Molecularly Imprinted Fluorescent-Shift Receptors Prepared with 2

Fluorescent spectra of the cinchona alkaloid exhibit a characteristic shift through binding to these polymer particles, originating most likely from t...
0 downloads 0 Views 71KB Size
Download PDF
Anal. Chem. 2000, 72, 3286-3290

Molecularly Imprinted Fluorescent-Shift Receptors Prepared with 2-(Trifluoromethyl)acrylic Acid Jun Matsui, Hiroyuki Kubo, and Toshifumi Takeuchi*

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

Methacrylic acid (MAA) and 2-(trifluoromethyl)acrylic acid (TFMAA) were used to prepare molecularly imprinted polymers exhibiting diastereoselectivity for cinchona alkaloids. Fluorescent spectra of the cinchona alkaloid exhibit a characteristic shift through binding to these polymer particles, originating most likely from the highly acidic residues in the imprinted polymers acting as a proton donator. Our results show that TFMAA based imprinted polymers can be used as polymer reagents for quantitating the cinchona alkaloid bound to the polymers without bound/free separation. Molecular imprinting has been known as a useful technique for preparing materials capable of molecular recognition. Most imprinted products are polymers that can be synthesized by crosslinking complexes of template molecules and functional monomers.1,2 Removal of the template molecule from the polymers results in binding sites formed by functional monomer-derived residues complementary to the template molecule. The molecularly imprinted polymer acts as an artificial receptor/antibody exhibiting tailor-made selectivity for the template molecule. A great number of molecularly imprinted polymers have been synthesized, and their molecular recognition properties have been utilized in a variety of separation and sensing techniques.3 Although imprinted polymers have drawn attention as chromatographic stationary phases for decades, they have recently been recognized as polymer reagents substitutive to antibodies and receptors for binding assays.4-7 In these applications, polymers are designed not only as recognition elements for particular molecules, i.e., substrates, but also as signal-generating elements, e.g., using fluorescent functional monomers to obtain imprinted polymers, displaying substrate-concentration-dependent fluorescence.8-10 * To whom correspondence should be addressed. Phone: +81-82-830-1603. Fax: +81-82-830-1610. E-mail: [email protected] (1) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (2) Sellergren, B. In Molecular and Ionic Recognition with Imprinted Polymers; Bartsch, R. A., Maeda, M., Eds.; American Chemical Society: Washington, DC, 1998; pp 49-80. (3) Takeuchi, T.; Haginaka, J. J. Chromatogr., B 1999, 728, 1-20. (4) Haupt, K.; Mosbach, K. Trends Biotechnol. 1998, 16, 468-475. (5) Andersson, L. I.; Nicholls, I. A.; Mosbach, K. In Immunoanalysis of Agrochemicals; Nelson, J. O, Karu, A. E. Wong, R. B., Eds.; American Chemical Society: Washington, DC, 1995; pp 89-96. (6) Haupt, K. React. Funct. Polym. 1999, 41, 125-131. (7) Takeuchi, T.; Dobashi, A.; Kimura, K. Anal. Chem. 2000, 72, 2418-2422. (8) Turkewitsch, P.; Wandelt, B.; Darling, G. D.; Powell, W. S. Anal. Chem. 1998, 70, 2025-2030. (9) Jenkins, A. L.; Uy, O. M.; Murray, G. M. Anal. Chem. 1999, 71, 373-378.

3286 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

Chart 1. Cinchona alkaloids imprinted. 1: (-)-cinchonidine. 2: (+)-cinchonine.

Other examples of reading out of direct substrate uptake by imprinted polymers have also been reported.11,12 We have previously reported on the effectiveness of a strongacid functional monomer, 2-(trifluoromethyl)acrylic acid (TFMAA), for developing high affinity and selectivity in molecular imprinting of Bro¨nsted base template molecules such as triazine herbicides,13 nicotine,14 and cinchona alkaloids.15 In this paper, we report on the shifting of fluorescence spectra of cinchona alkaloids (Chart 1) bound to cinchona-imprinted TFMAA based polymers. The optical properties of these imprinted polymers allow for the quantification of free and bound fluorescent substrate, thus enabling direct batch binding tests without prior bound/free separation. Results achieved with the imprinted polymers further exemplified the potential of molecularly imprinted polymers as assay and sensing materials. EXPERIMENTAL SECTION Materials. Cinchonidine, cinchonine, ethylene glycol dimethacrylate (EGDMA), methacrylic acid (MAA) were purchased from Wako Pure Chemical Industry (Osaka, Japan). 2-(Trifluoromethyl)acrylic acid (TFMAA) was obtained from Tokyo Chemical Industry (10) Cooper, M. E.; Hoag, B. P.; Gin, D. L. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1997, 38, 209-210. (11) Matsui, J.; Higashi, M.; Takeuchi, T. J. Am. Chem. Soc. 2000, 122, 52185219. (12) Wolfbeis, O. S.; Terpetschnig, E.; Piletsky, S.; Pringsheim, E. In Applied Fluorescence in Chemistry, Biology and Medicine; Rettig, W., Strehmel, B., Schrader, S., Seifert, H., Eds.; Springer-Verlag: Berlin, 1999; pp 277-296. (13) Matsui, J.; Miyoshi, Y.; Takeuchi, T. Chem. Lett. 1995, 1007-1008. (14) Matsui, J.; Doblhoff-Dier, O.; Takeuchi, T. Anal. Chim. Acta 1997, 343, 1-4. (15) Matsui, J.; Nicholls, I. A.; Takeuchi, T. Anal. Chim. Acta 1998, 365, 8993. 10.1021/ac000106c CCC: $19.00

© 2000 American Chemical Society Published on Web 06/16/2000

Table 1. Imprinted and Blank Polymers Prepared in This Study polymer

template (mmol)

PM(CD) PM(CN) PM(BL) PF(CD) PF(CN) PF(BL) PFM(CD)

cinchonidine (2.45) cinchonine (2.45) none cinchonidine (2.45) cinchonine (2.45) none cinchonidine (2.45)

PFM(CN)

cinchonine (2.45)

PFM(BL)

none

PB(CD) PB(BL)

cinchonidine (2.45) none

functional monomer (mmol) MAA (9.87) MAA (9.87) MAA (9.87) TFMAA (9.87) TFMAA (9.87) TFMAA (9.87) MAA (4.94) TFMAA (4.94) MAA (4.94) TFMAA (4.94) MAA (4.94) TFMAA (4.94) none none

spectrophotometer, Biochrom 4060 (Pharmacia-LKB Biochem, Cambridge, England), were used for spectroscopic studies. Polymer particles (5.0 mg) were incubated with a varied amount of cinchonidine dissolved in chloroform-acetonitrile (17:83, v/v, 2.0 mL) in a quartz cell (exth × intw, 48 mm × 10 mm). After the incubation for 18 h, the suspension was removed, and fluorescent measurements of it were carried out. Analysis of Cinchonidine in the Supernatants. After the fluorescent measurements, the supernatants were transferred to polypropylene tubes by a syringe with a PTFE filter (Whatman, 4-mm-diameter, 0.2-µm-pore) for flow injection determination of cinchona alkaloids in the supernatants. The flow injection analysis was performed by the same system as the chromatographic tests with a carrier solution of methanol/100 mM sulfuric acid (9:1, v/v). Free cinchonidine was subtracted from the initial cinchonidine to obtain the amount of cinchonidine bound to the polymer.

(Tokyo, Japan). Chloroform, acetone, acetic acid, acetonitrile, and methanol were purchased from Katayama Chemical (Osaka, Japan). Chloroform, MAA, and EGDMA were used after purification according to common procedures.16 Preparation of Imprinted Polymers. Cinchonidine-Imprinted Polymer PF(CD). As the template species, cinchonidine (721 mg, 2.45 mmol) was added into 250 mL of chloroform containing TFMAA (1380 mg, 9.87 mmol) as the functional monomer, EGDMA (13.7 g, 69.1 mmol) as the cross-linking agent, and 2,2′azobisisobutyronitrile (100 mg) as the initiator. The mixture was placed in a screw-capped Pyrex test tube and sparged with nitrogen gas for 5 min. The glass tube was sealed and placed under UV light (XX-15L, UVP, Upland, CA) for 16 h at 4 °C. The polymer block obtained was ground by an automatic mortar (AMN 1000, Nittokagaku, Nagoya, Japan), sieved (32-63 µm) in methanol-water, and packed in a stainless steel tube (100 mm × 4.6 mm, i.d.). The column was washed with methanol-acetic acid (7:3, v/v) overnight at a flow rate of 0.4 mL min-1 to extract the template from the polymer matrix and then conditioned with methanol. The polymer was used as the stationary phase in the chromatographic experiments or removed from the column tube for batch-binding tests. Cinchonine-imprinted polymer PF(CN) was prepared using cinchonine in place of cinchonidine. A nonimprinted blank polymer PF(BL) was prepared without addition of the template species to the polymerization mixture. Other imprinted and blank polymers were prepared in an identical fashion using the corresponding template compound (2.45 mmol) and functional monomer (9.87 mmol) as listed in Table 1. Chromatographic Tests. Chromatographic experiments were carried out using a Gulliver HPLC system (JASCO, Tokyo, Japan), which consists of a pump (PU-980), a degasser (DG-980-50), an automatic sample injector (AS-980-10), a UV detector (UV-975), and a fluorescent detector (FP-920). The flow rate was 1.0 mL min-1. Elutions were monitored by UV absorbance at 280 or 308 nm. The sample volume was 20 µL, and the concentration was 0.42 mM. Acetone was used as a void marker. The samples were injected independently in triplicate. Direct Fluorescence Measurements. A fluorescent spectrophotometer, F-2500 (Hitachi, Tokyo, Japan), and a UV-visible

RESULTS AND DISCUSSION Binding Behaviors in the Imprinted Polymers. Cinchonidine-imprinted and cinchonine-imprinted polymers (Table 1) were prepared using methacrylic acid (MAA) and 2-(trifluoromethyl)acrylic acid (TFMAA) as the functional monomer(s). The polymers were used as stationary phases for liquid chromatography to examine their affinity and diastereoselectivity. According to the molecular recognition principle, the solvent used for the polymerization could be the best eluent to provide the performance of the resultant imprinted polymers.17 As no adequate elution was observed with chloroform and dichloromethane due to the strong binding, we selected a number of alternative eluents for the chromatographic tests. As listed in Table 2, all the imprinted polymers showed diastereoselectivity for the template species over its antipode, while the nonimprinted blank polymers exhibited almost no diastereoselective retention to both isomers. The acetonitrile/acetic acid based solvent eluted the cinchona alkaloids faster than the dichloromethane-based solvent. This is very likely due to polar and protic eluents being unfavorable for electrostatic interaction and hydrogen bonding, which are supposed to be essential in the imprinting and recognition processes. The functional monomer used for imprinting is also influential on the retention characteristics of the resultant polymers. TFMAA based polymers exhibit strong binding, while polymers prepared using both TFMAA and MAA, such as PFM, exhibited moderately strong binding. The higher acidity of TFMAA seems to be favorable for rebinding the basic template molecule in the polymer network. The polymers prepared with TFMAA also exhibited significant retention of the antipode, while PM showed almost no retention. Polymers Prepared in the Absence of Functional Monomers. When no cinchonidine was added to the imprinted polymer suspended in the chloroform/acetonitrile solution, an emission maximum was observed around 400 nm (Figure 1a). This means that the template species could not be removed completely in the washing stage and remained in the imprinted polymers, as such fluorescence was not observed using the nonimprinted blank polymer prepared in the absence of cinchonidine. Currently, it is

(16) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory Chemicals, 3rd ed.; Pergamon Press: Oxford, 1988.

(17) Spivak, D.; Gilmore, M. A.; Shea, K. J. J. Am. Chem. Soc. 1997, 119, 43884393.

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

3287

Table 2. Chromatographic Evaluation of the Diastereoselectivity

Table 3. Retention Property of the Polymers Having No Functional Monomer

k′

k′

polymer

eluenta

CD

CN

polymer

cinchonidine

cinchonine

R

PM(CD) PM(CN) PM(BL) PF(CD) PF(CN) PF(BL) PFM(CD) PFM(CN) PFM(BL) PM(CD) PM(CN) PM(BL) PF(CD) PF(CN) PF(BL) PFM(CD) PFM(CN) PFM(BL) PM(CD) PM(CN) PM(BL) PF(CD) PF(CN) PF(BL) PFM(CD) PFM(CN) PFM(BL)

1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3

4.51 0 0 NCEb 2.47 0.08 8.52 0.39 0 0 0.17 0 NCEb 0.67 1.37 5.31 0.02 0.23 0.29 0 0 4.17 0.18 1.77 1.99 0 0.75

0 0.42 0 4.56 NCEb 0.07 1.42 4.67 0 1.91 0 0 1.54 9.00 1.41 0.28 1.33 0.23 0 0.29 0 0.72 2.79 1.88 0.16 1.23 0.76

PB(CD) PB(BL)

1.04 0.93

1.24 1.11

0.84 0.84

a eluent 1, dichloromethane/acetic acid (60:40, v/v); eluent 2, acetonitrile/acetic acid (85:15); eluent 3, acetonitrile/water/acetic acid (80:15:5). b No clear elution was observed.

Figure 1. Fluorescence spectra of PF(CD) suspension incubated with cinchonidine (a, 0; b, 0.005; c, 0.02; d, 0.05; e, 0.10; f, 0.15; g, 0.25; h, 0.50 mM) in chloroform/acetonitrile (λex, 330 nm).

uncertain whether the template molecule is covalently bound to the polymer chains or is tightly trapped in the network during polymerization. Polymers were prepared without any functional monomer, to examine whether the residual template species are involved in the diastereoselective retention property. As shown in Table 3, PB(CD) showed no cinchonidine-selective retention characteristics. Furthermore, the retention behaviors are quite similar to those of PB(BL). Residual cinchonidine does, therefore, not seem to be involved in significant interaction with the substrate tested, and the cinchonidine selectivity observed is most likely due to 3288 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

arrangements of the functional monomer(s) complementary to the template species caused by the imprint effects. Because of the poor solubility of free cinchonine in chloroform, a corresponding experiment using a cinchonine-imprinted polymer, lacking the functional monomer, could not be performed. Direct Fluorescence Measurement. Fluorescent measurements were conducted on various concentrations of cinchonidine incubated with suspensions of the imprinted polymers PM(CD) and PF(CD) in chloroform/acetonitrile. As it is known that heterogeneous populations of polymer particles may unfavorably influence the resultant spectra, the solvent was carefully chosen to be of the same density as the polymer tested. The emission spectra of PF(CD) are shown in Figure 1. Net fluorescence of cinchonidine in the PF(CD) suspension, calculated by subtracting the background fluorescence from the fluorescence observed with a range of concentrations of cinchonidine added, is shown in Figure 2A. The fluorescence spectra showed a blue shift in accordance with an increase of total concentration of cinchonidine with an emission maximum around 390 nm at lower concentrations and around 360 nm at higher concentrations. Free cinchonidine dissolved in the chloroform/ acetonitrile solution without the addition of polymers shows an emission maximum around 360 nm, and bound cinchonidine has an emission maximum near 390 nm, suggesting that the observed fluorescent spectra shift in PF(CD) is due to a balance of the spectra of free and bound cinchonidine. Fluorescence spectra of the supernatants after removal of the polymer particles by filtration showed emission maxima near 360 nm. This means that the imprinted polymer did not affect the fluorescence of free cinchonidine in the suspension and the observed shift was caused by the binding of cinchonidine to the polymers. In contrast, no significant shift was observed in the spectra when the MAA based polymer, PM(CD), was used, showing that MAA residues have no influences on the fluorescence of bound cinchonidine (Figure 2B). The polymers prepared by simultaneous use of the two functional monomers, PFM(CD) and PFM(BL), were also examined. The polymers exhibited a more moderate shift of fluorescence spectra as compared with those of PF polymers (Figure 2C), supporting the idea that TFMAA is the monomer responsible for shifting the fluorescence of cinchonidine. The wavelength of the emission maximum is plotted as a function of the total concentration of cinchonidine (Figure 3). The concentration-dependent fluorescence spectra suggest that TFMAA based polymers can be used for assaying cinchonidine. PFM(CD), that was prepared with the combined use of TFMAA and MAA, showed a sensitive red shift to the total cinchonidine concentration compared with PFM(BL), while no significant differences were observed between PF(CD) and PF(BL) that were prepared with the sole use of TFMAA (Figure

Figure 3. Shift of wavelength in emission maxima of the polymer suspensions incubated with various concentrations of cinchonidine in chloroform/acetonitrile (λex, 330 nm); 0 PF(CD), 9 PF(BL), O PFM(CD), b PFM(BL), 4 PM(CD), 2 PM(BL).

Figure 4. A bound/free profile obtained by the determination of free cinchonidine in the supernatants of PFM(CD) suspensions incubated with various concentrations of cinchonidine in chloroform/acetonitrile.

Figure 2. Fluorescence spectra of the polymer suspensions incubated with cinchonidine (a, 0.005; b, 0.02; c, 0.05; d, 0.10; e, 0.15; f, 0.25; g, 0.50 mM) in chloroform/acetonitrile (λex, 330 nm); A, PF(CD); B, PM(CD); C, PFM(CD).

3), although the imprinted polymers had exhibited larger capacity factors than the blank polymer in the chromatographic tests. This could be due to the less polar, aprotic solvents employed for the batch-binding tests, in which TFMAA residues in the blank polymers can make significant contribution to the nonspecific binding of cinchonidine. To block this unfavorable binding, an acid competitive with acidic residues of the binding sites, such as acetic acid, is usually added to the solvent and the chromatographic condition is the case. In this study, however, this approach was not adopted, as the addition of acid influences the fluorescence spectra of the free substrate.

Thus, PFM(CD) was selected for further assessment by batchbinding tests employing the flow injection analysis of supernatants. Currently, it is unknown whether both functional monomers were cooperatively allocated for one template molecule or worked independently in the imprinting process. The chromatographic and spectroscopic results, however, demonstrated that the combined use of two different functional monomers is effectual for compromising the strong binding character, the good imprint effects, and the sensitive fluorescence shift. Fluorescence and Substrate Uptake. To quantitate the relation of the substrate uptake and the fluorescent shift, the amount of cinchonidine bound to PFM(CD) was estimated by analyzing the supernatants. The results showed that the amount of cinchonidine bound to the polymer is proportional to the total cinchonidine concentration. The plot of bound/free values versus the concentration of total cinchonidine added (Figure 4) is similar to the emission maximum shift shown in Figure 3, strongly supporting the idea that the fluorescence shift is due to the uptake of cinchonidine by the imprinted polymer. Although further quantitative processing of fluorescence spectra has not been addressed, the fluorescent shift reflecting the binding of cinchonidine suggests that the direct fluorescent measurements of polymer suspension can also be used for assessing the affinity of imprinted polymers, such as in Scatchard analysis. Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

3289

showed red-shift in accordance with the increase of the concentration of TFMAA, while no significant shift was observed upon adding MAA. The apparent difference between the two functional monomers is acidity; pKa values of MAA18 and TFMAA19 are 4.6 and 2.3, respectively. Cinchonidine is expected to form hydrogenbonded or a proton-transfer complexes with these monomers, and the binding mode could be governed by the acidity of the monomers. A superior proton donor, such as TFMAA, would be capable of proton transfer with cinchonidine to generate a protonated cinchonidine species that may show a distinct fluorescence.20 This assumption agrees well with the considerably stronger binding property of PF(CD) as compared with PM(CD). Cinchonidine dissolved in aqueous hydrochloric acid also showed an emission maximum around 410 nm, supporting the hypothesis that proton donation is the predominant cause for the fluorescence shift. The single isosbestic point observed in fluorescence of cinchonidine titrated with TFMAA suggests that the shift is due to a protonation of one nitrogen atom in the cinchonidine structure. CONCLUSIONS Molecularly imprinted polymers prepared using TFMAA exhibited the ability to shift the fluorescence spectra of the substrate cinchonidine when being bound by the polymers. Such polymers have considerable potential as antibody/receptor in simplified analytical applications, avoiding bound/free separation procedures by direct monitoring of the fluorescence spectra shift of the analyte binding to the substrate. We are currently expanding this type of assay to other imprinted polymers and further fluorescent and fluorescence-labeled molecules, to develop novel competitive binding assays.

Figure 5. Fluorescence spectra of cinchonidine (0.1 mM) in various concentrations of TFMAA and MAA. (A) TFMAA (a, 0; b, 0.82; c, 1.64; d, 4.09; e, 8.18; f, 12.3; g, 16.4; h, 40.9; i, 61.3 mM; each concentration is corresponding to 0, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0, 25.0, 37.5 equivalents, respectively, of TFMAA used in the polymer preparation); (B) MAA (a, 0; b, 0.17; c, 0.85; d, 1.69; e, 8.45 mM; each concentration is corresponding to 0, 0.1, 0.5, 1.0, 5.0 equivalents, respectively, of MAA used in the polymer preparation). Thick lines (a) are the spectra of cinchonidine alone.

Mechanism of Fluorescent Shift. To verify that the observed fluorescence shift is due to the interaction of cinchonidine with the acidic residues in the polymer, fluorescent spectra of cinchonidine were measured in solution in the absence of the polymers and in the presence of monomers (MAA and TFMAA). The amount of the monomers added corresponded to 0-37.5 molar equivalents of the carboxylic residues existing in 5.0 mg of the polymer. As shown in Figure 5, the fluorescence spectra

3290

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

ACKNOWLEDGMENT The authors acknowledge the financial support from The Ministry of Education, Science, Sports and Culture, Japan. This work is also supported by a grant of the Research for the Future Program of JSPS. We thank Dr. Otto Doblhoff-Dier, University of Agricultural Sciences, Vienna, for his valuable suggestions. We also appreciate Mr. Toshihiro Mizukami, Sysmex Corporation (Kobe, Japan), and Ms. Akiko Omote, Visiting Scholar of Hiroshima City University, for their technical assistance.

Received for review February 3, 2000. Accepted April 29, 2000. AC000106C (18) Dean, J. A. Lange’s Handbook of Chemistry; McGraw-Hill: New York, 1998. (19) This is an estimated value obtained by a Hammet and Taft Theory-based calculation with a software pKalc of 3.2 (CompuDrug International, Inc.). (20) Guilbault, G. G. Practical Fluorescence, 2nd ed.; Marcel Dekker: New York, 1990.