Synthesis and evaluation of molecularly imprinted polymers for enalapril and lisinopril, two synthetic peptide anti-hypertensive drugs

Molecularly imprinted polymers (MIPs) for the recognition of enalapril and lisinopril were prepared using 4-vinylpyridine as the functional monomer. Following thermal polymerisation the resulting materials were crushed, ground and sieved. First generation MIPs were produced in protic polar porogenic solvents (mixture of methanol (MeOH) and acetonitrile (ACN)). These MIPs were used and validated as sorbents for solid phase extraction and binding assays. Second generation MIPs were produced with polar aprotic porogenic solvent (DMSO). These polymers were packed in HPLC columns in order to investigate their molecular recognition properties in a dynamic mode. The study of the mobile phase composition included two major parameters: organic modifier content and pH value. Retention factors illustrate selective binding of the template from the imprinted polymers, compared to structurally related compounds.     

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.     

Retention behavior of phenoxyacetic herbicides on a molecularly imprinted polymer with phenoxyacetic acid as a dummy template molecule

Molecular imprinted polymers (MIPs) binding with phenoxyacetic acid (PA) as a dummy template molecule were synthesized via thermal initiation in aqueous medium. The retention behaviors of benzoic acid (BA), PA, 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-chlorophenoxyacetic acid (4-CPA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on this MIP column indicate that this material can selectively retain phenoxyacetic herbicides. To investigate these recognition mechanisms, the interactions between the functional monomer 4-vinylpyridine (4-VP) and PA or 2,4-D were investigated by computational modeling. (1)H NMR spectroscopy of 2,4-D titrated by 4-VP was recorded. The chemical shift of the 2,4-D acidic proton (12.15-14.32ppm) shows the existence of the ion-pair interaction. This kind of polymers could be useful as stationary phases to extract 2,4-D, 4-CPA or MCPA and avoid leakage of a trace amount of target analyte remaining in the MIPs.     

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.     

Protein-responsive imprinted polymers with specific shrinking and rebinding

Stimuli-responsive protein imprinted polymers were obtained via a combination of molecular imprinting and reversible stimuli-responsive polymer using lysozyme or cytochrome c as template, N-isopropylacrylamide (NIPA) as major monomer, methacrylic acid (MAA) and acrylamide (AAm) as functional co-monomers, and N,N-methylenebisacrylamide (MBAAm) as crosslinker. The molecularly imprinted polymers (MIPs) can respond not only to external stimuli such as temperature and salt concentration, but also to the corresponding template protein with significant specific volume shrinking. This specific shrinking behavior was attributed to the synergistic effect of multiple-site weak interactions (electrostatic force, hydrogen bonding and hydrophobic interaction) and the cavity effect. The MIPs showed highly selective adsorption of template proteins with specific shrinking compared with the non-imprinted polymers. The results indicated that the MIPs seemed to change shape to accommodate the conformation of the template protein leading to the formation of a shape complementary cavity.     

Molecularly imprinted polymer using-p-hydroxybenzoic acid, p-hydroxyphenylacetic acid and p-hydroxyphenylpropionic acid as templates.

Molecularly imprinted polymers (MIPs) using p-hydroxybenzoic acid (p-HB), p-hydroxyphenylacetic acid (p-HPA) and p-hydroxyphenylpropionic acid (p-HPPA) as templates were synthesized. The performance of the templates and their analogues on polymer-based high performance liquid chromatography (HPLC) columns was studied. The imprinting effect of the MIP using p-HB as template is more obvious than that of MIP using either p-HPA or p-HPPA as template, and the mixture of p-HB and p-HPA can be well separated on the MIP using p-HB as template, but not on the blank. Interestingly, the recognition of MIP (p-HB as the template) to p-HB showed a synergistic effect. The retention factor of p-HB is not the sum of those of phenol and benzoic acid. We also found that the imprinting effect decreased when increasing the concentration of acetic acid in mobile phase. The possible reason is that acetic acid molecules occupied the binding sites of the polymer, thereby decreasing the concentration of binding sites. Furthermore, polymers, which showed specificity to 3,4-dihydroxybenzoic acid, can be prepared with p-HB as template. It is thus possible to synthesize a specific polymer for a compound that is either expensive or unstable by using a structurally similar compound as template.     

Direct rapid synthesis of MIP beads in SPE cartridges

Selecting optimal compositions for non-covalent molecularly imprinted polymers (MIPs) and screening for appropriate rebinding conditions necessitates synthesising a large number of polymers. This is extremely labour-intensive and usually results in very limited "optimisation" in studies of MIPs. Here, a new method is proposed for rapid synthesis of MIPs in a beaded form that can be used directly in many different performance evaluation studies. The method is based on synthesis of spherical particles by suspension polymerisation in liquid fluorocarbon [Mayes, A., Mosbach, K., 1996. Molecularly imprinted polymer beads: suspension polymerisation using a liquid perfluorocarbon as the dispersing phase. Anal. Chem. 68, 3769-3774]. The polymers were directly polymerised under UV light in solid phase extraction (SPE) cartridges, then washed and extracted in the same cartridges where they had been synthesised, resulting in a rapid and automatable process that requires no transfer or manipulation of the polymer particles. The particles were similar in terms of size, morphology and functional performance to particles obtained by suspension polymerisation in fluorocarbon solvent using a conventional reactor. In this initial study, 36 polymers were synthesised to study the effect of a variation in the type and amount of four different functional monomers, methacrylic acid (MAA), acrylic acid (AA), hydroxyethyl methacrylate (HEMA) and 2-vinylpyridine (2-VPy), for the imprinting of propranolol and morphine. The performance of polymers synthesised using MAA was as expected, but those synthesised with AA as functional monomer showed more surprising rebinding properties as a function of monomer to cross-linker ratios, demonstrating the potential value of pragmatic synthesis and screening approaches to polymer optimisation.     

Molecular recognition of indole derivatives by polymers imprinted with indole-3-acetic acid: A QSPR study

Three molecularly imprinted polymers (MIPs) were prepared using the phytohormone indole-3-acetic acid (IAA) as a template molecule, 4-vinylpyridine (MIP-1 and MIP-2) or N,N-dimethylaminoethyl methacrylate (MIP-3) as functional monomers, ethylenglycol dimethacrylate as a cross linker and acetonitrile (MIP-1), a methanol–water mixture (MIP-2) or chloroform (MIP-3) as porogens. Retention factors for IAA and 29 indole derivatives were determined by high-performance liquid chromatography, using the molecularly imprinted polymers as stationary phases and acetonitrile as an eluent. High correlations between selectivity factors of above mentioned polymers indicate that their retention mechanisms are basically the same. A quantitative structure–property relationships analysis revealed that the presence of the terminal carboxyl group on the 3-side chain plays an essential role in the binding of the indole derivatives to the polymers. The derivatives without the carboxyl group exhibit a drastically lower affinity toward the polymers. Another factor which favors the binding is electronic density of indole nucleus. Substituents with electro-withdrawing properties enhance the binding, while electro-donating substituents have the opposite effect. The length of the 3-side chain also affects the binding. Indole-3-carboxylic acid having the carboxyl group directly attached to the ring as well as the derivatives whose side chain is longer than that of IAA bind to the polymers with a lower affinity.     

Mobile phase effects on enantiomer resolution using molecularly imprinted polymers

The aim of this study was to rationalise retention behaviour of a chiral solute on molecularly imprinted polymer (MIP) HPLC stationary phases in terms of variation of the mobile phase. It is generally held that the most important interaction governing the separation of enantiomers on such materials is H-bonding, and that retention times increase with decreasing H-bonding potential of the mobile phase. Previous studies have largely concerned mobile phases containing chloroform with acetic acid as a polar modifier. Boc-L-Phenylalanine (Boc-L-Phe-OH) MIPs were prepared, processed, and packed into HPLC columns, which were then used to investigate the retention characteristics of Boc-L-Phe-OH and Boc-D-Phe-OH with a range of mobile phases. The main observations were as follows: (1) in chloroform-based mobile phases there was generally a linear relationship between the H-bond donator factor of the polar modifier and capacity (K′). Results also indicated a hydrogen bond donor parameter value for a polar modifier at which retention became concentration independent; (2) For given values of K′_L, K′_D varied depending on the polar modifier, indicating that enantiomer resolution was solvent dependent; (3) Using mobile phases based on solvents of lower polarity/H-bonding potential than chloroform, substantial increases in K′ were observed, although enantioselectivity was greatly reduced. Chirality 9:238–242, 1997. © 1997 Wiley-Liss, Inc.     

Effect of the template and functional monomer on the textural properties of molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) for zearalenone analysis have been synthesized using the template mimics cyclododecyl 2,4-dihydroxybenzoate (CDHB), resorcinol and resorcylic acid. The MIPs are photochemically prepared from 2-(diethylamino)ethyl methacrylate (2-DAEM), 4-vinylpyridine (VIPY), 2-hydroxyethyl methacrylate (HEMA) or 1-allylpiperazine (1-ALPP) as the functional monomers, trimethylolpropane trimethacrylate (TRIM) as cross-linker, azobis(isobutyronitrile) as initiator and acetonitrile as porogen. Non-imprinted polymers have been also synthesized for reference purposes. The textural properties of the novel polymers (BET areas, pore volumes and pore size distributions) have been determined from nitrogen adsorption-desorption isotherms. These parameters have shown to be strongly dependent on the presence of the template and the monomer nature. Scanning electron microscopy and solvent uptake experiments support these findings. Microporosity contributes less than 7% to the total pore volume for all the polymers prepared. Interestingly, a 3.5 nm pore opening is observed for all the polymers and additional pore apertures in the 20-40 nm region for VIPY-, HEMA- and 2-DAEM-based MIPs whereas a much wider opening size distribution has been measured for the 1-ALPP-based MIP. Molecular modeling and, particularly, (1)H NMR experiments demonstrate the strong (2:1) complex formed between 1-ALPP and the diphenolic CDHB (K(11)=4.7 x 10(4)M(-1) and K(12) = 2.6 x 10(2)M(-1) in acetonitrile) that make the corresponding MIP the most suitable for zearalenone recognition in real samples.     

Molecular imprinting and solid phase extraction of flavonoid compounds

Molecularly imprinted polymers (MIPs) for quercetin have been successfully prepared by a thermal polymerization method using 4-vinylpyridine (4-VP) and ethylene glycol dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The obtained molecularly imprinted polymers were evaluated by HPLC using organic eluents, with respect to their selective recognition properties for quercetin and related compounds of the flavonoid class. Two equivalent control polymers, a blank polymer and a polymer imprinted with a structural analogous template, were synthesized, in order to confirm the obtained results. Furthermore, preliminary experiments confirm the applicability of the prepared MIPs for solid phase extraction (SPE), as rapid and facile clean-up of wine samples for HPLC analysis is an envisaged field of application. The successful preparation of molecularly imprinted polymers for flavones provides an innovative opportunity for the development of advanced separation materials, with applications in the field of wine and fermentation analysis.     

Comparison of monofunctional and multifunctional monomers in phosphate binding molecularly imprinted polymers

In this study, molecularly imprinted polymers (MIPs) prepared using a multifunctional and a monofunctional monomer were compared with respect to their affinities, selectivities, and imprinting efficiencies for organophosphates. This is of interest because multifunctional monomers have higher affinities than traditional monofunctional monomers for their target analytes and thus should yield MIPs with higher affinities and selectivities. However, polymers containing multifunctional monomer may also have a higher number of unselective, non-templated binding sites. This increase in background binding sites could lead to a decrease in the magnitude of the imprinting effect and in the selectivity of the MIP. Therefore, phosphate selective imprinted and non-imprinted polymers (NIPs) were prepared using a novel multifunctional triurea monomer. The binding properties of these polymers were compared with polymers prepared using a monofunctional monourea monomer. The binding affinities and selectivities of the monomers, imprinted polymers, and NIPs were characterized by NMR titration, binding uptake studies, and binding isotherms. MIPs prepared with the triurea monomer showed higher binding affinity and selectivity for the diphenylphosphate anion in organic solvents than the MIPs prepared with the monofunctional monomer. Surprisingly, the binding properties of the NIPs revealed that the polymers prepared using the multifunctional and monofunctional monomers were very similar in affinity and selectivity. Thus, the multifunctional monomers increase not only the affinity of the MIP but also enhance the imprinting effect.     

Preparation of molecularly imprinted polymers for artemisinin based on the surfaces of silica gel

Artemisinin is an effective antimalarial drug isolated from the herbal medicine Artemisia annua L. Molecular imprinting is a technique of preparing molecularly imprinted polymers (MIPs) which can specifically recognize the imprinted template molecules. In this work, silica gel were used as supporting matrix, and vinyltriethoxysilane (VTES) was grafted onto its surface. The preparation of MIPs for artemisinin was performed on the surfaces of the modified silica gel using artemisinin as the template, acrylamide (AM) and methacrylic acid (MAA) as the functional monomers, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and 2,2'-azo-bis-isobutyronitrile (AIBN) as the initiator. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and pore size analysis were used to characterize the prepared MIPs. The adsorption kinetic curve, adsorption isotherm and selective adsorption were measured by static method. The adsorption reached equilibrium at about 10 h, while fast adsorption took place during the first 2-3 h. The maximum adsorption capacity has been found to be 37.13 mg/g according to calculation with Langmuir-Freundlich isotherm. The electivity coefficients of MIPs for artemisinin with respect to artemether and arteether were 2.88 and 3.38, respectively. The results showed that the MIPs possessed good specific adsorption capacity and selectivity for artemisinin.     

Monodispersed, molecularly imprinted polymers as affinity-based chromatography media

This review article deals with preparation methods for spherical and monodispersed molecularly imprinted polymers (MIPs) in micrometer sizes. Those methods include suspension polymerization in water, liquid perfluorocarbon and mineral oil, seed polymerization and dispersion/precipitation polymerization. The other methods are the use of beaded materials such as a spherical silica or organic polymer for grafting MIP phases onto the surfaces of porous materials or filling the pores of silica with MIPs followed by dissolution of the silica. Furthermore, applications of MIP microspheres as affinity-based chromatography media, HPLC stationary phases and solid-phase extraction media, will be discussed for pharmaceutical, biomedical and environmental analysis.     

Isolation and detection of steroids from human urine by molecularly imprinted solid-phase extraction and liquid chromatography

Naturally occurring steroids such as progesterone, testosterone and 17β-estradiol were analyzed in this study. These bio-identical molecules paradoxically can be either beneficial or harmful. Unfortunately as growth promoters can be toxic and cancerogenic at elevated levels. Due to difficulty in monitoring at trace quantities of these hormones in biological matrices specific adsorption materials molecularly imprinted polymers (MIPs) were used for preconcentration and clean up in sample preparation step. A non-covalent imprinting approach was used for bulk polymerization of progesterone, testosterone and 17β-estradiol imprinted polymers. Synthesis of MIPs was achieved by thermal, UV and γ irradiation initiated polymerization whereby were used methacrylic acid (MAA), 4-vinylpyridine (4-VP) as functional monomers, ethylene glycol dimethacrylate (EDMA), trimethylolpropane trimethacrylate (TRIM) as cross-linking agents and acetonitrile, isooctane–toluene (1:99, v/v) and chloroform as porogen solvents. It was also used as initiator 2,2′-azobis(2-methylpropionitrile) (AIBN) or benzyl methyl ether (BME). The MIPs were applied as selective sorbents in solid-phase extraction (SPE). Molecularly imprinted solid-phase extraction (MISPE) considered as hyphenated technique were applied in extraction step before HPLC-DAD analysis of steroids from human urine.     

Molecularly imprinted polymer-based optical immunosensors

Molecularly imprinted polymers (MIPs) are artificial antibodies for a target molecule. The review focuses mainly on mechanistic steps involved in forming MIPs and the role of co-monomers and porogen. In addition, the electronic transition between different energy levels is explained with the help of the Jablonski diagram. Diverse receptor and target molecules for anchoring artificial MIPs are discussed, accentuating the synergetic effects obtained. The binding efficiency, selectivity, and sensitivity of various optical sensors are discussed intensively. In addition to this, we focused on synthesis, physical forms, characterization techniques, and microorganism detection of imprinted polymers. A brief investigation on the use of MIPs in cancer diagnosis is also included, and attention is extended to the important challenges faced in using imprinted polymers.     

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.     

Molecularly imprinted polymers for the determination of a pharmaceutical development compound in plasma using 96-well MISPE technology

The use of molecularly imprinted polymers (MIPs) as sorbents for the solid phase extraction (SPE) of a pharmaceutical compound in development, prior to quantitative analysis was investigated. Three MIPs were synthesised using a structural analogue as the template molecule. Each polymer was prepared with different monomers and porogens. The MIPs were then tested for their performance both in organic and aqueous environments, the final aim being to load plasma directly onto the polymers. At an early development stage, there is a limited amount of compound available. Due to this limitation, reducing the amount of template required for imprinting was investigated. A MIP capable of extracting the analyte directly from plasma was produced. The specificity of the polymer allowed the method to be validated at a lower sensitivity than a more conventional SPE assay. For the first time, MIPs were packed into 96-well blocks enabling high throughput analysis. The analytical method was fully validated for imprecision and inaccuracy down to 4 ng/ml in plasma.     

A simple approach to prepare molecularly imprinted polymers from nylon‐6

A convenient and simple approach for the preparation of molecularly imprinted polymers (MIPs) based on polyamide (nylon‐6) was developed. The polymer matrix formation occurred during the transition of nylon from dissolved to solid state in the presence of template molecules in the initial solution. 2,2,2‐Trifluoroethanol (TFE) was chosen as a main solvent for the polyamide. It provides a high solubility of nylon and does not significantly change the structure of biopolymers. The alteration of the polymer matrix structure after the addition of different types of porogens in the nylon/TFE solution was investigated. The structured polymers in the form of films and microparticles were prepared in the chosen optimal conditions. Different model biomolecular templates (of low‐ and high‐molecular weight) were used for the preparation of MIPs, which were shown to specifically recognize these molecules upon binding experiments. The binding of the template molecules to MIPs was monitored using spectrophotometric, radioisotopic, or fluorometric detection. The selectivity coefficients of the MIPs were estimated to be 1.4–4.6 depending on the type of templates and conditions of the polymer matrix formation. Copyright © 2013 John Wiley & Sons, Ltd.