Application of Molecular Imprinting to the Development of Aqueous

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Anal. Chem. 1996, 68, 111-117

Application of Molecular Imprinting to the Development of Aqueous Buffer and Organic Solvent Based Radioligand Binding Assays for (S)-Propranolol Lars I. Andersson*

Pure and Applied Biochemistry, Chemical Center, P.O. Box 124, S-221 00 Lund, Sweden

Antibody mimics have been prepared by molecular imprinting of (S)-propranolol and applied in the development of radioligand binding assays for the imprint species. In the assays, polymer particles, radioligand, and analyte were incubated either in an organic solvent or an aqueous buffer, with similarly high sensitivity under both set of conditions. Optimization of the assay conditions led to 100-1000-fold improvements in the limit of determination and 100-1000-fold reductions of the amount of imprinted polymer used, compared with previous studies of this novel type of assay. The present assay uses only 10-50 µg of polymer, and the limit of determination is about 6 nM. The toluene-based assay showed excellent enantioselectivity, the cross-reactivity of the R enantiomer being only 1%, which is better than that demonstrated by biological antibodies. The aqueous buffer based assay showed high substrate selectivity for propranolol in the presence of structurally similar β-blockers. The different selectivity profiles obtained are due to a different balance between hydrophobic and polar interactions in toluene and water, since polar interactions, such as hydrogen bonds, are strong in apolar solvents and hydrophobic interactions are strong in water. Molecular imprinting1-6 is increasingly becoming recognized as a technique for the ready preparation of polymeric materials containing recognition sites of predetermined specificity. Such molecularly imprinted polymers (MIPs) may be applied in a range of applications requiring selective ligand binding, such as in the areas of selective detection, separation and purification, directed synthesis, and catalysis. The majority of previous studies have focused on enantiomeric separation problems where the MIPs were used as chiral stationary phases in HPLC. A wide range of chiral compounds have been resolved on imprinted columns, * Present address: Astra Pain Control, S-151 85 So ¨derta¨lje, Sweden. (1) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9-14. (2) Nicholls, I. A.; Andersson, L. I.; Mosbach, K.; Ekberg, B. Trends Biotechnol. 1995, 13, 47-51. (3) Andersson, L. I.; Nicholls, I. A.; Mosbach, K. Immunoanalysis of Agrochemicals: Emerging Technologies; Nelson, J. O., Karu, A. E., Wong, R. B., Eds.; ACS Symposium Series 586; American Chemical Society: Washington, DC, 1995; pp 89-96. (4) Wulff, G. In Polymeric Reagents and Catalysts; Ford, W. T., Ed.; ACS Symposium Series 308; American Chemical Society: Washington, DC, 1986; pp 186-230. (5) Wulff, G. Trends Biotechnol. 1993, 11, 85-87. (6) Shea, K. J. Trends Polym. Sci. 1994, 2, 166-173. 0003-2700/96/0368-0111$12.00/0

© 1995 American Chemical Society

including drug compounds,7,8 amino acid derivatives,9-15 sugars, and sugar derivatives.16-19 Baseline resolution has often been achieved, and in one case, a separation value (R) as high as 18 was achieved for the separation of the enantiomers of a dipeptide.20 Recently, it was demonstrated that MIPs can also be substituted for antibodies in immunoassay protocols.21,22 In the first study of this type, theophylline and diazepam imprints were successfully used in a radioligand binding assay for the accurate determination of concentrations of these drugs in human serum.21 Prior to the actual assay, performed under optimized conditions using organic solvents, the analyte was extracted from the serum using standard protocols. An excellent correlation was demonstrated between the results obtained using the imprint-based assay and a commercial immunoassay technique for the determination of theophylline. In this, and the subsequent studies on anti-morphine and anti-enkephalin MIPs,22 the imprints showed binding affinities approaching those demonstrated by antigen-antibody systems. The MIPs were highly specific, and selectivity profiles comparable to those of the corresponding antibodies against the same compounds were observed. Despite the increasing interest in the use of imprinted polymers, their use has necessitated that the rebinding occurred in organic solvents or mixtures with buffers composed primarily of an organic solvent. Very recently it was demonstrated that efficient imprint ligand rebinding is indeed possible in aqueous buffers,22 although the affinities and selectivities obtained were (7) Fischer, L.; Mu ¨ ller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360. (8) Kempe, M.; Mosbach, K. J. Chromatogr. 1994, 664, 276-279. (9) Sellergren, B.; Lepisto ¨, M.; Mosbach, K. J. Am. Chem. Soc. 1988, 110, 58535860. (10) O’Shannessy, D. J.; Ekberg, B.; Mosbach, K. Anal. Biochem. 1989, 177, 144-149. (11) Andersson, L. I.; O’Shannessy, D. J.; Mosbach, K. J. Chromatrogr. 1990, 513, 167-179. (12) Andersson, L. I.; Mosbach, K. J. Chromatrogr. 1990, 516, 313-322. (13) Ramstro¨m, O.; Andersson, L. I.; Mosbach, K. J. Org. Chem. 1993, 58, 75627564. (14) Kempe, M.; Fischer, L.; Mosbach, K. J. Mol. Recognit. 1993, 6, 25-29. (15) Sellergren, B.; Shea, K. J. J. Chromatrogr. 1993, 635, 31-49. (16) Wulff, G., Minarik, M. J. Liq. Chromatogr. 1990, 13, 2987-3000. (17) Wulff, G.; Schauhoff, S. J. Org. Chem. 1991, 56, 395-400. (18) Wulff, G.; Haarer, J. Makromol. Chem. 1991, 192, 1329-1338. (19) Mayes, A.; Andersson, L. I.; Mosbach, K. Anal. Biochem. 1994, 222, 483488. (20) Ramstro¨m, O.; Nicholls, I. A.; Mosbach, K. Tetrahedron: Assymetry 1994, 5, 649-656. (21) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645-647. (22) Andersson, L. I.; Mu ¨ ller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4788-4792.

Analytical Chemistry, Vol. 68, No. 1, January 1, 1996 111

Table 1. Polymer Preparations

polym

imprinted enantiomer/ mmol

cross-linking monomera/ mmol

methacrylic acid (mmol)

specific surface areab (m2/g)

pore volc (mL/g)

pore diamd (Å)

A B C D

S/1.13 R,S/1.13 S/0.2 S/1.0

EGDMA/45 EGDMA/45 TRIM/1.6 TRIM/8.0

9.0 9.0 1.6 8.0

193 197 73 188

0.66 0.60 0.35 0.83

186 174 233 258

a EGDMA, ethylene glycol dimethacrylate; TRIM, trimethylolpropane trimethacrylate. b Determined using the BET model. c Average of BJH cumulative adsorption and desorption pore volume of pores between 17 and 3000 Å. d BJH adsorption average pore diameter (4 × pore volume/ surface area) of pores between 17 and 3000 Å.

Figure 1. Structures of the compounds studied: propranolol (1), metoprolol (2), atenolol (3), and timolol (4).

less than those for ligand binding in an optimal organic solvent. In this present study, the influences of several parameters affecting ligand binding in water were studied. As a pilot study, we decided to imprint (S)-propranolol (Figure 1), which is one of a number of β-adrenergic antagonists (β-blockers) used extensively in the treatment of cardiovascular disorders such as hypertension and angina pectoris. Like most β-blockers used in therapy, propranolol was launched as a racemate. However, the enantiomeric forms have different pharmacological and pharmacokinetic properties,23-28 and the in vitro antagonist action of (S)-propranolol is about 100fold more potent than that of the R isomer.23,27 Pure enantiomer imprinting enabled us to study both substrate selectivity and enantioselectivity of the resultant MIPs, with the concomitant potential for the development of an enantiospecific ligand binding assay for the determination of (S)-propranolol. MATERIALS AND METHODS Preparation of Polymers. The compositions of the polymerization mixtures are summarized in Table 1. Imprint species and initiator (0.7 mol % of the total amount of polymerizable methacrylate units) were weighed into borosilicate test tubes and mixed with methacrylic acid, cross-linking monomer, and sodiumdried toluene (the volume ratio of solvent to monomers was 4:3). 2,2′-azobis(2-isobutyronitrile) (AIBN) was used as initiator for (23) Barrett, A. M.; Cullum, V. C. Br. J. Pharmacol. 1968, 34, 43-55. (24) Rahn, K. H.; Hawlina, A.; Kersting, F.; Planz, G. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1974, 286, 319-323. (25) Morris, T. H.; Kaumann, A. J. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1984, 327, 176-179. (26) Walle, T.; Webb, J. G.; Bagwell, E. E.; Walle, U. K.; Daniell, H. B.; Gaffney, T. E. Biochem. Pharmacol. 1988, 37, 115-124. (27) Stoschitzky, K.; Lindner, W.; Rath, M.; Leitner, C.; Uray, G.; Zernig, G.; Moshammer, T.; Klein, W. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1989, 339, 474-478. (28) Egginger, G.; Lindner, W.; Brunner, G.; Stoschitzky, K. J. Pharm. Biomed. Anal. 1994, 12, 1537-1545.

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polymers A, B, and C, and 2,2′-azobis(2,4-dimethylvaleronitrile) (ABDV) for polymer D. The solutions were cooled on ice and sparged with nitrogen. The test tubes were then placed under a UV source (366 nm) at 4 °C (polymerizations initiated by AIBN) or kept in a water bath at 45 °C (polymerizations initiated by ABDV) for 16 h. The hard bulk polymers were ground in a mechanical mortar and wet-sieved through a 25 µm sieve. The grinding and sieving were repeated four times until all the material passed through the sieve. The fines were removed by repeated sedimentation from ethanol. The polymer particles were then carefully washed on a sintered glass funnel with NH4OAc (1 M) dissolved in a mixture of ethanol, acetic acid, and water (40:25: 35; v/v/v, 500 mL) in several aliquots, followed by acetic acid in ethanol (1:3; v/v, 200 mL) and methanol (200 mL). Finally, the particles were dried under vacuum and stored at ambient temperature until use. Radioligand Binding in Organic Solvent. Polymer particles were suspended in the incubation solvent, which was toluene containing 0.5% (v/v) acetic acid, and from this stock suspension, kept uniform by vigorous stirring, appropriate volumes were distributed to Eppendorf test tubes. Incubations were set up containing 50 µg of polymer A or 25 µg of polymer C, 0.3 ng of [3H]-(S)-propranolol [specific activity 784.4 GBq/mmol (21.2 Ci/ mmol)] and the desired concentrations of (S)-propranolol, (R)propranolol, (R,S)-atenolol, (R,S)-metoprolol, or (R,S)-timolol in a total volume of 1 mL. The final concentrations of the unlabeled competing ligands were in the range from 10-2 to 106 ng/mL, depending on their ability to inhibit binding of the radioligand. The samples were placed on a rocking table for 16 h. After centrifugation, 700 µL of the supernatant was measured by liquid scintillation counting. Radioligand Binding in Buffers. The precise conditions for the optimization experiments, in which the effects of pH, ethanol concentration, buffer concentration, and ionic strength on ligand binding were studied, are described in the legends to Figures 4-7. In the ligand binding assays, 50 µg of polymer A, 25 µg of polymers B or C, or 20 µg of polymer D was incubated with 0.3 ng of [3H]-(S)-propranolol [specific activity 784.4 GBq/mmol (21.2 Ci/mmol)] and the desired concentrations of unlabeled competing ligand in a total volume of 1 mL. The incubation buffer was sodium citrate (25 mM, pH 6.0) containing 2% (v/v) ethanol. The competing ligands were (S)-propranolol, (R)-propranolol, (R,S)atenolol, (R,S)-metoprolol, and (R,S)-timolol at final concentrations in the range from 10-2 to 106 ng/mL, depending on their ability to inhibit binding of the radioligand. The samples were placed on a rocking table for 16 h, and after centrifugation, 800 µL of the supernatant was measured by liquid scintillation counting.

Table 2. IC50 Values for Inhibition of [3H]Propranolol Binding to the MIPs ligand (S)(R)(R,S)(R,S)- (R,S)assay propranolol propranolol metoprolol timolol atenolol MIP solvent (µM) (µM) (µM) (µM) (µM) A

water toluene water water toluene water

B C D a

0.52 0.29 0.43 2.6 0.16 0.91

3.0 20 1.22 4.7 3.3 2.1

76 4.6 64 77 1.5 86

170 770 38 1.6 250 1010 121 1720a 8.1 0.51 106 960

Extrapolated value.

RESULTS Imprints were prepared by copolymerization of methacrylic acid and ethylene glycol dimethacrylate (polymers A and B) or trimethylolpropane trimethacrylate29 (polymers C and D) in the presence of (S)-propranolol. In one case (polymer B), the imprint species was instead rac-propranolol. After preparation and workup of the polymers, it was estimated that g99% of the imprint molecules were removed, since in all instances, elemental analysis of nitrogen failed to detect any remaining imprint species. Initial studies showed that the use of toluene as the solvent of polymerization yielded a higher titer of high-affinity imprinted sites per unit weight of polymer compared with polymers made using methylene chloride or chloroform (data not shown). All further studies were undertaken on MIPs prepared with toluene as the solvent of polymerization. The optimizations of both the solvent and the water based assays were performed using MIP A. Ligand Binding Analysis in Organic Solvents. The strength of [3H]-(S)-propranolol binding to MIP A increased with decreasing polarity of the solvent of incubation, since less polymer was required to bind a specified amount (50%) of the radioligand added (data not shown). This observation is consistent with previous findings.21,22 Nonspecific binding could be suppressed by addition of low concentrations of acetic acid, and toluene containing 0.5% acetic acid was found to combine high avidity with negligible nonspecific binding. The nonspecific binding was measured as the binding, under identical conditions, to a nonimprinted reference polymer. Under these conditions the assays required only 50 µg of polymer particles/mL of incubation solvent and at this very low polymer concentration no nonspecific binding could be detected. Even at reference polymer concentrations as high as 1.6 mg/mL, the reference polymer bound only 7% of the added radioligand. Binding of radiolabeled (S)-propranolol in the absence and presence of varying concentrations of competing ligands was analyzed under conditions where the number of polymeric binding sites was limited, in a manner analogous to the conceptually identical competitive format of immunoassay.30 The IC50 values (the concentrations of competing ligand required to displace 50% of the specifically bound radioligand) for unlabeled (S)- and (R)propranolol and some related structures (Figure 1) are presented in Table 2. The values were calculated from the X intercepts after log-logit transformation.30 MIP A showed excellent selectivity (29) Kempe, M.; Mosbach, K. Tetrahedron Lett. 1995, 36, 3563-3566. (30) Price, C. P., Newman, D. J.. Eds. Principles and Practice of Immunoassay; Stockton: New York, 1991.

Figure 2. Displacement of [3H]-(S)-propranolol binding to MIP A in toluene containing 0.5% (v/v) acetic acid by increasing concentrations of competing ligand. B/B0 is the ratio of the amount of radiolabeled (S)-propranolol bound in the presence of displacing ligand, B, to the amount bound in the absence of displacing ligand, B0. Displacing ligands: (open squares) (S)-propranolol; (filled squares) (R)-propranolol; (filled circles) (R,S)-metoprolol; (open triangles) (R,S)-timolol; (open circles) (R,S)-atenolol.

for the imprinted enantiomer over its optical antipode (Figure 2); the cross-reaction of the R enantiomer was only 1.4% relative to the S form. The optical purities of the displacing ligands (analyzed by HPLC using cellulase CBH I immobilized on silica as the chiral stationary phase31) were found to be better than 99.9% (both the free bases used here and the hydrochloride salts used in the aqueous experiments below), and the purity of the radioligand was better than 98%. Atenolol and metoprolol showed significant cross-reactivities (18 and 6.3%) whereas timolol showed low crossreactivity (0.8%) (Table 2). The limit of determination, defined as the concentration of unlabeled (S)-propranolol able to displace 10% of the radioligand binding, was found to be 8.6 nM. The Scatchard plots were nonlinear, due to a heterogeneous population of sites with various affinities for the imprint molecule. The apparent equilibrium association constant (KA) was estimated by nonlinear least-squares fitting with the EBDA and LIGAND programs (Biosoft).32,33 The estimation was performed via approximation of the data to a two-site model for high- and lowaffinity binding sites. The apparent KA values for (S)-propranolol binding to MIP A were found to be (2.5 ( 1.4) × 107 (KD ) 40 nM) and (4.4 ( 1.6) × 104 M-1 (KD ) 23 µM), associated with site populations of 2.0 ( 0.47 and 38 ( 6.6 µmol/g, respectively. Although this model probably has little physical relevance, the calculated values may still provide a valid estimate of the levels of affinity achievable with imprinted polymers. MIP C, made using TRIM as the cross-linking agent,29 rebound the imprint species more strongly than MIP A, as demonstrated by the lower IC50 value (Table 2). Its enantioselective ability was poorer, however (4.8% cross-reactivity was recorded for the R enantiomer), and the other β-blockers interfered to a higher extent with the radioligand binding than was recorded for MIP A. The limit of determination was found to be 5.5 nM. Ligand Binding Analysis in Aqueous Buffers. The dependence of ligand binding on pH was investigated over the range (31) Marle, I.; Erlandsson, P.; Hansson, L.; Isaksson, R.; Pettersson, C.; Pettersson, G. J. Chromatogr. 1991, 586, 233-248. (32) McPherson, G. A. J. Pharmacol. Methods 1985, 14, 213-228. (33) Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107, 220-239.

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Figure 3. Binding of propranolol to the MIP A (circles) and the corresponding nonimprinted reference polymer (made under identical conditions and with the same composition except that the imprint molecule was omitted) (squares) as a function of pH. Radiolabeled (S)-propranolol (1.2 pmol) and 25 µg of polymer particles were incubated in 1 mL of 25 mM buffers containing 2 (v/v; open symbols) and 5% (v/v; filled symbols) ethanol. The buffers were citrate (pH 3.0-6.0), phosphate (pH 7.0-8.0), and carbonate (pH 9.0). B/T is the ratio of the amount of radioligand bound (B) to the total amount (T) added to the test tubes. For each pH the precise total activity, T, was determined, in tubes without polymer but otherwise treated identically to samples, in order to confirm that the ligand did not adsorb to the tube walls and was found constant over the whole pH interval.

from pH 3 to 9 (Figure 3). The binding of radiolabeled (S)propranolol to MIP A increased with increased pH as did the binding to the corresponding nonimprinted reference polymer. The pH profile was recorded for two ethanol concentrations (2 and 5% v/v). At both ethanol concentrations, the maximal difference between specific and nonspecific binding occurred at pH 6.0 (Figure 3). This pH was used in subsequent experiments. Under more acidic conditions virtually no binding to the reference polymer was observed. Nonspecific binding was still reasonably low at pH 6.0, but became significant at higher pH values. The increase in nonspecific binding at higher pH is probably due to an ion exchange mechanism involving randomly distributed carboxylate ions of the polymer and amino groups of propranolol. An investigation of the effect of addition of ethanol to the incubation mixture demonstrated that the binding strength decreased with increased ethanol content (Figure 4). Addition of some ethanol to the incubation mixture was necessary, however, since at very low ethanol concentrations (below 2% v/v) propranolol was adsorbed to the plastic walls of the Eppendorf tubes (data not shown). This is due to the hydrophobic nature of propranolol. The specific portion of the total binding (i.e., the difference between the binding to MIP A and the reference polymer) was constant over the range 2-16% (v/v) of ethanol (Figure 4) but decreased at higher and lower ethanol concentrations. In all subsequent experiments, the incubation buffer contained 2% (v/v) ethanol. The binding of (S)-propranolol increased with decreasing buffer concentration (Figure 5). The imprinted and nonimprinted polymers were, however, affected equally, at least for sodium citrate buffer (pH 6) stronger than 5 mM. Thus, the specific part of the total binding was constant. In all subsequent experiments, a buffer concentration of 25 mM was used in order to have sufficient buffer capacity in the incubation mixtures. High ionic 114 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996

Figure 4. Binding of propranolol to MIP A (open squares) and the corresponding nonimprinted reference polymer (filled squares) as a function of ethanol concentration. Radiolabeled (S)-propranolol (1.2 pmol) and 25 µg of polymer particles were incubated in 1 mL of citrate buffer (25 mM, pH 6.0) containing varying concentrations of ethanol. The precise total activity, T, was determined for each ethanol concentration, in tubes without polymer but otherwise treated identically to the samples. At 0 and 1% ethanol, some adsorption to the Eppendorf test tube walls was observed, as the T values were lower at these ethanol concentrations. The specific binding (open circles) is B/T on the imprinted polymer minus B/T on the nonimprinted polymer.

Figure 5. Binding of propranolol to MIP A (open squares) and the corresponding nonimprinted reference polymer (filled squares) as a function of buffer concentration. Radiolabeled (S)-propranolol (1.2 pmol) and 25 µg of polymer particles were incubated in 1 mL of citrate buffer (pH 6.0) at varying concentrations. The buffers contained 2% (v/v) ethanol. The specific binding (open circles) is B/T on the imprinted polymer minus B/T on the nonimprinted polymer.

strengths also weakened the binding, as shown in binding experiments where increasing amounts of sodium chloride were added to the incubation mixture (Figure 6). At sodium chloride concentrations below 0.1 M, the decrease in binding was equal for both polymers, which indicates that only the nonspecific part of the total binding was affected below this concentration. The cross-reactivity pattern of structurally related compounds in the aqueous assay was different from incubations using toluene as the solvent (Table 2). The enantioselectivity was reduced: A cross-reactivity of 17% of the R enantiomer was recorded when the incubation was performed using sodium citrate buffer. In contrast, substrate selectivity for propranolol in the presence of other β-blockers was higher than in toluene (Figure 7). The cross-

for the other β-blockers. The limit of determination of (S)propranolol was 10 nM. The enantioselectivities of MIPs C and D, made using TRIM as the cross-linking monomer, were lower than that recorded for the EGDMA-based MIP A (Table 2). The cross-reactivity values of MIPs C and D for the R form, 30 and 45%, respectively, were closer to those of MIP B than MIP A. Both polymers showed substrate selectivity patterns similar to that of MIP A. The limits of determination were found to be 42 and 24 nM for assays performed using polymers C and D, respectively.

Figure 6. Binding of propranolol to MIP A (open squares) and the corresponding nonimprinted reference polymer (filled squares) as a function of ionic strength. Radiolabeled (S)-propranolol (1.2 pmol) and 25 µg of polymer particles were incubated in 1 mL of 25 mM citrate buffer (pH 6.0) containing 2% (v/v) ethanol and varying concentrations of NaCl. The specific binding (open circles) is B/T on the imprinted polymer minus B/T on the nonimprinted polymer.

Figure 7. Displacement of [3H]-(S)-propranolol binding to MIP A in 25 mM citrate (pH 6.0) containing 2% (v/v) ethanol, by increasing concentrations of competing ligand. B/B0 is the ratio of the amount of radiolabeled (S)-propranolol bound in the presence of displacing ligand, B, to the amount bound in the absence of displacing ligand, B0. Displacing ligands: (open squares) (S)-propranolol; (filled squares) (R)-propranolol; (filled circles) (R,S)-metoprolol; (open triangles) (R,S)timolol; (open circles) (R,S)-atenolol.

reactivities of metoprolol, timolol, and atenolol were in all cases below 1%. The limit of determination for the (S)-propranolol assay was found to be 6.0 nM. Scatchard plot analysis yielded apparent equilibrium association constants of (2.5 ( 0.20) × 108 (KD ) 4.0 nM) and (2.4 ( 1.1 × 105) M-1 (KD ) 4.1 µM), associated with binding site populations of 0.63 ( 0.62 and 28 ( 4.5 µmol/g, respectively. MIP B, made in the presence of (R,S)-propranolol, showed an approximately 2-fold reduction in enantioselectivity, as measured by unlabeled propranolol displacement of [3H]-(S)-propranolol binding, compared with MIP A (Table 2). In this instance, the R enantiomer showed 35% cross-reactivity. Whereas the IC50 value for (S)-propranolol remained about the same as that recorded on MIP A, the IC50 value for (R)-propranolol decreased; i.e., MIP B exhibits higher affinity than MIP A for (R)-propranolol. Levels of cross-reactivity equal to those obtained for MIP A were recorded

DISCUSSION It has only very recently been demonstrated that imprinted polymers actually show selective ligand binding in aqueous buffers.22 The objective of this study was to further investigate aqueous ligand binding and to improve the performance of the aqueous-based assay. The influences on the ligand binding of critical parameters, such as pH, ionic strength, buffer concentration, and content of organic modifier, were studied. Such knowledge forms a basis for strategies for further optimization of the conditions for high-affinity, highly specific ligand binding. The conclusions of the present findings are that the workable region for efficient (S)-propranolol binding are the following: the presence of 2-16% ethanol, a buffer concentration above 5 mM, and pH between 5 and 8, at least when the level of nonspecific binding is recorded. Similarly high sensitivity was obtained with the aqueous assay as with the solvent-based assay, with limits of determination as low as 6 nM. The concentration ranges covered by the calibration graphs could be shifted to 1000- and 100-fold lower concentrations for the water and toluene assays, respectively, compared with previous achievements using MIPs.21,22 With regard to the ability to detect low concentrations of (S)-propranolol, the improvements made here place the aqueous and organic solvent based assays using MIPs on the same level as immunoassays using biological antibodies.34-38 Further work to improve the sensitivity of the aqueous assay may be based on the observation that the ratio of specific to nonspecific binding increases with increasing concentrations of ethanol and buffer (Figures 4 and 5). Hence, despite the fact that the total response is reduced, the portion that is sensitive to analyte concentration increases, which probably lead to an improvement of the overall assay performance. This study emphasizes, however, the possibility of using MIPs in ligand binding experiments using buffers with low concentrations of organic modifier, and for propranolol the use of 2% of ethanol was necessary to avoid its adsorption to the surfaces of tubes, pipet tips, etc. The amounts of MIP used per assay have been reduced dramatically, from 1-10 mg21,22 to 50 µg and below. Hence, a standard batch size MIP preparation which yields approximately 5 g of polymer is sufficient for more than 100 000 individual assays. The key consideration of MIP synthesis is the availability of the imprint molecule in sufficient quantity and its solubility in the polymerization mixture. As the MIP production cost is divided (34) Kawashima, K.; Levy, A.; Spector, S. J. Pharmacol. Exp. Ther. 1976, 196, 517-523. (35) Mould, G. P.; Clough, J.; Morris, B. A.; Stout, G.; Marks, V. Biopharm. Drug Dispos. 1981, 2, 49-57. (36) Al-Hakiem, M. H. H.; White, G. W.; Smith. D. S.; Landon, J. Ther. Drug Monit. 1981, 3, 159-165. (37) Eller, T. D.; Knapp, D. R.; Walle, T. Anal. Chem. 1983, 55, 1572-1575. (38) Sahui-Gnassi, A.; Pham-Huy, C.; Galons, H.; Warnet, J.-M.; Claude, J.-R.; Duc, H.-T. Chirality 1993, 5, 448-454.

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over a very high number of analyses, the cost of the imprint species is a minor issue. Considering the ease with which such a preparation is made, the availability of MIPs with consistent quality should not cause any problems in the implementation of a MIP-based ligand binding assay. The key step of noncovalent molecular imprinting is the polymerization of monomers around an imprint molecule.1-3 A rigid bulk polymer is obtained which, after removal of the imprint species, yields a material containing imprints that exhibit a “memory”, in terms of complementarity of both shape and chemical functionality, for the original imprint molecule. The main constituent of the polymerization mixture is the cross-linking monomer that forms the bulk of the resultant polymer backbone. Previous descriptions of the preparation of noncovalent molecular imprints have predominantly involved the use of ethylene glycol dimethacrylate (EGDMA) as the cross-linking monomer. Recently, a cross-linking monomer, trimethylol propane trimethacrylate (TRIM) containing three polymerizable units was utilized in noncovalent molecular imprinting.29 When used as a chiral stationary phase in liquid chromatography, such polymers were shown to exhibit improved load capacity, selectivity, and resolving capacity compared to EGDMA-based MIPs. In this present study, the enantioselectivity of the EGDMA-derived MIPs was superior to the TRIM-type MIPs. Chromatography is a dynamic experiment in contrast to a radioligand binding assay, which measures the equilibrium situation, and the MIP syntheses should be optimized accordingly. Chromatographic separations rely on multiple binding and release events using easily accessible imprinted sites and require sorbents with good flow characteristics and low diffusion resistance. Selective and sensitive ligand binding assays necessitate the presence of high-affinity, highly selective imprints. The MIPs exhibit sufficient affinity to allow the development of a radiolabeled-ligand binding assay for the detection of clinically significant levels of propranolol. In pharmacokinetic studies, the relevant concentrations are within 5-200 nM,27,28,35 which lie within the calibration graph of this assay (Figures 2 and 7). The fact that the incubation of polymer particles, radioligand, and analyte can be performed equally well using an organic solvent or an aqueous buffer adds flexibility to the development of the assay method. In many environmental and bioanalytical methods, a solvent extraction step is included in the sample workup process. In such situations, the assay described here can be performed directly after the extraction, thus eliminating the need for evaporation of the solvent and reconstitution of the analyte in the assay buffer. Furthermore, some analytes are very poorly soluble in water, causing problems with adsorption to surfaces exposed to the sample, such as pipet tips, vessels, etc. With these difficulties in mind, a few promising attempts have been made to use antibodies in organic solvents,39-42 although with limited success as yet. The solvent-based assay showed excellent enantioselectivity, the R enantiomer exhibiting just 1% cross-reactivity in the best case (Figure 2). Several propranolol immunoassays have been (39) Russell, A. J.; Trudel, L. J.; Skipper, P. L.; Groopman, J. D.; Tannenbaum, S. R.; Klibanov, A. M. Biochem. Biophys. Res. Commun. 1989, 158, 80-85. (40) Sto ¨cklein, W.; Gebbert, A.; Schmid, R. D. Anal. Lett. 1990, 23, 1465-1476. (41) Weetall, H. H. J. Immunol. Methods 1991, 136, 139-142. (42) Matsuura, S.; Hamano, Y.; Kita, H.; Takagaki, Y. J. Biochem. 1993, 114, 273-278.

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described previously.34-38 A few of these are stereoselective for the active enantiomer,34,38 but less so than our novel MIP assay. The cross-reactivities of the R enantiomer in these assays are between 5 and 7%. In contrast, the MIP buffer based assay was highly substrate selective for propranolol in the presence of structurally very similar substances (Figure 7), whereas the enantioselectivity under these conditions was low. The different selectivity profiles obtained are due to a different balance between hydrophobic and polar interactions in solvent and water. The aminopropanediol part of the structure is identical for all β-blockers, and the individual drugs differ by their ether substituent (Figure 1), which is R-naphthyl for propranolol. Enantiorecognition requires sensing the configuration of the hydroxy, aminomethyl, and oxymethyl functionalities on the chiral carbon. This involves hydrogen bonding between the chemical functionalities surrounding the chiral carbon and methacrylic acid residues of the polymer. Such polar interactions are strong in apolar solvents, and consequently, the best enantioselectivity is obtained for the solvent-based assay. Substrate recognition requires confirmation of the presence of the aminopropanediol part and the naphthyl ring structure. The interactions of the polymer with the aromatic naphthyl ring system are primarily hydrophobic interactions. These are strong in the presence of water and weak in solvents, which explains the higher substrate selectivity of the water-based assay. In addition, steric factors may contribute to both types of recognition. A MIP was made against the racemate of propranolol in order to investigate the stereospecific binding of labeled S enantiomer to a population of equal numbers of R- and S-selective sites. High enantioselectivity was recorded, although this MIP was less selective than the pure S-enantiomer-imprinted MIP. Whereas the IC50 value of the S enantiomer remained virtually the same, the cross-reactivity of the R form increased 2-fold. This demonstrates that enantioselective ligand binding is possible on a mixture of imprints, as long as the radioligand is optically pure. The radioligand preferentially binds to the imprints that are complementary to the S configuration, and hence the competition events occur primarily in these sites. This finding may be utilized in situations where the pure enantiomer is difficult to obtain in, for instance, gram-scale amounts for imprinting. Due to the small quantities of radioligand required, this may, on the other hand, be more easily obtained. The findings presented here demonstrate it is possible to apply molecular imprinting to the development of radioligand binding assays, which are conceptually similar to immunoassay. MIPs provide a combination of polymer mechanical and chemical robustness with highly selective molecular recognition comparable to biological systems. The selectivity of a MIP is predetermined by the choice of imprint species used during its preparation. The incubation of imprinted antibody mimics, radioligand, and analyte can be performed equally well using a toluene-based solvent or an aqueous buffer, which may enable extension of immunoassay technology to new environments and substances insoluble in water. Optimization of the assay conditions for both the solventand water-based assays led to 2-3 orders of magnitude improvements in assay sensitivity, compared with previous achievements with MIP-based ligand binding assays. Further increases in assay sensitivity may follow the use of other labeling techniques and detection systems, such as those based on recording fluorescence and enzymatic reactions.

Imprint-derived antibody mimics may be valuable complements to biological antibodies in many circumstances. Antibody preparation against low molecular weight compounds, so-called haptens, necessitates conjugation of the hapten to a carrier protein, which occasionally presents a synthetic challenge, before injection into the animal. Conjugation often changes the structural properties of the antigen exposed to the immune system, and the antibodies elicited may be directed to a different structure than desired. The MIP preparation avoids the need for derivatization of haptenic antigens, which may result, as shown here, in superior enantioselectivity of the artificial system.

ACKNOWLEDGMENT We thank Dr. R. Ansell for linguistic advice. This investigation was supported by the Swedish National Board for Laboratory Animals (CFN), the Swedish Fund for Research without Animal Experiments, and the Bertil Andersson Fund. Received for review July 6, 1995. Accepted October 16, 1995.X AC950668+ X

Abstract published in Advance ACS Abstracts, December 1, 1995.

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