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.     

Towards the rational design of molecularly imprinted polymers

Efforts to elucidate the mechanisms underlying the formation of binding sites in molecularly imprinted polymers (MIPs) and of MIP-ligand binding events are presented in the context of a thermodynamic treatment of MIP recognition phenomena.     

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.     

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 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.     

新型青霉素生物传感器的研究

以青霉素为模板分子,采用溶胶-凝胶法合成分子印迹膜,以浸泡的方法移除印迹分子,制备青霉素分子印迹膜电极。本印迹电极能有效地避免类似物对其测定的干扰。通过循环伏安法研究传感器对青霉素的响应特性,结果表明:富集时间为200 s,在0.1~1.8μg/L质量浓度范围内,青霉素在磷酸缓冲液(PBS)中的电流强度与其浓度呈良好的线性关系。吸附后的膜电极用甲醇洗脱后再生,可以重复利用3次,可以应用到实际检测中。     

Biosensor for the determination of sorbitol based on molecularly imprinted electrosynthesized polymers

Despite the increasing number of applications of biosensors in many fields, the construction of a steady biosensor remains still challenging. The high selectivity and stability of molecularly imprinted polymers for the template molecule make them ideal alternatives as recognition elements for sensors. In this work, the fabrication and characterization of biosensor based on molecularly imprinted electrosynthesized polymers is reported as the first case of imprinting sorbitol. A relevant molecularly imprinted film is prepared by o-phenylenediamine (o-PD) using the electrochemical method. Quartz crystal microbalance is employed as a sensitive apparatus of biosensor for the determination of sorbitol. An equation is deduced to characterize the interaction between molecularly imprinted films and the template. A linear relationship between the frequency shift and the concentration of analyte in the range of 1-15 mM was found. The detection limit is about 1mM.     

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.     

Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of tryptophan andN-CBZ-phenylalanine enantiomers

Monolithic molecularly imprinted columns were designed and prepared by anin-situ thermal-initiated copolymerization technique for rapid separation of tryptophan andN-CBZ-phenylalanine enantiomers. The influence of polymerization conditions and separation conditions on the specific molecular recognition ability for enantiomers and diastereomers was investigated. The specious molecular recognition was found to be dependent on the stereo structures and the arrangement of functional groups of the imprinted molecule and the cavities in the molecularly imprinted polymer (MIP). Moreover, hydrogen bonding interactions and hydrophobic interactions played an important role in the retention and separation. Compared to conventional MIP preparation procedures, the present method is very simple, and its macroporous structure has excellent separation properties.     

Design and synthesis of molecularly imprinted polypyrrole based on nanoreactor SBA-15 for recognition of ascorbic acid

Molecular imprinting is an attractive technique for preparing mimics of natural and biological receptors. Nevertheless, molecular imprinting for aqueous systems remains a challenge due to the hydrogen bonding between templates and functional monomers destroyed in the bulk water. The hydrogen bonding between templates and monomers are the most crucial factor governing recognition, particularly in non-covalent molecularly imprinted polymers. Using mesoporous materials for molecular imprinting is an effective approach to overcome this barrier and to remove the limitations of the traditional molecularly imprinted polymers which include incomplete template removal, small binding capacity, slow mass transfer, and irregular materials shape. Here, SBA-15 was used as a mesoporous silica material for synthesis of molecularly imprinted polypyrrole. The pyrrole monomers and template molecules were immobilized onto the SBA-15 hexagonal channels, and then polymerization occurred. The resulting nanocomposites were characterized by Fourier transform infrared (FT-IR) analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) methods. In batch rebinding tests, the imprinted nanocomposites reached saturated adsorption within 100min and exhibited significant specific recognition toward the ascorbic acid (AA) with high adsorption capacity (83.7mgg(-1)). To further illustrate the recognition property of the imprinted nanocomposites, binary competitive and non-competitive adsorption experiments were performed with ascorbic acid, dopamine, paracetamol and epinephrine. The imprinting factors for these compounds in non-competitive adsorption experiments were 3.2, 1.5, 1.4 and 1.3, respectively. The results showed that the imprinted nanocomposites exhibited significant adsorption selectivity for the ascorbic acid against the related compounds.     

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.     

Study on the recognition of templates and their analogues on molecularly imprinted polymer using computational and conformational analysis approaches

A simplified computational model was proposed to simulate the synthesis of molecularly imprinted polymers (MIP), removal of template and recognition of the template and its analogues by MIP. The MIPs with nicotinamide and iso-nicotinamide as templates were prepared using methacrylic acid as functional monomer. Based on our computational model, the interaction energies between the monomer and the template or its analogues were calculated, which were well correlated with the retention factors and imprinting factors obtained on HPLC columns packed with the corresponding MIP particles. The imprinting effects of the template and its analogues were also investigated from the viewpoint of conformational analysis. The computational data were successfully used to predict the chromatographic behaviour of some chemicals in separation on HPLC columns. We believe that the computational method will find application in designing monomers for MIP synthesis and in studying recognition of templates and their analogues on MIP.     

Composite of CdTe quantum dots and molecularly imprinted polymer as a sensing material for cytochrome c

A newly designed molecularly imprinted polymer (MIP) material was fabricated and successfully utilized as recognition element to develop a quantum dots (QDs) based MIP-coated composite for selective recognition of the template cytochrome c (Cyt). The composites were synthesized by sol-gel reaction (imprinting process). The imprinting process resulted in an increased affinity of the composites toward the corresponding template. The fluorescence of MIP-coated QDs was stronger quenched by the template versus that of non-imprinted polymer (NIP)-coated QDs, which indicated the composites could recognize the corresponding template. The results of specific experiments further exhibited the recognition ability of the composites. Under optimum conditions, the linear range for Cyt is from 0.97 μM to 24 μM, and the detection limit is 0.41 μM. The new composites integrated the high selectivity of molecular imprinting technology and fluorescence property of QDs and could convert the specific interactions between imprinted cavities and corresponding template to the obvious changes of fluorescence signal. Therefore, a simple and selective sensing system for protein recognition has been realized.     

Chiral separation using molecularly imprinted heteroaromatic polymers

Novel molecularly imprinted polymer systems utilizing 4-vinylpyridine and 1-vinylimidazole as functional monomers have been developed for enantioselective recognition of carboxylic and N-protected amino acids. Non-covalent interactions between the functional monomers and the template molecules were the source of the subsequent recognition sites in the resultant polymers. The capacity of the polymers for molecular recognition was investigated by using them as stationary phases in the HPLC mode. Polymers prepared with 4-vinylpyridine were found to be more efficient in racemic resolution than those prepared with 1-vinylimidazole. When applying a racemic mixture of the template molecule, the polymers showed highest affinity for the enantiomer used as template. Imprints of a racemic template molecule, as expected, did not exhibit enantioselectivity. The optimal molar ratio of 4-vinylpyridine to the template Cbz-L -Asp-OH in the polymerization mixture was determined to be 12:1. In addition to enantioselectivity, the investigated polymers demonstrated ‘ligand selectivity’ e.g., a Cbz-L -Asp-OH-imprinted polymer was able to separate Cbz-D ,L -Asp-OH, but was unable to separate Cbz-D ,L -Glu-OH.     

Design and evaluation of thin and flexible theophylline imprinted polymer membrane materials

The aim of this work was to produce a thin, flexible and diffusion able molecularly imprinted polymeric matrix with good template accessibility. Membranes were prepared using a non‐covalent molecular imprinting approach and their physical characteristics and binding capabilities investigated. Two materials were used, a poly(tri‐ethyleneglycol dimethyacrylate‐co‐methyl methacrylate‐co‐methacrylic acid) copolymer containing 14% cross‐linker and a monomer (g) to porogen (ml) ratio of 1:0.5 (A), and a blend of poly(TEGMA‐co‐MAA) and polyurethane (B). The polyurethane was added to improve membrane flexiblity and stability. The polymers were characterized using AFM, SEM and nitrogen adsorption, whilst binding was evaluated using batch‐rebinding studies. For all membranes the specific surface area was low (<10 m²/g). MIP (A) films were shown to bind specifically at low concentrations but specific binding was masked by non‐specific interactions at elevated concentrations. Selectivity studies confirmed specificity at low concentrations. K_D approximations confirmed a difference in the population of binding sites within NIP and MIP films. The data also indicated that at low concentrations the ligand‐occupied binding site population approached homogeneity. Scanning electron microscopy images of membrane (B) revealed a complex multi‐layered system, however these membranes did not demonstrate specificity for the template. The results described here demonstrate how the fundamental parameters of a non‐covalent molecularly imprinted system can be successfully modified in order to generate flexible and physically tolerant molecularly imprinted thin films. Copyright © 2009 John Wiley & Sons, Ltd.     

Piezoelectric sensors based on molecular imprinted polymers for detection of low molecular mass analytes

Biomimetic recognition elements employed for the detection of analytes are commonly based on proteinaceous affibodies, immunoglobulins, single-chain and single-domain antibody fragments or aptamers. The alternative supra-molecular approach using a molecularly imprinted polymer now has proven utility in numerous applications ranging from liquid chromatography to bioassays. Despite inherent advantages compared with biochemical/biological recognition (which include robustness, storage endurance and lower costs) there are few contributions that describe quantitative analytical applications of molecularly imprinted polymers for relevant small molecular mass compounds in real-world samples. There is, however, significant literature describing the use of low-power, portable piezoelectric transducers to detect analytes in environmental monitoring and other application areas. Here we review the combination of molecularly imprinted polymers as recognition elements with piezoelectric biosensors for quantitative detection of small molecules. Analytes are classified by type and sample matrix presentation and various molecularly imprinted polymer synthetic fabrication strategies are also reviewed.     

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.     

Role of surface adsorption and porosity features in the molecular recognition ability of imprinted sol-gels

Organically modified molecularly imprinted silicas (MIS) for nafcillin recognition were prepared using a simple sol-gel procedure. Molecular recognition of the template was observed by tuning the chemical and structural properties of the MIS. The relative amounts of organically modified alkoxysilane precursors were found to be key in the textural and morphological characteristics of the MIS as well as for developing an imprinting effect in the materials. The recognition properties of the imprinted materials were found to be strongly influenced by the hydrolytic stability of the alkoxysilanes and their inductive effects during sol-gel hydrolysis/condensation stages. The concept was to combine properties of organic groups with those of glass-like materials in order to develop synergetic properties through variations in the composition. Results from batch rebinding experiments as well as from the thorough study of the N(2) adsorption properties and the textural and structural characteristics of the MIS revealed that an imprint effect could be attributed to the presence of the template during the synthesis of MIS.     

Theoretical and computational strategies for rational molecularly imprinted polymer design

The further evolution of molecularly imprinted polymer science and technology necessitates the development of robust predictive tools capable of handling the complexity of molecular imprinting systems. A combination of the rapid growth in computer power over the past decade and significant software developments have opened new possibilities for simulating aspects of the complex molecular imprinting process. We present here a survey of the current status of the use of in silico-based approaches to aspects of molecular imprinting. Finally, we highlight areas where ongoing and future efforts should yield information critical to our understanding of the underlying mechanisms sufficient to permit the rational design of molecularly imprinted polymers.