Melamine Acoustic Chemosensor Based on Molecularly Imprinted

Anal. Chem. 2009, 81, 10061–10070

Melamine Acoustic Chemosensor Based on Molecularly Imprinted Polymer Film Agnieszka Pietrzyk,† Wlodzimierz Kutner,*,†,‡ Raghu Chitta,§ Melvin E. Zandler,§ Francis D’Souza,*,§ Francesco Sannicolo`,| and Patrizia R. Mussini⊥ Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland, Faculty of Mathematics and Natural Sciences, School of Science, Cardinal Stefan Wyszynski University in Warsaw, Dewajtis 5, 01-815 Warsaw, Poland, Department of Chemistry, Wichita State University, 1845 Fairmount, Wichita, Kansas 67260-0051, Department of Organic and Industrial Chemistry, University of Milan, Via Venezian 21, 20133 Milan, Italy, and Department of Physical Chemistry and Electrochemistry, University of Milan, Via Golgi 19, 20133 Milan, Italy A melamine piezomicrogravimetric (acoustic) chemosensor using a molecularly imprinted polymer (MIP) film has been devised and tested. The MIP films were prepared by electropolymerization of the melamine complexed by the functional monomer of the bis(bithiophene) derivative bearing an 18-crown-6 substituent 4. The structure of the MIP-melamine complex was visualized by the DFT B3LYP/3-21G(*) energy optimization calculations. The sensitivity and selectivity of the MIP film was improved by cross-linking the polymer with the bithianaphthene monomer 5 and the presence of the porogenic ionic liquid in the prepolymerization solution. After electropolymerization, the melamine template was extracted from the MIP film with an aqueous strong base solution. The measurements of UV-vis spectroscopy, X-ray photoelectron spectroscopy (XPS), DPV, and EIS as well as scanning electrochemical microscopy (SECM) imaging confirmed extraction of the melamine template from the MIP film and then rebinding of the melamine analyte while the film relative roughness and porosity was determined by atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging, respectively. The analytical as well as kinetic and thermodynamic parameters of the chemosensing were assessed under flow-injection analysis (FIA) conditions with piezoelectric microgravimetry (PM) detection. The linear concentration range for melamine detection was 5 nM to at least 1 mM with a limit of detection of ∼5 nM. The chemosensor successfully discriminated the cyanuric acid, cyromazine, and ammeline interfering agents.

for a protein level in, e.g., food products.1 Fatally, melamine was recently detected in baby milk formulas, pet food, animal feed, and protein sources including wheat and corn gluten as well as a rice protein concentrate.2 Melamine can form lethal kidney stones,2 especially when combined with cyanuric acid 3, due to precipitation of insoluble melamine cyanurate. High and prolonged dietary exposure to melamine results in the formation of bladder stones and increase incidence of urinary bladder tumors in male rats.3 Therefore, determination of melamine is of biological, clinical, and food industry importance. The permissible concentration of melamine in milk products should not exceed 2.5 ppm (20 µM) in the USA and the E.U. and 1 ppm (8 µM) in infant formulas in China.4 Unfortunately, the current melamine determination methods, although mostly accurate and sensitive, are quite expensive, requiring advanced and sophisticated instrumentation operated by trained personnel, a complicated matrix, and, in many instances, an extensive sample pretreatment including extraction, preconcentration, or derivatization.4,5 The melamine determination using an enzymatic essay kit6 is simple but is not very selective because of the use of the not too highly selective enzyme, melamine deaminase.7 Molecular imprinting8 is a well established approach to develop artificial recognition systems capable of mimicking features of the corresponding biological systems. This imprinting is a relatively inexpensive procedure for preparation of the MIP synthetic receptors with appreciable affinity, selectivity, and toughness.9 Hence, the development of a chemosensor featuring analyte polymeric recognition elements, prepared by molecular imprinting, could offer an inexpensive yet sensitive and selective method for melamine determination. This has been accomplished here where the MIP chemosensor prepared by an easy and straightforward

Melamine 1 (Scheme 1), a plant metabolite of cyromazine 2 pesticide, can artificially inflate with its 66% nitrogen, the reading

(1) (a) Bradley, E. L.; Boughtflower, V.; Smith, T. L.; Speck, D. R.; Castle, L. Food Addit. Contam. 2005, 22, 597–606. (b) Kawai, S.; Nagano, H.; Maji, T. J. Chromatogr. 1989, 477, 467–470. (c) Lund, K. H.; Petersen, J. H. Food Addit. Contam. 2006, 23, 948–955. (d) Martin, R. E.; Hizo, C. B.; Ong, A. M.; Alba, O. M.; Ishiwata, H. J. Food Prot. 1992, 55, 632–635. (2) Brown, C. A.; Jeong, K.-S.; Poppenga, R. H.; Puschner, B.; Miller, D. M.; Ellis, A. E.; Kang, K.-I.; Sum, S.; Cistola, A. M.; Brown, S. A. J. Vet. Diagn. Invest. 2007, 19, 525–531. (3) (a) http://www.inchem.org/pages/sids.html; OECD Screening Information Data Set (SIDS) Analysis: Melamine, United Nations Environment Program, 2002; (b) In 73; (IARC), I. A. f. R. o. C., Ed.: Lyon, France, 1999. (4) Zhu, L.; Gamez, G.; Chen, H.; Chingin, K.; Zenobi, R. Chem. Commun. 2009, 559–561.

* Corresponding author. E-mail: [email protected] (W.K.); Francis.DSouza@ wichita.edu (F.D.S.). Phone: +48 22 343 32 17 (W.K.); +1 316-978-7380 (F.D.S.). Fax: +48 22 343 33 33 (W.K.); +1 316-978-3431 (F.D.S.). † Polish Academy of Sciences. ‡ Cardinal Stefan Wyszynski University in Warsaw. § Wichita State University. | Department of Organic and Industrial Chemistry, University of Milan. ⊥ Department of Physical Chemistry and Electrochemistry, University of Milan. 10.1021/ac9020352 CCC: $40.75  2009 American Chemical Society Published on Web 11/19/2009

Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

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Scheme 1. Structural Formula of Melamine 1, Its Protonated Form 1a, and Its Protonated Imine Counterpart 1a′ of the Acid-Base Equilibrium in Aqueous Solution as well as Those of the Cyromazine 2, Cyanuric Acid 3, and Ammeline 7 Interfering Compounds and the Bis(2,2′-bithienyl)-benzo-[18-crown-6]methane 4 Functional Monomer Used for Preparation of MIP A, MIP B, and MIP C, and the 3,3′-Bis[2,2′-bis(2,2′-bithiophene-5-yl)]thianaphthene 5 Crosslinking Monomer Used for Preparation of MIP B and MIP C, and Also of the Trihexyl(tetradecyl)phosphonium tris(pentafluoroethyl)-trifluorophosphate Ionic Liquid 6 Used for Preparation of MIP C

way is shown to be capable of selective determining of melamine at an appreciably low, nanomolar concentration level. (5) (a) Toth, J. P.; Bardalaye, P. C. J. Chromatogr., A 1987, 408, 335–340. (b) Litzau, J.; Mercer, G.; Mulligan, K.; FDA Laboratory Information Bulletin, LIB No. 4423; 2008; Vol. 24. (c) Fligenzi, M. S.; Tor, E. R.; Poppenga, R. H.; Aston, L. A.; Puschner, B. Rapid Commun. Mass Spectrom. 2007, 21, 4027– 4032. (d) Turnipseed, S.; Casey, Ch.; Nochetto, C.; Heller, D. N.; FDA Laboratory Information Bulletin, LIB No. 4421; 2008; Vol. 24. (e) Smoker, M., Krynitsky, A. J.; FDA Laboratory Information Bulletin, LIB No. 4422; 2008; Vol. 24. (f) Kim, B.; Perkins, L. B.; Bushway, R. J.; Nesbit, S.; Fan, T.; Sheridan, R.; Greene, V. J. AOAC Int. 2008, 91, 408–413. (g) Cook, H. A.; Klampfl, C. W.; Buchberger, W. Electrophoresis 2005, 26, 1576– 1583. (h) Campbell, J. A.; Wunschel, D. S.; Petersen, C. E. Anal. Lett. 2007, 40, 3107–3118. (i) Huang, G. M.; Zheng, O. Y.; Cooks, R. G. Chem. Commun. 2009, 5, 556–558. (j) Yang, S. P.; Ding, J. H.; Zheng, J.; Hu, B.; Li, J. Q.; Chen, H. W.; Zhou, Z. Q.; Qiao, X. L. Anal. Chem. 2009, 81, 2426–2436. (k) Vail, T.; Jones, P. R.; Sparkman, O. D. J. Anal. Toxicol. 2007, 31, 304– 312. (l) Garber, E. A. E. J. Food Prot. 2008, 71, 590–594. (m) Ishiwata, H.; Inoue, T.; Yamazaki, T.; Yoshihira, K. J. Assoc. Off. Anal. Chem. 1987, 70, 457–460. (n) Mauer, L. J.; Chernyshova, A. A.; Hiatt, A.; Deering, A.; Davis, R. J. Agric. Food Chem. 2009, 57, 3974–3980. (o) Tseng, C.-H.; Mann, Ch. K.; Vickers, T. J. Appl. Spectrosc. 1994, 48, 421–541. (p) Wang, Z.; Chen, D.; Gao, X.; Song, Z. J. Agric. Food Chem. 2009, 57, 3464–3469. (6) Rinard, P.; Mattern, P. http://www1.umn.edu/news/news-releases/2009/ UR_CONTENT_123750.html; 2009. (7) Seffernick, J. L.; De Souza, M. L.; Sadowsky, M. J.; Wackett, L. P. J. Bacteriol. 2001, 183, 2405–2410. (8) Haupt, K. Anal. Chem. 2003, 75, 376 A383 A. (9) (a) Blanco-Lo´pez, M. C.; Lobo-Castanoń, M. J.; Miranda-Ordieres, A. J.; Tunoń-Blanco, P. Trends Anal. Chem. 2004, 23, 36–48. (b) Piletsky, S. A.; Turner, A. P. F. Electroanalysis 2002, 14, 317–323. (c) Henry, O. Y. F.; Cullen, D. C.; Piletsky, S. A. Anal. Bioanal. Chem. 2005, 382, 947–956.

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For MIP film preparation, we have employed electropolymerization10 since this technique by proper selection of conditions, viz., the amount of charge passed, solution acidity, and the nature of solvent, supporting electrolyte, as well as functional and crosslinking monomer, can conveniently tune the thickness, viscoelastic properties, and morphology (mostly porosity) of the resulting film. Here, a bis(bithiophene)methane bearing a benzo18-crown-6 substituent11 4 was used as the functional monomer. Its complex with the melamine template (Scheme S1 in the Supporting Information) was electrochemically copolymerized with the cross-linking monomer 5 of a 3,3′-bithianaphthene-based backbone under cyclic voltammetry (CV) conditions, in the presence of a phosphonium-phosphate ionic liquid 6. Subsequently, the melamine was extracted with a strong base solution leaving molecular cavities within the film complementary in size and shape to those of the melamine molecules. The reason to use 5 was to afford three-dimensionality of the polymer network12 and to retain structural integrity of the binding sites (Scheme 2). Moreover, 6, a less polar solvent,13 was used to stabilize the prepolymerization complex and yield a MIP film of higher (10) Malitesta, C.; Losito, I.; Zambonin, P. G. Anal. Chem. 1999, 71, 1366– 1370. (11) Kryatova, O. P.; Kolchinski, A. G.; Rybak-Akimova, E. V. Tetrahedron 2003, 59, 231–239. (12) Bonometti, V.; Noworyta, K.; Kutner, W.; Mussini, P. R.; Sannicolo, F., in preparation. (13) www.merck.de/servlet/PB/menu/1320990/index.html.

Scheme 2. Excerpt of Structural Formula of the MIP C Film Prepared by Electropolymerization of the Melamine-4 Complex, Self Organized in Solution, in the Presence of 5 and 6

porosity. Both the rate constants of the analyte rebinding and stability constants of the MIP-melamine complexes as well as the chemosensor analytical performance in the presence of some common interfering compounds are investigated herein under flow-injection analysis (FIA) conditions by piezoelectric microgravimetry (PM) with the quartz crystal microbalance (QCM) detection. The employed PM, due to its subnanogram limit of detection,14 seems to perform well for the present melamine-MIP chemosensor. EXPERIMENTAL SECTION Chemicals. Melamine 1, cyromazine 2, ammeline 7, and acetonitrile were procured from Sigma-Aldrich. Cyanuric acid 3, trifluoroacetic acid (TFA), and tetra-n-butylammonium perchlorate [(TBA)ClO4] were from Fluka. The trihexyl(tetradecyl)phosphonium tris(pentafluoroethyl)-trifluorophosphate ionic liquid 6 was purchased from Merck. The functional monomer, bis(2,2′-bithienyl)-benzo-[18-crown-6]methane 4, was prepared according to the earlier described procedure.15 The crosslinking monomer, 3,3′-bis[2,2′-bis(2,2′-bithiophene-5-yl)]thianaphthene 5,12 is unknown in the literature. Its synthesis and the analytical and structural characterization will be described elsewhere.12 Instrumentation, Techniques, and Procedures. An AUTOLAB computerized electrochemistry system of Eco Chemie, equipped with expansion cards of the PGSTAT 12 potentiostat and the FRA2 frequency response analyzer and controlled by the GPES 4.9 software of the same manufacturer, was used for the (14) (a) A´vila, M.; Zougagh, M.; Rıós, A´.; Escarpa, A. Trends Anal. Chem. 2008, 27, 54–65. (b) Tombelli, S.; Mascini, M. Anal. Lett. 2000, 33, 2129–2151. (c) Uludag, Y.; Piletsky, S. A.; Turner, A. P. F.; Cooper, M. A. FEBS J. 2007, 274, 5471–5480. (15) Pietrzyk, A.; Suriyanarayanan, S.; Kutner, W.; Chitta, R.; D’Souza, F. Anal. Chem. 2009, 81, 2633–2643.

CV, DPV, and EIS measurements. The Randles type equivalent circuit,16 comprising solution resistance (Rs) in series to parallel combination of double layer capacitance (Cdl) and impedance of the faradaic reaction (RCT), was used to fit experimental data of complex-plane impedance. A ∼0.5 mL three-electrode onecompartment V-shaped glass electrochemical minicell was used. A Pt disk of 1 mm in diameter sealed in soft glass tubing, Ag|AgCl, and coiled Pt wire was used as the working, reference, and auxiliary electrode, respectively. For PM experiments performed both under the analytical steady-state and flow conditions, a model EQCM 5710 and EQCM 5610,17 respectively, of the quartz crystal microbalance of IPC PAS was used, under control of the EQCM 5710-S2 software of the same manufacturer. The 5 mm diameter and 100 nm thick Pt film electrodes, sputtered onto the AT-cut, plano-plano, 10 MHz quartz crystal resonators of 14 mm in diameter, were simultaneously used both as the working electrodes and chemosensor substrates. Before electrodeposition of the polymer films, the resonators were cleaned for 30 s with a “piranha” solution (H2O2/H2SO4; 1:3, v/v; Caution: the “piranha” solution is dangerous if contacting skin or eyes). A Pt ring or Pt coil as well as an AgCl film-coated Ag ring or Ag wire was used as the counter and pseudoreference electrode, respectively. The resonant frequency changes were measured with 1 Hz resolution. The FIA experiments with the EQCM 5610 quartz crystal holder were performed at a 150 µm distance between the nozzle of the inlet capillary and surface of the resonator.17 The computational calculations for the MIP-melamine complexes were performed by DFT B3LYP/3-21G(*) methods with the GAUSSIAN 0318 software package on high speed PCs. The UV-visible spectra were recorded by the use of a Shimadzu UV 2501-PC spectrophotometer. The X-ray photoelectron spectroscopy (XPS) spectra were recorded with Al KR (hν ) 1486.6 eV) X-ray radiation using an Escalab-210 spectrometer of VG Scientific and were analyzed by the Avantage Data System software of Thermo Electron. The scanning electrochemical microscopy (SECM) images of the films were recorded with the CHI900B scanning electrochemical microscope of CHI housed in a Faraday cage. A three-electrode SCEM cell contained a 10 µm diameter Pt (RG ≈ 7) disk microelectrode, Ag|AgCl|KCl(sat.), and Pt wire as the working (SECM tip), reference, and counter electrode, respectively. A 1 mM K4Fe(CN)6 in 0.1 M KNO3 solution was used for the SECM imaging. The tip was biased at 0.50 V, i.e., at the potential characteristic of the diffusion rate controlled electrooxidation of Fe(CN)64-. The current was recorded as a function of lateral (16) Randles, J. E. Discuss. Faraday Soc. 1947, 1, 11–19. (17) Kochman, A.; Krupka, A.; Grissbach, J.; Kutner, W.; Gniewinska, B.; Nafalski, L. Electroanalysis 2006, 18, 2168–2173. (18) Frisch, M. J. T. G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 03; Gaussian, Inc.: Pittsburgh PA, 2003.


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Table 1. Composition of Solutions for Preparation of Imprinted (MIP) and Non-Imprinted (NIP) Polymer Films polymer MIP A MIP B MIP C NIP C

mole ratio of 1:4:5

presence of 6

1:3:0 1:3:3 1:3:3 0:3:3

no no yes yes

position of the tip placed ∼10 µm above the MIP surface.19 The 200 × 200 µm2 area was scanned with the tip velocity of 25 µm s-1. The MIP films were imaged by atomic force microscopy (AFM) using a Multimode NS3D instrument, equipped with the 10 × 10 µm2 scanner, of the Digital Instruments/Veeco Metrology Group. The films were FE scanning electron microscopy (SEM) imaged with a low-energy SEM SUPRA microscope of Zeiss using 2.00 kV energy of the accelerated electron beam. Chemosensor Fabrication. The preparation of all MIP films involved electropolymerization performed under CV conditions over the potential range of 0.50 to 1.50 V and the potential scan rate of 50 mV/s. The growth of the films, on the Pt/quartz electrodes, was controlled by the number of CV cycles and monitored with EQCM by simultaneous measurement of changes of the resonant frequency and dynamic resistance. Compositions of the solutions used for preparation of all films are compiled in Table 1. In contrast to the formerly devised PZ histamine MIP chemosensor,15 importantly, no poly(bithiophene) blocking underlayer was necessary for proper operation of the presently devised melamine chemosensor (vide infra). After electropolymerization, all MIP films were rinsed with abundant acetonitrile solvent in order to remove excess of the supporting electrolyte solution. Then, the melamine template was extracted with 0.01 M NaOH for 12 h. Completeness of this extraction was confirmed by the UV-vis spectroscopy, XPS, and electrochemical experiments. Moreover, a control nonimprinted polymer (NIP C) film was prepared by the use of the procedure similar to that used for preparation of the MIP C film, however, in the absence of the melamine template. The MIP A, MIP B, and MIP C films were grown to reach the same mass, determined by the use of the Sauerbrey equation. Flow Injection Analysis (FIA). Analytical performance of both the MIP and NIP films coating the Pt/quartz resonators as well as both kinetics and thermodynamics of the melamine analyte rebinding were examined under FIA conditions using the flowthrough EQCM 5610 holder. Toward that, a 1 mM HCl carrier solution was pumped through the holder with the model KDS100 syringe pump of KD Scientific. The FIA experiments were carried out at the 35 µL/min flow rate. A model 7725i rotary six-port valve of Rheodyne was used to inject samples of the test solutions of either the 100 µL or 1 mL volume. Samples of the analyte were dissolved in the solution of the same composition as that of the carrier solution, i.e., 1 mM HCl. RESULTS AND DISCUSSION The preformed in solution complex of melamine with the functional monomer 4 was electropolymerized (MIP A) or (19) Mirkin, M. V.; Horrocks, B. R. Anal. Chim. Acta 2000, 406, 119–146.

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copolymerized with the cross-linking monomer 5 in the absence (MIP B) or presence (MIP C) of the ionic liquid 6 to result in the melamine-templated MIP films on the Pt/quartz electrodes. For electropolymeric imprinting, an acidic nonaqueous solvent solution was used. In this solution, the amine-imine equilibrium of the protonated primary amine group of melamine is totally shifted to its amine form and, therefore, it is conceivable that all these amine groups are protonated.20 Moreover, melamine is not electrooxidized in the course of the electropolymerization (vide infra). In the determination step, the melamine analyte was rebound by the template-extracted MIP film in an acidic aqueous solution. In this solution, only one melamine aliphatic amine group is protonated 1a.20,21 Both kinetic and thermodynamic parameters of the binding as well as the analytical performance of the chemosensor were evaluated under FIA conditions with PM detection at QCM. Electropolymeric Preparation of the Melamine-Templated MIP and Nontemplated NIP Films. The CV curves of the first two cycles for melamine in 0.1 M (TBA)ClO4, in the acetonitrile solution of pH ) 3.0 (TFA) at the Pt disk electrode (curves 1 and 2 in Figure S1 in the Supporting Information) do not show any anodic peak. This is because the protonated in this solution all three aliphatic amine groups of melamine are virtually nonelectrooxidizable.22 Therefore, advantageously, there was no need, as before, to use a barrier film preventing the template from electrooxidation.15 The multiscan CV curves in Figure S2a in the Supporting Information correspond to electropolymerization of 4 in the presence of the melamine template, in 0.1 M (TBA)ClO4, 0.9 mM TFA (pH ) 3.0), in acetonitrile. This electropolymerization leads to formation of the MIP A film. The anodic peak at ∼1.03 V corresponding to electropolymerization of 4 decreases in subsequent cycles. Most likely, this electropolymerization is hampered due to progressively hindered diffusion of the monomer to the electrode surface through the film of increasing thickness. A simultaneously recorded decrease of the resonant frequency change manifests the growth of the MIP A film (Figure S2b in the Supporting Information). At the same time, the dynamic resistance change (Figure S2c in the Supporting Information) is insignificant, meaning that the MIP A film rigidity remains virtually unaltered during the electropolymerization. In search for the MIP film of the best performance, the complex of melamine and 4 was electrooxidatively copolymerized with 5. The aim of this copolymerization was to produce MIP of a relatively open but fixed 3D structure ensuring enhanced accessibility of its binding sites for the melamine analyte in the determination step of this procedure. This 3D configuration of MIP B, as opposed to the 2D structure of MIP A, was afforded by the use of a properly designed cross-linker 5. The anodic peak at ∼1.03 and that at 1.10 V (Figure S3a in the Supporting Information) corresponds to electrooxidation of 4 and 5, respectively. A simultaneous resonant frequency decrease is due to electropolymeric deposition of the MIP B film (Figure S3b in the (20) Jahagirdar, D. V.; Kharwadkar, R. M. Indian J. Chem., Sect. A: Inorg., Bioinorg., Phys., Theor. Anal. Chem. 1981, 26, 635–637. (21) Tashiro, T. J. Heterocycl. Chem. 2002, 39, 615–622. (22) Steckhan, E. In Organic Electrochemistry, Fourth ed., Revised and Expanded; Lund, H., Hammerich, O., Eds.; Marcel Dekker, Inc.: New York, 2001, pp 545-588.

Figure 1. Simultaneously recorded curves of (a) cyclic voltammetry as well as potential dependence of (b) resonant frequency change and (c) dynamic resistance change for electropolymerization (MIP C) of 0.3 mM 4 and 0.3 mM 5, in the presence of 0.1 mM melamine, in the ionic liquid 6/acetonitrile (1:1, v:v) solution, which was 0.9 mM in trifluoroacetic acid (pH ) 3.0), on the Pt/quartz electrode. The potential scan rate was 50 mV/s.

Supporting Information). However, visco-elasticity remains nearly unchanged in the course of the MIP B film growth since there is not any appreciable shift in the simultaneously recorded dynamic resistance change (Figure S3c in the Supporting Information). For preparation of the MIP film of a superior performance, the melamine template was imprinted, by electrochemical copolymerization of 4 and 5, in the presence of the ionic liquid 6. Here, the ionic liquid played a role of both the porogenic solvent and supporting electrolyte. A broad current-potential shoulder at ∼1.10 V in the CV curve (Figure 1a) corresponds to electrochemical copolymerization of the monomers. A simultaneously recorded distinct (3.05 kHz) drop in the resonant frequency accounts for a rapid growth of the MIP C film (Figure 1b). This drop is accompanied by a 350 Ω increase in the dynamic resistance change (Figure 1c). This increase can be estimated as a ∼25 Hz resonant frequency change due to the rigidity loss of the film (∆fvis), if approximate eq 1 is adopted.

∆fvis ) -

k2Rf0 πA(2µQFQ)1/2

(1)

In this equation, derived for a resonator wetted by a viscous liquid,23 k2 ) 7.74 × 10-3 A2 s2 cm2 is the electromechanical coupling factor for the quartz resonator, R is the dynamic resistance of the quartz resonator, f0 is the fundamental (23) Thompson, M.; Stone, D. C. In Chemical analysis: a series of monographs on analytical chemistry and is application; Winefordner, J. D., Ed.; Wiley: New York, 1997; Vol. 144.

frequency of the resonator (10 MHz), A is the acoustically active area of the resonator (0.2827 cm2), µQ is the shear modulus of quartz (2.947 × 1011 g s-2 cm-1), and FQ is the quartz density (2.648 g cm-3). A control NIP C film, corresponding to the MIP C film but synthesized in the absence of melamine, was also prepared (Figure S4 in the Supporting Information). In the recorded CV curves (Figure S4a in the Supporting Information), the anodic peak at ∼1.15 V, which is due to electropolymerization of the monomers, increases in subsequent cycles. At the same time, the resonant frequency change decreases indicating growth of the NIP C film (Figure S4b in the Supporting Information). A simultaneous 83 Ω increase in the dynamic resistance change (Figure S4c in the Supporting Information) evidences a very small loss of rigidity (∆fvis ≈ 6 Hz) of this film during electrodeposition. Geometry Optimization of the MIP-Melamine Complex. In order to visualize the structure of the MIP-melamine complex, DFT calculations at the B3LYP/3-21G(*) level18 were performed using one molecule of triprotonated melamine and three molecules of 4. To keep the calculations simple, neither cross-linking monomer nor ionic liquid, used in the fabrication of MIP C, was considered. A stable structure on the Born-Oppenheimer potential energy surface was obtained (Figure 2). That is, all of the protonated amine groups of melamine could be accommodated using three benzo-[18-crown-6] moieties without major steric hindrance. The association energy was calculated as 460 kcal/ mol, suggesting formation of a stable complex. Similar calculations, performed for the complex of diprotonated and monoprotonated melamine with the two and one functional monomer 4 ligand, respectively, also resulted in stable structures but with lower association energy of 235 and 80.5 kcal/mol, respectively. The proportionally higher value of association energy for the complex of higher coordination number suggests some cooperativity, presumably the π-π type and (crown ether)-(quarternarized aliphatic amine) interactions. However, one should keep in mind that computations at the B3LYP/3-21G(*) level result in somewhat higher energy values than those obtained with the use of larger basis sets due to the basis set superposition error. Electrochemical, Microscopic, and Spectroscopic Characterization of the MIP Films Coating the Resonator Transducers. After electropolymeric deposition, the MIP C film was rinsed with copious acetonitrile to remove physisorbed species. The volume of the MIP C film was estimated as ∼1 nL, by recording the frequency decrease during its electropolymerization and assuming that density of the film is 1.1 g/cm3, i.e., the same as that of the poly(bithiophene) film.24 After the melamine removal, the resonant frequency change was measured. The determined from this change thickness of the film served to estimate effective concentration of the molecular cavities in the MIP C film, i.e., the concentration of those accessible for rebinding of the melamine analyte. This concentration appeared to be, advantageously, high and equal to ∼0.90 M. This value was almost twice as high as that for the MIP B film (0.46 M) and almost three times higher than that for the MIP A film (0.31 M). The UV-vis spectroscopy and XPS measurements as well as SECM imaging, performed before and after extrac(24) Skompska, M.; Jackson, A.; Hillman, A. R. Phys. Chem. Chem. Phys. 2000, 2, 4748–4757.


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Figure 2. B3LYP/3-21G(*) optimized structure of the complex of the triprotonated melamine template with three prepolymerized bis(2,2′bithienyl)-benzo-[18-crown-6]methane monomers.

Figure 3. UV-vis spectra for the 0.01 M NaOH solution of (1) 1 µM melamine, (2A, 2B, and 2C) the first 2.5 mL extract of the melamine-templated MIP A, MIP B, and MIP C film, respectively, as well as (3) the twentieth 2.5 mL extract of the melamine-templated MIP C film. Conditions of preparation of the MIP A, MIP B, and MIP C film as in Figures S2 and S3 in the Supporting Information and Figure 1, respectively.

tion (see below in this section), were used to ascertain completeness of removal of the melamine template from the MIP C film. Once extracting the melamine-templated MIP C film with 2.5 mL portions of 0.01 M NaOH, we detected the melamine in the extracts by UV-vis spectroscopy. The resulting spectrum (curve 2C in Figure 3) shows a melamine band at ∼217 nm (curve 1 in Figure 3). A complete melamine removal from this film was 10066


confirmed by the absence of this band (curve 3 in Figure 3) after the twentieth consecutive extraction. Similar UV-vis spectroscopy measurements were performed both for the MIP A (curve 2A in Figure 3) and MIP B (curve 2B in Figure 3) film. It appeared that out of the first extracts of all three films the highest melamine UV-vis absorbance revealed that of the MIP C film. Apparently, the melamine load in MIP C was the highest or melamine could most easily leave just this film like if it was most porous, or both. The melamine-templated MIP films, deposited on the Au/ glass electrode, were imaged with AFM after extraction of the template (Figure S5 in the Supporting Information), and their surface roughness was determined. This roughness was expressed as relative surface area (RSA), i.e., the ratio of the area in threedimensional space, A3D, to that projected into two-dimensional space, A2D. As expected, RSA increases in the order MIP A (1.016), MIP B (1.021), and MIP C (1.039). The highest RSA value for MIP C confirms formation of the most porous film. The FE SEM image of the deposited on the Pt/quartz electrode melamine-templated MIP C film (Figure S6 in the Supporting Information), taken after extraction of melamine, shows the presence of distinct pores of different shape and size. Therefore, the MIP C film is enormously permeable enabling diffusion of the analyte and facilitating its access to the imprinted cavities. Importantly, neither surface of the MIP A nor MIP B film, imaged by FE SEM (not shown), is so porous. Electrochemical characterization with the K4Fe(CN)6 redox probe provided information on some morphological features of the MIP A, MIP B, and MIP C films. A reversible electron transfer for the Fe(CN)64-/Fe(CN)63- system at the bare Pt disk electrode is not hindered, as indicated by a well-defined DPV peak (curve 1 in Figure 4a). For the melamine template loaded MIP film, the DPV peak is practically absent (curves 2A, 2B, and 2C for the MIP A, MIP B, and MIP C film, respectively,

Figure 4. Differential pulse voltammograms for 1 mM K4Fe(CN)6 in 0.1 M KNO3 at (1) the bare Pt disk electrode as well as the Pt disk electrode coated with the melamine-templated MIP A, MIP B, and MIP C film (2A, 2B, and 2C) before and (3A, 3B, and 3C) after melamine extraction with 0.01 M NaOH, respectively. The step potential was 5 mV; the pulse amplitude was 25 mV; the pulse width was 50 ms.

in Figure 4a) meaning that diffusion of the probe through the film to the electrode surface is effectively prevented. However, the extraction of the melamine template from these films results in quite well developed peaks (curves 3A, 3B, and 3C for the MIP A, MIP B, and MIP C film, respectively, in Figure 4a). Apparently, the K4Fe(CN)6 electrooxidation is facilitated, in this case, as the redox probe diffusion through the vacated imprinted cavities is enabled. For the melamine-extracted MIP films of the same mean thickness of 50 nm, the highest DPV peak for K4Fe(CN)6 of the same 1 mM concentration in solution was for the MIP C film. Most likely, the Fe(CN)64- probe more easily diffuses through the MIP C film than through the other two films. Next, EIS was used for further electrochemical characterization of the films. The complex-plane impedance plots for the melamine template loaded MIP films coating the Pt disk electrodes in 1 mM K4Fe(CN)6 in 0.1 M KNO3 before and after melamine extraction are shown in Figure 5a and 5b, respectively. An unhindered electron transfer, whose rate is solely controlled by the rate of diffusion of the Fe(CN)64- probe to the bare Pt disk electrode, is depicted as a straight line with the π/4 slope (curve 1′ in Figure 5a). The plots for the electrodes coated with the melamine template loaded MIP A, MIP B, and MIP C film are presented in Figure 5a, by large arcs 2A, 2B, and 2C, respectively, related to the charge transfer resistance.25 Solid arcs are the curves fitted to the experimental data points based on the Randles type equivalent circuit. The charge transfer resistance is lower for MIP C (1.8 MΩ) than that for MIP A (2.25 MΩ) and that for MIP B (2.1 MΩ) but much higher than that for the bare Pt disk electrode (0.80 kΩ). Apparently, the presence of the melamine-containing MIP film hinders the Fe(CN)64-/Fe(CN)63- charge transfer. Importantly, if 6 is used as a porogenic solvent to make the MIP C film, the charge transfer resistance is even lower. The (25) Lasia, A. In Modern Aspects of Electrochemistry; White, R. E., Conway, B. E., Bockris, J., Eds.; Kluwer Academic/Plenum, New York, 1999; Vol. 32, pp 143-248.

Figure 5. Complex-plane impedance plots of electrochemical impedance spectroscopy for 1 mM K4Fe(CN)6 in 0.1 M KNO3 at (1′) the bare Pt disk electrode as well as at the Pt disk electrode coated with the melamine-templated (2A, 2B, and 2C) before and (3A, 3B, and 3C) after melamine extraction with 0.01 M NaOH of the MIP A, MIP B, and MIP C film, respectively. The EIS frequency ranged from 0.1 Hz to 10 kHz. Conditions of preparation of the MIP A, MIP B, and MIP C films as in Figures S2 and S3 in the Supporting Information and Figure 1, respectively.

plots, recorded just after the extraction of the melamine template from the MIP A, MIP B, and MIP C film, show arcs and straight line segments at the high and low frequency range, respectively, (curves 3A, 3B, and 3C in Figure 5b). However, the diameters of these arcs are smaller than those in curves 2A, 2B, and 2C, implying lower charge transfer resistances of the template-free MIP film coated electrodes being 1.85, 1.6, and 1.1 MΩ for the MIP A, MIP B, and MIP C film, respectively. The slopes of the straight line segments of curves 3A, 3B, and 3C are lower than that in curve 1′, as if open pores were left in the MIP films when the melamine template was removed.26 Apparently, the more porous the film is, the easier the diffusion of the analyte and its access to the MIP molecular cavities in the film. Microscopic information on some morphological features of the MIP A, MIP B, and MIP C films coating the resonator transducers was also provided by SECM imaging. During scanning the microelectrode across the quartz surface of the resonator to the adjacent surface of the melamine template loaded MIP C film coating the Pt film electrode, the anodic current was small and virtually constant27 (Figure 6a). This was because of the negative feedback resulting from hindered diffusion of the (26) de Levie, R. In Advances in Electrochemistry and Electrochemical Engineering; Delahay, P., Tobias, C. W., Eds.; John Wiley: New York, 1967; Vol. 6, pp 329-397. (27) Wittstock, G.; Burchardt, M.; Pust, S. E.; Shen, Y.; Zhao, Ch. Angew. Chem., Int. Ed. 2007, 46, 1584–1617.


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Figure 6. SECM image of the melamine-templated MIP C film at the edge of the Pt film electrode of the quartz resonator (a) before and (b) after melamine exhaustive extraction with 0.01 M NaOH.

Fe(CN)64- probe near both the bare quartz and the melamine template loaded MIP C film insulating surface. However, the latter current was markedly higher when scanning across the surface of the melamine-extracted MIP C film (Figure 6b). This effect resulted from the positive feedback when the tip was scanned above the extracted MIP C film. That was because the Fe(CN)63- product of the probe electrooxidation at the microelectrode could diffuse through the film to the Pt/quartz substrate electrode for electroreduction. Finally, when scanning across the surface of the melamine-rebound MIP C film, the current was slightly lower (Figure S7 in the Supporting Information), as compared to that at the melamine-extracted MIP C film (Figure 6b) confirming that the imprinted molecular cavities became partially occupied by the analyte molecules. Extraction of the melamine template from the MIP B film caused only a slight increase of the SECM current (not shown). However, this current was the smallest for the melamine-extracted MIP A film (not shown). Apparently, the redox probe most easily permeates through the melamine-extracted MIP C film because 10068


of its largest porosity. Evidently, results of SECM imaging are consistent with those of the DPV and EIS experiments. In the XPS measurements, binding energy of N 1s was used as a marker, confirming the melamine presence or absence in the MIP C film. Figure S8a and 8b in the Supporting Information shows the binding energy profile recorded for the MIP C film containing the melamine template imprinted in nonaqueous solution and that rebound in acidic aqueous solution, respectively. The composed band at around 401 eV corresponds to electrons of nitrogen atoms in different environments of melamine present in the MIP C film. The band in the deconvoluted spectrum at 401.2 and that at 403.0 eV (Figure S8a in the Supporting Information) can be attributed to the protonated (-NH3+) primary aliphatic amine group and aromatic (sNd) nitrogen atom of the 1,3,5-triazine ring, respectively.28 Nearly 1:1 intensity ratio of the bands stands for the presence of three quaternarized primary aliphatic amine groups in the template. These bands are slightly shifted to lower binding energy values as compared to the literature values for the free base, presumably due to different atomic environments.28 In the XPS spectrum of the MIP C film (not shown), recorded after melamine extraction, there is neither a band corresponding to the nitrogen atoms of the melamine template nor even the acetonitrile solvent left in the film. Apparently, the melamine template was completely removed from the MIP C film. As the analyte, melamine was herein determined using the MIP C film in an aqueous solution because real melamine sample solutions are aqueous solutions. Acidity of this solution was kept as low as pH ) 3.0 to increase solubility of melamine. In an acidic aqueous solution, the amine-imine equilibrium of melamine is shifted to the imine form 1a′ 20,21 and, therefore, only one proton can be accepted (pKa ) 5.0).29 Hence, only one aliphatic amine group of melamine can be protonated. This inference was confirmed by the XPS result (Figure S8b in the Supporting Information). That is, deconvolution of the XPS spectrum for the melamine-rebound MIP C film results in a band at 400.0, 401.2, and 403.0 eV, which is ascribed to the N 1s electron of the sNH2, sNH3+, and sNd moiety, respectively. The respective approximate band intensity ratio of 2:1:3 confirms protonation of only one primary aliphatic amine group of melamine 1a under these solution conditions. Analytical Performance of the MIP/QCM Chemosensor for Melamine. After extraction of the template from the melamineimprinted polymer film, subsequent rebinding of the melamine analyte for its determination was examined under FIA with PM detection conditions. The matched stereo geometry of the melamine molecule and the imprinted molecular cavity of MIP as well as affinity of the crown ether binding sites to the sNH3+ groups of the melamine analyte were crucial in this rebinding. Since the melamine analyte can be rebound in its protonated form 1a, 1 mM HCl (pH ) 3.0) was used as a carrier solution. The analyte solution, injected to the carrier solution, flowed over the melamine-extracted MIP C film causing a reversible (28) Bradley, R. H.; Ling, X.; Sutherland, I.; Beamson, G. Carbon 1994, 32, 185–186. (29) (a) Blank, W. J.; He, Z. A.; Hessell, E. T.; Abramshe, R. A. Polym. Mater. Sci. Eng. 1997, 77, 391–392. (b) Albert, A.; Goldacre, R.; Phillips, J. J. Chem. Soc. 1948, 455, 2240–2249. (c) Konakahara, T.; Kishimoto, K.; Sato, H.; Takagi, Y.; Sato, K. Bull. Chem. Soc. Jpn. 1988, 61, 4289–4294.

Figure 7. Resonant frequency changes with time due to repetitive FIA melamine injections, for a MIP C film, and for a NIP C film in inset, deposited by electropolymerization on the bare Pt/quartz electrode. Sample volume of the injected 1 mM HCl solution of melamine was 100 µL. Melamine concentration is indicated with a number at each curve. The flow rate of the 1 mM HCl carrier solution was 35 µL/min. Conditions of preparation of the MIP C and NIP C film are as in Figure 1 and Figure S4 in the Supporting Information, respectively.

decrease and increase of the resonant frequency change of the film (Figure 7). It means that the analyte ingress to and egress from the film was reversible, advantageously for chemosensing. Each injection of the melamine solution caused, first, a resonant frequency decrease due to the melamine interaction with the MIP C binding sites. The sensor response signal increased linearly with the increase of the melamine concentration. Then, the melamine was washed away relatively quickly from the film with the excess of the carrier solution increasing the resonant frequency nearly to its initial value. The response and recovery time was ∼3 and ∼7 min, respectively. Generally, the strength of the melamine-MIP interactions and the mass transfer resistance of MIP influenced the residence time and, hence, recovery of the chemosensor. Interactions of melamine with the NIP C binding sites were much weaker causing a much smaller drop in the resonant frequency change (inset to Figure 7). Sensitivity and Detectability. On the basis of the resonant frequency changes recorded under FIA conditions, the melamine calibration plots were constructed (Figure 8). For each MIP film, the frequency change linearly decreases with the increase of concentration of melamine in the injected sample solution. Noticeably, a negative slope of the resonant frequency decrease vs concentration lines, i.e., the chemosensor sensitivity (Table 2), is higher for the film of MIP C (line 1 in Figure 8) than that of MIP B (line 2 in Figure 8) and that of MIP A (line 5 in Figure 8). Apparently, the sensitivity of the chemosensor featuring the MIP film, built solely of the functional monomer (MIP A), was markedly increased if the cross-linking monomer was present in the polymer structure (MIP B) and even more increased if the ionic liquid was added to the solution for electropolymerization (MIP C). Importantly, the sensitivity of the chemosensor featuring a control NIP C film (line 4 in Figure 8) was 4 times lower than

Figure 8. Melamine calibration plots for the film of (1, 3) MIP C, (2) MIP B, (4) NIP C, and (5) MIP A. The flow rate of the 1 mM HCl carrier solution was 35 µL/min, and the injected sample volume of the melamine solution was (1, 2, 4 and 5) 100 µL as well as (3) 1 mL. Conditions of preparation of the NIP C, MIP A, MIP B, and MIP C film as in Figures S4, S2, and S3 in the Supporting Information and Figure 1, respectively. Inset represents initial resonant frequency changes with time (used to construct calibration plot 3) due to FIA melamine injections at the nanomolar concentration levels, for the MIP C film, deposited by electropolymerization on the Pt/quartz electrode. The flow rate of the 1 mM HCl carrier solution was 35 µL/min. Sample volume of the injected solution of melamine in the 1 mM HCl carrier solution was 1 mL. The melamine template was extracted from the MIP C film with 0.01 M NaOH before injecting the melamine analyte.

Table 2. Values of Sensitivity with Respect to Melamine and Those of Stability Constant, Ks, for the Complexes of Melamine with the NIP C and Melamine-Templated MIP Filmsa sensor architecture

sensitivity (± st.d.) (Hz/mM)

Ks (± st.d.) (M-1)

quartz/Pt/(MIP C) quartz/Pt/(MIP B) quartz/Pt/(MIP A) quartz/Pt/(NIP C)

0.42 ± 0.010 0.28 ± 0.010 0.05 ± 0.001 0.10 ± 0.002

1813 ± 32 1061 ± 28 810 ± 6 333 ± 12

a

The films were 130 nm thick.

that of the MIP C film (Table 2). Evidently, the presence of imprinted cavities in MIPs is decisive for sensitive analyte determination. The dynamic linear concentration range was appreciably broad extending from 5 nM to at least 1 mM melamine. Detectability of the chemosensor with the MIP C film, determined under the most favorable FIA conditions, i.e., at the flow rate as low as 35 µL/ min and the volume of the melamine sample as high as 1 mL (line 3 in Figure 8 and inset to Figure 8), was ∼5 nM melamine at the signal-to-noise ratio of 3. Notably, this detection limit is lower than that of the ABRAXIS device.30 Selectivity of the chemosensor was estimated by examining under FIA conditions cross-reactivity of the melamine-templated MIP C film with respect to structurally similar interfering compounds, such as cyromazine 2, cyanuric acid 3, and ammeline (30) ABRAXIS, Melamine ELISA (Microtiter Plate), Product No. 50005B.


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marized in Table 2. Apparently, the melamine-extracted MIP C film rebinds 5.44, 2.25, and 1.7 times stronger the melamine analyte than the nonimprinted NIP C, the noncross-linked MIP A, and the cross-linked MIP B film, respectively. Therefore, the most selective melamine chemosensor, using the MIP C film, was employed to determine melamine in real samples of a diary product, such as melamine spiked kefir from a local supermarket in Warsaw (Figure S11 in the Supporting Information). Before the FIA injection, the kefir sample was diluted 25 times with distilled water and spun. CONCLUSIONS

Figure 9. FIA calibration plots for (1) melamine 1, (2) ammeline 7, (3) cyanuric acid 3, and (4) cyromazine 2 for the melamine-templated MIP C film. Volume of the injected sample solution was 100 µL. The flow rate of the 1 mM HCl carrier solution was 35 µL/min. The melamine template was extracted from the MIP C film with 0.01 M NaOH before the determinations.

7. That is, 1 mM HCl sample solutions of these compounds were injected onto the melamine-extracted MIP C chemosensor. Respective calibration plots are presented in Figure 9. The chemosensor is sensitive to melamine almost 10 times more than to cyromazine, 4 times more than to cyanuric acid, and 1.5 times more than to ammeline (Table S1 in the Supporting Information), demonstrating appreciable selectivity with respect to these common interfering agents with the melamine determination. Values of the stability constant, Ks, for the MIP and guest (G) complexes (MIP-G) were determined from FIA kinetic studies15,31 for quantitative characterization of the analyte binding by MIPs. The determination from the slope and intercept ratio of the linear dependence of the observed association rate constant, kobs, on the guest concentration (Figure S9 in the Supporting Information) stability constant of the (MIP C)-melamine complex was over 3 times higher than that of the (MIP C)-ammeline complex, over 5 times higher than that of the (MIP C)-(cyanuric acid) complex, and over 11 times higher than that of the (MIP C)-cyromazine complex (Table S1 in the Supporting Information). Evidently, structurally complementary molecular cavities of the melamine-templated MIP C film rebind molecules of the melamine analyte stronger than those of the cyromazine, cyanuric acid, and ammeline interfering compounds. The plot of kobs linearly varies with the melamine concentration for the film of NIP C, MIP A, MIP B, and MIP C (curve 1, 2, 3 and 4, respectively, in Figure S10 in the Supporting Information). The determination from these plots Ks values for the NIP-melamine and MIP-melamine complexes are sum(31) Skladal, P. J. Braz. Chem. Soc. 2003, 14, 491–502.

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Combination of the melamine-templated MIP recognition film and the piezoelectric transducer of the bulk-acoustic-wave resonator results in an exceptionally sensitive and selective melamine piezomicrogravimetric chemosensor of appreciable detectability. A relatively simple electropolymeric procedure of MIP preparation developed herein allows for the control of the film thickness and morphology. The chemosensor performance was tremendously improved if both the tailored 3D cross-linking monomer and the ionic liquid were used for pore making in the MIP film. Under carefully selected FIA conditions, i.e., at a relatively low solution flow rate and large volume of the injected sample solution, the concentration limit of detection was as low as ∼5 nM melamine. The chemosensor was significantly more selective to melamine than to structurally similar interfering compounds, such as cyromazine, cyanuric acid, and ammeline. The determined stability constants of complexes of MIPs with melamine, cyanuric acid, or ammeline, suggest that the imprinted cavities fit the size and shape of the melamine molecule used for imprinting. ACKNOWLEDGMENT We thank Dr. Eng. O. Chernyayeva and M.Sc. A. Bilinski (IPC PAS, Warsaw, Poland) for their help with the XPS measurements, M.Sc. W. Nogala (IPC PAS, Warsaw, Poland) for his help with SECM imaging, M.Sc. I. Obraztsov (IPC PAS, Warsaw, Poland) for his help with AFM imaging, and Dr. A. Presz (CBW Unipress PAS, Warsaw, Poland) for his help with the FE SEM imaging. The authors acknowledge European Regional Development Fund (ERDF, POIG.01.01.02-00-008/08 2007-2013 to W.K.), and National Science Foundation (CHE 0804015 to F.D.S.) for financial support. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review September 9, 2009. Accepted October 30, 2009. AC9020352

Melamine Acoustic Chemosensor Based on Molecularly Imprinted

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