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Journal of Electroanalytical Chemistry 495 (2001) 87–97

Ion-channel-mimetic sensor for trivalent cations based onself-assembled monolayers of thiol-derivatized 4-acyl-5-pyrazolones

on gold

Takashi Ito *Department of Chemistry, Science Uni6ersity of Tokyo, Shinjuku-Ku, Tokyo 162-8601, Japan

Received 10 August 2000; received in revised form 20 September 2000; accepted 23 September 2000

Abstract

Self-assembled monolayers (SAMs) of thiol-derivatized 4-acyl-5-pyrazolone on gold electrodes were used for ion-channel-mimetic sensing of inorganic cations. The SAMs on gold electrodes were characterized with reductive-desorption and capacitancemeasurements. The pH dependence of the cyclic voltammograms (CVs) of [Fe(CN)6]3− and [Ru(NH3)6]3+ as electroactivemarkers suggested the protonation not only of the b-diketone part but also of the nitrogen moiety in the pyrazolone. With regardto the voltammetric responses to metal cations, an increase and a decrease in redox current of [Fe(CN)6]3−/4− and [Ru(NH3)6]3+/

2+ , respectively, were observed with increasing concentrations of the trivalent cations examined (La3+, Gd3+, Yb3+ or Al3+)from 10−6–10−4 up to 10−2 M at pH 5.5, at which the chelating group is present as its deprotonated form. In contrast, suchresponses were negligible in the presence of up to 10−2 M divalent cation (Mg2+, Ca2+, Sr2+ or Ba2+), Li+ or Na+. The orderof the magnitudes of the responses was Al3+\Yb3+:Gd3+\La3+�divalent cations, which is quite similar to that of thestability of 1:1 and 1:2 complexes between a b-diketonate-type chelate and the metal ions. The highly selective responses totrivalent cations seem to reflect the selectivity of the chelating group as well as the large change in the surface charge induced bythe complexation. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Chemically modified electrode; Ion-channel mimetic sensor; 4-Acyl-5-pyrazolone; Self-assembled monolayers (SAMs); Trivalent metalcations

1. Introduction

Since the mid-1970s, chemical modification of elec-trode surfaces with thin membranes such as monolayersand polymers has been performed in electrochemistryto develop chemical sensors (chemically modified elec-trodes) [1,2]. The membranes attached on the surfacesaccelerate the rate of redox reactions of the analytes,concentrate specific chemical species in the membranes,or change their electrochemical properties after thecomplexation with the analytes. The selectivities of theresulting sensors depend on the complexation abilitiesof the membranes with the analytes.

In 1987, Sugawara et al. reported a type of electro-chemical sensor based on chemically modified elec-

trodes [3]. This type of sensor works on the basis ofanalyte-induced changes in membrane permeability ofredox-active ions or molecules, often referred to asmarkers, due to steric exclusion of markers or electro-static attraction–repulsion between the receptor–ana-lyte complexes and the markers [4,5]. These sensorshave been called ‘ion-channel (mimetic) sensors’ be-cause of the similarity of their working principle to thatof ligand-gated ion-channel proteins in biomembranes.The working principle of ion-channel sensors suggeststhat the sensors allow the electrochemical detection ofredox-inactive species as well as the amplification of thesignal induced by the complexation. So far, artificialion-channel sensors, which were based on electrodesmodified with Langmuir–Blodgett (LB) monolayers,polymers or self-assembled monolayers (SAMs), havebeen reported for inorganic cations [3,6–10], inorganicanions [3,11], biomolecules [12–15] and other organic

* Tel.: +81-3-32604271; fax: +81-3-32352214.E-mail address: tito@ch.kagu.sut.ac.jp (T. Ito).

0022-0728/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0022 -0728 (00 )00398 -3

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–9788

compounds [16,17]. In particular, ion-channel sensorsthat function on the basis of the electrostatic mode areadvantageous for the detection of redox-inactive multi-valent ions: polyions such as redox-inactive protamine[14] and heparin [15] can be detected with ion-channelsensors based on SAM-modified electrodes at high se-lectivity and sensitivity, because their multiple charges(:+20 for protamine and :−70 for heparin) resultnot only in their strong binding to the receptor mono-layers on the electrode surfaces but also in largerchanges in surface charge upon their binding to thesurface monolayers. In addition, several ion-channelsensors showed higher selectivity and sensitivity fortrivalent metal ions as compared with divalent metalions [7,10].

In the present study, ion-channel mimetic sensing formetal ions was examined on gold electrodes modifiedwith SAMs of thiol-derivatized 4-acyl-5-pyrazolones.4-Acyl-5-pyrazolone is a type of b-diketone and isknown to be a good extracting reagent, having twooxygen atoms as coordinating centers for hard ionssuch as trivalent lanthanoid ions [18–21]. So far, severalchelator-modified electrodes have been reported forelectrochemical detection of metal ions on the basis ofstripping voltammetry [22,23] and ac impedance spec-troscopy [24]. Ion-channel sensors based on chelator-modified electrodes, as examined in this study, seem toallow the selective detection of trivalent metal cationson the basis of not only the high affinity of the chelatorbut also the advantage of the ion-channel sensors forthe multivalent-ion detection.

2. Experimental

2.1. Reagents

The following reagents were of analytical reagentgrade and used without further purification:La(NO3)3·6H2O, Gd(NO3)3·6H2O, Sr(NO3)2, Ba(NO3)2,LiNO3, NaNO3, KNO3, KOH, lithium acetate, aceticacid, nitric acid, K4[Fe(CN)6], and K3[Fe(CN)6] werepurchased from Kanto Chemical (Tokyo, Japan).

Yb(NO3)3·4H2O, Al(NO3)3·9H2O, Mg(NO3)2·6H2O andCa(NO3)2·4H2O were purchased from Wako PureChemical (Osaka, Japan). [Ru(NH3)6]Cl3 was purchasedfrom Aldrich Chemical (Milwaukee, WI). Deionizedwater purified (specific resistance \18 MV cm) with aMilli-Q water system (Millipore, Bedford, MA) wasused throughout all experiments. 1-Phenyl-3-methyl-4-octadecanoyl-5-pyrazolone (1; Fig. 1) was synthesizedfrom 1-phenyl-3-methyl-5-pyrazolone (Tokyo KaseiKogyo, Tokyo, Japan) and stearic acid (Wako) accord-ing to the literature [25], and purified with silica gel(Wako) column chromatography using CH2Cl2–n-hexane (4:1) and with subsequent recrystallizationfrom methanol. 1H-NMR (CDCl3): d 0.86 (t, 3H,�CH2�CH3), 1.41–1.23 (m, 28H, the other �CH2�), 1.72(m, 2H, �CO�CH2�CH2�), 2.15 (s, 3H, C�CH3), 2.71 (t,2H, �CO�CH2�), 7.26 (t, 1H, 4-Ar), 7.42 (t, 2H, 3,5-Ar), 7.81 (d, 2H, 2,6-Ar). Anal. Calc for C28H44N2O2:C, 76.32; H, 10.06; N, 6.36. Found: C, 76.08; H, 9.90;N, 6.36.

2.2. Syntheses of thiol-deri6atized 4-acyl-5-pyrazolones

2.2.1. 1-Phenyl-3-methyl-4-(1-bromohexanoyl)-5-pyrazolone (2; Fig. 1)

1-Phenyl-3-methyl-5-pyrazolone (1.72 g; 9.9 mmol)was dissolved in 2 ml distilled 1,4-dioxane at 50°C, andCa(OH)2 (0.74 g; 9.9 mmol; Wako) was added to thestirred solution. Then, a 1,4-dioxane solution (2 ml) ofBr(CH2)5COCl, which was prepared from 6-bromohex-anoic acid (2.01 g; 10.3 mmol; Wako) and SOCl2, wasadded dropwise and the reaction mixture was refluxedfor 2 h. After 50 ml of 3 M HCl was added to thereaction mixture, the yellow solid obtained was filteredand washed with dilute HCl and water. The crudeproduct was purified by silica gel (Walco) column chro-matography using CH2Cl2, and then recrystallized frommethanol to give a light pink solid. Yield: 1.25 g (36%).1H-NMR (CDCl3): d 1.55, 1.77 and 1.91 (m, 6H, theother �CH2�), 2.46 (s, 3H, C�CH3), 2.75 (t, 2H,�CO�CH2�), 3.42 (t, 2H, �CH2�Br), 7.26 (t, 1H, 4-Ar),7.43 (t, 2H, 3,5-Ar), 7.80 (d, 2H, 2,6-Ar).

2.2.2. 1-Phenyl-3-methyl-4-(1-mercaptohexanoyl)-5-pyrazolone (3; Fig. 1)

1-Phenyl-3-methyl-4-(1-bromohexanoyl)-5-pyrazolone(2) (0.82 g; 2.3 mmol) and thiourea 1.08 g (14.2 mmol;Wako) were dissolved in 10 ml ethanol, and the reactionmixture was refluxed for 22 h. Suspension of Ca(OH)2

(4.1 g; 55.3 mmol) in water (25 ml) was added to thestirred reaction mixture, and then it was refluxed furtherfor 1.5 h. After 50 ml of 4 M HCl was added, the solidobtained was filtered. The crude product was purifiedwith silica gel (Wako) column chromatographyusing CH2Cl2, and then recrystallized from methanol.

Fig. 1. Chemical structures of 1-phenyl-3-methyl-4-acyl-5-pyrazolones1–5 used in the present study.

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–97 89

Yellow solid. Yield: 0.46 g (66%). 1H-NMR (CDCl3): d

1.36 (t, 1H, �SH), 1.53, 1.69 and 1.77 (m, 6H, the other�CH2�), 2.48 (s, 3H, N�CH3), 2.57 (q, 2H, �CH2�S),2.76 (t, 2H, �CO�CH2�), 7.29 (t, 1H, 4-Ar), 7.45 (t, 2H,3,5-Ar), 7.83 (d, 2H, 2,6-Ar). Anal. Calc forC16H20N2O2S: C, 63.13; H, 6.62; N, 9.20; S, 10.53.Found: C, 63.23; H, 6.60; N, 9.13; S, 9.93.

2.2.3. 1-Phenyl-3-methyl-4-(1-bromoundecanoyl)-5-pyrazolone (4; Fig. 1)

The procedures for the synthesis were similar tothose for 2, in which 1-phenyl-3-methyl-5-pyrazolone(1.00 g; 5.8 mmol) and Br(CH2)10COCl, which wasprepared from 11-bromoundecanoic acid (1.47 g; 5.5mmol; Wako), were used to give a pink solid. Yield:1.74 g (72%). 1H-NMR (CDCl3): d 1.29–1.84 (m, 16H,the other �CH2�), 2.45 (s, 3H, C�CH3), 2.71 (t, 2H,�CO�CH2�), 3.39 (t, 2H, �CH2�Br), 7.26 (t, 1H, 4�Ar),7.42 (t, 2H, 3,5�Ar), 7.81 (d, 2H, 2,6�Ar).

2.2.4. 1-Phenyl-3-methyl-4-(1-mercaptoundecanoyl)-5-pyrazolone (5; Fig. 1)

The procedures for the synthesis were similar tothose for 3, in which 1-phenyl-3-methyl-4-(1-bro-moundecanoyl)-5-pyrazolone (4) 0.63 g (1.5 mmol) andthiourea 0.59 g (7.8 mmol) were used to give anotherpink solid. Yield: 0.38 g (68%). 1H-NMR (CDCl3): d

1.23–1.61 (m, 17H, �SH and the other �CH2�), 2.48 (s,3H, N�CH3), 2.52 (q, 2H, �CH2�S), 2.73 (t, 2H,�CO�CH2�), 7.27 (t, 1H, 4-Ar), 7.45 (t, 2H, 3,5-Ar),7.83 (d, 2H, 2,6-Ar). Anal. Calc for C21H30N2O2S: C,67.34; H, 8.07; N, 7.48; S, 8.56. Found: C, 67.22; H,8.05; N, 7.40; S, 8.60.

2.3. Electrode preparation

Gold disk electrodes (Bioanalytical Systems (BAS),West Lafayette, IN, USA) were used for all experi-ments. The electrodes were polished with wet diamondslurry (6 and 1 mm) and 0.5 mm alumina slurry (BAS)on a felt pad in this order, and rinsed repeatedly withwater. The polished electrodes were then dipped in 0.1M KOH solution and the potential was cycled between0 and −1.3 V versus Ag � AgCl until cyclic voltam-mograms (CVs) indicated a perfectly clean electrodesurface. The electrode area was determined bychronoamperometry in 4 mM [Fe(CN)6]4− [26]. Afterbeing dried in a stream of Ar, the gold electrodes wereimmersed in a 1 mM ethanolic solution of 4 or 5 for1–2500 min, and then washed with EtOH and water.

2.4. Electrochemical measurements

Electrochemical experiments were carried out in astandard three-electrode cell containing a Ag � AgCl 3M NaCl (type RE-1B, BAS) reference electrode and a

Pt auxiliary electrode with an ALS-model 600A electro-chemical analyzer (ALS, Tokyo, Japan). All measure-ments were performed with solutions deoxygenated bybubbling and purging with Ar at 2591°C. The solu-tion pH was measured by using a pH meter modelHM-5S (TOA Electronics, Tokyo, Japan) equippedwith a pH glass electrode (Type GST-5211C, TOA).Capacitance measurements were performed by usingfast scan voltammetry, i.e. by cycling the potentialbetween −0.4 and +0.3 V versus Ag � AgCl at 5 Vs−1 in 0.1 M KNO3 [8,27]. CVs for reductive desorp-tion were measured at 0.1 V s−1 in 0.5 M KOHdeoxygenated with Ar [28]. The effects of the pH andmetal ion concentration on membrane permeabilitywere evaluated from CVs of 1 mM K3[Fe(CN)6] or[Ru(NH3)6]Cl3 as electroactive marker in solutions con-taining 0.1 M KNO3 as a supporting electrolyte. 1 mMacetate buffer (pH 5.5) was added into the solutionswhen the metal-ion dependence of CVs was investi-gated. Upon changes in the composition of solutions,the solutions were deoxygenated by bubbling and purg-ing with Ar, and stirring for at least 3 min. For thedetermination of the pH dependence of CVs, the pHwas adjusted by adding a HNO3 solution containingKNO3 and electroactive marker. Metal concentrationswere adjusted by adding aliquots of the metal solution(1×10−4 or 1×10−2 M) containing 1 mM electroac-tive marker, 0.1 M KNO3 and 1 mM acetate buffer (pH5.5). CVs obtained at the first potential cycle are dis-cussed for all cases.

2.5. Measurements of surface pressure–molecular area(p–A) isotherms

A Langmuir film balance (model HBM, KyowaKaimenkagaku, Tokyo, Japan) equipped with a plat-inum Wilhelmy plate and a Teflon-coated trough (14×70 cm) was used for the measurement of p–Aisotherms. The temperature of the water subphase waskept at 25.0°C for all experiments. Monolayers wereobtained by spreading 140 ml of a 1.05 mM chloroformsolution of 1 on distilled water. The monolayer wasallowed to stand for 30 min to ensure complete evapo-ration of chloroform, and then a p–A isotherm wasrecorded at a compression velocity of 14 cm2 min−1.

3. Results and discussion

3.1. Monolayer formation of thiol-deri6atized4-acyl-5-pyrazolones on gold electrodes and theirelectrochemical characterization

The proper reaction time for the formation of theSAMs of thiol-derivatized 4-acyl-5-pyrazolones on gold

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–9790

Fig. 2. (A) Voltammetric desorption curves as a function of reactiontime in a 1 mM ethanolic solution of 3: immersion times; (a) 0 min;(b) 1 min; (c) 30 min; (d) 2500 min; electrolyte, 0.5 M KOH; scanrate, 0.1 V s−1; electrode area, 2.1 mm2. (B) Relation betweenreaction time for self-assembly and molecular area calculated fromthe charge density consumed by the reductive desorption of 3 in 0.5M KOH as shown in (A). (C) Relation between reaction time forself-assembly and membrane capacitance at 0 V versus Ag � AgCl in0.1 M KNO3.

electrodes was determined first, and then the SAMswere electrochemically characterized.

The proper reaction time for SAM formation wasdetermined on the basis of the change in the reductive-desorption peak of the SAMs observed in 0.5 M KOH[28]. Fig. 2(A) shows voltammetric desorption curves of3-SAMs prepared for various reaction times (0, 1, 30and 2500 min). Except for the 0 min reaction, a ca-thodic peak around −1.1 V versus Ag � AgCl wasobserved, and assigned as the peak due to the reductivedesorption of 3. The peak potential for reductive de-sorption (Erd) appeared at −1.054, −1.097 and−1.114 V versus Ag � AgCl for 1, 30 and 2500 min asreaction times, respectively. Erd shifted in a negativedirection as the reaction time increased, and reached ca.−1.11 V for reaction times larger than 500 min. Thischange in Erd to negative position reflects the improve-ment of the molecular packing in the SAMs, as re-ported by Widrig et al. that Erd is dependent on notonly the strength of the gold–sulfur interaction but alsothe extent of the intermolecular interactions betweenadjacent adsorbates [28].

The above conclusion is supported by the change inthe charge consumed by the reductive desorption (Qrd).In Fig. 2(A), Qrd increased with increasing reactiontime, i.e. 11.5, 19.9 and 25.4 mC cm−2 for 1, 30 and2500 min as reaction times, respectively. Fig. 2(B) sum-marizes the dependence of molecular area calculatedfrom the Qrd on the reaction time. With increasingreaction time, the estimated area per molecule graduallydecreased and then reached a minimum at around0.6590.01 nm2 molecule−1 for more than 500 min ofreaction, indicating that the SAM formation was com-pleted by immersing the solution for more than 500min. This area is larger than that obtained from thep–A isotherm of 1-phenyl-3-methyl-4-octadecanoyl-5-pyrazolone (1) on deionized water (Fig. 3). The molecu-lar area, as obtained from extrapolation to zero surfacepressure, was 0.43290.005 nm2 molecule−1 deter-mined in 6 measurements. This value is quite similar tothat estimated from CPK model (ca. 0.4 nm2

molecule−1) as obtained when the phenyl group of thechelator orients to the aqueous phase. Thus, we canconclude from the larger molecular area of 3 on goldelectrodes that 3 cannot form SAMs with this densely-packed structure on gold electrodes. On the other hand,the relatively low collapse pressure shown in Fig. 3(:5 mN m−1) suggests that this orientation of 1 in theLB monolayer is not so stable, as estimated from thefacing of the relatively hydrophobic phenyl group tothe aqueous phase as well as the presence of the hy-drophilic b-diketone moiety in the hydrophobic envi-ronment. The instability of the densely packedorientation seems to be one of the reasons for the lowcoverage of 3 in the SAMs on gold electrodes. Inaddition, the lower coverage in 3-SAMs seems to be

Fig. 3. Surface pressure-molecular area (p–A) isotherm for 1-phenyl-3-methyl-4octadecanoyl-5-pyrazolone (1). Measured at 25.090.1°Cwith distilled water as a subphase.

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–97 91

also due to weak hydrophobic interaction betweenshort alkyl chains of 3 in the SAMs. This is supportedby voltammetric desorption curves measured for theSAMs of 5, which has a longer alkyl chain (data notshown). For 5-SAMs at maximum packing, the Erd wasobserved at −1.18 V versus. Ag � AgCl, which is morenegative than that of 3-SAMs, and the molecular areaobtained in 4 measurements was 0.4490.05 nm2

molecule−1, which almost corresponds to that in thedensely-packed LB monolayer of 1. This desorptionbehavior for 5-SAMs suggests that the longer alkylchain of 5 allows the formation of a densely packedmonolayer on gold electrode on the basis of the lateralhydrophobic interactions between alkyl chains in theSAMs.

As described above, the SAM formation of 3 wascompleted when a gold electrode was immersed in anethanolic solution of 3 for more than 500 min. Asimilar trend was also obtained for the change in thetotal interfacial capacitance at 0 V versus Ag � AgCl in0.1 M KNO3 (Fig. 2(C)). The capacitance decreasedwith increasing reaction time, and was almost constant(1692 mF cm−2) for a 500 min reaction. This indicatesthat the SAM formation can be also evaluated bymeasurements of the capacitance. The capacitance ofelectrodes modified with 3-SAMs at the highest cover-age was a little higher than that for 5-SAMs (1291 mFcm−2), and this can also be explained by the differencein the thickness and the packing of the SAMs.

Subsequently, the CVs of two hydrophilic electro-chemical markers [Fe(CN)6]3− and [Ru(NH3)6]3+ were

compared for three kinds of electrodes: a bare goldelectrode and the gold electrodes modified with 3-SAMsor 5-SAMs at their maximum coverage. Fig. 4(A) and(B) show CVs of 1 mM [Fe(CN)6]3− and [Ru(NH3)6]3+

, respectively, observed for the three electrodes at pH5.5. On a bare electrode, the cathodic and anodicpeaks, assigned to the reduction and oxidation of[Fe(CN)6]3−/4− and [Ru(NH3)6]3+/2+, were observed,and the separations of these peak potentials (DEp) were:68 mV for [Fe(CN)6]3−/4− and :60 mV for[Ru(NH3)6]3+/2+. These DEp values indicate that al-most reversible reactions occurred on a bare gold elec-trode for both markers. In contrast, the gold electrodesmodified with the SAMs of 3 or 5 gave no peaks for[Fe(CN)6]3− (Fig. 4(A)), whereas they gave cathodicand anodic peaks that can be assigned to the reductionand oxidation of [Ru(NH3)6]3+/2+ (Fig. 4(B)). Thedifferent behavior of the two markers in CVs, whichwas similarly observed for gold electrodes modifiedwith COOH-terminated SAMs [8], can be explained bythe charge of the markers and the SAM surface. Uponthe immersion of the modified electrodes in an aqueoussolution of pH 5.5, the surface charge of the SAMs wasnegative due to the deprotonation of the chelatinggroups, which is supported by the pH dependence ofthe CVs as discussed below. As a result, the cationicmarkers can permeate through the negatively chargedSAMs to the electrode surface due to electrostaticattraction, whereas the permeation of the anionic mark-ers is inhibited by electrostatic repulsion. The DEp

values for [Ru(NH3)6]3+/2+ were :83 and :123 mVfor electrodes modified with 3- and 5-SAMs, respec-tively (Fig. 4(B)). This indicates that steric blocking ofthe SAMs inhibits the permeation of the cationicmarker even in the presence of electrostatic attraction.The wider DEp for 5-SAMs suggests that the largerthickness and better packing of the 5-SAMs (videsupra) results in the larger prevention of the access ofthe marker to the electrode.

In the following sections, the effects of the binding ofinorganic cations to the chelating groups on CVs wereinvestigated only with gold electrodes modified with3-SAMs at the maximum coverage, because gold elec-trodes modified with 5-SAMs gave very little change inCVs of [Fe(CN)6]3− as estimated from the abovediscussions.

3.2. pH dependence of CVs for gold electrodes modifiedwith 3-SAMs

It is well known that the extractability of metal ionsfrom aqueous into organic phases with chelatingreagents such as 4-acyl-5-pyrazolones is strongly af-fected by the pH of the aqueous phase, because onlythe deprotonated chelates can form the uncharged,hydrophobic metal complexes [18–21]. The pKa of 4-

Fig. 4. Cyclic voltammograms of (A) 1 mM [Fe(CN)6]3− and (B) 1mM [Ru(NH3)6]3+ in 0.1 M KNO3 on a bare gold electrode and goldelectrodes modified with 3- or 5-SAMs (pH 5.5). Scan rate, 0.1 V s−1;electrode area, 2.1 mm2.

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–9792

Fig. 5. pH dependence of cyclic voltammograms of (A) 1 mM [Fe(CN)6]3− and (B) 1 mM [Ru(NH3)6]3+ in 0.1 M KNO3 on gold electrodesmodified with 3-SAMs. The pH of the sample solution was adjusted by adding 0.1 M HNO3 containing 1 mM [Fe(CN)6]3− or [Ru(NH3)6]3+,respectively. Scan rate, 0.1 V s−1; electrode area, 2.1 mm2. (C) Schematic illustration of the pH responses observed with the cationic and anionicmarkers on the gold electrodes modified with 3-SAMs.

acyl-5-pyrazolone is known to be ca. 4 for its b-dike-tone moiety [29], whereas that for its nitrogen moiety is,to our knowledge, unknown. Therefore, we evaluatedthe extent of the protonation of the 4-acyl-5-pyrazolonemoiety in the 3-SAMs from CVs of the cationic andanionic markers at various pH values, before examiningion-channel mimetic sensing for several metal cationswith the 3-SAM-modified electrodes.

Fig. 5(A) and (B) show the CVs of 1 mM[Fe(CN)6]3− and [Ru(NH3)6]3+, respectively, at differ-ent pH values obtained with gold electrodes modifiedwith 3-SAMs. At pH 5.5 and 5.0, no peaks wereobserved for [Fe(CN)6]3− as a marker (Fig. 5(A))whereas cathodic and anodic peaks were observed for[Ru(NH3)6]3+ (Fig. 5(B)), as discussed in the formersection. In the case of [Fe(CN)6]3−, the reduction and

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–97 93

oxidation currents increased with decreasing pH, and atpH 1.9, a cathodic peak around −0.1 V versusAg � AgCl was observed (Fig. 5(A)). This increase in thecurrents indicates that the electrostatic repulsion be-tween the negatively charged surface of the SAM andthe anionic marker was reduced as the pH decreaseddue to the protonation of the chelating moieties (Fig.5(C)). In contrast, when [Ru(NH3)6]3+ was used as amarker, the reduction and oxidation currents decreasedwith decreasing pH (Fig. 5(B)), indicating the decreasein the electrostatic attraction between the negativelycharged SAM surface and the cationic marker withdecreasing pH, again due to the protonation of thechelating moiety (Fig. 5(C)).

The protonation of the chelating groups not onlyaffects the surface charge but also seems to induce thechange in the molecular arrangement in the SAMs,because the binding of the cations probably reduces theelectrostatic repulsion between the negatively-charged,deprotonated chelating groups of the SAMs. Thechange in the molecular arrangement probably leads tothe change in the extent of the steric blocking of theSAMs, which results in increases or decreases in theredox currents of both cationic and anionic markers.However, the increases and decreases in the currents of[Fe(CN)6]3−/4− and [Ru(NH3)6]3+/2+ with decreasingpH as shown in Fig. 5(A) and (B), respectively, suggestthat the effect of the molecular arrangement on the CVchange seems to be smaller in comparison with that ofsurface charge.

At pH 1.9, redox peaks were observed for[Fe(CN)6]3−/4− whereas no peak was observed for[Ru(NH3)6]3+/2+ (Fig. 5(A) and (B), respectively).From the viewpoint that [Ru(NH3)6]3+/2+ has a muchhigher rate constant of electron transfer [30] than[Fe(CN)6]3−/4− [31], the presence and absence of theredox peaks of [Fe(CN)6]3−/4− and [Ru(NH3)6]3+/2+,respectively, strongly suggest that the surface of theSAMs has a positive charge at that pH. If the protona-tion occurred only at the b-diketone moiety with in-creasing H+ concentration [18–21], the surface chargeof the SAMs should be neutral even at low pH. Thus,it can be concluded that the protonation occurred notonly at the b-diketone moiety but also at the nitrogenmoiety of the 4-acyl-5-pyrazolone in 3-SAMs at low pH(Fig. 5(C)). Indeed, the nucleophilicity of the pyra-zolone nitrogen is also shown by PbII-coordination atthe nitrogen part in 1-phenyl-3-methyl-4-acyl-5-pyra-zolone complexes of PbII [32].

Zhao et al. reported that the surface pKa of func-tional groups of the SAM surface can be estimatedfrom the pH dependence of CVs of [Fe(CN)6]3− [33].In that report, the peak current of the marker observedwith a gold electrode modified with thioctic acidchanged sigmoidally over a quite narrow range of pH(pH 4–6). In contrast, we could not estimate the sur-

face pKa of the chelating group on the SAMs from theresults shown in Fig. 5(A) and (B) in the same way,because the peak current changed gradually over quitea wide range of pH (pH 2–5). This may also supportthe gradual protonation of the nitrogen moiety as wellas the protonation of the b-diketone moiety of 3.

The changes in CVs occurred within 3 min for allcases, suggesting that the protonation of the surfacechelating groups occurs quite fast. In addition, thechanges in CVs were reversible even after the measure-ments were performed at acidic pHs, indicating that adecomposition of the SAMs did not occur over a shorttime scale. However, the absolute value of the changein the redox currents of the electroactive markers in-creased (ca. 30%) after the electrode was immersed in asolution of acidic pH (pH:2) for long time (]6 h).This may be due to the decomposition of the mono-layer or the chelating group and/or an irreversiblechange in the SAM structure. Very similar trends werealso observed in the investigation of the influence ofmetal-ions on CVs discussed in the next section.

3.3. Dependence of CVs on concentration of metal ionsfor gold electrodes modified with 3-SAMs

In the previous section, it was shown that a pHchange resulted in the increase or decrease of redoxcurrents of the markers for the electrodes modified with3-SAMs. Thus, in the presence of a 1 mM acetatebuffer of pH 5.5, we investigated the effect of metal-ionconcentration on CVs of 1 mM [Fe(CN)6]3− and[Ru(NH3)6]3+ for 3-SAM modified electrodes. At thatpH, the chelating group on the SAM surface is depro-tonated (vide supra), and as a result, can form strongmetal-complexes. Trivalent cations (La3+, Gd3+ andYb3+ as lanthanoid ions, and Al3+), alkaline earthions (Mg2+, Ca2+, Sr2+ and Ba2+), Na+ and Li+

were used here as metal cations.Fig. 6(A) shows a typical result of the influence of

[Al3+] on CVs of 1 mM [Ru(NH3)6]3+ for a goldelectrode modified with 3-SAM. Cathodic and anodicpeaks of [Ru(NH3)6]3+/2+, which were clearly observedin the absence of Al3+, gradually decrease with increas-ing concentration of Al3+ up to 0.01 M. The decreasein the current reflects the decrease in the negativecharge at the SAM surface (Fig. 6(C)), similarly to thepH dependence mentioned above, but the suppressionof the anodic peak is smaller than that for H+ at thesame concentration (cf. Fig. 5(B)). This suggests rela-tively lower stability of the complexes between Al3+

and the surface chelating groups as compared with thestability of the surface chelating groups to proton.

The effect of metal ion concentration on CV was alsoinvestigated for 1 mM [Fe(CN)6]3−. Fig. 7(A) shows atypical result of the influence of [Al3+] on CVs of 1mM [Fe(CN)6]3− for a gold electrode modified with

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–9794

3-SAM. In the absence of Al3+, no peaks were ob-served due to electrostatic repulsion between the nega-tively charged marker and SAM surface (vide supra).With increasing [Al3+], reduction currents of[Fe(CN)6]3− gradually increased from 0 to 10−2 M,whereas oxidation currents of [Fe(CN)6]4− increasedfrom 0 to 10−4 M and then decreased at higher concen-tration up to 10−2 M. Again, the increase in thereduction current reflects the decrease in the negativecharge due to the complexation between Al3+ and thesurface chelating group (Fig. 7(C)). The latter strangebehavior of the change in the oxidation current of[Fe(CN)6]4− was similarly observed for the other triva-lent ions examined, and was probably due to the forma-tion of insoluble complexes between the trivalentcations and [Fe(CN)6]4− [34]. In contrast to pH depen-dence (Fig. 5(A)), no reduction peak of [Fe(CN)6]3−

appears even in a solution containing 0.01 M Al3+,suggesting that the negative net charge of the SAMsurface was not completely cancelled under the above

condition and that excess positive charge was notpresent on the SAM surface.

The changes in CVs shown in the above occurredwithin 3 min for all cases, suggesting that the complex-ation of the trivalent ions to the surface chelatinggroups is quite fast. In addition, very similar voltam-metric responses were observed repeatedly when theinterval of the measurements was within 2 h. However,the magnitudes of the voltammetric responses to triva-lent cations increased for both the anionic and cationicmarkers (ca. 10–50%) after the electrode was immersedin a solution of 0.01 M trivalent ion for a long time(:2 h). This is probably due to an irreversible changein the SAM structure.

The dependence of the reduction currents of 1 mM[Ru(NH3)6]3+ and [Fe(CN)6]3− on the concentration ofthe divalent and trivalent ions is summarized in Figs.6(B) and 7(B), respectively. Fig. 6(B) shows that thereduction current of 1 mM [Ru(NH3)6]3+ at −0.180 Vversus Ag � AgCl decreased with increasing concentra-

Fig. 6. (A) Influence of Al3+ concentration on cyclic voltammograms of 1 mM [Ru(NH3)6]3+ in 0.1 M KNO3 and 1 mM acetate buffer (pH 5.5)on a gold electrode modified with 3-SAM. Scan rate, 0.1 V s−1; electrode area, 2.0 mm2. (B) Dependence of the reduction current of[Ru(NH3)6]3+ at −0.180 V versus Ag � AgCl on the concentration of trivalent and divalent cations, obtained on gold electrodes modified with3-SAMs. The results are the mean and standard deviation of at least three separate experiments. (C) Schematic illustration of the voltammetricresponses of electrodes modified with 3-SAMs to trivalent metal ions (M3+) for the cationic marker.

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–97 95

Fig. 7. (A) Influence of Al3+ concentration on cyclic voltammogramsof 1 mM [Fe(CN)6]3− in 0.1 M KNO3 and 1 mM acetate buffer (pH5.5) on a gold electrode modified with 3-SAM. Scan rate, 0.1 V s−1;electrode area, 2.4 mm2. (B) Dependence of the reduction current of[Fe(CN)6]3− at −0.200 V versus Ag � AgCl on the concentration oftrivalent and divalent cations, obtained on gold electrodes modifiedwith 3-SAMs. The results are the mean and standard deviation of atleast three separate experiments. (C) Schematic illustration of thevoltammetric responses of electrodes modified with 3-SAMs to triva-lent metal ions (M3+) for the anionic marker.

Yb3+:Gd3+]La3+�Ba2+, Sr2+, Ca2+, Mg2+,and the voltammetric responses were observed above10−5 M for Al3+ and above 10−4 M for the othertrivalent cations up to 10−2 M. The selectivity of thevoltammetric responses with [Ru(NH3)6]3+ was almostthe same as that with [Fe(CN)6]3−, but the magnitudeof the voltammetric responses was larger for[Ru(NH3)6]3+ (D(current density)max:170 mA cm−2 inFig. 6(B)) than for [Fe(CN)6]3− (D(current den-sity)max:100 mA cm−2 in Fig. 7(B)). Actually, theshape of CVs changed more drastically for[Ru(NH3)6]3+ (Fig. 6(A)) than for [Fe(CN)6]3− (Fig.7(A)). This suggests that the cationic marker is moresensitive to the binding of trivalent cations to the SAMsthan the anionic marker. The reason for the highsensitivity for the cationic marker is unclear, but thechange in the membrane permeability of the markersdue to the change in the molecular arrangement in theSAMs upon the complexation between trivalent cationand the surface chelating groups may explain the aboveresults.

The above selectivity of the voltammetric responsesseems to reflect not only the higher charge number ofthe trivalent cations but also selective binding of thetrivalent cations over alkaline earth ions to b-diketone-type 4-acyl-5-pyrazolone fixed on electrode surfaces.Because of the steric factor that the chelating groups ofthe SAMs are distributed two-dimensionally on theelectrode surface, they can form most likely only 1:1 or1:2 complexes between the trivalent cation and theligands, which are less stable than 1:3 complexes thatare often formed in an organic solution after solventextraction [35]. The above order is quite similar to thatof the stability of 1:1 and 1:2 complexes between metalions and acetylacetone (Al3+\Yb3+]Gd3+\La3+

�Mg2+) [35]. Interestingly, the order of the magnitudeof the voltammetric responses is also quite similar tothat of the extractability of the metal ions with 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone [19,21] and 1-phenyl-3-methyl-4-acyl-5-pyrazolone [18,20], althoughthe order obtained in this study probably reflects thepossible 1:1 and 1:2 complexation between the ions andthe surface chelating groups. On the other hand, it iswell known that Al3+ forms polyions such asAl2(OH)2

4+ in an aqueous solution of neutral pH [36].From this point of view, the high stability of the 1:1 or1:2 complexes between Al3+ and the surface chelatinggroups may not be a main reason for the large voltam-metric responses to Al3+. Instead, the large responsesmay reflect strong binding of the polyions to the nega-tively charged surface due to their multiple charges, asexpected from the advantage of the ion-channel sensorfor multivalent-ion detection (vide supra).

The above order of the selectivity of the voltammet-ric responses is quite different from those of previously

tion of the trivalent cations examined, whereas thecurrents decreased negligibly when the concentration ofalkaline earth ions was increased up to 0.01 M. Themagnitude of the decrease in the current of[Ru(NH3)6]3+ was in the order of Al3+]Yb3+:Gd3+\La3+�Ba2+, Sr2+, Ca2+, Mg2+. Suchvoltammetric responses were observed above 10−6 Mfor Al3+, Yb3+ and Gd3+, and above 10−4 M forLa3+ up to 10−2 M. Fig. 7(B) shows that the reductioncurrents of [Fe(CN)6]3− at −0.200 V versus Ag � AgClincreased with increasing concentration of the trivalentcations examined. In contrast to the trivalent cations,the currents increased slightly even in the presence of0.01 M alkaline earth ions, and no change in CVs of 1mM [Fe(CN)6]3− was observed in solutions containing0.01 M Li+ or Na+. The magnitude of the increase inthe current of [Fe(CN)6]3− was in the order of Al3+\

T. Ito / Journal of Electroanalytical Chemistry 495 (2001) 87–9796

reported ion-channel sensors for trivalent and divalentions. Takehara et al. reported that the voltammetricresponse for the reduction current of 0.5 mMFe(CN)6

3− for a gold electrode modified with a glu-tathione SAM was in the order of La3+\Eu3+\Lu3+�Ba2+\Sr2+\Ca2+\Mg2+, and theresponses for the trivalent and divalent ions were ob-served around 10−6 M and 10−3 M, respectively [6,7].Takaya et al. reported that the response of the reduc-tion current of 1 mM [Fe(CN)6]3− with a gold elec-trode modified with a phosphate ester monolayer wasin the order of Al3+:La3+\Ba2+\Sr2+\Ca2+\Mg2+, and the detection ranges were between 10−7

and 10−5 M for the trivalent ions and above 10−4 Mfor the divalent ions [10]. The relatively poor detectionlimit (above 10−6 M) for the 3-modified electrodesseems to reflect the lower permeability of the markersthrough 3-SAMs due to their better packing as com-pared with the SAMs of glutathione and the phosphateester [6,7,10]. Indeed, Aoki et al. [17] and Takaya et al.[10] pointed out that the detection limits of ion-channelsensors may be more favorable for electrodes modifiedwith thinner receptor monolayers, because the denselypacked layers of molecules hinder marker permeationboth in the presence and absence of analytes. The widerdetection range (10−6–10−2 M) for the 3-modifiedelectrodes may reflect the 1:2 and subsequent 1:1 com-plexation of the chelating groups to the metal ions [37].

4. Conclusions

This study showed that ion-channel sensors based onelectrodes modified with thiol-derivatized 4-acyl-5-pyra-zolone gave highly selective responses to trivalentcations as compared with alkaline and alkaline earthcations. The high selectivity of the voltammetric re-sponses to trivalent cations seems to be attributed notonly to the high affinity of the deprotonated chelatinggroup but also to the well-known advantage of ion-channel mimetic sensing for multivalent-ion detection.The detection range of the sensor was from 10−6 M to10−2 M for the trivalent cations, which is wider ascompared with ion-channel sensors reported previously.This seems to reflect the properties of the SAMs such ascomplexation ability of the ligand, surface charge andmolecular packing of the SAMs. The results obtainedin this study show that ion-channel sensors based onchelator-modified electrodes are advantageous for theselective detection of multivalent metal cations.

Acknowledgements

The author thanks Professor Yuko Hasegawa, De-partment of Chemistry, Science University of Tokyo,

and Professor Yoshio Umezawa, Department of Chem-istry, School of Science, the University of Tokyo, fortheir continuous encouragement and support. He grate-fully acknowledges Dr Philippe Buhlman, Departmentof Chemistry, School of Science, the University ofTokyo, for his helpful discussions and comments. Theauthor also thanks Professor Kazuo Miyamura, De-partment of Chemistry, Science University of Tokyo,for lending him the Langmuir balance for p–Aisotherm measurements. This work was supported by aGrant-in-Aid for Encouragement of Young Scientists(no. 11740413) from the Ministry of Education, Sci-ence, Sports and Culture, Japan.

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