Epitope imprinting of iron binding protein of Neisseria meningitidis bacteria through multiple monomers imprinting approach

Epitope imprinting is a promising technique for fabrication of novel diagnostic tools. In this study, an epitope imprinted methodology for recognition of target epitope sequence as well as targeted protein infused by bacterial infection in blood samples of patients suffering from brain fever is developed. Template sequence chosen is a ferric iron binding fbp A protein present in Neisseria meningitidis bacteria. To orient the imprinting template peptide sequence on gold surface of electrochemical quartz crystal microbalance (EQCM), thiol chemistry was utilized to form the self‐assembled monolayer on EQCM electrode. Here, synergistic effects induced by various noncovalent interactions extended by multiple monomers (3‐sulfopropyl methacrylate potassium‐salt and benzyl methacrylate) were used in fabricating the imprinting polymeric matrix with additional firmness provided by N,N‐methylene‐bis‐acrylamide as cross‐linker and azo‐isobutyronitrile as initiator. Extraction of template molecule was carried out with phosphate buffer solution. After extraction of epitope molecules from the polymeric film, epitope molecularly imprinted polymeric films were fabricated on EQCM electrode surface. Nonimprinted polymers were also synthesized in the similar manner without epitope molecule. Detection limit of epitope molecularly imprinted polymers and imprinting factor (epitope molecularly imprinted polymers/nonimprinted polymers) was calculated 1.39 ng mL^?1 and 12.27 respectively showing high binding capacity and specific recognition behavior toward template molecule. Simplicity of present method would put forward a fast, facile, cost‐effective diagnostic tool for mass health care.     

Insight into molecular imprinting in precipitation polymerization systems using solution NMR and dynamic light scattering

Molecular imprinting is a powerful synthetic technique for generating template-defined binding sites in cross-linked polymers. One scientific challenge in molecular imprinting research is to understand the intermolecular interactions leading to molecular complexation and the process of binding site formation during polymerization. In this work, we present a novel method for studying the molecular imprinting process in precipitation polymerization systems. This method employs solution (1) H NMR and dynamic light scattering (DLS) to investigate the association of template molecules with colloidal particles and the dynamic process of particle growth. Under precipitation polymerization conditions, the colloidal particles formed did not interfere with NMR signals from the soluble components, allowing unreacted monomers and free template to be easily quantified. To examine the process of particle nucleation and growth, DLS was used to measure the hydrodynamic particle size at different reaction times. To corroborate the interpretation of the NMR and DLS results, imprinted nanoparticles were collected at different reaction times and their binding characteristics were evaluated using radioligand-binding analysis. Our experimental results provide new insights into the molecular imprinting process that will be useful in the development of new imprinted nanoparticles.     

Preparation and evaluation of a molecularly imprinted polymer for the selective recognition of testosterone--application to molecularly imprinted sorbent assays

Biomimetic testosterone receptors were synthesized via molecular imprinting for use as antibody mimics in immunoassays. As evaluated by radioligand binding assays, imprinted polymers prepared in acetonitrile were very specific for testosterone because the nonimprinted control polymers bound virtually no radiolabeled testosterone. The polymers present an appreciable affinity, with association constants of K(a) = 3.3 x 10(7) M(- 1) (high-affinity binding sites). The binding characteristics of the polymers were also evaluated in aqueous environment to study their viabilities as alternatives to antibodies in molecularly imprinted sorbent assays. Compared with the testosterone-specific antibodies present in commercial kits, our molecularly imprinted polymers are somewhat less sensitive but show a high selectivity.     

Some new developments and challenges in non-covalent molecular imprinting technology

The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules via the use of templates. Molecularly imprinted polymers have been applied in an increasing number of applications where molecular binding events are of interest. These include the use of molecularly imprinted polymers as tailor-made separation materials, antibody and receptor binding site mimics in recognition and assay systems, enzyme mimics for catalytic applications and as recognition elements in biosensors. The stability and low cost of molecularly imprinted polymers make them advantageous for use in analysis as well as in industrial-scale production and application.     

Fabrication of a surface imprinted hydrogel shell over silica microspheres using bovine serum albumin as a model protein template

Surface imprinting is an effective approach to improve the template transfer efficiency in applications of molecularly imprinted polymers as biosensors and separation materials. In this paper, we tried to fabricate a surface imprinted hydrogel over silica microspheres for selective recognition of bovine serum albumin by covalent immobilization of a water-soluble UV sensitive initiator onto the surface of silica beads. The polymerization was initiated by UV radiation with N-[3-(dimethylamino)propyl]methacrylamide and N-isopropylacrylamide as the functional monomer and assistant monomer, respectively, and a thin coat of stimuli-responsive hydrogel yielded over the silica gels. The surface imprinted hydrogels exhibited specific affinity toward the template protein with an association constant (K_a) of 2.2 × 10⁵ L mol⁻¹ and a maximum binding capacity (Q_max) of 27.3 mg g⁻¹ in Tris–HCl buffer (pH 7.0). The rebinding and desorption kinetics of the surface imprinted hydrogels were determined and proven to be extremely fast (about 1 min compared to 3 h for the previously prepared bulk imprinted hydrogel). Besides, the hydrogel-silica core-shell particles inherit both the stimuli-responsive property of the hydrogel and the good mechanical strength of the silica beads based on the on-line evaluation with high-performance liquid chromatography. The above comprehensive merits of the obtained surface imprinted hydrogel suggest the presented approach an attractive and broadly applicable way of developing biosensors and high-performance protein separation materials.     

Building fluorescent sensors for carbohydrates using template-directed polymerizations

The ability to custom-make fluorescent sensors for different analytes could have a tremendous impact in a variety of areas. Template-directed polymerization or molecular imprinting seems to be a promising approach for the preparation of high-affinity and specific binding sites for different template molecules. However, the application of molecular imprinting in the preparation of fluorescent sensors has been hampered by the lack of suitable fluorescent tags, which would respond to the binding event with significant fluorescence intensity changes. We have designed and synthesized a fluorescent monomer (1) that allows for the preparation of fluorescent sensors of cis diols using molecular imprinting methods. This monomer has been used for the preparation of imprinted polymers as sensitive fluorescent sensors for D-fructose. The imprinted polymers prepared showed significant fluorescence intensity enhancement upon binding with the template carbohydrate.     

Oxytocin receptor mimetics prepared by molecular imprinting

Summary Oxytocin receptor mimetics were prepared by molecular imprinting using Z-oxytocin as the template. Comparative binding studies with reference polymers showed that the imprinted polymers recognized both Z-oxytocin and unprotected oxytocin selectively. The dissociation constants were 47 μM and 102 μM, respectively, and the density of binding sites was 12 μmol/g. The synthetic oxytocin receptors were easily prepared, possessed high mechanical and chemical stability, and were reused without loss of selectivity and capacity after regeneration by extraction. Abbreviations: B_max, number of binding sites; CLEAR, Cross-Linked Ethoxylate Acrylate Resin; EDMA, ethylene glycol dimethacrylate; FABMS, fast atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high-performance liquid chromatography; K_D, dissociation constant; MAA, methacrylic acid; MIP, molecularly imprinted polymer; SPPS, solid-phase peptide synthesis; TRIM, trimethylolpropane trimethacrylate; Z, benzyloxycarbonyl. Abbreviations used for amino acids and the designation of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977–983]. All amino acids were of thel-configuration.     

Aqueous batch rebinding and selectivity studies on sucrose imprinted polymers

Polymers imprinted with sucrose and corresponding non-imprinted polymers are prepared photo-chemically at 3 °C and thermally at 65 °C. The pre-polymerization complex formation in dimethyl sulfoxide between sucrose and methacrylic acid via hydrogen bonding was investigated through ¹H NMR titration. The imprinting effect and the selectivity of the imprinted polymers in water are studied by batch rebinding studies with different mono and disaccharides and fitted to the Freundlich isotherm. Based on the calculated numbers of binding sites and average affinity, it is concluded that sucrose has been successfully imprinted at 3 and 65 °C. The polymer imprinted at 3 °C possesses the best recognition properties. The imprinted polymers are selective towards sucrose in water.     

Ion association reactions with biological membranes,studied with the fluorescent dye 1-anilino-8-naphthalenesulfonate

Summary (1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs⁺ binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs⁺>Rb⁺>K⁺>Na⁺>Li⁺. The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2mm ^–1. The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.     

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.     

Supported imprinted nanospheres for the selective recognition of cholesterol

The preparation of innovative polymeric systems using molecular imprinting technology for application in extracorporeal blood purification is described. Membranes based on a methylmethacrylate-co-acrylic acid copolymer, produced through the phase inversion method, were modified introducing into their structure specific binding sites for cholesterol molecule by adding molecularly imprinted nanoparticles in the membrane matrix. Membranes prepared are intended to selectively remove cholesterol from the blood by using interactions at a molecular level, between the membrane/nanoparticles devices and the template, created during the preparation of polymers. Three polymeric systems in form of nanoparticles were prepared differing in the polymerisation solvent (a mixture of acetonitrile and ethanol (1:1) or pure ethanol), and the molar ratio between the functional monomer and the cross-linker (2.3:1 and 1:1). Two out of three of the prepared polymers showed a very good template rebinding capacity both in phosphate buffer solution (pH 6.9) and in ethanol. In particular the nanoparticles rebound 115.4 mg cholesterol/g polymer in buffer solution, and 57 mg cholesterol/g polymer in ethanol.

The deposition of the nanoparticles on the surface of the phase inversion membranes produced devices with interesting rebinding performances towards cholesterol in buffer solution: a specific recognition of 14.09 mg cholesterol/g system (membrane and nanoparticles) was detected, indicating maintained binding capacity of supported particles as well.     

Adsorption of Ni2+ on the surface of molecularly imprinted adsorbent from Penicillium chysogenum mycelium

The adsorption capacity for Ni²⁺ on to the surface molecular imprinting adsorbent on Penicillium chysogenum mycelium (the surface-imprinted adsorbent) was 40–45 mg g^–1 (using 200 mg Ni²⁺ l^–1), two times of the mycelium adsorbent. The surface-imprinted adsorbent had good stability at pH 28. The optimal concentration of EDTA for desorption was 0.1 to 0.5 g l^–1. The surface imprinted adsorbent could be reused 15 times without losing its uptake.     

Carbon nanotube array: A new MIP platform

Here we demonstrate that a free-standing carbon nanotube (CNT) array can be used as a large surface area and high porosity 3D platform for molecular imprinted polymer (MIP), especially for surface imprinting. The thickness of polymer grafted around each CNT can be fine-tuned to imprint different sizes of target molecules, and yet it can be thin enough to expose every imprint site to the target molecules in solution without sacrificing the capacity of binding sites. The performance of this new CNT–MIP architecture was first assessed with a caffeine-imprinted polypyrrole (PPy) coating on two types of CNT arrays: sparse and dense CNTs. Real-time pulsed amperometric detection was used to study the rebinding of the caffeine molecules onto these CNT-MIPPy sensors. The dense CNT-MIPPy sensor presented the highest sensitivity, about 15 times better when compared to the conventional thin film, whereas an improvement of 3.6 times was recorded on the sparse CNT. However, due to the small tube-to-tube spacing in the dense CNT array, electrode fouling was observed during the detection of concentrated caffeine in phosphate buffer solution. A new I–V characterization method using pulsed amperometry was introduced to investigate the electrical characterization of these new devices. The resistance value derived from the I–V plot provides insight into the electrical conductivity of the CNT transducer and also the effective surface area for caffeine imprinting.     

Towards the rational development of molecularly imprinted polymers: 1H NMR studies on hydrophobicity and ion-pair interactions as driving forces for selectivity

The preparation of molecularly imprinted polymers (MIP) based on non-covalent interactions has become a widely used technique for creating highly specific sorbent materials predominantly used in separation chemistry. A crucial factor in a successful imprinting protocol is the optimisation of the template/functional monomer interaction in the pre-polymerisation mixture, eventually leading to a maximum of high-affinity binding sites in the resulting polymer matrix. In order to develop more efficient preparation technologies for imprinted polymers, two separate pre-polymerisation complexes were investigated by NMR spectroscopic techniques in order to identify the types of interactions occurring in the pre-polymerisation mixture, and their implications for the subsequently formed imprinted polymer. In particular, hydrophobic effects have been followed by NMR spectroscopy and their contribution to the selectivity of the resulting MIP has been investigated. The 2,4-D imprint system is used as an example to fundamentally study whether observations at the pre-polymerisation stage correlate with properties of the finally prepared MIP, and which parameters govern success of an imprinting protocol.     

Designing of MIP based QCM sensor having thymine recognition sites based on biomimicking DNA approach

Quartz crystal microbalance (QCM) sensors coated with molecular imprinted polymers (MIP) have been developed for the determination of thymine. In this method, methacryloylamidoadenine (MA-Ade) have used as a new monomer and thymine template for inspiration of DNA nucleobases interaction. The thymine can be simultaneously hydrogen binding to MA-Ade and fit into the shape-selective cavities. Thus, the interaction between nucleobases has an effect on the binding ability of the QCM sensors. The binding affinity of the thymine imprinted sensors has investigated by using the Langmuir isotherm. The thymine imprinted QCM electrodes have shown homogeneous binding sites for thymine (K_a: 1.0 × 10⁵ M⁻¹) while heterogeneous binding sites for uracil. On the other hand, recognition selectivity of the QCM sensor based on thymine imprinted polymer toward to uracil, ssDNA and ssRNA has been reported in this work.     

Synthetic cinchonidine receptors obtained by cross-linking linear poly(methacrylic acid) derivatives as an alternative molecular imprinting technique

A molecular imprinting approach to construct synthetic receptors was examined, wherein a linear pre-polymer bearing functional groups for intermolecular interaction with a given molecule is cross-linked in the presence of the molecule as a template, and subsequent removal of the template from the resultant network-polymer is expected to leave a complementary binding site. Poly(methacrylic acid) (PMAA) derivatized with a vinylbenzyl group as a cross-linkable side chain was utilized as the pre-polymer for the molecular imprinting of a model template, (-)-cinchonidine. Selectivity of the imprinted polymer was evaluated by comparing the retentions of the original template, (-)-cinchonidine and its antipode (+)-cinchonine in chromatographic tests, exhibiting a selectivity factor up to 2.4. By assessment of the imprinted polymers in a batch mode, a dissociation constant at 20 degrees C for (-)-cinchonidine was estimated to be K (d) = 2.35 x 10(-6) M (the number of binding sites: 4.54 x 10(-6) mol/g-dry polymer). The displayed affinity and selectivity appeared comparable to those of an imprinted polymer prepared by a conventional monomer-based protocol, thus showing that the pre-polymer, which can be densely cross-linked, is an alternative imprinter for developing template-selective materials. (-)-Cinchonidine-imprinted polymers were prepared and assessed using the pre-polymers bearing different densities of the vinylbenzyl group and different amounts of the cross-linking agent to examine the appropriate density of the cross-linking side chain that was crucial for developing the high affinity and selectivity of the imprinted polymers.     

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.     

Binding properties of an aminostyrene-based polymer imprinted with glutamylated monascus pigments

An aminostyrene-based polymer imprinted with glutamylated monascus pigments specifically bound the template but not other analogous pigments even when present in a mixture. The overall number of binding sites in the imprinted polymer and the equilibrium binding constant for the glutamylated pigment were determined as 0.023 mmol g^–1 polymer and 31 mM^–1 in ethylacetate, respectively.     

Virtual imprinting as a tool to design efficient MIPs for photosynthesis-inhibiting herbicides

Molecular modelling and computational screening were used to identify functional monomers capable of interacting with several different photosynthesis-inhibiting herbicides. The process involved the design of a virtual library of molecular models of functional monomers containing polymerizable residues and residues able to interact with the template through electrostatic, hydrophobic, Van der Waals forces and dipole-dipole interactions. Each of the entries in the virtual library was probed for its possible interactions with molecular models of the template molecules. It was anticipated that the monomers giving the highest binding score would represent good candidates for the preparation of affinity polymers. Strong interactions were computationally determined between acidic functional monomers like methacrylic acid (MAA) or itaconic acid (IA) with triazines, and between vinylimidazole with bentazone and bromoxynil. Nevertheless, weaker interactions were seen with phenylureas. The corresponding blank polymers were prepared using the selected monomers and tested in the solid phase extraction (SPE) of herbicides from chloroform solutions. A good correlation was found between the binding score of the monomers and the affinities of the corresponding polymers. The use of computationally designed blanks can potentially eliminate the need for molecular imprinting, (adding a template to the monomer mixture to create specific binding sites). Data also showed that some monomers have a natural selectivity for some herbicides, which can be further enhanced by imprinting. Thus, in regard to retention on the blank polymer, we can estimate if the resulting imprinted polymer will be effective or not.     

Comparative study of the molecularly imprinted polymers prepared by reversible addition–fragmentation chain transfer “bulk” polymerization and traditional radical “bulk” polymerization

Bisphenol A (BPA) and propranolol‐imprinted polymers have been prepared via both reversible addition–fragmentation chain transfer “bulk” polymerization (RAFTBP) and traditional radical “bulk” polymerization (TRBP) under similar reaction conditions, and their equilibrium binding properties were compared in detail for the first time. The chemical compositions, specific surface areas, equilibrium bindings, and selectivity of the obtained molecularly imprinted polymers (MIPs) were systematically characterized. The experimental results showed that the MIPs with molecular imprinting effects and quite fast binding kinetics could be readily prepared via RAFTBP, but they did not show improved template binding properties in comparison with those prepared via TRBP, which is in sharp contrast to many previous reports. This could be attributed to the heavily interrupted equilibrium between the dormant species and active radicals in the RAFT mechanism because of the occurrence of fast gelation during RAFTBP. The findings presented here strongly demonstrates that the application of controlled radical polymerizations (CRPs) in molecular imprinting does not always benefit the binding properties of the resultant MIPs, which is of significant importance for the rational use of CRPs in generating MIPs with improved properties. Copyright © 2013 John Wiley & Sons, Ltd.