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.     

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.     

Molecularly imprinted polymer-assisted sample clean-up of ochratoxin A from red wine: merits and limitations

A new analytical method for the determination of the carcinogenic mycotoxin ochratoxin A (OTA) in red wines has been developed involving a two-dimensional solid-phase extraction (SPE) clean-up protocol on C18-silica and a target-selective molecularly imprinted polymer (MIP). Prior removal of the interfering acidic matrix compounds by C18 solid-phase extraction was crucial for a successful clean-up as direct sample loading onto the MIP led to poor recoveries. The combined solid-phase extraction protocol afforded extracts suitable for sensitive ochratoxin A quantification by HPLC-fluorescence detection. Preliminary validation of the method performance with spiked (0.033-1.0 ng OTA/ml) and commercial red wines provided recoveries >90% and < 10%, with limit of detection (LOD) and limit of quantification (LOQ) of 0.01 and 0.033 ng/ml. However, a similarly favorable performance characteristics was observed in control experiments in which the MIP was replaced by the corresponding non-imprinted polymer (NIP). These findings provide evidence that under the employed experimental conditions specific analyte binding to imprinted binding sites plays a minor role in selective OTA retention. In the framework of this study, other problems inherent to MIP-based solid-phase extraction have been addressed. These include the reproducible preparation of MIP materials with consistent molecular recognition characteristics, the potential for repeated use of MIP, unfavorable polymer swelling in application-relevant solvents, potential sample contamination by template bleeding, and slow analyte binding kinetics.     

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.     

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.     

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.     

Molecularly imprinted polymers (MIP) in electroanalysis of proteins

The review summarizes current knowledge on the main approaches used for creation of high affinity polymer analogs of antibodies (known as molecularly imprinted polymers, MIP) applicable for electroanalysis of functionally important proteins such as myoglobin, troponin T, albumin, ferritin, lysozyme, calmodulin. The main types of monomers for MIP preparation as well as methods convenient for analysis of MIP/protein interactions, such as surface plasmon resonance (SPR), nanogravimetry with the use of a quartz crystal resonator (QCM), spectral and electrochemical methods have been considered. Special attention is paid to experimental data on electrochemical registration of myoglobin by means of o-phenylenediaminebased MIP electrodes. It was shown that the imprinting factor calculated as a ratio of the myoglobin signal obtained after myoglobin insertion in MIP to the myoglobin signal obtained after myoglobin insertion in the polymer lacking the molecular template (NIP) is 2–4.     

‘Bite-and-Switch’ approach using computationally designed molecularly imprinted polymers for sensing of creatinine

A method for the selective detection of creatinine is reported, which is based on the reaction between polymerised hemithioacetal, formed by allyl mercaptan, o-phthalic aldehyde, and primary amine leading to the formation of fluorescent isoindole complex. This method has been demonstrated previously for the detection of creatine using creatine-imprinted molecularly imprinted polymers (MIPs) Since MIPs created using traditional methods were unable to differentiate between creatine and creatinine, a new approach to the rational design of a molecularly imprinted polymer (MIP) selective for creatinine was developed using computer simulation. A virtual library of functional monomers was assigned and screened against the target molecule, creatinine, using molecular modelling software. The monomers giving the highest binding score were further tested using simulated annealing in order to mimic the complexation of the functional monomers with template in the monomer mixture. The result of this simulation gave an optimised MIP composition. The computationally designed polymer demonstrated superior selectivity in comparison to the polymer prepared using traditional approach, a detection limit of 25 μM and good stability. The ‘Bite-and-Switch’ approach combined with molecular imprinting can be used for the design of assays and sensors, selective for amino containing substances.     

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.     

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.     

Plastic antibody for the recognition of chemical warfare agent sulphur mustard

Molecularly imprinted polymers (MIPs) known as plastic antibodies (PAs) represent a new class of materials possessing high selectivity and affinity for the target molecule. Since their discovery, PAs have attracted considerable interest from bio- and chemical laboratories to pharmaceutical institutes. PAs are becoming an important class of synthetic materials mimicking molecular recognition by natural receptors. In addition, they have been utilized as catalysts, sorbents for solid-phase extraction, stationary phase for liquid chromatography and mimics of enzymes. In this paper, first time we report the preparation and characterization of a PA for the recognition of blistering chemical warfare agent sulphur mustard (SM). The SM imprinted PA exhibited more surface area when compared to the control non-imprinted polymer (NIP). In addition, SEM image showed an ordered nano-pattern for the PA of SM that is entirely different from the image of NIP. The imprinting also enhanced SM rebinding ability to the PA when compared to the NIP with an imprinting efficiency () of 1.3.     

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.     

Molecularly imprinted polymer anchored on the surface of denatured bovine serum albumin modified CdTe quantum dots as fluorescent artificial receptor for recognition of target protein

A new type of molecularly imprinted polymer (MIP)-based fluorescent artificial receptor was developed by anchoring MIP on the surface of denatured bovine serum albumin (dBSA) modified CdTe quantum dots (QDs) using the surface molecular imprinting process. The approach combined the merits of molecular imprinting technology and the fluorescent property of the CdTe QDs. The dBSA was used not only to modify the surface defects of the CdTe QDs, but also as assistant monomer to create effective recognition sites. Three different proteins, namely lysozyme (Lyz), cytochrome c (Cyt) and methylated bovine serum albumin (mBSA), were tested as the template molecules and then the receptors were synthesized by sol-gel reaction (imprinting process). The results of fluorescence and binding experiments demonstrated the recognition performance of the receptors toward the corresponding template. Under optimum conditions, the linear range for Lyz was from 1.4×10(-8) to 8.5×10(-6) M, and the detection limit was 6.8 nM. Moreover, the new artificial receptors were applied to separate and detect Lyz in real samples. This fluorescent artificial receptor may serve as a starting point in the design of highly effective synthetic fluorescent receptor for recognition of target protein.     

Syntheses of chitin-based imprinting polymers and their binding properties for cholesterol

A novel chitin derivative, cholesteryl chitin carbonate (Chitin-Chol), was synthesized from chitin and cholesteryl chloroformate. This product was characterized by Fourier transform infrared (FTIR) spectroscopy and solid-state ¹³C nuclear magnetic resonance (¹³C NMR), and was used as a covalently bound template precursor for imprinting cholesterol. After cross-linking with toluene 2,4-diisocyanate, it was efficiently cleaved hydrolytically to afford a guest-binding site accompanying the easy and efficient removal of a sacrificial spacer. The selectivity and efficacy of a chitin-based imprinting polymer for steroid binding were assessed by a chromatographic screening process. The results of binding experiments showed that this molecular imprinting polymer (MIP) has a high binding capacity with cholesterol. The target discrimination towards cholesterol over its close structural analogue suggested that the polymer recognition site was possible on the basis of the inversion of configuration of a single hydroxyl group. In addition, non-covalent imprinting was done using chitin as a precursor and its binding properties for cholesterol were also evaluated.     

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

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

Fluorescent competitive flow-through assay for chloramphenicol using molecularly imprinted polymers

It is a fact that molecular imprinting techniques have reached tremendous importance in the research of new artificial recognition systems. These methods resemble the mechanism of natural recognition, generally based on non-covalent interactions, but improving their stability by means of a simple and inexpensive technique. Molecular imprinting polymers (MIPs) are easily obtained by copolymerisation of suitable functional monomers and crosslinkers in the presence of the print molecule. Removal of the template leaves a polymer that selectively recognises it. In this work, different imprinted polymers for chloramphenicol (CAP) obtained using different monomers and polymerisation conditions were tested in a HPLC system, in order to obtain a highly selective material for CAP. The optimised MIP was then used as recognition phase in a fluorescent competitive flow assay to determine chloramphenicol.     

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.     

Molecularly imprinted cryogel cartridges for the selective recognition of tyrosine

Molecularly imprinted polymers are used for creating a specific cavity and selective recognition sites for the structure of a target molecule in a polymeric structure. In this study, specific molecularly imprinted cryogel cartridges were synthesized using two distinct functional monomers to compare imprinting efficiency for the selective recognition of Tyrosine (Tyr). Tyr-imprinted cryogel cartridge (MIP1) was prepared using metal-chelate coordination for the imprinting process by free-radical bulk polymerization under frozen conditions, and Tyr-imprinted cryogel cartridge (MIP2) was prepared in the same way using hydrophobic effects for imprinting. After the characterization of the cryogel cartridges was carried out, the optimum adsorption conditions of both were determined according to the different parameters such as flow rate (0.5–2.5 ml/min), pH of the medium (4.0–8.0), initial Tyr concentration (0.1–3.0 mg/ml), and temperature (4–45°C). Selectivity experiments of Tyr-imprinted and non-imprinted cryogel cartridges were carried out by using phenylalanine, tryptophan, and cysteine. Besides, the eluted Tyr from MIP1 and MIP2 cryogel cartridge were applied to FPLC system. Also, the reusability experiments of Tyr-imprinted cryogel cartridges was observed no significant decrease in the adsorption capacity.     

A molecularly imprinted polymer on indium tin oxide and silicon

Molecular imprinting is a technique for creating artificial receptor sites in a polymer. Molecularly imprinted polymers (MIPs) are produced by forming a polymer around a molecule that is used as the template. Upon removal of the template, molecular holes remain which are specific in shape and size to the target molecule. In this research, a MIP was formed for theophylline using a copolymer of methacrylic acid and ethylene glycol dimethacrylate. The theophylline MIP was formed on two platforms: indium tin oxide (ITO) and silicon, which were used as the working electrode for cyclic voltammetry measurements. The presence of theophylline was measured using cyclic voltammetry and corresponded to the peak current on the cyclic voltammograms. The results of this research agreed with previous results of MIPs immobilized on an ITO platform. The peak currents of the MIP in the presence and absence of theophylline were compared to the blank polymer for each platform. The ratio of peak currents on ITO increased by a factor of 9.5 for the MIP compared to the non-imprinted polymer. Similarly, the ratio of peak currents on silicon increased by a factor of 6 compared to the non-imprinted polymer. This research demonstrated a procedure for evaluating a MIP layer on two different platforms.     

Substitution of antibodies and receptors with molecularly imprinted polymers in enzyme-linked and fluorescent assays

A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid (APBA), 3-thiopheneboronic acid (TBA) and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in enzyme linked immunosorbent assay (ELISA).