Anal. Chem. 1998, 70, 3936-3939
Herbicide Assay Using an Imprinted Polymer-Based System Analogous to Competitive Fluoroimmunoassays Karsten Haupt,* Andrew G. Mayes,† and Klaus Mosbach
Department of Pure and Applied Biochemistry, Chemical Center, Lund University, P.O. Box 124, S-22100 Lund, Sweden
A fluorescent ligand displacement assay has been developed for the herbicide 2,4-dichlorophenoxyacetic acid based on a molecularly imprinted polymer. The format of the assay is analogous to competitive fluoroimmunoassay formats and uses a coumarin derivative as a nonrelated fluorescent probe. This avoids the use of radiolabels commonly employed in imprinted polymer-based assays and, thus, the handling of radioactive material. Specificity and sensitivity of the assay are on a par with a radioligand displacement assay using the same molecularly imprinted polymer and radiolabeled 2,4-dichlorophenoxyacetic acid. The assay can be used both in aqueous buffer and in organic solvents, the detection limit being about 100 nM. Antibodies are routinely employed as analytical reagents in clinical and research laboratories. One of their most common applications is in immunoassays.1 These are based on the exquisite recognition properties of an antibody for the antigen, such that the antibody can differentiate between a specific antigen and other structurally closely related compounds which bind with lower affinity. The design and synthesis of biomimetic receptor systems capable of binding a target molecule with similar affinity and specificity to antibodies is a long-term goal of bioorganic chemistry. One technique which is being increasingly adopted for the generation of artificial macromolecular receptor systems is molecular imprinting of synthetic polymers.2-5 During the polymerization process, the target molecule functions as a template or imprint molecule. Monomers carrying certain functional groups are arranged specifically around the template and are then “immobilized” by polymerization with a high degree of crosslinking. Subsequent removal of the template leaves cavities complementary in size, shape, and functionality. These highly specific receptor sites are capable of rebinding the target molecule in preference to other closely related structures.
In their present configurations, molecularly imprinted polymers (MIPs) are larger than antibodies, rigid, and insoluble and, therefore, cannot compete with antibodies for use in techniques in which they are used in their soluble form, e.g., immunodiffusion, immunoelectrophoresis, or immunoblotting. However, for techniques based on immobilized antibodies, such as immunoaffinity chromatography, immunoassays, and immunosensors, MIPs appear to offer a potential alternative.3 Indeed, it has been shown on several occasions that molecularly imprinted polymers can be employed instead of antibodies in immunoassay-type binding assays.6-8 The assay format mainly used with imprinted polymers is the radioligand displacement assay. This format is analogous to the first reported solid-phase immunoassay, a competitive radioassay for human growth hormone,9 except that the polymer-bound antibody is replaced by a MIP. However, radioassays involve the handling of radioactive materials, which is often undesirable. Alternative formats for imprinted polymer-based assays could, much like immunoassays, use fluorescence or an enzyme reaction for detection. In the present paper, we report the development of an imprinted polymer-based fluorescent ligand displacement assay. As a model analyte, we have chosen the herbicide and synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), for which we have recently developed a MIP-based radioassay.10 EXPERIMENTAL SECTION 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), 7-carboxymethoxy-4-methylcoumarin (CMMC), and 2,4-dichlorophenoxyacetic acid-carboxy-14C (14C-2,4-D; specific activity 15.7 mCi/ mmol) were from Sigma (St. Louis, MO). All other chemicals were of analytical grade, and solvents were of HPLC quality. Preparation of Polymers. Polymers molecularly imprinted with 2,4-D were prepared as described in a previous publication,10 using 4-vinylpyridine as the functional monomer, ethyleneglycol dimethacrylate as the cross-linker, and methanol/water (4:1) as
* To whom correspondence should be addressed. Fax: 46-46-2224611. E-mail:
[email protected]. † Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom. (1) Hage, D. S. Anal. Chem. 1995, 67, 455R-462R. (2) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (3) Mosbach, K.; Ramstro¨m, O. Bio/Technology 1996, 14, 163-170. (4) Shea, K. J. Trends Polym. Sci. 1994, 2, 166-173. (5) Vidyasankar, S.; Arnold, F. H. Curr. Opin. Biotechnol. 1995, 6, 218-224.
(6) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645-647. (7) Muldoon, M.; Stanker, L. J. Agric. Food Chem. 1995, 43, 1424-1427. (8) Andersson, L. I. Anal. Chem. 1996, 68, 111-117. (9) Catt, K.; Niall, H. D.; Tregear, G. W. Nature 1967, 25, 825-827. (10) Haupt, K.; Dzgoev, A.; Mosbach, K. Anal. Chem. 1998, 70, 628-631.
3936 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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© 1998 American Chemical Society Published on Web 08/20/1998
the porogen. A control polymer was prepared using the same recipe but without the addition of the template 2,4-D. The bulk polymers were ground, sieved, and sedimented to obtain fine particles with an average diameter of 1 µm which were used in all experiments. Ligand Displacement Assays. The polymer particles were suspended in the incubation solvent, and appropriate volumes were added to 1.5-mL polypropylene test tubes, followed by the radioligand 14C-2,4-D (0.26 nmol) or a fluorescent ligand (CMMC; 0.64 nmol), varying amounts of a solution of a competing ligand if appropriate, and solvent to a total volume of 1 mL. The samples were incubated on a rocking table for 2 h, followed by a centrifugation step to sediment the particles. For radioligand displacement assays, radioactivity in the supernatant was measured by liquid scintillation counting as described previously.10 For fluorescent ligand displacement assays, the fluorescence intensity in the supernatant was measured at excitation and emission wavelengths of 323 and 385 nm, respectively. When the assay was performed in acetonitrile, the supernatants were diluted 1:1 with 25 mM phosphate buffer, pH 7, prior to the fluorescence measurements. RESULTS AND DISCUSSION Preparation of the Imprinted Polymer. The imprinted polymer particles used in the present work were similar to the ones used in a previously reported radioligand displacement assay for 2,4-D.10 The particles were porous and had an average diameter of 1 µm. The imprinted polymer was prepared in the presence of the polar protic solvents methanol and water, which is rather unusual for noncovalently imprinted polymers. 4-Vinylpyridine was used as the functional monomer. A good selectivity of the polymer for 2,4-D over closely related compounds was demonstrated. Fluorescence Assay. Fluorescence immunoassays are often based on a fluorescence-labeled antigen which can bind to the antibody.11 Unlabeled antigen present in the solution competes with the labeled antigen for the binding sites of the antibody, allowing a calibration curve to be recorded and the concentration of antigen in an unknown sample to be determined. A fluorescence immunoassay developed for 2,4-D uses fluorescein as a label, which is bound to the carboxyl group of 2,4-D via a diamine spacer.12 With imprinted polymers, one could imagine several possible approaches to a competitive fluorescence assay: (i) The polymer is imprinted with the target analyte, and for detection a fluorescencelabeled derivative of the analyte is used. (ii) The polymer is imprinted with the target analyte, whereas for detection a nonrelated probe which can bind to the polymer is used. (iii) The polymer is imprinted with the fluorescence-labeled or otherwise derivatized analyte, and upon analysis the unlabeled analyte competes with the labeled analyte for the binding sites in the polymer. The first two approaches have the advantage that, since the target analyte was used as the template, the assay should have the highest selectivity for the analyte that can be obtained with the imprinting protocol used. On the other hand, there is the potential problem that, taking into account the high selectivi(11) Hemmila¨, I. Clin. Chem. 1985, 31, 359-370. (12) Lunskaya, I. M.; Eremin, S. A.; Egorov, A. M.; Kolar, V.; Franek, M. Agrokhimiya 1993, 2, 113-118 (in Russian).
ties usually observed with imprinted polymers, the labeled analyte or the nonrelated probe might not bind to the imprinted sites in the polymer. The third approach avoids this potential shortcoming, but this might, in some cases, be at the expense of selectivity, since the template was structurally different from the target analyte. Other assays could use direct formats, where either the analyte is fluorescent itself or the polymer may contain fluorescent reporter groups, the fluorescence of which is altered upon analyte binding. Both formats have been suggested for sensor applications.13,14 Aiming to develop a detection system analogous to competitive fluoroimmunoassays, we initially attempted to use 2,4-D labeled with a fluorescent moiety. Fluorescein isothiocyanate was coupled to the carboxyl group of 2,4-D via ethylenediamine or 1,6-diaminohexane spacers. However, no specific binding of the labeled analyte to the 2,4-D-imprinted polymer was observed. Even though under certain conditions the imprinted polymer did bind the fluorescence-labeled analyte, competition by unlabeled 2,4-D did not take place. The observed binding could, therefore, only be attributed to nonspecific interactions of the fluorescein moiety with the polymer. Thus, it was not possible to use fluorescein labeling in our system, although others have reported success with the same or a similar labeling strategy.15,16 We have opted for a different approach, using a nonrelated fluorescent probe which itself may bind to the imprinted polymer, but which can subsequently be displaced upon specific analyte binding. Coumarin derivatives seemed to be promising candidates due to their small size. After preliminary binding assays, 7-carboxymethoxy-4-methylcoumarin (CMMC, 1) was chosen. Due to
its phenoxyacetic acid moiety, it slightly resembles 2,4-D (2) and could be expected to bind to the polymer to some extent. The fluorescence excitation and emission maxima of CMMC are at 323 and 385 nm, respectively, under the conditions used in our experiments. Figure 1 shows a titration experiment comparing 14C-2,4-D and CMMC binding to the molecularly imprinted polymer, both in aqueous buffer and in acetonitrile. The same buffer as in the radioligand binding assay10 was used in the fluorescence assay, to ensure optimal binding of 2,4-D. As can be seen, the bound fraction of both ligands increases with polymer concentration in both solvents. In aqueous buffer, 500 µg of polymer is needed to bind 50% of the added CMMC, as compared to 100 µg for 14C2,4-D. At the same time, only very low binding to the nonimprinted control polymer was observed. In acetonitrile, 230 and (13) Kriz, D.; Ramstro¨m, O.; Svensson, A.; Mosbach, K. Anal. Chem. 1995, 67, 2142-2144. (14) Cooper, M. E.; Hoag, B. P.; Gin, D. L. Polym. Preprints 1997, 38, 209-210. (15) Piletsky, S. A.; Piletskaya, E. V.; Levi, R.; Yano, K.; Karube, I. Anal. Lett. 1997, 30, 445-455. (16) Levi, R.; McNiven, S.; Piletsky, S. A.; Cheong, S. H.; Yano, K.; Karube, I. Anal. Chem. 1997, 69, 2017-2021.
Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Figure 1. Binding of 14C-2,4-D (squares) and CMMC (circles) to the imprinted (filled symbols) and control polymers (open symbols) in (a) 20 mM phosphate buffer, pH 7, 0.1% Triton X-100 and (b) acetonitrile.
200 µg of polymer bound 50% of the added 14C-2,4-D and CMMC, respectively. Here, at concentrations >500 µg/mL, the control polymer bound a significant amount of both ligands, though still much less than the imprinted polymer. To verify whether the CMMC binding to the polymer occurs in the imprinted sites, a competitive binding experiment was done. Figure 2 shows the competition of unlabeled 2,4-D and CMMC with 14C-2,4-D for binding to the imprinted polymer. The results clearly show that CMMC competes with 14C-2,4-D, although binding is much weaker as compared to that of unlabeled 2,4-D. The binding of CMMC to the polymer in buffer was determined to be 6% as compared to that of 2,4-D. This is due to the poor resemblance between the two compounds and explains the higher amount of polymer needed to bind 50% of the added CMMC in the titration experiment. In acetonitrile, the difference in binding between 2,4-D and CMMC is smaller, which again is in concordance with the titration experiment. Taking into account that neither 2,4-D nor CMMC binds to the control polymer at the polymer concentrations used in our assays, it can safely be assumed that binding occurs in the imprinted sites. Thus, CMMC should be useful as a fluorescent probe in a competitive assay for 2,4-D. 3938 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
Figure 2. 14C-2,4-D displacement by 2,4-D (9) and CMMC (b) in (a) 20 mM phosphate buffer, pH 7, 0.1% Triton X-100 and (b) acetonitrile.
Standard Curve for 2,4-D and Cross-Reactivities. Figure 3 shows the competition by 2,4-D of CMMC binding to the imprinted polymer in buffer as well as in acetonitrile. Typical sigmoid standard curves similar to those observed in competitive immunoassays were obtained. The useful concentration range for detection of 2,4-D is from 100 nM to 50 µM and from 100 nM to 10 µM in buffer and acetonitrile, respectively. The standard curve for the fluorescence assay in buffer was similar to the one obtained with the radioassay reported earlier10 with the detection limit in the same range. To evaluate the specificity of the imprinted polymer, binding to the imprinted polymer of several compounds with related structures was studied (Table 1, Figure 3a). In buffer, the crossreactivities measured were very similar to the ones obtained in the radioassay described earlier.10 With the exception of 2,4-DB, 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). In the case of POEtOH, which has no chlorine substituents and no charged group, binding is completely suppressed. 2,4-DB binds to the polymer nearly as well as the original template. In acetonitrile, the situation is somewhat different (Table 1, Figure 3b). The cross-reactivities obtained for CPOAc
Table 1. Cross-Reactivities with Respect to 2,4-D for Different Related Compounds Binding to the 2,4-D-Imprinted Polymer cross-reactivity with 2,4-D (%) radioligand displacement fluorescence fluorescence assay in assay in assay in monoclonal compound buffera buffer acetonitrile ELISAb 2,4-D 2,4-DB 2,4-D-OMe CPOAc POAc DPAc CPAc POEtOH a
Figure 3. Standard curves for 2,4-D. Competition of CMMC binding to the imprinted polymer by 2,4-D (9), CPOAC (b), and POAc (2) in (a) 20 mM phosphate buffer, pH 7, 0.1% Triton X-100 and (b) acetonitrile.
and POAc are slightly higher, whereas all other compounds have lower cross-reactivities than in buffer. Surprisingly, 2,4-DB shows much lower cross-reactivity with 2,4-D in acetonitrile than in buffer. It seems that, in the organic solvent, the distance between the aromatic ring and the carboxyl group of the analyte is more important for recognition, whereas changes in the ring structure of the analyte affect the binding to a lesser extent than in buffer. A possible explanation could be a change in swelling and flexibility of the polymer in acetonitrile as compared to those in aqueous buffer, as well as a different balance of the interactions involved in the binding. Different specificities in organic and aqueous solvents of an imprinted polymer have been reported earlier.8 When compared to immunoassays for 2,4-D, our assay using imprinted polymer particles is on a par with or better than indirect (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) Lukin, Y. V.; Dokuchaev, I. M.; Polyak, I. M.; Eremin, S. A. Anal. Lett. 1994, 27, 2973-2982. (20) Fra´nek, M.; Kola´r, V.; Grana´tova´, M.; Nevora´nkova´, Z. J. Agric. Food Chem. 1994, 42, 1369-1374.
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