on 11.7 Hb [g=dL], value from the technicaldata sheet accompanying the controlled blood samples).

Fig. 6. Storage stability of the two enzymes inside the carbon paste. Theblack solid line indicates the average value of 417� 37 nA.

Table 1. Current values (in nA) for 10mM NADPH obtained with elevenbienzyme electrodes from four different batches of enzyme-modi®edcarbon paste.

Electrode Batch A Batch B Batch C

1 412� 50[a] 365� 39[b] 299� 282 382� 54 345� 31 283� 363 379� 44 336� 32 271� 21[c]4 338� 35 285� 25[b] ±Average 380� 57 328� 43 284� 28

All current values shown are the average of n � 3, with the exception of[a] n � 4; [b] n � 2, and [c] n � 12.

Fig. 7. Plot of current response vs. G6PDH activity. The currentresponse (nA=min) was obtained in the presence of 200 mM pHB,800 mM G6P and 400 mM NADP� and in pH 6.2 phosphate buffer.

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Electroanalysis 1999, 11, No. 5

The G6PDH activity of 4 different blood samples was mea-sured with both the biosensor and spectrophotometric method.The correlation between the two methods was quite good (with acorrelation coef®cient of 0.98 ). The results obtained by thebiosensor ranged from 90%±102% (with an average % of 96� 6)when compared to the values obtained using the spectro-photometer. The biosensor method is a good alternative to theoptical-based method because it can be easily miniaturized andmade into the form of a hand held device. This is more costeffective (the cost of reagents and cofactors can be greatlyreduced) and useful for the monitoring and screening of G6PDHde®ciency in third-world and developing countries.

4. Conclusions

The sensitive detection of NADPH is demonstrated using thebienzyme CPE containing HBH and laccase. The successfulmeasurement of G6PDH activity in buffer and in control bloodsamples shows that it is possible to utilize this sensor for appli-cations which may require NADPH detection at low micromolarconcentration. The low applied potential minimizes the effect ofpossible interferants. The good stability exhibited by the bien-zyme CPE shows carbon paste as a good immobilizing matrix forenzyme based sensors. This new p-hydroxybenzoate=laccasebienzyme sensor (for NADPH detection), together with the sal-icylate hydroxylase=tyrosinase bienzyme system (for NADHdetection) reported previously [19], can provide the general basisfor developing biosensors utilizing both the NADH- andNADPH-dependent dehydrogenases.

5. Acknowledgements

The authors would like to thank the Fonds der ChemischenIndustrie (Project 400137) for ®nancial support. T. Huang wasthe recipient of an DAAD (Deutscher Akademisher Aus-tauschdienst) annual research grant (Budgetary sec. 331-4-03-001, No. A=97=05745) for the 1997±98 academic year. Specialthanks are given to Nenad Gajovic and the Scheller researchgroup for helpful discussions.

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