detection of dopamine in the presence of excess ascorbic acidat physiological concentrations through...

ORIGINAL PAPER

Detection of dopamine in the presence of excess ascorbic acidat physiological concentrations through redox cyclingat an unmodified microelectrode array

Anupama Aggarwal & Mengjia Hu & Ingrid Fritsch

Received: 31 August 2012 /Revised: 2 December 2012 /Accepted: 12 January 2013 /Published online: 9 February 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The electrochemical behavior of dopamine wasexamined under redox cycling conditions in the presenceand absence of a high concentration of the interferent ascorbicacid at a coplanar, microelectrode array where the area of thegenerator electrodes was larger than that of the collectorelectrodes. Redox cycling converts a redox species betweenits oxidized and reduced forms by application of suitablepotentials on a set of closely located generator and collectorelectrodes. It allows signal amplification and discriminationbetween species that undergo reversible and irreversible elec-tron transfer. Microfabrication was used to produce 18 indi-vidually addressable, 4-μm-wide gold band electrodes, 2 mmlong, contained in an array having an interelectrode spacing of4 μm. Because the array electrodes are individually address-able, each can be selectively biased to produce an overalloptimal electrochemical response. Four adjacent microbandswere shorted together to serve as the collector, and wereflanked on each side by seven microbands shorted as thegenerator (a ratio of 1:3.5 of electroactive area, respectively).This configuration achieved a detection limit of 0.454±0.026 μM dopamine at the collector in the presence of100 μM ascorbic acid in artificial cerebrospinal fluid buffer,concentrations that are consistent with physiological levels.Enhancement by surface modification of the microelectrodearray to achieve this detection limit was unnecessary. Theresults suggest that the redox cycling method may be suitablefor in vivo quantification of transients and basal levels ofdopamine in the brain without background subtraction.

Keywords Dopamine . Ascorbic acid . Electrochemistry .

Redox cycling . Microelectrode array

Introduction

The catecholamine dopamine (DA) is an important neuro-transmitter in the brain.Dopaminergenic systems are involvedin a multitude of functions, including neurocognition, motorcontrol, motivation, punishment, and reward [1–8]. Dysfunc-tion of DA production and uptake rates plays a vital role invarious illnesses, such as Parkinson’s disease [9, 10],Huntington’s disease [11], substance abuse [10, 12, 13],Tourette’s syndrome [14], schizophrenia [15], and atten-tion deficit–hyperactivity disorders [12, 16]. The need tostudy DA for neurological and medical purposes is welldocumented in the literature.

Facile oxidation of DA to dopamine quinone (DAQ)renders it suitable for electroanalysis. Electrochemical de-tection has been used successfully to investigate DA directlyin the brain to determine its implications in processingmotor, motivational, and sensory information [17], anticipa-tory learning [18], the effects of addictive drugs such ascocaine [19] and nicotine [20], and general function [21].However, there remains a need to differentiate between theelectrochemical signal arising from DA and that arisingfrom ascorbic acid (AA), as well as other electrochemicallyinterfering species, including other catecholamines. This isbecause the oxidation of AA occurs at approximately+0.2 V versus Ag/AgCl (4 M NaCl), which is similar to thepotential for DA and other catecholamines [22, 23]. Also, itis a challenge to measure the low concentrations of DA in thepresence of high concentrations of AA and other reactivespecies. Extracellular DA concentrations from evoked re-lease in the striatum, for example, are reported to be single-digit micromolar concentrations [21, 24–26], whereas the

Published in the topical collection Bioelectroanalysis with guesteditors Nicolas Plumeré, Magdalena Gebala, and WolfgangSchuhmann.

A. Aggarwal :M. Hu : I. Fritsch (*)Department of Chemistry and Biochemistry,University of Arkansas, Fayetteville, AR 72701, USAe-mail: [email protected]

Anal Bioanal Chem (2013) 405:3859–3869DOI 10.1007/s00216-013-6738-z

concentrations of AA (100–500 μM) are 100 times to 1,000times those of DA [10, 22, 23, 27–30].

Several electrochemical methods have been used formeasuring DA in vivo. Much of the earlier work employeddifferential pulse voltammetry. It has poor temporal resolu-tion (more than 30 s) and allows selectivity for simultaneousmeasurement of analytes with oxidation peaks only morethan 100 mV apart [31]. Chronoamperometry (CA) offerssubmillisecond temporal resolution. It has been used todetect DA and other catecholamine neurotransmitters[32–35], but has poor chemical selectivity. Fast-scan cyclicvoltammetry (CV) [23, 36–38] at carbon-fiber electrodes(CFEs) is currently the most widely used electrochemicalmethod for making DA measurements in vivo. It has hightemporal resolution (100 ms), although not as good as CA.At fast scan rates the current from AA oxidation is negligi-ble. However, the rapidly changing voltage causes a largebackground current due to double-layer charging that over-takes the faradaic current. Because the background currentin vivo is stable for only short times, fast-scan CV is typi-cally not used for measurements exceeding 1 min withoutreestablishing the background signal [23]. Thus, it is diffi-cult with fast-scan CV to measure resting or static DAconcentrations. This is where redox cycling may ultimatelyhave an advantage.

Because DA is electrochemically reversible, it can bedetected by redox cycling, where two or more closelyspaced electrodes, the generator and the collector, are heldat oxidative (anodic) potentials and reductive (cathodic)potentials, respectively. Charging current is not an issuebecause it drops to zero within nanoseconds to microsec-onds after the initial application of the potential at a micro-electrode. Upon arrival at the generator, DA undergoesoxidation. The DAQ product diffuses away, where somemolecules reach the collector across the gap with a certainefficiency and reduce back to DA; the cycle continues.Thus, current at both the generator and the collector can beused to determine the DA concentration. AA, however, alsooxidizes at the generator, adding to the measured anodiccurrent. The dehydroascorbic acid (DHAA) product thenundergoes subsequent rapid hydrolysis to a nonelectroactiveform (rate constant of 1.4×103s−1 [39]) prior to reaching thecollector, appearing silent at the cathode on the timescale ofthis experiment [40, 41]. Therefore, the redox cycling meth-od offers improved signal-to-background ratio and real-timemonitoring of DA without requiring high-speed scanningelectronics. It also provides elimination from AA interfer-ence when the current is monitored at the cathodic collector[42, 43].

Redox cycling to detect DA using the closely spacedelectrodes of a microelectrode array (MEA) has been previ-ously reported [42–49]. Low nanomolar concentrationshave been measured with redox cycling involving flow

channels or electrode modification [42, 44, 45, 47, 48,50–52]. However, none of those studies demonstrate simul-taneous detection of DA in the presence of a large excess ofAA (100 times) at physiological concentrations, which isimportant for in vivo detection, without requiring an additionalcoating to concentrate DA and exclude AA.

To perform the in vitro redox cycling studies describedhere, a MEAwas microfabricated. It is important to considerits advantages and disadvantages compared with those ofCFEs when considering it for in vivo measurements. Thestate-of-the-art fabrication procedure for CFEs is lengthy,manually intensive, and irreproducible. Also, the single sens-ing site of a CFE limits the scope of mapping DA in the brain.In an attempt to resolve that complication, Zachek et al. [53]evenly spaced and manually mounted four CFEs as an arrayinto a holder to perform simultaneous in vivo DA measure-ments by fast-scan CV in spatially distinct locations. Micro-fabrication of multiple electrodes on one substrate, in contrast,can provide greater control over electrode size, spacing, andflexibility in electrode design, and thus allows the more ad-vanced electrochemical methods, such as redox cycling, ana-lyte mapping, and multianalyte sensing, to be possible withoutextensive manual manipulation [54].

Two other issues to consider in comparing microfabricatedMEAs with CFEs are the suitability of the electrode materialand the probe size for in vivo analysis. Gold served as theelectrode material of our array. This is because metals can bemore easily microfabricated onto substrates than carbon withhigh spatial resolution [55–57]. Nonetheless, carbon electro-des patterned with relatively large features on probes for tissueinsertion have been reported [58, 59]. Metal electrodes (goldand platinum), however, generally have a smaller inert poten-tial range compared with carbon electrodes, which needs to bekept in mind in setting a voltage window for analysis. Withrespect to probe size, the insertion of CFEs (5–15-μm diam-eter) and metal electrodes of small diameters in the brainavoids ischemia and glial proliferation [60]. This is in contrastto the degeneration of tissue observed from insertion of muchlarger microdialysis probes (approximately 220 μm or greaterin diameter) [24, 26, 60–63], which affects the analyte con-centrations in the sampled fluid [21, 25, 64]. Thus, it isdesirable to construct microfabricated arrays to be as smallas possible, at least narrower than microdialysis probes. Al-though a systematic study of tissue damage has not yet beenperformed onmicrofabricated microelectrodes of intermediatewidth (approximately 100 μm at the sensing element), in vivoresults suggest that tissue damage is indeed less than for thelarger microdialysis probes [57, 58, 65, 66]. Similar probedimensions of 100 μm (and smaller) for the substrate arecertainly achievable for interdigitated microband arrays suchas the MEA used here for redox cycling. A next-generationredox-cycling MEA design and the effect on signal for probesto be used in vivo are discussed further in “Conclusions.”

3860 A. Aggarwal et al.

To our knowledge, this is the first report of quantification ofDAwith redox cycling in the presence of up to 100 times itsconcentration of AA at physiologically relevant levels, with-out using polymer coatings that concentrate the analyte andexclude the interferent. A MEA was used with an electrodeassignment having a small central collector region flanked bytwo larger generator regions to enhance the discriminationagainst AA. To further challenge the redox cycling method,artificial cerebrospinal fluid (aCSF) was chosen as the elec-trolyte, because its composition better mimics extracellularbrain fluid [67] than the more commonly used pristine buffersused for in vitro studies, such as phosphate-buffered saline,2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid(HEPES)-buffered saline, and 4-amino-2-(hydroxymethyl)-propane-1,3-diol (Tris) [49, 68–70].

Experimental

Chemicals and materials

All chemicals were used as received unless otherwise stated.Hexaammineruthenium(III) chloride, dopamine hydrochloride,calcium chloride, sodium dihydrogen phosphate monohydrate,

L-(+)-AA, HEPES, and Tris (all ACS grade) were obtainedfrom Alfa Aesar (Ward Hill, MA, USA). Magnesium sulfateand sodium chloride were obtained from EMD Chemicals(Gibbstown, NJ, USA). Sodium hydrogen carbonate wasobtained from J.T. Baker (Phillipsburg, NJ, USA). Sodiumsulfate was obtained from Aldrich Chemical (St. Louis, MO,USA). D(+)-glucose (anhydrous) and potassium chloride wereobtained from BDH/VWR (Radnor, PA, USA). Water (ACSreagent grade, 18 MΩ cm or greater) was obtained fromRicca Chemical (Arlington, TX, USA).

The MEA

Details of the design of the MEA and one of the two electrodeconfigurations are shown in Fig. 1. The devices contain 18,parallel, coplanar band electrodes (expanded view in Fig. 1b)and two large gold features (4.00mm×4.00mm and 4.00mm×2.00 mm), of which the larger one served as an auxiliaryelectrode and the smaller one was not used (left at open circuit).Each of the electrodes in the array is individually addressablevia contact pads. The microbands have an average length of2.00±0.01 mm, a width of 4.0±0.1 μm, and are separated bygaps of 4.0±0.1 μm. Electrochemical characterization of theMEAs using the model compound hexaammineruthenium(III)chloride is reported elsewhere [71].

MEA fabrication is also reported in detail elsewhere [71].In brief, a 50-nm-thick layer of gold was deposited onto a5-nm-thick chromium adhesion layer on a silicon dioxidecoated (2-μm) silicon wafer. The metal layers were patterned

to define electrodes, leads, and contact pads. The MEAs werethen coated by an insulation layer of benzocyclobutene, whichwas subsequently patterned to define the active electrode areasthat would be exposed to solution and the contact pads thatwould be inserted into the edge connector (solder contact,20/40 position, 0.05-in. pitch, Sullins Electronics, San Marcos,CA, USA).

Electrochemical methods and instrumentation

All experiments were performed with a fresh solution eachtime to avoid carryover from the previous electrochemistry.A bipotentiostat equipped with a Faraday cage and apicoamp booster (CHI 760, CH Instruments, Austin, TX,USA) was used.

The electrode configuration that was used had one collectorregion, formed by shorting either two or four microbandelectrodes together. It was flanked by two larger generatorregions, each formed by shorting eight or seven microbandelectrodes together, respectively. Figure 1c illustrates the latter

- Collector

DAQ DA

(b)

(c)

DA

AA AAO

DAQ

(d) DA

AA AAO

DAQ

+ Generator + Generator Diffusion Diffusion

1

(a)

Contact Pads

Auxiliary

4 mm x 4 mm

Array(2 mm long)

50 m 2 mm

Fig. 1 Electrode chip design and assignment of microband electrodesas generators and collectors. a Photographic image showing a full viewof the microelectrode-array-containing chip. b An expanded view ofthe array region (cropped in length) showing 4-μm-wide microbandelectrodes with 4-μm gaps. c The configuration with the large gener-ator regions flanking a single, smaller collector region used for thecalibration studies. The generator electrodes are shown in black and thecollector electrodes in gray (not drawn to scale). d Redox cyclingbetween the reduced form dopamine (DA) and dopamine quinone(DAQ), the oxidized form of dopamine, and the oxidation of thereduced form of ascorbic acid (AA) to a form that is unable to re-reduceat the collector electrodes (AAO)

Detection of dopamine in the presence of excess ascorbic acid 3861

configuration used to produce the calibration curves for DAobtained by CA in the presence and absence of AA.

Either CV (by sweeping from −0.100 to +0.500 V andback) or CA (at a constant oxidizing potential of +0.500 V)versus a Ag/AgCl (saturated KCl) reference electrode wasperformed at the generator electrodes. In the redox cyclingexperiments, the collector electrodes were held at a constantreducing potential (−0.100 V for CV studies and −0.200 Vfor CA studies). The potentiostat applies a potential at theelectrode(s) for 2 s (the quiet time) prior to commencing theCV and CA waveforms. (Data are not acquired during thequiet time.) Those potentials during the quiet time for CVwere −0.100 Vat the generator and −0.100 Vat the collector.Those potentials for CA were 0.000 V at the generator and−0.200 V at the collector.

Solutions containing DA and AAwere made fresh beforeuse each day in Tris buffer (15 mM Tris, 140 mM NaCl,3.25 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.25 mMNaH2PO4, 2.0 mM Na2SO4) or in aCSF buffer (100 mMNaCl, 5.0 mM KCl, 1.2 mM NaH2PO4, 5.0 mM NaHCO3,10 mM glucose, 2.5 mM HEPES, 1.2 mM MgSO4, 1.0 mMCaCl2) at 7.4 pH. Stock solutions were purged under argongas to prevent air oxidation, but the experiments them-selves were conducted with solutions exposed to air.

The limit of detection was calculated as three times thestandard deviation of the blank signal, divided by the slopeof the calibration curve. The standard deviation of the limitof detection was obtained from propagation of error. Thecollection efficiencies, unless stated otherwise, wereobtained by taking the ratio of the steady-state current atthe collector electrode to the steady-state current at thegenerator during redox cycling. The amplification factors,unless otherwise stated, were obtained by taking the ratio ofthe current at the generator electrode undergoing redoxcycling to that at the generator electrode without redoxcycling.

Results and discussion

Characterization of 1 μM DA in 100 μM AA by CV in Trisbuffer

Redox cycling of DA (1 μM) was evaluated by slow-scanCV (0.010 Vs−1) in the presence and absence of a 100-foldexcess of AA in Tris buffer. These concentrations are withinthe ranges of those found in the brain. Figure 1d illustratesthe general redox cycling process and how DA may bedetected at the collector electrode (via reduction of DAQ)while simultaneously discriminating against AA. The redoxcycling results are shown in Fig. 2.

The procedure involved holding the potential of the collectorat a constant reducing potential of −0.100Vwhile sweeping the

potential of the generator at a scan rate of 0.010Vs−1, starting ata reducing potential of −0.100 V, where there should be no orlittle faradaic current, to +0.500 V, which is sufficient to oxidizeDA and AA, producing an anodic current. The potential at thegenerator was then scanned back to −0.100 Vat the same rate.The collector showed a cathodic current when the generatorreached a potential that was anodic enough to produce anoxidized species that diffused to the collector, where it wasreduced. The slow scan was necessary to ensure that a steadystate was achieved for the redox cycling. All of the CVresponses in Fig. 2 are plotted as a function of the potentialapplied to the generator in the forward and reverse sweeps; thecurrent in plots a, c, and e in Fig. 2 was measured at thecollector and the current in plots b, d, and f in Fig. 2 wasmeasured at the generator.

The configuration of the electrodes used for redox cyclingusing the CVapproach involved two large generator regions ofeight microbands each, flanking a much smaller collector regionof two electrodes. This configuration was chosen because it isknown that AA undergoes a spontaneous reaction with DAQ,converting it back to DA, which diminishes the amount of DAQarriving at the collector electrode, resulting in a loss of signal atthe collector, although the kinetics are slow [43, 48]. Thisarrangement, as opposed to the more popular alternating con-figuration of equally sized generator and collector electrodes,takes advantage of the shielding effect of neighboring generators[71, 72] to diminish the concentration of AA near the collector.Niwa et al. [42] described the problem of recovering DAQ at thecollector with increasing concentrations of AA at an alternatinggenerator–collector configuration of interdigitated microbandelectrodes. They showed that in the presence of a concentrationof AA that is six times that of DA, the cathodic current for DAQat the collector drops to one third (for 10-μm widths and 5-μmgaps) and two thirds (for 3-μm widths and 2-μm gaps), com-pared with the DAQ signal in the absence of AA. We wouldexpect the DAQ signal for an alternating configuration of ourarray, which has intermediate dimensions (4-μm widths and4-μm gaps), to decrease to a similar value (between one thirdand two thirds). For a solution where the concentration of AA is100 times that of DA, the decrease in the cathodic current shouldbe far worse or even immeasurable. Thus, a large generator-to-collector area was used for our studies here to consume a largeramount of AA and allow more DAQ molecules to survive thetrip to the collector from the generator.

Plot e in Fig. 2 shows an easily measured cathodiccurrent at the collector in a mixture containing 1 μM DAand 100 μM AA when the potential at the generator wassufficiently anodic (about +0.150 to +0.500 V) to produceDAQ. Its magnitude (when the generator was at +0.450 V)was 1.68 nA. This is a magnitude similar to that for 1 μMDA alone (1.55 nA; Fig. 2, plot a).

There is no contribution to the collector current from theexcess 100 μM AA because the oxidized product DHAA is


quickly hydrolyzed to an electrochemically irreversible form.However, as can be seen in plot c in Fig. 2, there is anoticeable signal at the collector (40 % of that when 1 μMDA is present) due to reduction of oxygen that is no longerdepleted at the generator electrodes when they are scanned topositive potentials. This explanation is further confirmed byredox cycling in buffer alone (not shown), where the collectorshowed similar behavior. This effect with oxygen providesfurther justification for exploiting large, flanking generatorelectrodes that “titrate” unwanted species (such as AA), andtherefore prevent undesired reactions with analyte speciesbefore they can be detected at the collector electrodes.

At the generator, because both DA and AA oxidize in asimilar voltage range, the CV response of the mixture shouldequal the sum of the responses for the individual components,unless there are reactions between them. Overall, the shape ofthe CVresponse for the mixture of 1 μMDA and 100 μMAA(Fig. 2, plot f) reflects the sum of the shapes for 1 μM DA(Fig. 2, plot b) and 100 μM AA (Fig. 2, plot d) alone. (Thisadditive effect at the generator is consistent with previousredox cycling results performed with DA and AA at a rela-tively high concentration of 1 mM each [49].) However, themagnitude of the generator current for the mixture at +0.450V(19.85 nA; Fig. 2, plot f) is slightly smaller than the sum of the

currents for 1 μMDA (2.92 nA in Fig. 2, plot b) and 100 μMAA (14.80 nA in Fig. 2, plot d). This suggests that there mightbe reactions between these species or their oxidized products.The fate of DA and DAQ in the presence of AA during redoxcycling is discussed in the next section in the context of dataobtained from studies using CA.

Calibration curves of DA in the presence and absenceof 100 μM AA in aCSF buffer obtained by CA

CA was used instead of CV to produce calibration curves,because although CV is more diagnostic, CA is more quan-titative and has better temporal resolution at a specifiedpotential. Ultimately, CA, not slow-scan CV, would be theelectrochemical method of choice for redox cycling for invivo measurements, where CA would allow observation ofboth transients and basal levels of DA. The response time ofthe collector is delayed from that of the generator by thetime required for DAQ to diffuse across the gap between thegenerator and the collector. That would be approximately33 ms under mass transfer conditions (based on the Einsteinequation, Eq. 1) [73],

Δ ¼ffiffiffiffiffiffiffiffi

2Dtp

; ð1Þ

Fig. 2 Comparison of cyclic-voltammetric responses at the collector(a, c, e) and the generator (b, d, f) during redox cycling at a scan rate of0.010 Vs−1 in 1 μM DA (a, b), 100 μM AA (b, c), and their mixture(d, e) in 4-amino-2-(hydroxymethyl)propane-1,3-diol buffer (pH7.4)using a configuration similar to that in Fig. 1c, but with only twomicroband electrodes serving as the collector and eight microbandelectrodes in each of the two large generator regions flanking the

smaller collector region. A triangular potential waveform was appliedto the generator, starting at −0.100 V, sweeping to +0.500 V at a scanrate of 0.010 Vs−1, and then switching direction to sweep back at thesame scan rate to +0.500 V. Meanwhile, the collector was held at aconstant reducing potential of −0.100 V. The current measured at thecollector is plotted as a function of the applied potential at the generator.All potentials are referenced to Ag/AgCl (saturated KCl)


assuming a gap between the generator and the collector of4 μm and using a diffusion coefficient of 2.4×10−6cm2s−1

[74] for DA in striatal extracellular space.The buffer was also changed to produce the calibration

curves, from Tris to aCSF (pH7.4). The latter more closelymimics the composition of extracellular fluid in the brain,further challenging the redox cycling method and providinga more realistic representation of results that might beexpected for in vivo brain studies.

In addition, the electrode configuration was changed.Four electrodes were shorted together to form the collectorregion, instead of two, so that a larger cathodic current wouldbe obtained, and therefore improve the sensitivity for DA.The number of microbands assigned as generators was de-creased to seven on each side of the collector region, keepingthe total number of active electrodes at 18. The electrodeconfiguration is shown in Fig. 1c. This change in electrodeconfiguration was based on studies (not reported here) with awell-behaved, reversible redox species. In a solution con-taining 5 mM Ru(NH3)6Cl3 and 0.5 M KCl, the collectorcurrent increased by 19 % (from 915.1 to 1,090 nA), thegenerator current increased by 3.5 % (from 2,232 to2,311 nA), collection efficiency increased by 15 % (from41.0 % to 47.2 %), and the amplification factor increased by8.2 % (from 1.35 to 1.46). (More information on how dif-ferent assignments of collector and generator electrodes af-fect current, collection efficiencies, and amplification factorsis available elsewhere [71].)

Examples of raw CA responses under these conditionsare shown in Fig. 3. The generator current (Fig. 3, plots b, c,e, f, h, and i) responds to the potential step (from theequilibrating potential of 0.000 V to a value of +0.500 V)as expected. A large anodic faradaic transient current atinitial times (from oxidation of DA and AA) drops offapproximately with the inverse square root of time until atlong times it reaches a pseudosteady state or steady statewhen the length of the diffusion layer exceeds the generatorwidth, when mass transfer is dominated by radial diffusion,or when redox cycling becomes established, respectively.Charging current should also be superimposed on the initialfaradaic current, but it falls off exponentially with time(within microseconds) and is not distinguishable in Fig. 3.As expected, when the collector is held at −0.200 V, the DAis recycled and the anodic current at the generator is ampli-fied (Fig. 3, plots b and h), compared with the case when thecollector is left at open circuit (no redox cycling; Fig. 3,plots c and i). There is no enhancement for the solutioncontaining only AA (Fig. 3, plot e vs plot f), because thehydrolyzed DHAA cannot be recycled by the collector.

The collector current (Fig. 3, plots a, d, and g) beginsnear zero when the generator is stepped to +0.500 V. This isbecause the collector is equilibrated for 2 s prior to the stepat the same potential at which it is held during the step

(−0.200 V), which is too far negative to oxidize DA andAA, and there is no DAQ yet to reduce. Thus, the collectoris not expected to show charging or an initial cathodicfaradaic transient, with the exception of reduction of resid-ual oxygen, which is most obvious when DA is absent(Fig. 3, plot d). A cathodic current then rapidly grows atthe collector upon arrival of DAQ from the generator(Fig. 3, plots a and g), and is generated at a high rate initiallyand then more slowly as the diffusion layer at the generatoris depleted and redox cycling achieves a steady state.

Calibration curves obtained from the current measured at15 s in the CA responses for DA over a range from 5 to100 μM in the presence and absence of 100 μM AA inaCSF are shown in Fig. 4, both with and without redoxcycling at the electrode configuration illustrated in Fig. 1c.The current is linear with DA concentration (all with R2≥0.990, except for DA at the collector in the absence of AA,where R2=0.927). The y-intercept of 35 nA for the plot ofthe generator current is due to oxidation of the 100 μM AA,as expected.

There was an 8 % increase in sensitivity to DA at thegenerator electrode during redox cycling in the presence of100 μMAA (0.697±0.024 nAμM−1; Fig. 4b) compared withDA alone (0.645±0.031 nAμM−1; Fig. 4a). This could be dueto the reduction of DAQ, before it reaches the collector, by theAA that has not been completely eliminated by the largegenerator electrode configuration. This interaction would re-sult in an increased concentration gradient of DA at thegenerator, thus amplifying the anodic current. To be consistentwith this hypothesis, a decrease would be expected in theslope of the calibration curve for DA obtained at the collectorin the presence of 100 μM AA compared with DA alone.However, unlike the generator sensitivities with redox cyclingin the presence and absence of AA, the corresponding collec-tor sensitivities are not significantly different from each otherat the 95 % or 90 % confidence level, and thus that trendcannot be confirmed (0.192±0.011 nAμM−1 compared with0.209±0.034 nAμM−1, respectively). A decrease in the cali-bration slope at the generator electrode without redox cyclingwith AA compared with DA alone could not be confirmedeither, for the same reason (0.521±0.028 nAμM−1 vs 0.525±0.016 nAμM−1, respectively).

The y-intercept and the slope of the calibration curves inFig. 4b allow one to separate the contribution to anodiccurrent at the generator from oxidation of AA and DA,respectively. Thus, it is possible to obtain the collectionefficiency for DA in the presence of AA simply by takingthe ratio of the slope of the calibration curve for the mixtureat the collector to that for the mixture at the generator withredox cycling. The value (27.5±1.9 %) is significantly low-er (at the 90 % confidence level) than that in the absence ofAA (32.4±5.5 %). This is consistent with the hypothesisthat reduction of some DAQ by residual AA occurs before it


reaches the collector. Also supporting this hypothesis is theamplification factor for DA in the presence of AA, obtainedby taking the ratio of the slope of the calibration curve forthe mixture at the generator with redox cycling to that forthe mixture at the generator without redox cycling (1.34±0.08), which is significantly higher (at the 90 % confidencelevel) than that in the absence of AA (1.23±0.07). Asexpected, the collection efficiencies and amplification fac-tors for this electrode configuration for DA are still lowerthan those for an electrolyte solution containing only thereversible model compound Ru(NH3)6

3− (47.2 % and 1.46,respectively), whose fate is not affected by intramolecularrearrangements or homogeneous chemical reactions on thetimescale of the experiment.

The detection limits for DA in the presence and absenceof 100 μM AA at the electrodes with and without redox

cycling are listed in Table 1. In the presence of 100 μM AA,the current at the collector that is unique to DA provided asubmicromolar detection limit (0.454±0.026 μM). That val-ue is within the error of the detection limit for DA at thecollector in the absence of AA (0.417±0.067 μM), high-lighting the discriminating capability of redox cycling. Atthe generator, for comparison, the detection limit for DA inthe absence of AA with redox cycling (0.163±0.008 μM)was better than at the collector, as would be expected,because of its enhanced sensitivity. However, without redoxcycling, the detection limit at the generator for DA in theabsence of AA (0.107±0.003 μM) was even better, despitethe unamplified current and therefore lower sensitivity there.This unexpected result can be explained by the greater noiseat the generator when the collector is activated, and isreflected in a larger standard deviation of the blank signal,

Fig. 3 Examples of chronoamperometric responses are shown forredox cycling at the collector (a, d, g) and the generator (b, e, h) andwithout redox cycling at the generator (c, f, i) for solutions of artificialcerebrospinal fluid (aCSF) buffer (pH7.4) containing 10 μMDA (solidcurve), 25 μM DA (thick dashed curve), 50 μM DA (thin dashedcurve), and 100 μM DA (thick dotted curve) (a, b, c), 100 μM AA (d,e, f), and their mixtures (g, h, i) using the electrode configuration in

Fig. 1c. The generator was stepped to +0.500 V (from an equilibrationpotential of 0.000 V). For redox cycling, the collector’s potential wasstepped to −0.200 V (from an equilibration potential of −0.200 V). Inthe absence of redox cycling, the collector was left at open circuit. Allpotentials are referenced to Ag/AgCl (saturated KCl). (Note that thespikes in plot a at approximately 2 s and in plot d at approximately 4 sand approximately 6 s are from electronic noise.)


which is used in the calculation of the limit of detection.(The standard deviations of the blank were 0.029 nA at thecollector, 0.035 nA at the generator with redox cycling, and0.019 nA at the generator without redox cycling.)

Although the submicromolar DA detection limits arequite respectable, compared with detection limits for redoxcycling previously reported in the literature, the detection

limits are not as optimal as they could be. One reason is alimitation of the potentiostat that was used here. Thepicoamp booster for the CHI 760 potentiostat does notsupport the secondary electrodes, and therefore does notfilter out noise at the collector. With instrumentation thatcan filter out noise at the electrodes, the detection limitsshould improve further. Also the collectors are held at apotential in the oxygen reduction regime. Thus, its reductionat the collector adds to error owing to variation fromexperiment to experiment, leading to poorer detectionlimits. One way to avoid this problem would be tolessen the overpotential at the collector electrode byapplying a less negative potential, e.g., 0.000 V versusAg/AgCl (saturated KCl).

Conclusions

Redox cycling with the unique MEA configuration de-scribed here was shown to be an effective electrochemicalmethod to detect DA and discriminate it from AA atphysiologically relevant concentrations in aCSF, withoutrequiring enhanced sensitivity through electrode modifica-tion, which can significantly decrease diffusion coeffi-cients and slow response times. The detection limitsreported here are sufficient to measure the submicromolarto micromolar concentrations of DA in vivo that havebeen found with other electrochemical detection methods[64]. However, redox cycling eliminates the need to sub-tract charging current, which restricts use of fast-scan CVto measuring DA transients. Thus, redox cycling is wellpoised for in vivo measurement of basal levels of DA inthe future.

There are several other electrochemically active com-pounds that can oxidize in a similar potential windowas DA and have various re-reduction behaviors at cath-odes. These include dihydroxyphenylacetic acid, seroto-nin, norepinephrine, epinephrine, and uric acid. In vitrostudies to detect DA in the presence of these and othercompounds using redox cycling are currently under way.

Ultimately, additional studies are needed to confirm thesuitability of redox cycling for in vivo DA measurements.Several interferences in natural samples that are difficult tomimic in vitro could affect the signal at the collector, in-cluding possible restricted diffusion in tissue [75], otherhomogeneous reactions that deplete DAQ, and current fromelectron transfer of other species at the cathodic collector.However, it is encouraging that DAQ can be detected withhigh concentrations of AA in vitro, suggesting thatadditional interfering reductants that are present in vivowould also need to have such high concentration orexhibit faster reaction kinetics to make a significantimpact on the collector signal from DAQ.

Fig. 4 Calibration curves of the steady-state chronoamperometricresponses as a function of DA concentration in solutions containing aaCSF buffer (pH7.4) and b 100 μM AA in aCSF buffer, shown for thegenerator current with (open squares, iG) and without (closed squares,iG/C) redox cycling, and for the collector current (closed circles, iC).The electrode configuration in Fig. 1c was used. The large y-intercept(35 nA) for the generator current in b, but not observed for thecollectors, is due to the oxidation of the 100 μMAA there. The averagecurrent taken at approximately 15 s from triplicate chronoamperomet-ric responses was used to produce the calibration curves. Error bars ateach marker represent ± one standard deviation

Table 1 The limit of detection (LOD) for dopamine (DA) in thepresence and absence of 100 μM ascorbic acid (AA) at the generatorand at the collector with and without redox cycling in artificial cere-brospinal fluid buffer, using chronoamperometry with the electrodeconfiguration in Fig. 1c. Each LOD was calculated as three times thestandard deviation of the blank signal, divided by the slope of thecalibration curve. The standard deviation of the LOD was obtainedfrom propagation of error

Generator withoutredox cycling

Generator withredox cycling

Collector withredox cycling

LOD (DA) 0.107±0.003 μM 0.163±0.008 μM 0.417±0.067 μM

LOD (DAwith AA)

0.108±0.006 μM 0.151±0.005 μM 0.454±0.026 μM


Investigations are also under way to scale down theMEA configuration onto probes having dimensions suit-able for insertion into brain tissue to implement redoxcycling for in vivo studies. The outer dimensions of theentire MEA used here were 140 μm in width and 2 mmin length. Simply compressing the overall width dimen-sions to 70 μm, by halving the gap and electrode widths,should not change the magnitude of the collector currentbecause the enhanced signal from the narrower gapmakes up for the loss in electrode area. (This assumesthat our electrode configuration would behave the sameway upon compression as an alternating configuration ofequally sized collector and generator electrodes [76].) Anelectroactive region that has a 70-μm width is similar tothat of previously fabricated silicon probes [59] and iswithin microfabrication capabilities. Decreasing the elec-trode length to 200 μm, a length similar to that of CFEsused for in vivo studies, would decrease the cathodiccurrent at the collector by a factor of 10 (to approxi-mately 0.1 nA for 1 μM DA in 100 μm AA). Improve-ments in instrumentation with filtering and propershielding to decrease electronic noise would allow similaror better detection limits to be obtained. This should beachievable, given that instrumentation used for fast-scanCV in vivo already allows magnitudes of faradaic currentof 0.1–10 nA to be successfully measured.

MEA devices with a smaller gap between generators andcollectors can be fabricated to offer further improvements.First, the time for DAQ and DA to diffuse across the gapshould decrease dramatically, because it depends on thesquare of the distance (Eq. 1). One benefit is that the timefor the collector to respond to changes in DA concentrationwould also decrease dramatically. Another is that longerresponse times from slowed mass transport in polymers thatcould be used to modify a MEA may be offset by the shorterdistances and yield a response time similar to that of a bareMEA. Thus, coatings (thinner than the gap) may be consid-ered, especially for long-term use in vivo, to eliminateadditional interferences and protect electrodes from fouling.Second, a narrower gap would cause an increase in themagnitude of the collector signal, not only because of anincreased concentration gradient of DAQ across the gap, buteven more so because less DAQ would be lost throughintramolecular cyclization (k=0.13±0.05 s−1 at physiologi-cal pH) [77, 78] and reactions with reductants (such asresidual AA) in that space.

Acknowledgments Funding was provided in part by the NationalScience Foundation (CHE-0719097) and the Arkansas BiosciencesInstitute, the major research component of the Arkansas TobaccoSettlement Proceeds Act of 2000. We thank Errol Porter for adviceon microfabrication. The use of the High Density Electronics Centermicrofabrication facilities is also acknowledged. We express ourappreciation to Adrian Michael of the University of Pittsburgh for

insightful discussions about applications. T.S. Hollingsworth isacknowledged for assistance in preparing this manuscript.

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