Journal of Electroanalytical Chemistry 650 (2010) 105–115

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier .com/locate / je lechem

Novel ultrasensitive and ultrafast voltammetric determinationof biological aminochromes on the copper nanodoped mercurymonolayer carbon fiber electrode

Grigore Munteanu a,⇑, Eithne Dempsey a, Tim McCormac b

a Centre for Research in Electroanalytical Technologies (CREATE), Department of Science, Institute of Technology Tallaght, Tallaght, Dublin 24, Irelandb Dundalk Institute of Technology, Dundalk Co. Louth, Ireland

a r t i c l e i n f o

Article history:Received 29 June 2010Received in revised form 24 August 2010Accepted 27 August 2010Available online 21 September 2010

Keywords:Biological aminochromesElectrocatalyticFast voltammetryMercury monolayer electrode

1572-6657/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.jelechem.2010.08.016

Abbreviations: AC, adrenochrome; ACs, aminochalbumin; CAs, catecholamines; CSF, cerebro-spinal flmine; DAC, dopaminechrome; DASQ, dopaminesemdroxy-L-phenylalanine; DOPAC, DOPAchrome; EP, epreduced; MMCFE, mercury monolayer carbon fiberchrome; NEP, norepinephrine; o-BQ, 1,2-benzoquinoPyr, pyrocatechol.⇑ Corresponding author. Fax: +353 1 4042700.

E-mail address: grigore.munteanu@ittdublin.ie (G.

a b s t r a c t

The influence of the biologically important aminochromes – dopaminechrome, adrenochrome, nor-adrenochrome and DOPAchrome – on the Cu2+/0 catalytic system by fast voltammetry (v P 200 V s�1)using a mercury monolayer carbon fiber electrode was studied. It was demonstrated for the first time thatfollowing the application of a short (0.1 ms) cathodic accumulation impulse the catalytic analytical signalof the Cu2+/0-system depends linearly on the aminochrome concentration over the range 1–200 pM. Itwas found that the analytical signal of the studied aminochromes was strongly amplified in the presenceof thiols (reduced glutathione and cysteine) and protein (bovine serum albumin) in the analyzed solu-tions; while the ascorbic acid and the reduced form of aminochromes (catecholamines) did not influencethis signal (the mechanism of such influence is discussed). The analytical applicability of the elaboratedprocedure of aminochrome quantification was confirmed by comparing the analytical signal of adreno-chrome spiked with the cerebro-spinal fluid with the one of adrenochrome spiked with the bovine serumalbumin.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

The biological important catecholamines, epinephrine, norepi-nephrine and dopamine with the general formula (I)

HO

HON

H R2

R1 R1=R2=H-dopamine

R1=OH, R2=CH3-epinephrine

R1=OH, R2=H-norepinephrine

R1=COOH, R2=H -DOPA(I)

and their precursor L-3,4-dihydroxyphenylalanine (L-DOPA),which act as neurotransmitters (chemicals which assure the

ll rights reserved.

romes; BSA, bovine serumuid; Cys, cysteine; DA, dopa-

iquinone; DOPA, 3,4-dihy-inephrine; GSH, glutathioneelectrode; NAC, noradreno-

ne; p-BQ, 1,4-benzoquinone;

Munteanu).

signal exchange between neurons and other cells) play animportant role in the normal function of the human central ner-vous system [1]. The content of these highly chemically activecompounds in blood plasma has a direct impact on the appear-ance of some neurological and heart diseases such as Parkinsondisease and postural tachycardia syndrome [2]. Thus the controlof catecholamine levels in biological fluids (blood plasma, cere-bro-spinal fluid, urine, etc.) is a significant resource in the treat-ment and the prevention of these diseases. The analyticalprocedures employed to achieve this control must possess highsensitivity and selectivity, considering that the normal level ofcatecholamines in these fluids is at nanomolar level and theirchemical properties are very similar.

Despite of the importance of monitoring the catecholaminecontent, it has been suggested that diseases caused by increased(or decreased) level of such amines can be better explained assum-ing that due to oxidative stress, part of these amines is converted toit’s oxidized form which is more toxic than the reduced form [3].Taking into account that this part contributes only a few percentof the catecholamine content the analytical procedure should beable to detect and to quantify the oxidized form of catecholamine(aminochromes (II)) at picomolar level.

NOO

R2

R1

(II)

R1=R2=H-dopaminechrome, R1=OH, R2=H-noradrenochromeR1=OH, R2=CH3-adrenochrome, R2=H, R1=COOH-DOPAchrome

106 G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115

The selectivity of aminochrome determination is assured by theuse of High Pressure Liquid Chromatography (HLPC) combinedwith electrochemical (amperometric) detection [4]. This methodcan assure picomolar level detection following extraction on batchalumina [2]. However, the limit of detection of approximately10 pM may be insufficient in the case where more exact measure-ments of the aminochrome level in biofluids is required (as in caseof epinephrine in CSF [5]). Therefore, the elaboration of a fast anddirect analytical procedure of aminochrome quantification withsubpicomolar detection limits is desirable in the presence of an ex-cess of the reduced catecholamine form and still remains an impor-tant analytical problem in clinical chemistry.

Recently [6–8], we have demonstrated that the mercury mono-layer carbon fiber electrode (MMCFE) is a powerful source of highlychemically active hydrogen intermediates – H0 (atomary hydrogen,a strong reductant) and Hþ2 (the molecular ion, a strong oxidant) inconditions when the fast voltammetry (v (potential scanrate) > 100 V s�1) is utilised. Combining this electrode (by meansof nanodoping of MMCFE by the metallic copper) with the Cu2+/0-system which is catalytically sensitive to the indicated hydrogenintermediates allows quantification of substances (species) con-suming or supplying these intermediates.

Taking into account that the aminochrome molecule is pro-duced from catecholamine [9] as a result of the loss by the latterof 4 protons and 4 electrons, this process can be understood asthe abstraction of 4 hydrogen atoms from the catecholamine mol-ecule. Consequently, assuming that under suitable conditions these4 hydrogen atoms can be restored to the oxidized form, the quan-tification of such forms becomes possible by means of the mea-surement of the quantity of consumed hydrogen atoms. In otherwords, the copper nanodoped MMCFE as the source of atomaryhydrogen coupled with the Cu2+/0-system – catalytically sensitiveto these atoms, can be utilised for a rapid and sensitive voltammet-ric determination of aminochromes, considering the fast kinetics ofthe chemical catalytic reaction (k = 104–107 s�1). This representsthe main goal of the proposed work which should compensatefor the absence of a sensitive voltammetric procedure for amino-chrome quantitation.

2. Experimental

2.1. Chemicals

All solutions were prepared using high purity distilled–deion-ized water (Milli-Q, 18.2 MX cm�1). Solutions of 15.7 mM Hg2+

and 0.94 mM Cu2+ were prepared by dissolution of high puritymetallic mercury (99.9999%) and Cu (99.999%) in nitric acid(Aldrich AR). A solution of 0.01 M NaCl (ACS, Aldrich) + 0.01 Mphosphate buffer (PB) (pH = 7.0) prepared from potassiumphosphate monobasic (TraceSELECT (>99.995%), Sigma–Aldrich)and potassium phosphate dibasic (TraceSELECT (>99.999%),Sigma–Aldrich) were used as background electrolyte. The [Hg2+]in background electrolytes was kept constant, at 2 lM duringgrowth of the mercury monolayer. In the presence of micro molarconcentrations of bovine serum albumin (BSA), cysteine (Cys) orglutathione reduced (GSH) the concentration of mercuric ionswas increased to 10–20 lM thus assuring the stability of MMCFE.

Dopamine (P98.5%), L-DOPA (P98.0%), (+/-)-EP (P98%),DL-NorEP (P98%), Adrenochrome (100%), Pyrocatechol (P99%),

p-Benzoquinone (P98%), potassium ferricyanide (P99%), L-Gluta-thione reduced (P99%), L-Cysteine BioUltra (P98.5%), Bovine ser-um albumin BioChemica (P98.0%) (BSA) were acquired fromSigma–Aldrich and used without additional purification, cerebro-spinal fluid was acquired from Gemini Bio-Products (West Sacra-mento, USA).

2.2. Electrodes

The cylindrical working microelectrode was prepared from highmodulus PAN based carbon fiber VPR-19C (diam = 7 lm). Fibers,whose lengths were measured by the aid of a micrometer, wereimmersed to a depth of 7.2 ± 0.1 mm (working electrode geometricarea 0.159 mm2). Experiments were conducted in a 5 ml, three-electrode electrochemical cell using a ‘‘non-leak” Cypress SystemsAg/AgCl reference electrode and a carbon rod (diam = 2 mm, AlfaAesar) as auxiliary electrode.

2.3. Apparatus

All electrochemical experiments were performed with a CHI630 a potentiostat (CH Instruments, Austin TX, USA) in a three-electrode configuration. The fast voltammograms were registeredusing a 32 K Hz i/E converter filter and 15 K Hz signal filter. All po-tential values are quoted with respect to saturated Ag/AgCl elec-trode. The solution was stirred by argon (1 bubble/5–6 s).

2.4. Procedure

Electrochemical activation of the indicator electrode prior tomercury deposition was performed by applying 100 rectangularpulses with a positive potential (1.5 V, 0.05 s) and with a negativepotential (�1 V, 0.01 s). The mercury monolayer was depositedelectrolytically in a solution of background electrolyte containing2.0 lM Hg2+ for 500 s at a potential of �1.2 V (step repeated 8times). In addition, the quantity of deposited metallic mercurywas controlled electrochemically by dissolution of the mercurylayer under voltammetric conditions (v = 0.005 V s�1) immediatelyafter finishing the experiment. Measurements were performed atroom temperature (22 ± 1 �C). The possible influence of adsorp-tion–desorption kinetics of the Cu2+ species on the fast voltammo-grams was considered by its registration in the format of 1–4successive cycles. For calculations and figures the 2nd cycle wasemployed following extraction of the charging (or background)current (where indicated).

2.5. Aminochromes preparation from aminocatechols

The studied aminochromes were prepared using a standardscheme of aminocatechol oxidation with ferricyanide (amino-chrome:ferricyanide = 1:4) in 0.01 M phosphate buffer (pH = 7.0).To a mixture of 5.25 ll of 1 M KH2PO4 and 7.25 ll of 1 M K2HPO4

was added 0.80 ml of 1.25 mM of freshly prepared aminocatecholsolution followed by the addition of 0.20 ml of 20 mM ferricyanidesolution. The red intensive colour appears immediately followingaddition of the oxidizer. Two–three minutes later the amino-chrome solution obtained was diluted to the required concentra-tion (the final concentration 5 nM) by step 1:100. The prepared

-1.2

0.0

1.2

2.4

3.6-0.4 -0.2 0.0

E/V

j/mA

cm

-2

a

bcd

Fig. 1. Influence of chloride ions on the voltammetric peaks of the Cu2+/0 catalytic2+ �

G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115 107

ACs standard solutions were placed on ice to slow down itsdecomposition.

2.6. Measurement of voltammetric signal of aminochromes spikedwith bovine serum albumin

This procedure was realised by utilisation of two similar elec-trochemical cells (cell I and cell II). The chemical composition ofsolutions in both cells was identical except for the aminochromecontent. Into cell I the studied aminochrome solution with fixedconcentration (0.5–2 nM) was spiked with 30 (15) lM of BSA whilethe cell II did not contain aminochrome. The calibration curve ofthe aminochrome being studied was registered by addition of var-iable volumes of solution from cell I to cell II. It should be notedthat the quantity of the catalyst (Cu2+) added to BSA (or thiols) con-taining solution was determined using a procedure similar to thetitration one. To these solutions, Cu2+ was added slowly (2 minintervals) by small steps (1–2 lM) until the small and stable sig-nals of Cu2+/0-system started to appear. For BSA this quantity ran-ged between one and two equivalents. Each calibration curve (CC)was measured 2–3 times. The variation of the slopes of thesecurves compiles 10–12%.

system. [Cu ] = 50 pM; [Cl ] (mM) – 2 (a, b), 5 (c), 10 (d); Ea = �1 V, ta (s) – 20 (a),50 (b–d); v = 1000 V s�1.

3. Results and discussion

The maximal efficiency of utilisation of the catalytically activehydrogen intermediates generated by MMCFE requires fulfilmentof some conditions due to its short lifetime. The exclusion or signif-icant diminution of the diffusion distance between interacting spe-cies – the substrate and the catalyst is mainly of importance. Thissituation can most completely be realised if all interacting speciesare adsorbed on the surface of MMCFE. Taking into account thatthis electrode is a combination of the carbon support and a mer-cury monolayer it is reasonable to use the well known effect ofmercury electrodes to facilitate adsorption of the studied specieson the surface of MMCFE. The most promising, from our point ofview, is the effect of anion-induced adsorption of metal ions [10]by the specifically adsorbed on mercury anions.

3.1. Chloride-induced adsorption of ions Cu2+

The mass transfer to/from microelectrodes is much more rapidrelative to macroelectrodes [11] implying the necessity to retainCu2+ ions (catalyst) on the surface of MMCFE following anodic dis-solution of the electrolytically deposited (during the accumulationstep) metallic copper. Despite the large number of possible ligandsable to adsorb on the mercury layer and to form a complexes withCu2+, we have chosen chloride ions (Cl�), which contribute also tothe stability of the mercury monolayer on the carbon support dueto binding of the free form of mercury ions Hg2+ in a complex thatdecreases substantially the rate of the disproportion reaction be-tween the metallic mercury and the ions Hg2+ through formationof calomel (Hg2Cl2).

It is well known that Cu2+ ions in the presence of chloride ionsare able to form complexes of general formula CuCl2�p

p where the pranges between 1 and 4 depending on the chloride concentration[12] and that for the micromolar solutions of Cu2+ containing10 mM of Cl� the complex CuCl+ is the predominant form in solu-tion (reaction (20)) due to it higher stability constant. The registra-tion of cyclic voltammograms of Cu2+/0-system (Fig. 1) indicatesthat chloride ions have a significant influence on the appearanceand the magnitude of the catalytic currents of this system(10 mM concentration of chloride ions was found to be optimal).

The graph of the dependence Ep on log [Cl�] for anodic peaks rep-resents a straight line with a slope amounting 0.063 V dec�1. This

value (with correction for the quasireversibility of the electrochem-ical process) means [13] that anodic dissolution of metallic copper isaccompanied by a chemical reaction on the surface of electroderesulting from the formation of the species CuCl2�p

p wherep = 0.9 � 1. Thus the data presented above (Fig. 1) allows the conclu-sion that chloride ions can influence the catalytic currents of theCu2+/0-system mainly via the following reactions (processes):

Cl�sol ! Cl�adsðHgÞ ð1Þ

Cu2þsol þ Cl�ads ! CuClþadsðHgÞ ð2Þ

Cu2þsol þ Cl�sol ! CuClþsol ! CuClþadsðHgÞ ð20 Þ

The subscripts sol refers to species in solutions, while ads forthe ones adsorbed on the electrode surface.

3.2. Open circuits experiments with the catecholamines andaminochromes

Preliminary voltammetric results have shown a certain catalyticactivity of amninochromes regarding the Cu2+/0-system whilst thecatecholamines were catalytically inactive. To clarify this differ-ence we have studied the influence of these compounds on theopen circuit potential (Eoc) of MMCFE which is sensitive to changesoccurring in the electrical double layer of the working electrode,mostly in conditions when some species dissolved in backgroundelectrolyte adsorb on the surface of this electrode. The results ofthis study (Fig. 2A and B) show a significant influence of the amino-chromes on the Eoc in comparison with the catecholamines. Suchsubstantial differences can be explained by the adsorption on thesurface of MMCFE of the former while the latter do not adsorb.Only dopamine at concentrations exceeding the micromolar level(Fig. 2A curve d) shows that it adsorbs on MMCFE, but such levelof concentrations is irrelevant according to the goal of this work.

On the contrary, the Eoc of MMCFE changes significantly in thepresence of aminochromes at nanomolar level of concentrations(Fig. 2B). Open circuit experiments also point to a significant differ-ence between para-benzoquinone (p-BQ) and ortho-benzoquinone(o-BQ), product of pyrocatechol oxidation (whose radical is a com-ponent of catecholamines) using ferricyanide (Fig. 2B, curves b and

100

120

140

160

1800 25 50 75 100

t/s

Eoc

/mV

a

b

c

d

e B90

120

150

1800 25 50 75 100

t/s

Eoc

/mV

a

b

cde

f

g

D

140

150

160

170

1800 25 50 75 100

t/s

Eoc

/mV

-100

-75

-50

-25

0

Eoc

/mV

-100

-30

40

110

1800 25 50 75 100

t/s

Eoc

/mV

a

b

c

d

e

CA

a

c

d

e

b

Fig. 2. Open circuit potential of MMCFE alone ((a) – A–D) and in the presence of: A – 10 lM EP (b), 1 lM Pyr (c), 1 lM DA (d), 20 lM GSH (e); B – 10 lM p-BQ (b), 0.1 lM o-BQ(c), 0.2 lM DAC (d), 0.2 lM AC (e); C – [GSH] (lM) – 20 (b, c, e), 20 lM Cys (d), [AC] (nM) – 10 (d), 200 (e); D – 30 lM BSA (b, c), 30 lM BSA + 10 lM Cu2+ (d), 30 lMBSA + 20 lM Cu2+ (e), 30 lM BSA + 30 lM Cu2+ (f), 30 lM BSA + 32 lM Cu2+ +1 nM DAC (g); [Cu2+](nM) – 0.05 (a (A–D)), (d, e (A)), (c (C)), (b (D)); 25 – (d, e (B)), 50 – (b, c (A));200 – (d (C)); 400 – (e (C)); [Hg2+](lM) – 2 (a–e (A), (a, b (C), a (B, D), (b–e (B), b (D)), 10 (c (C)), 20 – (e (C), c–g (D)).

108 G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115

c). In this case, the first compound para-benzoquinone, is catalyti-cally inactive regarding the Cu2+/0-system in contrast to ortho-benzoquinone.

The biosample matrix varies both in chemical composition andconcentration level of the components. Considering the presence ofCu2+ (catalyst) and Hg2+ in the background electrolyte which en-sure the stability of the mercury monolayer on the carbon support,it is important to know the influence of this matrix (its main com-ponents) on the concentration of these ions, which control theappearance and the magnitude of catalytic currents of the Cu2+/0-system, as well on the process of catalyst adsorption on the surfaceof MMCFE. Such useful information (in addition to voltammetricstudies) can also be obtained from the monitoring of Eoc.

In order to achieve this, we have chosen bovine serum albumin(BSA) as the protein component of the matrix, whose chemicalcomposition and binding capacity of ions Cu2+ is very close to hu-man serum albumin (HSA) [14], the reduced form of glutathione(GSH) and cysteine (Cys), present in such matrices at concentra-tions exceeding the micromolar level. These compounds interactstrongly with the indicated ions and can specifically adsorb onthe mercury layer via their sulfur atom(s).

The results of measurement of Eoc of MMCFE in the presence ofthese main matrix components and variable concentration of Cu2+

and Hg2+ are presented in Fig. 2C and D. The open circuit potentialof MMCFE in solutions containing 20 lM of GSH or Cys (Fig. 2C,curves b and d) changes considerably – its value decreased by morethan by 100 mV. In so doing the influence of GSH is more accentu-ated, which can be explained by its stronger adsorption on MMCFE,

as well as by the difference between the effective charges of thesecompounds (�1.02 in the case of GSH (pH = 7.0) and �0.04 for Cys– the effective charge was calculated as sum of all charges of iono-genic groups, including their signs, considering the dissociationconstants of these groups). The Eoc reached positive values in thecase when the Hg2+ concentration (Fig. 2C, curve c) or the Cu2+ con-centration (Fig. 2C, curve e) increases. A reasonable explanation ofthis effect is the binding of ions Cu2+ and Hg2+ by adsorbed thiols, afact which should be considered upon optimisation of the experi-mental conditions for registration of the aminochrome analytical(voltammetric) signal.

The influence of bovine serum albumin on the open circuit po-tential of MMCFE is similar to that observed for the studied thiols(Fig. 2D), which can be explained by the presence in its chemicalstructure [15] of functional groups, similar to these of GSH andCys. On the other hand, it can be observed that the variation ofEoc is less pronounced than for thiols, despite the bigger effectivecharge of BSA molecule [16] (ca. �8). This apparent contradictioncan be easily explained considering the dimensions of thiol andBSA molecules and of the compact Helmholtz layer, wherespecifically adsorbed species (ions or molecules) [17] are located.The radius of the BSA molecule (macromolecule) is about one orderbigger than of Cys and GSH molecules. Consequently, the numberof BSA molecules that can penetrate the compact Helmholtz layeris significantly smaller than that of Cys and GSH, an effect whichattenuates the difference between its effective charges.

The registered differences in open circuit experiments betweencatecholamines, aminochromes, o-BQ and p-BQ can be explained

G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115 109

taking into account the role of Cu2+ in (adsorption) processeswhich have a strong influence on the value of Eoc. Namely, this roleis determined by the capacity of Cu2+ ions to form simple, one li-gand complexes as well the mixed-ligand complexes [18]. Consid-ering our studies and the anion-induced adsorption of ions Cu2+ onthe surface of MMCFE the most realistic explanation of these differ-ences is the assumption that aminochromes adsorb on the MMCFEdue to its complexation with the Cu2+ ions adsorbed there. Infavour of this assumption are the following well known facts.Harley-Mason [19] has proposed a zwitterion form (III)

NO

-O

R2

R1

(III)

+

of aminochromes which better reflect the chemical properties ofthese compounds. The structure (III) differs from (II) only in elec-tron distribution. We presume that structure (III) facilitates thecomplexation of aminochromes by the Cu2+ ions adsorbed on theelectrode as a result of reaction (3).

+N

OO

R2

R1N

OO

R2

R1Cu

+Cu

2++

ads

(sol) (ads)

2N

O-

O

R2

R1+

ð3Þ

The dissociation constants (pK) of hydroxyl (catechol) groups ofcatecholamines are bigger than 9 [20] and at pH = 7.0 these groupsare undissociated, thus hindering the complexation process withCu2+. The different effect of o-BQ and p-BQ on the Eoc suggests thatthe ortho configuration of the quinone radical is preferable for Cu2+

complexation, which can explain the observed selectivity of o-qui-none determination using tyrosinase [21], a copper-containingenzyme.

3.3. Cyclic voltammetry of aminochromes

The voltammetric catalytic activity of aminochromes suggeststhat these compounds undergo a (electro)chemical reductionprocess as a result of the application of a short (0.1 ms) cathodicimpulse (Ea = �1.8 V) before registration of the cyclic voltammo-gram of the catalytic Cu2+/0-system. It was demonstrated [22] thatchemical oxidation of a catecholamine molecule by ferricyanide atpH = 7 involve transfer of four electrons and the release of 5 pro-tons (the 5th proton from the amino group) indicating formationof the aminochrome (III). The aminochrome formation pathway in-cludes formation of intermediates with different lifetime as a re-sult of the consecutive [4] reactions (4).

The reaction (4) presented above indicates that the reductionof aminochrome (structure III) can result in formation of inter-mediates (Ia)-(Id) as well as initial catecholamine (I). In otherwords, the reduction process can involve participation of 1–3or 4 electrons and protons (that is equivalent to 1–3 or 4 hydro-gen atoms). To clarify this fact we have compared the influenceon the Cu2+/0-sytem of similar compounds (o-BQ, able to accepttwo hydrogen atoms) or a product of soft dopamine oxidation(using Hg2+ ions [23]) having a yellow colour which differs fromthe red coloured DAC and is therefore able to accept less thanfour electrons (hydrogen atoms). The voltammograms of thesecompounds differ substantially (Fig. 3). The product of DA oxida-tion by Hg2+ has the smallest influence on the voltammetricpeaks of the Cu2+/0-sytem (Fig. 3, curve a), while the o-BQ vol-tammetric signal is only about two times smaller than that ofdopaminochrome (Fig. 3, curves b and c). These results can beexplained assuming that DAC reduction on the MMCFE results

in regeneration of the initial dopamine, accompanied by acquisi-tion of four electrons and four protons (or four hydrogen atoms).The weak influence of the product of dopamine oxidation bymercuric ions is probably caused by formation of DASQ (Ia)which binds strongly to Hg2+ (during the oxidation reaction) ina complex similar to that presented in reaction (3) hinderingits reduction on the MMCFE.

Thus, the appearance of catalytic voltammetric signal of amino-chromes on MMCFE can be explained by the occurrence of the fol-lowing chemical and electrochemical reactions taking into accountthat the cations Cu2+ catalyze the reaction of catecholamine oxida-tion to aminochromes [8]:

Acuumulation step (cathodic impulse)

Hþads þ e� ! H0ads ð5Þ

ð4Þ

+N

OO

R2

R1N

OO

R2

R1Cu

+

(ads)

+ 2e-

(ads)N

O-

O

R2

R1+2 + Cu

0(Hg) ð6Þ

ð7Þ

ð8Þ

-1.50

-0.75

0.00

0.75

1.50

-0.6 -0.4 -0.2 0.0 0.2E/V

j/ m

A cm

-2

a

b

c

Fig. 3. The influence of the oxidant on the catalytic currents of oxidized dopamine(a, c) and pyrocatechol (b) (background current extracted). The oxidant – 10 mMsolution of Hg2+ (a), 4 mM solution of K3[Fe(CN)6] (b, c), Ox:Red = 5:1 (a), 4:1 (b, c).[DASQ] = 200 nM, [o-BQ] = 50 nM, [DAC] = 20 nM; [Cu2+] (nM) – 25 (a, c), 50 (b);Ea = �1.8 V, ta = 0.1 ms, v = 1000 V s�1.

-4

-2

0

2

4

6-0.6 -0.4 -0.2 0.0 0.2

E/V

j/ m

A c

m-2

a

b

c

d

-1.4

-0.7

0.0

0.7

1.4

2.1

j/ m

A c

m-2

Fig. 4. The effect of glutathione reduced on the catalytic currents of adrenochrome.[AC](pM) – 2000 (a), 20 (b, d), 0 (c); [Cu2+](nM) – 25 (a), 500 (b-d), [GSH](lM) – 0(a), 20 (b–d), background current extracted (a, b), original voltammograms (c, d).Ea = �1.8 V, ta = 0.1 ms, v = 200 V s�1.

110 G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115

The chemical reactions (10) and (12) contribute to an additional(in parallel with reactions (5) and (8)) regeneration of the reducedand oxidized catalyst species as a result the voltammetric peakcurrents of these species increase in comparison with the ACs lesssolutions.

3.3.1. Cyclic voltammetry of aminochromes in thiol containingsolutions

Taking into consideration compounds present in biological fluidsin addition to aminochromes, we have studied the influence of thiols(GSH, Cys) on the catalytic signal in view of their specifically adsorp-

G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115 111

tion on MMCFE, capacity to bind both Cu2+ and Hg2+ and protolyticactivity [8]. These experiments gave unexpected results consideringthe thiol’s capacity to reduce aminochromes chemically [24]. Thestudied thiols, at concentrations comparable with those in biofluids,instead of decreasing the analytical signal, their presence increasedit by about two orders (Fig. 4, curves a, b). This apparent contradic-tion can be explained making allowance for all ‘‘actors” presentand their roles in generation of the voltammetric catalytic signal.Namely we should consider the presence of Cu2+ and specifically ad-sorbed thiols on the MMCFE surface, species that have oppositeinfluence on aminochromes. The Cu2+ ions contribute to conversionof reduced catecholamines (reaction (10)) to aminochromes, whilethiols can reduce these compounds, possibly to leukoaminochrome(structure Ic) [23]. We can assume that the significantly increasedcatalytic signal of aminochromes in the presence of thiols (RSH) iscaused by the repetition of the following fast chemical reactionswhich promote recycling of the aminochrome.

ð13Þ

ð14Þ

In addition, the possibility of the occurrence of reaction (15) shouldbe considered which can prevent (or slow) the further cyclisation ofthe thiol-catecholamine adduct [25]. Exclusion of one chemicalreaction (step) can contribute to the increase of the overall rate ofthe catalytic chemical reaction(s).

ð15Þ

3.3.2. Cyclic voltammetry of aminochromes in protein (BSA)containing solutions

The presence of protein (BSA) in the background electrolyte hasa similar effect to that of thiol (Fig. 5A, curves a, b). Such a similar-ity appears to be due to the chemical composition of the BSA mol-ecule, which contains one sulfhydryl group –SH, a functional groupcharacteristic for thiols. It is well known [26] that proteins de-crease the hydrogen evolution overpotential on mercury elec-trodes, consequently the quantity of atomary hydrogen onMMCFE with adsorbed BSA increases also considering the Vol-mer–Tafel mechanism of hydrogen evolution [27]. Our open circuitexperiments have indicated towards strong binding of the catalyst(Cu2+) ions by BSA, consequently taking into account the capacityof these ions to form complexes with aminochromes from solutionthe quantity of the later on the electrode surface can increase also.The possibility that BSA could bind an aminochrome through sulf-hydryl functional group [15] should also be considered. Thus, whenadsorbed on the MMCFE surface BSA serves as catalytic matrix –concentrating the species generated there and amplifying the vol-tammetric signal of the aminochromes. A similar catalytic effect for

epinephrine oxidation in the presence of BSA and Cu2+ was ob-served previously [28].

The cathodic voltammetric peak of the Cu2+/0-system in thepresence of BSA surpasses significantly the anodic one, an effectthat can be attributed mainly to the considerable augmentationof the quantity of atomary hydrogen on the surface of MMCFE.As a result, the process of anodic oxidation of copper slows downbecause this hydrogen intermediate is a relative strong reductant.

The significant increase in the quantity of the atomary hydrogenin the presence of BSA (thiols) on the surface of MMCFE also cre-ates conditions to promote the occurrence of reaction (7) after(10) in parallel with reaction (14) due to the extremely small quan-tity of ACs on the electrode surface. However, the influence of BSAchanges notably in conditions when the aminochromes are spikedwith a protein excess before registration of its signal. As a resulttheir voltammetric electrocatalytic signal increases by approxi-mately 4–5 times (Fig. 5A, curves c, d) in comparison with nonspiked aminochromes. In this case the difference between theadrenochrome prepared by oxidizing EP (Fig. 5A, curve c) and thecommercially available adrenochrome (Fig. 5A, curve d) is insignif-icant. This unexpected positive effect of protein increases not onlysensitivity but also the linear region of the calibration curve (CC)(presented below).

This significant increase in sensitivity can be explained con-sidering the intrinsic property of the protein to change overtime, following interaction with the aminochromes. Despite thelarge number of protein properties, such as numerous specificgroups (dissociated or non), large molecular mass, large numberof different amino acids residues, we suppose that only thechange of the conformational structure of the protein contributeto this effect. This hypothesis is in agreement with the previousexperimental facts [29–31] that have demonstrated that thehydrogen catalytic activity of BSA increases as a result of itunfolding. Thus, unfolded (denaturated) BSA, probably due toits stronger adsorption, in comparison with the unfolded form,facilitates the access of its catalytically (protolytic) active groupsto surface of MMCFE which further increases the quantity ofatomary hydrogen there.

We can deduce that increasing the linear region of the calibra-tion curve for aminochromes in the presence of protein (BSA)results from the significant decrease of diffusion of these com-pounds to the surface, due to enhanced adsorption favoured bythe adsorbed BSA in addition to the increased quantity of atomaryhydrogen. The presence of BSA in the analyzed solutions results inan increase of the analytical signal for studied compounds (Fig. 5B).In addition, spiking of aminochrome solutions with BSA has similareffect on ACs signals of all studied aminochromes. The effects ofBSA are weakly pronounced for o-BQ which allows the voltammet-ric determination of aminochromes on MMCFE in the presence ofan excess of such quinones.

3.3.3. Influence of potential scan rateThe potential scan rate (v) is an important parameter which has

a strong influence on the sensitivity and selectivity of voltammet-

0.0

1.5

3.0

4.5

6.0

0 1 2 3 4

log v(Vs-1)

j p/ mA

cm

-2

0

-10

-20

-30

-40

j p/ mA

cm

-2

a b

A

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4

log v(Vs-1)

Qca

tal/Q

diff

x 1

0-7

a

b

B

Fig. 6. Influence of potential scan rate on the Cu2+/0 peak currents (A) and catalytic efficiency (B) in the system MMCFE-Cu2+-BSA-AC. [Cu2+] = 39 lM, [BSA] = 30 lM,[AC] = 500 pM; a – currents (efficiency) in anodic scan, b – currents (efficiency) in cathodic scan; Ea = �1.8 V, ta = 0.1 ms.

-6

-4

-2

0

2

4-0.6 -0.4 -0.2 0.0 0.2 0.4

E/V

j/ m

A c

m-2

ab

c

d

-6

-3

0

3

6-0.6 -0.4 -0.2 0.0 0.2

E/V

j/ m

A c

m-2

a

b

c

d

A B

Fig. 5. A – Spiking effect of adrenochrome with BSA on it catalytic currents (background current extracted) ([AC] = 50 pM, obtained by oxidizing of EP – (a, c), from Sigma–Aldrich (d); [GSH] = 20 lM (a); added to 30 lM BSA (b), spiked with 30 lM BSA (c, d)); B – comparison of 200 pM solution of all aminochromes (adrenochrome (a),DOPAchrome (b), noradrenochrome (c), dopaminechrome (d)). Ea = �1.8 V, ta = 0.1 ms, v = 200 V s�1.

112 G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115

ric analytical procedures. The role of this parameter increases sig-nificantly in conditions when the electrode reaction generating avoltammetric analytical signal is coupled with one or more chem-ical reactions [8] (similar to reactions (7) and (10) in our case).Therefore, correlation of the experimental time window with thecharacteristic lifetime of chemical reactions and its products bymeans of this parameter can contribute to an increase in the sen-sitivity for voltammetric determination. Registration of cyclic vol-tammograms of Cu2+/0-system in a large scan rate range v (1–5000 V s�1) confirms the data presented above. The cathodic andanodic peaks currents (Fig. 6A) have a comparable magnitude inthe relatively slow (1–50 V s�1) potential scan rate range, a domainwhere the influence of hydrogen intermediates is relatively weak

due to its short lifetime. Further increases in v result in distinct dif-ferences between these peaks: the cathodic one rising abruptlywhile the anodic peak current remains practically constant(Fig. 6A, curves a, b). The continued increase in the cathodic peakcurrent without reaching a plateau indicates to an unusually highrate of chemical reactions which have a strong contribution to thecatalytic signal generated.

The evaluation of the rate constant of chemical reaction(s)(kcatal) is a non trivial problem because we consider that at least foursuch chemical reactions (10, 12–14) take place influencing both thevoltammetric peak current and the peak potential (Ep) of catalyticCu2+/0-system. The value of kcatal can be roughly evaluated fromthe difference between Ep measured in the presence of BSA and in

G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115 113

it absence (Ep,n) utilising the Laviron procedure (Eq. (12) [32])assuming that chemical reactions are of first order. This differencefor v = 200 V s�1 is approximately 54 mV for the cathodic peakwhich results in kcatal = 1.4 � 108 s�1 for a reversible electrochemi-cal process (n = 4, reactions (7) and (10)). Taking into account thatthe electrochemical process at v = 200 V s�1 is irreversible (assum-ing that the cathodic transfer coefficient a = 0.5) the value of kcatal

becomes equal to 0.7 � 108 s�1. We can conclude then that registra-tion of voltammetric signal of ACs at picomolar level is due, mainly,to the unusual fast chemical (catalytic) reactions coupled to elec-trode reaction.

It is interesting to estimate the catalytic efficiency of theproposed voltammetric procedure for aminochrome determinationin comparison with a diffusion limited electrochemical process,considering the strong influence of the fast chemical (catalytic)reaction(s). To this end we have calculated the ratio of themeasured electric charge of the voltammetric peaks to the calcu-lated value for a diffusion limited process taking place during theapplied accumulation step (impulse) [33]. The results of suchcalculations (Fig. 6B) confirm the strong influence of the chemicalreaction(s) on the measured peak currents resulting in a catalyticefficiency of an order 107 which is in agreement with the evaluatedvalue of the rate constant of the chemical (catalytic) reaction(s).The data presented in Fig. 6B indicate that maximal catalytic effi-

Table 1Comparison of metrological characteristics of calibration curve of aminochromes and oxid

Compound Protolytic (binding) agent CC slope (A nM�1 cm�2 s�1)

DAC Not 1.22 ± 0.01a

0.92 ± 0.01c

DASQd Not 0.013 ± 0.001a

0.008 ± 0.001c

o-BQe Not 0.58 ± 0.01a

0.39 ± 0.01c

o-BQe 30 lM BSA 0.56 ± 0.01a

1.63 ± 0.03c

o-BQe 30 lM BSA (spiked) 0.77 ± 0.02a

3.40 ± 0.03c

DAC 30 lM BSA 30 ± 1a

253 ± 7c

NAC 30 lM BSA 121 ± 13a

258 ± 1c

NAC 30 lM BSA (spiked) 412 ± 17a

967 ± 16c

DOPAC 36 ± 1a

132 ± 3c

AC Not 3.79 ± 0.04a

2.01 ± 0.02c

AC Notf 2.21 ± 0.03a

1.48 ± 0.02c

AC 20 lM GSH 164 ± 1a

166 ± 1c

AC 10 lM GSH + 10 lM Cysg 118 ± 10a

164 ± 9c

AC 30 lM BSA 44.3 ± 0.3a

145 ± 1c

AC 30 lM BSA (spiked) 65 ± 6a

704 ± 12c

AC 30 lM BSA (spiked)h 200 ± 4a

566 ± 7c

AC 30 lM BSAi (spiked) 205 ± 9a

852 ± 19c

a Anodic.b In nM.c Cathodic.d Product of DA oxidation by Hg2+.e Product of Pyr oxidation by FeðCNÞ3�6 .f In the presence of 2 lM EP.g 100 lM ascorbic acid.h 6 lM EP.i Adrenochrome (Sigma–Aldrich).

ciency for both anodic and cathodic processes are registered for vranging between 50 and 200 V s�1. In this case, the lower limit re-fers to the anodic process while the upper one for the cathodic pro-cess. This difference, probably, is caused by an abrupt increase ofthe surface density of atomary hydrogen in the presence of protein(BSA) that hinders anodic oxidation of copper, diminishing the sur-face concentration of catalyst (Cu2+) during the narrowing of theexperimental time window.

3.4. Calibration curve of aminochromes

The registration of the calibration curve allows evaluation ofboth the metrological characteristics of the studied analyticalprocedure as well as examining the application of the method ofstandard additions for analytical determinations in complicatedanalytical matrices. The data presented above data indicates thatthe sensitivity of aminochrome determination is stronglyinfluenced by the compounds usually accompanying the analytein biological samples. Consequently we have measured the calibra-tion curves for different solution compositions. The results are pre-sented in Table 1.

The calibration curve under optimal conditions presents astraight line passing through the origins of coordinates (for exam-ple the equation of CC of NAC is jp/A nM�1 cm�2 s�1 =

ized forms of catechols (Ea = �1.8 V, ta = 0.1 ms, v = 200 V s�1; P = 0.95).

Linearity (pM) LOD (pM) Catalytic efficiency � 10�7

50–5000 28 0.00233 0.002

50–1000b 187 0.0000387 0.00003

0.5–20b 38 0.00153 0.001

0.2–40b 59 0.00655 0.002

0.2–40b 88 0.01160 0.003

1–300 0.07 0.370.08 5.08

1–400 0.31 1.930.04 4.86

1–200 0.12 7.600.05 18.2

1–300 0.12 0.460.07 2.27

50–2000 31 0.00430 0.003

50–2000 41 0.00441 0.003

1–50 0.37 0.920.36 1.19

1–10 0.26 5.160.17 5.05

1–500 0.02 0.620.02 2.30

1–200 0.28 0.560.05 14.2

1–200 0.06 3.040.04 11.4

1–200 0.13 3.210.07 15.0

114 G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115

(967 ± 16)c + 489 for peak currents and Qp/C nM�1 cm�2 s�1 =(1.03 ± 0.02)c � 0.89 for peak electric charges) which confirmsthe application of standard addition method. Nevertheless the sen-sitivity and the linearity domain (dynamic range) are stronglydependent on the composition of analyzed solutions (Table 1).The slope of the calibration curve in the absence of the binding(protolytic) agents (thiols, BSA) range between 1 and 4 A n-M�1 cm�2 s�1 being greater for anodic currents relative to cathodic.The limit of detection (LOD) in this case is approximately 30 pM(calculated using three S/N criteria). The slope of the curve changesconsiderably for thiol containing solutions. In the case of adreno-chrome the CC slope becomes approximately 80 times bigger andit value is comparable for both anodic and cathodic currents. Thisresults in a LOD of 1–5 pM.

If the analyzed solution contains 15–30 lM of protein (BSA) theslope of CC further increases and this slope for cathodic currentssurpasses that of anodic currents about 3–8 times. The LOD inthe presence of BSA decreases to subpicomolar level. In conditionswhen the aminochrome solutions are spiked with BSA the slope ofthe calibration curve increases relative to non spiked solutions ofBSA. In so doing the slope based on the cathodic peaks increasesdistinctly while for the the anodic peak currents this increase isless pronounced. This effect is most probably a consequence ofthe augmentation of the quantity of atomary hydrogen involvedin chemical catalytic reactions (reactions (7) and (13)). Such a dif-ference makes it more preferable to use the cathodic peak currentsfor quantification.

It should be noted that the sensitivity obtained for nor-adrenochrome corresponds to the registration of a signal of 6molecules of aminochrome on the MMCFE electrode with a sur-face area of 0.00159 cm2, assuming that the process of its elec-trochemical transformation (reduction) is a diffusion limitedprocess.

The slopes of the calibration curves for all aminochromes inves-tigated are very close due to their similar chemical properties andutilisation of the same catalytic (Cu2+/0) system for signal registra-tion. As a result it is problematic to propose an analytical proce-dure for separate quantification of aminochromes from itsmixture based on the differences of these slopes. In this case it ispreferable to apply chromatography or microfluidic systems forthe preliminarily separation stage.

3.4.1. Interference of catecholamines on the signal of aminochromesThe quantification of the aminochromes examined on the

MMCFE by means of the electrocatalytic signal of the Cu2+/0-systemrequired a study of the influence of catecholamines, a mixture ofaminochromes and other interfering compounds on the signal ofthis system. Considering this, two clearly different situations exist,the presence of binding (protolytic) agent such as thiols or BSA andthe absence of these compounds. For the first case the possible

Table 2Recovery of adrenochrome spiked with cerebro-spinal fluid (n = 4; P = 0.95).

Added (pM) Measuredtotal (pM)

Spiked with CSFrecovered (%)

Spiked with CSF Spiked with BSA

10 0 8.0a 800 12.0b 120

20 20 42.0a 11020 39.4b 97

30 40 68.0a 9340 70.5b 101

Mean 100 ± 14

a Cathodic peak current.b Anodic peak current.

interferences can influence the Cu2+/0-system only after additionof a small excess of free (non bound to thiol or BSA) Cu2+ions. Inother words, this interference has not retroactive character andthe interfering compounds present in the analyzed solution cannotinfluence directly the signal of the analyzed aminochromes. In sodoing, a big excess (6 lM) of epinephrine added to analyzed solu-tion before addition of Cu2+ in the presence of 30 lM of BSA doesnot influence the signal of adrenochrome over the range 1–300 pM (compare CC slope 704 ± 12 A nM�1 cm�2 s�1 without EPand 593 ± 5 A nM�1 cm�2 s�1 in the presence of 6 lM of EP). How-ever, if the reduced form of aminochrome (catecholamine) is addedafter titration of BSA with Cu2+ this form has a strong interferenceon the signal of adrenochrome. If the concentration of catechol-amine exceeds the adrenochrome one by 2–5 times the interfer-ence results in a decrease of the signal of aminochrome. If thisexcess is greater, the signal of the catecholamine becomes predom-inant. In this case BSA should be replaced by a thiol (GSH for exam-ple), which is unable to bind the catecholamines and as a result theinterference effect of these compounds may be excluded.

We have also studied the influence of the reduced form of cat-echolamines (epinephrine) and of ascorbic acid at physiologicallevels on the slope of the calibration curve (Table 1) in the absenceof the binding (protolytic) agent. The results of this study indicatethat ascorbic acid has an insignificant influence, whilst epinephrinecontributes to a certain decrease in the slope. This effect can beattributed to a disproportion reaction (16) taking place in solution[34] which results in a decrease in aminochrome concentration.However, this reaction is slow due to a large difference betweenconcentrations of interacting species and it influence can be furtherdiminished by dilution of solutions containing an excess ofcatecholamines.

ð16Þ

Upon investigating the possibility of quantification of a mixtureof two or more aminochromes we have shown that the analyticalsignal generated by a mixture of two or three aminochromes isnot equal to the sum of signals of individual aminochromes, whichcan be explained by the diminishing of the mole fraction of amino-chromes in the mixture resulting in a decrease of its adsorption. Asimilar effect was observed in the presence of BSA as well. This ef-fect indicates competition between aminochromes for bindingsites of BSA in the first case and Cu2+ in the absence of BSA. Thus,correct quantification of the mixture of aminochromes becomespractically impossible considering that the slopes of the calibrationcurves are similar (Table 1).

The results presented above demonstrate that the proposed vol-tammetric procedure for individual aminochrome determinationcan be successfully applied to quantification of these biologicallyimportant compounds in biological fluids due to its high sensitiv-ity, speed and selectivity regarding catecholamines and ascorbicacid.

3.5. Analytical applicability

We have tested the analytical applicability of this voltammetricprocedure taking into account the features of the analytical signalgeneration, namely the strong influence of the protein concentra-tion on this signal and the necessity to titrate the protein withCu2+ to create a small quantity of these ions unbound to protein.

G. Munteanu et al. / Journal of Electroanalytical Chemistry 650 (2010) 105–115 115

3.5.1. Registration of combined calibration curve of adrenochromespiked with cerebro-spinal fluid

0.100 ml of CSF was spiked with 0.040 ml of 5 nM adreno-chrome and the volume was adjusted to 0.200 ml. 0.050 ml of thissolution was added to the background electrolyte (5 ml) containing30 lM of BSA, 34 lM Cu2+ and 20 lM of Hg2+ followed by the reg-istration of cyclic voltammogram of Cu2+/0-catalytic system. Thenext addition of spiked CSF was realised after two consecutiveadditions of adrenochrome standard solution (the same volumeand concentration) (spiked with 30 lM BSA) and registration ofthe analytical signal. The signals of three additions of spiked CSFset on the same linear calibration curve of Adrenochrome (CCequation jp/A nM�1 cm�2 s�1 = (305 ± 10)c + 153 for cathodic peakcurrents and jp/A nM�1 cm�2 s�1 = (37 ± 2)c + 54 for anodic peakcurrents), that allows calculating the recovery of AC spiked withCSF. The results of this calculus are presented in Table 2.

4. Conclusions

The analysis of biological important aminochromes – dop-aminechrome, adrenochrome, noradrenochrome and DOPAchrome– on the copper nanodoped mercury monolayer carbon fiber elec-trode has demonstrated that the voltammetric Cu2+/0 catalytic sys-tem in the presence of studied aminochromes exhibit an increaseof the peak currents which depends linearly on the aminochromesconcentration in solution. The open circuit experiments as well fastcyclic voltammetry have contributed to elucidation of the catalyticmechanism of aminochromes signal generation.

Cu2+ ions adsorb on the electrode inducing aminochromesadsorption due to complex formation. The adsorbed amino-chromes are reduced to catecholamines under the action of atom-ary hydrogen resulting from the application of a short cathodicimpulse. Cu2+ catalyses the re-oxidation of the catecholamines toaminochromes resulting in an increase in the voltammetric peakcurrents of the Cu2+/0-system proportional to the aminochromesconcentration.

The presence of thiols and/or protein in the solution contributesto an increase in the aminochromes signal by about two ordersassuring a subpicomolar detection limit for these compounds andenlarging the dynamic range of determinations. The picomolarsensitivity, selectivity to major components accompanying theaminochromes in biosamples and the speed of the proposedvoltammetric procedure for aminochromes quantification guaran-tees it success as a powerful tool in clinical chemistry.

The analytical applicability of elaborated catalytic voltammetricprocedure of aminochromes quantification has been confirmed bypractical 100% recovery of the adrenochrome spiked with the cere-bro-spinal fluid.

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