Gold Electrode Incorporating Corrole as anIon-Channel Mimetic Sensor for Determination ofDopamine

Katarzyna Kurzatkowska,† Eduard Dolusic,‡ Wim Dehaen, Karolina Sieron-Stołtny,§

Aleksander Sieron,§ and Hanna Radecka*,†

Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Tuwima Street 10,10-747 Olsztyn, Poland, University of Leuven, Chemistry Department, Celestijnenlaan 200F, B-3001 Leuven, Belgium,and Silesian Medical University, Department of Internal Medicine, Batorego Street 15, 41-902 Bytom, Poland

Here, we report on an ion-channel mimetic sensor usingself-assembly monolayers deposited onto gold electrodesfor electrochemical determination of dopamine. The dif-ferent compositions of the modification solution consistof corrole-SH and other thiol derivatives used as the“background compounds” such as 1-dodecanethiol (DDT),6-mercapto-1-hexanol (HS(CH2)6OH), or 11-mercapto-1-undecanol (HS(CH2)11OH) were explored to find thebest self-assembled monolayer (SAM) suitable fordopamine determination. Among them, the mixed SAMconsisting of corroles with the -SH group and 6-mer-capto-1-hexanol (HS(CH2)6OH) in the molar ratio 1:10was the most sensitive. The signals generated by theformation of a complex between the corrole host andthe dopamine guest were measured by Osteryoungsquare-wave voltammetry (OSWV) and electrochemicalimpedance spectroscopy (EIS) with [Ru(NH3)6]3+ asan electroactive marker. The developed sensor was freeof interferences of components of human plasma suchas ascorbic acid, creatinine, creatine, and uric acid.The detection limits observed by EIS in buffer solutionand in the presence of centrifuged human plasma 80times diluted with a 0.9% NaCl containing 0.01 Mborate buffer solution of pH 7.0 were 3.3 × 10-12 and5.3 × 10-12 M, respectively.

Dopamine (DA) is one of the most significant neurotransmit-ters because of its role in the functioning of the cardiovascular,renal, hormonal, and central nervous system. An abnormaldopamine transmission has been linked to several neurologicaldisorders, e.g., schizophrenia, Huntington’s disease, Parkinson’sdisease, and also to the HIV infection.1-3 Dopamine is also used

as the therapeutic drug to reduce the risk of renal failure byincreasing renal blood flow4,5 and to increase of the splanchnicblood flow and splanchnic oxygen uptake in patients with septicshock.6

Hence, there is a great interest in the monitoring of theconcentration of dopamine using a reliable method with goodsensitivity and selectivity. Spectrophotometry7-9 and chromatogra-phy10-13 are the most frequently applied. While these methodsrequire sophisticated and expensive instrumentation, methodsbased on electrochemical measurements offer advantages in thatthey are simple, rapid, and easy to apply, still providing enoughsensitivity to detect submicromolar concentrations of analytes.

Ion-selective electrodes modified with hexahomotrioxacalix[3]-arene,14 crown ether,15 or -cyclodextrin16 were applied forpotentiometric determination of dopamine. The main disadvantageof these methods is the sensitivity in the range of 0.01-0.1 mM.Therefore, the ISEs could be applied mainly for determination ofDA in drugs.

The majority of electrochemical sensors for dopamine deter-mination exploit its ease of oxidation. However, the oxidativeapproaches suffer from interferences caused by other electroactivesubstances existing in the physiological samples. One of the maininterferences is ascorbic acid (AA). The concentration of DA is

* Corresponding author. Phone: +48895234636. Fax: +48895240124. E-mail:hanna.radecka@pan.olsztyn.pl.

† Institute of Animal Reproduction and Food Research of the Polish Academyof Sciences.

‡ University of Leuven.§ Silesian Medical University.

(1) Cooper, J. R.; Bloom, R. H.; Roth, R. H. The Biochemical Basis ofNeuropharacology; Oxford University Press, Oxford, UK, 1982.

(2) Damier, P.; Hirsch, E. C.; Agid, Y.; Graybiel, A. M. Brain 1999, 122, 1437–1448.

(3) Ali, S. R.; Ma, Y.; Parajuli, R. R.; Balogun, Y.; Lai, W. Y. C.; He, H. Anal.Chem. 2007, 79, 2583–2587.

(4) Bellomo, R.; Chapman, M.; Finfer, S.; Hickling, K.; Myburgh, J. Lancet2000, 356, 2139–2143.

(5) Brienza, N.; Malcangi, V.; Dalfino, L.; Trerotoli, P.; Guagliardi, C.; Bortone,D.; Facond, G.; Ribezzi, M.; Ancona, G.; Bruno, F.; Firore, T. Crit. CareMed. 2006, 34, 707–714.

(6) Meier-Hellmann, A.; Bredle, D. L.; Specht, M.; Spies, C.; Hannemann, L.;Reinhart, K. Intens. Care Med. 1997, 23, 31–37.

(7) Nagaraja, P.; Vasantha, R. A.; Sunitha, K. R. Talanta 2001, 55, 1039–1046.(8) Nagaraja, P.; Vasantha, R. A.; Sunitha, K. R. J. Pharm. Biomed. Anal. 2001,

25, 417–424.(9) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 1566–1571.

(10) Ji, Ch.; Li, W.; Ren, X.; El-Kattan, A. F.; Kozak, R.; Fountain, S.; Lepsy, Ch.Anal. Chem. 2008, 80, 9195–9203.

(11) Carrera, V.; Sabater, E.; Vilanova, E.; Sogorb, M. A. J. Chromatogr., B 2007,847, 88–94.

(12) Yoshitake, M.; Nohta, H.; Ogata, S.; Todoroki, K.; Yoshida, H.; Yoshitake,T.; Yamaguchi, M. J. Chromatogr., B 2007, 858, 307–312.

(13) Yoshitake, T.; Yoshitake, S.; Fujino, K.; Nohta, H.; Yamaguchi, M.; Kehr,J. J. Neurosci. Methods 2004, 140, 163–168.

(14) Saijo, R.; Tsunekawa, S.; Murakami, H.; Shirai, N.; Ikeda, S.; Odashima, K.Bioorg. Med. Chem. Lett. 2007, 17, 767–771.

(15) Othman, A. M.; Rizka, N. M. H.; El-Shahawi, M. S. Anal. Sci. 2004, 20,651–655.

(16) Lima, J. L. F. C.; Montenegro, M. C. B. S. M. Mikrochim. Acta 1999, 131,187–190.

Anal. Chem. 2009, 81, 7397–7405

10.1021/ac901213h CCC: $40.75 2009 American Chemical Society 7397Analytical Chemistry, Vol. 81, No. 17, September 1, 2009Published on Web 07/28/2009

extremely low (0.01-1 µM) while that of AA is as high as 0.1mM in biological systems.3,17 Moreover, at almost all electrodesmaterials, DA and AA are oxidized at nearly the same potential,which results in overlapped voltammetric response. Therefore, itis very important to eliminate AA and other interfering com-pounds, or to use a special modification layer allowing one tosimultaneously detect both DA and AA at different potentials. Inorder to solve this problem, various modified electrodes have beenconstructed such as a carbon fiber electrode,18-20 electrodesmodified with polymers,17,21-24 electrodes modified with nano-materials,3,25-29 and electrodes modified with biomolecules suchas DNA or peroxidase,30-34 just to name a few. Among thesenumerous methods, the use of self-assembled monolayers (SAMs)carrying different functional groups were identified as the veryuseful approach for the modification of the electrode surface,destined for the simultaneous electrochemical determination ofDA and AA. Pioneering work in this field was done by Malemand Mandler.35 They applied ω-mercaptocarboxylic acids SAM toseparate the oxidation potential of DA and AA. A COOH-terminated SAM formed by 3,3′-dithiopropionic acid on the goldelectrode displaying a detection limit in the micromolar range wassuitable for DA determination in pharmaceutical formulations.36

Raj et al. reported a cationic SAM modified gold electrodecontaining 2,2′-dithiobisethaneamine (CYST) and 6,6′-dithiobish-exaneamine (DTH) for the square-wave voltammetric detectionof DA in the presence of AA.37 The voltammetric behavior of DAon a gold electrode modified with SAM of N-acetylcysteine hasbeen investigated by Liu et al.38 They observed that the oxidationpeak current increased linearly with the concentration of DA inthe range of 1.0 × 10-6 to 2.0 × 10-4 M. Hu et al. used anL-cysteine self-assembled modified electrode for the detec-tion of DA by the chronoamperometric method in the presence

of AA.39 The detection limit obtained with this modificationwas 2.0 × 10-8 M DA.

Although these sensors allow determination of DA based onits redox property, none of them are totally free from interferencescoming from AA or other electrochemically active substances.Also, detection limits are still insufficient for the direct determi-nation of DA occurring in the physiological samples at very lowconcentration 0.01-1 µM.

Here, we report a different approach for the detection of DAbased on an ion-channel mimetic sensor. Binding of analytes toreceptors immobilized on electrode surfaces facilitates or sup-presses the access of an anionic (cationic) marker ion to themodified surface due to electrostatic attraction or repulsion of themarker and/or distortion of the modification layer arrangement.This leads to the changes of the electron transfer rate betweenthe marker and electrode surface through the modification layer.Because the working principle of these sensors is similar to thatof ligand gated ion-channel proteins in biomembranes, they arenamed “ion-channel”. These types of electrochemical sensors havebeen introduced and developed by Umezawa and co-workers.40,41

Gold electrodes modified with macrocyclic polyamines, work-ing as “ion-channel” sensors have been successfully applied forthe voltammetric detection of adenine nucleotides42,43 and volta-mmetric discrimination of maleic and fumaric acids.44

It has been already reported that corrole derivatives formcomplexes with dihydroxybenzene derivatives through a NH · · ·OHhydrogen bond.45 Thus, this compound has been selected as asuitable receptor for dopamine in the study presented.

Scheme 1 illustrates the working principle of the sensorproposed. The corrole host molecules have been covalentlyattached on the surface of gold electrodes through Au-S bonds.In the measuring condition (pH 7.0), corrole exists as anuncharged molecule.45 The monolayer created on the electrodesurface is quasi permeable for a redox marker existing in theaqueous solution. Upon addition of dopamine, the corrole-DAcomplex is formed and the monolayer gained the positive charge,which repealed the positive charged redox marker. This leads tothe decreasing of the electron transfer rate between the markerand electrode surface through the modification layer (Scheme 1).

The electroanalytical signals generated based on the corrole-DAcomplex formed at the electrode surface were explored usingOsteryoung square-wave voltammetry (OSWV) and electrochemi-cal impedance spectroscopy (EIS) in the presence of [Ru(NH3)6]3+

as an electroactive marker, which does not interfere with theoxidation of the DA or AA.

(17) Ghita, M.; Arrigan, D. W. M. Electrochim. Acta 2004, 49, 4743–4751.(18) Gonon, F. G.; Fomarlet, C. M.; Buda, M. J.; Pujol, J. F. Anal. Chem. 1981,

53, 1386–1389.(19) Heien, M. L. A. V.; Phillips, P. E. M.; Stuber, G. D.; Seipel, A. T.; Wightman,

R. M. Analyst 2003, 128, 1413–1419.(20) Hermans, A.; Keithley, R. B.; Kita, J. M.; Sombers, L. A.; Wightman, R. M.

Anal. Chem. 2008, 80, 4040–4048.(21) Tu, X.; Xie, Q.; Jiang, S.; Yao, S. Biosens. Bioelectron. 2007, 22, 2819–

2826.(22) Lin, X.; Zhang, Y.; Chen, W.; Wu, P. Sens. Actuators, B 2007, 122, 309–

314.(23) Yi, S.-Y.; Chang, H.-Y.; Cho, H.; Park, Y. C.; Lee, S. H.; Bae, Z.-U. J.

Electroanal. Chem. 2007, 602, 217–225.(24) Kumar, S. A.; Tang, C.-F.; Chen, S.-M. Talanta 2008, 74, 860–866.(25) Ali, S. R.; Ma, Y.; Parajuli, R. R.; Balogun, Y.; Lai, W. Y.-C.; He, H. Anal.

Chem. 2007, 79, 2583–2587.(26) Boo, H.; Jeong, R.; Park, S.; Kim, K. S.; An, K. H.; Lee, Y. H.; Han, J. H.;

Kim, H. C.; Chung, T. D. Anal. Chem. 2006, 78, 617–620.(27) Bustos, E.; Garcia Jimenez, M. G.; Diaz-Sanchez, B. R.; Juaristi, E.; Chapman,

T. W.; Godinez, L. A. Talanta 2007, 72, 1586–1592.(28) Huang, X.; Li, Y.; Wang, P.; Wang, L. Anal. Sci. 2008, 24, 1563–1568.(29) Alarcon-Angeles, G.; Perez-Lopez, B.; Palomar-Pardave, M.; Ramırez-Sliva,

M. T.; Algret, S.; Merkoci, A. Carbon 2008, 46, 898–906.(30) Castilho, T. J.; del Pilar Taboada Sotomayor, M.; Kubota, L. T. J. Pharm.

Biomed. Anal. 2005, 37, 785–791.(31) Moccelini, S. K.; Fernandes, S. C.; Vieira, I. C. Sens. Actuators, B 2008,

133, 364–369.(32) Lin, X.; Kang, G.; Lu, L. Bioelectrochemistry 2007, 70, 235–244.(33) Lin, X. Q.; Lu, L. P.; Jiang, X. H. Microchem. Acta 2003, 143, 229–235.(34) Lu, L. P.; Lin, X. Q. Anal. Sci. 2004, 20, 527–530.(35) Malem, F.; Mandler, D. Anal. Chem. 1993, 65, 37–41.(36) Codognoto, L.; Winter, E.; Paschola, J. A. R.; Suffredini, H. B.; Cabral, M. F.;

Machado, S. A. S.; Rath, S. Talanta 2007, 72, 427–433.(37) Raj, C. R.; Tokuda, K.; Ohsaka, T. Bioelectrochemistry 2001, 53, 183–191.(38) Liu, T.; Li, M.; Li, Q. Talanta 2004, 63, 1053–1059.

(39) Hu, G.; Liu, Y.; Zhao, J.; Cui, S.; Yang, Z.; Zhang, Y. Bioelectrochemistry2006, 69, 254–257.

(40) Sugawara, M.; Kojima, K.; Sazawa, H.; Umezawa, Y. Anal. Chem. 1987,59, 2842–2846.

(41) Umezawa, Y.; Aoki, H. Anal. Chem. 2004, 76, 321A–326A.(42) Szymanska, I.; Radecka, H.; Radecki, J.; Pietraszkiewicz, M.; Pietraszkiewicz,

O. Comb. Chem. High Throughput Screening 2000, 3, 509–519.(43) Radecka, H.; Szymanska, I.; Pietraszkiewicz, M.; Pietraszkiewicz, O.; Aoki,

H.; Umezawa, Y. Anal. Chem. (Warsaw, Poland) 2005, 50, 85–102.(44) Radecki, J.; Szymanska, I.; Bulgariu, L.; Pietraszkiewicz, M. Electrochim.

Acta 2006, 51, 2289–2297.(45) Radecki, J.; Stenka, I.; Dolusic, E.; Dehaen, W.; Plavec, J. Comb. Chem.

High Throughput Screening 2004, 7, 375–381.

7398 Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

EXPERIMENTAL SECTIONSynthesis of Corrole. The corrole (HSCOR) was synthesized

in adaptation of our reported procedures46-48 from 4-(10-undece-nyloxy)benzaldehyde.

4-(11-Thioacetoxyundecyl)benzaldehyde. Aldehyde 4-(10-unde-cenyloxy)benzaldehyde (7.450 g; 27.15 mmol), thioacetic acid (4mL; 56.23 mmol), and azo-bis-isobutyronitrile (229 mg; 1.37 mmol)were dissolved in 90 mL of toluene (p.a.). The mixture wasdegassed with a stream of argon and then refluxed for 6.5 h. Thereaction was quenched with 5% aqueous NaHCO3 (400 mL) andextracted three times with ethyl acetate. The combined organiclayers were washed with 5% aqueous NaHCO3 and then brine

and dried over MgSO4. Upon filtration and removal of thesolvent under reduced pressure, the yellow solid residue waschromatographed on silica eluting with a gradient of 5:1 to 1:1light petroleum ether/ethyl acetate. The crude product ob-tained was recrystallized from methanol to afford 4.086 g (43%)of the title aldehyde as a white powder. 1H NMR (300 MHz)9.88 (s, 1H, aldehyde H), 7.83 (d, J 8.6, 2H, aryl H), 6.99 (d, J8.6, 2H, aryl H), 4.04 (t, J 6.6, 2H, undecenyl 1-H), 2.86(t, J 7.3, 2H, undecenyl 11-H), 2.32 (s, 3H, CH3), 1.81 (quintet,J 7.3, 2H, undecenyl 2-H), 1.46-1.28 (m, 16H, 8CH2).

meso-5,15-Bis(2,6-dichlorophenyl)-10-(4-(11-thioacetoxy-1-dodec-yloxy) phenyl)corrole. meso-(2,6-Dichlorophenyl)dipyrromethane(1.522 g; 5.23 mmol) and the previous aldehyde (606 mg; 1.73mmol) were dissolved in 146 mL of dichloromethane. The reactionflask was wrapped in aluminum foil and placed in an ice bath.Argon was bubbled through the solution for 15 min. TFA (10 µL;0.13 mmol) was added, and the mixture was stirred under anargon atmosphere for ∼48 h.

The ice bath was removed, and 935 mg (3.77 mmol) ofp-chloranil was added. After an additional 1 h at room temperature,the mixture was evaporated with silica and chromatographed twicein a 1.5:1 mixture of dichloromethane and n-heptane to afford 452mg (29%) of the title corrole. 1H NMR 9.00 (d, J 4.1, 2H, 2-Hand 18-H of corrole), 8.62 (d, J 4.7, 2H, 8-H and 12-H of corrole),8.53 (d, J 4.7, 2H 7-H, and 13-H of corrole), 8.42 (d, J 4.1, 2H,3-H and 17-H of corrole), 8.08 (d, J 8.5, 2H, aryl o-H), 7.78 (d,J 7.7, 4H, dichlorophenyl m-H), 7.66 (dd, J1 7.7, J2 8.7, 2H,dichlorophenyl p-H), 7.27 (d partially overlapped with CHCl3,J 8.5, 2H, aryl m-H), 4.24 (t, J 6.5, 2H, OCH2), 2.89 (t, J 7.3, 2H,SCH2), 2.32 (s, 3H, CH3), 1.98 (quintet, J 7.4, 2H, -CH2),1.62-1.36 (m, 16H, 8CH2), -2.30 (br. s, 3H, NH); δC 196.0,158.9, 138.6 (quaternary C), 137.4 (quaternary C), 135.5 (CH),134.0 (quaternary C), 130.3 (dichlorophenyl p-C), 128.0 (dichlo-rophenyl m-C), 127.2 (pyrrole CH), 125.7 (pyrrole CH), 120.8(C-3 and C-17 of corrole), 116.2 (C-2 and C-18 of corrole), 113.2(CH), 111.7 (quaternary C), 108.8 (quaternary C), 68.3, 30.6,29.6, 29.5, 29.1, 28.8, 26.2; UV-vis 410.1 (1.35 × 105), 566.1(0.22 × 105); ESI-MS 907 (MH+).

meso-5,15-Bis(2,6-dichlorophenyl)-10-(4-(11-mercapto-1-dodecyl-oxy)phenyl)corrole. Acetyl protected corrole (130 mg; 0.14 mmol)was dissolved in 7.5 mL of THF and 3 mL of methanol. The flaskwas placed in an ice bath, and 1 mL of a 1.65 M solution ofCH3ONa in methanol (1.65 mmol of CH3ONa) was added. Thereaction mixture was stirred at 0 °C for 30 min and then pouredinto diluted aqueous HCl (∼0.02 M). This solution wasextracted with ethyl acetate (some brine had to be added fora better separation) and then washed with brine and distilledwater and dried over MgSO4. Upon filtration and removal ofthe solvent under reduced pressure, 26 mg (21%) of the productwas isolated by chromatography in mixtures of CH2Cl2 andhexane (1.5:1, column and 1:1, preparative plate). 1H NMR 8.97(d, J 3.9, 2H, 2-H and 18-H of corrole), 8.61 (d, J 4.5, 2H, 8-Hand 12-H of corrole), 8.51 (d, J 4.5, 2H, 7-H and 13-H of corrole),8.39 (d, J 3.9, 2H, 3-H and 17-H of corrole), 8.07 (d, J 8.2, 2H,aryl o-H), 7.72 (m, 4H, dichlorophenyl m-H), 7.59 (dd, J1 8.3,J2 8.3, 2H, dichlorophenyl p-H), 7.24 (d partially overlappedwith CHCl3, J 8.2, 2H, aryl m-H), 4.20 (t, J 6.4, 2H, OCH2), 2.50(t, J 6.8, 2H, SCH2), 1.93 (quintet, 2H, -CH2), 1.60 (m, 4 H, 2

(46) Asokan, C. V.; Smeets, S.; Dehaen, W. Tetrahedron Lett. 2001, 42, 4483–4485.

(47) Rohand, T.; Dolusic, E.; Ngo, T. H.; Maes, W.; Dehaen, W. Arkivoc 2007,307–324.

(48) Maes, W.; Ngo, T. H.; Vanderhaeghen, J.; Dehaen, W. Org. Lett. 2007, 9,3165–3168.

Scheme 1. Schematic Illustration of AmperometricResponse of a Gold Electrode Coated with HSCOR/HS(CH2)6OH SAM Generated in the Presence ofDopamine

7399Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

CH2), 1.39-1.26 (m, 13H, 6CH2 and SH), ∼-1.9 (br. s, 3H,NH);13C NMR 158.8 (CO), 142.1 (9-C and 11-C of corrole), 139.8(6-C and 14-C of corrole), 138.5, 137.4, 135.5, 134.6, 134.0, 130.7,130.3, 128.0 (3-C and 5-C of 5,15-dichlorophenyl), 127.1, 125.7(8-C and 12-C of corrole), 120.8 (A and D ring -pyrrole C),116.2 (A and D ring -pyrrole C), 113.2 (A and D ring -pyrroleC), 111.7 (5-C and 15-C of corrole), 108.9 (10-C of corrole);ESI-MS 865 (MH+).

Reagents and Materials. 1-Dodecanethiol (DDT), 6-mercapto-1-hexanol (HS(CH2)6OH), 11-mercapto-1-undecanol (HS(CH2)11-OH), [Ru(NH3)6]Cl3, dopamine hydrochloride (DA), creatinine,creatine, uric acid, chloroform were purchased from Sigma-Aldrich (Poznan, Poland). KOH, KNO3, NaCl, borate buffer,L(+)ascorbic acid (AA), ethanol were obtained from ABChem,Gliwice, Poland. All aqueous solutions were prepared withdeionized and charcoal-treated water (resistivity of 18.2 MΩcm) purified with a Milli-Q reagent grade water system(Millipore, Bedford, MA).

Electrode Preparation. Gold disk electrodes of 2 mm2 area(Bioanalytical Systems (BAS), West Lafayette, IN) used for theexperiments were polished with wet 0.3 and 0.05 µm aluminaslurry (Alpha and Gamma Micropolish, Buehler, Lake Bluff,IL) on a flat pad for at least 10 min and rinsed repeatedly withwater and finally in a sonicator (30 s). The polished electrodeswere then dipped in 0.5 M KOH solution deoxygenated bypurging with argon for 15 min, and the potential was cycledbetween -400 and -1200 mV (versus Ag/AgCl referenceelectrode) with a scan rate of 100 mVs-1 until the CV no longerchanged.

The electrochemically polished electrodes were dipped intodifferent modification solutions in chloroform at room temperaturefor 30 min (Table 1). The modification solutions were put intothe tubes (8 mm diameter, with no flat bottom). After theelectrodes were dipped, the tubes were sealed with Teflon tapein order to avoid the solvent evaporation. The modified electrodeswere stored in buffer solution (0.1 M KNO3, 0.01 M borate buffer,pH ) 7.0) until used.

Electrochemical Measurements. All electrochemical mea-surements were performed with a potentiostat-galvanostat AutoLab(Eco Chemie, Utrecht, Netherlands) with a three-electrode con-figuration. Potentials were measured versus the Ag/AgCl elec-trode, and a platinum wire was used as an auxiliary electrode.Osteryoung square-wave voltammetry (OSWV) was performedwith a potential from 0 to -500 mV, and with a step potential of5 mV, a square-wave frequency of 100 Hz, and an amplitude of 50mV for 1.0 mM [Ru(NH3)6]Cl3.

Electrochemical impedance spectroscopy (EIS) measurementswere recorded within the frequency range of 0.1 Hz to 10 kHz atthe formal potential of the redox couple [Ru(NH3)6] 3+/2+ (-0.20V) with an amplitude of 10 mV. The dependence of theOsteryoung square-wave voltammograms and electrochemicalimpedance spectra on the pH was measured in a solutioncontaining 0.1 M KNO3 and 0.01 M borate buffer. The pH ofthis solution was adjusted with 0.1 M KOH or 0.1 M HNO3.

Determination of Dopamine in Diluted Human Plasma.Gold electrodes modified with HSCOR/HS(CH2)6OH (modifica-tion no. 5, Table 1) were used for voltammetric and impedimetricdetermination of DA in human plasma. Natural human plasma T

ab

le1

.Co

mp

osi

tio

no

fth

eC

hlo

rofo

rmS

olu

tio

ns

Use

dfo

rth

eG

old

Ele

ctr

od

eM

od

ific

ati

on

an

dT

he

irR

esp

on

ses

To

wa

rds

DA

com

posi

tion

ofm

odifi

catio

nso

lutio

nde

tect

ion

limit

[M]

inbu

ffer

solu

tion

dete

ctio

nlim

it[M

]in

hum

anpl

asm

ab

no.

HSC

OR

[mM

]D

DT

[mM

]H

S(C

H2)

6OH

[mM

]H

S(C

H2)

11O

H[m

M]

linea

rra

nge

[M]

buffe

rso

lutio

nahu

man

plas

maa

,bO

SWV

EIS

OSW

VE

IS1

0.01

1.0

(1.0

×10

-9 )-

(1.0

×10

-5 )

Y 1)

-3.

6lo

g(x)

+59

.72.

6×

10-

108.

3×

10-

10

r2)

0.96

8,σ)

0.96

Y 2)

10.0

log(

x)+

104.

1r2

)99

1,σ)

0.54

20.

011.

0(3

.2×

10-

11)-

(1.0

×10

-7 )

Y 1)

-3.

6lo

g(x)

+61

.56.

1×

10-

119.

5×

10-

11

r2)

0.99

3,σ)

0.66

Y 2)

13.1

log(

x)+

138.

2r2

)0.

983,

σ)

1.31

30.

011.

0(1

.0×

10-

10)-

(1.0

×10

-6 )

40.

051.

0(1

.0×

10-

11)-

(5.6

×10

-9 )

50.

101.

0(3

.2×

10-

12)-

(3.2

×10

-10

)Y 1

)-

4.5

log(

x)+

43.8

Y 1)

-15

.0lo

g(x)

-94

.82.

4×

10-

123.

3×

10-

121.

5×

10-

125.

3×

10-

12

r2)

0.86

8,σ)

1.07

r2)

0.91

2,σ)

1.44

Y 2)

16.2

log(

x)+

193.

1Y 2

)56

.1lo

g(x)

+69

5.6

r2)

0.90

6,σ)

1.15

r2)

0.98

5,σ)

3.10

61.

0(1

.0×

10-

9 )-(1

.0×

10-

5 )Y 1

)-

6.3

log(

x)+

24.9

1.7

×10

-11

9.5

×10

-11

r2)

0.95

9,σ)

0.44

Y 2)

28.6

log(

x)+

309.

6r2

)0.

929,

σ)

13.8

97

1.0

(1.0

×10

-9 )-

(1.0

×10

-5 )

aT

helin

ear

rela

tions

hip:

Y 1)

OSW

Vre

spon

se(i

p/i 0

)×

100%

;i0,

OSW

Vpe

akcu

rren

tin

the

abse

nce

ofan

alyt

es,i

p,O

SWV

peak

curr

enti

nth

epr

esen

ceof

agi

ven

conc

entr

atio

nof

anal

ytes

.Y2)

EIS

resp

onse

(Ri-

R0/

R0)

×10

0%;R

0,E

ISre

sist

ance

inth

eab

senc

eof

anal

ytes

,Ri,

EIS

resi

stan

cein

the

pres

ence

ofa

give

nco

ncen

trat

ion

ofan

alyt

es.x

)co

ncen

trat

ion

ofan

alyt

e;r2 ,c

orre

altio

nco

effic

ient

;σ,

aver

age

stan

dard

devi

atio

n,n)

5.b

Hum

anpl

asm

aaf

ter

filtr

atio

nan

dce

ntri

fuga

tion

for

30m

inat

1400

0grc

fwith

Mill

ipor

eM

icro

con

Ultr

acel

YM

-3an

dw

asdi

lute

d80

times

with

0.9%

NaC

l+0.

01M

bora

tebu

ffer

pH7.

0;th

evo

lum

ein

elec

troc

hem

ical

cell:

2.0

mL.

7400 Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

obtained from the Regional Center of Blood-Donation in Olsztynwas filtered with a Millipore Micron Ultracel YM-3 and centrifugedfor 30 min at 14 000g rcf in order to remove proteins with amolecular weight above 3 kDa. The filtered human plasma wasdiluted 80 times with a 0.9% NaCl + 0.01 M borate buffer solutionof pH 7.0. A 2.0 mL amount of thus prepared human plasmasamples were spiked with a known amount of DA and analyzedwith OSWV and EIS.

In order to check the influence of the centrifugation andfiltration on the dopamine concentration, 500 µL of human plasmawas spiked with 0.1 mM DA. This sample was centrifuged for 30min at 14 000g rcf. The concentration of DA in the supernatantwas determined by UV-vis spectroscopy.

UV-Visible Confirmation of Dopamine-Corrole ComplexFormation. A stock solution of 1.0 × 10-3 M was prepared bydissolving DA in DMSO. The working solution of HSCOR forspectrophotometric measurements in DMSO was 1.0 × 10-5

M. UV-vis spectra of 1.0 × 10-5 M of HSCOR in the presenceof different concentrations of DA: (a) 0, (b) 2.5 × 10-5, (c) 1.0× 10-4, (d) 5.0 × 10 -4, and (e) 7.5 × 10-4 M were recordedwith an UV mini-1240 spectrophotometer (Shimadzu, Kyoto,Japan).

RESULTS AND DISCUSSION

Formation and Characterization of Corrole IncorporatingSAMs. It has been already discovered that corrole formscomplexes with the hydroxy function of phenols via the formationof a hydrogen bond.45 The corrole-dopamine interaction wasproven by UV-vis measurements (Figure 1).

Therefore HSCOR was selected as the host molecule for theelectrochemical determination of DA and was used for chemicalmodification of gold electrodes via Au-S bond formation. In orderto show the influence of the SAM composition used for the goldelectrode modification on the sensitivity of DA determination, fivedifferent mixed SAMs were explored (Table 1). Mixed SAMs wereused in order to prevent mutual interactions between corrolemacrocycles. The same strategy was applied for the creation of

macrocyclic polyamine SAMs43,44 and dipyrromethene SAMs.49

1-Dodecanethiol (DDT), 6-mercapto-1-hexanol (HS(CH2)6OH),and 11-mercapto-1-undecanol (HS(CH2)11OH) were applied for“dilution” of corrole in mixed monolayers.

HSCOR has a longer alkyl chain than HS(CH2)6OH; therefore,in these type of SAMs, corrole “heads” were distributed overthe surface of HS(CH2)6OH, which creates a well orderedmonolayer through hydrogen bond between -OH groups(Table 1, modification nos. 2, 4, and 5). The lengths of the alkylchains of DDT, HS(CH2)11OH, and HSCOR are similar, so thecorrole “heads” were surrounded by OH or CH3 groups inmodification nos. 1 and 3 (Table 1).

The corroles, because of their unusually high N-H acidityrelative to porphyrins, could exist, depending on the pH, in aneutral, deprotonated and protonated form.45,50 The binding ofprotons by corroles’ receptors, in relation to the pH of the aqueousphase, changes the surface charge from neutral (pH 8.0) topositive (pH 4.0), thereby suppressing the access of positivelycharged [Ru(NH3)6]3+ and the subsequent electron transfer ratebetween the electrode and maker. The OSWV and EISperformed at pH 4.0 and at pH 8.0 were used as a simple testfor checking the SAMs quality for every type of modification.All of the modifications incorporating HSCOR gave similarresults. A typical pH test for HSCOR-HS(CH2)6OH SAM no. 5is shown in Figure 2. The pH tests for modification nos. 1 and 3are shown in the Supporting Information (Figure S-1,A,B). Onlythese electrodes that displayed good response toward protonshave been used for the determination of DA.

A similar pH test was used for confirmation of the presenceof macrocyclic polyamine43,44 and dipyrromethene49 on the surfaceof gold electrodes.

The responses toward pH were also recorded for SAMsconsisting only of DDT (modification no. 6) and HS(CH2)6OH(modification no. 7; Table 1). For these SAMs, pH responseswere not observed. The representative OSWV and EIS areillustrated in the Supporting Information, Figure S-1,C,D.

Response of Corrole Mixed SAMs toward Dopamine. Thesensing of DA molecules by gold electrodes modified with corrolehas been examined with Osteryoung square-wave voltammetryand electrochemical impedance spectroscopy techniques. Themeasurements were done in 0.1 M KNO3 and 0.01 M boratebuffer (pH 7.0).

Among the following marker candidates: [Fe(CN)6]3-,[Ru(NH3)6]3+, and [IrCl6]2-, the most suitable for study of theinteractions between neutral corrole host immobilized on thegold electrode surface and DA guest existing in the aqueoussolution as the protonated species was [Ru(NH3)6]3+. Theoxidation potential for DA and AA on every type of modificationusing a mixed monolayer was at 0.380 ± 0.015 and 0.060 ± 0.012V, respectively. Ruthenium complex oxidation was observedat -0.204 ± 0.01 V. Thus, when [Ru(NH3)6]3+ was used as theredox marker in the sample solutions, oxidation of DA as wellas AA did not occur.

(49) Szymanska, I.; Orlewska, C.; Janssen, D.; Dehaen, W.; Radecka, H.Electrochim. Acta 2008, 53, 7932–7940.

(50) Mohammed, A.; Weaver, J. J.; Gray, H. B.; Abdelas, M.; Gross, Z.Tetrahedron Lett. 2003, 44, 2077–2079.

Figure 1. The absorption spectra of 1.0 × 10-5 M HSCOR in DMSOin the presence of different concentrations of DA: (a) 0, (b) 2.5 ×10-5, (c) 1.0 × 10-4, (d) 5.0 × 10-4, and (e) 7.5 × 10-4 M.

7401Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

The responses toward DA of all modified gold electrodes(Table 1, Figure 3) were checked in order to find the most suitableone. On the basis of the results obtained, the best compound fordilution of HSCOR in a mixed SAM is HS(CH2)6OH. Therefore,for this type of modification, the molar ratio between HSCORand HS(CH2)6OH was optimized. Among the compositionsstudied (Table 1; modification nos. 2, 4, and 5), this one with aHSCOR/HS(CH2)6OH molar ratio equal to 1:10 was the mostsensitive toward DA. With an increasing concentration of DAin the range from 3.2 × 10-12 to 3.2 × 10-10 M, the peak currentsof OSWV decreased and the peak potentials shifted morenegatively (Figure 3A). Taking into account a signal-to-noise ratioof 3, the detection limit was estimated as 2.4 × 10-12 M.

The responses of the gold electrodes modified with HSCOR/HS(CH2)6OH were also measured using the EIS technique. Themeasurements were carried out under the same conditions asfor OSWV. Representative EIS curves obtained for Au-HSCOR/HS(CH2)6OH electrodes in the presence of DA are illustrated inFigure 3A. The diameter of Nyquist circles increases with theincreasing concentration of DA. All the impedance spectra were fittedto the Randles equivalent circuit in order to obtain the values ofcharge transfer resistance for each concentration of DA studied. Inorder to compare the different electrodes measured in the sameconditions, the relative values: [(Rct - Rct(0))/Rct(0)] × 100% wereused as analytical signals. The detection limit was estimated as3.3 × 10-12 M, defined from a signal-to-noise ratio of 3. EIS is moresensitive in comparison to OSWV. The slope of the calibrationcurves obtained for modification no. 5 with OSWV and EIS were4.5 [%] M-1 and 16.2 [%] M-1, respectively (Table 1).

Representative OSWV and EIS curves obtained for Au-HS-COR/HS(CH2)11OH (modification no. 3, Table 1) are shown inFigure 3B. This modification was less sensitive toward DA. Thesurrounding of corrole macrocycle by tightly packed OH groupsmakes the binding sites more rigid and less accessible for DA.This is probably the main reason for the lower response.

The corrole macrocycles were surrounded by hydrophobicCH3 groups in the case of modification no. 1 (Table 1). Thesensitivity of this modification toward DA was quite good (Table

1), but unfortunately, this modification was not selective. A controlexperiment proved that responses of DDT SAM (no. 6) towardDA are even better than the one obtained using Au-HSCOR/DDT(Table 1; Figure 3C,E). In this case, unspecific hydrophobicinteractions between DDT SAM and DA occurred. Therefore,DDT is not a suitable compound for the formation of mixedmonolayers with HSCOR destined for DA determination.

A similar control experiment was performed with aHS(CH2)6OH SAM (no. 7). This monolayer showed no responsetoward DA in the buffer solution (Figure 3D). HS(CH2)6OHmolecules formed well-packed monolayers through hydrogenbonds between OH groups. This eliminates unspecific interac-tions with DA.

The above results allow us to state that among the modifica-tions studied, this with HSCOR/HS(CH2)6OH in a molar ratioof 1:10 was the most suitable for determination of DA. Thegold electrodes modified with this composition, after measuringthe DA response, were restored by conditioning in the buffersolution. They could be used five times to explore DAresponses with the same sensitivity. Also, storing of this typeof electrode in buffer solution in the refrigerator during 1 monthhas no influence on its sensitivity toward DA. Therefore, thismodification was used for the future experiments.

Effect of Human Plasma Components on Dopamine De-termination by Gold Electrode Modified with HSCOR/HS(CH2)6OH SAM. In order to demonstrate the applicabilityof the sensor proposed, the influence of main components ofhuman plasma on the electrochemical determination of DA withAu-HSCOR/HS(CH2)6OH were checked. The following com-pounds were explored: ascorbic acid (AA), creatinine, creatine,and uric acid.

With 1.0 × 10-4 M AA in the buffer solution, the data pointsin the calibration curve are parallel to points in the curveobtained for DA in the buffer solution without AA (Figure S-2,Supporting Information). These results demonstrate the near-complete elimination of interference from ascorbic acid. Similarresults were obtained in the presence of other components ofhuman plasma. Thus, it might be concluded that the Au electrode

Figure 2. The OSWV and EIS of HSCOR/HS(CH2)6OH (1:10) modified gold electrode in the electrolyte solution (a) pH 5.0 and (b) pH 8.0. Theelectrolyte composition: 0.1 M KNO3, 0.01 M borate buffer, 1.0 mM [Ru(NH3)6]Cl3; the pH of the solution was adjusted with 0.1 M NaOH or 0.1M HNO3. The OSWV was performed with step potential: 5 mV, square-wave frequency 100 Hz, and square-wave amplitude 50 mV. The EISmeasurements were recorded within the frequency range of 0.1 Hz to 10 kHz at the formal potential of the redox couple [Ru(NH3)6]3+/2+ (-0.20V) with ac amplitude of 10 mV.

7402 Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

Figure 3. The OSWV and EIS responses of gold electrodes modified with (A) HSCOR/HS(CH2)6OH, (B) HSCOR/HS(CH2)11OH, (C) HSCOR/DDT, (D) HS(CH2)6OH, and (E) DDT toward DA in buffer solution. The electrolyte composition: 0.1 M KNO3, 0.01 M borate buffer pH 7.0, 1.0mM [Ru(NH3)6]Cl3; concentration of DA: (A) (3.2 × 10-12)-(3.2 × 10-10); (B-E) (1.0 × 10-10)-(1.0 × 10-6) M. The parameters of OSWV andEIS measurements, see Figure 2.

7403Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

modified with HSCOR/HS(CH2)6OH SAM with [Ru(NH3)6]3+ asthe redox marker in the sample solutions could be used fordirect electrochemical determination of DA in human plasma.

Determination of Dopamine in Diluted Human Plasma.The sensitivity of gold electrodes modified with HSCOR/HS(CH2)6OH in a molar ratio of 1:10 is sufficient forcontrolling the safe concentration level of DA or its overdosein human plasma.3-6 The applicability of the proposed sensorwas tested using the OSWV and EIS techniques. Beforemeasurements, the samples of human plasma were passedthrough a Millipore Micron Ultracel YM-3 and centrifuged for30 min at 14 000g rcf in order to remove the proteins withmolecular weight over 3 kDa. In order to check the influenceof the centrifugation and filtration on dopamine concentration,500 µL sample of human plasma was spiked with 0.1 mM ofdopamine and centrifuged for 30 min at 14 000g rcf. Theconcentration of dopamine in the supernatant, determined byUV-vis spectroscopy, was the same as the concentration ofdopamine added to the plasma. This result confirms that thecentrifugation and filtration does not influence the dopamineconcentration.

The presence of centrifuged and undiluted human plasmacaused a strong matrix influence. Therefore, the voltammetricand impedimetric responses of the modified electrode studiedtoward DA were explored in the presence of 80 times dilutedhuman plasma with 0.9% NaCl containing 0.01 M borate bufferpH 7.0 and 1.0 mM [Ru(NH3)6]Cl3. The appropriate dilutiondegree was found experimentally. Representative OSWV andEIS curves are shown in Figure 4A,B. The presence of the

diluted human plasma influenced electrode sensitivity towardDA very little. The linear range of response was from 3.2 ×10-12 to 3.2 × 10-10 M. The detection limit estimated in thepresence of the human plasma defined from a signal-to-noiseratio of 3 was 1.5 × 10-12 and 5.3 × 10-12 M, respectively, forthe OSWV and EIS measurement techniques (Table 1).

The ion channel mimetic sensor having HSCOR as thesensitive element displayed better sensitivity, in the 10-12 Mrange, toward DA in comparison to electrochemical sensorsbased on separation of the oxidation peaks for DA and AA.17-29

Also, the proposed sensor is free from the interferences comingfor the components of human plasma.

The applicability of the proposed sensor was checked byperforming the recovery test. A volume of 2.0 mL of humanplasma was centrifuged, 80 times diluted with 0.9% NaClcontaining 0.01 M borate buffer pH 7.0 and 1.0 mM[Ru(NH3)6]Cl3 (see Experimental Section), and spiked witha known amount of DA. Next, the DA concentration wasdetermined by Au-HSCOR/HS(CH2)6OH using both tech-niques, OSWV and EIS, on the basis of a calibration equationobtained in the presence of human plasma prepared asdescribed above. The recovery test performed for DAdetermination in the presence human plasma (Table 2)indicated that the gold electrode coated with mixed HSCOR/HS(CH2)6OH SAM could be applied for the direct electro-chemical determination of DA in physiological samples inthe very low concentration range 10-12-10-11 M.

Figure 4. The OSWV (A) and EIS (B) response of gold electrodes modified with HSCOR/HS(CH2)6OH (1:10) toward DA in an 80-fold dilutedhuman plasma solution. Concentrations of DA: (a) 0, (b) 3.2 × 10-12, (c) 5.6 × 10-12, (d) 1.0 × 10-11, (e) 1.8 × 10-11, (f) 3.2 × 10-11, (g) 5.6× 10-11, (h) 1.0 × 10-10, (i) 1.8 × 10-10, and (j) 3.2 × 10-10 M. The solution composition: human plasma centrifuged and 80-fold diluted with0.9% NaCl, 0.01 M borate buffer pH 7.0, 1.0 mM [Ru(NH3)6]Cl3. The volume in the measuring cell was 2.0 mL (see Experimental Section). Theparameters of OSWV and EIS measurements, see Figure 2.

Table 2. Recovery Test Performed with HSCOR/HS(CH2)6OH Gold Electrode (Modification Number 5) in thePresence of 80-Fold Diluted Human Plasmaa

OSWV EIS

dopamine added (mol/L) dopamine determined (mol/L) recovery (%) dopamine determined (mol/L) recovery (%)7.4 × 10-12 7.0 × 10-12 (±0.13) 94.6 (±1.8) 8.0 × 10-12 (±0.07) 108.1 (±1.0)1.5 × 10-11 1.4 × 10-11 (±0.03) 93.3 (±1.8) 1.6 × 10-11 (±0.02) 106.7 (±1.4)2.2 × 10-11 2.2 × 10-11 (±0.03) 100.0 (±1.5) 2.3 × 10-11 (±0.04) 104.5 (±1.9)

a Measuring conditions, see Table 1; n ) 3).

7404 Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

To our knowledge, the only ion channel mimetic approach forDA determination has been reported by Shervedani et al.51 TheDA accumulated on the surface of a gold electrode modified with4-formylphenylboronic acid covalently attached to the amino groupof the cysteamine was determined by cyclic voltammetry in thepresence of redox marker [Fe(CN6)]3-/4-. The detection limitobserved in the PBS buffer was in the range 10-9 M. Measure-ments in the presence of human plasma were not performed.

Mechanisms of Amperometric Response of HSCOR/HS(CH2)6OH Coated Gold Electrode toward Dopamine.In the present study, the sensing of DA was performed based onthe formation of the DA-HSCOR complex on the electrode/aqueous interface. OSWV and EIS measurements were carriedout in the presence of a solution of 0.1 M KNO3 + 0.01 M boratebuffer pH 7.0 or in the presence of centrifuged human plasma80 times diluted with 0.09% NaCl + 0.01 M borate buffer pH7.0. All sample solutions contained 1.0 mM [Ru(NH3)6Cl3.

At pH 7.0, DA, because of the presence of the amino group, ispresent in the cationic form (pKa ) 8.9),36 whereas corrolemolecules immobilized on gold electrode are still neutral.45,50

The partial positive charging of the neutral surface of Au-HSCOR/HS(CH2)6OH upon interaction with the cationic form of DAincreases repulsion between the electrode and the positivelycharged redox marker [Ru(NH3)6]3+ (Scheme 1), as could beexpected according to the general idea of ion-channel mimeticsensors.40-44 This caused a decrease of the electron transfer ratebetween the marker and the electrode surface, which was linearrelated to increasing DA concentration.

The interaction between dopamine and corrole moleculeswithin one phase, in DMSO solution, was also proven by UV-visspectrophotometry. Upon an increase in the DA concentration,the peaks of corrole absorption spectra at 430, 449, and 631 nmdecreased. On the other hand, the peaks at 588 and 758 nmincreased and slightly shifted toward higher value (Figure 1).

CONCLUSIONSA thio-derivative of corrole has been immobilized on the

surface of gold electrodes via covalent Au-S bonds acting as aselective receptor for dopamine. The complex formation on theelectrode surface between the corrole host and dopamine guestvia hydrogen bonding was detected by Osteryoung square-wavevoltammetry and by electrochemical impedance spectroscopyusing [Ru(NH3)6]Cl3 as the redox marker. Under the measuringconditions, within the potential window from 0 to -500 mV,the oxidation of dopamine and ascorbic acid, the main interfer-ing compounds, does not exist.

The proposed sensor was effective regarding the followingparameters: very good sensitivity toward DA (detection limit in10-12 M range by using both EIS and OSWV techniques), verygood selectivity (human plasma components at 80-fold dilutionhave no influence on dopamine determination), very simple,and quick procedure of electrode preparation. Therefore, thegold electrodes modified with mixed HSCOR/HS(CH2)6OHwork as “ion-channel mimetic sensors” in the presence of[Ru(NH3)6]Cl3 as the redox marker and could be applied forthe determination of dopamine in clinical analysis.

ACKNOWLEDGMENTThis work was supported by a grant from the Polish Ministry

of Science and Higher Education Grant No. 105/6.PR UE/ 2007/7and the statutory fund of the Institute of Animal Reproductionand Food Research of Polish Academy of Sciences, Olsztyn,Poland.

SUPPORTING INFORMATION AVAILABLEAdditional information as noted in text. This material is

available free of charge via the Internet at http://pubs.acs.org.

Received for review June 4, 2009. Accepted July 13, 2009.

AC901213H(51) Shervedani, R. K.; Bagherzadeh, M. Electroanalysis 2008, 5, 550–557.

7405Analytical Chemistry, Vol. 81, No. 17, September 1, 2009