Combinatorial Molecular Imprinting: An ... - American Chemical Society

AC Research

Anal. Chem. 1999, 71, 285-290

Accelerated Articles

Combinatorial Molecular Imprinting: An Approach to Synthetic Polymer Receptors Toshifumi Takeuchi,* Daigo Fukuma, and Jun Matsui

Laboratory of Synthetic Biochemistry, Faculty of Information Sciences, Hiroshima City University, 3-4-1 Ozuka-higashi, Asaminami-ku, Hiroshima 731-3194, Japan

A novel method for synthesizing and evaluating artificial receptors is demonstrated, combining a molecular imprinting concept and a combinatorial chemistry strategy. Combinatorial libraries of molecularly imprinted polymers (MIPs) were prepared and screened for high affinity and selectivity to the original template by a newly developed semiautomatic system. The preparation of MIPs was automatically performed using programmed liquid-handling equipment with a new in situ molecular imprinting protocol whereby MIP is prepared on the bottom surface of each glass vial, followed by an automated discrete binding test with the programmed liquid handler. MIPs as artificial receptors for triazine herbicides, ametryn and atrazine, were prepared by the combinatorial molecular imprinting using a diverse amount of two functional monomers, methacrylic acid (MAA) and 2-(trifluoromethyl)acrylic acid (TFMAA). Examining the MIP libraries, it appears that, depending upon the functional monomer used, the imprinting efficiency is different for each triazine herbicide; MAA is preferred for the atrazine receptor preparation and TFMAA for the ametryn receptor preparation. The results suggest that the proposed highthroughput combinatorial molecularly imprinting technique is a promising method for finding optimal conditions of MIP preparation for given molecules.

Design and synthesis of artificial receptor molecules have been a focal research area for understanding the molecular recognition phenomena in biological systems and for developing novel materials mimicking biological functions usable in analytical applications. Molecular imprinting1-4 is recognized as a powerful * Corresponding author: (phone) +81-82-8301603; (fax) +81-82-8301610; (email) [email protected]. (1) Takeuchi, T.; Matsui, J. Acta Polym. 1996, 47, 471-480. 10.1021/ac980858v CCC: $18.00 Published on Web 12/11/1998

© 1999 American Chemical Society

technique to synthesize polymer-type artificial receptors. In this technique, functional monomers and cross-linkers are polymerized in the presence of a template molecule, which is followed by the template removal from the resultant polymer network to leave a template-fitted cavity. The functional monomers used are expected to be laid out in the cavity as complementary to the chemical functionality of the template molecule, because the functional monomers are bound with the template molecule during the polymerization. Consequently, resultant polymers exhibit the template-selective binding capacity. Intuitively, the most critical point in the molecular imprinting concept is, especially when the functional monomers are bound to a template by noncovalent bonding, whether functional monomers form a steady complex with a template molecule. Therefore, the selection of appropriate functional monomers and the determination of their stoichiometry applied are most important in the design of a molecularly imprinting system for given target molecules. To date, one of successful molecular imprinting protocols has employed bulk polymerization to obtain glassy polymer blocks to be used as powder after being crushed, ground, and sieved. Due to these tedious and time-consuming experimental steps, it normally takes several days to complete the whole procedure to prepare and evaluate molecularly imprinted polymers (MIPs), though a polymer preparation itself is simple and prompt. Therefore, it has not been easy to find an optimal functional monomer system by examining a number of MIPs prepared under different conditions. Accordingly, a method involving a combinatorial chemistry-based approach5,6 has been desired, which can readily perform the preparation and evaluation of MIPs, to establish an optimal functional monomer system in a short time. (2) Ansell, R. J.; Kriz, D.; Mosbach, K. Curr. Opin. Biotechnol. 1996, 7, 8994. (3) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (4) Shea, K. J. Trends Polym. Sci. 1994, 2, 166-173. (5) Czarnik, A. W. Anal. Chem. 1998, 70, 378A-386A. (6) Kyranos, J. N.; Hogan, J. C., Jr. Anal. Chem. 1998, 70, 389A-395A.

Analytical Chemistry, Vol. 71, No. 2, January 15, 1999 285

Table 1. Members of the Ametryn-Imprinted Polymer Librarya MAA (equiv)

0

1

2

TFMAA (equiv) 3

4

5

6

0 1 2 3 4 5 6

P(AME00) P(AME10) P(AME20) P(AME30) P(AME40) P(AME50) P(AME60)







a Amount of each functional monomer added in the polymerization mixture: 0, no corresponding functional monomer; 1, 1.95 µmol; 2, 3.90 µmol; 3, 5.85 µmol; 4, 7.80 µmol; 5, 9.75 µmol; 6, 11.7 µmol. ATZ, is used for the atrazine-imprinted polymers instead of AME for the ametrynimprinted polymers.

We have recently reported in situ molecular imprinting,7,8 where polymerization is directly carried out inside a stainless steel tube and the resultant column filled with an imprinted polymer rod can be immediately used as affinity media for liquid chromatography (LC) without the tedious and time-consuming experimental steps. Although the technique has simplified the whole imprinting procedure, the polymer rods can be applied only for LC. Since the higher affinity polymers result in longer analysis time, the technique is not very adequate for quick evaluation of MIPs. The study presented in this paper is focused upon the establishment of a rapid and facile method for preparing and screening a library of various imprinted polymers as artificial receptors. A new in situ molecular imprinting protocol is engaged for introducing an automated procedure using programmable liquid-handling equipment, where molecularly imprinted polymers are prepared on the bottom surface of glass vials and are directly assessed by discrete binding tests. To prove the feasibility of this method, combinatorial libraries of the polymers imprinted against a triazine herbicide, ametryn or atrazine, are prepared, using a diverse amount of two functional monomers, methacrylic acid (MAA) and 2-(trifluoromethyl)acrylic acid (TFMAA). EXPERIMENTAL SECTION Materials. Ametryn, MAA, methyl methacrylate (MMA), and ethylene glycol dimethacrylate were purchased from Wako Pure Chemical Industries (Osaka, Japan), and TFMAA was obtained from Tokyo Chemical Industry (Tokyo, Japan). Chloroform and acetonitrile were from Katayama Chemical (Osaka, Japan). Atrazine was kindly donated by Nissan Chemical Industries, Ltd. (Tokyo, Japan). Chloroform, MAA, MMA, and ethylene glycol dimethacrylate were purified by distillation prior to use. HPLC Analysis. Quantification of triazine herbicides in acetonitrile was performed with a HPLC system consisting of a HPLC pump (Hitachi, L-6200), a degasser (Hitachi, L-5090), a UV detector (Hitachi, L-3000), an automatic sample injector (Gilson, 232XL), a controller-data processor Unipoint (Gilson), and a reversed-phase column, Supelco LC-8-DB (4.6 mm × 150 mm, i.d.). Acetonitrile-water (7:3, v/v) was used as the eluent at a flow rate of 1.0 mL min-1. The herbicides were detected by UV (7) Matsui, J.; Miyoshi, Y.; Matsui, R.; Takeuchi, T. Anal. Sci. 1995, 11, 10171019. (8) Matsui, J.; Kato. T.; Takeuchi, T.; Suzuki, M.; Yokoyama K.; Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2223-2224.

286

Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

absorption at 263 nm. The sample volume was 20 µL. Quantification of triazine herbicides in chloroform was carried out by a flow injection system using chloroform as the carrier at the flow rate of 1.0 mL min-1. Semiautomated Polymer Preparation. Glass vials (1.5 mL) were placed in a polyethylene bag for 15 min under the stream of nitrogen gas and were sealed with silicon caps. Reagents for the molecular imprinting, ametryn or atrazine as a template (1.95 µmol), MAA and/or TFMAA as the functional monomer (Table 1), ethylene glycol dimethacrylate as a cross-linker (30.0 mg), 2,2′azobisisobutyronitrile (747 µg) as a polymerization initiator, and chloroform as a pore former were dispensed into the vials by a programmed liquid handler, model 232XL (Gilson). All the reagents, except ethylene glycol dimethacrylate, were dissolved in chloroform for the dispensation. Chloroform in total was 59.0 µL in each vial. After being incubated with nitrogen gas, the glass vials with the prepolymerization mixtures were placed under the long-wave UV irradiation (UVP, XX-15L) for 12 h in a thermostatic water bath at 5 °C (Taitec, CP-80F). Time Course of Ametryn Desorption. Acetonitrile (1.5 mL) was dispensed into a glass vial of P(AME60) (see Table 1) with a magnetic stirring bar. The vial was placed in the automatic injector and incubated with continuous stirring. Injection of the supernatant was automatically carried out at appropriate intervals. Instant First Screening. After the polymer preparation protocol, acetonitrile (1.5 mL) was dispensed into each vial. After incubation for 24 h with continuous rotation (Taitec, RT-50), the supernatants were analyzed by the reversed-phase HPLC to quantify the free template species. Regular Screening. Into the glass vials, 1.5 mL of a washing solvent, methanol-acetic acid-water (7:1:2, v/v/v), was dispensed and removed after incubation for 2 h. This washing procedure was repeated 10 times, and additional washing was carried out 3 times with chloroform. The polymers were incubated with 1.5 mL of ametryn solution (500 µM) for 24 h. After the incubation, the supernatants were analyzed by the flow injection system to quantify the concentration of free ametryn. Amounts of ametryn bound were obtained by subtracting free ametryn from the initial amount. Average data of three sets of the MIP libraries were plotted in Figure 5 and Figure 6. Selectivity tests were also performed in the identical fashion.

Figure 1. Illustration of the high-throughput preparation and screening of the molecularly imprinted polymers by the batch-type in situ protocol using liquid-handling equipment.

Figure 2. Time course experiment to observe the desorption of the template ametryn from the MIP obtained by the batch-type in situ preparation.

RESULTS AND DISCUSSION New in Situ Molecular Imprinting. A batch-type in situ molecular imprinting was newly adopted in this study for enabling a high-throughput polymerization and evaluation of molecularly imprinted polymers performed by a programmed liquid-handling equipment (Figure 1). This protocol consists of (1) automatic dispense of the reagents into a glass vial with silicon sealing, (2) polymerization under long-wave UV to leave a thinly coated polymer on the bottom inner surface of the vial, and (3) automatic washing by repeated dispensing and removal of the washing solvent without polymer grinding or sieving.

Two classes of assessments of the MIP library members are carried out, namely, “instant first screening” and “regular screening”. The instant first screening is performed prior to the washing step 3 in order to roughly estimate affinity of the template to the resultant polymers by quantifying the template bound when the polymer is incubated in acetonitrile. Regular screenings are carried out, after completion of the washing, to make more precise evaluation of the affinities and selectivities of the MIP library members, which consists of three steps: conditioning, substrate dispensing, and quantification of the substrate bound. Both assessments employ the programmed liquid handler as a sample dispenser and a HPLC/FIA sample injector. Thus, all the procedures except the UV irradiation step are automated by the “combinatorial molecular imprinting robot”. As a preliminary examination of the new in situ protocol, a time course experiment was carried out to ensure that the template can be released from the thinly coated polymers and to estimate how long it takes for the equilibrium to be established. The concentration of free template in the supernatant was traced as a function of time when a prepared polymer is incubated with acetonitrile. As shown in Figure 2, the desorption of ametryn was observed, and it took ∼20 h for the equilibrium to be reached in the ametryn extraction, while the time to establish equilibrium was several minutes in the previous work using polymer powders.9 The diffusion process would be rate-controlling because the thinly coated polymers are directly used without grinding. According to these results, conditions for the washing step 3 and the (9) Matsui, J.; Miyoshi, Y.; Doblhoff-Dier, O.; Takeuchi, T. Anal. Chem. 1995, 67, 4404-4408.


287

Figure 3. Structures of the template molecules and the functional monomers: 1, MAA; 2, TFMAA; 3, ametryn; 4, atrazine.

incubation time were determined; the solvent was replaced every 2 h in the washing step and the incubation was conducted for 24 h. Molecularly Imprinted Polymer Library. In molecular imprinting, the complementary intermolecular interactions between a template molecule and functional monomers are important for precise molecular recognition. Therefore, a large number of functional monomers have been studied for various template molecules. While some functional monomers are tailored for a specific template species,3,10,11 a methacrylic acid family has demonstrated a general utility for a wide range of compounds.2,12,13 These “widely applicable monomers” are appealing because they have simple structures and plainly illustrate the characteristic feature of molecular imprinting; i.e., plural functional monomers are arranged to work cooperatively to attain a highly specific recognition of the given template while each monomer provides only weak and nonspecific interactions. The generality, however, makes it difficult to select one among the widely applicable monomers suitable for a given template. Usually the selection of functional monomers needs time-consuming trial and error. Therefore, for the development of highperformance imprinting polymers, a high-throughput polymer preparation and evaluation system such as the robot system we proposed here should be required. To demonstrate the effectiveness of our high-throughput system, the suitability of two widely applicable functional monomers is examined on the triazine herbicide imprinting. Using MAA (1) and a methacrylic acid-analogue having stronger acidity (TFMAA, 2) as the functional monomers, MIP libraries for ametryn (3) and atrazine (4) were prepared (see Figure 3). Each library consists of 49 members of MIPs prepared with various stoichiometries of the two functional monomers (Table 1). MMA having no significant hydrogen bond interaction was added to adjust the molar ratio, cross-linker/monomers, to be constant, avoiding diverse rigidity of the resultant polymer network. Factors concerning cross-link and solvents were fixed in this study, though they would also affect the performance of the resultant polymers.14-16 (10) Tanabe, K.; Takeuchi, T.; Matsui, J.; Yano, K.; Ikebukuro, K.; Karube, I. J. Chem. Soc., Chem. Commun. 1995, 2303-2304. (11) Yano, K, Nakagiri, T., Takeuchi, T., Matsui, J., Ikebukuro, K., Karube, I. Anal. Chim. Acta 1997, 357, 91-98. (12) Matsui, J.; Miyoshi, Y.; Takeuchi, T. Chem. Lett. 1995, 1007-1008. (13) Fischer, L.; Mu ¨ ller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360.

288 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

Figure 4. Instant screening of the ametryn-imprinted polymer library.

Instant First Screening. The instant first screening was examined using the ametryn-imprinted polymer library, where the MIPs were incubated in acetonitrile without any washing step after the polymerization, and the concentration of free template released to the supernatant was measured to quantify the ametryn remaining in the polymer. This method is able to make a rough estimation of the affinity to the template of each library member, because the initial amount of the template added is identical in each vial and the amount bound can be calculated by subtracting free ametryn from the initial amount (Figure 4). A trend is observed that TFMAA-rich library members exhibit higher affinity to ametryn. The results show that TFMAA works well with the substrate ametryn, suggesting that TFMAA is suitable as the functional monomer for imprinting ametryn. MAA also showed a slightly positive effect on the binding ability of the library members; the members of 6 equiv of MAA, P(AME60)P(AME66), showed higher ametryn adsorption over the members of no MAA, P(AME00)-P(AME06). At this stage, we are not sure whether MAA is suitable for ametryn imprinting, however, it is clear that the apparent affinity of MAA-rich members is lower than that of TFMAA-rich members under the conditions employed. The reproducibility for preparation and screening was investigated. The results from 10 batches of P(AME60) prepared and evaluated by the same procedure showed the coefficient of variation as 7.4% (n ) 10). Thus, the first screening using this method appeared to be possible for making a rough selection of the affinity-rich polymers. Currently, HPLC analysis is required to quantify the free template in this screening because the supernatant contained some impurities of starting materials. Although this reduces the utility of this screening method due to the long analysis time, this instant screening could still have merit because the liquid handler can also work as an autosampling injector and is connected with the HPLC system, making the assays automatic. A faster analysis protocol using fluorescenceor chromophore-labeled template analogues, which can be analyzed without interference by the impurities, is currently being investigated. (14) Sellergren, B.; Shea, K. J. Chromatogr. 1993, 635, 31-49. (15) Yoshizako, K.; Hosoya, K.; Iwakoshi, Y.; Kimata, K.; Tanaka, N. Anal. Chem. 1998, 70, 386-389. (16) Matsui, J.; Kubo, H.; Takeuchi, T. Anal. Sci. 1998, 14, 699-702.

Figure 5. Regular screening of the ametryn-imprinted polymer library, P(AMExy): (a) binding of the original template ametryn; (b) binding of the reference triazine herbicide atrazine; (c) selectivity factors.

Figure 6. Regular screening of the atrazine-imprinted polymer library, P(ATZxy): (a) binding of the original template atrazine; (b) binding of the reference triazine herbicide ametryn; (c) selectivity factors.

Regular Screening. For detailed assessment of the MIP libraries, a regular screening was conducted whereby polymers were exhaustively washed and incubated with chloroform solution containing the original template or a reference triazine herbicide as a substrate. The initial substrate solution (500 µM, 1.5 mL) contains 750 nmol of the substrate, which corresponds to 38.5% of the theoretical number of maximum total binding sites that was

calculated as the amount of template used for each polymer preparation (1.95 µmol). Figure 5a shows the amounts of ametryn adsorbed to the ametryn-imprinted polymers. The trend showed no significant differences, as compared to those in the instant screening binding tests; more use of TFMAA leads to the polymers exhibiting stronger binding capacity to ametryn. The difference in the apparent amount of ametryn bound between the two screening Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

289

systems would be due to the differences in the solvents used and the total amount of ametryn in a vial: 1.95 µmol in the instant first screening and 750 nmol in the regular screening. In the regular screening, MAA-based members gave positive results and MAA also appeared to work as the functional monomer; in contrast, clear results have not been obtained in the first screening. This discrepancy could be due to the solvent used for the assay; acetonitrile was used in the instant first screening, while chloroform was used for the regular screening. It may be better to use a less polar solvent for evaluating hydrogen bondingbased affinity. However, we made a compromise with the use of acetonitrile since it can be directly injected to the reversed-phase LC employed and the trend of binding behavior could be roughly estimated though acetonitrile may interfere in the hydrogen bond formation. Selectivity of the ametryn-imprinted polymers was evaluated by examining the binding behavior of atrazine as a reference substrate. As shown in Figure 5b, the amounts of atrazine bound were significantly smaller than those of ametryn, showing that the ametryn-selective binding was induced in the ametrynimprinted polymers. To identify the best selective member in the library, a selectivity factor is defined as the relative amount of the original template ametryn bound versus that of atrazine. As shown in Figure 5c, more use of TFMAA resulted in better selectivity factors while MAA make little contribution for developing ametryn selectivity. Finally, a conclusion can be drawn that TFMAA is a more effective functional monomer over MAA to develop the ametryn-receptor MIPs. Despite the structural analogy between ametryn and atrazine, the atrazine-imprinted polymer library showed a dissimilar profile in the preference of the functional monomers (Figure 6a). When no MAA is engaged in the polymer preparation, TFMAA seems effective for developing the affinity to atrazine. However, less effectiveness of TFMAA can be observed among the members prepared with a sufficient amount of MAA, for instance, P(ATZ60)-P(ATZ66). Furthermore, the use of TFMAA reduced the selectivity to atrazine (Figure 6c), when the selectivity was evaluated by the selectivity factors, ratios of amount of atrazine bound (Figure 6a) to that of ametryn (Figure 6b). It could be due to the inherent strong interaction between TFMAA and ametryn, which may have caused the strong nonspecific binding of ametryn by the atrazine-imprinted polymers. Accordingly, MAA appeared to be the better functional monomer to imprint atrazine, especially when the receptors are required to be selective to atrazine over ametryn. The trends observed in this study agree with that obtained by our previous study9,13 employing the conventional approach where a block polymer is ground, sieved, and evaluated in LC mode. Ametryn bears a SCH3 group while atrazine possesses a Cl group, as shown in Figure 3. The two triazine herbicides show different basicity due to the substituents, governing the fashion of the interaction between the triazine herbicide and the functional monomers.17 This is a plausible reason that they prefer a different

functional monomer. As can be seen, it is, however, still difficult at the current stage to predict what functional monomer is suitable for imprinting a given template molecule without experimental work, suggesting the necessity of high-throughput combinatorial chemistry-based approaches for obtaining high-performance MIP receptors.

(17) Dauwe, C.; Sellergren, B. J. Chromatogr., A 1996, 753, 191-200.

AC980858V

290


CONCLUSIONS In this study, a semiautomated system for the preparation and screening of MIP receptors has been established using programmed liquid-handling equipment. Two systems, instant first screening and regular screening, performed well, as we expected; the regular screening provided detailed information whereas the instant first screening gave us preliminary results. These two screenings can be performed successively and we can decide whether the following regular screening should be continuously performed after the evaluation of the first screening. Only the primal screening tests are addressed in this paper; however, further screening can be easily conducted, e.g., saturation binding tests and more detailed selectivity examinations. Although two functional monomers were examined in this study, a molecular imprinting system, which consists of plural functional monomers, would be also interesting to investigate. There are also other agents to be examined, such as solvents and cross-linkers, which have significant influence on the performance of the resultant polymers. Furthermore, polymerization temperature would also make a difference in recognition ability. The effects of these agents and conditions employed have come to be understood to some degree. Nevertheless, it is still hard to predict the best recipe for imprinting a given template molecule. Therefore, the current best way for acquiring high-performance molecularly imprinted receptors is to examine thousands of various polymers prepared with different combinations and amounts of the agents under different polymerization conditions. The new in situ molecular imprinting can provide an extremely useful tool, employing the programmed liquid-handling equipment, for the preparation and evaluation of a number of different MIPs in a short time. The combinatorial molecular imprinting with the streamlined procedure presented here is currently employed to seek for new MIP receptors in this laboratory. Because the proposed technique would be applicable for other functional polymer preparations, it could open a new strategy in the field of polymer science. ACKNOWLEDGMENT The work is supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for Encouragement of Young Scientists from The Ministry of Education, Science, Sports and Culture, Japan. The authors also thank The 1st Toyota High-tech Research Grant Program for financial support. Received for review August 3, 1998. Accepted November 3, 1998.

Combinatorial Molecular Imprinting: An ... - American Chemical Society

Recommend Documents