Influence of Composition and Morphology on Template Recognition

(13) (template, functional-, and cross-linking monomer molar ratio 1:12:55), and we ... at the prepolymerization stage and final MIP morphology on rec...
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Influence of Composition and Morphology on Template Recognition in Molecularly Imprinted Polymers Kerstin Golker,† Björn C. G. Karlsson,† Gustaf D. Olsson,† Annika M. Rosengren,† and Ian A. Nicholls*,†,‡ †

Bioorganic and Biophysical Chemistry Laboratory, Linnæus University Centre for Biomaterials Chemistry, Linnaeus University, SE-391 82 Kalmar, Sweden ‡ Department of Chemistry - BMC, Uppsala University, SE-751 23 Uppsala, Sweden S Supporting Information *

ABSTRACT: A combination of theoretical and experimental studies has provided correlations between molecularly imprinted polymer composition, morphology, and recognition behavior obtained using a series of bupivacaine-imprinted methacrylic acid (MAA)−ethylene glycol dimethacrylate copolymers differing in molar ratios of the respective monomers. Results extracted from analysis of molecular dynamics (MD) trajectory data demonstrated that stability and frequency of interactions between bupivacaine and the monomers in the prepolymerization phase were strongly affected by minor changes in polymer composition, which in turn affected binding site affinity and heterogeneity of the imprinted polymers. Moreover, through the characterization of polymer morphology, we show that higher molar fractions of MAA resulted in polymeric materials with increased pore size, a feature that enhanced the binding capacity of the polymers. Furthermore, the results presented point at the strength of MD for predicting MIP-template binding capacity and affinity.

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INTRODUCTION Molecularly imprinted polymers (MIPs) are synthetic recognition materials that display predetermined selectivity towards a chosen template or target structure. The recognition properties of MIPs can be compared to those demonstrated by biological counterparts such as antibodies, enzymes, or membrane receptor proteins.1−4 Their physical and chemical stability in combination with a relative low cost of production5 make these materials attractive as ligand-selective materials for use in separation or purification processes or in biomimetic recognition systems.6−9 Despite the large and growing volume of literature in the field, our understanding of the mechanisms underlying MIP formation and ligand recognition is still limited, and our appreciation of morphology on recognition is even less established. Previous studies have shown that the nature and extent of template complexation at the prepolymerization stage are crucial for the formation of high affinity sites in MIPs synthesized via the noncovalent approach of molecular imprinting.6,10,11 As a consequence, in order to improve the performance of MIPs, a complete elucidation of the physical mechanisms underlying binding site formation at the prepolymerization stage is highly motivated. Recently, the potential of molecular dynamics (MD) simulation studies for providing detailed descriptions of the physical mechanisms involved at the prepolymerization stage of MIP synthesis,12−15 and their use in MIP rational design,10,16 has provided a molecular level picture explaining the bindingsite heterogeneity typically observed in noncovalently prepared MIPs.13 © 2013 American Chemical Society

Here we have built upon the insights from studies of the bupivacaine MIP system described by Karlsson et al.13 (template, functional-, and cross-linking monomer molar ratio 1:12:55), and we report on the results from MD simulations of a series of ten all-component prepolymerization mixtures, covering a wide range of relative stoichiometries between the monomers. The polymer systems investigated were methacrylic acid (MAA)−ethylene glycol dimethacrylate (EGDMA) copolymers imprinted with the local anesthetic bupivacaine and were chosen for this study due to the high number of previous studies using bupivacaine as a template.13,17−26 To date, attempts to correlate imprinted polymer composition with morphology have been few.27−35 Moreover, to the best of our knowledge, no attempts have been made to employ all-component MD simulations to examine the interplay of MIP composition with polymer performance. Here, we correlate, for the first time, the effects of the extent of template-monomer complexation found in a broad range of varying relative molar ratios of monomers of all-component MD simulations at the prepolymerization stage and final MIP morphology on recognition behavior. Correlations are highlighted and important aspects for optimizing the bupivacaine MIP composition are presented. Collectively, results presented here demonstrate that minor modifications of MIP prepolymerization mixture composition affect binding site affinity and heterogeneity in final MIPs by influencing the stability and Received: November 23, 2012 Revised: January 17, 2013 Published: February 6, 2013 1408

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the binned number of atoms in a volume segment dependent on the bin width, δr, used.

extent of hydrogen bond contacts between functionalities of bupivacaine, MAA, and EGDMA at the prepolymerization stage. Importantly, the results presented point to the strength of MD as a tool for predicting MIP-template binding capacity and affinity and to the importance of studies of the influence of MIP morphology on performance.

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g (r ) =

ρij (r ) ⟨ρj ⟩

=

nij(r ) ⟨ρj ⟩4πr 2δr

(1)

Polymer Synthesis. Ten bupivacaine imprinted polymers, and their corresponding nonimprinted reference (REF) polymers, with molecular compositions and molar ratios representative of those simulated by MD were prepared (Table 1) as described previously.20

EXPERIMENTAL SECTION

Chemicals. [3H]-(R,S)-Bupivacaine (specific activity 2.7 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA). (R,S)Bupivacaine hydrochloride and methacrylic acid (MAA) were purchased from Sigma-Aldrich (Steinheim, Germany). Toluene was purchased from Merck (Solna, Sweden), and (R,S)-ethycaine hydrochloride and (R,S)-pentycaine hydrochloride were from AstraZeneca R&D (Södertälje, Sweden). Ethylene glycol dimethacrylate (EGDMA) was obtained from Fluka (Buchs, Switzerland) and 2,2′-azobis(2-methylpropionitrile) (AIBN) from Janssen Chimica (Geel, Belgium). All chemicals were of analytical grade and the water used was of Millipore quality (Millipore AB, Sweden). Molecular Dynamics Simulations. All-atom MD simulations were carried out using the AMBER (v. 10.0 UCSF, San Francisco, CA) platform of programs36,37 and a method described previously by Karlsson et al.13 The simulated systems, however, were initially designed using the PACKMOL software38,39 to obtain random starting geometries as presented by Olsson and co-workers.14 For information regarding the molecular composition of the simulated all-component prepolymerization mixtures as well as for equilibration and production run data, see Supporting Information, Tables S1 and S2, respectively. All systems (10 MIP- and their corresponding reference prepolymerization mixtures) were simulated in quintuplicate, each covering 10 ns of recorded trajectory data for each mixture (totally 50 ns). Final equilibrated trajectories were analyzed using the PTRAJ module implemented in AmberTools. 37 All hydrogen bond interactions were extracted from the trajectories using a cutoff distance and angle of 3.0 Å and 120°, respectively. The structures of the analyzed molecular species and the interacting atoms potentially participating in hydrogen bond interactions can be found in Chart 1.

Table 1. Composition of the Molecularly Imprinted Polymers Prepared (in mmol)a polymer MIP MIP MIP MIP MIP MIP MIP MIP MIP MIP

0 1 2 3 4 5 6 7 8 9

bupivacaine

MAA

EGDMA

AIBN

toluene

molar ratiob

1.39 1.39 1.39 1.39 1.39 1.39 1.39 1.39 1.39 1.39

0.00 3.30 7.23 12.51 16.68 20.1 22.24 25.02 30.90 64.20

77.4 78.1 74.5 77.4 77.4 82.3 77.4 77.4 82.6 76.2

2.00 2.10 2.03 2.18 2.23 2.40 2.30 2.30 2.55 2.80

219.8 226.0 221.5 236.4 240.9 259.4 246.9 251.4 274.0 298.4

1:0:56 1:2:56 1:5:54 1:9:56 1:12:56 1:14:59 1:16:56 1:18:56 1:22:59 1:46:55

a

Nonimprinted reference polymers were also prepared, though in the absence of bupivacaine. bBupivacaine:MAA:EGDMA.

Prepolymerization mixtures were placed under a UV source (Model UVGL-58, Upland, 365 nm) at 8 °C for 24 h. The resultant bulk polymers were manually ground and sieved through a 63 μm sieve. Fine particles were removed by wet sieving (acetone, 600 mL) through a 25 μm sieve prior to repeated sedimentation from acetone (200 mL, minimum of 3 × 15 min), giving a final particle size between 25 and 63 μm. The polymer particles (2−3 g) were slurry packed with acetone in an HPLC column, and thereafter the template was removed using a previously described washing protocol.20 Polymers were then air-dried before drying in an oven at 60 °C for 24 h. The corresponding REF polymers were prepared and treated identically to the MIPs, though synthesized in the absence of the template, bupivacaine. Polymer Titration. Binding to both the MIP and the REF polymers was studied at increasing polymer concentrations (0.025−4.5 mg/mL). The radioligand, [3H]-(R,S)-bupivacaine (15 pmol/mL), and amounts of polymer suspended in toluene were mixed to provide a specific polymer concentration in a total volume of 1 mL. The samples were incubated on a rocking table at 293 K for 3 h. Thereafter, they were centrifuged (7000g for 5 min), and the supernatant (600 μL) was mixed with scintillation cocktail (2 mL, Beckman Ready safe). The activity was then measured by scintillation counting (Packard TriCab 2100TR liquid scintillation counter, 2 min). Control samples containing no polymer were also prepared and treated identically. All samples were analyzed in triplicate, and data are presented as ± standard error of the mean. Displacement-Binding Experiments. For each polymer system, the amount of polymer equivalent to the PC50 (Table 2), i.e., the polymer concentration required for 50% binding of the maximal amount bound radioligand, was incubated together with the radioligand [3H]-(R,S)-bupivacaine (15 nM) and unlabeled (R,S)bupivacaine or the competing unlabeled analogues (R,S)-ethycaine and (R,S)-pentycaine (100 nM−100 μM). Morphology Characterization. Polymer surface areas and porosities were studied through nitrogen sorption studies by the Brunauer, Emmett, and Teller (BET)40 and the Barrett, Joyner, and Halenda (BJH)41 methods. Prior to the measurements, samples were degassed at 323 K for 24 h to remove adsorbed gases and moisture. BET surface areas were calculated from the adsorption data using 0.162 nm2 as the molecular cross-sectional area for adsorbed nitrogen molecules.42 The BJH method was applied to calculate the pore size

Chart 1. Molecular Structures and Atoms Studied on (A) Bupivacaine, (B) MAA and (C) EGDMA

For the average hydrogen bond occupancies for all analyzed atom pairs in each system and the corresponding averaged lifetimes see Table S3. In order to examine the local distribution of molecular species at a range close enough to the template to be viable for interaction, RDF analyses were performed (Figures S1−S6) and local densities of specified atom pairs were computed (Chart 1 and Figure S7). Here, the RDF (g(r)) is described by eq 1 as the ratio between the observed number density, ρij, of a specified solvent atom at a certain distance (r) from a solute atom (i) and the average bulk atom number density of the solvent, ⟨ρj⟩. The radial shell number, nij(r), is 1409

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Table 2. Polymer Concentration Required for 50% Binding of the Maximal Amount Bound Radioligand polymer

MIP 1

MIP 2

MIP 3

MIP 4

MIP 5

MIP 6

MIP 7

MIP 8

MIP 9

PC50 (mg/mL)

3.7

2.1

0.93

1.4

0.75

0.93

1.02

0.72

0.75

distributions from desorption branches of the isotherms. All measurements were performed on an ASAP 2004 instrument (Micromeritics, Norcross, GA) at 77 K.

bupivacaine complexation through hydrogen bonding was found to decrease with an increase in the molar fraction of MAA in the systems, the observed averaged hydrogen bond interaction occupancy between EGDMA and bupivacaine was on overall higher than the occupancy of MAA−bupivacaine interactions (Figure 1 and Table S3). Analysis of the nature and extent of hydrogen bonding between other components in the prepolymerization mixtures revealed an increase in the dimerization of MAA with increased molar fraction of MAA in the systems for both the simulated MIP and REF prepolymerization mixtures. Further, analysis of hydrogen bond interactions formed between MAA and EGDMA showed that these interactions were comparable to those observed between the MAA molecules, i.e., similar with regard to both frequency and stability, as well as displaying increasing extents when increasing the molar fraction of MAA. Template dimerization in the mixtures was found to be negligible, and contacts between the template and the initiator AIBN, as investigated earlier in chloroform,13 did not show any evidence for hydrogen bond formation between these components. Closer examination of the functional groups engaged in hydrogen bonding revealed clear optima in the extent of hydrogen bonding to all three major functionalities present on bupivacaine, with the most frequent hydrogen bonding observed between the acidic proton (HAA) of MAA and the carbonyl oxygen (O) of bupivacaine (Chart 1 and Figure 2A), in agreement with a previous study of a single system stoichiometry.13 A further detailed analysis of this interplay was obtained through extraction of hydrogen bond lifetimes (τ), which again demonstrated that the strongest interaction between MAA and bupivacaine involved the carbonyl oxygen functionality of bupivacaine and the acidic proton of MAA with an associated lifetime of τO‑HAA ∼ 5−7 ps (Figure 2C). The hydrogen bond behavior of the various prepolymerization components revealed that the bupivacaine amide proton functionality (HAB) interacts with EGDMA rather than with MAA (Figure 2B), explaining the high occupancy observed between EGDMA and bupivacaine seen in Figure 1. This can be related to the larger numbers of EGDMA present in the simulated systems, relative to the numbers of MAA, and also to

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RESULTS AND DISCUSSION A series of all-component prepolymerization mixtures, differing in composition through variation of the relative molar ratio of the monomers, MAA and EGDMA, and bupivacaine was initially studied by MD simulations to predict the nature and extent of monomer and template complexation present at the prepolymerization stage. Data extracted from MD simulations indicated an increase in extent of MAA−bupivacaine complexation through hydrogen bonding with an increasing molar fraction of MAA. Interestingly, an optimum in MAA−bupivacaine hydrogen bonding was found at a bupivacaine:MAA:EGDMA molar ratio of 1:14:56 (Figure 1). Moreover, although EGDMA−

Figure 1. Hydrogen bonds formed between functionalities of bupivacaine, MAA, and EGDMA for the series of prepolymerization mixtures studied. Template-averaged hydrogen bonding in each mixture is presented as percentage (% occupancy) of the total simulation time of 10 ns. Contacts observed in mixtures representing MIPs are denoted with (+B) and contacts observed in mixtures representing REFs with (−B). In the case of MAA−EGDMA and MAA−MAA hydrogen bonding is calculated per EGDMA and MAA molecule, respectively. Error bars correspond to the standard error of the mean obtained from five simulations of each system.

Figure 2. Functional groups of bupivacaine interacting via hydrogen bonding with the functional groups of (A) MAA and (B) EGDMA as a percentage of total simulation time (10 ns) and (C) corresponding observed average lifetimes, τ, for the interactions. Error bars correspond to the standard error of the mean resulting from five simulations of each system. See Chart 1 for denotation of the atoms. 1410

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Further, obtained desorption pore volume distributions (Figure 5) of the prepared polymers demonstrate that the

the high tendency of MAA to form dimers. Moreover, although displaying a high frequency, the hydrogen bond interactions between EGDMA and bupivacaine were observed to be rather unstable (τHAB‑OAE/OEF ∼ 1 ps) compared to those observed between MAA and bupivacaine (Figure 2C). In order to determine the average number of MAA and EGDMA molecules found at a range close enough to bupivacaine to be viable for interaction, radial distribution functions (RDFs) were calculated (Figures S1−S6). The results, obtained from integrating RDFs up to 3 Å from the selected reference atom of bupivacaine, showed clear optima for MAA accumulation at all three functionalities of bupivacaine when plotting the atomic densities as a function of the molar fraction MAA in the simulated systems (Figure S7A). The observed optima correspond to an average of ∼0.34 molecules of MAA per bupivacaine molecule around bupivacaine’s carbonyl functionality (O) at a bupivacaine:MAA:EGDMA molar ratio of 1:14:59. The corresponding numbers of MAA molecules accumulating around bupivacaine’s amine functionality (N) and amide proton (HAB) were found to be ∼0.18 and ∼0.08 at molar ratios of 1:18:56 and 1:16:56, respectively. Importantly, the number of EGDMA molecules accumulating at the amide proton functionality of bupivacaine was in all simulated systems with ∼0.30−0.45 observed to be significantly higher than the corresponding number of MAA found at this range close enough to be viable for interaction (Figure S7B). In order to characterize the physical properties of the bulk polymers, MIPs and REFs with molar ratios corresponding to those of the simulated systems were prepared and their morphologies were investigated by detailed nitrogen sorption studies. Analysis of polymer surface areas with respect to the molar fraction of MAA used during synthesis displayed a trend where an increasing molar fraction of MAA led to a decreasing surface area (Figure 4A and Table S4). The surface areas were in the range of 80−550 m2/g and either similar for MIPs and corresponding REFs or in some cases higher for the MIPs. The same trend was observed for the relationship between the pore volume of the polymers and the molar fraction of MAA (Figure 4B).

Figure 5. BJH desorption pore volume distributions for the investigated polymer series: (A) MIPs and (B) REFs.

decreasing pore volume is associated with increasing pore diameters since the curves declined and the distributions shifted towards larger pore diameters as the molar fraction MAA in the polymers increased. These trends may be explained by considering the average number of the pores and the surface sizes of these materials. A higher molar fraction of MAA yielded materials with lower numbers of large pores (macropores >500 Å) and smaller surface areas. The opposite situation was found to be true at lower molar fractions of MAA where high fractions of pores with smaller diameters in the mesopore range, 20−500 Å, were found to be associated with larger surface areas. In order to investigate the influence of the template complexation during the prepolymerization stage (obtained from MD simulations) and the physical characteristics (obtained from nitrogen sorption studies) on the polymers capacity to bind bupivacaine and to discriminate between structurally similar compounds, a series of radioligand binding studies were performed. Initially, the binding capacity of the polymers was examined through polymer titrations, which allow the comparison of systems with large differences in binding. Results from these experiments demonstrated a general trend towards an increased binding of bupivacaine with increasing molar fraction of MAA, for both the MIPs and the REFs (Figure 6A, B) as indicated by the results from MD simulations. The increase in binding can be explained with the increasing portion of macropores in the materials with respect to the total pore volume as a result of the increasing fraction MAA. The rebinding of bupivacaine to the series of MIPs investigated was, with exception of MIP 0, for all polymer concentrations higher than that observed for the corresponding REFs, thus indicating the presence of bupivacaine-imprinted

Figure 4. (A) Surface area and (B) pore volume of the studied polymers as a function of the molar fraction MAA: (■) MIPs and (□) REFs. 1411

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Figure 6. Binding to the bupivacaine imprinted polymers (A), to the nonimprinted reference polymers (B), and the resulting specific binding (C), which is defined as the difference between the binding to the imprinted and the nonimprinted reference polymers. B/T is the fraction radioligand bound to the polymer. Error bars obtained from analyzing the samples in triplicate correspond to the standard error of the mean.

binding sites in the MIPs. Importantly, the difference in binding of bupivacaine to the MIPs and to the REFs, usually defined as the specific binding, was found to be highest for MIP 5, at a bupivacaine:MAA:EGDMA molar ratio of 1:14:59 (Figure 6C). This polymer corresponded to the simulated prepolymerization mixture that displayed maximal template−MAA hydrogen bond interactions. Furthermore, MIP 5, with a bupivacaine:MAA:EGDMA molar ratio of 1:14:59 and MIP 8−9, with bupivacaine: MAA:EGDMA molar ratios of 1:22:59 and 1:46:55, respectively, were identified as those polymers with the lowest PC50 of 0.72−0.75 mg/mL, i.e., the polymer concentration required for 50% binding of the maximal amount bound radioligand (Table 2). This result reflects higher affinity for bupivacaine in these MIPs and correlates yet again well with the results obtained from MD simulation. Polymer 0, which contained no MAA (bupivacaine: MAA:EGDMA molar ratio 1:0:56), displayed no discernible binding, neither to the MIP nor to the REF. An explanation for this behavior may be found when taking the results from the MD simulations into account. Although EGDMA was observed to interact with bupivacaine via hydrogen bonding over about 45% of the total simulation time, the hydrogen bond contacts formed were as described earlier rather unstable, as reflected by the short average lifetime of these interactions (Figure 2C). This result suggests strong interactions between monomers and template in the prepolymerization phase being necessary, as those observed between MAA and bupivacaine, in order for a MIP to be capable of molecular recognition. However, we suggest EGDMA−bupivacaine complexation plays a role in binding site formation in polymers containing MAA, in accordance with results presented earlier by Dourado and coworkers.43 To examine possible influences on polymer−ligand selectivity, a series of radioligand displacement studies were performed. These studies demonstrated MIP preference for the template, bupivacaine, over the competing analogues ethycaine and pentycaine in all cases. Obtained IC50 values, i.e., the concentration of the unlabeled ligand needed to displace 50% of [3H]-bupivacaine binding, were in the micromolar range and of the same order of magnitude as observed previously by Karlsson et al.20 (Chart 2, Table 3, and Figure S8). Interestingly, although evidence was found correlating both template complexation in the simulated prepolymerization mixtures and physical properties (pore size and surface area) with template binding capacity, no significant differences were

Chart 2. Structure of Bupivacaine and the Competing Analogues Ethycaine and Pentycaine

observed when using template analogues quite closely related to the template structure. This suggests that more detailed MD analyses of prepolymerization mixtures and polymer morphological studies may be required in order to establish correlations. Collectively, within the molar ranges tested the binding capacity of the polymers increased with increasing pore size and the ligand affinity was observed to vary even with small changes in the relative stoichiometry between template, functional- and cross-linking monomers used during synthesis. This can be explained by the nature and extent of monomer−template complexation observed in the simulated all-component prepoymerization mixtures. Importantly, the stoichiometry dependent optima observed in the MD studies of monomer− template interactions (Figures 1 and 2A) are strongly reminiscent of deviations from Raoult’s law, e.g., azeotropes, where well-defined component stoichiometries produce solution structures with unexpected properties44 such as the stoichimetry-dependent optima reported here.

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CONCLUSIONS In this study, we correlate, for the first time, the extent of template- and monomer complexation found in a series of MD simulated all-component prepolymerization mixtures with MIP composition, morphology, and recognition behavior. The results point to the relative stoichiometries of the key components in the systems studied (bupivacaine, MAA, and EGDMA) as steering both the morphology and ligand recognition properties in MIPs. MIPs with a higher fraction of MAA were observed to display higher binding capacities and form fewer but larger pores with associated smaller surface areas. Our results obtained from MD simulations indicate that increasing hydrogen bond contact between bupivacaine and MAA in combination with increased MAA dimerization is related to these effects. The results demonstrate that even minor changes in stoichiometry induce changes in binding site affinity and heterogeneity in the final imprinted polymers. The 1412

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Table 3. IC50 Values for Displacement of [3H]-Bupivacaine Binding to the Studied MIPs log IC50 [r2]a polymer MIP MIP MIP MIP MIP MIP MIP MIP MIP

1 2 3 4 5 6 7 8 9

bupivacaine −5.33 −5.35 −5.46 −5.03 −5.54 −5.10 −4.91 −5.37 −5.16

[1.00] [1.00] [0.99] [0.99] [1.00] [0.99] [1.00] [0.98] [0.99]

ethycaine −4.97 −5.18 −4.99 −4.69 −5.29 −4.80 −4.69 −5.17 −4.99

[0.99] [1.00] [0.98] [1.00] [0.99] [0.99] [1.00] [1.00] [1.00]

IC50 (μM) pentycaine −5.02 −5.25 −5.13 −4.77 −5.35 −4.89 −4.78 −4.97 −5.03

bupivacaine

ethycaine

pentycaine

4.6 4.4 3.5 9.4 2.9 7.9 12.2 4.3 6.9

10.6 6.6 10.1 20.3 5.1 16.0 20.3 6.8 10.2

9.7 5.7 7.4 16.9 4.4 13.0 16.7 10.8 9.3

[1.00] [0.99] [1.00] [0.96] [0.97] [1.00] [1.00] [0.99] [0.99]

a 2

r is the coefficient of determination obtained from linear regression of the logit transformation. (6) Andersson, H. S.; Karlsson, J. G.; Piletsky, S. A.; Koch-Schmidt, A.-C.; Mosbach, K.; Nicholls, I. A. J. Chromatogr., A 1999, 848, 39−49. (7) Martin, P.; Wilson, I. D.; Jones, G. R. J. Chromatogr., A 2000, 889, 143−147. (8) Theodoridis, G.; Manesiotis, P. J. Chromatogr., A 2002, 948, 163−169. (9) Zhang, H. Q.; Ye, L.; Mosbach, K. J. Mol. Recognit. 2006, 19, 248−259. (10) Nicholls, I. A.; Andersson, H. S.; Golker, K.; Henschel, H.; Karlsson, B. C. G.; Olsson, G. D.; Rosengren, A. M.; Shoravi, S.; Suriyanarayanan, S.; Wiklander, J. G.; Wikman, S. Anal. Bioanal. Chem. 2011, 400, 1771−1786. (11) Baggiani, C.; Giovannoli, C.; Anfossi, L.; Passini, C.; Baravalle, P.; Giraudi, G. J. Am. Chem. Soc. 2012, 134, 1513−1518. (12) O’Mahony, J.; Karlsson, B. C. G.; Mizaikoff, B.; Nicholls, I. A. Analyst 2007, 132, 1161−1168. (13) Karlsson, B. C. G.; O’Mahony, J.; Karlsson, J. G.; Bengtsson, H.; Eriksson, L. A.; Nicholls, I. A. J. Am. Chem. Soc. 2009, 131, 13297− 13304. (14) Olsson, G. D.; Karlsson, B. C. G.; Shoravi, S.; Wiklander, J. G.; Nicholls, I. A. J. Mol. Recognit. 2012, 25, 69−73. (15) Karlsson, B. C. G.; Rosengren, A. M.; Naslund, I.; Andersson, P. O.; Nicholls, I. A. J. Med. Chem. 2010, 53, 7932−7937. (16) Nicholls, I. A.; Andersson, H. S.; Charlton, C.; Henschel, H.; Karlsson, B. C. G.; Karlsson, J. G.; O’Mahony, J.; Rosengren, A. M.; Rosengren, K. J.; Wikman, S. Biosens. Bioelectron. 2009, 25, 543−552. (17) Andersson, L. I. Analyst 2000, 125, 1515−1517. (18) Andersson, L. I.; Abdel-Rehim, M.; Nicklasson, L.; Schweitz, L.; Nilsson, S. Chromatographia 2002, 55, 65−69. (19) Andersson, L. I.; Hardenborg, E.; Sandberg-Stall, M.; Moller, K.; Henriksson, J.; Bramsby-Sjostrom, I.; Olsson, L. I.; Abdel-Rehim, M. Anal. Chim. Acta 2004, 526, 147−154. (20) Karlsson, J. G.; Andersson, L. I.; Nicholls, I. A. Anal. Chim. Acta 2001, 435, 57−64. (21) Karlsson, J. G.; Karlsson, B.; Andersson, L. I.; Nicholls, I. A. Analyst 2004, 129, 456−462. (22) Rosengren, A. M.; Golker, K.; Karlsson, J. G.; Nicholls, I. A. Biosens. Bioelectron. 2009, 25, 553−557. (23) Rosengren, A. M.; Karlsson, J. G.; Andersson, P. A.; Nicholls, I. A. Anal. Chem. 2005, 77, 5700−5705. (24) Schweitz, L.; Andersson, L. I.; Nilsson, S. J. Chromatogr., A 1997, 792, 401−409. (25) Courtois, J.; Fischer, G.; Schauff, S.; Albert, K.; Irgum, K. Anal. Chem. 2005, 78, 580−584. (26) Courtois, J.; Fischer, G.; Sellergren, B.; Irgum, K. J. Chromatogr. A 2006, 1109, 92−99. (27) Holland, N.; Duggan, P.; Owens, E.; Cummins, W.; Frisby, J.; Hughes, H.; McLoughlin, P. Anal. Bioanal. Chem. 2008, 391, 1245− 1253. (28) Holland, N.; Frisby, J.; Owens, E.; Hughes, H.; Duggan, P.; McLoughlin, P. Polymer 2010, 51, 1578.

molecular-level events and their influence on the bulk properties of the resultant materials may be compared to the molecular-level events leading to deviations from Raoult’s law, e.g., azeotropes, as shown in the nonlinearity of the templatefunctional monomer stoichiometry profile derived from the MD studies. Finally, the results presented herein point at the potential of the MD method for predicting MIP-template binding capacity and affinity in MIPs prepared using noncovalent imprinting strategies and highlight the role of morphology on MIP performance.

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ASSOCIATED CONTENT

S Supporting Information *

Composition of simulated prepolymerization mixtures; equilibration and production run data; summary of hydrogen bonding results; radial distribution function analyses results for bupivacaine−MAA, bupivacaine−EGDMA, MAA−MAA, and bupivacaine−bupivacaine interactions; graph of atomic densities at Rcut = 3.0 Å of studied atoms of MAA and EGDMA around studied atoms of bupivacaine; displacement binding study results; BET analyses data with regard to surface areas and pore volumes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the Swedish Research Council (VR), the Knowledge Foundation (KKS), Carl Trygger Foundation, the European Union (WATERMIM), and Linnaeus University is most gratefully acknowledged.

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REFERENCES

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