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.     

Towards molecularly imprinted polymers selective to peptides and proteins. The epitope approach

In this paper, we describe the epitope approach to molecular imprinting. The applicability of molecular imprinting, a method that allows the preparation of biomimetic compounds (artificial receptors and antibodies), is extended by this approach. Our approach makes it possible to obtain imprinted polymers selective to peptides and proteins whereas, to date, molecular imprinting has been used primarily for the preparation of polymers that selectively bind to relatively low molecular weight substances. The epitope approach is based on using (as a template) a short peptide that represents only part of a larger peptide or protein (as an epitope represents an antigen), which in turn can be recognized by the synthesized polymer. It is demonstrated that although other parts of peptides can influence the process of molecular recognition, the polymers imprinted with a short peptide efficiently recognize both the template and larger peptides (for example, oxytocin) that possess the same C-terminal part of the structure.     

Computational strategies for understanding the nature of interaction in dioxin imprinted nanoporous trappers

A new computational model capable of understanding the nature of interactions in signature complexes formed between the template (2,3,7,8‐tetrachlorodibenzo‐p dioxin (TCDD)) and the functional monomers (methacrylic acid (MAA)) using density functional theory (DFT) has been designed. The polymer precursors were optimized for geometries in polymerization media, computing the interaction energies between template molecules and functional monomers of transient pre‐polymerized complexes (PPC), and structural and vibrational properties reference to theoretical infrared spectra were computed using DFT of B3LYP/6 311+G(d,p) hybrid functional method. Atom in molecule theory was used to analyze the hydrogen‐bonding characteristics of PPC of MAA–TCDD. Considering the theoretical titrations conducted in a virtual solvent box, it was found that the 1:4 molar ratio was required to form the most stable PPC in a given solvent system. The electron density plots indicate strong hydrogen bonding as shown by the 2pz dominant highest occupied molecular orbital (HOMO) character that could be the preferable sites of binding for target molecule, TCDD. Considering HOMO approach, the active adsorption sites in molecularly imprinted polymer was modeled to get insight on molecular recognition property for targeted molecule, TCDD. The proposed computational protocol is simple, accurate, and novel to design the polymer and is useful to predict the properties of polymer systems than the conventional theoretical analysis of template–monomer interactions. Copyright © 2015 John Wiley & Sons, Ltd.     

Insights into the origins of binding and the recognition properties of molecularly imprinted polymers prepared using an amide as the hydrogen-bonding functional group

Molecularly imprinted polymers (MIPs) prepared using an amide hydrogen-bonding functional monomer (acrylamide) exhibited efficient enantiomeric recognition properties in both organic and aqueous media in the HPLC mode. The results indicate that the amide functional groups formed strong hydrogen-bonding interactions with the template molecule, and specific recognition sites were created within the polymer matrix during the imprinting process. When Boc-L-Trp was used as the template, an MIP prepared in a polar organic solvent (acetonitrile) using acrylamide as the functional monomer showed better enantiomeric recognition of Boc-Trp than the MIPs prepared in the same solvent using an acidic (methacrylic acid) or a basic (2-vinylpyridine) functional monomer or a combination of an acidic and a basic functional monomer (methacrylic acid + 2-vinylpyridine). Our results indicate that in organic media the degree of retention of the sample molecule on the imprinted polymer was controlled by hydrogen-bonding interactions between the sample molecule and the polymer, while in aqueous media it was determined to a considerable extent by hydrophobic interactions. In both media the shape, size and the nature of the hydrogen-bonding groups of the sample molecules were all important factors in determining the enantiomeric and substrate selectivity. In the aqueous media, however, the hydrophobicity of the sample molecules was also found to play an important role.     

Class-specific molecularly imprinted polymers for the selective extraction and determination of sulfonylurea herbicides in maize samples by high-performance liquid chromatography–tandem mass spectrometry

A novel method based on the molecularly imprinted solid-phase extraction (MISPE) procedure has been developed for the simultaneous determination of concentrations of sulfonylurea herbicides such as chlorsulfuron (CS), monosulfuron (MNS), and thifensulfuron methyl (TFM) in maize samples by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). The molecularly imprinted polymer (MIP) for sulfonylurea herbicides was synthesized by precipitation polymerization using chlorsulfuron as the template molecule, 2-(diethylamino)ethyl methacrylate (DEAMA) as the functional monomer, and trimethylolpropane trimethacrylate (TRIM) as the cross-linker. The selectivities of the chlorsulfuron template and its analogs on the molecularly imprinted polymer were evaluated by high-performance liquid chromatography (HPLC). The extraction and purification procedures for the solid-phase extraction (SPE) cartridge with a molecularly imprinted polymer as the adsorbent for the selected sulfonylurea herbicides were then established. A molecularly imprinted solid-phase extraction method followed by high-performance liquid chromatography–tandem mass spectrometry for the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl was also established. The mean recoveries of these compounds in maize were in the range 75–110% and the limits of detection (LOD) of chlorsulfuron, monosulfuron, and thifensulfuron methyl were 0.02, 0.75, and 1.45 μg kg⁻¹, respectively. It was demonstrated that the MISPE–HPLC–MS/MS method could be applied to the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl in maize samples.     

Quantification and confocal imaging of protein specific molecularly imprinted polymers

We have employed FITC--albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labeled template is reported, with subsequent characterization of the smart material to show that the HydroMIP possesses a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC--albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of the imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine hemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols and document the use of fluorescence as a useful tool in template labeling/detection and novel imaging strategies.     

A circular dichroism study of DNA-basic peptides associations in the absence or in the presence of Ca++.

The interaction of DNA with basic peptides (Lys methyl ester*, Lys2, (Lys)2methyl ester) has been studied by circular dichroism. The changes of the DNA CD spectra in the presence of peptides are interpreted as a transconformation from the B form to the C form of DNA. The presence of Ca++ in the mixture induces a supplementary transconformation. These observations suggest Ca++-basic peptides-DNA complexes as a structural model for chromatin.     

Monitoring of cAMP-imprinted polymer by fluorescence spectroscopy

Conventional functional monomers together with fluorescent monomer, trans-4-[p-(N,N-dimethylamino)styryl]-N-vinylbenzylpyridinium chloride (vb-DMASP), were copolymerised in the presence of a target molecule, nucleotide-cAMP that acted as a molecular template. The polymer was copolymerised in thin-layer films. After removal of the template the functionalised cavities that exist in the fluorescent material are able to specifically bind the template. Subsequent adsorption of the template-cAMP causes quenching of fluorescence of the polymer. The specific photochemical processes accompanying the template adsorption are discussed further. The imprinted polymers monitored by both steady-state and time-resolved fluorescence techniques show specificity and selectivity of binding of the template on the imprinted functionalised cavities.     

Imprinted polymer layer for recognizing double-stranded DNA

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.     

Direct detection of analyte binding to single molecularly imprinted polymer particles by confocal Raman spectroscopy

We describe the use of Raman spectroscopy to detect and quantify, for the first time, the presence of the imprinting template in single molecularly imprinted polymer microspheres. The polymers were imprinted with the β-blocking drugs propranolol and atenolol, and precipitation polymerization was used to obtain spherical particles of diameters of 200 nm and 1.5 μm. The size of the Raman laser spot being between 1 μm and a few μm, the nanoparticles were used for bulk detection whereas with micrometer-sized particles, quantitative measurements on single particles were possible. The laser power, and consequently the acquisition times, needed to be adapted as a function of the polymer and template used in order to avoid burning. Analyte quantification from Raman spectra is straightforward by determining the peak height of a typical Raman band of the analyte, and by using a typical polymer peak for normalization. Relatively low detection limits down to 1 μM have been reached for the detection of S-propranolol through bulk measurements on MIP nanoparticles.     

Effect of the solvent on recognition properties of molecularly imprinted polymer specific for ochratoxin A

A molecularly imprinted polymer specific for the mycotoxin ochratoxin A has been synthesised using a non-covalent approach. The polymer has shown an excellent affinity and specificity for the target template in aqueous solutions. The binding experiments, NMR study and molecular modelling have proven that the template recognition by polymer originates from the shape complementarity of binding sites. The binding mechanism is critically depended on factors that affect the polymer conformation. Thus the variation in buffer concentration, pH and presence of organic solvent, which affect the polymer swelling or shrinking, had a profound effect on the polymer recognition properties.     

Molecularly imprinted 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.     

Influence of polymerization temperature on the molecular recognition of imprinted polymers

This paper aimed at investigating the influence of polymerization temperature on the molecular recognition of molecularly imprinted polymers (MIPs) based on multiple non-covalent interactions. 3-l-Phenylalanylaminopyridine (3-l-PheNHPy) imprinted polymers were prepared using azobisnitriles as either thermal initiators or photoinitiators at various temperatures of 10, 40 and 60 degrees C, respectively. These polymers were subsequently evaluated in the high-performance liquid chromatographic (HPLC) mode for enantioselectivity. An unexpected result shows that polymer prepared at 40 degrees C has the highest enantioselectivity, but not the polymer prepared at lower temperature of 10 degrees C. Further, the effect of elution temperature and sample load on the selectivity of polymers was investigated in detail. In order to get a better understanding of the "exception", the influence of polymerization temperature on the polymerization extent and polymer morphology was studied by FT-IR spectrum test, cross-polarization magic angle spinning (CP-MAS) (13)NMR spectra experiment and pore analysis. Based on these results we attribute this "exception" to that there is a tradeoff between the extent of polymerization and stabilization of the template-functional monomer complexes. And an optimal polymerization temperature can be found for each combination of template and monomer.     

Automatic sequence design of major histocompatibility complex class I binding peptides impairing CD8+ T cell recognition

An automatic protein design procedure was used to compute amino acid sequences of peptides likely to bind the HLA-A2 major histocompatibility complex (MHC) class I allele. The only information used by the procedure are a structural template, a rotamer library, and a well established classical empirical force field. The calculations are performed on six different templates from x-ray structures of HLA-A0201-peptide complexes. Each template consists of the bound peptide backbone and the full atomic coordinates of the MHC protein. Sequences within 2 kcal/mol of the minimum energy sequence are computed for each template, and the sequences from all the templates are combined and ranked by their energies. The five lowest energy peptide sequences and five other low energy sequences re-ranked on the basis of their similarity to peptides known to bind the same MHC allele are chemically synthesized and tested for their ability to bind and form stable complexes with the HLA-A2 molecule. The most efficient binders are also tested for inhibition of the T cell receptor recognition of two known CD8(+) T effectors. Results show that all 10 peptides bind the expected MHC protein. The six strongest binders also form stable HLA-A2-peptide complexes, albeit to varying degrees, and three peptides display significant inhibition of CD8(+) T cell recognition. These results are rationalized in light of our knowledge of the three-dimensional structures of the HLA-A2-peptide and HLA-A2-peptide-T cell receptor complexes.     

Detection of oxytocin,atrial natriuretic peptide,and brain natriuretic peptide using novel imprinted polymers produced with amphiphilic monomers

The cystine‐bridged cyclic peptide hormones (CBCPHs) represent signature structural feature as well as unique biological activity. In this study, three CBCPHs have been identified and characterized, namely, oxytocin, atrial natriuretic peptides (ANPs), and brain natriuretic peptides (BNPs). Because research has shown that ANPs and BNPs are powerful diagnostic biomarkers for heart disease, a highly laudable endeavor would be to develop a novel sensor for detecting ANP or BNP levels. Therefore, an amphiphilic monomer Acr‐His‐NHNH‐Fmoc was synthesized to form molecularly imprinted polymers (MIPs) for targeted CBCPH detection. First, oxytocin, a cardiovascular hormone and a CBCPH, was used as a template to fabricate MIPs on quartz crystal microbalance (QCM) chips. On the other hand, fabricated selected ANP segment or BNP segment as an epitope is able to construct epitope‐mediated MIPs (EMIPs) for ANP or BNP. The developed oxytocin or ANP sensor reached a detection limitation of 0.1nM with the dissociation constants being 30pM for oxytocin and 20pM for ANP. Moreover, BNP sensor achieved a detection limitation of 2.89pM with an even lower K_d value as 2pM. Compared with the performance of EMIPs, the imprinted films showed high affinity and selectivity in special binding to CBCPHs. The developed MIPs‐QCM biosensors thus provide an improved sensing platform using an amphiphilic monomer and may be useful for applications toward cyclotides, cystine knot motifs, or insulin‐like peptides.     

Theophylline molecularly imprinted polymer dissociation kinetics: a novel sustained release drug dosage mechanism

The template release kinetics of theophylline molecularly imprinted polymers has been examined with a view to determining their potential as a controlled release drug dosage form. The basis for the ligand selectivity of these polymers has been shown through the demonstration of pre-polymerization template-monomer complexation and HPLC studies of the product polymer ligand selectivities. The release kinetics shows a dependence upon template loading and pH. Small differences in release characteristics between imprinted and non-imprinted (reference) polymers have been observed.     

Chromatin models. The ionic strength dependence of model histone-DNA interactions: circular dichroism studies of lysine-leucine polypeptide-DNA complexes.

The ionic strength dependence of the complexes between DNA and both random, (Lysx, Leuy)n, and block copolymers, (Lysx)n(Leuy)m, of lysine and leucine, with different amino acid compositions, was studied using circular dichroism (CD) as the probe to detect conformational differences in these complexes relative to native DNA. It was found that the CD spectra of complexes of both the random (Lys84, Leu16)n and block (Lys85)n(Leu15)m copolymers with DNA show a very sharp ionic strength dependence. The maximum altered CD spectrum for the complexes with the block copolymer was found to occur at the same ionic strength as that for poly(L-lysine)-DNA complexes, while the maximum CD change for the random copolymer complex occurred at a slightly lower ionic strength. This sharp dependence of the CD change on the ionic strength was found to be independent of the polymer/DNA ratio, r, for each individual copolymer. The CD spectra for these complexes at optimum NaCl concentration resemble those of the psi spectra of DNA [Jordan, C. F., Lerman, L.S., and Venable, J.H. (1972), Nature (London), New Biol. 236, 67]. The complexes of the random copolymer, (Lys68, Leu32)n, with DNA (r=0.25) at 0.15 M NaCl and below have CD spectra that resemble the A-form DNA spectra. The ionic strength dependence of the CD spectra of this complex is not as sharp as observed with the above polymers and has a broad positive plateau. It is suggested that both the CD spectra of these complexes reflect the phenomena of DNA condensation into a higher order asymmetric structure (folded and compact). The block copolymer, (Lys77)n(Leu23)m, complexes with DNA show very slight alterations in the CD spectra, with respect to native DNA. It appears that the long Leu sequence at one end of such copolymers may be unpropitious for causing the polypeptide-DNA complex to condense into a higher order asymmetric structure. Thus the importance of the distribution of hydrophobic residues, in the copolypeptides of Lys, is shown for causing condensation of complexes with DNA. The relevance of these findings to histone-DNA complexes in chromatin is discussed.     

Face-to-face porphyrin moieties assembled with spacing for pyrazine recognition in molecularly imprinted polymers

A strategy for arranging two porphyrin moieties in a face-to-face fashion in polymeric material was demonstrated by molecular imprinting, whereby porphyrin Zn(II) complex monomers were cross-linked with ethylene glycol dimethacrylate in the presence of pyrazine or 1,5-naphthyridine as a template molecule. In chromatographic studies using the resultant imprinted polymers as stationary phase, both the polymers showed selectivity for the original template molecule, suggesting that two zinc porphyrin moieties were immobilized in the face-to-face fashion, and were center-aligned for pyrazine recognition and offset-arranged for 1,5-naphthyridine recognition. The imprinted polymer with porphyrin moieties also showed a decrease in its fluorescence intensity in response to the concentration of the target molecule, suggesting the potential utility as sensing material.     

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.     

Molecular imprinting of nitrophenol and hydroxybenzoic acid isomers: effect of molecular structure and acidity on imprinting

Three nitrophenol isomer-imprinted polymers were prepared under the same conditions using 4-vinylpyridine as a functional monomer. Different recognition capacities for template molecules were observed for the three polymers. Another imprinting system with stronger acidity than nitrophenol isomers, 2-hydroxybenzoic acid (salicylic acid) and 4-hydroxybenzoic acid, was imprinted using 4-vinylpyridine or acrylamide as functional monomer respectively. Both 4-hydroxybenzoic acid-imprinted polymers using the two monomers showed recognition ability for the template molecule. However, when acrylamide was chosen as functional monomer, the salicylic acid-imprinted polymer showed very weak recognition for the template molecule, whereas strong recognition ability of the resultant polymer for salicylic acid was observed with 4-vinylpyridine as functional monomer. It seems that the structure and acidity of template molecules is responsible for the difference in recognition, by influencing the formation and strength of interaction between template molecule and functional monomer during the imprinting process. An understanding of the mechanism of molecular imprinting and molecular recognition of MIPs will help to predict the selectivity of MIPs on the basis of template molecule properties.