A direct comparison of the performance of ground, beaded and silica-grafted MIPs in HPLC and turbulent flow chromatography applications

Spherical molecularly imprinted polymers (MIPs) specific to the beta-blocker propranolol have been synthesised using two different approaches and compared to traditional ground monolithic MIPs in HPLC and TFC applications. TFC is a LC technique used for rapid extraction of compounds directly from complex matrices. It can be easily coupled to HPLC and MS for automation of an extraction/analysis procedure. Spherical MIP beads were produced using a suspension polymerisation technique and silica/MIP composite beads by grafting MIP to spherical silica particles using a surface-bound initiator species. Synthesis of both beaded and silica-grafted MIPs was more practical than using the traditional grinding method and yields of spherical particles of the required size between 80 and 100% were routinely achieved. Under HPLC conditions, beaded and ground MIP materials showed a degree of chiral separation for all of the nine beta-blockers tested. The beaded MIP, however, showed much better flow properties and peak shape than the ground material. Silica-grafted MIP showed some separation in five of the drugs and a large improvement in peak shape and analysis times compared with both ground and beaded MIPs. The materials prepared were also used in extraction columns for Turbulent Flow Chromatography (TFC). Although no imprinting effect was observed under typical TFC conditions, beaded polymer materials showed promise for use as TFC extraction columns due to the good flow properties and clean extracts obtained.     

In situ synthesis of molecularly imprinted nanoparticles in porous support membranes using high-viscosity polymerization solvents

There is a growing need in membrane separations for novel membrane materials providing selective retention. Molecularly imprinted polymers (MIPs) are promising candidates for membrane functionalization. In this work, a novel approach is described to prepare composite membrane adsorbers incorporating molecularly imprinted microparticles or nanoparticles into commercially available macroporous filtration membranes. The polymerization is carried out in highly viscous polymerization solvents, and the particles are formed in situ in the pores of the support membrane. MIP particle composite membranes selective for terbutylazine were prepared and characterized by scanning electron microscopy and N? porosimetry. By varying the polymerization solvent microparticles or nanoparticles with diameters ranging from several hundred nanometers to 1 μm could be embedded into the support. The permeability of the membranes was in the range of 1000 to 20,000 Lm?2 hr?1 bar?1. The imprinted composite membranes showed high MIP/NIP (nonimprinted polymer) selectivity for the template in organic media both in equilibrium-rebinding measurements and in filtration experiments. The solid phase extraction of a mixture of the template, its analogs, and a nonrelated compound demonstrated MIP/NIP selectivity and substance selectivity of the new molecularly imprinted membrane. The synthesis technique offers a potential for the cost-effective production of selective membrane adsorbers with high capacity and high throughput.     

Towards water compatible MIPs for sensing in aqueous media

When synthesizing molecularly imprinted polymers (MIPs), a few fundamental principles should be kept in mind. There is a strong correlation between porogen polarity, MIP microenvironment polarity and the imprinting effect itself. The combination of these parameters eventually determines the overall binding behavior of a MIP in a given solvent. In addition, it is shown that MIP binding is strongly influenced by the polarity of the rebinding solvent. Because the use of MIPs in biomedical environments is of considerable interest, it is important that these MIPs perform well in aqueous media. In this article, various approaches are explored towards a water compatible MIP for the target molecule l-nicotine. To this end, the imprinting effect together with the MIP matrix polarity is fine-tuned during MIP synthesis. The binding behavior of the resulting MIPs is evaluated by performing batch rebinding experiments that makes it possible to select the most suitable MIP/non-imprinted polymer couple for future application in aqueous environments. One method to achieve improved compatibility with water is referred to as porogen tuning, in which porogens of varying polarities are used. It is demonstrated that, especially when multiple porogens are mixed, this approach can lead to superior performance in aqueous environments. Another method involves the incorporation of polar or non-polar comonomers in the MIP matrix. It is shown that by carefully selecting these monomers, it is also possible to obtain MIPs, which can selectively bind their target in water.     

Binding site characteristics of 17beta-estradiol imprinted polymers

The variety of applications utilizing molecularly imprinted polymers (MIPs) requires synthetic strategies yielding different MIP formats including films, irregular particles, or spheres, along with precise knowledge on the specific material characteristics, such as binding capacity and binding efficiency of these materials. In response to this demand, MIPs are prepared in different formats by variation of the polymerization methodology. It is commonly agreed that micro- and sub-microspheres are particularly advantageous MIP formats, due to their monodispersity and facile synthesis procedures in contrast to conventional imprinted polymers prepared by bulk polymerization. However, the differences in actual rebinding characteristics of different MIP formats based on molecular interactions under a variety of binding/rebinding conditions have not been studied in detail to date. Consequently, the present work details an analytical strategy generically applicable to MIP systems for rebinding studies including equilibrium binding, non-equilibrium binding, and release experiments enabling more profound understanding on the molecular interactions between the imprinted materials and the template molecules. In this study, three MIP formats were considered for the same template molecule, 17beta-estradiol: irregularly shaped particulate polymers prepared by bulk polymerization and grinding, microspheres, and sub-microspheres. The latter two formats were synthesized via precipitation polymerization using different processing strategies. The morphologies and porosities of the resulting imprinted materials were characterized by scanning electron microscopy (SEM) and Brunauer-Emmett-Teller (BET) analysis, respectively. The obtained results indicate that microspheres prepared by precipitation polymerization provide superior rebinding properties during equilibrium binding in contrast to bulk polymers and sub-microspheres, and that the rebinding properties are different during equilibrium binding versus non-equilibrium binding. The median binding affinity constant determined during non-equilibrium rebinding is higher than the values obtained from equilibrium rebinding. Furthermore, the binding site distribution appears more homogeneous thief derived from non-equilibrium rebinding, as reflected in a heterogeneity index of m=0.725. Moreover, it is hypothesized that the specific interactions between template and monomers are related to the porosity of the imprinted polymers, which implies that the amount of binding sites and the pore sized distribution of the imprinted materials are a critical factor in achieving the desired MIP performance in various analytical applications. The BET results indicate that particles prepared with lower cross-linker-to-template ratio have a reduced surface area. Furthermore, it can be expected that there are less specific binding sites available at particles with reduced surface area and pore volume given similar distribution of the binding sites, as confirmed by the equilibrium binding isotherm studies. The pore size distribution results reveal that control of the pore size in the range of 100-180 A is essential to obtain the desired retention properties and Gaussian peak shape during HPLC analysis of small molecules.     

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.     

A computational approach to study functional monomer‐protein molecular interactions to optimize protein molecular imprinting

Molecular imprinting has become a promising approach for synthesis of polymeric materials having binding sites with a predetermined selectivity for a given analyte, the so‐called molecularly imprinted polymers (MIPs), which can be used as artificial receptors in various application fields. Realization of binding sites in a MIP involves the formation of prepolymerization complexes between a template molecule and monomers, their subsequent polymerization, and the removal of the template. It is believed that the strength of the monomer‐template interactions in the prepolymerization mixture influences directly on the quality of the binding sites in a MIP and consequently on its performance. In this study, a computational approach allowing the rational selection of an appropriate monomer for building a MIP capable of selectively rebinding macromolecular analytes has been developed. Molecular docking combined with quantum chemical calculations was used for modeling and comparing molecular interactions among a model macromolecular template, immunoglobulin G (IgG), and 1 of 3 electropolymerizable functional monomers: m‐phenylenediamine (mPD), dopamine, and 3,4‐ethylenedioxythiophene, as well as to predict the probable arrangement of multiple monomers around the protein. It was revealed that mPD was arranged more uniformly around IgG participating in multiple H‐bond interactions with its polar residues and, therefore, could be considered as more advantageous for synthesis of a MIP for IgG recognition (IgG‐MIP). These theoretical predictions were verified by the experimental results and found to be in good agreement showing higher binding affinity of the mPD‐based IgG‐MIP toward IgG as compared with the IgG‐MIPs generated from the other 2 monomers.     

Novel biphasic separations utilising highly selective molecularly imprinted polymers as biorecognition solvent extraction agents

Molecularly imprinted polymers (MIPs) represent a class of artificial receptors that promise an environmentally robust alternative to naturally occurring biorecognition elements of biosensing devices and systems. However, in general, the performance of conventional MIPs in aqueous environments is poor. In the study reported here, this limitation has been addressed by the novel application of MIPs as a solvent extraction solid phase in a biphasic solvent system. This paper describes a previously unreported use of MIPs as solvent extraction reagents, their successful application to aqueous sample media and the opportunities for utilisation of this unique system in novel biosensing and separation procedures. This study demonstrates the development of a novel biphasic solvent system utilising MIP in the extracting phase to enhance both efficiency and selectivity of a simple two phase liquid extraction. Monodisperse propranolol imprinted polymer microspheres [p(divinylbenzene-co-methacrylic acid)] were prepared by precipitation polymerisation. Initially, the affinity of the polymers for (R,S)-propranolol was assessed by established techniques whereby the MIP demonstrated greater affinity for the template than did the non-imprinted control polymer (NIP). Importantly, MIP performance was also assessed using the novel dual solvent system. The depletion of (R,S)-propranolol from the aqueous phase into the polymer containing organic phase was determined. When compared to control extractions containing no polymer the presence of MIP in the extracting solvent phase resulted in an increased extraction of (R,S)-propranolol from the aqueous phase. Importantly, this extraction was significantly greater in the presence of MIP when compared to NIP. This unique principle generates opportunities for MIP based extractions and chemical enrichments in industrial applications, offering commercial, ecological and practical advantages to traditional solvent extraction techniques. The technique is readily transferable to analytical microsystems utilising MIP recognition elements generating promising opportunities for MIP based sensing of aqueous sample media.     

Molecularly imprinted polymer films for reflectometric interference spectroscopic sensors

Reflectometric interference spectroscopic measurements were performed on molecularly imprinted polymer (MIP) films with the herbicide atrazine as the template molecule. A conventional imprinting protocol was used relying on non-covalent interactions between the functional monomers and the template. The MIPs were deposited on glass transducers by two different methods: spin-coating followed by in situ polymerization of thin films of monomers containing a sacrificial polymeric porogen, and autoassembly of MIP nanoparticles with the aid of an associative linear polymer. Reproducible results were obtained upon measurements of atrazine solutions in toluene with both films. Atrazine concentrations as low as 1.7 ppm could be detected with the autoassembled particle film. No or very little analyte adsorption was observed onto non-imprinted control films made by spin-coating and by particle assembly, respectively. We believe that these MIP layers in combination with the general reflectrometric transduction scheme could be a versatile sensing tool for the detection of environmentally important and other analytes.     

Fluorescence anisotropy studies of molecularly imprinted polymers.

A molecularly imprinted polymer (MIP) is a biomimetic material that can be used as a biochemical sensing element. We studied the steady-state and time-resolved fluorescence and fluorescence anisotropy of anthracene-imprinted polyurethane. We compared MIPs with imprinted analytes present, MIPs with the imprinted analytes extracted, MIPs with rebound analytes, non-imprinted control polymers (non-MIPs) and non-MIPs bound with analytes to understand MIP's binding behaviour. MIPs and non-MIPs had similar steady-state fluorescence anisotropy in the range 0.11-0.24. Anthracene rebound in MIPs and non-MIPs had a fluorescence lifetime of tau = 0.64 ns and a rotational correlation time of phi(F) = 1.2-1.5 ns, both of which were shorter than that of MIPs with imprinted analytes present (tau = 2.03 ns and phi(F) = 2.7 ns). The steady-state anisotropy of polymer solutions increased exponentially with polymerization time and might be used to characterize the polymerization extent in situ.     

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.     

Evaluation of water-compatible molecularly imprinted polymers as solid-phase extraction sorbents for the selective extraction of sildenafil and its desmethyl metabolite from plasma samples

The evaluation of molecularly imprinted polymers (MIPs) as selective sorbents for the solid-phase extraction of sildenafil and its principal metabolite, desmethylsildenafil, was investigated. Two MIPs were synthesised using structural analogues of sildenafil as templates, and a comparison of the performance of the two MIP sorbents in organic and aqueous media was performed. Additionally, the feasibility of applying molecularly imprinted solid-phase extraction (MISPE) to the clean-up of plasma samples containing sildenafil and desmethylsildenafil was investigated. A preliminary, quantitative MISPE for the determination of both compounds in plasma was also performed. The results showed that the MIPs used for the selective extraction of sildenafil gave better compound recovery when aqueous samples were used in comparison to organic-based samples. A preliminary, quantitative MISPE of sildenafil and desmethylsildenafil indicated that the imprinted materials could be used successfully as SPE sorbents for sample pre-treatment for the determination of sildenafil, and related compounds, in plasma.     

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.     

Preparation of molecularly imprinted polymers using nitroxide-mediated living radical polymerization

The use of molecularly imprinted polymers (MIPs) in chemical and bioanalytical applications has been gaining in interest in recent years. Compared to their biological receptor counterparts, MIPs are easy to prepare, have long shelf stability and can be used under a variety of harsh conditions. The majority of MIPs currently used are produced by traditional free radical polymerization. One drawback with the use of standard free radical initiators is that little control can be exerted over the chemical processes that form the final imprinted cavities. In this study we set out to investigate the application of controlled (living) free radical polymerization to the preparation of MIPs. This was exemplified by the synthesis of cholesterol-imprinted bulk polymers by nitroxide-mediated polymerization (NMP). A sacrificial covalent bond was employed to maintain imprinting fidelity at elevated temperature. Selective uptake of cholesterol from solutions in hexane was studied with imprinted polymers prepared under different conditions. The imprinted hydrolyzed MIP prepared by NMP displayed higher selective cholesterol binding than that prepared by a traditional radical polymerization.     

Molecularly imprinted polymer formats for capillary electrochromatography

The research aimed towards the adaptation of molecularly imprinted polymers (MIPs) to the capillary format and the use of these highly selective matrices for capillary electrochromatography (CEC) is reviewed in this article. The MIP is prepared by incorporation of a template molecule into a polymerization protocol. After polymerization and extraction of the template from the resulting polymer a highly selective material with recognition cavities complementary to the template in size, shape and chemical functionality is obtained. MIPs have been used as recognition elements in several different analytical techniques. In combination with CEC a novel separation system with a unique selectivity towards a predetermined target (the template) is achieved. The merge of molecular imprinting technology (MIT) and CEC have introduced several interesting polymer formats, due to the adaptation of the MIP to the miniaturized capillary format. The polymer formats can be classified according to their preparation protocols and appearance into three conceptually different categories, i.e. the monolith, the coating and the nanoparticles. The preparation protocols, characteristics and applications of these formats will be discussed.     

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.     

Reading Biochips by Raman and Surface-Enhanced Raman Spectroscopies

Biochips are a rapidly increasing research field, driven by the versatility of sensing devices and the importance of their applications. The regular approaches for creating biochips and for reading them suffer from some limitations, motivating development of miniature biochips and label-free formats. To push forward these challenges, we have chosen to combine the methods of printing of droplets of synthetic receptors by pipettes or nanofountain pens with detection by Raman spectroscopy or its surface-assisted plasmon variant, namely, surface-enhanced Raman spectroscopy (SERS). The selected receptors included molecularly imprinted polymers (MIPs), produced by polymerization of functional and cross-linking monomers around a molecular template, the β-blocking drug propranolol. The measured Raman and SERS spectra of the MIP constituents enabled identification of the template presence and consequently chemical imaging of individual and multiple dots in an array. This concept, combining nanolithography techniques with SERS paves the road toward miniaturized arrayed MIP sensors with label-free, specific and quantitative molecular recognition.     

Water‐compatible silica sol–gel molecularly imprinted polymer as a potential delivery system for the controlled release of salicylic acid

Molecularly imprinted polymers (MIPs) for salicylic acid were synthesized and evaluated in aqueous environments in the aim to apply them as drug delivery carriers. One organic MIP and one inorganic MIP based on the sol–gel process were synthesized. The organic MIP was prepared by radical polymerization using the stoichiometric functional monomer, 1‐(4‐vinylphenyl)‐3‐(3,5‐bis(trifluoromethyl)phenyl)urea, which can establish strong electrostatic interactions with the –COOH of salicylic acid. The sol–gel MIP was prepared with 3‐(aminopropyl)triethoxysilane and trimethoxyphenylsilane, as functional monomers and tetraethyl orthosilicate as the crosslinker. While the organic MIPs bound the target specifically in acetonitrile, they exhibited lower binding in the presence of water, although the imprinting factor increased under these conditions, due to reduced non‐specific binding. The sol–gel MIP has a high specificity and capacity for the drug in ethanol, a solvent compatible with drug formulation and biomedical applications. In vitro release profiles of the polymers in water were evaluated, and the results were modelled by Fick's law of diffusion and the power law. Analysis shows that the release mechanism was predominantly diffusion‐controlled. Copyright © 2014 John Wiley & Sons, Ltd.     

Retentivity and enantioselectivity of uniformly sized molecularly imprinted polymers for d-chlorpheniramine and -brompheniramine in hydro-organic mobile phases

Uniformly sized molecularly imprinted polymers (MIPs) for d-chlorpheniramine (CP) and -brompheniramine (BP) have been prepared by a multi-step swelling and polymerization method using methacrylic acid (MAA) or 2-(trifluoromethyl)acrylic acid (TFMAA) and ethylene glycol dimethacrylate (EDMA) as a functional monomer and cross-linker, respectively. The retentive and enantioselective properties of CP, BP and their structurally related compounds on the MIPs were evaluated using hydro-organic mobile phases. CP and BP enantiomers were retained the most as a monovalent cation on MAA-co-EDMA polymers and a divalent cation on TFMAA-co-EDMA polymers. Ion exchange and hydrophobic interactions could mainly work for the retention and enantioseparation of CP and BP on both MAA-co-EDMA and TFMAA-co-EDMA polymers in hydro-organic mobile phases. Though the respective MIPs gave the highest enantioselectivity for the template molecule, cross-reactivity for CP and BP was observed with them.     

HPLC-based bioseparations using molecularly imprinted polymers

HPLC-based separations of amino acids and peptides, nucleotide bases, drugs, sugars and steroids using molecularly imprinted polymers (MIPs) have been reviewed in this article. The molecular recognition mechanisms of the template molecules on the MIPs in organic and aqueous eluents were discussed. Furthermore, new polymerization methods suitable for preparations of HPLC columns and packing materials using molecular imprinting techniques, and their applications to HPLC-based separations are also dealt with.     

Understanding the relationships between molecule structure and imprinting effect of two acetyl‐nitrogen heterocyclic compounds

The molecularly imprinted polymers (MIPs) for two structural analogs, 1,3,5‐triacetyl‐1,3,5‐triazacyclohexane (TRAT) and 1,3,5,7‐tetraacetyl‐1,3,5,7‐tetraazacyclooctane (TAT), have been synthesized respectively under the same conditions. The TAT‐MIP showed excellent imprinting effect, whereas the TRAT‐MIP did not. To understand the different imprinting effects of the MIPs prepared from these two templates, the geometric structures and energetic properties of complexes formed around TAT and TRAT were studied computationally. The results indicate that in liquid phase, for the complexes formed with TAT and its nearest neighbor molecules, the magnitude of the binding energy increases with the number of surrounding TAT, methacrylic acid, and acetonitrile (ACT), whereas for the cases of TRAT, the magnitude of the binding energy increases with the number of surrounding TRAT and trimethylolpropane trimethacrylate. The studied systems form stronger and thus more stable networks encapsulating TAT than with TRAT. ACT may also play an important role in the polymerization phase in stabilizing the shapes of the cavities that TATs reside in. We propose these as the major factors that affect the different imprinting effects of the two MIPs. Copyright © 2016 John Wiley & Sons, Ltd.