Molecularly imprinted polymer-assisted refolding of lysozyme

For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein.     

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 catalysis of a Diels-Alder reaction

A series of synthetic polymers were designed and synthesized for enhancing the rate of the Diels-Alder cycloaddition reaction of 1,3-butadiene carbamic acid benzyl ester (1) and N,N-dimethyl acrylamide (2), to yield the corresponding endo- (3) and exo- (4) reaction products. Putative transition state analogues (TSAs) for the endo- (5) and exo- (6) reaction pathways were used as templates for the synthesis of molecularly imprinted methacrylic acid (MAA)–divinylbenzene (DVB) copolymers. The polymer system utilized was selected based upon a series of ¹H NMR studies of complex formation between template and a functional monomer analogue (K_d (app) ≈ 70 mM, d₈-toluene, 293 K). Batch binding studies revealed that the imprinted polymers were selective for the TSA corresponding to the template used in the polymer synthesis. Studies on the influence of the polymers on the catalysis of the reaction of 1 and 2 demonstrated a 20-fold enhancement of the rate of the reaction relative to the solution reaction. A surprising temperature dependence of the reaction of 1 and 2 in the presence of the polymers was observed, which provides support for the role of template-functional monomer complexes in the catalysis of the Diels-Alder reaction.     

Molecularly imprinted polymer as storage medium for an analyte

A molecularly imprinted polymer (MIP) able to bind theophylline with a considerable degree of selectivity was prepared through a noncovalent route. The polymer after equilibrating with the template molecule (theophylline) and storage for various periods of time, the amount of theophylline desorbed from the polymer was found to be effectively quantitative indicating that MIPs could be used as a storage matrix for analytes. The stored analyte could be desorbed and analysed conveniently.     

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 hydrophilic polymer sorbents for selective sorption of erythromycin

New hydrophilic polymer sorbents comprising reactionary sites which are complementary to a molecule of antibiotic erythromycin were synthesized by the method of molecular imprinting. A series of similar sorbents without reactionary sites was used for comparison of sorption characteristics. Sorption of erythromycin on both types of polymer sorbents synthesized was studied in a wide range of pH and ionic strength. Selectivity of erythromycin sorption on molecularly imprinted cross-linked polymers was shown to depend on the specific interaction of target molecule with polymer matrix. This type of sorbent is perspective for the development of antibiotic purification directly from a culture medium Saccharopolyspora erythreus.     

Molecularly imprinted polymer microspheres for solid-phase extraction of chloramphenicol residues in foods

Preparation of molecularly imprinted polymer microspheres (MIPMs) for chloramphenicol (CAP) by aqueous suspension polymerization is reported for the first time in this study. The resulting MIPMs had the ability to specifically adsorb CAP, and the molecularly imprinted solid phase extraction (MISPE) based on the MIPMs was shown to be applicable for clean-up and preconcentration of trace CAP in milk and shrimp samples with high recoveries of 92.7% and 84.9%, respectively. Combined with MISPE, the conventional HPLC-UV analysis sensitivity for CAP in foods could be significantly increased.     

Molecularly imprinted polymer with salicylaldehyde-Cu(OAc)2 as template

In the report molecularly imprinted polymer (MIP) with salicylaldehyde-Cu(OAc)(2) as the template was synthesized and characterized by SEM, porosity and elemental analysis. Copper acetate was introduced since salicylaldehyde alone cannot display imprinting effect for its intramolecular hydrogen bond. The strong coordination interaction between salicylaldehyde and copper acetate made the complex have high retention on the HPLC column based on the SAD-Cu(OAc)(2) imprinted polymer. Several structural analogues such as salicylaldoxime, sulfosalicylic acid, p-hydroxybenzaldehyde and their complexes with copper acetate were chosen to study the selectivity of the MIPs. The influence of acetic acid and H(2)O in methanol mobile phase was studied. The experimental results showed that small amount of either acetic acid or H(2)O in mobile phase would weaken the interaction between the complex and the polymer, therefore, the retention of the complex was lowered to a large extent, but that of salicylaldehyde remained almost unchanged. The polymer imprinted with the complex showed high selectivity to both the acetate and copper (II). In addition, the MIP showed an enhanced selectivity to its template compared with the polymer prepared without copper acetate.     

Molecularly imprinted polymers in pseudoimmunoassay

Immunoassays are a class of analytical techniques based on the selective affinity of a biological antibody for its antigen. Competitive binding assays, of which the radioimmunoassay (RIA) was the first example, are based on the competition between analyte and a labelled probe for a limited number of binding sites. Molecularly imprinted polymers (MIPs) have been shown to be suitable replacements for biological antibodies in such techniques. Molecularly imprinted sorbent assays (MIAs) similar to RIA have been developed for a range of analytes of clinical and environmental interest. Limits of detection and selectivities of such assays are often similar to those using biological antibodies. Some assays have been used for measurements directly in biological fluids. The field is reviewed and it is shown that some perceived disadvantages of MIPs do not hinder their application in competitive binding assays: many MIAs have been demonstrated in aqueous solvents, and it has been shown that the quantity of template required to prepare imprinted polymers can be drastically reduced, and that binding site heterogeneity is not a problem as long as the sites which bind the probe most strongly are selective. Finally, recent developments including assays in microtitre plates, the use of enzyme-labelled probes, flow-injection assays and a scintillation proximity MIA are discussed.     

Molecularly imprinted polymers: synthesis and characterisation

This short review aims to present, in clear English, a summary of the principal synthetic considerations pertaining to good practice in the polymerisation aspects of molecular imprinting, and is primarily aimed at researchers familiar with molecular imprinting methods but with little or no prior experience in polymer synthesis. It is our hope that this will facilitate researchers to plan their own syntheses of molecular imprints in a more logical and structured fashion, and to begin to appreciate the limitations of the present synthetic approaches in this molecularly complex area, as well as the scope for rationally designing improved imprinted materials in the future.     

Molecularly imprinted polymer diffraction grating as label-free optical bio(mimetic)sensor

Micropatterned molecularly imprinted polymer (MIP) transmissive 2D diffraction gratings (DGs) are fabricated and evaluated as label-free antibiotic bio(mimetic)sensors. Polymeric gratings are prepared by using microtransfer molding based on SiO(2)/Si molds. The morphology of the MIP gratings is studied by optical and atomic force microscopes. MIP 2D-DGs exhibit 2D optical diffraction patterns, and measurement of changes in diffraction efficiency is used as sensor response. The refractive index of the micropatterned MIP material was estimated, via solvent index matching experiments, to be 1.486. Immersion of a MIP 2D-DG in different solutions of target-antibiotic enrofloxacin leads to significant variations in diffraction efficiency, demonstrating target-molecule detection. On the other hand, no significant response is observed for both control experiments: MIP grating exposed to a non-retained analyte and an equivalent non-imprinted polymer grating exposed to the target analyte, showing highly specific antibiotic label-free optical recognition.     

Molecularly imprinted polymer sensors for detection in the gas, liquid, and vapor phase

Fast, reliable, and inexpensive analytical techniques for detection of airborne chemical warfare agents are desperately needed. Recent advances in the field of molecularly imprinted polymers have created synthetic nanomaterials that can sensitively and selectively detect these materials in aqueous environments, but thus far, they have not been demonstrated to work for detection of vapors. The imprinted polymers function by mimicking the function of biological receptors. They can provide high sensitivity and selectivity but, unlike their biological counterparts, maintain excellent thermal and mechanical stability. The traditional imprinted polymer approach is further enhanced in this work by the addition of a luminescent europium that has been introduced into the polymers to provide enhanced chemical affinity as well as a method for signal transduction to indicate the binding event. The europium in these polymers is so sensitive to the bound target; it can distinguish between species differing by a single methyl group. The imprinted polymer technology is fiber optic-based making it inexpensive and easily integratable with commercially available miniature fiber optic spectrometer technologies to provide a shoebox size device. In this work, we will describe efforts to apply these sensors for detection of airborne materials and vapors. Successful application of this technology will provide accurate low level vapor detection of chemical agents or pesticides with little to no false positives.     

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.     

Molecularly imprinted poly(ethylene-co-vinyl alcohol) membranes for the specific recognition of phospholipids

In this paper we concentrated on the possibility of adopting molecular imprinting technology for the preparation of polymeric membranes imprinted with phosphatidylcholine, one of the main phospholipids found in the cell membrane and lipoproteins, via phase inversion, with the intention of applying this method in the ongoing research into the regression of atherosclerosis. The polymer matrix was based on poly(ethylene-co-vinyl alcohol) with an ethylene molar content of 44% and the amount of template molecule was varied so as to obtain three different kinds of membrane. We found that they possessed elevated binding capabilities (78.6% of the initial amount of phosphatidylcholine was found to be adsorbed by the membrane) united with a very high selectivity. Similar phospholipids (phosphatidylinositol and phosphatidylethanolamine) were found to be adsorbed only in very small quantities and mostly due to the porosity of the membrane itself and not to molecular imprinting.     

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.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

Molecularly imprinted polymers for histamine recognition in aqueous environment

Amino Acids - Molecularly imprinted polymers (MIP) for histamine using methacrylic acid were developed and recognition mechanisms were thoroughly characterized for the first time in this study. The...     

Separation/enrichment of active natural low content protein using protein imprinted polymer

We describe a new type of protein-imprinted polymer for separation/enrichment of active natural protein present at a relatively low level in cell extracts, with a cloned bacterial protein as template. In this work, cloned pig cyclophilin 18 (pCyP18) was used as template. The template protein was selectively assembled with assistant recognition polymer chains (ARPCs) from their library, which consists of numerous limited length polymer chains with randomly distributed recognition and immobilizing sites. These assemblies of protein and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, the synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 200 times. The assay of peptidyl-prolyl cis-trans-isomerase (PPIase) activity showed that natural pCyP18 is more active than cloned pCyP18 and, in particular, it is much more sensitive to the suppressant cyclosporine A (CsA).