Anal. Chem. 1998, 70, 628-631
Assay System for the Herbicide 2,4-Dichlorophenoxyacetic Acid Using a Molecularly Imprinted Polymer as an Artificial Recognition Element Karsten Haupt,* Anatoli Dzgoev, and Klaus Mosbach
Department of Pure and Applied Biochemistry, Chemical Center, Lund University, P.O. Box 124, S-22100 Lund, Sweden
Noncovalent molecular imprinting of a synthetic polymer with the herbicide 2,4-dichlorophenoxyacetic acid has been achieved in the presence of the polar solvents methanol and water. Formation of the prearranged complex relied on hydrophobic and ionic interactions between the template and the functional monomer 4-vinylpyridine. The polymer obtained binds the original template with an appreciable selectivity over structurally related compounds. The potential use of micrometersized imprinted polymer particles as the recognition element in a radioligand binding assay for 2,4-dichlorophenoxyacetic acid is demonstrated. Molecular imprinting is becoming increasingly recognized as a powerful technique for the preparation of synthetic polymers containing tailor-made recognition sites for certain target molecules.1-4 The imprinting process is performed by copolymerizing functional and cross-linking monomers in the presence of a molecular template. After elution of the template, complementary binding sites are revealed within the polymer network that allow rebinding of the template with a high specificity, sometimes comparable to that of antibodies.5,6 The artificial receptors so obtained may be used in applications that demand specific ligand binding, such as for the analytical and preparative separations of closely related compounds,7-9 as recognition elements in sensors,10 in immunoassay-type binding assays,5,11,12 and in solid-phase extraction protocols.13,14
Owing to their highly cross-linked polymeric nature, molecularly imprinted polymers are resistant against physical and chemical stresses, e.g., heat, organic solvents, acids and bases, etc. They can be stored in the dry state at ambient temperatures for several years and, if necessary, regenerated and reused many times without loss of their molecular memory. Furthermore, polymers can be imprinted with substances against which natural antibodies are difficult to raise, e.g., immunosuppressive drugs and nonimmunogenic or small compounds. The latter have to be coupled to a carrier molecule in order to raise natural antibodies, which often changes their antigenic properties considerably. Therefore, artificial receptors prepared by molecular imprinting can provide an attractive alternative or complement to natural antibodies and receptors in many applications. Among the different methods available for the preparation of molecularly imprinted polymers, the so-called noncovalent approach, which uses only noncovalent interactions between the template and the functional monomers, is probably the most flexible in terms of the choice of functional monomers and possible template molecules and has, therefore, been the most widely adopted. However, it does have some limitations. The bonds formed during prearrangement, e.g., hydrogen bonds or other electrostatic interactions, are relatively weak. Therefore, conditions must be chosen to shift the equilibrium toward complex formation. As a result, noncovalent imprinting has been performed mostly in apolar organic solvents, since in the presence of polar solvents, especially water, the prearranged complex is destabilized. It has, however, been shown that, using metal chelation, molecularly imprinted polymers can be prepared in rather polar solvents.15 In an attempt to develop a detection system for the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), we imprinted this compound in the “classical” way in the presence of nonpolar solvents. We had only limited success with this approach, which was probably due to the carboxyl group being the only potential point of interaction with the functional monomer. We therefore investigated whether specific noncovalent molecular imprints can be obtained in the presence of polar solvents using a combination of the hydrophobic effect and ionic interactions. 2,4-D is a good
(1) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (2) Shea, K. J. Trends Polym. Sci. 1994, 2, 166-173. (3) Vidyasankar, S.; Arnold, F. H. Curr. Opin. Biotechnol. 1995, 6, 218-224. (4) Mosbach, K.; Ramstro¨m, O. Bio/Technology 1996, 14, 163-170. (5) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645-647. (6) Ramstro¨m, O.; Ye, L.; Mosbach, K. Chem. Biol. 1996, 3, 471-477. (7) Fischer, L.; Mu ¨ ller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360. (8) Matsui, J.; Kato, T.; Takeuchi, T.; Suzuki, M.; Yokoyama, K.; Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2223-2224. (9) Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 11791183. (10) Piletsky, S. A.; Piletskaya, E. V.; Elgersma, A. V.; Yano, K.; Karube, I.; Parhometz, Y. P.; El’skaya, A. V. Biosens. Bioelectron. 1995, 10, 959-964. (11) Andersson, L. I. Anal. Chem. 1996, 68, 111-117. (12) Muldoon, M. T.; Stanker, L. H. J. Agric. Food Chem. 1995, 43, 1424-1427. (13) Sellergren, B. Anal. Chem. 1994, 66, 1578-1582.
(14) Matsui, J.; Okada, M.; Tsuruoka, M.; Takeuchi, T. Anal. Commun. 1997, 34, 85-87. (15) Vidyasankar, S.; Ru, M.; Arnold, F. H. J. Chromatogr. A 1997, 775, 51-63.
628 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
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model template for this alternative approach, owing to its hydrophobic aromatic ring and the ionizable carboxyl group. EXPERIMENTAL SECTION Ethyleneglycol dimethacrylate (EDMA) and 4-vinylpyridine (4VP) were from Merck (Darmstadt, Germany). 2,4-Dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenoxybutyric acid (2,4-DB), 2,4-dichlorophenoxyacetic acid methyl ester (2,4-D-OMe), 4-chlorophenoxyacetic acid (CPOAc), 2,4-dichlorophenylacetic acid (DPAc), 4-chlorophenylacetic acid (CPAc), phenoxyacetic acid (POAc), phenoxyethanol (POEtOH), and 2,4-dichlorophenoxyacetic acid-carboxy-14C (14C-2,4-D; specific activity 15.7 mCi/mmol) were from Sigma (St. Louis, MO). 2,2′-Azobis(2,4-dimethylvaleronitrile) (ABDV) was from Wako (Osaka, Japan). All other chemicals were of analytical grade, and solvents were of HPLC quality. Preparation of Polymers. For the imprinted polymer (A), 20 mmol of EDMA, 4 mmol of 4-VP, 1 mmol of 2,4-D (template), and 0.31 mmol of polymerization initiator (ABDV) were weighed into glass test tubes and dissolved in 4 mL of methanol and 1 mL of ultrapure water. The solutions were then sonicated, sparged with nitrogen for 2 min, and placed in a thermostated water bath at 45 °C for 4 h, followed by 2 h at 60 °C. Control polymers were prepared using the same recipe but without the addition of the template (B) or containing 1 mmol of toluene and 1 mmol of acetic acid (C) or 1 mmol of POEtOH (D) instead of the 2,4-D. The resultant hard bulk polymers were ground in a mechanical mortar and wet-sieved in acetone through a 25-µm sieve. The particles were washed by incubation in methanol/acetic acid (7:3) (2×), acetonitrile/acetic acid (9:1) (2×), acetonitrile (1×), and methanol (2×) for 2 h each time, followed by centrifugation. The particles were then resuspended in acetone and allowed to settle for 4 h. The ones that remained in suspension (fines) were collected, and the procedure was repeated four times. The solvent was removed by centrifugation, and the particles were dried in vacuo. In this way, fine particles were obtained with a total yield of ∼60%. These particles had an average diameter of 1 µm (determined by transmission electron microscopy) and were used in all further experiments. The porosity of the particles was determined by nitrogen adsorption/desorption porosimetry on a Micrometrix ASAP 2400M instrument (Atlanta, GA). Radioligand Binding Assays. The polymer particles were suspended in the incubation solvent, and appropriate volumes were added into 1.5-mL polypropylene test tubes, followed by the radioligand 14C-2,4-D (0.26 nmol), varying amounts of a solution of a competing ligand if appropriate, and solvent to give a total volume of 1 mL. The samples were incubated on a rocking table for 2 h. After centrifugation, 700 µL of supernatant was withdrawn and measured by liquid scintillation counting. RESULTS Preparation of the Polymers. The imprinted polymer (A) was prepared by copolymerization of a cross-linking monomer (EDMA) with a functional monomer (4-VP) in the presence of 2,4-D as the template. Given that the goal was to prepare and use the imprinted polymers in water-containing solvents, complex formation had to rely on hydrophobic and ionic interactions which, unlike hydrogen bonding, are not, or are at least to a lesser extent, disturbed in the presence of water. 4-VP was chosen as the
Figure 1. Binding of 14C-2,4-D to polymers A and B as a function of pH. Conditions: 150 µg of polymer/1 mL of assay; 20 mM buffer (pH 3, sodium formate; pH 4 and 5, sodium acetate; pH 6, 7, and 8, sodium phosphate; pH 9, sodium carbonate); 0.1% Triton X-100.
functional monomer to allow for ionic interaction with the carboxyl group of the template as well as for the hydrophobicity of its aromatic ring. The imprinting was performed in methanol/water (4:1) since EDMA and 2,4-D were only poorly soluble in pure water. In contrast to other reports on binding assays with imprinted polymers,5,11,12 in the present work we used fine particles with a diameter of about 1 µm, which are normally discarded. These particles were porous, with a specific surface area of 64 m2 g-1, a total pore volume of 0.25 mL g-1 (determined at a relative pressure of P/P0 ) 0.9926), and an average pore diameter (by BET) of 154 Å. We found that micrometer-sized particles had the same binding characteristics for the target molecule as the 25-µm particles normally used. Furthermore, not only were the incubation times reduced due to shorter diffusion distances, but fines were found to be more practical for binding assays, as they stayed in suspension longer, were easier to pipet, and were easier to remove by centrifugation, as they formed a denser pellet. Rebinding of 14C-2,4-D. Initially, all four polymers were tested for rebinding of 14C-2,4-D in the original imprinting solvent. The imprinted polymer (A) could rebind the radiolabeled template in methanol/water (4:1). Only 200 µg of polymer was needed to adsorb ∼50% of the added radioligand. The control polymers B, C, and D showed only very low binding of the radioligand. As the polymers were intended for use in aqueous buffer, the conditions for aqueous binding assays had to be optimized. In initial binding assays, we found that the addition of small amounts of a nonionic surfactant (0.02-0.1% Triton X-100) increased the wettability of the polymer and prevented adsorption of the hydrophobic analytes on test tubes, pipet tips, etc. but also improved the mixing of the polymer particles in the test tubes. Therefore, we added 0.1% Triton X-100 to all binding assays. Also of importance is the pH of the buffer used. Figure 1 shows binding of 14C-2,4-D to polymers A and B in different buffers from pH 3 to 9. Binding to polymer A was approximately constant between pH 3 and 7 and then decreased with further increase in pH. The local minimum at pH 5 can be attributed to the fact that, at this pH, both the pyridine group and the 2,4-D are partially charged, which renders them less hydrophobic. Binding should now be due mostly to ionic interactions where the buffer ions can more effectively act as competitors. The nonimprinted control Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
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Figure 2. (a) Binding of radioligand relative to polymer concentration for the polymers A-D and (b) radioligand displacement curves with unlabeled 2,4-D as competitor for the polymers A and B, at 150 µg polymer/1 mL assay. Conditions: 20 mM sodium phosphate buffer pH 7; 0.1% Triton X-100.
polymer B showed highest binding at pH 3, which rapidly decreased to remain at a constant low level above pH 6. All subsequent experiments were performed in phosphate buffer at pH 7, where binding to the control polymer was minimized. As can be seen from Figure 2a, polymer A was able to rebind radiolabeled 2,4-D in 20 mM phosphate buffer at pH 7, and 150 µg/mL of polymer was needed to bind 50% of the added radioligand. At this polymer concentration, very low binding to the control polymers was observed. It has to be added that rebinding of the radiolabeled template was only observed with polymers having 4-VP as the functional monomer. Polymers prepared with methacrylic acid did not specifically bind 14C-2,4-D, either in buffer at different pH or in toluene or acetonitrile. Competitive Binding Assays. Figure 2b shows the competition of 14C-2,4-D binding to polymers A and B by unlabeled 2,4-D. A typical sigmoid standard curve similar to those observed in competitive immunoassays was obtained for the imprinted polymer A. The useful concentration range for detection of 2,4-D is from 30 ng/mL (135 nM) to 10 µg/mL (45 µM). Cross-Reactivity Studies. To assess the specificity of the imprinted polymer, competition of 14C-2,4-D binding by structurally related compounds was studied. By comparing the concentration that yields 50% inhibition of 14C-2,4-D binding (IC50 value) for the different competitors to that of 2,4-D, the cross-reactivities of the related compounds can be estimated. The structures of the 630 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
Figure 3. Structures of the different compounds used as competitors in the radioligand binding assays and corresponding crossreactivities. Cross-reactivities were calculated by dividing the IC50 value for 2,4-D by that of the other compounds. IC50 values were obtained from the competition curves by nonlinear regression.
different competitors and the corresponding cross-reactivities are shown in Figure 3. All evaluated compounds exhibited lower binding to the polymer than the original template. The lowest cross-reactivities were obtained with compounds not having a charged group (2,4-D-OMe, POEtOH), whereas 2,4-dichlorophenoxybutyric acid showed almost the same binding as 2,4-D. To assess whether the added surfactant Triton X-100 had an influence on the binding of the different compounds to the polymer, binding was also studied in the same buffer containing 10% ethanol instead of the surfactant. The cross-reactivities obtained were very similar to or identical with those measured in the presence of surfactant. DISCUSSION Molecular imprinting of polymers using only noncovalent interactions between the template and the functional monomers has until now been limited to apolar environments. In some cases, however, the use in aqueous buffer of polymers initially imprinted in nonpolar solvents has been demonstrated to be feasible. It has been suggested that the interaction between the molecule of interest and the polymer is governed by different molecular forces in nonpolar solvents and in aqueous buffer.11 This would mean that the interactions that are predominant in the latter case, mainly hydrophobic interactions and ionic bonds, are strong enough to allow for complex formation in a polar environment. The use of the solvent system methanol/water in our experiments was dictated by the low solubility of the cross-linker and the template
in pure water. We believe, however, that other templates, used in conjunction with water-soluble cross-linkers, may work even better at higher water concentrations. A similar solvent system (methanol/water 95:5) has been successfully employed by another group for imprinting of adenine.16 From a comparison of polymer A (imprinted with 2,4-D) and polymer B (control polymer, no template) in rebinding of the template, it can be concluded that polymer A is clearly templated. To confirm the templating effect, two more control polymers were synthesized where 2,4-D was replaced by 1 molar equiv each of toluene and acetic acid (polymer C) or by POEtOH (polymer D). The fact that neither of these polymers bound 14C-2,4-D indicates that an ionizable group has been positioned close to a hydrophobic pocket in polymer A through templating with 2,4-D. This welldefined juxtaposition of polymer-bound functional group and hydrophobic pocket is much less likely to occur when toluene and acetic acid are used simultaneously as templates (polymer C) or if the template has no carboxyl group (polymer D). Hydrophobic interactions are strong in water and should thus make the biggest contribution to adsorption; however, they are generally nonspecific. Therefore, it was of interest to investigate the specificity of polymer A by use of related compounds as competitors for 14C-2,4-D-binding in radioligand binding assays. The cross-reactivities obtained between the different competitors and 2,4-D are surprisingly low. Moreover, these data provide additional information about the contribution of different forces to the interaction. Comparing 2,4-D and 2,4-D-OMe, it is obvious that canceling out the effect of the negative charge greatly reduces binding (7% cross-reactivity, Figure 3). Removing one (CPOAc) or two (POAc) chlorine atoms from the ring also progressively weakens the affinity (24 and 2% cross-reactivity, respectively), as does the removal of the ether function (DPAc, CPAc). In the case of POEtOH, which has no chlorine and no charged group, binding is completely suppressed. On the other hand, 2,4-DB binds to the polymer nearly as well as the original template. (16) Mathew, J.; Buchardt, O. Bioconjugate Chem. 1995, 6, 524-528. (17) Hall, J. C.; Deschamps, R. J. A.; Krieg, K. K. J. Agric. Food Chem. 1989, 37, 981-984. (18) Dzgoev, A.; Mecklenburg, M.; Larsson, P. O.; Danielsson, B. Anal. Chem. 1996, 68, 3364-3369. (19) Fra´nek, M.; Kola´r, V.; Grana´tova´, M.; Nevora´nkova´, Z. J. Agric. Food Chem. 1994, 42, 1369-1374.
When compared to immunoassays for 2,4-D based on antibodies, the radioligand binding assay using imprinted polymer particles is about as sensitive as indirect ELISA or indirect RIA,17 even though much more sensitive immunoassays have been developed using other detection methods.18 The sensitivity of our assay seems to be limited by the specific activity of the radioligand, rather than by the affinity of the polymer. Concerning the crossreactivities with related compounds, it is higher for the imprinted polymer than for monoclonal antibodies against 2,4-D if the structure of the aromatic ring is altered.19 On the other hand, the polymer shows much less cross-reactivity for the 2,4-D-methyl ester than the antibodies (30-107%), which is not surprising as the polymer was imprinted against free 2,4-D and not against a haptene-carrier conjugate which had to be used to raise the antibodies. CONCLUSION In the present work, a polymer has been imprinted with 2,4-D in aqueous methanol. This showed that molecular imprinting in the presence of polar protic solvents using only noncovalent interactions is possible, depending on the nature of the template molecule and the functional monomers. It was also demonstrated that an appreciable binding specificity for the original template can be obtained. These findings extend the potential applicability of noncovalent molecular imprinting, particularly to cases where either the use of polar solvents and especially water may be required or the target molecule may lack the functionalities required for imprinting in nonpolar solvents. The possible use of this imprinted polymer as the recognition element instead of antibodies in binding assays for 2,4-D was also demonstrated. ACKNOWLEDGMENT The authors thank Dr. Peter Cormack for linguistic advice. Financial support of K.H. by the EU Human Capital and Mobility program is gratefully acknowledged. Received for review August 15, 1997. Accepted November 4, 1997.X AC9711549 X
Abstract published in Advance ACS Abstracts, December 15, 1997.
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