Subpicomolar Sensing of δ-Opioid Receptor Ligands by Molecular

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Subpicomolar Sensing of δ-Opioid Receptor Ligands by Molecular-Imprinted Polymers Using Plasmon-Waveguide Resonance Spectroscopy Savitha Devanathan,† Zdzislaw Salamon,† Anoop Nagar,‡ Subhash Narang,‡ Donald Schleich,§ Paul Darman,| Victor Hruby,†,⊥ and Gordon Tollin*,†,⊥

Department of Biochemistry and Molecular Biophysics and Department of Chemistry, University of Arizona, Tucson, Arizona 85721, Product Development Laboratory, SRI International, Menlo Park, California 94025, and Nasotronics Inc., Palo Alto, California 94303

Here we report, for the first time, the formation of a biomimetic covalently imprinted polymeric sensor for a target ligand, the δ-opioid G-protein coupled receptor agonist DPDPE, which reproducibly exhibits subpicomolar binding affinity in an aqueous environment. In addition to having a well-defined and homogeneous binding site, the imprinted polymer template is quite stable to storage in both the dry and wet states and has at least 6 orders of magnitude higher affinities than exhibited by similar peptide-based molecular-imprinted polymers (MIPs) thus far. A highly sensitive optical detection methodology, plasmon-waveguide resonance spectroscopy, was employed, capable of measuring binding in real time and discriminating between ligand molecules, without requiring labeling protocols (fluorophores or radioisotopes). The DPDPE-imprinted polymer showed a broad structureactivity relationship profile, not unlike that found for protein receptors. Such sensitivity and robustness of MIPs suggests potential applications ranging from biowarfare agent detection to pharmaceutical screening. Host-guest binding chemistries have been applied in the design of synthetic materials selectively recognizing biological molecules1-3 for applications as antibody mimics,4,5 for enantiomeric separations,6 to create artificial enzyme transition-state * Corresponding author. E-mail: [email protected]. Phone: (520) 6213447. Fax: (520) 621-9288. † Department of Biochemistry and Molecular Biophysics, University of Arizona. ‡ SRI International. § Nasotronics Inc. | This paper is dedicated to Paul Darman, who died unexpectedly following an accident, and was the first to suggest that PWR spectroscopy might be used to examine the interactions of bioactive peptide ligands with imprinted polymers that would act as “receptors”. ⊥ Department of Chemistry, University of Arizona. (1) Zimmerman, S. C.; Wendland, M. S.; Rakow, N. A.; Zharov, I.; Suslick, K. S. Nature 2002, 418, 399-403. (2) Shi, H.; Tsai, W.-B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593-597. (3) Looger, L. L.; Dwyer, M. A.; Smith, J. J.; Hellinga, H. W. Nature 2003, 423, 185-193. (4) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645-647. (5) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (6) Ekberg, B.; Mosbach, K. Trends Biotechnol. 1989, 7, 92-96. 10.1021/ac048476e CCC: $30.25 Published on Web 03/11/2005

© 2005 American Chemical Society

analogues,7 and to function in chemical and biological sensing assays8 and in the development of biomedical materials.9,10 Molecular imprinting of polymeric materials has become a rapidly growing technique for mimicking such molecular recognition systems.11,12 This process involves polymerization and cross-linking of functional monomers in the presence of a target molecule. The memory or “imprint” of this compound in terms of complementarity of chemical functionality and shape is conserved after the polymerization and cross-linking and subsequent removal of the “imprinted” ligand. The molecular cavity, having exposed functional recognition sites that are structurally stabilized by the crosslinked polymeric matrix, retains the ability to rebind the imprinted species, ideally with high specificity and affinity. Molecular imprinted polymers (MIPs) can have several binding sites of varying size and are rigid and often insoluble. Most previous studies with MIPs have been in the vapor phase or have used organic solvent environments. Furthermore, such sensors have been limited by low selectivities and sensitivities. Other sensors exhibiting good sensitivities are limited by the thickness of their polymer membranes (several micrometers thick), and additionally by exhibiting a heterogeneous population of binding sites with different affinities, which affects the response time of binding.13,14 MIPs have a long and successful history as the foundation for detection of both organic and inorganic molecules.12 They are designed by employing a qualitative consideration of the potential recognition sites on a target molecule. In general terms, one determines the likely “keys” on a target and then rationally designs complementary “locks”. In previous work in our laboratories aimed at designing a MIP for a chemical warfare precursor, thiodiglycol, we were able to unambiguously detect the target of interest while at the same time rejecting very closely related structural challengers such as thioxane (Narang, S., unpublished data). Thio(7) Lerner R. A.; Benkovic, S. J.; Schultz, P. G. Science 1991, 252, 659-667. (8) Byfield, M. P.; Abuknesha, R. A. Biosens. Bioelectron. 1994, 9(4-5), 373400. (9) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 837-850. (10) Ratner B. D. J. Mol. Recognit. 1996, 9, 617-625. (11) Mosbach, K.; Ramstrom, O. Bio-technology 1996, 14, 163-170. (12) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495-2504. (13) Sergeyeva, T. A.; Piletsky, S. A.; Brovko, A. A.; Slinchenko, E. A.; Sergeeva, L. M.; Panasyuk, T. L.; El’skaya, A. V. Analyst 1999, 124, 331-334. (14) Jenkins, A. L.; Manuel Uy O.; Murray, G. M. Anal. Chem. 1999, 71, 373378.

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diglycol was detected at 13 ppb while the interference thioxane was barely seen at 60 ppm, a concentration almost 5000 times higher. Other interferences, differing in some cases by only one atom, were not seen at all. Thus, we have created in the polymer a cavity the approximate size and shape solely of the specific target that also contains recognition elements, or binding sites, in the proper three-dimensional spatial disposition, closely resembling biological sensors such as antibodies or receptors. An important point to note is that the MIPs for thioglycol were developed for the vapor phase, whereas in the present work, a similar highly effective imprinting strategy was adopted for an aqueous environment. Understanding molecular recognition phenomena15 in biological systems is key in the building of novel materials mimicking biological functions and in the design and synthesis of artificial receptor molecules or analyte detecting sensors. In the present study, we have used as a model a G-protein coupled receptor (GPCR), the human δ-opioid receptor, a member of the superfamily of 7-transmembrane helix integral membrane proteins that are the targets for numerous peptide hormones, neurotransmitters, and other critical molecules required for intercellular communication in many functions critical to life, such as the fearflight response, stress response, and glucose homeostasis. The human δ-opioid receptor is present in the brain and is involved in modulation of pain perception and addiction, mediating analgesic responses to endogenous enkephalins, and a variety of other pharmacophores.16-18 It is well known that ∼50% of pharmaceutical drugs presently target the GPCRs. We have used a highly sensitive detection platform called plasmon-waveguide resonance (PWR) spectroscopy,19-24 that allows kinetic, thermodynamic, and structural information to be rapidly obtained using small sample sizes and a wide concentration range from subpicomolar to millimolar or higher. It is based upon intrinsic molecular properties (refractive index, optical extinction coefficient, film thickness), and unlike many other methods of investigating receptor-ligand interactions, molecular labeling strategies are not required. PWR uses a polarized continuous wave laser (electric vector either perpendicular, p-polarization, or parallel, s-polarization, to the resonator surface) to excite electromagnetic oscillations in a silver and silica layer deposited onto a glass prism. Interaction of molecules immobilized at the outer silica surface with this field changes the resonance characteristics and allows the characterization of anisotropic optical properties of oriented molecules, thereby permitting structural and confor(15) Cram, D. J. Science 1988, 240, 760-767. (16) Iyengar, R., Hildebrandt, J. D., Eds. G-protein Pathways; Methods in Enzymology Vols. 343, 344, and 345; Academic Press: New York, 2002. (17) Hruby, V. J.; Al-Obeidi, F.; Kazmierski, W. M. Biochem. J. 1990, 268, 249262. (18) Hruby, V. J.; Mosberg, H. I. In Delta Receptor, Ligands, Pharmacology and Physiology, Chang, K. I., Porreca, F., Woods, J. H., Eds.; Mercel Dekker: New York, 2003; pp 159-174. (19) Salamon, Z.; Macleod, H. A.; Tollin, G. Biophys. J. 1997, 73, 2791-2797. (20) Salamon, Z.; Tollin, G. Encyclopedia of Spectroscopy and Spectrometry; Academic Press: New York, 1999; Vol. 3, pp 2311-2319. (21) Salamon, Z.; Tollin, G. Encyclopedia of Spectroscopy and Spectrometry; Academic Press: New York, 1999; Vol. 3, pp 2294-2302. (22) Salamon, Z.; Brown, M. F.; Tollin, G. Trends Biochem. Sci. 1999, 24, 213219. (23) Salamon, Z.; Cowell, S.; Varga, E.; Yamamura, H. I.; Hruby, V. J.; Tollin, G. Biophys. J. 2000, 79, 2463-2474. (24) Salamon, Z.; Hruby, V. J.; Tollin, G.; Cowell, S. M. J. Pept. Res. 2002, 60, 322-328.

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Table 1. Chemical Structures of Ligands

mational changes to be clearly distinguished from mass density changes. Two PWR spectra (plots of reflected light intensity vs incident angle) can be obtained using these two polarizations that can thereby characterize the anisotropic optical properties of the immobilized molecules. Spectral changes caused by molecular immobilization at the resonator surface can be followed in real time, and with high sensitivity (only femtomole quantities of material are needed), and binding constants can be directly determined. In previous studies, we have used this technique to determine structure-activity relations for opiate ligand binding to the human δ-opioid receptor incorporated into lipid bilayers.23-25 These have provided a benchmark for comparison to the present studies utilizing MIPs. The goal of the present study was to design a MIP that can closely mimic the overall traits of such biological macromolecules. The specific opiate ligand chosen for imprinting, DPDPE, is a synthetic cyclic analogue of enkephalin that is conformationally and topographically constrained, with high potency and selectivity for the δ-opioid receptor and used widely in the study of pain behavior26 (Table 1). As will be demonstrated below, we have been successful in creating a unique MIP with a thickness on the order of a few hundred Å, that is capable of recognizing DPDPE with very high selectivity, reproducibility, and affinity. MATERIALS AND METHODS Engineering a Template for Specific DPDPE Recognition. DPDPE (10 mg) was dissolved in deionized water (3.33 gm) via (25) Alves, I. D.; Cowell, S.; Salamon, Z. S.; Devanathan, S.; Tollin, G.; Hruby, V. J. Mol. Pharmacol. 2004, 65, 1248-1257. (26) Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.; Burks, T. F. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5871-5874.

sonication (Cole Parmer, 8890 Sonicator) for 10 min. Separately, the imprinting polymer mixture (9.5 mg) (comprising n-vinylpyrollidone, 1-hexadecene, and 1-decene; 1:0.8:0.2 mol %) and (3acryloxypropyl)trimethoxysilane (0.5 mg) were dissolved in 1-butanol (16.67 g). The long-chain alkenes were chosen to obtain the correct hydrophobic/hydrophilic balance between the hydrophilic pyrollidone ring and the hydrophobic alkane pendant groups, keeping in mind the 1:1 mole ratio of pyrollidone to alkane pendant groups. Shorter alkyl chains led to coatings that were too hydrophilic and thus swelled too much and delaminated quickly in the sensing environment. Longer alkyl chains provided coatings that were excessively hydrophobic and would show low or no interaction with the aqueous sensing environment. A crosslinking agent such as ethylene glycol diacrylate (2 mol %) was added to the monomer mix to improve the mechanical integrity of the films, along with Irgacure-1700 (2 wt %) (Ciba Specialty Chemicals), which was used as the photoinitiator. Silyl coupling agents in the mixture provided improved adhesion of the films to the silica substrate of the prism surface due to covalent coupling. This is accomplished by allowing the monomer solution to age overnight, whereby the silyl agent starts to slowly polymerize. The DPDPE solution and the polymer solution were mixed with stirring for 30 min. The prisms were cleaned by washing with chloroform and deionized water, dried under ambient conditions, and treated with an oxygen plasma (Harrick, PDC-3XG) for 1 min. The prism was mounted on a custom-made polysiloxane mold. The prism-mounted mold was in turn mounted on the vacuum chuck of a spin coater (Integrated Technologies Inc., model P-6000). The prism was spun at 1500 rpm, and the imprinted polymer solution (100 µL) was applied dropwise at the center of the spinning prism. Spinning continued for an additional 90 s, followed by drying for 1 h at room temperature in a dust-free environment. The monomer mixture was photopolymerized using low-intensity UV radiation for several minutes (Figure 1a). The rotational speed and polymeric conditions were chosen after trial with several combinations to obtain the best optical quality films, with thicknesses on the order of a few hundred Å. The imprinted ligand (Figure 1b) was leached out of the films by repeated flushing of the imprinted polymer film by buffer solution prior to the addition of a specific ligand to be bound. This resulted in swelling of the film and an increase of accessible polymer pores to remove the imprinting molecule. PWR spectra were obtained for the PWR resonator prisms before and after coating with the polymer, and the leaching process was monitored as a function of changes in s- and p-polarized PWR spectra until no further changes were obtained. In addition to having a well-defined and homogeneous binding site, the imprinted polymer was quite stable to storage in both dry and wet states. Ligands. The ligands were obtained as follows: DPDPE (American Peptide Co.), deltorphin-II (Tocris Inc., Ellisville, MO), DADLE and (-) isoproterenol (Sigma Chemical Co., St. Louis, MO), TIPP-psi (obtained from NIDA to V.H.), Tan67 (Toray Industries, Kumakura, Japan). NDP-R MSH,27 TMT-Tic,28 and [Gln4]-deltorphin II were synthesized in the Hruby laboratory. The structures of these ligands are presented in Table 1. (27) Sawyer, T. K.; Sanfilippo, P. J.; Hruby, V. J.; Engel, M. H.; Heward, C. B.; Burnett, J. B.; Hadley, M. E. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 57545758.

Figure 1. (a) Schematic synthesis route of DPDPE-MIP. (b) Schematic representation of DPDPE-MIP.

PWR Spectroscopy. The theoretical principles and experimental implementation of plasmon-waveguide resonance spectroscopy have been thoroughly described in previous publications.19-22 Briefly, PWR spectra can be described by three parameters, the spectral position, the spectral half-width, and the resonance depth. These experimental features depend on the optical properties of the polymeric film along the two dimensions (parallel and perpendicular to the resonator surface). This allows the characterization of anisotropic systems that are deposited on the resonator surface and changes in conformation occurring as a consequence of interactions. The area of the prism probed by the laser beam at a given time corresponds to ∼0.2 mm cross section. PWR spectra for s- and p-polarization were measured as a plot of reflectance versus incident angle using a prototype instrument obtained from Proterion Corp. (Piscataway, NJ) with an angular resolution of 1 mdeg. The imprinted polymer on the resonator surface was first equilibrated with an aqueous buffer in the PWR sample cell. Equilibration occurred over a period of 30 min and was observed as changes in the resonance spectral angle with time (not shown). The equilibration process can be likened to a swelling phenomenon due to buffer interaction with the polymeric pores, which suggests the existence of a degree of flexibility and dynamic movements within the imprinted polymeric network. After equilibration was achieved, ligand binding profiles to the polymeric matrix were obtained by measuring PWR Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

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RESULTS AND DISCUSSION PWR resonance spectra obtained for the buffer-equilibrated MIP and after subsequent binding of saturating amounts of DPDPE are shown in Figure 2. Binding was observed as spectral shifts to larger angular positions for both p- and s-polarization, indicative of an increase in film mass (i.e., refractive index) and thickness upon DPDPE addition. The magnitude of the shifts of the p-polarized resonance minimums were larger than those for the s-polarized resonances, which reflects an increase in structural anisotropy within the polymer matrix.29 Ligand binding curves for DPDPE are shown in Figure 3a. The subpicomolar binding constant (Table 2) was obtained by fitting the p- and s-polarization data to a hyperbolic function characterizing a 1:1 binding interaction and averaging the two values. Since the spectral shift is directly proportional to the bound ligand, and the amount of ligand bound is much smaller than the bulk ligand concentration, this process yields a true thermodynamic binding constant. The hyperbolic fit for both polarizations was quite good and can be attributed to a homogeneous population of binding sites. Nonspecific binding was not observed in these

studies even with DPDPE concentrations that were increased into the millimolar range. DPDPE binding to the imprinted polymeric thin films was tested with five separate prisms having the same polymer formulation and were found to be reproducible with a range of KD values from 0.37 to 0.42 pM, under the same experimental conditions. The on-rate of ligand binding was quite rapid (106 1.9 ( 3.9 17.2 ( 5.9 8.1 ( 2.0 11.9 ( 3.9 3.05 ( 0.35 8.13 ( 2.35 114.5 ( 24.7

16 ( 4 1.2 ( 0.2 0.06 ( 0.02 520 ( 30 3.4 ( 1.3 2.8 ( 0.3 2.6 ( 0.2b ndc nd

5.2 ( 1.2

nd

a K values for the MIPs are an average between hyperbolic curves D obtained with s- and p-polarization. References 23-25. b Reference 34. c nd, not determined since these are not biologically relevant to this receptor.

of the binding specificity rather than in an elimination of binding of the nonimprinted ligand. CONCLUSIONS The results described above demonstrate the ability of a macromolecular template to generate artificial high-affinity binding (33) Andersson, L. I.; Mu ¨ ller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. 1995, 92, 4788-4792. (34) Law, P.-Y.; Hom, D. S.; Loh, H. H. J. Biol. Chem. 1985, 260 (6), 35613569.

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sites in a well-defined binding pocket that mimics those found in native proteins. The binding affinities observed are as high or even higher than those found with the natural δ-opioid receptor. Previous results utilizing MIPs imprinted to enkephalin33 yielded heterogeneous sites with multiple binding affinities in the low- to high-micromolar range. Future extensions of this work provide a unique opportunity to explore innovative avenues in molecular sensing, such as application-oriented MIPs that can be used as arrays, as well as disposable sensor chips for field use. Such platforms have wide potential applications, most notably in environmental screening, drug development, therapeutics, and medical monitoring. Abbreviations: DPDPE,c-[D-Pen2, D-Pen5]enkephalin; GPCR, G-protein coupled receptor; MIP, molecular-imprinted polymer; NDP-RMSH, (Ac-[Nle4-DPhe7]R-melanotropin-NH2); PWR, plasmon-waveguide resonance; TIPPpsi, H-L-Tyr-L-Tetrahydroisoquinoline-3-carboxylic acid-[CH2-NH]-Phe-Phe-OH; TMT-Tic, (2S,3R) β-methyl-2′,6′-dimethyltyrosyl-tetrahydroisoquinoline-3-carboxylic acid. ACKNOWLEDGMENT This work was supported in part by a contract from DST/ATP (2001-H557900-000) to V.J.H, G.T., and S.N.. We thank Dr. Scott Cowell for the synthesis of the Gln4-deltorphin II ligand, Isabel Alves for providing the other ligands used in this study, and Philippe Guerit for Figure 1b.

Received for review October 14, 2004. Accepted February 4, 2005. AC048476E