Molecular Imprinting under Molecular Crowding Conditions: An Aid to

Jan 20, 2007 - Jun Matsui,*,†,‡ Shou Goji,‡ Takashi Murashima,†,‡ Daisuke Miyoshi,† Satoshi Komai,‡. Aiko Shigeyasu,‡ Takuho Kushida,â...
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Anal. Chem. 2007, 79, 1749-1757

Correspondence

Molecular Imprinting under Molecular Crowding Conditions: An Aid to the Synthesis of a High-Capacity Polymeric Sorbent for Triazine Herbicides Jun Matsui,*,†,‡ Shou Goji,‡ Takashi Murashima,†,‡ Daisuke Miyoshi,† Satoshi Komai,‡ Aiko Shigeyasu,‡ Takuho Kushida,‡ Toshifumi Miyazawa,‡ Takashi Yamada,‡ Katsuyuki Tamaki,‡ and Naoki Sugimoto*,†,‡

Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan, and Department of Chemistry, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan

Molecular crowding, an important feature of the molecular environments in biological cells, was applied to the synthesis of antibody-mimic polymers selective for a group of biologically active compounds, the triazine herbicides. Synthesis of these polymers was conducted using molecular imprinting under molecular crowding conditions, whereby atrazine (a template molecule) was complexed with methacrylic acid (a functional monomer) in the presence of a macromolecular crowding agent (either poly(methyl methacrylate) (PMMA) or polystyrene (PS)) followed by cross-linking with ethylene glycol dimethacrylate. After removal of atrazine from the polymer matrix, the retention properties and selectivity of the resultant polymers were assessed by chromatographic tests. The addition of a crowding-inducing agent resulted in polymers with superior retention properties and excellent selectivity for triazine herbicides, as compared to polymers prepared without addition of a crowding-inducing agent. An imprinted polymer prepared in the presence of PS as a crowding agent exhibited a retention factor for atrazine an order of magnitude larger than that of an imprinted polymer prepared in the absence of a crowding agent. NMR results suggest that the crowding agent is capable of promoting hydrogen bond formation between atrazine and methacrylic acid, which could account for the effect of crowding on molecular imprinting. Living systems continue to inspire chemists in the creation of new biofunctional materials in terms of functions, structures, and mechanisms. For example, the high specificity of enzymatic and immune reactions introduced chemists to the concept of hostguest chemistry and molecular recognition, leading to the design * Address correspondence to either author. E-mail: [email protected] (J.M.); [email protected] (N.S.). † Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University. ‡ Department of Chemistry, Faculty of Science and Engineering, Konan University. 10.1021/ac060441m CCC: $37.00 Published on Web 01/20/2007

© 2007 American Chemical Society

and synthesis of new artificial receptors and enzymes.1 These functional molecules are particularly useful, for example, in the field of bioanalytical chemistry, where they are coupled with various reporter moieties to build molecular sensors2 and immobilized on solid supports for affinity separations.3 In addition, structural aspects of biological molecules have yielded important insights used in the design of new functional molecules. Since Lehn proposed the concept of a “supramolecule”, weak bonds such as hydrogen bonds and stacking interactions, which play important roles in composing the highly ordered structures of biomolecules, have been key to the successful design of artificial receptors and other molecular devices.4 Biology was also important in inspiring Pauling in his work on artificial antibodies, in which artificial receptors were proposed to be synthesized as macromolecules (1) (a) Pedersen, C. J. J. Am. Chem. Soc. 1967, 89, 7017-7036. (b) Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40, 486-516. (c) Andrews, P. C.; Kennedy, A. R.; Mulvey, R. E.; Raston, C. L.; Roberts, B. A.; Rowlings, R. B. Angew. Chem., Int. Ed. 2000, 39, 1960-1962. (d) Antonisse, M. M. G.; Reinhoudt, D. N. Chem. Commun. 1998, 443-448. (e) Izatt, R. M.; Pawlak, K.; Bradshaw, J. S.; Bruening, R. L. Chem. Rev. 1995, 95, 25292586. (f) Izatt, R. M.; Pawlak, K.; Bradshaw, J. S. Chem. Rev. 1991, 91, 1721-2085. (g) Merz, T.; Wirtz, H.; Vo ¨gtle, F. Angew. Chem., Int. Ed. 1986, 25, 567-568. (2) (a) Martinez-Manez, R.; Sancenon, F. Chem. Rev. 2003, 103, 4419-4476. (b) Yun, S.; Kim, Y.-O.; Kim, D.; Kim, H. G.; Ihm, H.; Kim, J. K.; Lee, C.W.; Lee, W. J.; Yoon, J.; Oh, K. S.; Yoon, J.; Park, S.-M.; Kim, K. S. Org. Lett. 2003, 5, 471-474. (c) Tong, H.; Wang, L.; Jing, X.; Wang, F. Macromolecules 2002, 35, 7169-7171. (d) Chen, C.-T.; Huang, W.-P. J. Am. Chem. Soc. 2002, 124, 6246-6247. (e) Gunnlaugsson, T.; Leonard, J. P.; Murray, N. S. Org. Lett. 2004, 6, 1557-1560. (f) Guo, X.; Qian, X.; Jia, L. J. Am. Chem. Soc. 2004, 126, 2272-2273. (g) Hanaoka, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 1247012476. (h) Yun, S.; Kim, Y.-O.; Kim, D.; Kim, H. G.; Ihm, H.; Kim, J. K.; Lee, C.-W.; Lee, W. J.; Yoon, J.; Oh, K. S.; Yoon, J.; Park, S.-M.; Kim, K. S. Org. Lett. 2003, 5, 471-474. (i) Ward, C. J.; Patel, P.; James, T. D. Org. Lett. 2002, 4, 477-479. (j) Fang, J.-M.; Selvi, S.; Liao, J.-H.; Slanina, Z.; Chen, C.-T.; Chou, P.-T. J. Am. Chem. Soc. 2004, 126, 3559-3566. (j) Gokel, G. W.; Leevy, W. M.; Weber, M. E. Chem. Rev. 2004, 104, 2723-2750. (3) (a) Lambert, J. B.; Liu, C.; Boyne, M. T.; Zhang, A. P.; Yin, Y. Chem. Mater. 2003, 15, 131-145. (b) Stalcup, A. M.; Gahm, K. H. Anal. Chem. 1996, 68, 1360-1368. (c) Gratz, S. R.; Stalcup, A. M. Anal. Chem. 1998, 70, 51665171. (d) Gong, Y.; Lee, H. K. Anal. Chem. 2003, 75, 1348-1354. (e) Mertz, E.; Zimmerman, S. C. J. Am. Chem. Soc. 2003, 125, 3424-3425.

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in the presence of a template molecule.5 Although the proposed strategy was not precisely the same as that used in biological antibody production, Mosbach proved that the concept is effective for synthesizing organic polymers that show excellent specificity comparable to a real antibody.6 This technique called molecular imprinting is now recognized as a useful approach for producing various categories of molecular recognition materials. These compounds, which range from inorganic to organic polymers, have been effectively utilized in analytical applications.7 The most extensively studied molecular imprinted material is a polymethacrylate-based plastic antibody (also called an artificial receptor).6 In the synthesis of a plastic antibody, a functional monomer (often methacrylic acid) complexed with a print molecule (i.e., a target molecule) is cross-linked (typically with ethylene glycol dimethacrylate) and immobilized around the print molecule (which has a complementary geometry). After extraction of the print molecule, the cross-linked polymer matrices possess a binding site selective for that print molecule. Whether molecular imprinting succeeds or not depends critically on the efficiency of complexation between the print molecule and the functional monomers. Therefore, studies have focused on increasing complexation efficiency, including selection of suitable functional monomers and optimization of the polymerization temperature and solvent. For efficient selection of appropriate functional monomers, computer-aided rational approaches and random selection methodologies have been developed.8 When the selected monomers engage in electrostatic interaction and hydrogen bonding, low temperatures and less polar solvents have been found to be preferable for stable complex formation.9 High pressure has also been examined as a means of stabilizing the complexes and resulted in high specificity.10 In spite of these (4) (a) Lehn, J.-M. Supramolecular Chemistry: Concepts and Perspectives; WileyVCH: Weinheim, 1995. (b) Mingos, D. M. P., Ed. Supramolecular Assembly Via Hydrogen Bonds I & II; Springer-Verlag: Berlin, 2004. (c) Glusker, J. P. Top. Curr. Chem. 1998, 198, 1-56. (d) Zimmerman, S. C.; Corbin, P. S. Struct. Bonding 2000, 96, 63-94. (e) Schalley, C. A.; Rebek, J., Jr. In Stimulating Concepts in Chemistry; Vo ¨gte, F., Stoddart, J. F., Shibaski, M., Eds.; Wiley-VCH: Weinheim, Germany, 2000; pp199-210. (f) Rebek, J., Jr. Acc. Chem. Res. 1999, 32, 278-286. (g) MacDonald, C.; Whitesides, G. M. Chem. Rev. 1994, 94, 2383-2420. (5) (a) Pauling, L. J. Am. Chem. Soc. 1940, 62, 2643-2657. (b) Pauling, L.; Campbell, D. H. J. Exp. Med. 1942, 76, 211-220. (6) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645. (7) (a) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O’Mahony, J.; Whitcombe, M, J. J. Mol. Recognit. 2006, 19, 106-180. (b) Andersson, L. I.; Nicholls, I. A. J. Chromatogr. B: Biomed. Sci. Appl. 2004, 804. (c) Mayes, A. G.; Whitcombe, M. J. Adv. Drug Delivery Rev. 2005, 57, 1742-1778. (d) Ye, L.; Haupt, K. Anal. Bioanal. Chem. 2004, 378, 1887-1897. (e) Komiyama, M., Takeuchi, T., Mukawa, T., Asanuma, H., Eds. Molecular ImprintingsFrom Fundamentals to Applications; Wilely-VCH: Weinheim, Germany, 2002. (f) Sellergren, B., Ed. Molecularly Imprinted Polymers: Man-Made Mimics of Antibodies and Their Application in Analytical Chemistry; Elsevier: Amsterdam, 2001. (g) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495-2504. (8) (a) Chianella, I.; Lotierzo, M.; Piletsky, S. A.; Tothill, I. E.; Chen, B.; Karim, K.; Turner, A. P. F. Anal. Chem. 2002, 74, 1288-1293. (b) Takeuchi, T.; Fukuma, D.; Matsui, J. Anal. Chem. 1999, 71, 285-290. (c) Dirion, B.; Cobb, Z.; Schillinger, E.; Andersson, L. I.; Sellergren, B. J. Am. Chem. Soc. 2003, 125, 15101-15109. (9) (a) O’Shannessy, D. J.; Ekberg, B.; Mosbach, K. Anal. Biochem. 1989, 177, 144. (b) Sellergren, B. Makromol. Chem. 1989, 190, 2703. (c) Piletsky, S. A.; Piletska, E. V.; Karim, K.; Freebairn, K. W.; Legge, C. H.; Turner, A. P. F. Macromolecules 2002, 35, 7499-7504. (10) (a) Sellergren, B.; Dauwe, C.; Schneider, T. Macromolecules 1997, 30, 2454-2459. (b) Piletsky, S. A.; Guerreiro, A.; Piletska, E. V.; Chianella, I.; Karim, K.; Turner, A. P. F. Macromolecules 2004, 37, 5018-5022.

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efforts, the molecular imprinting technique is still useful only in limited cases, although it is, in principle, capable of customizing a host molecule to any given guest molecule. This limitation is mainly due to a lack of general procedures for obtaining stable complexes in pre-polymerization mixtures; therefore, a new, generally applicable approach for stabilizing the complex is required. Our inspiration for addressing this problem was again obtained from biological systems. As mentioned above, molecular imprinting has successfully utilized the functions (e.g., recognition), structures (e.g., hydrogen bonding), and mechanisms (e.g., tailormade synthesis) of biological systems in devising new design and synthetic methodologies of biofunctional polymers. However, a unique environmental feature of biomolecules in living systems has yet to be utilized. Recently, considerable attention has focused on the fact that many solutes (e.g., proteins and nucleic acids) are present at high concentration (up to 400 mg mL-1) in biological cells, while chemical reactions in vitro are usually conducted in dilute solutions.11 Recent studies have shown that molecular crowding in biological cells affects the stability of higher-order structures of biopolymers and promotes the association of biomolecules.12,13 Thus, we envisioned that molecular crowding can shift the equilibrium of a print molecule reacting with functional monomers in the direction of complex formation side, promoting molecular imprinting more efficiently, and producing artificial receptors with greater capacity and selectivity. Herein, we demonstrate molecular imprinting in the presence of a macromolecular co-solute as a crowding-inducing agent in order to synthesize polymeric artificial receptors for triazine herbicides. EXPERIMENTAL SECTION Chemicals and Instruments. Atrazine (2-chloro-4-ethylamino6-isopropylamino-1,3,5-triazine), simazine (2-chloro-4,6-bis(ethylamino)-1,3,5-triazine), propazine (2,4-bis(isopropylamino)-6-chloro1,3,5-triazine), cyanazine (2-[[4-chloro-6-ethylamino-1,3,5-triazine2-yl]amino]-2-methylpropionitrile), ametryn (2-ethylamimo-4-isopropylamino-6-methylthio-1,3,5-triazine), prometryn (2,4-bis(isopropylamino)-6-methylthio-1,3,5-triazine), metribuzin (4-amino-6(11) (a) Zhang, W.; Horoszewski, D.; Decatur, J.; Nuckolls, C. J. Am. Chem. Soc. 2003, 125, 4870-4873. (b) Lonhienne, T. G. A.; Winzor, D. J. Biochemistry 2001, 40, 9618-9622. (c) Lonhienne, T. G. A.; Winzor, D. J. Biochemistry 2002, 41, 6897-6901. (d) Morar, A. S.; Pielak, G. J. Biochemistry 2002, 41, 547-551. (e) Martin, J. Biochemistry 2002, 41, 5050-5055. (f) Shtilerman, M. D.; Ding, T. T.; Lansbury, P. T., Jr. Biochemistry 2002, 41, 3855-3860. (g) Goobes, R.; Minsky, A. J. Am. Chem. Soc. 2001, 123, 12692-12693. (h) Morar, A. S.; Wang, X.; Pielak, G. J. Biochemistry 2001, 40, 281-285. (i) Goodrich, G. P.; Helfrich, M. R.; Overberg, J. J.; Keating, C. D. Langmuir 2004, 20, 10246-10251. (j) Evans, W. J.; Kozimor, S. A.; Nyce, G. W.; Ziller, J. W. J. Am. Chem. Soc. 2003, 125, 13831-13835. (k) Vergara, A.; Annunziata, O.; Paduano, L.; Miller, D. G.; Albright, J. G.; Sartorio, R. J. Phys. Chem. B. 2004, 108, 2764-2772. (l) Flaugh, S. L.; Lumb, K. J. Biomacromolecules 2001, 2, 538-540. (m) Schlarb-Ridley, B. G.; Mi, H.; Teale, W. D.; Meyer, V. S.; Howe, C. J.; Bendall, D. S. Biochemistry 2005, 44, 6232-6238. (n) Goobes, R.; Kahana, N.; Cohen, O.; Minsky, A. Biochemistry 2003, 42, 2431-2440. (o) Vergara, A.; Paduano, L.; Vitagliano, V.; Sartorio, R. Macromolecules 2001, 34, 9911000. (p) Cayley, S.; Record, M. T., Jr. Biochemistry 2003, 42, 1259612609. (12) Minton, A. P. J. Biol. Chem. 2001, 276, 10577-10580. (13) (a) Miyoshi, D.; Matsumura, S.; Nakano, S.; Sugimoto, N. J. Am. Chem. Soc. 2004, 126, 165-169. (b) Nakano, S.; Karimata, H.; Ohmichi, T.; Kawakami, J.; Sugimoto, N. J. Am. Chem. Soc. 2004, 126, 14330-14331. (c) Miyoshi, D.; Nakao, A.; Sugimoto, N. Biochemistry 2002, 41, 1501715024.

tert-butyl-3-methylthio-as-triazine-5(4H)-one), anilazine (4,6-dichloroN-(2-chlorophenyl)-1,3,5-triazin-2-amine), alachlor (2-chloro-2′,6′diethyl-N-(methoxymethyl)acetanilide), mepronil (2-methyl-N-[3(1-methylethoxy)(1-phenyl)benzamide], flutolanil (R,R,R-trifluoro3′-isopropoxy-o-toluanilide), asulam (methyl [(4-aminophenyl)sulfonyl]carbamate), terbucarb (MBPMC, 2,6-di-tert-butyl-4methylphenyl methylcarbamate), diazinon (O,O-diethyl-O-[6-methyl-2-(1-methylethyl)-4-pyrimidinyl]phosporothioate), chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)phosporothioate), diuron (N′-(3,4-dichlorophenyl)-N,N-dimethylurea), chlorothalonil (TPN, tetrachloroisophthalonitrile), pendimethalin (N-(1-ethylpropyl)-3,4dimethyl-2,6-dinitrobenzenamine), triflumizole (1-[(1E)-1-[[4-chloro2-(trifluoromethyl)phenyl]imino]-2-propoxyethyl]-1H-imidazole), ethylene glycol dimethacrylate (EDMA), methacrylic acid (MA), 2,2′-azobis(2,4-dimethivaleronitrile) (ADVN), methanol, acetonitrile, and acetic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan). Metolachlor (2-chloro-N-(2-ethyl-6methyl(2-phenyl)-N-(2-(2-methoxy-1-methylethyl)acetamide) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Poly(methyl methacrylate) (PMMA, average Mw: 100 000) and polystyrene (PS, average Mw: 350 000) were purchased from SigmaAldrich Japan (Tokyo, Japan). Chloroform, EDMA, and MA were purified by standard procedures prior to use in order to remove water and stabilizers. ADVN was recrystalized from ethanol before use. Images of polymers were obtained using a USB-connectable microscope (MIC-D, Olympus, Tokyo, Japan). Chromatographic experiments were performed with a high-performance liquid chromatography (HPLC) system consisting of a UV absorbance detector (Waters 2487) and a separation module (Waters 2690). A spectrophotometer (Multispec-1500, Shimadzu, Kyoto, Japan) was used for detecting PS. Polymerization was conducted using a temperature-controllable bath (Advantec, LCH- 3000) (Tokyo, Japan). Column packing was conducted with an LC pump (L-7100, Hitachi, Tokyo, Japan). 1H NMR spectra were recorded with Varian INOVA (500 MHz) using CDCl3 as the solvent. Atrazine-Imprinted Polymer Preparation. In a typical preparation, atrazine (72 mg, 0.33 mmol), MA (115 mg, 1.33 mmol), EDMA (1.85 g), and ADVN (24 mg) were dissolved in chloroform medium (5.0 mL), in which PMMA (1.54 g) was dissolved. The mixture was sparged with nitrogen gas, sealed in a test tube, and placed in a water bath (60 °C). After 12 h, the resulting polymer IP2m (Table 1) was immersed in chloroform to remove the crowding agent and potential unreacted monomers. After drying, the polymer was ground with a mortar and pestle and wet-sieved to collect particles with diameters in the range of 32-63 µm. These polymer particles were packed in a stainless steel tube (50 × 4.6 mm, i.d.) and washed with methanol-acetic acid (95:5, v/v) in order to remove the template. Other imprinted polymers were similarly prepared using varying amounts of PS or PMMA (Table 1). Non-imprint polymers (NP) were also prepared without addition of the atrazine template. Chromatographic Assessment of the Imprinted Polymers. The polymer particles were suspended in chloroform-acetonitrile (1:1, v/v) and packed in stainless steel columns (50 mm × 4.6 mm i.d.). After thorough washing with methanol-acetic acid (9: 1, v/v), each column was connected to an HPLC instrument and conditioned with acetonitrile until a stable baseline was obtained.

Table 1. Imprinted and Nonimprinted Blank Polymers Prepared in This Study polymer IP/NP0 IP/NP02m IP/NP04m IP1m IP/NP2m IP/NP02s IP/NP04s IP/NP1s IP0c IP/NP1scb

crowding amount CHCl3 imprinting agent (g) (mL)a k′ (IP) k′ (NP) factorb PMMA PMMA PMMA PMMA PS PS PS

0.197 0.377 0.865 1.54 0.197 0.377 0.865

PS

0.865

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 3.85 3.8

1.2 1.1 1.4 2.3 8.0 1.5 2.4 3.4 1.9 13.5

0.22 0.51 0.84 0.74 0.84 0.26 0.68 0.94

5.6 2.2 1.6 3.3 9.5 5.7 3.5 3.6

0.94

14.4

a The shown figure is the amount of a mixture of chloroform and the corresponding crowding agent. b Imprinting factors were calculated as k′ (IP)/k′ (NP).

Chromatographic measurements were conducted using acetonitrile as the eluent at a flow rate of 0.2-1.0 mL min-1 at 30 °C, and the eluent was monitored at 280 nm. Each 20 µL (0.5 mM) sample was independently injected. A retention factor (k′) was calculated using the equation k′ ) (tR - t0)/ t0, where tR is the retention time of a sample and t0 is the elution time of the void marker, acetone. Batch Binding Test of the Imprinted Polymers. The polymer particles (IP1sc and IP0) were repeatedly washed with methanol-acetic acid (8:2, v/v) until HPLC analysis of the supernatant showed no peak of atrazine. After being thoroughly washed with methanol and dried under reduced pressure, the polymer (10 mg) was immersed in a 1.0-mL chloroform solution of atrazine with various concentrations ranging from 28 µM to 4.5 mM (3.0 mM in the case of IP0) at 293 K. After the incubation for 20 h, the sample tubes were centrifuged. Aliquots of the supernatant were taken and analyzed by HPLC to quantify the concentration of free atrazine, F, and subsequently the amount of atrazine bound to the polymer, B. Three independent batches were tested for each concentration. The average data were used for subsequent analysis. For Scatchard analysis, B/F is plotted versus B according to the equation, B/F ) (Bmax - B)/Kd, where Kd is the equilibrium dissociation constant and Bmax is the theoretical maximum number of binding sites. RESULTS AND DISCUSSION Macromolecular Crowding-Inducing Agents. Biological cells contain high concentrations of macromolecules such as proteins and nucleic acids, resulting in peculiar molecular environment called “molecular crowding”. Although many details about molecular crowding are still unknown, several reports have shown that crowding affects the association of biomolecules. For example, the self-associations of proteins such as spectrin, fibrinogen, and FtsZ are enhanced by molecular crowding conditions.12 Polymerization and fibrillation of diease related proteins such as R-synuclein and apolipoprotein C-II are also accelerated under molecular crowding conditions. Furthermore, molecular crowding affects the structure and stability of DNA duplexes, triplexes, and quadruplexes.13 Assuming that some of these effects could be validated in organic solvents, we envisioned that intermolecular interactions between the template molecule and functional monoAnalytical Chemistry, Vol. 79, No. 4, February 15, 2007

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Figure 1. Schematic representation of molecular imprinting under molecular crowding conditions. Atrazine (a template molecule) is mixed with MA (a functional monomer) in chloroform with PMMA or PS as a macromolecular co-solute (a); cross-linking with EDMA is achieved by heating, forming a three-dimensional polymer matrix (b); atrazine is extracted from the matrix to leave behind a binding site (c).

mers in molecular imprinting could be stabilized by molecular crowding. Instead of biomacromolecules, we used the synthetic polymers, PMMA and PS, as crowding-inducing macromolecular agents. These agents were chosen because (i) they have good solubility in chloroform that was adopted as the solvent (diluent) used for the synthesis of molecularly imprinted polymers (MIPs); (ii) PMMA has functionalities similar to ethylene glycol dimethacrylate (EDMA) used as the cross-linker (e.g., methyl groups, ester bonds and alkylene chains), which reduces a potential concern about unexpected influences of residual crowding agent on the retention property of MIPs; (iii) PS bears no hydrogen-bonding functionality that may interfere with the complexation of atrazine and MA. Synthesis and Assessment of Atrazine-Imprinted Polymers. Using atrazine as a proof-of-principle target,14 molecularly (14) (a) Chappell, M. A.; Laird, D. A.; Thompson, M. L.; Li, H.; Teppen, B. J.; Aggarwal, V.; Johnston, C. T.; Boyd, S. A. Environ. Sci. Technol. 2005, 39, 3150-3156. (b) Abate, G.; Masini, J. C. J. Agric. Food Chem. 2005, 53, 1612-1619. (c) Ibanez, M.; Sancho, J. V.; Pozo, O. J.; Hernandez, F. Anal. Chem. 2004, 76, 1328-1335. (d) Blume, E.; Bischoff, M.; Moorman, T. B.; Turco, R. F. J. Agric. Food Chem. 2004, 52, 7382-7388. (e) Abate, G.; Penteado, J. C.; Cuzzi, J. D.; Vitti, G. C.; Lichtig, J.; Masini, J. C. J. Agric. Food Chem. 2004, 52, 6747-6754. (f) Barriuso, E.; Koskinen, W. C.; Sadowsky, M. J. J. Agric. Food Chem. 2004, 52, 6552-6556. (g) Acosta, E. J.; Steffensen, M. B.; Tichy, S. E.; Simanek, E. E. J. Agric. Food Chem. 2004, 52, 545-549. (h) Silva, E.; Fialho, A. M.; Sa-Correia, I.; Burns, R. G.; Shaw, L. J. Environ. Sci. Technol. 2004, 38, 632-637. (i) Feng, J.; Shan, G.; Maquieira, A.; Koivunen, M. E.; Guo, B.; Hammock, B. D.; Kennedy, I. M. Anal. Chem. 2003, 75, 5282-5286. (j) Shoji, R.; Takeuchi, T.; Kubo, I. Anal. Chem. 2003, 75, 4882-4886. (k) Lim, T.-k.; Oyama, M.; Ikebukuro, K.; Karube, I. Anal. Chem. 2000, 72, 2856-2860.

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imprinted polymers were prepared with varying concentrations of the crowding agents (Table 1) and were subjected to chromatographic analysis. Molecular imprinting with atrazine has been reported independently by several groups.15 Therefore, our synthesis was conducted according to a formerly reported procedure,15g adopting MA, EDMA, and chloroform as the functional monomer, the cross-linker, and the diluent, respectively (Figure 1). Chloroform was adopted as the solvent (diluent) because the complexation of atrazine and MA is based on noncovalent hydrogen bonds, which chloroform cannot disrupt seriously.16 Although lower temperature polymerization is favorable for such complexations,9 thermal polymerization was adopted because of a concern that crowding conditions may cause lower UV permeability and thus heterogeneous polymerization. The resulting polymers were ground and sieved to collect MIP particles and packed in a stainless steel column. Retention of atrazine by (15) (a) D’Agostino, G.; Alberti, G.; Biesuz, R.; Pesavento, M. Biosens. Bioelectron. 2006, 22, 145-152. (b) Sun, H.; Fung, Y. Anal. Chim. Acta 2006, 576, 67-76. (c) Svenson, J.; Zheng, N.; Fo ¨hrman, U.; Nicholls, I. A. Anal. Lett. 2005, 38, 57-69. (d) Ka´tay, G.; Mincsovics, E.; Szokan, G.; Tyiha´k, E. J. Appl. Polym. Sci. 2005, 98, 362-372. (e) Cacho, C.; Turiel, E.; MartinEsteban, A.; Pe´rez-Conde, C.; Ca´mara, C. J. Chromatogr. B 2004, 804, 83. (f) Chapuis, F.; Pichon, V.; Lanza, F.; Sellergren, B.; Hennion, M.-C. J. Chromatogr. B 2004, 804, 93-101. (g) Matsui, J.; Miyoshi, Y.; DoblhoffDier, O.; Takeuchi, T. Anal. Chem. 1995, 67, 4404-4408. (h) Siemann, M.; Andersson, L. I.; Mosbach, K. J. Agric. Food Chem. 1996, 44, 141145. (i) Muldoon, M. T.; Stanker, L. H. Anal. Chem. 1997, 69, 803-808. (16) (a) Wu, L.; Zhu, K.; Zhao. M.; Li, Y. Anal. Chim. Acta 2005, 549, 39-44. (b) Shea, K. J.; Spivak, D. A.; Sellergren, B. J. Am. Chem. Soc. 1993, 115, 3368-3369.

Figure 2. Retention factor of atrazine exhibited by the atrazineimprinted (filled circle) and non-imprinted (open circle) polymers prepared with varying amounts of PS (solid line) or PMMA (broken line).

the atrazine-imprinted and non-imprinted polymers was plotted as a function of the amount of a crowding agent used for the synthesis (Figure 2). As shown in Figure 2, both crowding agents resulted in significant increase in retention. In the case of PMMA as the crowding agent, IP2m exhibited the largest retention factor (k′ ) 8.0), approximately 6.5 times larger than that of IP0 prepared conventionally under non-crowding conditions. In order to confirm that the enhanced retention was not simply due to the decreased amount of chloroform used in the synthesis, we examined an atrazine-imprinted polymer, IP0c, as a control (Table 1). Addition of crowding agent to the pre-polymerization mixture decreases the concentration of chloroform because all the imprinted polymers (except IP1sc and NP1sc) were prepared in an identical volume (5 mL) of the media consisting of chloroform and the crowding agent. For example, the polymerization mixture for IP2m involved ca. 3.85 mL of chloroform, while IP0 was in 5.00 mL. Thus, the concentration in the pre-polymerization mixture for IP2m is about 1.5 times higher than that for IP0, if the atrazine concentration is defined as atrazine (mole) per chloroform (vol). Because this difference may have affected the imprinting process, we prepared IP0c using 3.85 mL of chloroform (without the crowding agent). Chromatographic analysis showed that atrazine was retained 2.9-min (k′ ) 1.89) by IP0c which is only a slightly longer retention than that by IP0, showing that the smaller amount of chloroform does not result in significant enhancement of the atrazine-retention property. This result highlights the effectiveness of the crowding strategy for enhancing the retention property of molecularly imprinted polymers. The other crowding agent examined in this study, PS, exhibited slightly more effective enhancement of the atrazine-retaining property of MIPs (compared with PMMA) when the amount of the crowding agent added was up to 0.865 g (Figure 2). The difference in the crowding effect of PS and PMMA can be partially accounted for by their structure: PS does not possess any hydrogen-bonding functionality, while PMMA bears carbonyl groups that can be involved in hydrogen bonding with atrazine

and MA. This hydrogen-bonding property of PMMA may interfere with hydrogen bonding between atrazine and MA, which is necessary for molecular imprinting. Thus, PS could be more conducive to complex formation between atrazine and MA, but it results in unfavorable stiffness of the resultant polymers. When more PS was added to the pre-polymerization mixture (results not shown), the resulting polymeric materials were sol-like rather than a brittle resin and thus were not suited to characterization by chromatography. Additionally, the shape and chemical functionality of the template molecule is unlikely to be “memorized” by such a fluid material. The imprinted polymer IP1s, showing slightly better retention than IP1m, also exhibited a somewhat soft structure; therefore, the MIP synthesis was conducted using less chloroform media (3.8 mL including 0.865 g of PS) in order to obtain a hard and brittle polymer. The resultant polymer, IP1sc, exhibited a retention factor of 13.5, the largest retention factor achieved in this study (Table 1). These results suggest that the optimal amount of PS should be determined on the basis of the degree of molecular crowding and the rigidity of the resultant polymers. To ensure that the observed long retention was due to the addition of both the template and the crowding agent, nonimprinted polymers were assessed as controls: they were prepared without addition of the template but using an identical amount of the crowding agent as was used in the synthesis of the imprinted polymers. As shown in Figure 2, the non-imprinted polymers exhibited poor retention, compared with the corresponding imprinted polymers. A non-imprinted polymer (NP1sc) prepared in a manner similar to IP1sc also exhibited shorter retention (k′ ) 0.94; Table 1). These results suggest that carboxyl moieties, if not oriented through interactions with the template molecule, can exhibit significantly lower affinity than the template-guided carboxyls. The short retentions can be also accounted for by dimer formation of MA via double hydrogen bonding between the carboxyl groups, which would result in carboxyl moieties that are less accessible to an analyte molecule after immobilization in the non-imprinted polymers. The imprinting factor, defined as the ratio of the retention factor of an imprinted polymer to that of the corresponding non-imprint polymer, was 5.6 (IP0 vs NP0) without the crowding agent, whereas it was 9.5 with PMMA (IP2m vs NP2m) and 14.4 with PS (IP1sc vs NP1sc). The increase in the imprinting factor by addition of a crowding agent implies that the agents allowed the imprinting process to proceed more efficiently. Selectivity of Atrazine-Imprinted Polymers. Using triazines and other categories of agrochemicals, the selectivities of the imprinted polymers, IP2m and IP1sc, were assessed by chromatography. The polymers, IP0, NP2m, and NP1sc, were examined as references in order to determine the influence of molecular crowding and template addition on selectivity. Relative retention times are summarized in Figure 3, where the retention time of atrazine is defined as 100. It can be seen that selectivity for atrazine against other triazines was enhanced by addition of the template and the crowding agent; the reference polymer IP0 exhibited larger retention for simazine, propazine, cyanazine, ametryn, prometryn, and metribuzin than for atrazine. In the case of the non-imprinted polymers, minute comparison of retention may be insignificant because the retention was too short to be compared (k′ < 1). However, selectivity appeared to be enhanced by Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

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Figure 3. Selectivity of the imprinted polymers prepared with the crowding agent (filled bar); left, IP2m; right, IP1sc. As controls, the imprinted polymers prepared without the crowding agent (gray bar) and the non-imprinted polymers (blank bar) are also shown; left, IP0 and NP2m; right, IP0 and NP1sc. The relative retention was obtained by dividing the retention time of each sample by the retention time of atrazine on each polymer, and multiplying by 100.

imprinting; NP2m and NP1sc also showed comparable or larger retention for ametryn and metribuzin, whereas IP2m and IP1sc clearly discriminate these compounds from atrazine. More distinct differences between the crowding-assisted MIPs and the reference polymers can be observed when the retention of triazines and that of other chemical species are compared. Whereas no clear distinction can be drawn between the triazines and the others in the case of IP0, NP2m, and NP1sc, it can be clearly seen that IP2m and IP1sc exhibited a group selectivity for triazines. The enhanced selectivity of IP2m and IP1sc suggests that molecular crowding conditions did not enhance nonspecific binding sites but rather engaged in the generation of selective binding sites. Batch Binding Test for Assessing Atrazine-Adsorption Property of the Crowding-Assisted Imprinted Polymer. Molecularly imprinted polymers, IP1sc and IP0, were subjected to batch binding tests for estimating the affinity and the theoretical number of binding sites for atrazine. The batch binding test is useful for characterizing the crowding-assisted imprinted polymer as sorbent material. Because molecularly imprinted polymers have been applied to sorbent assays as well as chromatography, this characterization would be important for assessing the usefulness of the imprinted polymers. In Figure 4a, the amount of atrazine bound to the imprinted polymers (IP1sc and IP0) in varied concentrations of atrazine was plotted. Scatchard plot was shown in Figure 4b, where dashed lines were drawn assuming that each polymer possesses two categories of binding sites (i.e., highaffinity binding sites and low-affinity ones). The Scatchard plot suggests that both polymers exhibited the affinity for atrazine 1754

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similar to each other; the high-affinity binding sites and low-affinity ones of IP1sc (IP0) showed dissociation constants (Kd) as 57 µM (55 µM) and 1.2 mM (1.1 mM) at 293 K, respectively. The difference in the dissociation constant between the high-affinity binding sites and low-affinity ones can be converted to ∆∆G approximately as 1.7 kcal mol-1. This value is roughly consistent with energy of the formation of one or two hydrogen bond(s),17 implying that the two categories of binding sites differ in the arrangement of the methacrylic acid moieties. Because one methacrylic acid can form two hydrogen bonds with atrazine, a possible interpretation would be that the high-affinity binding sites and low-affinity ones consist of two and one methacrylic acid moieties, respectively. On the other hand, there was significant difference in the theoretical number of binding sites (Bmax) between IP1sc and IP0. The number of high-affinity binding sites in IP1sc and IP0 were estimated as 10 and 8 µmol g-1, respectively. As for the low-affinity binding sites, also, IP1sc exhibited the larger number of binding sites (81 mol g-1) as compared to IP0 (25 mol g-1). The increased number of binding sites in IP1sc suggests the higher capacity of the crowding-assisted imprinted polymer that would be an advantageous feature when the imprinted polymer is applied as sorbent material. Origin of Crowding Effects Stage 1: Pre-polymerization. Molecular imprinting primarily consists of three stages: (1) prepolymerization, (2) polymerization, and (3) post-polymerization. (17) Sugimoto, N.; Nakano, S.; Yoneyama, M.; Honda, K. Nucleic Acids Res. 1996, 24, 4501-4505.

Figure 4. Binding isotherm (A) and Scatchard plot (B) of the crowding-assisted imprinted polymer (IP1sc) and the non-assisted imprinted polymer (IP0) at 293 K. Dashed lines in the Scatchard plot were drawn by a linear least-squares fit assuming that binding sites can be classified into two groups: high-affinity binding sites and lower affinity ones.

Therefore, the origin of enhanced retention caused by the addition of crowding agents should be explored in terms of each of these three stages. At the pre-polymerization stage, a high concentration of macromolecular cosolutes was expected to promote complexation of atrazine with MA. To examine this assumption, an NMR study was conducted with a pseudo-pre-polymerization mixture consisting of atrazine, chloroform, and MA. The initiator ADVN was omitted because of its insignificant role in complex formation. The cross-linker EDMA was also omitted because its carbonyl group can be involved in hydrogen bonding with atrazine and/or MA, making the system too complicated to observe complexation between atrazine and MA. Due to the same concern with PMMA, PS was chosen as the crowding agent for the NMR spectral measurements. The concentrations of atrazine and MA were the same as those used in the polymerization of IP1s. When MA was added to the solution of atrazine, a peak derived from an amino proton of atrazine exhibited a downfield shift (Figure 5a,b), suggesting the formation of hydrogen bonds between atrazine and MA.18 Upon the addition of PS to the pseudo-pre-polymerization mixture, the amino proton peak was shift even further downfield, as shown in Figure 5, panels b and c. A second amino proton peak was not clearly observed due to the broad PS peak; titration experiments where PS was gradually added were also unsuccessful because of this broad PS peak. However, the results strongly suggest that the origin of the enhanced atrazine-retention property is, at least in part, due to the equilibrium shift in the direction of (18) (a) Quaglia, M.; Chenon, K.; Hall, A. J.; De Lorenzi, E.; Sellergren, B. J. Am. Chem. Soc. 2001, 123, 2146-2154. (b) Spivak, D.; Gilmore, M. A.; Shea, K. J. J. Am. Chem. Soc. 1997, 119, 4388-4393. (c) Welhouse, G. J.; Bleam, W. F. Environ. Sci. Techol. 1993, 27, 494-500. (d) Welhouse, G. J.; Bleam, W. F. Environ. Sci. Techol. 1993, 27, 500-505. (e) Sellergren, B.; Lepistoe, M.; Mosbach, K. J. Am. Chem. Soc. 1988, 110, 5853-5860.

the atrazine-MA complex formation. 1H NMR spectra were also measured without MA (Figure 5d), showing that the addition of PS resulted in a downfield shift, although the shift was moderate as compared to that observed with MA. This downfield shift can be accounted for by the promotion of atrazine dimerization based on double hydrogen bonding. Because the equilibrium shift toward the complex formation between atrazine and MA would result in a higher concentration

Figure 5. 1H NMR spectra in CDCl3 with the pseudo-pre-polymerization: (a) atrazine; (b) atrazine + methacrylic acid (MA); (c) atrazine + MA + polystyrene (PS); (d) atrazine + PS.

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Figure 6. Microscopic images of the imprinted polymers before and after sonication in chloroform; (a) the imprinted polymer prepared with PMMA (IP2m), (b) one without the crowding agent (IP0).

of the complexes, which presumably produce a larger number of binding sites in the imprinted polymer, the results of NMR study appeared to be consistent with those of the batch binding test showing that the crowding-assisted polymer has a larger number of binding sites. The increased capacity of the crowding-assisted imprinted polymers could be a cause for the longer retention of atrazine observed in the chromatographic tests. Origin of Crowding Effects Stage 2: Polymerization. Another possible source of the enhanced imprinting effect is that the crowding agent may act as a polymeric diluent and thus affect the polymerization process. It is known that the product polymer’s morphology depends on the property of diluents used for the synthesis19 and may influence its behaviors as chromatographic stationary phase and sorbent.20 In our MIP synthesis, chloroform was used as the primary diluent as it can solvate the product polymer chains. Generally, such a diluent tends to cause a homogeneous and porous bulk polymer. However, some polymeric diluents favor the production of microsphere aggregates with lower porosity.17b Therefore, the addition of a crowding agent to the polymerization mixture (i.e., reducing the amount of chloroform) may result in fewer micropores and more macropores. Formation of microsphere aggregates upon the addition of crowding agents was supported by microscopic images as shown in Figure 6: IP2m was dispersed into microspheres when immersed and sonicated in chloroform, while IP0 was intact against the same treatment. It is believed that molecular imprinting creates binding sites associated with both micropores and mesopores. Currently, it is unclear how the distribution of these binding sites affects retention property because the number of the former binding sites is usually larger due to the larger surface area, while (19) Viklund, C.; Ponten, E.; Glad, B.; Irgum, K.; Horstedt, P.; Svec, F. Chem. Mater. 1997, 9, 463-471. (20) (a) Schmidt, R. H.; Haupt, K. Chem. Mater. 2005, 17, 1007-1016. (b) Zimmerman, S. C.; Lemcoff, N. G. Chem. Commun. 2004, 5-14.

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the accessibility of the latter binding sites would be greater because of higher diffusion. In addition, in our case it is currently unknown how morphological differences affect atrazine retention. A slight increase in retention of the blank polymers (NP1s and NP2m in Figure 2) with increasing concentration of crowding agent could imply that polymer morphology resulting from the use of the crowding agent is slightly favorable for atrazine retention. Shape of diluent molecules is also known to affect the selectivity of imprinted polymers; it has been reported that selectivity for a given analyte molecule can be induced using a porogen that is structurally similar to the analyte molecule, in which the porogen molecules are believed to work as print molecules.21 Therefore, higher retention factors attained with PS could be partially attributed to structural difference in the side chain between PMMA and PS, given the similarity in size between a phenyl group and a triazine ring. Origin of PMMA Effects Stage 3: Post-polymerization. In addition to the pre-polymerization and polymerization processes, the macromolecular co-solute may affect the atrazine re-binding process if it remains in the cross-linked polymer after the washing procedures, although we employed PMMA to avoid this complication. For instance, residual co-solute may interact with atrazine or affect the hydrophobicity of the atrazine-binding site. Therefore, to determine if the crowding agent can be completely removed from the resultant MIPs, the polymers NP1m and NP1sc were thoroughly washed with chloroform, and the recovery rate of the crowding agent was estimated by analyzing the wash wastes. The wash wastes from NP1m and NP1sc were dried and weighed, and examined by UV spectroscopy (262.5 nm) to estimate the amount of extracted crowding agent. The weights of the residues allowed (21) Yoshizako, K.; Hosoya, K.; Iwakoshi, Y.; Kimata, K.; Tanaka, N. Anal. Chem. 1998, 70, 386-389.

matrix. The slight increase of PS implies that residual PS could fill the pores in the imprinted polymer, reducing the number of accessible binding sites. CONCLUSIONS

Figure 7. Effect on retention behavior of residual crowding agent in the imprinted polymers; square, IP2m (PMMA); circle, IP1sc (PS). The fraction of extracted crowding agent was calculated by dividing the amount (g) of extracted PMMA or PS by the initial amount (g) of the agent in the corresponding IP packed in a column. Estimation of the initial amount was based on the assumption that MA and EDMA react stoichiometrically to yield imprinted polymers. In this graph, a fraction of zero does not mean that each IP involved 100% of the initial amount of crowding agent, because some of the agent had likely already been lost during the wet sieving and packing process. The amount of agent lost during the process has not been estimated due to the difficulty of isolating the crowding agent from the sieving and packing waste (which would also include unreacted monomers and/ or oligomeric products).

the recovery rate from NP1m and NP1sc to be estimated at 118% and 124%, respectively. Recovery rates larger than 100% suggest that the extracts also include other compounds such as unreacted monomers and oligomeric products. UV absorbance also allowed the recovery rate from NP1sc to be estimated at ca. 94%. Removal of PMMA could not be precisely quantified by UV spectroscopy because of poor UV absorbance of PMMA. These results show that almost all of the crowding agent added can be recovered. To further confirm the ineffectiveness of residual crowding agent on the retention property, the imprinted polymers IP2m and IP1sc were column chromatographed with alternative use of acetonitrile (for measuring the retention of atrazine) and chloroform (for extraction of the crowding agent) as the eluent. The retention of atrazine was measured each time after the intermittent extraction of the crowding agent. Chloroform was used to remove the crowding agent because acetonitrile used as the eluent was ineffective at extracting the crowding agent. The retention factor displayed no drastic decrease upon removal of the crowding agent (Figure 7). These results suggest that residual crowding agent, if any, was not significantly involved in atrazine retention at the post-polymerization stage. Accordingly, it would be reasonable to conclude that the crowding agents engaged significantly in molecular imprinting processes. Comparing the results of IP1m and IP1sc, the removal of PMMA resulted in a slight decrease in the retention factor and that of PS led to a slight increase, although both changes were moderate. The opposite trends could have derived from the different properties of the crowding agents. PMMA bears a carbonyl group that can interact with atrazine; therefore, the removal of PMMA could reduce the interaction points, though the interaction would be as weak as that by EDMA-based polymer

This study demonstrated that molecular crowding can be utilized for synthesizing molecularly imprinted sorbents with enhanced capacity and selectivity. The imprinted polymer IP1sc, which was prepared in the presence of PS (ca. 0.26 g L-1) as the crowding agent, marked a retention factor of 13.5 in acetonitrile. This retention factor was approximately 11 times as large as that of IP0, which was prepared under non-crowding conditions. Furthermore, the crowding-assisted MIPs exhibited superior retention compared to a previously reported MIP photopolymerized at a lower temperature, which was more favorable for hydrogen bonding between atrazine and MA.15g In this study, two kinds of linear polymers, PS and PMMA, were shown to be effective as crowding agents for enhancing retention of MIP sorbents. It is currently difficult to predict which macromolecule would be most useful for molecular crowdingassisted imprinting because which characteristics of polymers govern their effectiveness remains poorly understood. In in-cell molecular crowding studies, the essential nature of molecular crowding and its effects on biomolecular reactions are still under investigation, although recent studies by our group have revealed that hydration plays a significant role.13 Furthermore, molecular crowding in organic solvents might be significantly different from that in aqueous solution. Therefore, in order to understand the mechanism by which molecular crowding assists molecular imprinting, it would be necessary to screen more macromolecular co-solutes for their effectiveness as crowding agents. At the same time, detailed characterization of the product polymers is also important in order to gain insights into the origin of the crowding effect in molecular imprinting. In the present study, such discussion is still at a preliminary stage. Future work should consider more detailed thermodynamic and physical properties of MIPs; this information would allow substantial conclusions to be drawn. However, whatever the mechanism of the crowding effects, this study suggests that the crowding strategy can be generally applied to molecular imprinting based on noncovalent interactions. In the past decade, a number of molecular imprinting studies have been reported utilizing noncovalent bonds between a template and functional monomers. We trust that our findings will allow significant progress to be made regarding molecular imprinting methodologies that will produce analytically useful polymeric reagents and materials. ACKNOWLEDGMENT This work was supported in part by Grants-in-Aid for Scientific Research and Academic Frontier Project (2004-2009) from MEXT (Ministry of Education, Culture, Sports, Science and Technology), Japan.

Received for review March 9, 2006. Accepted December 9, 2006. AC060441M Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

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