María Pilar Marco

Immunochemical techniques for environmental analysis II. Antibody production and immunoassay development

Abstract Antibody production is the key step of any immunochemical technique. The antibody determines to a large extent the specificity and sensitivity of the resulting immunochemical technique. These features may be modulated by judicious design of the immunogen and by rational immunoassay development. Criteria for hapten design and the steps involved in the obtention of antibodies and the development of competitive assays are described.

Immunochemical techniques for environmental analysis I. Immunosensors

Abstract Immunoassays are analytical techniques based on the avidity and specificity of the antigen-antibody reaction. However, new immunochemical techniques have emerged recently as a consequence of the incorporation of scientific advances and knowledge from other areas such as molecular biology, microelectronics and chemistry. The principles of these techniques and their application to environmental analysis are presented.

Immunoassay for folic acid detection in vitamin-fortified milk based on electrochemical magneto sensors.

An immunoassay-based strategy for folic acid in vitamin-fortified milk with electrochemical detection using magneto sensors is described for the first time. Among direct and indirect competitive formats, best performance was achieved with an indirect competitive immunoassay. The immunological reaction for folic acid (FA) detection was performed, for the first time on the magnetic bead as solid support by the covalent immobilization of a protein conjugate BSA-FA on tosyl-activated magnetic bead. Further competition for the specific antibody between FA in the food sample and FA immobilized on the magnetic bead was achieved, followed by the reaction with a secondary antibody conjugated with HRP (AntiIgG-HRP). Then, the modified magnetic beads were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with electrochemical detection using magneto sensors was successfully evaluated using spiked-milk samples and compared with a novel magneto-ELISA based on optical detection. The detection limit was found to be of the order of microgl(-1) (13.1 nmoll(-1), 5.8 microgl(-1)) for skimmed milk. Commercial vitamin-fortified milk samples were also evaluated obtaining good accuracy in the results. This novel strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological and food samples.

Validation of two immunoassay methods for environmental monitoring of carbaryl and 1-naphthol in ground water samples

Abstract Two formats (microtiter plates and magnetic particles) of sensitive enzyme-linked immunosorbent assays (ELISA) for carbaryl determination have been compared to the EPA method 531.1 (liquid chromatography-post-column derivatization-fluorescence detection, LC-PCR-FD). An immunoassay developed for 1-naphthol determination has also been evaluated to monitor previous exposure to carbaryl in natural water samples. Matrix effect of this kind of water on the immunoassays is negligible and no sample preparation has found to be necessary other than buffering the samples. It is possible to perform a high number of analyses in a short period of time. A close correspondence was found for the results obtained when spiked and well water samples were split for analysis by ELISA and by LC-PCR-FD. Both methods are compared in terms of precision, reliability, reproducibility and their utility as screening tools. Application of those methods to the direct determination of carbaryl and 1-naphthol in ground water samples of the aquifer Campo de Nijar (Almeŕia, Spain) is reported.

Detection of pesticide residues using an immunodevice based on negative dielectrophoresis.

The detection of atrazine using a novel optical immunosensing technique based on negative dielectrophoresis (n-DEP) in microfluidic channels is described. Atrazine is a toxic triazine herbicide within the most frequently used. Polystyrene microparticles (6 microm diameters) modified with bovine serum albumin conjugated with atrazine (atrazine-BSA) were manipulated and captured when subjected to intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 10 V(peak) and frequencies over 1 MHz were applied to the electrodes. The immunological reaction occurring on the particles for detecting atrazine is based on an indirect competitive assay using a secondary anti-mouse immunogloburin G (IgG) antibody labeled with fluorescein isothiocyanate. The microfluidic device, with three-dimensional microelectrodes, was fabricated comprising two caged areas, allowing two simultaneous measurements inside the same microfluidic channel. The performance of this n-DEP immunosensing technique was evaluated using wine samples. The immunodevice showed a limit of detection for atrazine in buffer samples of 0.11 microgL(-1) and in pre-treated wine samples of 6.8 microg L(-1); these detection limits are lower than the maximum residue level (MRL) established by the European Community for residues of this herbicide in wine (50 microg L(-1)). This methodology offers great promise for rapid, simple, cost effective, and on-site analysis of biological, foods and beverages, and environmental samples.

Reversible immunosensor for the automatic determination of atrazine. Selection and performance of three polyclonal antisera

Abstract The development of an immunosensor for the highly sensitive, rapid and automatic analysis of the herbicide atrazine is presented. The immunosensor used the principles of competitive direct enzyme immunoassays, and protein A/G covalently bound to an azlactone-activated polymeric support as immunocomplex affinity binder. The immunosensor was completely automated, and was able to carry out a complete analysis, including surface regeneration, in less than 25xa0min, having a complete autonomy for more than 72xa0h. Three antisera have been tested and their performance compared in terms of sensitivity and cross-reactivity, and only slight differences in behavior have been found. The best antiserum for sensitivity (R-11) led to a competitive calibration curve with an I 50 value of 0.053xa0μgxa0l −1 (0.24xa0nM), a limit of detection (90% of blank signal) of 0.007xa0μgxa0l −1 , and a dynamic range from 0.014 to 0.232xa0μgxa0l −1 . The best antiserum in terms of cross-reactivity (R-10) led to a system that was also very sensitive ( I 50 0.101xa0μgxa0l −1 and limit of detection 0.011xa0μgxa0l −1 ) and free of interferences except from propazine. The immunosensor using all the three antisera has been used to determine atrazine in reference river water samples, and results were compared to ELISA and chromatography measurements. The immunosensor based on R-10 antiserum produced the best results when compared to those obtained with reference methods (101.4% and 104.8% mean recoveries for chromatography and ELISA, respectively), although a good correlation is achieved with all the three antisera.

Performance of two immunoassays for the determination of atrazine in sea water samples as compared with on-line solid phase extraction-liquid chromatography-diode array detection

Two immunoassay formats, magnetic particles-based assay (Atrazine RaPID assay and Atrazine High-Sensitivity RaPID assay) and microtiter plate based assay (Department of Entomology and Environmental Toxicology, University of California in Davis) were evaluated for the determination of atrazine in sea water samples. The results obtained were compared and validated with those obtained by using on-line solid phase extraction followed by liquid chromatography-diode array detection (on-line SPE-LC-DAD). The correlation between both techniques was good when analyzing levels of atrazine ranging from 0.01 to 5 μg/l in samples showing salt concentration values varying from 0 to 35 g/l and pH values from 2 to 10. One of these immunoassays (Atrazine High-Sensitivity RaPID assay) was employed to directly analyze atrazine in real estuarine and coastal water samples. The same samples were analyzed after filtration and C18 Empore disks extraction.

Evaluation of a field-test kit for triazine herbicides (SensioScreen® TR500) as a fast assay to detect pesticide contamination in water samples

Abstract A field-test kit (SensioScreen® TR500) for the determination of triazine herbicides in water samples has been evaluated. The test is based on an ELISA method performed on a membrane that allows the visual estimation of the presence of triazine herbicides in

Electronic Anabolic Steroid Recognition with Carbon Nanotube Field-Effect Transistors

A proof of concept of the electronic detection of two anabolic steroids, stanozolol (Stz) and methylboldenone (MB), was carried out using two specific antibodies and arrays of carbon nanotube field-effect transistors (CNTFETs). Antibodies specific for Stz and MB were prepared and immobilized on the carbon nanotubes (CNTs) using two different approaches: direct noncovalent bonding of antibodies to the devices and bonding the antibodies covalently to a polymer previously attached to the CNTFETs. The results indicated that CNTFETs bonded to specific antibodies covalently or noncovalently are able to detect the presence of steroids. Statistically significant changes in the threshold voltage and drain current were registered in the transistors, allowing the steroids to be recognized. On the other hand, it was determined that the specific antibodies do not detect other steroids other than Stz and MB, such as nandrolone (ND) because, in this case, statistically significant changes in the transistors were not detected. The polymer prevents the aggregation of antibodies on the electrodes and decreases the transistor hysteresis. Nevertheless, it is not able to avoid the nonspecific adsorption of streptavidin, meaning that nonspecific adsorption on CNTs remains a problem and that this methodology is only useful for purified samples. Regarding the detection mechanism, in addition to charge transfer, Schottky barrier, SB, modification, and scattering potential reported by other authors, an electron/hole trapping mechanism leading to hysteresis modification has been determined. The presence of polymer seems to hinder the modulation of the electrode-CNT contact.

Reusable conductimetric array of interdigitated microelectrodes for the readout of low-density microarrays

Low-density protein microarrays are emerging tools in diagnostics whose deployment could be primarily limited by the cost of fluorescence detection schemes. This paper describes an electrical readout system of microarrays comprising an array of gold interdigitated microelectrodes and an array of polydimethylsiloxane microwells, which enabled multiplexed detection of up to thirty six biological events on the same substrate. Similarly to fluorescent readout counterparts, the microarray can be developed on disposable glass slide substrates. However, unlike them, the presented approach is compact and requires a simple and inexpensive instrumentation. The system makes use of urease labeled affinity reagents for developing the microarrays and is based on detection of conductivity changes taking place when ionic species are generated in solution due to the catalytic hydrolysis of urea. The use of a polydimethylsiloxane microwell array facilitates the positioning of the measurement solution on every spot of the microarray. Also, it ensures the liquid tightness and isolation from the surrounding ones during the microarray readout process, thereby avoiding evaporation and chemical cross-talk effects that were shown to affect the sensitivity and reliability of the system. The performance of the system is demonstrated by carrying out the readout of a microarray for boldenone anabolic androgenic steroid hormone. Analytical results are comparable to those obtained by fluorescent scanner detection approaches. The estimated detection limit is 4.0 ng mL(-1), this being below the threshold value set by the World Anti-Doping Agency and the European Community.