Miguel Ángel González-Martínez

Development of a highly sensitive enzyme-linked immunosorbent assay for atrazine Performance evaluation by flow injection immunoassay

Specific polyclonal antibodies to the herbicide atrazine (6-chloro-N-ethyl-N′-isopropyl-1,3,5-triazine-2,4-diamine) have been raised by immunizing three New Zealand rabbits. With the antisera (As) a highly sensitive enzyme-linked immunosorbent assays (ELISA) has been developed to determine atrazine in water samples. Several usable competitive immunoassays have been obtained by screening a battery of nine enzyme tracers (ETs) and three antisera. The optimized ELISA presents an IC50 of 0.28 nM (60 ng l−1) and a detection limit of 0.043 nM (9 ng l−1). Cross-reactivity studies have proved that the immunoassay is specific for atrazine while other triazine compounds are only detected on a minor extent. The flow injection immunoanalysis (FIIA) method has an IC50 of 2 nM (0.47 μg l−1) reaching a detection limit of 0.35 nM (75 ng l−1). The performance of both methods has been evaluated by analyzing water samples containing mixtures of atrazine and other pesticides at the ppb level. For this purpose two candidate reference materials have been used (A and B) and a spiked sample stored on Empore disks (sample C). A close correspondence was found between the results obtained with both immunochemical techniques.

On-line immunoanalysis for environmental pollutants: from batch assays to automated sensors

Abstract The development of antibody-based sensors has grown steadily during recent years, and their use as routine instruments in pollution control programmes might become reality in the 21st century. The conversion from batch immunoassays to practical immunosensors has not been easy, owing to the sometimes different principles employed in these methodologies. In this review we illustrate the main difficulties found in the development and implementation of on-line immunosensors, as applied to the determination of organic pollutants such as pesticides. Some solutions to these problems are proposed, and their validities discussed critically.

Dual-Polarization Interferometry: A Novel Technique To Light up the Nanomolecular World

Nanomolecular World Jorge Escorihuela,† Miguel Ángel Gonzaĺez-Martínez,† Jose ́ Luis Loṕez-Paz,† Rosa Puchades,† Ángel Maquieira,† and David Gimenez-Romero*,‡ †Department of Chemistry, Institute of Molecular Recognition and Technological Development, Universitat Politec̀nica de Valeǹcia, Camino de Vera s/n, 46022 Valeǹcia, Spain ‡Physical Chemistry Department, Faculty of Chemistry, Universitat de Valeǹcia, Avenida Dr. Moliner 50, 46100 Burjassot, Valeǹcia, Spain

Development of an automated controlled-pore glass flow-through immunosensor for carbaryl

The application of controlled-pore glass (CPG) as solid support for immobilization of immunoreagents in order to develop flow-through immunosensors is described. Monoclonal antibodies (MAbs) to carbaryl were site-directed immobilized on CPG through covalent attachment of their oxidized carbohydrate moieties to amine groups generated on the surface of silanyzed CPG. The automated immunosensor system is based on the LIB-CNA36 MAb in a direct competitive assay format, with horseradish peroxidase as enzyme label and fluorimetric detection. The dynamic range of the sensor is 0.05–1 μg l−1, with a detection limit of 0.029 μg l−1, being sensitive enough to be applied to drinking water samples without preconcentration. The immobilized antibody reactor is able to run a whole assay in 20 min, and is reusable for more than 100 of consecutive assay cycles without significant loss of performance. The recognition of l-naphthol — the main metabolite of carbaryl — and other N-methylcarbamate insecticides are also studied, none of these compounds showing cross-reactivity higher than 7%. A preliminary validation of the immunosensor, carried out by analysing real samples spiked with carbaryl, shows good results for bottled water and for commercial honey diluted with PBS (1 gl−1) as the only sample pretreatment.

A comparative study by the enzyme-linked immunofiltration asssay of solid phases used in the development of flow immunosensors

The application of an inert membrane-based, enzyme-linked immunofiltration assay (ELIFA) to the characterization of immunosorbents suitable for flow immunosensor development is described. For direct assays, eight monoclonal antibodies (MAb) raised against the insecticide carbaryl were immobilized on three sorbents, namely, controlled pore glass (CPG), hydrazide derivatized agarose beads and a hydrophilic polymer with immobilized Protein A/G. The interaction between immobilized antibodies and antigen was directly detected using a carbaryl hapten conjugated to horseradish peroxidase. Immunosorbent characterization was based on both sensitivity and re-usability. Optimal immunosorbent regeneration was achieved using 0.1 M glycine/HCl, pH 2.0 as the desorbent solution. The best covalent immunosorbent was obtained by immobilizing LIB-CNA36 MAb on hydrazide derivatized agarose beads. The best immunosorbent obtained by reversible immobilization was LIB-CNH45 MAb on Protein A/G. Using this support the eventual irreversible denaturation of covalently immobilized MAbs was overcome. For indirect assays, N-hydroxisuccinimide derivatized agarose beads and glutaraldehyde-activated CPG were used as sorbents for hapten immobilization via the amino groups of a carrier protein. In this format, antigen-MAb interactions were detected using a peroxidase-conjugated rabbit anti-mouse immunoglobulin. The highest sensitivity was achieved by LIB-CNH45 MAb in combination with derivatized agarose beads. All these results demonstrated the suitability of ELIFA as a fast, precise and easy-to-use technique for immunosorbent selection.

Antibiotic immunosensing: Determination of sulfathiazole in water and honey

The development of two sensitive and selective immunosensors for sulfathiazole, using immunoreagents - haptens, polyclonal antibodies, enzyme conjugates - previously obtained and characterized, is presented. One of them is based on the competitive immunocomplex capture format making use of an immobilized protein A/G sorbent, while the other employs a restricted access support in a novel homogeneous-heterogeneous (HH) assay mode. Maximum sensitivity, achieved with a total assay time of 18min for the capture sensor, is traduced in a dynamic range from 0.4 to 24microgL(-1), with a lower limit of detection of 0.11microgL(-1), increasing to 1.2microgL(-1) when employing an accelerated capture assay protocol that yields a sampling rate of 7 cycles per hour. The HH sensor shows the fastest response, performing each whole assay in only 2min, with a limit of detection of 0.85 and a measurement interval of 3.9-181.0microgL(-1), and with no need of support regeneration. Immunosensors are selective for sufathiazole, and only sulfamethoxypyridazine, sulfamethizole and sulfapyridine show non-negligible cross-reactivity, the same as in ELISA batch immunoassay. The application of the developed systems to the analysis of water, with no sample treatment, as well as honey samples after solid-phase extraction, demonstrate the reliability of the immunosensing for the monitoring of this type of pollutants.

High density Microarrays on Blu-ray discs for massive screening

The potential and the capabilities of Blu-ray technology (discs and drives) for massive screening applications are presented. High density microarrays are fabricated onto a Blu-ray disc and applied for the determination of microcystin residues and pathogenic microorganisms. Specific probes were physisorbed onto the BD surface and the biorecognition event was displayed using labeled secondary antibody solution and subsequent signal amplification. The attenuation of the reflected light caused by the reaction product is detected by the Blu-ray drive and inversely correlated with analyte concentration. BDs preserve the optical properties according to Blu-ray specifications, ensuring maximum accuracy and sensitivity of the drive during disc scanning. Detection limits of 0.4 μg/L for microcystin LR and 10(0) and 10(1) cfu/mL for Salmonella typhimurium and Cronobacter sakazakii respectively, were achieved, improving considerably the DVD performances and reaching similar sensitivity as real-time quantitative PCR. Blu-ray technology adapted to the analysis of high density arrays highlights the enormous capabilities (high sensitivity, speed-scanning, optical resolution, portability) for point-of-care settings, diagnostics, and high-throughput screening applications.

Analysis of Atrazine in Water and Vegetables Using Immunosensors Working in Organic Media

Flow immunosensors using a competitive capture format have been studied and applied to the analysis of atrazine in extracts of water and vegetables containing high percentages of organic solvents. Four organic mixtures have been assayed: M1 50% methanol-50% buffer, M2 25% isopropanol-25% methanol-50% buffer, M3 25% acetonitrile-75% buffer, and M4 10% ethyl acetate-25% methanol-65% buffer. Three polyclonal antisera and two haptens conjugated to horseradish peroxidase and alkaline phosphatase as enzyme tracers have been studied with each mixture. Although sensitivity is better in aqueous medium, good results have been obtained with the four organic mixtures tested, being best in M1, with a limit of detection of 0.15 μg L–1 for the sensor employing peroxidase as label. Selectivity, expressed as cross-reactivity, and precision of the assays have been shown to be better in organic media than in aqueous one. More than 400 assay cycles can be run with the same immunosensor. Good recoveries have been obtained when the methanolic extracts of atrazine-spiked water and vegetables were analysed after solid phase extraction on C18 cartridges. These results show the potential of organic immunoassays.

Automated immunosensing system for 3,5,6-trichloro-2-pyridinol: Application to surface water samples

The development of immunosensors for 3,5,6-trichloro-2-pyridinol is presented. The sensors are based on the principles of the direct competitive enzyme-linked immunosorbent assay, and use a specific monoclonal antibody (mAb) raised against the analyte. Immunoreagents are orientedly immobilized on both a derivatized agarose ester and alkylamined controlled-pore glass. Another immunosensor carries out the competition reaction in solution, being the immunocomplexes captured by Protein A/G. The competition is established, in all cases, between the analyte and a hapten conjugated to horseradish peroxidase. Fluorimetric detection is applied. The performance of the three sensors is compared in terms of assay sensitivity and reactor reusability. All the sensors developed are sensitive enough to detect the analyte in the sub-ppb range. Interferences from related compounds are negligible (cross-reactivity less than 1%) except for 2,4,5-trichlorophenol (cross-reactivity 9.28%). The application to the determination of the analyte in tap and lake water samples is discussed.

INSEL: an in silico method for optimizing and exploring biorecognition assays

A practical in silico method for optimizing and exploring biointeraction-based events is developed.