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Background-subtraction of fast-scan cyclic staircase voltammetry at

Biosensors &Bioelectronics 13(1998)

1297–1305

protein-modiﬁed carbon-ﬁber electrodes

Mark A.Hayes

a,*

,Eric W.Kristensen b ,Werner G.Kuhr

Department of Chemistry and Biochemistry,Arizona State University,Tempe,AZ 85287-1604,USA

Abbott Labs,Chicago,USA

Department of Chemistry University of California,Riverside,USA Received 16February 1998;received in revised form 8June 1998;accepted 30June 1998

Abstract

Background-subtraction techniques were applied to the voltammetry of nicotinamide adenine dinucleotide (NADH)at protein-modiﬁed carbon-ﬁber microelectrodes.The background currents at carbon-ﬁber electrodes were stable and voltammetric scans immediately before or after the analyte were effectively used for background subtraction.Digital step-potential waveforms were used to excite these carbon-ﬁber electrodes,where the resulting voltammetric analysis assessed the optimal switching and initial potentials and the electrochemical response time was determined.The initial potential was 0.0V and the switching potential 1.1V (versus Ag/AgCl)and the response time was approximately 300ms.Some sensitivity to NADH was lost and voltammetric prescans were required at protein-modiﬁed electrodes to obtain a stable baseline.Current versus time was assessed by the average current of the faradaic region from each voltammogram and by differential current;the average current minus the current from a non-faradaic potential range.Differential current assessme

nts discriminated against artifacts caused by pH (as high as 1.0pH unit)and ionic strength ﬂux (100mM).These background-subtraction techniques allowed the faradaic information to be obtained quickly and conveniently while maximizing sensitivity and maintaining selectivity.©1998Elsevier Science S.A.All rights reserved.

Keywords:Avidin–biotin;Background-subtracted voltammetry;Carbon-ﬁber electrode;Fast-scan cyclic voltammetry;NADH voltammetry;Protein modiﬁed microelectrodes

1.Introduction

Fast-scan cyclic staircase voltammetry (FSCSV)at microelectrodes has become a well established tech-nique.This technique has led to an exceptional degree of spatial (Յ5␮m)and temporal (Յ20ms)resolution,especially for monitoring stimulated release of easily oxidized neurotransmitters and metabolites in vivo (O’Neil,1974;Wightman et al.,1988).One compli-cation of a fast scan rate is the concomitant increase in the background capacitive current (Bard and Faulkner,1980).However,this background current at carbon elec-trode surfaces is remarkably stable.The stability of this current from scan to scan (given a consistent local buffer environment)offers a convenient and simple source of background scans for subtraction schemes.This scheme

碳化硅纳米线*Corresponding author.Tel:ϩ16029652566;Fax:ϩ16029652747;E-mail:mhayes@asu.edu

leads to the improvement of detection limits (Wiedemann et al.,1991;Kawagoe et al.,1993b).In this paper,this method is applied to nicotinamide adenine dinucleotide (NADH)voltammetry associated with enzyme-modiﬁed carbon-ﬁber electrodes.This class of modiﬁed electrodes provides a method to monitor non-electroactive species in physiologically relevant time-scales and volumes (Kuhr et al.,1993).The enzymes that use NADH as a co-factor include some 200species and each may be coupled to this class of biosensors via an avidin–biotin linkage system (Kuhr et al.,1993).NADH/NAD +-linked electrochemical probes offer new types of enzyme-based biosensor for a large number of analytes,but the fundamental operating parameters must be investigated.

Background-subtraction can digitally minimize or remove the background current.For a FSCSV experi-ment,the processed data is described as a background-subtracted cyclic voltammogram (BSCV)in which a full cyclic voltammogram is generated each 200ms

1298M.A.Hayes et al./Biosensors&Bioelectronics13(1998)1297–1305

(Wightman and Wipf,1990;Kuhr et al.,1993).For this background-subtraction procedure,the analytical per-formance of the FSCSV measurement is enhanced in numerous ways,most notably by addressing the trade-off between temporal resolution and current signal-to-noise ratio(while retaining qualitative information). Digital background-subtraction techniques permit sensi-tive measurements over a short time course without decreasing the scan rate to diminish background capaci-tive currents.Separation of the faradaic information of the analyte from the background ensures that the inherent selectivity of the FSCSV measurement is also retained.This selectivity is quantitated by the position of the oxidative and/or reductive peak potential which differentiates the species on the basis of their electron transfer kinetics.This technique allows this qualitative information to be recorded in a fast,sensitive manner (Wightman and Wipf,1990).

Fast-responding,enzyme-modiﬁed carbon-ﬁber microelectrodes,which transduce the non-electroactive analytes into an electroactive species through the interac-tion of cofactors have been developed(Pantano and Kuhr,1993).This microelectrode utilizes avidin–biotin interaction to immobilize enzymes onto the electrode surface.The dehydrogenase family of enzymes is parti-cularly attractive for this purpose since their activity is linked to the electroactive cofactor,NADH.The cofactor generated in this manner acts as an electron-transfer mediator that can be monitored by FS

CSV at carbon-ﬁber surfaces.While the FSCSV for the oxidation of NADH produces high faradaic currents and low overpot-ential at bare carbon-ﬁber microelectrodes,the response at an enzyme-modiﬁed surface is diminished(Pantano and Kuhr,1993;Hayes and Kuhr,1998a).The dimin-ished FSCSV response occurs because the carbon-ﬁber surface is both the site of electron transfer and of enzyme-immobilization.A quantitative balance between these two tasks has been attempted and,because these are competing activities,some voltammetric perform-ance has necessarily been sacriﬁced(Pantano and Kuhr, 1993;Hayes and Kuhr,1998a,b).尼龙抛光轮

Factors were evaluated that control the quality of a BSCV generated at a dehydrogenase-modiﬁed carbon-ﬁber microelectrodes to allow full interpretation of the available information.Near the detection limit for NADH the results of these background-subtraction para-meters were most pronounced.

A digitally generated potential-step waveform was used to excite carbon-ﬁber electrodes to generate a BSCV.The carbon-ﬁber electrodes required an electro-chemical pretreatment to provide low overpotential and high faradaic currents for the oxidation of NADH.These pretreated electrodes were used to characterize the back-ground-subtraction technique.First,the switching and initial potentials were determined and the response time was then characterized.Background currents were found to

drift more at protein-modiﬁed electrodes than freshly polished electrodes,but stable background currents were obtained after a series of voltammetric prescans were performed.To obtain qualitative voltammetric infor-mation,the scans used for background subtraction must be chosen,both in number and position.These choices were characterized for sensitivity and stability.Improve-ment of the signal-to-noise ratio was obtained by both full-scan summing and averaging,and averaging each three adjoining data points within a single scan.Current artifacts caused by pHﬂux and ionic strength changes were eliminated by using information available within each scan.Current arising in non-faradaic potential regions was used to compensate those within faradaic, or information-containing,regions.These data manipu-lations reduced sensitivity,but could compensate for pH ﬂux up to1.0pH units and ionic strength changes of 100mM.These digital excitation and data manipulation techniques provide a convenient and powerful method to obtain information quickly and with high sensitivity for the electrochemical measurement of NADH.

2.Experimental

2.1.Chemicals

Glutamate dehydrogenase(GDH,40units/mg,E.C.

1.4.1.3),NADH,ExtrAvidin,and1-ethyl-3-((dimethylamino)propyl)carbodiimide(EDC)(Sigma Chemical Co.,St Louis,MO,USA);sulfo-NHS-LC-biotin(Pierce Chemical Co.,Rockford,IL,USA),and poly(oxyalkylene)diamine(Jeffamine™ED-2001;Tex-aco Chemical Co.,Houston,TX,USA)were used as they were received.Phosphate buffer(PBS;0.15M NaCl,0.10M Na2HPO4,pH8.5)was prepared with reagent grade chemicals in water puriﬁed by a Milli-Q water puriﬁcation system(Millipore,Bedford,MA, USA).All FSCSV measurements were conducted in pH 8.5phosphate buffer.

2.2.Carbon-ﬁber microelectrodes

The fabrication of carbon-ﬁber microelectrodes has been described previously(Pantano and Kuhr,1993).All 10␮m diameter carbon-ﬁber microelectrodes(Thornel P-55S;Amoco Performance Products,Greenville,SC, USA)were bevelled at a30°angle for10min on a pol-ishing wheel covered with1␮m diamond paste(Metadi II;Buehler,Lake Bluff,IL,USA).Residual polishing materials were removed by sonicating the electrodes in hot toluene and then in de-ionized water for10s.Unless otherwise noted,all10␮m diameter carbon-ﬁber microelectrodes were electrochemically treated in1.0M HCl by a3s,50Hz cyclic potential waveform generated betweenϪ0.2V and1.8V(versus Ag/AgCl).All32

1299 M.A.Hayes et al./Biosensors&Bioelectronics13(1998)1297–1305

␮m diameter carbonﬁber(Textron Specialty Materials, Lowell,MA,USA)microelectrode surfaces were pol-ished on a glass wheel embedded with8–10␮m dia-mond particles(Sutter Glass,Novato,CA,USA).These 32␮m diameter carbon-ﬁber electrodes were further activated with aϪ0.2to2.0V(versus Ag/AgCl),50 Hz waveform in pH8.5PBS(Hayes and Kuhr,1998a). The derivatization of carbon-ﬁber microelectrodes with biotinylated-GDH has been described in detail (Pantano and Kuhr,1993).In brief,the modiﬁed egg-white protein,ExtrAvidin,serves to link biotinylated-(sulfo-NHS-LC-biotin)GDH to a biotinylated-(sulfo-NHS-LC-biotin)hydrophilic tether(Jeffamine ED-2001) which is covalently bound(EDC linkage)to the carbon-ﬁber surface.

2.3.Instrumentation

Fast-scan cyclic-staircase voltammetry was performed with an EI-400potentiostat(Cypress Systems,Lawr-ence,KS,USA)which was designed to accommodate placement of the working electrode pre-ampliﬁer inside a faraday cage.All staircase cyclic voltammetric wav-eforms were generated,and currents acquired,with an 80486PC microcomputer utilizing a12-bit,20kHz A/D–D/A interface(Labmaster DMA;Scientiﬁc Sol-utions,Solon,OH,USA).All potentials reported were referenc

ed to a0.4mm o.d.Ag/AgCl electrode.A400 kHz digital oscilloscope(Nicolet instruments Model 310;Madison,WI,USA)was used to acquire the stair-case waveform shown in Fig.1(a);the waveform was acquired with1ms(Fig.1(a)inset)and200ms(Fig. 1(a))oscilloscope time constants.All currents were recorded with a faraday cage with aﬂow-injection analy-sis(FIA)system previously described,where buffer(1.5 ml/min)was now controlled by a peristaltic pump (Model203;Scientiﬁc Industries,Bohemia,NY,USA) (Kuhr et al.,1993).All FSCSV measurements acquired wereﬁltered within a1–3kHz range by the two-pole, low passﬁlter(3db between1.5Hz and15kHz)of the EI-400potentiostat.

艾叶油胶丸2.4.Data manipulation and presentation

FIA data may be presented in a variety of formats. Qualitative data for BSCV in cyclic voltammetric-format (CV)was obtained by subtracting background scans from analyte scans.The background scans may be chosen from data sets before or after the analyte plug. Time versus current information is presented in two distinct formats:average current and differential current. The average current versus time plots is the average cur-rent of a potential range from each cyclic voltammog-ram-format data set(typically including the peak poten-tial(E p)of the analyte).This potential range for averaged current may be varied and was investigated.Differential current plots subtracted the averag

e current from a potential range where faradaic analyte current is absent from the average current data.This data manipulation allows changes in buffer composition which inﬂuence electrode capacitive currents to be compensated.The current changes caused by capacitive inﬂuence will be reﬂected equally in faradaic and non-faradaic regions. Therefore,this non-faradaic current region provides a background-subtraction source for compensation of these artifact currents.Plots may also be created in a three-dimensional view.Some data was exported as an ASCIIﬁle and manipulated in Lotus1-2-3(Lotus Devel-opment Corp.,Cambridge,MA,USA)or Excel (Microsoft,Redmond,WA,USA).

3.Results and discussion

氢键受体The exceptional spatial and temporal resolution of a fast-scan voltammetric measurement is made possible by the rapid electrochemical response of microelectrodes (approximately a1␮s time constant)(Wightman and Wipf,1990).An experiment may be performed at a scan rate of100V/s,when the oxidative and reductive scans across a1V region are completed in20ms.When these scans are repeated at100Hz,the time course of the measurement provides sub-second temporal resolution (approximately200ms),where selective and sensitive data can be recorded in the same measurement (Wightman et al.,1988).

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Digitally generated step-potential waveforms are instrumental for these measurements(Bilewicz et al., 1989;Murphy et al.,1989;Karpinski and Osteryoung, 1993).With fast linear scan rates,the capacitive (residual)current(i c)increases linearly with an increas-ing scan rate,whereas the faradaic current(i f)follows only a square root dependence.At high scan rates,the capacitive current is dominant.The use of step-potential waveforms and time-delay digital data acquisition min-imize the contributions of capacitive currents at fast scan rates,thus allowing the relative amount of faradaic cur-rent to increase.

A cyclic staircase potential waveform(Fig.1(a))is described by its potential step-height and its potential step-width,in which the scan rate(V/s)is determined by their ratio(Howell et al.,1986).The advantage of this technique arises from the temporal control of the current measurement.Digital sampling provides for acquisition of the current at any point along the potential step width.Since the decay of capacitive currents are fast(e−t/Rc,where t is time,R is the electrode resistance and C is the electrode capacitance),and the decay of the faradaic current follows a slower time dependence(t1/2), the current is recorded at the end of the potential step (arrows,Fig.1(a)inset).

The background current at a carbon-ﬁber microelec-

1300M.A.Hayes et al./Biosensors&Bioelectronics13(1998)1297–1305

Fig.1.Fast-scan cyclic staircase voltammetry(100V/s,100prescans)at a polished and electrochemically pretreated,10␮m diameter carbon-ﬁber microelectrode.(a)Cyclic staircase potential waveforms,in which the oxidative and reductive scans across a1.2V region(tϭ22ms)were repeated every200ms.(a,Inset)An individual potential step was18.30mV high and0.18ms long.All currents were sampled at the end of each potential step(arrows).(b)Three-dimensional view of the oxidative portion,plotted in an upward direction,of the FSCSV experiment.(c)Average oxidative current versus time when the time course for the appearance of NADH is ascertained by monitoring the oxidative current between714 and934mV versus Ag/AgCl.(d)Cyclic staircase voltammograms(average of10scans)acquired during(boxes)and after(triangles)the4s FIA injection of100␮M NADH.(e)BSCV created by the digital subtraction of the two voltammograms from(c).

1301 M.A.Hayes et al./Biosensors&Bioelectronics13(1998)1297–1305

trode contains not only capacitive components but also faradaic components.The faradaic component of the residual background current stems from the carbon-oxy-gen moieties that are localized on the carbon surface, most notably quinones and hydroquinones(Wiedemann et al.,1991;Kawagoe et al.,1993a).While the use of digital staircase waveforms discriminates against capaci-tive currents,the background current is signiﬁcantly affected by these surface-bound faradaic

processes (Howell et al.,1986;Kawagoe et al.,1993b).Since the background current is only partially discriminated against with this technique,further improvements are needed.三苯基氢氧化锡

Far greater results for improving i f/i c and the signal-to-noise ratios have been reported with background-current subtraction(Howell et al.,1986).For this technique,FIA provides the stable background-currents because the environment of the microelectrode surface is constant, except for the introduction of the sample bolus (Engstrom et al.,1988).This stable background current is necessary to prevent distortion of the faradaic infor-mation(Howell et al.,1986;Kawagoe et al.,1993a,b).

A well-formed electrochemical response to a100␮M bolus of NADH and a stable background current is shown in Fig.1(b and c)at a polished carbon-ﬁber microelectrode.This demonstrates a consistent back-ground current,and the introduction of analyte does not produce artifacts(odd peak shape,hysteresis,etc.).

To optimize FSCV further for the electrochemical measurement of NADH when performed at carbon-ﬁber microelectrodes,several electrochemical pre-treatments were explored.These were examined for their ability to optimize the quality and reproducibility of the NADH response for FSCV.A mild ano

dic treatment in HCl pro-duced low overpotential and high faradaic currents for oxidation of NADH(Pantano and Kuhr,1993)(Fig.1(d and e))at10␮m diameter carbon-ﬁber electrodes, whereas32␮m carbonﬁbers required treatment in pH 8.5PBS to produce similar results(Hayes and Kuhr, 1998a).While low overpotential and high faradaic cur-rents can be observed at freshly polished carbon-ﬁber surfaces,there was considerable variability from elec-trode to electrode.The BSCV shown in Fig.1(e)was generated at a pretreated electrode by subtracting vol-tammetric scans acquired after the FIA injection(Fig. 1(c),triangles)from the voltammetric scans acquired during the injection(Fig.1(c),boxes).This result dem-onstrates a BSCV where the facile electron-transfer information is retained through the subtraction process. Measurement of the response time of this system was accomplished by monitoring the current from800to 1100mV(faradaic current from oxidation of NADH) versus time(Fig.1c).The response observed to this square-wave input was an electrochemical measurement characteristic of the temporal response of the sensing system.The response of a pretreated carbon-ﬁber microelectrode to the injection of NADH in a FIA-FSCSV experiment is quite rapid(Fig.1(c));the time required to reach63%of the steady-state current is approximately300ms.

3.1.Selection of initial and switching potentials While FSCSV of the NADH at a polished,electro-che

mically pretreated carbon-ﬁber microelectrode typi-cally exhibits an oxidative peak potential at800mV ver-sus Ag/AgCl(Fig.1(e)),this anodic wave is shifted more than300mV positive following the enzyme-modiﬁ-cation procedure(Pantano and Kuhr,1993;Hayes and Kuhr,1998b).A switching potential of1100mV(versus Ag/AgCl)was used because it was the maximum poten-tial allowed without signiﬁcant interference from the oxidation of the background buffer.The possibility of using the reductive NADH current to improve sensitivity is eliminated since oxidation of NADH at carbon elec-trodes is chemically irreversible(Moiroux and Elving, 1979,1980).

The choice of an initial potential is inﬂuenced by other factors that are associated with the use of these probes in vivo.In previous in vivo FSCSV determinations for stimulated release of catecholamines,aϪ400mV (versus Ag/AgCl)initial potential was required (Wightman et al.,1988).This value was chosen to ensure the complete reduction of the oxidized quinone present on numerous biological molecules of interest generated during the oxidative voltammetric scan.Fluc-tuations in the concentrations of these species could interfere with the analysis of NADH.Nonetheless,even if no detectable quinone/hydroquinone redox couple were present in solution,quinone and hydroquinones on the carbon-ﬁber surface itself would always be present (Kawagoe et al.,1993a).An initial potential of0.0V (versus Ag/AgCl)was chosen to eliminate the quinone/hydroquinone redox couple,thus avoiding poss-ible artifacts from this process altogether.

3.2.Background current drift

Repetitive cycling of a potential waveform will improve the stability of the background current,when this cycling allows the electrode surface to approach a steady state(Kinoshita,1988).In vivo voltammetry experiments typically require a10min cycling of the potential waveform after implantation before any data is acquired(Wightman et al.,1988).The majority of this voltammetric degradation occurs within theﬁrst few minutes after implantation.With carbon-ﬁber surfaces, the sensitivity is diminished,but the background currents will remain stable for the remainder of the experiment and therefore background-subtraction techniques may be employed.

A carbon-ﬁber surface with immobilized enzymes

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