Insights into the origins of binding and the recognition properties of molecularly imprinted polymers prepared using an amide as the hydrogen-bonding functional group

Molecularly imprinted polymers (MIPs) prepared using an amide hydrogen-bonding functional monomer (acrylamide) exhibited efficient enantiomeric recognition properties in both organic and aqueous media in the HPLC mode. The results indicate that the amide functional groups formed strong hydrogen-bonding interactions with the template molecule, and specific recognition sites were created within the polymer matrix during the imprinting process. When Boc-L-Trp was used as the template, an MIP prepared in a polar organic solvent (acetonitrile) using acrylamide as the functional monomer showed better enantiomeric recognition of Boc-Trp than the MIPs prepared in the same solvent using an acidic (methacrylic acid) or a basic (2-vinylpyridine) functional monomer or a combination of an acidic and a basic functional monomer (methacrylic acid + 2-vinylpyridine). Our results indicate that in organic media the degree of retention of the sample molecule on the imprinted polymer was controlled by hydrogen-bonding interactions between the sample molecule and the polymer, while in aqueous media it was determined to a considerable extent by hydrophobic interactions. In both media the shape, size and the nature of the hydrogen-bonding groups of the sample molecules were all important factors in determining the enantiomeric and substrate selectivity. In the aqueous media, however, the hydrophobicity of the sample molecules was also found to play an important role.     

Towards the rational development of molecularly imprinted polymers: 1H NMR studies on hydrophobicity and ion-pair interactions as driving forces for selectivity

The preparation of molecularly imprinted polymers (MIP) based on non-covalent interactions has become a widely used technique for creating highly specific sorbent materials predominantly used in separation chemistry. A crucial factor in a successful imprinting protocol is the optimisation of the template/functional monomer interaction in the pre-polymerisation mixture, eventually leading to a maximum of high-affinity binding sites in the resulting polymer matrix. In order to develop more efficient preparation technologies for imprinted polymers, two separate pre-polymerisation complexes were investigated by NMR spectroscopic techniques in order to identify the types of interactions occurring in the pre-polymerisation mixture, and their implications for the subsequently formed imprinted polymer. In particular, hydrophobic effects have been followed by NMR spectroscopy and their contribution to the selectivity of the resulting MIP has been investigated. The 2,4-D imprint system is used as an example to fundamentally study whether observations at the pre-polymerisation stage correlate with properties of the finally prepared MIP, and which parameters govern success of an imprinting protocol.     

Capacitive sensor for environmental monitoring based on thin films of molecularly imprinted polymers. Computer modeling for optimization of the composition of synthetic analogs of bioreceptors

A capacitive sensor for environmental monitoring based on thin films of desmetryn-selective molecularly imprinted polymer (MIP) was developed. The method of modification of gold electrodes with the thin film of herbicide-selective MIP using the grafting polymerization approach was developed. The method of computational modeling was used to optimize the composition of desmetryn-selective MIPs. It was shown that 2-acrylamido-2-methyl-1-propan-sulfonic acid is the optimal functional monomer for desmetryn. Formation of synthetic binding sites in MIPs was demonstrated to be determined by the binding energy between the template and functional monomers as well as the number of functional groups taking part in the recognition of the template molecule. Electrochemical processes occurring at the MIP-modified electrode were analyzed. The detection limit for desmetryn comprised 100 nM. High selectivity of the capacitive sensor towards structural analogues of desmetryn as well as high operational and storage stabilities was demonstrated.     

Size-selective recognition of catecholamines by molecular imprinting on silica-alumina gel

The preparation of a catecholamine receptor was carried out using a molecular imprinting method with silica-alumina gel to form complementary structures for template recognition. The molecularly imprinted polymer (MIP) was synthesized by the condensation of silicate from tetraethyl orthosilictate (TEOS) under hydrothermal conditions at 60 degrees C. Aluminum chloride was added as a functional monomer to increase the material's rebinding ability. The selectivity of the MIP receptor prepared with different ratios of template to Si and Al, was examined with seven analytes including: dopamine, epinephrine, norepinephrine, ascorbic acid, homovanillic acid, uric acid, and l-tyrosine. The results showed a size selective effect for the receptors with respect to the recognition of the catecholamines. Some factors affecting the recognition ability were investigated including: the solution pH of analytes, surface capping on the MIP, and the imprinting pH of the silica-alumina solution. Also, the catecholamine MIP films on quartz crystal microbalance (QCM) electrodes were fabricated as sensors for in situ monitoring of the analytes in a 2-propanol solution.     

Molecular imprinting of nitrophenol and hydroxybenzoic acid isomers: effect of molecular structure and acidity on imprinting

Three nitrophenol isomer-imprinted polymers were prepared under the same conditions using 4-vinylpyridine as a functional monomer. Different recognition capacities for template molecules were observed for the three polymers. Another imprinting system with stronger acidity than nitrophenol isomers, 2-hydroxybenzoic acid (salicylic acid) and 4-hydroxybenzoic acid, was imprinted using 4-vinylpyridine or acrylamide as functional monomer respectively. Both 4-hydroxybenzoic acid-imprinted polymers using the two monomers showed recognition ability for the template molecule. However, when acrylamide was chosen as functional monomer, the salicylic acid-imprinted polymer showed very weak recognition for the template molecule, whereas strong recognition ability of the resultant polymer for salicylic acid was observed with 4-vinylpyridine as functional monomer. It seems that the structure and acidity of template molecules is responsible for the difference in recognition, by influencing the formation and strength of interaction between template molecule and functional monomer during the imprinting process. An understanding of the mechanism of molecular imprinting and molecular recognition of MIPs will help to predict the selectivity of MIPs on the basis of template molecule properties.     

New biomedical devices with selective peptide recognition properties. Part 1: Characterization and cytotoxicity of molecularly imprinted polymers

Molecular imprinting is a technique for the synthesis of polymers capable to bind target molecules selectively. The imprinting of large proteins, such as cell adhesion proteins or cell receptors, opens the way to important and innovative biomedical applications. However, such molecules can incur into important conformational changes during the preparation of the imprinted polymer impairing the specificity of the recognition cavities. The "epitope approach" can overcome this limit by adopting, as template, a short peptide sequence representative of an accessible fragment of a larger protein. The resulting imprinted polymer can recognize both the template and the whole molecule thanks to the specific cavities for the epitope. In this work two molecularly imprinted polymer formulations (a macroporous monolith and nanospheres) were obtained using the protected peptide Z-Thr-Ala-Ala-OMe, as template, and Z-Thr-Ile-Leu-OMe, as analogue for the selectivity evaluation, methacrylic acid, as functional monomer, and trimethylolpropane trimethacrylate and pentaerythritol triacrylate (PETRA), as cross-linkers. Polymers were synthesized by precipitation polymerization and characterized by standard techniques. Polymerization and rebinding solutions were analyzed by high performance liquid chromatography. The highly cross-linked polymers retained about 70% of the total template amount, against (20% for the less cross-linked ones). The extracted template amount and the rebinding capacity decreased with the cross-linking degree, while the selectivity showed the opposite behaviour. The PETRA cross-linked polymers showed the best recognition (MIP 2-, alpha=1.71) and selectivity (MIP 2+, alpha'=5.58) capabilities. The cytotoxicity tests showed normal adhesion and proliferation of fibroblasts cultured in the medium that was put in contact with the imprinted polymers.     

Development of molecularly imprinted polymers for the binding of nitrofurantoin

Novel molecularly imprinted polymers (MIPs) for the recognition of nitrofurantoin (NFT) were prepared by photoinitiated polymerisation in polar solvent using 2,6-bis(methacrylamido) pyridine (BMP) as the functional monomer and carboxyphenyl aminohydantoin (CPAH) as the analogue of the template. The binding constants of the complex between BMP and nitrofurantoin or CPAH in DMSO were determined with 1H NMR titration to be 630 ± 104 and 830 ± 146 M⁻¹, respectively. To study the influence of the functional monomer, two polymer compositions were prepared containing the template, the functional monomer and the crosslinker in the molar ratio 1:1:12 for MIP1 and 1:4:20 for MIP2, respectively. The imprinting factor at saturation concentration of nitrofurantoin, which is the ratio of the amount bound to the MIP and the non-imprinted control polymer (NIP), was determined to be 2.47 for MIP1 and 2.49 for MIP2. The cross reactivity of the imprinted polymers seems to be determined by the ability to form hydrogen bonds to the functional monomer while the shape of the molecule has no real influence.     

Matrine-imprinted monolithic stationary phase for extraction and purification of matrine from Sophorae flavescentis Ait

A matrine-imprinted monolithic stationary phase (MIP monolith) was prepared by in situ polymerization for extraction and purification of matrine from Sophorae flavescentis Ait. Matrine was used as the template molecule, methacrylic acid as the function monomer, ethylene glycol dimethacrylate as the cross-linking agent, and toluene and dodecanol as the porogenic solvents. Scanning electron microscope study revealed that a monolithic structure with mesopores and 36 μm diameter nodules was obtained. The molecular recognition process and the effect of varying chromatographic conditions on separation were examined by high-performance liquid chromatography (HPLC). Hydrogen bonding, electrostatic, hydrophobic interactions and the molecular shape matching in MIP monolith cavities were proposed to be responsible for the recognition mechanism. The use of MIP monolith as a solid-phase extraction (SPE) sorbent for extraction and purification of matrine from S. flavescentis Ait was investigated. The extraction yield was 89.2% (for 3.0 mmol l(-1) matrine) with enrichment factor 29.     

Grafting of molecularly imprinted polymers on iniferter-modified carbon nanotube

Molecularly imprinted polymers (MIPs) were grafted on iniferter-modified carbon nanotube (CNT). Tween 20 was first immobilized on CNT by hydrophobic interactions. The hydroxyl-functionalized CNT was modified by silanisation with 3-chloropropyl trimethoxysilane. The iniferter groups were then introduced by reacting the CNT-bound chloropropyl groups with sodium N,N-diethyldithiocarbamate. UV light-initiated copolymerization of ethylene glycol dimethacrylate (crosslinking agent) and methacrylic acid (functional monomer) resulted in grafting of MIP on CNT for theophylline as a model template. MIPs grafted on CNT were characterized with elemental analysis, scanning electron microscopy, and thermogravimetric analysis. The theophylline-imprinted polymer on CNT showed higher binding capacity for theophylline than non-imprinted polymer on CNT and selectivity for theophylline over caffeine and theobromine (similar structure molecules). The data of theophylline and caffeine binding into the theophylline-imprinted polymer correlated well with the Scatchard plot. These MIPs on CNT can potentially be applied to probe materials in biosensor system based on CNT field effect transistor.     

Surface molecular imprinting by atom transfer radical polymerization

Results are presented that demonstrate the successful preparation of ultrathin (< 10 nm), surface-confined, molecularly imprinted polymer (MIP) films on model gold substrates using atom transfer radical polymerization (ATRP). 2-Vinylpyridine (2Vpy) was investigated as the functional monomer, and ethylene glycol dimethacrylate (EGDMA) was the cross-linking monomer. Fluorescently labeled N,N'-didansyl-L-cystine and N,N'-didansyl-L-lysine were used as the template molecules to form the MIPs. Spectroscopic and ellipsometric results are presented that follow film formation and growth rates. Results are also presented from fluorescence experiments used to quantify and compare the adsorption capacities of MIP surface films and nonimprinted (NIP) control films. MIP films exhibited higher binding capacities than the control NIP films at all solution concentrations of N,N'-didansyl-L-cystine and N,N'-didansyl-L-lysine. Furthermore, template removal from these imprinted films appears to be 100% efficient. Selectivity studies showed that the MIPs display some cross-reactivity between these two molecules; nevertheless, MIPs prepared against one template showed selectivity for that template. A selectivity coefficient of 1.13 was achieved for MIP surfaces prepared against N,N'-didansyl-L-lysine; a value of 1.51 was observed for MIP surfaces prepared against N,N'-didansyl-L-cystine.     

Chiral imprinted polymers as enantiospecific coatings of stir bar sorptive extraction devices

This paper reports the design of Molecularly Imprinted Polymers (MIP) with affinity towards (S)-citalopram using computational modeling for the selection of functional monomers and monomer:template ratio. Acrylamide was selected as functional monomer and the final complex functional monomer/template resulted in a 3:1 ratio. The polymer was synthesized by radical polymerization initiated by UV onto magnetic stir-bars in order to obtain a stir bar sorptive extraction (SBSE) device capable of selective enantiomeric recognition. After successful template removal, the parameters affecting the SBSE procedure (sample volume, ionic strength, extraction time and pH) were optimized for the effective rebinding of the target analyte. The resultant chirally imprinted polymer based stir-bar was able to selectively extract (S)-citalopram from a racemic mixture in an aqueous media with high specificity (specificity factor 4) between 25 and 500 μgL(-1). The MIP coated stir-bars can have significance for enantiospecific sample pre-concentration and subsequent analysis without the need for any chiral chromatographic separation.     

Effective removal of rhodamine B from contaminated water using non-covalent imprinted microspheres designed by computational approach

Molecular dynamics simulations and computational screening were used to identify functional monomers capable of interacting with rhodamine B (RhB). A library of 24 kinds of common functional monomers for preparing molecular imprinted polymer (MIP) was built and their interactions with RhB in acetonitrile were calculated using the molecular dynamics software (Gromacs 3.3). It was anticipated that the monomers giving the highest binding energy are suitable for preparing the affinity polymers. According to the theoretical calculation results, the MIP microspheres with RhB as template was prepared by reverse microemulsion polymerization method using acrylamide (AAm) as functional monomer and divinylbenzene as cross-linker in acetonitrile. Microspheres have been characterized by scanning electron microscopy (SEM). The proper adsorption and selective recognition ability of the MIP were studied by an equilibrium-adsorption method. The MIP showed outstanding affinity towards RhB in aqueous solution and the optimum pH value for binding has been found around neutral range. The molecular recognition of RhB was analyzed in detail by using molecular modeling software (Gaussian03). In addition, the MIP reusability without obviously deterioration in performance was demonstrated at least five repeated cycles.     

Novel chiral recognition elements for molecularly imprinted polymer preparation

The use of a novel chiral functional monomer system in molecular imprinting protocols is described. The monomer, dibenzyl (2R,3R)-O-monoacryloyl tartrate, possesses a hydroxyl moiety which can be used to direct template-functional monomer interactions during molecular imprinting polymerization. This system simultaneously positions benzyl ester-protected carboxyl groups in close proximity to the template, which upon deprotection yield recognition sites with stronger ligand-binding capacities. Furthermore, the inherent chirality of the monomer engenders the polymer with an inbuilt preference for a given stereoisomer. Application of the system to the molecular imprinting of the cinchonidine alkaloids (+)-cinchonine and (-)-cinchonidine yielded stereoselective polymers. The effect of imprinting (+)-cinchonine produced a polymer which more than reversed the inherent chiral selectivity of the chiral monomer residues present in the matrix.     

A computational approach to studying monomer selectivity towards the template in an imprinted polymer

A computational approach was proposed to study monomer–template interactions in a molecularly imprinted polymer (MIP) in order to gain insight at the molecular level into imprinting polymer selectivity, regarding complex formation between template and monomer at the pre-polymerisation step. This is the most important step in MIP preparation. In the present work, chlorphenamine (CPA), diphenhydramine (DHA) and methacrylic acid (MAA), were chosen as the template, non-template, and monomer, respectively. The attained complexes were optimised, and changes in the interaction energies, atomic charges, IR spectroscopy results, dipole moment, and polarisability were studied. The effects of solvent on template–monomer interactions were also investigated. According to a survey of the literature, this is the first work in which dipole moment and polarisability were used to predict the types of interactions existing in pre-polymerisation complexes. In addition, the density functional tight-binding (DFTB) method, an approximate version of the density functional theory (DFT) method that was extended to cover the London dispersion energy, was used to calculate the interaction energy.     

Combinatorial screening of polymer precursors for preparation of benzo[α] pyrene imprinted polymer: an ab initio computational approach

A combinatorial screening procedure was used for the selection of polymer precursors in the preparation of molecularly imprinted polymer (MIP), which is useful in the detection of the air pollution marker molecule benzo[a]pyrene (BAP). Molecular imprinting is a technique for the preparation of polymer materials with specific molecular recognition receptors. The preparation of imprinted polymers requires polymer precursors such as functional monomer, cross-linking monomer, solvent, an initiator of polymerization and thermal or UV radiation. A virtual library of functional monomers was prepared based on interaction binding scores computed using HyperChem Release 8.0 software. Initially, the possible minimum energy conformation of the monomers and BAP were optimized using the semi-empirical (PM3) quantum method. The binding energy between the functional monomer and the template (BAP) was computed using the Hartree-Fock (HF) method with 6-31 G basis set, which is an ab initio approach based on Moller-Plesset second order perturbation theory (MP2). From the computations, methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were selected for preparation of BAP imprinted polymer. The larger interaction energy (ΔE) represents possibility of more affinity binding sites formation in the polymer, which provides high binding capacity. The theoretical predictions were complimented through adsorption experiments. There is a good agreement between experimental binding results and theoretical computations, which provides further evidence of the validity of the usefulness of computational screening procedures in the selection of appropriate MIP precursors in an experiment-free way.     

Molecularly imprinted ligand-exchange recognition assay of glucose by quartz crystal microbalance

Molecular imprinted polymers (MIP) as a recognition element for sensors are increasingly of interest and MIP-quartz crystal microbalance (QCM) have started to appear in the literature. In this study, we have combined quartz crystal microbalance with MIP to prepare a sensor using the ability of glucose to chelate of copper (II) ion of methacrylamidohistidine (MAH) monomer to create ligand exchange (LE) assembled monolayer which is suitable for glucose determination. The study includes the measurement of binding interaction of molecularly imprinted QCM sensor via ligand interaction, investigation of the pH effect on frequency shift and recognition selectivity studies of glucose-imprinted polymer with respect to methyl-alpha-d-glucopyranoside and sucrose. Bmax (number of binding sites) and K(D) (dissociation constant of the metal-chelate copolymer) were also calculated using Scathard plot and the detection limit was found as 0.07 mM. MIP showed higher glucose-binding affinity than a well-known glucose binding protein, conconavalin A.     

Biodegradable molecularly imprinted polymers based on poly(ε-caprolactone)

Novel biodegradable molecularly imprinted polymers (MIPs) based on poly(ε-caprolactone) (PCL) were prepared by combining two important properties required of ideal biomaterials, biodegradability (with biocompatibility) and molecular recognition properties. Acrylate or methacrylate end-capped PCL macromers were synthesized through the reaction of PCL diol or triol with acryloyl or methacryloyl chloride. The synthesis of acrylate or methacrylate end-capped macromers was confirmed using FT-IR and H NMR spectroscopic techniques. These macromers were used to prepare biodegradable crosslinked networks by photopolymerization with functional monomer (acrylic acid) and a model template (theophylline). The theophylline-imprinted polymer showed higher binding capacity for theophylline compared with non-imprinted polymer (NIP), and also showed selectivity for theophylline over caffeine (similar structure molecules). PCL-based MIP degraded 8% of the initial weight in 30 days in phosphate-buffered saline (PBS) solution (pH 7.4) and over 90% of the initial weight within 24 h in 1 N NaOH at 37°C.     

N-isopropylacrylamide as a functional monomer for noncovalent molecular imprinting

Although N-isopropylacrylamide (NIPAM) has previously been used in molecular imprinting, it has mostly been considered as an 'inert' monomer, or included for its temperature-responsive nature, rather than as a functional monomer responsible for the interactions with the template at the recognition site. A comparative study of NIPAM and other traditional, functional monomers for the imprinting of a hydrogen bond donor template, bisphenol A (BPA), is reported. Nuclear magnetic resonance titration data suggest that NIPAM forms a stronger complex with BPA than either acrylamide or methacrylic acid but a weaker complex than vinylpyridine. Molecular imprinted polymers (MIPs) were prepared using each functional monomer and compared as stationary phases for the separation of BPA from structural analogues. The NIPAM-containing MIP bound BPA with better selectivity than those prepared using acrylamide or methacrylic acid. Using NIPAM also reduces the nonspecific binding, which is found with MIPs using vinylpyridine as functional monomer.     

Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.     

Separation of ursodeoxycholic acid from its isomeric mixture using core–shell molecular imprinting polymer

In order to separate ursodeoxycholic acid (UDCA) from its isomeric mixture, the molecular imprinting polymers (MIPs) were synthesized by using core–shell emulsion polymerization. In the porous imprinting polymer, ursodeoxycholic acid was used as imprinting molecule, acrylamide (AM) and α-methacrylic acid (MAA) were functional monomers, and CaCO₃ was used for the porogen in the polymerization to obtain large pore. Characterization of the MIP structure with IR spectra demonstrated the expected MIPs. Through adsorption and selectivity assays, AM as the functional monomer showed better separation efficiency than MAA, and nonspecific and specific adsorption capacities of MIP with AM were 43.52 and 13.93 mg/g, respectively. The separation factor of MIP with AM for UDCA was 2.20. Furthermore, MIP with AM could be applied to separate UDCA from the isomeric mixture by column chromatography successfully.