Juan J. Manclús

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 microg l(-1) for carbaryl and TCP, respectively. The sensitivity attained (I(50) value) was around 30 microg l(-1) for both compounds. Linear working ranges were 15-53 microg l(-1) for carbaryl and 13-83 microg l(-1) for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.

Development of Monoclonal Immunoassays for the Determination of Triazole Fungicides in Fruit Juices

Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for the detection of triazole fungicides have been developed. With this aim, hapten-protein conjugates, containing the common triazole and chlorinated aromatic moieties, were prepared. From mice immunized with these conjugates, several monoclonal antibodies (MAbs) with the ability to sensitively bind several triazoles with different specificity were obtained. Both analyte- and class-specific ELISAs were developed. The hexaconazole-specific immunoassay can determine this fungicide with a limit of detection of 0.3 mug/L in standard buffer. The so-called triazole-specific immunoassay allowed for the detection of tetraconazole, penconazole, cyproconazole, and myclobutanil, with limits of detection in the 0.1-0.7 mug/L range. These immunoassays were applied to the determination of triazoles in spiked fruit juices. Samples were adequately diluted to minimize the matrix effects. Coefficients of variation were below 30%, and recoveries ranged from 62 to 135%. Therefore, the developed immunoassays can determine triazole fungicides in fruit juices down to the maximum residue limits currently legislated, without any sample treatment other than dilution.

FLUORESCENCE POLARIZATION IMMUNOASSAY FOR THE INSECTICIDE DDT AND ITS METABOLITES

ABSTRACT Two fluorescence polarization immunoassays (FPIA) for detection of the insecticide DDT (DDT-specific FPIA) and the sum of DDT isomers and metabolites (DDT class-specific FPIA) were developed. Various fluorescein-labeled tracers were synthesized and studied in order to achieve a high sensitivity using different monoclonal antibodies. For DDT-specific assay the sensitivity, estimated as limit of detection, was 12 ng/mL, with a linear working range between 20 and 1000 ng/mL. For DDT class-specific assay the sensitivity was 3 ng/mL, with a linear working range between 5 and 1000 ng/mL. Regarding specificity to other chlorinated pesticides, both immunoassays showed no cross-reaction (<0.1%) with 2,4-D, endosulfan, diuron and hexachlorocyclohexanes. Both FPIAs were successfully applied to the analysis of DDT in spiked tap water samples. The FPIAs for DDT are simple, rapid and homogeneous (without separation and washing steps). Total time of analysis is less than 1 min per sample. The developed FPIAs could be used for rapid screening of water samples for contamination with DDT and its isomers and metabolites.

Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well–known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I50 value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

High-frequency phase shift measurement greatly enhances the sensitivity of QCM immunosensors.

In spite of being widely used for in liquid biosensing applications, sensitivity improvement of conventional (5-20MHz) quartz crystal microbalance (QCM) sensors remains an unsolved challenging task. With the help of a new electronic characterization approach based on phase change measurements at a constant fixed frequency, a highly sensitive and versatile high fundamental frequency (HFF) QCM immunosensor has successfully been developed and tested for its use in pesticide (carbaryl and thiabendazole) analysis. The analytical performance of several immunosensors was compared in competitive immunoassays taking carbaryl insecticide as the model analyte. The highest sensitivity was exhibited by the 100MHz HFF-QCM carbaryl immunosensor. When results were compared with those reported for 9MHz QCM, analytical parameters clearly showed an improvement of one order of magnitude for sensitivity (estimated as the I50 value) and two orders of magnitude for the limit of detection (LOD): 30μgl(-1) vs 0.66μgL(-1)I50 value and 11μgL(-1) vs 0.14μgL(-1) LOD, for 9 and 100MHz, respectively. For the fungicide thiabendazole, I50 value was roughly the same as that previously reported for SPR under the same biochemical conditions, whereas LOD improved by a factor of 2. The analytical performance achieved by high frequency QCM immunosensors surpassed those of conventional QCM and SPR, closely approaching the most sensitive ELISAs. The developed 100MHz QCM immunosensor strongly improves sensitivity in biosensing, and therefore can be considered as a very promising new analytical tool for in liquid applications where highly sensitive detection is required.

Robotic sample pretreatment-immunoassay determination of chlorpyrifos metabolite (TCP) in soil and fruit.

A fully automated method for leaching of TCP (3,5,6-trichloro-2-pyridinol, the major degradation product of the widely used chlorpyrifos insecticides) from soil and fruit with subsequent determination by ELISA is reported. The automation of the weighing and leaching steps enables unattended development of sample preparation. The determination of the target analyte, traditionally performed by liquid chromatography, has been substituted by specific immunoassay using a non-commercial monoclonal antibody which enables the determination of TCP within the range 0.01-7 ng 1(-1) with an R.S.D. of 8.6%.

Application of a monoclonal-based immunoassay for the determination of imazalil in fruit juices

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of imazalil [(RS)-1-(β-allyloxy-2,4-dichlorophenylethyl)imidazole] in apple, tomato and orange juice samples. From an imazalil hapten, which mimics the analyte structure, several monoclonal antibodies were obtained. An ELISA in the conjugate-coated format was developed and optimized using the antibody showing the highest sensitivity. For standards, the detection limit of the ELISA was 0.2 nM (0.06 ng ml−1), with an I 50 value of 1.6 nM (0.5 ng ml−1). The study of the influence of matrices on assay reliability indicated that the ELISA could determine imazalil in fruit juices at the low ng ml−1 level simply by diluting the sample, without any clean-up or concentration step. Recovery and precision of the method were evaluated by spiking juice samples with imazalil in the 10–500 ng ml−1 range. The mean recovery from fruit juices was 97% and the mean coefficient of variation was ∼20%. In addition to being precise and accurate, the method has proved to be simple and sensitive, with a quantification limit well below the maximum residue limits for imazalil in these matrices.

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.

Development and application of recombinant antibody-based immunoassays to tetraconazole residue analysis in fruit juices.

Tetraconazole is currently used as a fungicide in fruit and vegetables. The aim of this work was the development of immunochemical techniques based on recombinant antibodies for the screening of tetraconazole residues in fruit juices. Recombinant antibodies were produced from a hybridoma cell line secreting a monoclonal antibody specific for tetraconazole and from lymphocytes of mice hyperimmunised with tetraconazole haptens conjugated to bovine serum albumin. From these antibodies, enzyme-linked immunosorbent assays in the conjugate-coated format were developed, which were able to detect tetraconazole standards down to 1ng/mL. From recovery studies with spiked samples, these immunoassays determined tetraconazole in orange and apple juices with acceptable reproducibility (coefficients of variation below 25%) and recoveries (ranging from 78% to 145%) for a screening technique. The analytical performance of RAb-based immunoassays was fairly similar to that of the MAb-based immunoassays. Due to their simplicity and high sample throughput, the developed recombinant-based immunoassays can be valuable analytical tools for the screening of tetraconazole residues in fruit juices at regulatory levels.

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(β-allyloxy-2,4-dichlorophenylethyl)imidazole (imazalil) were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.