Molecularly-Imprinted Materials for Potentiometric Transduction: Application to the Antibiotic Enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mV/Δlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL−1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ± 0.7%), fast response, good sensitivity (47 mV/Δlog([ENR]/M), wide linear range (1.0 × 10−5–1.0 × 10−3 M), low detection limit (0.9 µg mL−1), and a stable baseline for a 5 × 10−2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.

Novel imprinted polymers as artificial enzymes

Derivatives of L-histidine were investigated as suitable models for the Asp-His couple found in the catalytic triad of serine proteases. A combination of molecular dynamics and IH NMR spectroscopy suggested that the most populous conformations of N-acetyl-L-histidine and the N-acetyl-L-histidine anion were predominated by those in which the carboxylate group was gauche to the imidazole ring overcoming steric and electrostatic repulsion, suggesting there is an interaction between the carboxylate group and the imidazole ring. Kinetic studies, using imidazole, N-acetyl-L-histidine and the N-acetyl-L-histidine anion showed that in a DMSO/H20 9: 1 v/v solution, the N-acetyl-L-histidine anion catalysed the hydrolysis of p-nitrophenyl acetate at a greater rate than using either imidazole or N-acetyl-L-histidine as catalyst. This indicates that the carboxylate group affects the nucleophilicity of the unprotonated imidazole ring. 31P MAS NMR spectroscopy was investigated as a new technique for the study of the template molecule environment within the polymer networks. It was found that it was possible to distinguish between template associated with the polymer and that which was precipitated onto the surface, though it was not possible to distinguish between polymer within imprinted cavities and that which was not. Attempts to study the effect of the carboxylate group/imidazole ring interaction in the imprinted cavity of a molecularly imprinted polymer network were hindered by the method used to follow the reaction. It was found though that in a pH 8.0 buffered solution the presence of imprinted cavities increased the rate of reaction for those polymers derived from L-histidine. Some preliminary investigations into the design and synthesis of an MIP which would catalyse the oxy-Cope rearrangement were carried out but the results were inconclusive.

Short columns with molecularly imprinted monolithic stationary phases for rapid separation of diastereomers and enantiomers

Three molecularly imprinted monolithic columns with different length but almost identical column volume had been prepared. It was observed that the separation factors of diastereomers and enantiomers were almost unaffected by column length. However, the short column with dimension of 38 mm x 8 mm W. showed much lower resistance to flow rate so that it could be operated at much higher flow rates. By combining stepwise gradient elution with elevated flow rate, the diastereomers of cinchonine and cinchonidine and the enantiomers of Cbz-DL-Trp and Fmoc-DL-Trp were successfully separated within 3 min on the short column with dimension of 38 mm. x 8 mm i.d.. Based on the above results, a cinchonine imprinted monolithic disk with dimension of 10 mm x 16 mm W. was further developed. The SEM image and the pore size distribution profile showed that large flow-through pores are present on the prepared monolith, which allowed mobile phase to flow through the disk with very low resistance. Chromatographic performances on the monolithic disk were almost unchanged compared with the long columns. A rapid separation of cinchonine and cinchonidine was achieved in 2.5 min at the flow rate of 9.0 ml/min. Furthermore, it was observed that there was almost no effect of the flow rate on the dynamic binding capacity at high flow rates. In addition, the effect of the loading concentration of analytes on the dynamic binding capacity, namely adsorption isotherm, was also investigated. A non-linear adsorption isotherm of cinchonine was observed on the molecularly imprinted monolith with cinchonine as template, which might be a main reason to result in the peak tailing of template molecule. (C) 2004 Elsevier B.V. All rights reserved.

Molecularly imprinted solid-phase extraction sorbent for removal of nicotine from tobacco smoke

A sorbent showing specific affinity for nicotine was prepared by molecular imprinting technique, using nicotine as the template, methacrylic acid (MAA) as the functional monomer, ethyleneglycol dimethacrylate (EDMA) as the crosslinker and chloroform as the porogen. UV spectroscopic analysis in the molecular imprinting prepolymerization stage confirmed that nicotine could complex with the functional monomer by electrostatic interaction (ionic interaction and hydrogen bonding). The affinity and the binding properties of the imprinted polymer towards nicotine were investigated by equilibrium rebinding experiments. The results indicated the presence of nicotine-specific binding sites in the imprinted polymer, and that the imprinted polymer had a good capacity (90 mumol/g polymer) for nicotine. The elution conditions were optimized on the column packed with the imprinted polymer to elute nicotine quantitatively. The imprinted polymer was used as a solid-phase extraction (SPE) material for the removal of nicotine from tobacco smoke. The results obtained showed that the imprinted polymer was superior in terms of removing nicotine in tobacco smoke, compared with the commercial filter tip.

Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers

The method for preparation of molecularly imprinted monolithic stationary phase has been improved to achieve liquid chromatographic separation of enantiomers and diastereomers. By adopting low polar porogenic solvents of toluene and dodecanol and optimal polymerization conditions, the molecularly imprinted monolithic stationary phases with good flow-through properties and high resolution were prepared. Enantiomers of amino acid derivatives and diastereomers of cinchona alkaloids were completely resolved using the monolithic stationary phases. The influence of porogenic composition, monomer-template ratio and polymerization conditions on the chromatographic performance was investigated. Some chromatographic conditions such as the composition of the mobile phase and the temperature were characterized. Scanning electron microscopy showed that the molecularly imprinted monolithic stationary phase has a large through-pore structure to allow the mobile phase to flow through the column at very low backpressure. Accelerated separations of enantiomers and diastereomers were therefore achieved at elevated flow rates. Finally, the chiral recognition performance of the prepared stationary phase in aqueous media was investigated. Hydrophobic interaction, and ionic and/or hydrogen bonding interactions were proposed to be responsible for the recognition mechanism. (C) 2002 Elsevier Science B.V. All rights reserved.

Water-compatible imprinted polymers for selective depletion of riboflavine from beverages

Artificial riboflavin receptors adapted to aqueous environments were studied for their ability to selectively extract riboflavine (Rf) from three types of beverages i.e. milk, beer and a multivitamin mixture. The basic receptor was first prepared by molecular imprinting in nonaqueous medium using a hydrogen-bond donor-acceptor-donor functional monomer (2,6-bis(acrylamido)pyridine), complementary to the imide motif of the template, riboflavin tetra-acetate as template and pentaerythritol triacrylate (PETA) as a hydrophilic cross-linking monomer. The polymer was then packed in columns and used for extraction of riboflavine from beverages. Riboflavine (Rf) was selectively removed from milk and an artificial vitamin mixture but the nonspecific binding was still significant, as judged from the binding of Rf to a control nonimprinted polymer. In order to suppress this nonspecific binding, attempts to hydrolytically hydrophilize the polymer matrix were performed. The preferred approach consisted in a controlled base hydrolysis of pendent unreacted acrylate groups, using hydroxides with differently sized counterions as reagents. This resulted in a decreased binding of Rf to both polymers, but to an equal extent implying a preferential suppression of the nonspecific contribution to the binding. The hydrophilized polymers, when subjected to beer, showed larger imprinting factors at lower phase ratios compared to the nontreated polymers and a maximum removal of 86% compared to 47% for the nonimprinted control polymer.

Molecularly imprinted polymer based on MWCNTs-QDs as fluorescent biomimetic sensor for specific recognition of target protein

A novel molecularly imprinted optosensing material based on multi-walled carbon nanotube-quantum dots (MWCNT-QDs) has been designed and synthesized for its high selectivity, sensitivity and specificity in the recognition of a target protein bovine serum albumin (BSA). Molecularly imprinted polymer coated MWCNT-QDs using BSA as the template (BMIP-coated MWCNT-QDs) exhibits a fast mass-transfer speed with a response time of 25 min. It is found that the BSA as a target protein can significantly quench the luminescence of BMIP-coated MWCNT-QDs in a concentration-dependent manner that is best described by a Stem-Volmer equation. The K-SV for BSA is much higher than bovine hemoglobin and lysozyme, implying a highly selective recognition of the BMIP-coated MWCNT-QDs to BSA. Under optimal conditions, the relative fluorescence intensity of BMIP-coated MWCNT-QDs decreases linearly with the increasing target protein BSA in the concentration range of 5.0 x 10(-7)-35.0 x 10(-7) M with a detection limit of 80 nM.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0mVdecade−1 and 1.0×10−5 to 2.7×10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Detection of cardiac biomarker proteins using a disposable based on a molecularly imprinted polymer grafted onto graphite

A low-cost disposable was developed for rapid detection of the protein biomarker myoglobin (Myo) as a model analyte. A screen printed electrode was modified with a molecularly imprinted material grafted on a graphite support and incorporated in a matrix composed of poly(vinyl chloride) and the plasticizer o-nitrophenyloctyl ether. The protein-imprinted material (PIM) was produced by growing a reticulated polymer around a protein template. This is followed by radical polymerization of 4-styrenesulfonic acid, 2-aminoethyl methacrylate hydrochloride, and ethylene glycol dimethacrylate. The polymeric layer was then covalently bound to the graphitic support, and Myo was added during the imprinting stage to act as a template. Non-imprinted control materials (CM) were also prepared by omitting the Myo template. Morphological and structural analysis of PIM and CM by FTIR, Raman, and SEM/EDC microscopies confirmed the modification of the graphite support. The analytical performance of the SPE was assessed by square wave voltammetry. The average limit of detection is 0.79 μg of Myo per mL, and the slope is −0.193 ± 0.006 μA per decade. The SPE-CM cannot detect such low levels of Myo but gives a linear response at above 7.2 μg · mL−1, with a slope of −0.719 ± 0.02 μA per decade. Interference studies with hemoglobin, bovine serum albumin, creatinine, and sodium chloride demonstrated good selectivity for Myo. The method was successfully applied to the determination of Myo urine and is conceived to be a promising tool for screening Myo in point-of-care patients with ischemia.

Label-free Detection of Microcystin-LR in Waters Using Real-Time Potentiometric Biosensors Based on Single-Walled Carbon Nanotubes Imprinted Polymers

Microcystin-LR (MC-LR) is a dangerous toxin found in environmental waters, quantified by high performance liquid chromatography and/or enzyme-linked immunosorbent assays. Quick, low cost and on-site analysis is thus required to ensure human safety and wide screening programs. This work proposes label-free potentiometric sensors made of solid-contact electrodes coated with a surface imprinted polymer on the surface of Multi-Walled Carbon NanoTubes (CNTs) incorporated in a polyvinyl chloride membrane. The imprinting effect was checked by using non-imprinted materials. The MC-LR sensitive sensors were evaluated, characterized and applied successfully in spiked environmental waters. The presented method offered the advantages of low cost, portability, easy operation and suitability for adaptation to flow methods.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0 mV decade−1 and 1.0 × 10−5 to 2.7 × 10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Molecularly imprinted silica films prepared by electroassisted deposition for the selective detection of dopamine

Dopamine (DA) can be detected by electrochemical oxidation in conventional electrodes. However, the presence of other oxidizable species (interferents) usually present in physiological fluids at high concentrations (like ascorbic acid) makes very difficult its electrochemical detection. In the present work, glassy carbon electrodes have been modified with molecularly imprinted silica (MIS) films prepared by electroassisted deposition of sol–gel precursors. The production of MIS films was performed by adding the template molecule (DA) to the precursor sol. The molecular impression of silica was assessed showing a high coherency allowing a filtering capacity in the molecular scale. The MIS-modified electrodes present a high selectivity for the detection of DA in neutral or acidic solutions. The MIS-modified electrodes allow the amperometric determination of dopamine in solutions containing ascorbic acid with molar ratios lower than 1:50,000.

Grafting of functional brushes on the surface of 5-Fluorouracil molecularly imprinted polymer particles through RAFT polymerization

The grafting of functional brushes on the surface of molecularly imprinted polymer (MIP). particles hás been explored in the last few years to synthesize materiais combining high molecular recognition capabilities and stimulation triggered by changes in the surrounding environment [1, 2]. In the present work, MIP particles for 5-fluorouracil (a drug used in câncer treatment) were produced by precipitation polymerization in acetonitrile, using either MAA or HEMA as imprinting fünctional monomers, and m the presence of different kinds of RAFT agents. In a second step, taking advantage of the RAFT groups present in the surface of the particles, different kinds of fiinctional polymer brushes were grafted on the MIPs considering a "grafting from" process in the presence of a RAFT agent.