Sergi Morais

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.

Multiplexed microimmunoassays on a digital versatile disk.

Multiplexed microimmunoassays for five critical compounds were developed using a digital versatile disk (DVD) as an analytical support and detecting technology. To this end, coating conjugates were adsorbed on the polycarbonate face of the disk; a pool of specific antibodies, gold labeled secondary antibodies, and silver amplification were addressed for developing the assays. The detection principle is based on the capture of attenuated analog signals with the disk drive that were proportional to optical density of the immunoreaction product. The multiplexed assay achieved detection limits (IC10) of 0.06, 0.25, 0.37, 0.16, and 0.10 microg/L, sensitivities of (IC50) 0.54, 1.54, 2.62, 2.02, and 5.9 microg/L, and dynamic ranges of 2 orders of magnitude for atrazine, chlorpyrifos, metolachlor, sulfathiazole, and tetracycline, respectively. The features of the methodology were verified by analyzing natural waters and compared with reference chromatographic methods, showing its potential for high-throughput multiplexed screening applications. Analytes of different chemical nature (pesticides and antibiotics) were directly quantified without sample treatment or preconcentration in a total time of 30 min with similar sensitivity and selectivity to the ELISA plate format using the same immunoreagents. The multianalyte capabilities of immunoassaying methods developed with digital disk and drive demonstrated the competitiveness to quantify targets that require different sample treatment and instrumentation by chromatographic methods.

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.

DNA microarraying on compact disc surfaces. Application to the analysis of single nucleotide polymorphisms in Plum pox virus

The potential of using compact discs as high throughput screening platforms for DNA microarraying is discussed and applied to discriminate genetic variations of Plum pox virus.

Detection of oligonucleotide systematic mismatches with a surface plasmon resonance sensor

The detection and parallel characterization of the hybridization event of 21-base, unlabeled DNA oligonucleotides with a monolayer of complementary DNA immobilized on a gold surface by surface plasmon resonance (SPR) is presented. A thiol modification on the probe DNA strand allowed for its attachment to the surface via self-assembly. For the hybridization of full match DNA a detection limit of 20 pM was determined. The change in SPR signal was always larger for the full match compared to the one-mismatch target DNA oligonucleotides when the mismatch was in the middle or at the proximal end of the target DNA. Hybridization conditions (buffer concentration, flow rate, and temperature) did not affect the ability of the sensor to discriminate for single nucleotide mismatches. To our knowledge this is the only work where a comparison on the surface hybridization efficiency is performed between proximal, distal, and middle mismatches and the effect of three hybridization parameters is studied with regard to the detection of single nucleotide mismatches using SPR.

Isothermal DNA amplification strategies for duplex microorganism detection

A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp. and Cronobacter spp., with excellent amplification yields (0.2-8.6 · 10(8) fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (10(1)-10(2)CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature.

Detection of food-borne pathogens with DNA arrays on disk

A DNA oligonucleotide array for duplex pathogen detection on a DVD platform is developed. The assay involves hybridization of PCR products and optical detection using compact disc technology. Different DNA array constructions for attachment of synthetic oligonucleotides on to DVD surface are evaluated, finding that streptavidin-biotin coupling method yielded the highest sensitivity in combination with enzymatic signal amplification. Issues of importance for the DNA array construction such immobilized probes design, PCR product labeling strategy and composition of the hybridization buffer were addressed. The methodology was proved scoring single nucleotide polymorphisms with high selectivity. The assay capability was also demonstrated by the identification of two pathogenic microorganisms in powder milk samples. In fifty minutes, the DVD-array system identifies Salmonella spp. and Cronobacter spp. (previously named Enterobacter sakazakii) precise and simultaneously with a sensitivity of 10(0) and 10(2) cfu/mL, respectively, in infant milk. Results were in good agreement with those obtained by quantitative real-time PCR.

Selection and characterisation of membranes by means of an immunofiltration assay. Application to the rapid and sensitive determination of the insecticide carbaryl

The characterisation and selection of membranes by means of an immunofiltration assay is described. The chemical composition of the membranes was: nitro-cellulose, polyamide, polyvinylidene difluoride, polyethersulfone, cellulose acetate, regenerated cellulose, cellulose nitrate, and glass fibre. In order to characterise the membranes according to their binding capacity, immobilisation stability, sensitivity and hydrodynamic properties, two basic immunofiltration formats were performed. In both formats, enzyme label (horseradish peroxidase, HRP) and colorimetric detection were used. In the immobilised antibody format, three monoclonal antibodies (mAb) against the insecticide carbaryl were immobilised on the membranes by passive adsorption. In the immobilised hapten format, two haptens conjugated to bovine serum albumin (BSA) were immobilised. Immobilon-P was the best membrane with regard to the characterisation criteria and permitted the filtration of large volume (5.0 ml) through the membrane without release of the receptor. The immobilisation of the receptor (antibody or haptenic conjugate) was pH dependent. Good results with regard to mAb-antigen recognition, were obtained using 50 mM carbonate/bicarbonate buffer, pH 9.6. However, the most sensitive assays were achieved using, 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCI (PBS), pH 7.4 as immobilisation buffer. Furthermore, all these results permit the choice of the best membrane for the rapid and sensitive determination of carbaryl. This study will assist the development of dipsticks, immunoelectrodes, membrane-based immunoreactors or immunoconcentration devices that are based on the use of membranes as immunosupports.

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.

Determination of microcystins in river waters using microsensor arrays on disk.

The development of simple, accurate, and rapid multisample analytical methodologies to find out critical targets in waters is highly demanded. Optical microsensor arrays to determine microcystins in river waters are developed on the polycarbonate side of compact discs. The working principle of the sensors relied on an indirect competitive microimmunoassay, where free microcystin LR (MC-LR) competes with immobilized conjugate for specific monoclonal antibody. The results of the immunoreaction are detected with a DVD drive, showing the readouts in minutes. The method reached a sensitivity (IC(50)) for MC-LR of 1.04 μg/L and a linear response in the range 0.12-2.00 μg/L, allowing its determination below the upper limit proposed by the World Health Organization in drinking water. The developed analytical approach shows simplicity, good sensitivity, high throughput capability, and rapidity (37 min) in field use. The optimized assay showed also high congener reactivity to MC-LY (144%), MC-LA (125%), MC-LF (119%), MC-LW (102%), MC-YR (83%), and nodularin (94%). Furthermore, the suitability of the disk biosensor to quantify MC-LR was successfully evaluated analyzing river water samples, obtaining excellent recoveries (78-113%). Precoated discs are stable for at least seven weeks without loosing their analytical performances. Also, the portability of the analytical system permits on-site analysis and quantification, saving time and other resources. To our knowledge, this is the only work where a portable, easy-to-use, array based system has been developed for on-site microcystin quantification and applied to simultaneously analyze 42 samples plus the calibration curve, reaching microgram per liter sensitivity.