Anal. Chem. 1995, 67,4404-4408
A Molecularly imprinted Synthetic Polymer Receptor Selective for Atrazine Jun Matsui, Yoko Miyoshi, Otto Doblhoff-Dier,tand Toshifumi Takeuchi" Laboratory of Synthetic Biochemistty, Faculty of Information Sciences, Hiroshima City University, 15 1-5Ozuka, Numata-cho, Asa-minami-ku,-Hiroshima 731-31, Japan
A synthetic polymer selective for atrazine was prepared by a molecular imprinting technique, and the binding characteristics of the atrazine-imprinted polymer were evaluated. Scatchard analysis showed that at least two classes of binding sites were formed in the imprinted polymer, and a dissociationconstant of the higher W t y binding sites was estimated to be 12 pM. The induced atbity and selectivity by atrazine imprintingwere examined chromatographically. The polymer gave more than 60 times longer retention for atrazine than the nonimprinted polymer with the same chemical composition. The selectivitywas evaluatedby capacity factors of atrazine and other pesticides, and atrazine showed the longest retention time among the tested compounds with 1,3,5triazine-basedstructure. Other herbicides and pesticides with unrelated structures were retained 1%or less, as compared to atrazine. Because the optimal binding performance was exhibited in organic solvents, the synthetic polymer receptor is expected to be a good material in lipophilic herbicide analysis. Synthetic polymer receptors have been increasingly developed using a molecular imprinting technique as mimics of natural molecular recognition The principle of this technique involves the formation of definable interactions between a given template molecule and polymerizable functional monomers during polymerization with cross-linking agents (see Figure 1). Subsequent removal of the template from the resulting polymer yields complementary binding sites which consist of functional groups with a particular arrangement fit for the corresponding templates. Many molecularly imprinted polymers (MIPS) have been prepared and utilized mainly as affinity chromatography ~
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On leave from Institute of Applied Microbiology, State University of Forestry & Agriculture, Vienna, Austria. (1) Wulff, G. ACS Symp. Ser. 1986, No. 308, 186-230. (2) Shea, K. J. Trends Polym. Sci. 1994,2, 166-173. (3) Mosbach, K Trends Biochem. Sci. 1994, 19, 9-14. (4) Matsui, J.; Doblhoff-Dier, 0.;Takeuchi, T. Chem. Lett. 1995,489. (5) Matsui, J.; Kato, T.; Takeuchi, T.; Suzuki, M.: Yokoyama. IC;Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2223-2224. (6) Nicholls, I. A; Ramstrom, 0.; Mosbach. K. J. Chromatogr. A 1995, 691, 349-353. (7) Ramstrom, 0.:Nicholls, I. A.; Mosbach. K Tetrahedron: Asymmetry 1994, 5, 649-656. (8) Fischer, L.: Muller, R.; Ekberg, B.; Mosbach, K.J. Am. Chem. SOC.1991, 213, 9358-9360. (9) Andersson, L. I.; O'shannessy, D. J.; Mosbach, K. J. Chromatogr. 1990, 513, 167-179. (10) Vlatakis, G.; Andersson, L. I.: Muller, R ; Mosbach, K. Nature 1993, 361, 645-647. ( 1 1 ) Muldoon, M. T.; Stanker, L. H.J. Agn'c. Food Chem. 1995,43,1424-1427. +
4404 Analytical Chemistry, Vol. 67, No. 23, December 7, 1995
MIP-based binding assays have also been developed in which MIPs are successfully used as antibody mimics.'OJ Molecular imprinting technology has been expanded to the field of environmental analytical c h e m i ~ t r y . ~Herbicides ,~~ are important compounds to be analy~ed'~,'~ because over the last years herbicide contamination of drinking water and agricultural products has become a major concern and the number of herbicides has been steadily increasing, with over 100 species registered at present. Currently herbicides are often preconcentrated or purified by liquid ~hromatography'~-'~ using organic solvents; therefore, easily prepared, low-cost, and stable affinity elements working in organic solvents such as MIPs would find novel applications in herbicide analysis. Recently, we reported a preliminary study on molecularly imprinted polymers for atrazine? which is one of the most widely used herbicides, and used them as affinity chromatography media. Here, we describe the preparation of synthetic polymer receptors for atrazine by a molecular imprinting technique using methacrylic acid, known as a common but potentially functional monomer. The detailed characteristics of the MIPs including the binding properties and selectivity are examined by Scatchard analysis, chromatographic behavior, and NMR studies, and the recognition mechanism in this system is also discussed. EXPERIMENTAL SECTION Materials. Atrazine [2c~or~(ethylamino)~(isopropylamino)1,3,5triazinel, simazine [2-chloro-4,6bis(ethylamino)-l,3,5triazine I, cyanazine 12-[ [4chloro6(ethylamino)-1,3,5triazine-2-y1]amino1-2-methylpropionitrile1, prometryn [2,4bis(isopropy1amino)6(methylthio)-l,3,5triazine I, metribuzin [4amino-6tert-butyl-3(methylthio)as-trih-5 (4H)-one], diuron [ N -(3,4dichlorophenyl)N,N-dimethylureal , alachlor [ 2-chloro-2',6'-diethyl-N-(methoxymethyl)acetanilidel, bentazon [3-isopropyl-lH-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide], MCP ethyl [ethyl (khloro-2-methy1phenoxy)acetatel and pendimethalin [N-(l-ethylpropyl)-3,4dimethyl-2,Wtrobenzenamjne] were all donated as bulk herbicide formulations from Nissan Chemical Industry (Tokyo, Japan). The herbicides were extracted from the bulk formulation with methanol and purified by silica gel chromatography using ethyl acetate and hexane as the eluent. All the other pesticides, ametryn ~
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(12) Berg, M.; Muller, S. R.: Schwarzenbach. R P. Anal. Chem. 1 9 9 5 , 6 7 , 18601865. (13) Tom-Moy, M.; Baer, R. L.; Spira-Solomon, D.; Doherty, T. P. Anal. Chem. 1995, 67, 1510-1516. (14) &a, D.S.; Thurman, E. M. Anal. Chem. 1993, 65. 2894-2898. (15) Molto, J. C.; Pico, Y.; Font, G.; Manes, J.J. Chromatogr. 1991, 555, 137145. (16) Cai, Z.; Ramanujam, V. M. S.;Giblin. D. E.; Gross, M. L.; Spalding, R. F. Anal. Chem. 1993, 65, 21-26.
0003-2700/95/0367-4404$9.00/0 0 1995 American Chemical Society
irradiation (UVF' Model XX-lSS). These conditions were maintained for 4 h. The obtained polymer was crushed in a mortar and ground to pass through a 45pm sieve. Fine particles were removed by decantation in acetonitrile. The resulting particles were used for the following studies. Scatchardhalysis. The sized and washed polymer particles (10.0 mg) were mixed with a l.0mL chloroform solution of atrazine of varied concentrations from 25 1 M to 3.0 mM. The mixture was incubated for 16 h with continuous stirring at room temperature (20 "C). Prior to the saturation experiments, it had been confirmed that the reaction of polymer and atrazine reaches equilibrium under these conditions. After the incubation, the sample tubes were centrifuged Tomy SRX-201 centrifuge, Rotor TA-1) and 501L samples of the chloroform layer taken. The aliquots were analyzed using the HPLC system with a reversedphase column (ODS2. GL Science Inc.) to quantify the concentration of free atrazine, [At].The eluent was methanoVwater (65 35, v/v). and detection was canied out at 263 nm. The amount of atrazine hound to the polymer, B, was calculated by subtracting [At] from the initial atrazine concentration. The average data of triplicated independent results were used for the Scatchard analysis. Binding data can be linearly transformed according to the Scatchard equation, B/[Atl = (Bm, - @/KO, where Kn is an equilibrium dissociation constant and B,, is an apparent maximum number of binding sites. When B/[Atl is plotted versus B, KO and B,, can be estimated from the slope and the intercept, respectively." NMR Study. NMR spectra study was carried out with ETNMR Model GSX-400 UEOL). Samples were prepared with a Figure 1. Schematic illustration of the molecular imprinting procek e d concentration of atrazine (30 mM) and varied molar ratio, dures in this study. (a)A template (atrazine)and functional monomers up to 16, of methacrylic acid in CDCh. TMS was used as an (methacrylic acid) are induced to form a complex at the prepolymerization stage. (b) Polymerization with cross-linking agents is carried internal standard. The measurements were carried out at 24 "C. out in the presence of the complex. (c) Subsequent removal of the Chromatographic Study. Chromatographic experiments template yields a binding site complementary to the template. were performed with an HPLC system (Hitachi) consisting of an LZ-(ethylamino)-4-~sopropylamino)-6(methylthio)-l,3,~tri~el, L6200 and a L 6 W gradient pump system, L5090 degasser, Metolachlor [2chloro-N-(2-ethyl~methyIphenyl)-N-(Z-metho~- L4200 UV/visible detector, and D-2500 integrator. Sample injec1-methy1ethyl)acetamidel, MBPMC (ZBdi-tert-butyI-4-meth~h~ tion was carried out with a 231-XL sampling injector (Gilson). The polymer particles were slurried in chloroform/acetonitrile (l:l, nyl methylcarbamate), flutolanil (a.u,a-triiluoro-3'-isopropoxy-ov/v) toluanilide) , chlorwrifos [ O , O d i e t h y l 0 ( 3 , 5 , 6 ~ c h l o r o - ~ ~ d ~ y l ) and packed in stainless steel column tubes (150 mm x 4.6 mm i.d.) using a Model 510 pump (Waters). To remove the phosphorothioatel, diazinon [O,Odiethyl O[Gmethyl-2-(l-methtemplate molecules, the columns were washed with methanol/ ylethyl)-4pyrimidinyl1 phosphorothioatel, isofenphos [isopropyl acetic acid (41, v/v). The column was then washed with @[ethoxy~sopropylamino)phosphinothioyll salicylate], TPN (tetacetonitrile at a flow rate of 1.0 mL/min until a stable baseline rachloroisophthalonitrile),and 1,3,5triazinewere purchased from was obtained. All the LC analysis was performed on both the commercial sources. Chloroform, ethylene glycol dimethaaylate. and methacrylic acid were dried and distilled prior to use in order imprinted polymer and the reference polymer with isocratic eluent to remove stabilizers. Other chemicals were used without further conditions at a flow rate of 1.0 mL/min and detection at 260 nm. purification. The injection volume was 20 pL, and the sample concentration Polymer Preparalions. For the preparation of atrazine was 0.2 mM. Each sample was injected independently. A capacity imprinted polymer P(At), 360 mg of the template atrazine was factor, k , was calculated by the equation, k' = ( t R - tn)/to, where dissolved in 25 mL of chloroform in an 80mL glass tube. Into f R is the retention time of a sample and tnis the time to elute the the solution were added 0.575 g of the functional monomer void marker acetic acid. For the evaluation of the selectivity, the methacrylic acid (MAA), 9.35 g of the cross-linking monomer relative capacity factors lOO(k'Jk3 was used, where k', is the ethylene glycol dimethacrylate (EDMA), and 120 mg of the capacity factor for a sample and k', is the capacity factor for initiator 2,2'-azobisisobutyronitrile (AIBN). A reference nonimatrazine. Tests were performed with acetic acid for void volume printed polymer P O was prepared using the same recipe, estimation. All the steps were carried out at room temperature. without addition of the template. After nitrogen gas was sparged into the solution for 5 min, the glass tubes were sealed and placed (17) Yamamua. H. I.: Enna, S. J.; Kuhar, M.J. NeurotmnsnriffaRee~PforBinding: Raven Press: New York. 1985: Chapter 3. in a water bath at 0 "C. Polymerization was initiated hy W light CI
Anaiytical Chemistly, Vol. 67,No. 23, December
1, 1995
4405
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RESULTS AND DISCUSSION
Affinity of the Atrazine-Imprinted Polymer. In order to investigate the binding performance of the atrazine-imprinted polymer P (At), saturation experiments and subsequent Scatchard analysis were carried out. In Figure 2, the amount of atrazine bound to P(At), B, is shown against varied initial concentrations of atrazine. In many receptor binding experiments, a plot of bound versus initial concentrations does not yield a typical saturation profile due to linearly increasing nonspecific binding.I8 The obtained plot, however, exhibited saturation at higher atrazine concentrations. This suggests that atrazine binds to the polymer more possibly due to the specific binding to a limited number of binding sites than due to the nonspecific adsorption to the polymer network. Thus, the nonspecific binding can be assumed to be small enough to be ignored in this concentration range. The obtained binding data were plotted to estimate the dissociation constant KD and the number of binding sites B,, As shown in Figure 3, the Scatchard plot was not linear, suggesting that the binding sites in P(At) are heterogeneous in respect to the aftinity for a t r a ~ i n e . ~Because ~ . ~ ~ there are two (18) Hulme. E. C. Receptor Biochemistry;IRL Press: New York, 1990: Appendix. (19) Shea. K J.: Spivac, D. A: Sellergren, B. J. Am. Chem. Soc. 1 9 9 3 , 115.33683369.
4406 Analytical Chemistry, Vol. 67, No. 23, December 7, 7995
Methacrylic acid/Atrazine Figure 4. Effects of the addition of methacrylic acid on the chemical shift of the amino protons of atrazine on 'H NMR in CDC13 at 24 "C. The sample concentration of atrazine was 30 mM.
distinct sections within the plot which can be regarded as straight lines, it would be reasonable to assume that the binding sites can be classified into two distinct groups with specific binding properties. Under this assumption the respective KD'Scan be calculated to be 12 p M and 100 p M , and the respective Bmax's 20 pmol and 40 pmol /g of dry polymer. The obtained values for B,, would therefore correspond to 12%and 24%of the theoretical total binding sites derived from the amount of the template used for the polymerization. Recognition Mechanism. (1) NMR Study. The study of the recognition mechanism of the polymer would be important to understand the imprinting and recognition phenomena. The reaction mixture before polymerization was investigated by IH NMR20,21Because the principle of molecular imprinting lays in the preservation of the prepolymerized host-guest structure into a polymer matrix, it is crucial that the template and the functional monomers form stable host-guest complexes in the prepolymerization mixture. Therefore, it is important to investigate the interactions between atrazine and the functional monomer methacrylic acid at the prepolymerization stage. Since the cross-linking monomer and the initiator would be much less important for the interaction of the template and the functional monomer, the NMR study was performed with diverse molar ratios of the template atrazine and MAA in CDC13. In this system, the two side-chain amino groups of atrazine presumably interact with the carboxyl group of MAA. As expected, the addition of MAA into the atrazine solution resulted in low-field shifts of the peaks derived from the ethylamino and the isopropylamino protons of atrazine (see Figure 4). This observation suggests that the hydrogens and/or nitrogens of the amino groups of the atrazine are involved in hydrogen-bonding formation. The three nitrogens of the triazine structure could also be involved in hydrogen bonding as hydrogen acceptors. It has been rep ~ r t e d ~that ~ , the * ~ nitrogen para to the chloro substituent is more reactive than the two ortho nitrogens and that the para nitrogen and the side-chain amino hydrogen cause cooperative hydrogen(20) Sellergren, B.; Lepisto, M.:Mosbach, K. J. Am. Chem. SOC.1988,110,58535860. (21) Shea, K J.; Sasaki, D. Y. J Am. Chem. Soc. 1 9 9 1 , 113, 4109-4120. (22) Welhouse. G. J.; Bleam, W. F. Enuiron. Sci. Technol. 1 9 9 3 , 27, 494-500. (23) Welhouse, G. J.; Bleam, W. F. Enuiron. Sci. Technol. 1 9 9 3 , 27, 500-505.
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Chloroform in the Acetonitrile Eluent (Om) Water in the Acetonitrile Eluent (%) Figure 5. Capacity factors of the atrazine-imprinted polymer P(At) (0)and the reference polymer P(Rf) (0)for atrazine at varying ratios of (a) acetonitrile/water and (b) acetonitrile/chloroform as the eluent. bonding formation with acetic acid. A proposed 1:l complexz3 could be one of the major complex species to be imprinted. Under the excess of MAA, however, the presence of other complex species can be also expected in the prepolymerization mixture, although the structures and the populations are currently unknown. Because it would be important to provide multipoint interacting binding, sites of high selectivity in the resulting polymer, excess (4equimolar) of MAA was added to the template for the polymer preparation in this study. Chloroform was carefully chosen as the solvent because it does not interfere with hydrogen bonding. (2) Eluent Study. The NMR study suggests the formation of hydrogen bonding between atrazine and MAA in the prepolymerization mixture. Thus, we decided to examine the polymer performance in the LC ~ y s t e m in ~ -order ~ to veriry the hydrogenbonding-based mechanism. In the chromatographic study, acetonitrile was used as a standard solvent, that is, commonly used to perform a chromatographic test with hydrogen-bonding-based molecular imprints? Acetonitrile/water and acetonitrile/chloroform were used as test solvent systems. The addition of water may interfere with hydrogen bonding6 and increase hydrophobic interaction due to the high polarity. In contrast, the polymer is expected to exhibit the binding profile based on hydrogen bonding in less polar solvent systems containing more chloroform. With increasing water content, the retention for atrazine significantly decreased (see Figure 5a). At 5% (v/v) water content in the eluent, the polymer lost most of its atrazine retention capacity. This suggests that hydrogen bonding is a dominant interaction for the binding. Because both polymers exhibited almost no retention with high content of water, hydrophobic interaction by the polymer network is negligable. These results would agree with the results of the saturation and NMR experiments. On the contrary, the addition of chloroform in the eluent resulted in increased capacity factors for atrazine in P(At), while practically no changes were observed in P o (see Figure 5b). P(At) demonstrated a capacity factor more than 60 times larger than that of P o at 75%chloroform eluent. Experiments were also performed at lOoO/o chloroform eluent, exhibiting the longest retention times. Under these conditions, however, the chromatographic peak could not be detected precisely, due to the broad peak shape. Those results are reasonable, because the optimal binding conditions could be achieved in the same solvent systems
R”HV/N7rNHR2 I
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4 R1 = CH(CH3)z Rz = CH(CH3)2
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5 Rl = CzH5
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X = SCH,
6 RI=H
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Figure 6. Structures of the 1,3,54riazinederivatives used in this study: 1, atrazine; 2, simazine; 3, cyanazine; 4, prometryn; 5, ametryn; and 6, 1,3,54riazine.Atrazine was used as the template for the polymer preparation.
as the prepolymerization system. In our system, chloroform was used as the solvent for the polymerization process; therefore, it could be the optimal solvent for atrazine binding to the imprinted polymer. Selectivity of the Atrazine-ImprintedPolymer. The selectivity test of P(At) was camed out using a series of herbicides and other pesticides as reference samples, which were selected either for their structural interest or their purpose of use. The chemical structures of atrazine and other 1,3,5triazineherbicides are shown in Figure 6. The relative capacity factors (see Experimental Section) are listed in Table 1. The P(At) polymer exhibited high selectivity for 1,3,5triazines, while other structurally unrelated compounds showed almost no retention. Moreover, atrazine 1 exhibited large retention compared to all the other tested 1,3,5triazine derivatives. In P O , atrazine was less retained than some other 1,3,5triazines, such as prometryn (3)and ametryn (5). Therefore it appears that the selectivity of P(At) for atrazine was clearly induced during the imprinting process. This induced recognition ability is particularly noteworthy, because the tested 1,3,5triazines have only small structural differences. The selectivity test results may also give aspects of the molecular recognition mechanisms. The base structure of atrazine, 1,3,5triazine (6),did not show long retention times. This result agrees with the presumption that the side-chain amino groups of atrazine play important roles in the recognition process. Since P(At) recognizes atrazine out of 1,3,5triazines, it is obvious Analytical Chemistry, Vol. 67, No. 23, December 1, 1995
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Table 1. Selectivity Test: Relative Capacity Factors. for Atrazine and Reference Pesticides
sample triazines atrazine (1) simazine (2) prometryn (3) cyanazine (4) ametryn (5) herbicides metolachlor diuron bentazon metribuzin pendimethalin alachlor MCP ethyl others MBPMC flutolanil anilazine chlorpyrifos diazinon isofenphos TPN 1,3,5-triazine(6)
P (At)
pm
100 78 30 65 32
The eluent in this study is chloroform/acetonitrile (l:l, v/v). * The data were calculated on the capacity factor of atrazine in P(At) as 100. Parenthesized data were on the capacity factor of atrazine in P(Rf) as 100. Although 1,3,5triazine is not used as an agrochemical, it is listed for its structural interest.
that not only the distance and angle of the amino groups were recognized but also their size and shape were selected by the three-dimensional polymer network. The chloro group also seems to be important for molecular recognition. Ametryn (5),bearing the exact same two side chains as atrazine (l), was retained less than simazine (2) or cyanazine (4). The size and shape recognition by the polymer network possibly accounts for the poor retention of 5, because it was retained longer than 1 in P(Rf) due to the higher basicity caused by the electron-donating effect of the methylthio substituent. Prometryn (3)also exhibited poor (24) Matsui, J.; Miyoshi, Y.; Takeuchi, T. Chem. Left..in press. (25) Tanabe, K: Takeuchi, T.; Matsui, J.; Yano, K; Karube, I.]. Chem. Soc., Chem. Commun.. in press.
4408 Analytical Chemistry, Vol. 67, No. 23, December 1, 1995
retention. The observed selectivity for the 1,3,5triazines with a chloro group is presumably caused by the template effects. The use of MAA as a functional monomer would be also important to develop the present selectivity, because the polymers exhibited selective binding to 1,3,5triazines with a methylthio group such as ametryn and prometryn when 2-(trifluoromethyl)acrylic acid was used as a functional monomer.24 CONCLUSIONS The d c i a l receptor for atrazine was prepared by a molecular imprinting technique. The obtained atrazine-imprinted polymer P(At) exhibited highly selective binding for atrazine and 1,3,5 triazine derivatives. Because P(At) is made of synthetic polymethacrylate resin, it is inherently stable and long-lived. The molecularly imprinted polymer, with optimum binding conditions in apolar organic solvent, could be a useful tool for analytical purposes, because most of the widely used herbicides are more soluble in organic solvents. The preparation of the polymer is simple and inexpensive, making these affinity media readily available. P(At) could be useful for sample prepurification without any complicated procedures associated with the immobilization of natural binders to support materials in affinity chromatography. Although the selectivity of P(At) looks comparable, the affinity of P(At) for atrazine is currently not as good as atrazine antibodies. However, the molecular imprinting technique is still in a preliminary stage, and the performance of molecularly imprinted polymers may be improved, e.g., by the use of novel functional monomers that provide spec& or strong interactions with templates.z4,zs ACKNOWLEDGMENT The authors thank Nissan Chemical Industries, Ltd. (Tokyo, Japan) for providing herbicides, and JEOL (Tokyo, Japan) for assistance with the NMR spectral measurements. This work was supported by The Ministry of Education, Science, Sports and Culture: Large-scale Research Projects under the New Program in Grants-in-Aid for Scienti6c Research. Received for review June 27, 1995. Accepted September
15, 19951.~ AC9506387 Abstract published in Advance ACS Abstracts, November 1, 1995.
