Carnitine tailored Sensors on Surface Molecular Imprinting based on Graphene layers

III Jornadas de Electroquímica e Inovação (Electroquímica e Nanomateriais), na Universidade de Trás-os-Montes e Alto Douro, Vila Real, 16 a 17 de Setembro de 2013

Host-Tailored Sensors for Carnitine Potentiometric Measurements based on Surface Molecular Imprinting

Graduate Student Symposium on Molecular Imprinting 2013, na Queen’s University, Belfast, United Kingdom, 15 a 17 de Agosto de 2013

Chiral separations of mandelic acid by HPLC using molecularly imprinted polymers

Styrene is used in a variety of chemical industries. Environmental and occupational exposures to styrene occur predominantly through inhalation. The major metabolite of styrene is present in two enantiomeric forms, chiral R- and S- hydroxy-1-phenyl-acetic acid (R-and S-mandelic acid, MA). Thus, the concentration of MA, particularly of its enantiomers, has been used in urine tests to determine whether workers have been exposed to styrene. This study describes a method of analyzing mandelic acid using molecular imprinting techniques and HPLC detection to perform the separation of diastereoisomers of mandelic acid. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting using (+) MA, (-) MA or (+) phenylalanine, (-) phenylalanine as templates. Methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were copolymerized in the presence of the template molecules. The bulk polymerization was carried out at 4ºC under UV radiation. The resulting MIP was grounded into 25~44¼m particles, which were slurry packed into analytical columns. After the template molecules were removed, the MIP-packed columns were found to be effective for the chromatographic resolution of (±)-mandelic acid. This method is simpler and more convenient than other chromatographic methods.

A new biomimetic sensor based on molecularly imprinted polymers for highly sensitive and selective determination of hexazinone herbicide

A new selective sensor based on molecularly imprinted polymers (MIPs) was developed for the determination of hexazinone (HXZ) in environmental samples. MIPs were synthesized using a non-covalent approach, and selection of the monomers employed in the polymerization reaction was carried out by molecular modeling. Three functional monomers with high (2-vinylpyridine (MP17)) and intermediate (methacrylic acid (MP12) and acrylamide (MP5)) energies of binding to the template (HXZ) were selected for preparation of the MIPs, in order to conduct comparative studies and validate the theoretical data. For sensor construction, carbon pastes were modified with each MIP or NIP (non-imprinted polymer), and HXZ determination was performed using differential pulse adsorptive cathodic stripping voltammetry (DPAdCSV). All parameters affecting the sensor response were optimized. In HCl at pH 2.5, the sensor prepared with MP17 (5% w/w in the paste) showed a dynamic linear range between 1.9 × 10−11 and 1.1 × 10−10 mol L−1, and a detection limit of 2.6 × 10−12 mol L−1, under the following conditions: accumulation time of 200 s at a potential of −0.5V, scan rate of 50 mVs−1, pulse amplitude of 60 mV, and pulse width of 50 ms. The sensor was selective in the presence of other similar compounds, and was successfully applied to the analysis of HXZ in river water samples.

Molecular recognition with nanostructures fabricated by photopolymerization within metallic subwavelength apertures

The first demonstration of fabrication of submicron lateral resolution molecularly imprinted polymer (MIP) patterns by photoinduced local polymerization within metal subwavelength apertures is reported. The size of the photopolymerized MIP features is finely tuned by the dose of 532 nm radiation. Rhodamine 123 (R123) has been selected as a fluorescent model template to prove the recognition capability of the MIP nanostructures, which has been evaluated by fluorescence lifetime imaging microscopy (FLIM) with single photon timing measurements. The binding selectivity provided by the imprinting effect has been confirmed in the presence of compounds structurally related to R123. These results pave the way to the development of nanomaterial architectures with biomimetic artificial recognition properties for environmental, clinical and food testing.

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.

Improved Selective Cholesterol Adsorption By Molecularly Imprinted Poly(methacrylic Acid)/silica (pmaa-sio₂) Hybrid Material Synthesized With Different Molar Ratios.

The present paper describes the synthesis of molecularly imprinted polymer - poly(methacrylic acid)/silica and reports its performance feasibility with desired adsorption capacity and selectivity for cholesterol extraction. Two imprinted hybrid materials were synthesized at different methacrylic acid (MAA)/tetraethoxysilane (TEOS) molar ratios (6:1 and 1:5) and characterized by FT-IR, TGA, SEM and textural data. Cholesterol adsorption on hybrid materials took place preferably in apolar solvent medium, especially in chloroform. From the kinetic data, the equilibrium time was reached quickly, being 12 and 20 min for the polymers synthesized at MAA/TEOS molar ratio of 6:1 and 1:5, respectively. The pseudo-second-order model provided the best fit for cholesterol adsorption on polymers, confirming the chemical nature of the adsorption process, while the dual-site Langmuir-Freundlich equation presented the best fit to the experimental data, suggesting the existence of two kinds of adsorption sites on both polymers. The maximum adsorption capacities obtained for the polymers synthesized at MAA/TEOS molar ratios of 6:1 and 1:5 were found to be 214.8 and 166.4 mg g(-1), respectively. The results from isotherm data also indicated higher adsorption capacity for both imprinted polymers regarding to corresponding non-imprinted polymers. Nevertheless, taking into account the retention parameters and selectivity of cholesterol in the presence of structurally analogue compounds (5-α-cholestane and 7-dehydrocholesterol), it was observed that the polymer synthesized at the MAA/TEOS molar ratio of 6:1 was much more selective for cholesterol than the one prepared at the ratio of 1:5, thus suggesting that selective binding sites ascribed to the carboxyl group from MAA play a central role in the imprinting effect created on MIP.

Molecularly Imprinted Solid Phase Extraction Of Fluconazole From Pharmaceutical Formulations.

This work encompasses a direct and coherent strategy to synthesise a molecularly imprinted polymer (MIP) capable of extracting fluconazole from its sample. The MIP was successfully prepared from methacrylic acid (functional monomer), ethyleneglycoldimethacrylate (crosslinker) and acetonitrile (porogenic solvent) in the presence of fluconazole as the template molecule through a non-covalent approach. The non-imprinted polymer (NIP) was prepared following the same synthetic scheme, but in the absence of the template. The data obtained from scanning electronic microscopy, infrared spectroscopy, thermogravimetric and nitrogen Brunauer-Emmett-Teller plot helped to elucidate the structural as well as the morphological characteristics of the MIP and NIP. The application of MIP as a sorbent was demonstrated by packing it in solid phase extraction cartridges to extract fluconazole from commercial capsule samples through an offline analytical procedure. The quantification of fluconazole was accomplished through UPLC-MS, which resulted in LOD≤1.63×10(-10) mM. Furthermore, a high percentage recovery of 91±10% (n=9) was obtained. The ability of the MIP for selective recognition of fluconazole was evaluated by comparison with the structural analogues, miconazole, tioconazole and secnidazole, resulting in percentage recoveries of 51, 35 and 32%, respectively.

Direct Solid-Phase Optical Measurements in Flow Systems: A Review

Measurements based on absorption, reflectance, or luminescence of molecular species or complex ions can be carried out directly on a solid support simultaneously to the retention of the analyte. The use of this strategy in flow-based systems is advantageous in view of the reproducible handling of solutions in retention and elution steps of the analyte. This approach can be exploited to increase sensitivity, minimize reagent consumption as well as waste generation, improve selectivity or for simultaneous determination based on selective retention or differences in sorption rates of the analytes. This review focuses on the main characteristics of direct solid-phase measurements in flow systems, including the discussion of advantages and limitations and practical guidelines to the successful implementation of this approach. Selected applications in diverse fields, such as pharmaceutical, food, and environmental analysis are discussed.

Desenvolvimento de Anticorpos Plásticos para um Biomarcador do Cancro

O trabalho descrito compreende o desenvolvimento de um anticorpo plástico (MIP, do inglês Molecularly Imprinted Polymer) para o antigénio carcinoembrionário (CEA, do inglês Carcinoembriogenic Antigen) e a sua aplicação na construção de dispositivos portáteis, de tamanho reduzido e de baixo custo, tendo em vista a monitorização deste biomarcador do cancro do colo-retal em contexto Point-of-Care (POC). O anticorpo plástico foi obtido por tecnologia de impressão molecular orientada, baseada em eletropolimerização sobre uma superfície condutora de vidro recoberto por FTO. De uma forma geral, o processo foi iniciado pela electropolimerização de anilina sobre o vidro, seguindo-se a ligação por adsorção do biomarcador (CEA) ao filme de polianilina, com ou sem monómeros carregados positivamente (Cloreto de vinilbenziltrimetilamónio, VB). A última fase consistiu na electropolimerização de o-fenilenodiamina (oPD) sobre a superfície, seguindo-se a remoção da proteína por clivagem de ligações peptídicas, com o auxílio de tripsina. A eficiência da impressão do biomarcador CEA no material polimérico foi controlada pela preparação de um material análogo, NIP (do inglês, Non-Imprinted Polymer), no qual nem a proteína nem o monómero VB estavam presentes. Os materiais obtidos foram caracterizados quimicamente por técnicas de Infravermelho com Transformada de Fourier (FTIR, do inglês, Fourier Transform Infrared Spectroscopy) e microscopia confocal de Raman. Os materiais sensores preparados foram entretanto incluídos em membranas poliméricas de Poli(cloreto de vinilo) (PVC) plastificado, para construção de sensores (biomiméticos) seletivos a CEA, tendo-se avaliado a resposta analítica em diferentes meios. Obteve-se uma boa resposta potenciométrica em solução tampão de Ácido 4-(2-hidroxietil)piperazina-1-etanosulfónico (HEPES), a pH 4,4, com uma membrana seletiva baseada em MIP preparada com o monómero carregado VB. O limite de deteção foi menor do que 42 pg/mL, observando-se um comportamento linear (versus o logaritmo da concentração) até 625 pg/mL, com um declive aniónico igual a -61,9 mV/década e r2>0,9974. O comportamento analítico dos sensores biomiméticos foi ainda avaliado em urina, tendo em vista a sua aplicação na análise de CEA em urina. Neste caso, o limite de deteção foi menor do que 38 pg/mL, para uma resposta linear até 625 pg/mL, com um declive de -38,4 mV/década e r2> 0,991. De uma forma geral, a aplicação experimental dos sensores biomiméticos evidenciou respostas exatas, sugerindo que os biossensores desenvolvidos prossigam estudos adicionais tendo em vista a sua aplicação em amostras de indivíduos doentes.

Electrochemical biosensor based on biomimetic material for myoglobin detection

A novel reusable molecularly imprinted polymer (MIP) assembled on a polymeric layer of carboxylated poly(vinyl chloride) (PVCsingle bondCOOH) for myoglobin (Myo) detection was developed. This polymer was casted on the gold working area of a screen printed electrode (Au-SPE), creating a novel disposable device relying on plastic antibodies. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and Fourier transform infrared spectroscopy (FTIR) studies confirmed the surface modification. The MIP/Au-SPE devices displayed a linear behaviour in EIS from 0.852 to 4.26 μg mL−1, of positive slope 6.50 ± 1.48 (kΩ mL μg−1). The limit of detection was 2.25 μg mL−1. Square wave voltammetric (SWV) assays were made in parallel and showed linear responses between 1.1 and 2.98 μg mL−1. A current decrease was observed against Myo concentration, producing average slopes of −0.28 ± 0.038 μA mL μg−1. MIP/Au-SPE also showed good results in terms of selectivity. The error% found for each interfering species were 7% for troponin T (TnT), 11% for bovine serum albumin (BSA) and 2% for creatine kinase MB (CKMB), respectively. Overall, the technical modification over the Au-SPE was found a suitable approach for screening Myo in biological fluids.

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.

Synthesis of an antibody-like material for the detection of Albumin

6th Graduate Student Symposium on Molecular Imprinting

Molecularly imprinted electrochemical sensor for ochratoxin A detection in food samples

A novel electrochemical sensor for ochratoxin A (OTA) detection was fabricated through the modification of a glassy carbon electrode (GCE) with multiwalled carbon nanotubes (MWCNTs) and a molecularly imprinted polymer (MIP). The MWCNTs dramatically promoted the sensitivity of the developed sensor, while polypyrrole (PPy) imprinted with OTA served as the selective recognition element. The imprinted PPy film was prepared by electropolymerization of pyrrole in the presence of OTA as a template molecule via cyclic voltammetry (CV). The electrochemical oxidation of OTA at the developed sensor was investigated by CV and differential pulse voltammetry (DPV). The developed MIP/MWCNT/GCE sensor showed a linear relationship, when using DPV, between peak current intensity and OTA concentration in the range between 0.050 and 1.0 μM, with limits of detection (LOD) and quantification of 0.0041 μM (1.7 μg/L) and 0.014 μM (5.7 μg/L) respectively. With the developed sensor precise results were obtained; relative standard deviations of 4.2% and 7.5% in the evaluation of the repeatability and reproducibility, respectively. The MIP/MWCNT/GCE sensor is simple to fabricate and easy to use and was successfully applied to the determination of OTA in spiked beer and wine samples, with recoveries between 84 and 104%, without the need of a sample pre-treatment step.

Molecularly imprinted sensor for voltammetric detection of norfloxacin

In this work, a norfloxacin selective modified glassy carbon electrode (GCE) based on a molecularly imprinted polymer (MIP) as electrochemical sensor was developed. A suspension of multi-walled carbon nanotubes (MWCNTs) was deposited on the electrode surface. Subsequently, a molecularly imprinted film was prepared by electropolymerization, via cyclic voltammetry of pyrrole (PPy) in the presence of norfloxacin (NFX) as the template molecule. A control electrode (NIP) was also prepared. Scanning electron microscopy (SEM) and cyclic voltammetry in a ferrocyanide solution were performed for morphological and electrochemical characterisation, respectively. Several experimental parameters were studied and optimised. For quantification purposes the MIP/MWCNT/GCE was immersed in NFX solutions for 10 min, and the detection was performed in voltammetric cell by square wave voltammetry. The proposed sensor presented a linear behaviour, between peak current intensity and logarithmic concentration of NFX between 1 × 10−7 and 8 × 10−6 M. The obtained results presented good precision, with a repeatability of 4.3% and reproducibility of 9% and the detection limit was 4.6 × 10−8 M (S/N = 3). The developed sensor displayed good selectivity and operational lifetime, is simple to fabricate and easy to operate and was successfully applied to the analysis of NFX in urine samples.