The microcontact imprinting of proteins: the effect of cross-linking monomers for lysozyme, ribonuclease A and myoglobin

The performance of molecularly imprinted polymers (MIPs) is of interest to researchers in the field of analytical chemistry, and in the pharmaceutical and food industries. Because the choice of the functional monomer(s) plays a key role in the selectivity of a MIP, the synthesis of an effective, tight-binding MIP can be difficult and time-consuming, involving the evaluation of the binding performance of MIPs of many different compositions. In this study, we report an express method combining molecular imprinting and microcontact printing techniques to prepare a polymer thin film as an artificial antibody. In addition to the microcontact printing technique, isothermal titration of monomers to proteins stamps was investigated to screen the functional monomer for MIPs. Finally, the importance of the choice of cross-linking monomers in MIPs was studied, and these studies suggest that monomers containing an optimal length PEG spacer give higher imprinting effectiveness. Several model antigens (lysozyme, ribonuclease A and myoglobin) were adsorbed on a cover glasses that were pretreated with hexamethyldisilazane (HMDS). These protein stamps were then contacted with different monomer solutions (cross-linking monomers) on a glass slide substrate. Photopolymerization yielded the molecularly imprinted polymer. This technique, analogous to microcontact printing, allows for the rapid, parallel synthesis of MIPs of different compositions, and requires very small volumes of monomers (ca. 4 microL). The technique also avoids potential solubility problems with the molecular targets. Of several cross-linking monomers screened, tetraethyleneglycol dimethacrylate (TEGDMA) gave the most selective lysozyme binding, while polyethyleneglycol 400 dimethacrylate (PEG400DMA) were most selective for ribonuclease A and myoglobin.     

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.     

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.     

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.     

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

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

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.     

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.     

Characterization of molecularly imprinted polymers using a new polar solvent titration method

A new method of characterizing molecularly imprinted polymers (MIPs) was developed and tested, which provides a more accurate means of identifying and measuring the molecular imprinting effect. In the new polar solvent titration method, a series of imprinted and non‐imprinted polymers were prepared in solutions containing increasing concentrations of a polar solvent. The polar solvent additives systematically disrupted the templation and monomer aggregation processes in the prepolymerization solutions, and the extent of disruption was captured by the polymerization process. The changes in binding capacity within each series of polymers were measured, providing a quantitative assessment of the templation and monomer aggregation processes in the imprinted and non‐imprinted polymers. The new method was tested using three different diphenyl phosphate imprinted polymers made using three different urea functional monomers. Each monomer had varying efficiencies of templation and monomer aggregation. The new MIP characterization method was found to have several advantages. To independently verify the new characterization method, the MIPs were also characterized using traditional binding isotherm analyses. The two methods appeared to give consistent conclusions. First, the polar solvent titration method is less susceptible to false positives in identifying the imprinting effect. Second, the method is able to differentiate and quantify changes in binding capacity, as measured at a fixed guest and polymer concentration, arising from templation or monomer aggregation processes in the prepolymerization solution. Third, the method was also easy to carry out, taking advantage of the ease of preparing MIPs. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Imprinted polymer-modified hanging mercury drop electrode for differential pulse cathodic stripping voltammetric analysis of creatine

The molecularly imprinted polymer [poly(p-aminobenzoicacid-co-1,2-dichloroethane)] film casting was made on the surface of a hanging mercury drop electrode by drop-coating method for the selective and sensitive evaluation of creatine in water, blood serum and pharmaceutical samples. The molecular recognition of creatine by the imprinted polymer was found to be specific via non-covalent (electrostatic) imprinting. The creatine binding could easily be detected by differential pulse, cathodic stripping voltammetric signal at optimised operational conditions: accumulation potential −0.01 V (versus Ag/AgCl), polymer deposition time 15 s, template accumulation time 60 s, pH 7.1 (supporting electrolyte ≤ 5 × 10⁻⁴ M NaOH), scan rate 10 mV s⁻¹, pulse amplitude 25 mV. The modified sensor in the present study was found to be highly reproducible and selective with detection limit 0.11 ng mL⁻¹ of creatine. Cross-reactivity studies revealed no response to the addition of urea, creatinine and phenylalanine; however, some insignificant magnitude of current was observed for tryptophan and histidine in the test samples.     

Molecularly imprinted polymer of 5-methyluridine for solid-phase extraction of pyrimidine nucleoside cancer markers in urine

Normal and modified urinary nucleosides represent potential biomarkers for cancer diagnosis. To selectively extract modified nucleosides, we developed a molecularly imprinted polymer (MIP) of 5-methyluridine as selective material for molecularly imprinted solid-phase extraction (MISPE). The MIPs were obtained from vinyl-phenylboronate ester derivative of the template, acrylamide and pentaerythritol triacrylate co-polymer, and were tested in batch and cartridge experiments with aqueous samples. Our results indicated that the imprinted polymer was selective for pyrimidine nucleosides with a K(d) and a B(max) of 46 microM and 18 micromol/g, respectively. Finally, a MISPE of the most common pyrimidine nucleoside cancer markers in urine sample was realized.     

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.     

Synthesis of a molecularly imprinted polymer for the solid‐phase extraction of betulin and betulinic acid from plane bark

Introduction – Plant extracts are usually complex mixtures of various polarity compounds and their study often includes a purification step, such as solid‐phase extraction (SPE), to isolate interest compounds prior analytical investigations. Molecularly imprinted polymers (MIPs) are a new promising type of SPE material which offer tailor‐made selectivity for the extraction of trace active components in complex matrices. Numerous specific cavities that are sterically and chemically complementary of the target molecules, are formed in imprinted polymers. A molecularly imprinted polymer (MIP) was synthesised in order to trap a specific class of triterpene, including betulin and betulinic acid from a methanolic extract of plane bark. Methodology – Imprinted polymers were synthesised by thermal polymerisation of betulin as template, methacrylic acid (MAA) or acrylamide (AA) as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and chloroform as porogen. Afterwards, MAA‐ and AA‐MIPs were compared with their non‐imprinted polymers (NIPs) in order to assess the selectivity vs betulin and its derivatives. Recovered triterpenes were analysed by HPLC during MIP‐SPE protocol. Results – After SPE optimisation, the MAA‐imprinted polymer exhibited highest selectivity and recovery (better than 70%) for betulin and best affinity for its structural analogues. Thus, a selective washing step (chloroform, acetonitrile) removed unwanted matrix compounds (fatty acids) from the SPE cartridge. The elution solvent was methanol. Finally, the MAA‐MIP was applied to fractionate a plane bark methanolic extract containing betulin and betulinic acid. Conclusion – This study demonstrated the possibility of direct extraction of betulin and its structural analogues from plant extracts by MIP technology. Copyright © 2009 John Wiley & Sons, Ltd.     

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

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

Determination of digoxin in serum samples using a flow-through fluorosensor based on a molecularly imprinted polymer

This work describes the development of a competitive flow-through FIA assay for digoxin using a molecularly imprinted polymer (MIP) as the recognition phase. In previous work, a number of non-covalent imprinted polymers were synthesised by “bulk” polymerisation. The digoxin binding and elution characteristics of these MIPs were then evaluated to obtain a highly selective material for integration into a sensor. The optimum MIP was synthesised by photo-initiated polymerisation of a mixture containing digoxin, MAA, EDGMA and AIBN in acetonitrile. The bulk polymer was ground and sieved and the template removed by Soxhlet extraction in MeOH/ACN. The MIP was packed into a flow cell and placed in a spectrofluorimeter to integrate the reaction and detection systems. The physical and chemical variables involved in digoxin determination by the sensor (nature and concentration of solution, flow rates, etc.) were optimised. Binding with the non-imprinted polymer (NIP) was also analysed. The new fluorosensor showed high selectivity and sensitivity, a detection limit of 1.7 × 10⁻² μg l⁻¹, and high reproducibility (R.S.D. of 1.03% and 1.77% for concentrations of 1.0 × 10⁻³ and 4.0 × 10⁻³ mg l⁻¹, respectively). Selectivity was tested by determining the cross-reactivity of several compounds with structures analogous to digoxin. Under the assay conditions used, in which the potential interfering compounds were in concentrations 100 times higher than that of the analyte, no interference was recorded. The proposed fluorosensor was successfully used to determine digoxin concentration of human serum samples.     

Molecular modelling of a template substitute and monomers used in molecular imprinting for aflatoxin B1 micro-HPLC analysis

The preparation of molecularly imprinted polymers (MIPs) involves the polymerisation of functional monomers in the presence of template molecules. 5,7-Dimethoxycoumarin (DMC) was found to be a structural analogue for aflatoxin B1 (AB1) and serves as its substitute in a grafting solution for the MIP synthesis. It was found that both methacrylic acid and allylamine are functional monomers which could provide a similar binding towards AB1 and DMC.     

Solid-phase extraction of tramadol from plasma and urine samples using a novel water-compatible molecularly imprinted polymer

In this study, a novel method is described for the determination of tramadol in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and tramadol as template molecule. The novel imprinted polymer was used as a solid-phase extraction (SPE) sorbent for the extraction of tramadol from human plasma and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for the MIP cartridges were studied. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of tramadol. The limit of detection (LOD) and limit of quantification (LOQ) for tramadol in urine samples were 1.2 and 3.5 μg L⁻¹, respectively. These limits for tramadol in plasma samples were 3.0 and 8.5 μg L⁻¹, respectively. The recoveries for plasma and urine samples were higher than 91%.     

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.