Subpicomolar Sensing of δ-Opioid ReceptorLigands by Molecular-Imprinted Polymers UsingPlasmon-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, andNasotronics Inc., Palo Alto, California 94303

Here we report, for the first time, the formation of abiomimetic covalently imprinted polymeric sensor for atarget ligand, the δ-opioid G-protein coupled receptoragonist DPDPE, which reproducibly exhibits subpicomo-lar binding affinity in an aqueous environment. In additionto having a well-defined and homogeneous binding site,the imprinted polymer template is quite stable to storagein both the dry and wet states and has at least 6 orders ofmagnitude higher affinities than exhibited by similarpeptide-based molecular-imprinted polymers (MIPs) thusfar. A highly sensitive optical detection methodology,plasmon-waveguide resonance spectroscopy, was em-ployed, capable of measuring binding in real time anddiscriminating between ligand molecules, without requir-ing labeling protocols (fluorophores or radioisotopes). TheDPDPE-imprinted polymer showed a broad structure-activity relationship profile, not unlike that found forprotein receptors. Such sensitivity and robustness ofMIPs suggests potential applications ranging from bio-warfare agent detection to pharmaceutical screening.

Host-guest binding chemistries have been applied in thedesign of synthetic materials selectively recognizing biologicalmolecules1-3 for applications as antibody mimics,4,5 for enantio-meric separations,6 to create artificial enzyme transition-state

analogues,7 and to function in chemical and biological sensingassays8 and in the development of biomedical materials.9,10

Molecular imprinting of polymeric materials has become a rapidlygrowing technique for mimicking such molecular recognitionsystems.11,12 This process involves polymerization and cross-linkingof functional monomers in the presence of a target molecule. Thememory or “imprint” of this compound in terms of complemen-tarity of chemical functionality and shape is conserved after thepolymerization and cross-linking and subsequent removal of the“imprinted” ligand. The molecular cavity, having exposed func-tional recognition sites that are structurally stabilized by the cross-linked polymeric matrix, retains the ability to rebind the imprintedspecies, ideally with high specificity and affinity. Molecularimprinted polymers (MIPs) can have several binding sites ofvarying size and are rigid and often insoluble. Most previousstudies with MIPs have been in the vapor phase or have usedorganic solvent environments. Furthermore, such sensors havebeen limited by low selectivities and sensitivities. Other sensorsexhibiting good sensitivities are limited by the thickness of theirpolymer membranes (several micrometers thick), and additionallyby exhibiting a heterogeneous population of binding sites withdifferent affinities, which affects the response time of binding.13,14

MIPs have a long and successful history as the foundation fordetection of both organic and inorganic molecules.12 They aredesigned by employing a qualitative consideration of the potentialrecognition sites on a target molecule. In general terms, onedetermines the likely “keys” on a target and then rationally designscomplementary “locks”. In previous work in our laboratories aimedat designing a MIP for a chemical warfare precursor, thiodiglycol,we were able to unambiguously detect the target of interest whileat the same time rejecting very closely related structural chal-lengers such as thioxane (Narang, S., unpublished data). Thio-

* Corresponding author. E-mail: gtollin@u.arizona.edu. Phone: (520) 621-3447. Fax: (520) 621-9288.

† Department of Biochemistry and Molecular Biophysics, University ofArizona.

‡ 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 usedto examine the interactions of bioactive peptide ligands with imprinted polymersthat 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.; Muller, 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.

(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), 373-

400.(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, 373-

378.

Anal. Chem. 2005, 77, 2569-2574

10.1021/ac048476e CCC: $30.25 © 2005 American Chemical Society Analytical Chemistry, Vol. 77, No. 8, April 15, 2005 2569Published on Web 03/11/2005

diglycol was detected at 13 ppb while the interference thioxanewas barely seen at 60 ppm, a concentration almost 5000 timeshigher. Other interferences, differing in some cases by only oneatom, were not seen at all. Thus, we have created in the polymera cavity the approximate size and shape solely of the specific targetthat also contains recognition elements, or binding sites, in theproper three-dimensional spatial disposition, closely resemblingbiological sensors such as antibodies or receptors. An importantpoint to note is that the MIPs for thioglycol were developed forthe vapor phase, whereas in the present work, a similar highlyeffective imprinting strategy was adopted for an aqueous environ-ment.

Understanding molecular recognition phenomena15 in biologi-cal systems is key in the building of novel materials mimickingbiological functions and in the design and synthesis of artificialreceptor molecules or analyte detecting sensors. In the presentstudy, we have used as a model a G-protein coupled receptor(GPCR), the human δ-opioid receptor, a member of the super-family of 7-transmembrane helix integral membrane proteins thatare the targets for numerous peptide hormones, neurotransmit-ters, and other critical molecules required for intercellular com-munication in many functions critical to life, such as the fear-flight response, stress response, and glucose homeostasis. Thehuman δ-opioid receptor is present in the brain and is involvedin modulation of pain perception and addiction, mediating anal-gesic responses to endogenous enkephalins, and a variety of otherpharmacophores.16-18 It is well known that ∼50% of pharmaceuticaldrugs presently target the GPCRs.

We have used a highly sensitive detection platform calledplasmon-waveguide resonance (PWR) spectroscopy,19-24 that al-lows kinetic, thermodynamic, and structural information to berapidly obtained using small sample sizes and a wide concentrationrange from subpicomolar to millimolar or higher. It is based uponintrinsic molecular properties (refractive index, optical extinctioncoefficient, film thickness), and unlike many other methods ofinvestigating receptor-ligand interactions, molecular labelingstrategies are not required. PWR uses a polarized continuous wavelaser (electric vector either perpendicular, p-polarization, orparallel, s-polarization, to the resonator surface) to excite electro-magnetic oscillations in a silver and silica layer deposited onto aglass prism. Interaction of molecules immobilized at the outersilica surface with this field changes the resonance characteristicsand allows the characterization of anisotropic optical propertiesof oriented molecules, thereby permitting structural and confor-

mational changes to be clearly distinguished from mass densitychanges. Two PWR spectra (plots of reflected light intensity vsincident angle) can be obtained using these two polarizations thatcan thereby characterize the anisotropic optical properties of theimmobilized molecules. Spectral changes caused by molecularimmobilization at the resonator surface can be followed in realtime, and with high sensitivity (only femtomole quantities ofmaterial are needed), and binding constants can be directlydetermined. In previous studies, we have used this technique todetermine structure-activity relations for opiate ligand bindingto the human δ-opioid receptor incorporated into lipid bilayers.23-25

These have provided a benchmark for comparison to the presentstudies utilizing MIPs.

The goal of the present study was to design a MIP that canclosely mimic the overall traits of such biological macromolecules.The specific opiate ligand chosen for imprinting, DPDPE, is asynthetic cyclic analogue of enkephalin that is conformationallyand topographically constrained, with high potency and selectivityfor the δ-opioid receptor and used widely in the study of painbehavior26 (Table 1). As will be demonstrated below, we have beensuccessful in creating a unique MIP with a thickness on the orderof a few hundred Å, that is capable of recognizing DPDPE withvery high selectivity, reproducibility, and affinity.

MATERIALS AND METHODSEngineering a Template for Specific DPDPE Recognition.

DPDPE (10 mg) was dissolved in deionized water (3.33 gm) via

(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, 249-

262.(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, 213-

219.(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.

(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.

Table 1. Chemical Structures of Ligands

2570 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

sonication (Cole Parmer, 8890 Sonicator) for 10 min. Separately,the imprinting polymer mixture (9.5 mg) (comprising n-vinylpy-rollidone, 1-hexadecene, and 1-decene; 1:0.8:0.2 mol %) and (3-acryloxypropyl)trimethoxysilane (0.5 mg) were dissolved in1-butanol (16.67 g). The long-chain alkenes were chosen to obtainthe correct hydrophobic/hydrophilic balance between the hydro-philic pyrollidone ring and the hydrophobic alkane pendantgroups, keeping in mind the 1:1 mole ratio of pyrollidone to alkanependant groups. Shorter alkyl chains led to coatings that weretoo hydrophilic and thus swelled too much and delaminatedquickly in the sensing environment. Longer alkyl chains providedcoatings that were excessively hydrophobic and would show lowor no interaction with the aqueous sensing environment. A cross-linking agent such as ethylene glycol diacrylate (2 mol %) wasadded to the monomer mix to improve the mechanical integrityof the films, along with Irgacure-1700 (2 wt %) (Ciba SpecialtyChemicals), which was used as the photoinitiator. Silyl couplingagents in the mixture provided improved adhesion of the films tothe silica substrate of the prism surface due to covalent coupling.This is accomplished by allowing the monomer solution to ageovernight, whereby the silyl agent starts to slowly polymerize.The DPDPE solution and the polymer solution were mixed withstirring for 30 min. The prisms were cleaned by washing withchloroform 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 vacuumchuck of a spin coater (Integrated Technologies Inc., modelP-6000). The prism was spun at 1500 rpm, and the imprintedpolymer solution (100 µL) was applied dropwise at the center ofthe spinning prism. Spinning continued for an additional 90 s,followed by drying for 1 h at room temperature in a dust-freeenvironment. The monomer mixture was photopolymerized usinglow-intensity UV radiation for several minutes (Figure 1a).

The rotational speed and polymeric conditions were chosenafter trial with several combinations to obtain the best opticalquality films, with thicknesses on the order of a few hundred Å.The imprinted ligand (Figure 1b) was leached out of the films byrepeated flushing of the imprinted polymer film by buffer solutionprior to the addition of a specific ligand to be bound. This resultedin swelling of the film and an increase of accessible polymer poresto remove the imprinting molecule. PWR spectra were obtainedfor the PWR resonator prisms before and after coating with thepolymer, and the leaching process was monitored as a functionof changes in s- and p-polarized PWR spectra until no furtherchanges were obtained. In addition to having a well-defined andhomogeneous binding site, the imprinted polymer was quite stableto 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 (TorayIndustries, Kumakura, Japan). NDP-R MSH,27 TMT-Tic,28 and[Gln4]-deltorphin II were synthesized in the Hruby laboratory. Thestructures of these ligands are presented in Table 1.

PWR Spectroscopy. The theoretical principles and experi-mental implementation of plasmon-waveguide resonance spec-troscopy have been thoroughly described in previous publica-tions.19-22 Briefly, PWR spectra can be described by threeparameters, the spectral position, the spectral half-width, and theresonance depth. These experimental features depend on theoptical properties of the polymeric film along the two dimensions(parallel and perpendicular to the resonator surface). This allowsthe characterization of anisotropic systems that are deposited onthe resonator surface and changes in conformation occurring asa consequence of interactions. The area of the prism probed bythe laser beam at a given time corresponds to ∼0.2 mm crosssection. PWR spectra for s- and p-polarization were measured asa plot of reflectance versus incident angle using a prototypeinstrument obtained from Proterion Corp. (Piscataway, NJ) withan angular resolution of 1 mdeg. The imprinted polymer on theresonator surface was first equilibrated with an aqueous buffer inthe PWR sample cell. Equilibration occurred over a period of 30min and was observed as changes in the resonance spectral anglewith time (not shown). The equilibration process can be likenedto a swelling phenomenon due to buffer interaction with thepolymeric pores, which suggests the existence of a degree offlexibility and dynamic movements within the imprinted polymericnetwork. After equilibration was achieved, ligand binding profilesto the polymeric matrix were obtained by measuring PWR

(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, 5754-5758.

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

Analytical Chemistry, Vol. 77, No. 8, April 15, 2005 2571

resonance spectral shifts for both p- and s-polarizations as afunction of increasing ligand concentration. Equilibrium dissocia-tion constants were obtained by fitting the experimental data to asingle hyperbolic function.

RESULTS AND DISCUSSIONPWR resonance spectra obtained for the buffer-equilibrated

MIP and after subsequent binding of saturating amounts ofDPDPE are shown in Figure 2. Binding was observed as spectralshifts to larger angular positions for both p- and s-polarization,indicative of an increase in film mass (i.e., refractive index) andthickness upon DPDPE addition. The magnitude of the shifts ofthe p-polarized resonance minimums were larger than those forthe s-polarized resonances, which reflects an increase in structuralanisotropy within the polymer matrix.29

Ligand binding curves for DPDPE are shown in Figure 3a.The subpicomolar binding constant (Table 2) was obtained byfitting the p- and s-polarization data to a hyperbolic functioncharacterizing a 1:1 binding interaction and averaging the twovalues. Since the spectral shift is directly proportional to the boundligand, and the amount of ligand bound is much smaller than thebulk ligand concentration, this process yields a true thermody-namic binding constant. The hyperbolic fit for both polarizationswas quite good and can be attributed to a homogeneous populationof binding sites. Nonspecific binding was not observed in these

studies even with DPDPE concentrations that were increased intothe millimolar range. DPDPE binding to the imprinted polymericthin films was tested with five separate prisms having the samepolymer formulation and were found to be reproducible with arange of KD values from 0.37 to 0.42 pM, under the sameexperimental conditions. The on-rate of ligand binding was quiterapid (<3 min), and not surprisingly, the off-rates were very slow(on the order of several hours) upon replacement of the ligand-containing buffer. However, after a single binding event (for allof the ligands used here), the polymeric film was no longerfunctional. This could be the result of disruption of the structureof the thin-film cavity upon recognition and release of the specificligand. During the testing phase of this project, some thickerpolymer formulations yielded larger anisotropy changes as wellas a greater magnitude of spectral shifts (∼25 mdeg) upon DPDPEbinding with nanomolar binding constants, and these formulationsallowed rebinding of the ligand at least three times with the samefilm, although the subsequent binding events resulted in incre-mentally smaller spectral shifts and larger KD values before finallybecoming nonfunctional.

The specificity of the DPDPE-imprinted template was testedby determination of the cross-reactivity to other structurally relatedopioid ligands (Table 1). The results are presented in Table 2. Asexpected, the tightest binding was observed for DPDPE, and arange of ∼103-fold in affinities (excluding deltorphin-II) wasobtained for interactions with the other ligands used here. It isinteresting that the overall range of affinities was comparable tothat observed for these ligands with the natural receptor (∼104-fold; Table 2), although the order of binding affinities was quitedifferent. This order difference is perhaps expected inasmuch asour polymer binding site was imprinted for the cyclically con-strained peptidomimetic DPDPE, whereas the biological receptorhas been optimized by evolution to bind to endogenous linearpeptides such as the enkephalins,30 rather than to a syntheticpseudopeptide ligand such as DPDPE.

The key recognition sites in DPDPE in the present MIP arelikely to be the tyrosine aromatic ring and its hydroxyl group,the phenyl ring of phenylalanine, and the positive charge on theamine nitrogen (Table 1), as they are in the δ-opioid receptor.18,31

These lead to cooperative multivalent interactions involvinghydrogen bonding, electrostatics, and hydrophobic interactionsto yield a large overall ligand binding affinity. Furthermore, vander Waals forces may also contribute to specific recognitionelements due to steric complementarity between the template andligand.

Two ligands not specific for the opioid receptor were used ascontrols to further test the cross-reactivity in binding. (-)Isoproterenol, a â-2 adrenergic receptor agonist,32 with a catecholfunctionality and an amine positive charge (Table 1), bound only∼15-fold less tightly than DPDPE, whereas a melanocortinreceptor peptide agonist NDP-RMSH27 bound to the imprintedcavity nearly 300 times more weakly. Note that this latter ligandcontains a Glu in the fifth position (Table 1), which may contributeto an unfavorable electrostatic interaction. An important point tonote about the recognition pattern is that the deltorphin II ligand

(28) Liao, S.; Lin, J.; Shenderovich, M. D.; Han, Y.; Hosohata, K.; Davis, P.; Qiu,W. Porreca, F.; Yamamura, H. I.; Hruby, V. J. Bioorg. Med. Chem. Lett. 1997,7, 3049-3052.

(29) Salamon, Z.; Tollin, G. Biophys. J. 2001, 80, 1557-1567.

(30) Hruby, V. J.; Gehrig, C. A. Med. Res. Rev. 1989, 9, 343-401.(31) Shenderovich, M. D.; Liao, S.; Qian, X.; Hruby, V. J. Biopolymers 2000, 53,

565-580.(32) Gether, U.; Kobilka, B. K. J. Biol. Chem. 1998, 273, 17979-17982.

Figure 2. PWR spectra obtained upon buffer addition and DPDPEbinding to MIP film. PWR spectra were obtained upon equilibrationof a deposited dry MIP film in buffer before (solid lines) and afterDPDPE binding (closed symbols s-polarization; open symbols p-polarization). Aqueous buffer used in the sample cell was 10 mM Tris,0.5 mM EDTA, 10 mM KCl, pH 7.3 at ambient temperature.

2572 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

did not bind to the MIP even at micromolar concentrations, insharp contrast to its high-affinity binding to the native receptor(Table 2). However, substitution of Glu by Gln in the fourthposition in this ligand resulted in a picomolar binding affinity tothe MIP (Table 2), clearly demonstrating the huge inhibitoryelectrostatic effect of the negative charge at this position.

Furthermore, additional control experiments with ligand bind-ing to blank prisms without the presence of a polymer matrix, aswell as to prisms coated with the polymer membrane without theimprint, also were done. In both cases, these resulted in nochanges in the PWR spectra, i.e., no detectable binding. Thisdemonstrates that the difference in binding specificities of theligands tested was not due to differences in adsorption directlyon to the prism surface as a result of the physicochemicalproperties of the ligands.

In all cases, the spectral shifts obtained using s-polarizationwere smaller than those obtained with p-polarized excitation,indicating that the structural changes induced in the polymer

matrix by ligand binding were anisotropic; i.e., the changesperpendicular to the plane of the film were larger than thoseparallel to the film plane. This is strong evidence for a nonrandomorientation of the ligand binding sites within the polymer film.Furthermore, the differences in the binding constants, the varia-tions in the magnitudes of the anisotropic spectral changes, andthe rapid on-rates and slow off-rates of ligand binding point tothe shape- and structure-specific recognition accompanying theinteractions with the MIP binding cavities. We suggest, therefore,that the processes of complexation and decomplexation of ligandsinvolve dynamic flexibility resembling that occurring in biologicalmolecules.

Additional cross-experiments with a polymer designed andimprinted for another ligand (deltorphin-II) resulted in tightbinding with a KD of 0.54 ( 0.08 pM. As noted above, deltorphin-II did not bind to the DPDPE-imprinted polymer. DPDPE bindingto the deltorphin-II-imprinted MIP resulted in a decrease inbinding affinity by 65-fold (data not shown), indicating a reversal

Figure 3. Effect of addition of various concentrations of ligands on PWR spectral positions upon binding to a DPDPE-imprinted polymertemplate. Aliquots of ligands dissolved in buffer (10 mM Tris, 0.5 mM EDTA, 10 mM KCl, pH 7.3) were added to the aqueous compartment ofthe PWR cell resulting in final bulk concentrations as indicated. Changes in anisotropy were indicated by p-polarized spectral shifts (closedsymbols) that were larger than s-polarized shifts (open symbols). Solid lines are hyperbolic fits to the experimental data, and the KD values arereported in Table 2.

Analytical Chemistry, Vol. 77, No. 8, April 15, 2005 2573

of the binding specificity rather than in an elimination of bindingof the nonimprinted ligand.

CONCLUSIONSThe results described above demonstrate the ability of a

macromolecular template to generate artificial high-affinity binding

sites in a well-defined binding pocket that mimics those found innative proteins. The binding affinities observed are as high or evenhigher than those found with the natural δ-opioid receptor.Previous results utilizing MIPs imprinted to enkephalin33 yieldedheterogeneous sites with multiple binding affinities in the low- tohigh-micromolar range. Future extensions of this work provide aunique opportunity to explore innovative avenues in molecularsensing, such as application-oriented MIPs that can be used asarrays, as well as disposable sensor chips for field use. Suchplatforms have wide potential applications, most notably inenvironmental screening, drug development, therapeutics, andmedical 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, plas-mon-waveguide resonance; TIPPpsi, H-L-Tyr-L-Tetrahydroiso-quinoline-3-carboxylic acid-[CH2-NH]-Phe-Phe-OH; TMT-Tic,(2S,3R) â-methyl-2′,6′-dimethyltyrosyl-tetrahydroisoquinoline-3-car-boxylic acid.

ACKNOWLEDGMENTThis 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. ScottCowell for the synthesis of the Gln4-deltorphin II ligand, IsabelAlves for providing the other ligands used in this study, andPhilippe Guerit for Figure 1b.

Received for review October 14, 2004. Accepted February4, 2005.

AC048476E

(33) Andersson, L. I.; Muller, 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), 3561-3569.

Table 2. Dissociation Constants for Ligand Binding toa DPDPE-Imprinted Polymer and to the Human δ-OpioidReceptor

ligand

KD(DPDPE-MIP),

pM

KD(δ-opioid receptor),a

nM

DPDPE 0.41 ( 0.05 16 ( 4deltorphin-II >106 1.2 ( 0.2TIPPpsi 1.9 ( 3.9 0.06 ( 0.02morphine 17.2 ( 5.9 520 ( 30TAN-67 8.1 ( 2.0 3.4 ( 1.3TMT-Tic 11.9 ( 3.9 2.8 ( 0.3DADLE 3.05 ( 0.35 2.6 ( 0.2b

Gln4-deltorphin II 8.13 ( 2.35 ndc

NDP-RMSH(melanotropin ligand)

114.5 ( 24.7 nd

(-) isoproterenol(â2-adrenergic ligand)

5.2 ( 1.2 nd

a KD values for the MIPs are an average between hyperbolic curvesobtained with s- and p-polarization. References 23-25. b Reference 34.c nd, not determined since these are not biologically relevant to thisreceptor.

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