Persona: Paniagua González, Gema
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Publicación A MIP-based flow-through fluoroimmunosensor as an alternative to immunosensors for the determination of digoxin serum samples(Springer, 2009-01-09) Durand Alegría, Jesús Senén; Paniagua González, Gema; Fernández Hernando, PilarThis work reports a comparative study of two automated flow-through fluorosensors for the determination of digoxin in serum samples: an immunosensor with an anti-digoxin polyclonal antibody as the reactive phase permanently immobilized on CPG, and a sensor with a selective reaction system based on a methacrylic molecularly imprinted polymer (MIP) synthesized by bulk polymerisation. The variables affecting the sensitivity and dynamic range of the sensors (e.g., the carrier and elution solutions, flow-rates, pH and reagent concentrations) were optimised and the binding characteristics of their reactive phases compared in a competitive fluorescent assay. Digoxin was reproducibly determined by both sensors at the mg L-1 level (detection limit= 1.20×10-3 mg L-1 and RSD= 4-7% for the immunosensor; detection limit= 1.7×10-5 mg L-1 and RSD= 1-2% for the MIP sensor). No cross reactivity with digoxinrelated compounds was seen for either sensor at a digoxin:interferent ratio of 1:100. The lifetime of the immunosensor was about 50 immunoassays; its shelf life, when unused, is about three months. The lifetime of the MIP sensor was over 18 months. Both sensors were used to determine the digoxin concentration of human serum samples with satisfactory results.Publicación A morphological study of molecularly imprinted polymers using the scanning electron microscope(Elsevier, 2006-01) Durand Alegría, Jesús Senén; Paniagua González, Gema; Fernández Hernando, PilarMolecular imprinting is an emerging technique for producing polymers with applications in affinity-based separation, in biomimetic sensors, in catalysis, etc. This variety of uses relies upon the production of polymers with different affinities, specificities, sensitivities and loading capacities. Research into the development of molecular imprinted polymers (MIPs) with new or improved morphologies – which involves modification of the polymerisation process – is therefore underway. This paper reports a comparative study of non-covalent MIPs synthesised by “bulk” polymerisation using digoxin as template. These were synthesised under different conditions, i.e., changing the functional monomers employed (methacrylic acid or 2-vinylpyridine), the porogens (acetonitrile or dichloromethane) used, and by altering the volume of the latter. The polymerisation process was allowed to proceed either under UV light or in a thermostat-controlled waterbath. The surface morphology (was determined by scanning electron microscopy) and the ability of the different polymers to selectively rebind the template was then evaluated.Publicación Determination of digoxin in serum samples using a flow-through fluorosensor based on a molecularly imprinted polymer(Elsevier, 2008-06-15) Durand Alegría, Jesús Senén; Paniagua González, Gema; Fernández Hernando, PilarThis work describes the development of a competitive flow-through FIA assay for digoxin using a molecularly imprinted polymer (MIP) as the recognition phase. In previous work, a number of non-covalent imprinted polymers were synthesised by “bulk” polymerisation. The digoxin binding and elution characteristics of these MIPs were then evaluated to obtain a highly selective material for integration into a sensor. The optimum MIP was synthesised by photo-initiated polymerisation of a mixture containing digoxin, MAA, EDGMA and AIBN in acetonitrile. The bulk polymer was ground and sieved and the template removed by Soxhlet extraction in MeOH/ACN. The MIP was packed into a flow cell and placed in a spectrofluorimeter to integrate the reaction and detection systems. The physical and chemical variables involved in digoxin determination by the sensor (nature and concentration of solution, flow rates, etc.) were optimised. Binding with the non-imprinted polymer (NIP) was also analysed. The new fluorosensor showed high selectivity and sensitivity, a detection limit of 1.7 × 10−2 μg l−1, and high reproducibility (R.S.D. of 1.03% and 1.77% for concentrations of 1.0 × 10−3 and 4.0 × 10−3 mg l−1, respectively). Selectivity was tested by determining the cross-reactivity of several compounds with structures analogous to digoxin. Under the assay conditions used, in which the potential interfering compounds were in concentrations 100 times higher than that of the analyte, no interference was recorded. The proposed fluorosensor was successfully used to determine digoxin concentration of human serum samples.Publicación Permanent oriented antibody immobilization for digoxin determination with a flow-through fluoroimmunosensor(Springer, 2003-02-28) Durand Alegría, Jesús Senén; Pérez Conde, Concepción; Fernández Hernando, Pilar; Paniagua González, GemaDigoxin is a very important compound in clinical chemistry and is indicated in the treatment of congestive heart failure and artery disease. The measurement of serum digoxin concentration is necessary owing to the narrow therapeutic range of this drug. Further, even with similar dosage regimens, the biological response of patients often results in very different concentrations of digoxin in serum. Concentrations of greater than 2.6 mmol/L are generally interpreted as toxic in adult patients. Most methods for digoxin determination are based on gas chromatography or radiochemical and enzymatic immunoassay techniques. However, some of these methods are tedious and difficult to automate. Nowadays, they are being replaced by more practical immunoassay techniques, involving, for example, fluorescent immunosensors that allow rapid, automated and selective digoxin determinations. This paper reports a new flow–through fluoroimmunosensor for digoxin determination, the function of which is based on antibodies immobilized on an inmunoreactor of controlled pore glass (CPG). The immunosensor has a detection limit of 1.20 μg/L and provides high reproducibility (RSD = 4.5% for a concentration of 0.0025 mg/L, and RSD = 6.7% for 0.01 mg/L). The optimum working concentration range was found to be 1.2×10-3 - 4.0×10-2 mg/L. The lifetime of the immunosensor was about 50 immunoassays, if stored unused its lifetime can be extended to three months. A sample speed of about 10–12 samples per hour can be attained. Possible interference from substances with structures similar to digoxin (morphine, heroine, tebaine, codeine, pentazocine and narcotine) was investigated. No cross-reactivity was seen at the highest digoxin:interferent ratio studied (1:100). The proposed fluoroimmunosensor was successfully used to determine digoxin concentrations in human serum samples.Publicación Occurrence of erythromycin residues in sheep milk. Validation of an analytical method(Elsevier, 2015-04) Soledad Rodríguez, Beatriz Elena; Durand Alegría, Jesús Senén; García Mayor, M Asunción; Paniagua González, Gema; Fernández Hernando, Pilar; Garcinuño Martínez, Rosa MªThe paper describes a new and selective analytical sample treatment for quantitative extraction and preconcentration of erythromycin in presence of other macrolide antibiotics in sheep milk samples. The methodology is based on the use of a molecular imprinted polymer (MIP) employed as solid phase extraction sorbent (MISPE). The synthesized material by bulk polymerization using erythromycin (ERY) as template was evaluated as solid phase extraction sorbent, in a novel sample treatment technique that can be coupled to high-performance liquid chromatography with diode-array detector (HPLC-DAD). MIP selectivity was studied for other macrolide antibiotics with similar structures, such as tylosin (TYL), spiramycin (SPI), josamycin (JOS), roxithromycin (ROX) and ivermectin (IVER) getting recoveries for these interferents lower than 35%, for all cases except for ROX, which recoveries were around 85%. The variables affecting the molecularly imprinted solid-phase extraction (MISPE) procedure were optimized to select the best conditions of selectivity and sensitivity to determine ERY at concentration levels established by EU legislation in sheep milk. Under the selected experimental conditions, quantification limit was 24.1 µg kg−1. Recoveries were higher than 98%, with RSDs between 0.7% and 2%. The proposed MISPE-HPLC method was validated and successfully applied to ERY analysis in sheep milk samples.Publicación An optical sensor for the determination of digoxin in serum samples based on a molecularly imprinted polymer membrane.(Elsevier, 2009-04) Durand Alegría, Jesús Senén; Paniagua González, Gema; Fernández Hernando, PilarThis paper reports the synthesis and testing of a molecularly imprinted polymer membrane for digoxin analysis. Digoxin-specific bulk polymer was obtained by the UV initiated co-polymerisation of methacrylic acid and ethylene glycol dimethacrylate in acetonitrile as porogen. After extracting the template analyte, the ground polymer particles were mixed with plasticizer polyvinyl chloride to form a MIP membrane. A reference polymer membrane was prepared from the same mixture of monomers but with no template. The resultant membrane morphologies were examined by scanning electron microscopy. The imprinted membrane was tested as the recognition element in a digoxin-sensitive fluorescence sensor; sensor response was measured using standard solutions of digoxin at concentrations of up to 4 × 10−3 mg L−1. The detection limit was 3.17 × 10−5 mg L−1. Within- and between-day relative standard deviations RSD (n = 5) were in the range 4.5–5.5% and 5.5–6.5% respectively for 0 and 1 × 10−3 mg L−1 digoxin concentrations. A selectivity study showed that compounds of similar structure to digoxin did not significantly interfere with detection for interferent concentrations at 10, 30 and 100 times higher than the digoxin concentration. This simply manufactured MIP membrane showed good recognition characteristics, a high affinity for digoxin, and provided satisfactory results in analyses of this analyte in human serum.