Mechanisms underlying molecularly imprinted polymer molecular memory and the role of crosslinker: resolving debate on the nature of template recognition in phenylalanine anilide imprinted polymers

A series of molecular dynamics simulations of prepolymerization mixtures for phenylalanine anilide imprinted co-(ethylene glycol dimethacrylate-methacrylic acid) molecularly imprinted polymers have been employed to investigate the mechanistic basis for template selective recognition in these systems. This has provided new insights on the mechanisms underlying template recognition, in particular the significant role played by the crosslinking agent. Importantly, the study supports the occurrence of template self-association events that allows us to resolve debate between the two previously proposed models used to explain this system's underlying recognition mechanisms. Moreover, the complexity of the molecular level events underlying template complexation is highlighted by this study, a factor that should be considered in rational molecularly imprinted polymer design, especially with respect to recognition site heterogeneity.     

Effect of constituting amino acid residue numbers on molecularly imprinted chiral recognition sites

In the present study, the effect of constituting amino acid residue numbers of oligopeptide derivatives, which are candidate materials to construct molecular recognition sites, on chiral recognition ability was investigated. Chiral recognition sites were formed from oligopeptide derivatives, of which constituting amino acid residue numbers were three to six, by adopting an alternative molecular imprinting. It was made clear that the number four, in other words, the tetrapeptide derivative, is the best candidate material to form a chiral recognition site.     

Synthesis of molecularly imprinted polymers using a functionalized initiator for chiral-selective recognition of propranolol

We present a new concept of synthesis for preparation of molecularly imprinted polymers using a functionalized initiator to replace the traditional functional monomer. Using propranolol as a model template, a carboxyl-functionalized radical initiator was demonstrated to lead to high-selectivity polymer particles prepared in a standard precipitation polymerization system. When a single enantiomer of propranolol was used as template, the imprinted polymer particles exhibited clear chiral selectivity in an equilibrium binding experiment. Unlike the previous molecular imprinting systems where the active free radicals can be distant from the template-functional monomer complex, the method reported in this work makes sure that the actual radical polymerization takes place in the vicinity of the template-associated functional groups. The success of using functional initiator to synthesize molecularly imprinted polymers brings in new possibilities to improve the functional performance of molecularly imprinted synthetic receptors.     

Investigation of disaccharide recognition by molecularly imprinted polymers

The selectivity of carbohydrate-imprinted polymers for several disaccharides, namely cellobiose, maltose, lactose and gentiobiose, is investigated. An ternary ligand–Cu(II)–carbohydrate complex was formed in alkaline solution and captured afterwards in the polymer. The accessibility of the polymer matrix for disaccharides was investigated by HPLC analysis, refractometry and ¹H NMR spectroscopy applying excess of the original template during rebinding experiments under saturation conditions in unbuffered, aqueous solution at neutral pH and 20°C. The selective discrimination of the - and -glycosidic linkage of cellobiose and maltose is demonstrated. It is further shown, that the disaccharide-imprinted polymers slightly distinguish between the 1,4-- and the 1,6--glycosidic linkage of cellobiose and gentiobiose, while cellobiose and lactose are not selectively recognized. Due to the weak apparent binding constant of the functional Cu(II) monomers with the targeted disaccharides at physiological pH, the recognition process is dominated by the shape of the created imprinted cavity under the applied conditions.     

A molecular imprinted polymer with recognition properties towards the carcinogenic mycotoxin ochratoxin A

A molecularly imprinted polymer which recognises the mycotoxin ochratoxin A was prepared using the mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L) -phenylalanine as a template. The polymer was obtained by dissolving the template, methacrylic acid and ethylendimethacrylate in chloroform and polymerising the mixture by thermal treatment at 60°C. The monolith obtained was crushed, sieved to 30–90 m and extensively washed till the template could no longer be found in the washing solution. The binding properties towards the template, ochratoxin A and several related molecules were measured by eluting with acetonitrile and chloroform a HPLC column packed with the imprinted polymer. The experimental results show that the polymer recognises not only the template well, but also the ochratoxin A. The specific molecular recognition effect is due to hydrogen bond interactions but in order to assure the full recognition effect adjunctive steric factors are necessary. The magnitude of these interactions can be controlled by the use of limited amounts of acetic acid in the mobile phase.From the measurement of the relative selectivity it was found that only the simultaneous presence of the carboxyl, the phenolic hydroxyl and certain peculiar substructures such as the chlorine atom assures the whole recognition of the template.     

Study of the factors influencing peak asymmetry on chromatography using a molecularly imprinted polymer prepared by the epitope approach

Investigations of the effect of sample load on peak asymmetry during chromatography on molecularly imprinted polymer prepared by the epitope approach showed that the shape of the peaks for the template Tyr-Pro-Leu-Gly-NH₂ and for acetyl-L-tyrosine ethyl ester changed considerably until a split was observed. In contrast, the asymmetry of the peaks corresponding to oxytocin, which possesses the same C-terminus tripeptide as the template and interacts with the imprinted polymer, remained essentially unaltered. The circular dichroism (CD) spectra of these peptides showed significant dependence on peptide concentration, and the dependence was nearly the same for all the tested peptides. The addition of acetic acid influenced the CD spectra of YPLG and oxytocin but had no influence on the spectrum of acetyl-L-tyrosine ethyl ester. The shape differences in the chromatographic peaks seem to be associated with a solvation mechanism rather than with solute-solute complexation in solution. However, the observed differences in peak asymmetry cannot be completely explained by the mechanisms that have been postulated previously. Our results suggest the formation of triple complexes between a solute molecule (or molecules), an already adsorbed solute molecule, and an adjacent region of the polymeric stationary phase. These triple complexes may influence the retention of analytes and contribute to peak asymmetry.     

Relationship between enantioselectivity of alternative molecularly imprinted polymeric membranes and species of amino acid residues composing chiral recognition sites

Molecularly imprinted polymeric membranes with tetrapeptide residue H-Asp(OcHex)-Asp(OcHex)-Asp(OcHex)-Asp(OcHex)-CH₂- (DDDD) or H-Glu(OBzl)-Glu(OBzl)-Glu(OBzl)-Glu(OBzl)-C H₂- (EEEE) were prepared during membrane preparation (casting) processing in the presence of print molecules. The Boc-L-Trp imprinted polymeric membranes thus obtained showed adsorption selectivity toward Ac-L-Trp from its racemic mixtures. From adsorption isotherms of Ac-Trp, the chiral recognition site, that had been formed by the presence of print molecules in the membrane preparation process, exclusively recognized Ac-L-Trp that possessed the same configuration of the print molecule. The affinity constants between chiral recognition sites in the membrane and Ac-L-Trp was determined to be 1.00 × 10⁴ mol^–1 dm³ and 1.08 × 10⁴ mol^–1 dm³ for the DDDD and EEEE membranes, respectively. Enantioselective electrodialysis could be attained by applying an optimum potential difference to give permselectivity, with a value close to its adsorption selectivity.     

Preparation and evaluation of a molecularly imprinted polymer for the selective recognition of testosterone--application to molecularly imprinted sorbent assays

Biomimetic testosterone receptors were synthesized via molecular imprinting for use as antibody mimics in immunoassays. As evaluated by radioligand binding assays, imprinted polymers prepared in acetonitrile were very specific for testosterone because the nonimprinted control polymers bound virtually no radiolabeled testosterone. The polymers present an appreciable affinity, with association constants of K(a) = 3.3 x 10(7) M(- 1) (high-affinity binding sites). The binding characteristics of the polymers were also evaluated in aqueous environment to study their viabilities as alternatives to antibodies in molecularly imprinted sorbent assays. Compared with the testosterone-specific antibodies present in commercial kits, our molecularly imprinted polymers are somewhat less sensitive but show a high selectivity.     

Application of a quartz crystal nanobalance to the molecularly imprinted recognition of phenylalanine in solution

A quartz crystal nanobalance (QCN) biosensor was developed for the selective determination of phenylalanine (Phe) in aqueous solutions. A Phe imprinted copolymer was synthesized using polyacrylonitrile and acrylic acid [poly(AN-co-AA)]. The copolymer was then coated on quartz crystal electrode to form complementary structures for the template recognition of Phe. The composite electrode was then used to determine Phe levels in solution. Determinations were based on frequency shifts of molecularly imprinted polymer (MIP) modified quartz crystal electrode caused by Phe adsorption. The frequency shifts were linearly dependent on Phe concentration over the range 50∼500 mgL⁻¹. The results obtained show that the imprinted poly(AN-co-AA) modified biosensor had higher sensitivity (0.5839 Hz/mgL⁻¹) than a non-molecularly imprinted copolymer (0.2724 Hz/mgL⁻¹). Furthermore, good reproducibility, R.S.D. = 1.84% (n = 7) was observed, and the detection limit was 45 mgL⁻¹. The selectivity of the imprinted poly(AN-co-AA) modified biosensor was examined using a number of analytes similar to Phe, i.e., dopamine (DA), ascorbic acid (AscA), vanillylmandelic acid (VMA), uric acid (UA), tryptophan (Trp), and tyrosine (Tyr), and the results obtained showed a size dependent selective effect.     

A brief review of coarse-grained and other computational studies of molecularly imprinted polymers

Molecular imprinting is an established method for the creation of artificial recognition sites in synthetic materials through polymerization and cross-linking in the presence of template molecules. Removal of the templates leaves cavities that are complementary to the template molecules in size, shape, and functionality. In recent years, various theoretical and computational models have been developed as tools to aid in the design of molecularly imprinted polymers (MIPs) or to provide insight into the features that determine MIP performance. These studies can be grouped into two general approaches-screening for possible functional monomers for particular templates and macromolecular models focusing on the structural characterization of the imprinted material. In this review, we pay special attention to coarse-grained models that characterize the functional heterogeneity in imprinted pores, but also cover recent advances in atomistic and first principle studies. We offer a critical assessment of the potential impact of the various models towards improving the state-of-the-art of molecular imprinting.     

A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method

In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme‐specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25–65°C), optimum pH (3–10), thermal stability (4–70°C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of the solvent on recognition properties of molecularly imprinted polymer specific for ochratoxin A

A molecularly imprinted polymer specific for the mycotoxin ochratoxin A has been synthesised using a non-covalent approach. The polymer has shown an excellent affinity and specificity for the target template in aqueous solutions. The binding experiments, NMR study and molecular modelling have proven that the template recognition by polymer originates from the shape complementarity of binding sites. The binding mechanism is critically depended on factors that affect the polymer conformation. Thus the variation in buffer concentration, pH and presence of organic solvent, which affect the polymer swelling or shrinking, had a profound effect on the polymer recognition properties.     

Performance analysis of molecularly imprinted polymers for carboxylate and aminophosphate templates using commercially available basic functional monomers

A survey of commercially available amine-based monomers for binding and selectivity of carboxylate and phosphonic acid templates has revealed that the best selectivity is found for the pyridine-based monomers, while the highest affinity was found for 2-(dimethylamino)ethyl methacrylate (2-DEMA, 1). In fact, a more general finding is that selectivity is higher for aromatic amine-based monomers even though affinity remains higher for aliphatic amine-based monomers. An attempt to combine the optimal properties of these two classes of amine monomers, i.e. 2-vinylpyridine (2-VPY, 2), and 2-DEMA by using both simultaneously in a single imprinted polymer resulted in an MIP whose properties were dominated by the aliphatic amine-based monomer 2-DEMA. A controversy between the two commercially available vinylpyridine monomers, 2-VPY and 4-vinylpyridine (4-VPY, 3), was investigated, revealing that neither monomer is generally better for molecular imprinting; rather, the choice of 2-VPY or 4-VPY is template specific (although the preponderance of data tends to frequently favor 4-VPY). Phosphonic acid templates proved to be less successful as templates for molecular imprinting versus carboxylate functionalized templates, although binding was obtained and shown to be controllable via an ion-exchange process.     

Selective recognition of neodymium (III) using ion imprinted polymer particles

Neodymium (III) ion-imprinted polymer (IIP) materials were prepared by the copolymerization of neodymium (III)-5,7-dichloroquinoline-8-ol-4-vinylpyridine ternary complex with styrene(monomer), divinyl benzene (crosslinking monomer) in the presence of 2,2'-azobisisobutyronitrile (initiator). The synthesis was carried out in 2-methoxy ethanol medium (porogen) and the resultant material was filtered, washed, dried and powdered to form unleached IIP particles. The imprint ion was removed by stirring the above particles with 50% (v/v) HCl for 6 h to obtain leached IIP particles with cavities in the polymer particles. Control polymer (CP) particles were similarly prepared without imprint ion, i.e. neodymium (III). CP, unleached and leached IIP particles were characterized by TLC, IR, microanalysis, XRD and UV-visible spectrophotometric studies. The preconcentration of 5-150 microg of neodymium (III) ions present in 500 ml of solution was possible with as little as 40 mg of neodymium (III) IIP particles in the pH range 7.5-8.0 with a detection limit of 50 ng/l. Five replicate determinations of 25 microg of neodymium (III) present in 500 ml of solution gave a mean absorbance of 0.120 with a relative standard deviation of 2.65%. The imprinting effect of IIP particles was noticed in all preconcentration and selectivity studies when compared with CP particles. Furthermore, the selectivity coefficients of neodymium (III) IIP particles were much higher compared with the reported separation factors for the best liquid-liquid extractants, viz. di-2-ethylhexyl phosphoric acid and 2-ethylhexyl-ethylhexyl phosphonate. Kinetic and isotherm studies during rebinding of neodymium (III) onto IIP particles were also carried out.     

Selective solid-phase extraction using molecularly imprinted polymer for analysis of methamidophos in water and soil samples

An analytical methodology for the analysis of methamidophos in water and soil samples incorporating a molecularly imprinted solid-phase extraction process using methamidophos-imprinted polymer was developed. Binding study demonstrated that the polymer exhibited excellent affinity and high selectivity to the methamidophos. Evidence was also found by FT-IR analysis that hydrogen bonding between the CO(2)H in the polymer cavities and the NH(2) and P=O of the template was the origin of methamidophos recognition. The use of molecularly imprinted solid-phase extraction improved the accuracy and precision of the GC method and lowered the limit of detection. The recovery of methamidophos extracted from a 10.0 g soil sample at the 100 ng/g spike level was 95.4%. The limit of detection was 3.8 ng/g. The recovery of methamidophos extracted from 100 mL tap and river water at 1 ng/mL spike level was 96.1% and 95.8%, and the limits of detection were 10 and 13 ng/L respectively. These molecularly imprinted solid-phase extraction procedures enabled selective extraction of polar methamidophos successfully from water and soil samples, demonstrating the potential of molecularly imprinted solid-phase extraction for rapid, selective, and cost-effective sample pretreatment.     

Synthesis of a molecularly imprinted polymer for the solid‐phase extraction of betulin and betulinic acid from plane bark

Introduction – Plant extracts are usually complex mixtures of various polarity compounds and their study often includes a purification step, such as solid‐phase extraction (SPE), to isolate interest compounds prior analytical investigations. Molecularly imprinted polymers (MIPs) are a new promising type of SPE material which offer tailor‐made selectivity for the extraction of trace active components in complex matrices. Numerous specific cavities that are sterically and chemically complementary of the target molecules, are formed in imprinted polymers. A molecularly imprinted polymer (MIP) was synthesised in order to trap a specific class of triterpene, including betulin and betulinic acid from a methanolic extract of plane bark. Methodology – Imprinted polymers were synthesised by thermal polymerisation of betulin as template, methacrylic acid (MAA) or acrylamide (AA) as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and chloroform as porogen. Afterwards, MAA‐ and AA‐MIPs were compared with their non‐imprinted polymers (NIPs) in order to assess the selectivity vs betulin and its derivatives. Recovered triterpenes were analysed by HPLC during MIP‐SPE protocol. Results – After SPE optimisation, the MAA‐imprinted polymer exhibited highest selectivity and recovery (better than 70%) for betulin and best affinity for its structural analogues. Thus, a selective washing step (chloroform, acetonitrile) removed unwanted matrix compounds (fatty acids) from the SPE cartridge. The elution solvent was methanol. Finally, the MAA‐MIP was applied to fractionate a plane bark methanolic extract containing betulin and betulinic acid. Conclusion – This study demonstrated the possibility of direct extraction of betulin and its structural analogues from plant extracts by MIP technology. Copyright © 2009 John Wiley & Sons, Ltd.     

Preparation of surface molecularly imprinted polymeric microspheres and their recognition property for basic protein lysozyme

The surface imprinting of basic protein lysozyme (Lys) was carried out by designing a new route. The copolymerization of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) was first conducted in an inverse suspension polymerization system, and the crosslinked copolymeric microspheres HEMA/NVP were prepared. Subsequently, the esterification reaction of methacryloyl (MAO) chloride with the hydroxyl groups on the surfaces of HEMA/NVP microspheres was performed, and the modified microspheres MAO–HEMA/NVP, on which a mass of polymerisable double bonds were introduced, were obtained. In the presence of lysozyme, by initiating of K₂S₂O₈–NaHSO₃, the monomer methacrylic acid (MAA) in the solution was crosslink-polymerized on the surfaces of MAO–HEMA/NVP microspheres, resulting in the surface imprinting of lysozyme. After removing the template molecules, the lysozyme molecule-surface-imprinted material MIP-HEMA/NVP was obtained. Because there were strong interactions between lysozyme and monomer MAA, electrostatic interaction and hydrogen bonding, the lysozyme molecule-surface imprinting was successfully realized. The MIP-HEMA/NVP microspheres have very high binding affinity for lysozyme, and the binding capacity gets up to 216 mg/g. It is more important that MIP-HEMA/NVP microspheres have specific recognition selectivity for lysozyme, and the selectivity coefficient for lysozyme with respect to bovine hemoglobin (BHb), which was used as a contrast protein in the experiments, actually reaches 31.07. In the respect of protein imprinting, the imprinting material with such high performance is unwonted.     

Protein-protein recognition: an experimental and computational study of the R89K mutation in Raf and its effect on Ras binding

Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf/Arg89 in the center of the binding interface plays an important role determining Ras-Raf binding affinity. We have investigated experimentally and computationally the Raf-R89K mutation, which abolishes signaling in vivo. The binding to [gamma-35S]GTP-Ras of a fusion protein between the Raf-binding domain (RBD) of Raf and GST was reduced at least 175-fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal/mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9+/-1.9 kcal/mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal/mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein-protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 A away, namely residues 37-41 in the conserved effector domain of Ras (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2-3 specific water molecules. This analysis will provide insights into the large experimental database of Ras-Raf mutations.     

Screening combinatorial libraries of molecularly imprinted polymer films casted on membranes in single-use membrane modules

A new and fast technique for screening combinatorial libraries of molecularly imprinted polymers is presented. The procedure is based on commercially available membrane modules which are rinsed with pre-polymerization imprinting mixtures. After the in situ polymerization and generation of MIP films on the PTFE membranes within the modules, the membranes are evaluated in terms of affinity towards the target molecule (template) R-(-)-phenylbutyric acid. Therefore, after template extraction from the freshly produced membranes a solution of this target molecule is flushed through the module. By analyzing the remaining analyte concentration in the permeate, the amount of analyte adsorbed on the membrane can be calculated and related to specific interactions with the molecular imprints. By this means, optimized recipes in terms of cross-linker to template ratios could be obtained in combination with the optimal porogen, when screening p-divinylbenzene or ethylene glycol dimethacrylate as cross-linker and porogens like acetonitrile, dimethylsulfoxide and methanol.     

New biosourced chiral molecularly imprinted polymer: Synthesis,characterization, and evaluation of the recognition capacity of methyltestosterone

New biosourced chiral cross‐linkers were reported for the first time in the synthesis of methyltestosterone (MT) chiral molecularly imprinted polymers (cMIPs). Isosorbide and isomannide, known as 1,4:3,6‐dianhydrohexitols, were selected as starting diols. The cMIPs were synthesized following a noncovalent approach via thermal radical polymerization and monitored by Raman spectroscopy. These cross‐linkers were fully characterized by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy and high‐resolution mass spectrometry. The cross‐polarization magic angle spinning ¹³C NMR, Fourier transform infrared spectroscopy, scanning electron microscopy, and specific surface areas following the Brunauer‐Emmett‐Teller (BET) method were used to characterize the cMIPs. The effect of stereochemistry of cross‐linkers on the reactivity of polymerization, morphology, and adsorption‐recognition properties of the MIP was evaluated. The results showed that the cMIP exhibited an obvious improvement in terms of rebinding capacity for MT as compared with the nonimprinted polymer (NIP). The highest binding capacity was observed for cMIP‐Is (27.298 mg g⁻¹) for high concentrations (500 mg L⁻¹). However, the isomannide homologue cMIP‐Im showed higher recovery—up to 65% and capacity for low concentrations (15 mg L⁻¹). The experimental data were properly fitted by the Freundlich adsorption isothermal model.