Analysis of alternariol and alternariol monomethyl ether in foodstuffs by molecularly imprinted solid-phase extraction and ultra-high-performance liquid chromatography tandem mass spectrometry.

Molecularly imprinted porous polymer microspheres selective to Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), were synthesized and applied to the extraction of both mycotoxins in food samples. The polymer was prepared using 4-vinylpiridine (VIPY) and methacrylamide (MAM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as cross-linker and 3,8,9-trihydroxy-6H-dibenzo[b,d]pyran-6-one (S2) as AOH surrogate template. A molecularly imprinted solid phase extraction (MISPE) method has been optimized for the selective isolation of the mycotoxins from aqueous samples coupled to HPLC with fluorescence (λex=258nm; λem=440nm) or MS/MS analysis. The MISPE method was validated by UPLC-MS/MS for the determination of AOH and AME in tomato juice and sesame oil based on the European Commission Decision 2002/657/EC. Method performance was satisfactory with recoveries from 92.5% to 106.2% and limits of quantification within the 1.1-2.8µgkg-1 range in both samples.

Multiresidue analysis of cephalosporin antibiotics in bovine milk based on molecularly imprinted polymer extraction followed by liquid chromatography-tandem mass spectrometry.

This work reports the preparation of molecularly imprinted polymers (MIPs) selective to cephalosporin (CF) antibiotics, and their application as molecularly imprinted solid-phase extraction (MISPE) sorbents for the determination of these antimicrobials in milk samples. Several functional monomers and cross-linkers have been screened to select the best combination that provides high selectivity for the simultaneous multiresidue extraction of cefthiofur (THIO), cefazolin (AZO), cefquinome (QUI), cephapirin (API), cephalexin (ALE) and cephalonium (ALO) from the samples. The novel MIPs were prepared by a non-covalent imprinting approach in the form of spherical microparticles using the synthetic surrogate molecule sodium 7-(2-biphenylylcarboxamido)-3-methyl-3-cepheme-4-carboxylate, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea (VPU) as functional monomer, and divinylbenzene (DVB) as crosslinking agent in a 1:2:20 molar ratio. The optimized MISPE method allowed the extraction of the target antimicrobials from raw cow milk samples using a selective washing with 5mL methanol/2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (0.1M, pH 7.5) (2:98, v/v) to remove the non-specifically retained compounds, followed by elution with 1mL of trifluoroacetic acid (TFA) in methanol (0.1:99.9, v/v). The extracts have been analysed by UHPLC-MS/MS and the analytical method has been validated according to EU guideline 2002/657/EC. The limits of quantification (S/N=10) were in the 1.7-12.5µgkg-1 range, well below the maximum residue limits (MRLs) currently established for the quantified cephalosporins in milk samples. The developed MIP allows mutiresidual determination of the six cephalosporin antibiotics mentioned above, significantly broadening the application to food analysis of MISPE methods.

Active food packaging based on molecularly imprinted polymers: study of the release kinetics of ferulic acid.

A novel active packaging based on molecularly imprinted polymer (MIP) was developed for the controlled release of ferulic acid. The release kinetics of ferulic acid from the active system to food simulants (10, 20, and 50% ethanol (v/v), 3% acetic acid (w/v), and vegetable oil), substitutes (95% ethanol (v/v) and isooctane), and real food samples at different temperatures were studied. The key parameters of the diffusion process were calculated by using a mathematical modeling based on Fick's second law. The ferulic acid release was affected by the temperature as well as the percentage of ethanol of the simulant. The fastest release occurred in 95% ethanol (v/v) at 20 °C. The diffusion coefficients (D) obtained ranged between 1.8 × 10(-11) and 4.2 × 10(-9) cm(2)/s. A very good correlation between experimental and estimated data was obtained, and consequently the model could be used to predict the release of ferulic acid into food simulants and real food samples.

Molecular recognition with nanostructures fabricated by photopolymerization within metallic subwavelength apertures.

The first demonstration of fabrication of submicron lateral resolution molecularly imprinted polymer (MIP) patterns by photoinduced local polymerization within metal subwavelength apertures is reported. The size of the photopolymerized MIP features is finely tuned by the dose of 532 nm radiation. Rhodamine 123 (R123) has been selected as a fluorescent model template to prove the recognition capability of the MIP nanostructures, which has been evaluated by fluorescence lifetime imaging microscopy (FLIM) with single photon timing measurements. The binding selectivity provided by the imprinting effect has been confirmed in the presence of compounds structurally related to R123. These results pave the way to the development of nanomaterial architectures with biomimetic artificial recognition properties for environmental, clinical and food testing.

Multiresidue analysis of fluoroquinolone antimicrobials in chicken meat by molecularly imprinted solid-phase extraction and high performance liquid chromatography.

This paper describes the synthesis of novel molecularly imprinted polymer (MIP) micro-beads for the selective extraction (MISPE) of six fluoroquinolone (FQ) antibiotics (enrofloxacin, ciprofloxacin, lomefloxacin, danofloxacin, sarafloxacin and norfloxacin) from chicken muscle samples and further analysis by high-performance liquid chromatography (HPLC) with fluorescence (FLD) or mass spectrometry (MS) detection. A combinatorial screening approach has been applied to select the optimal functional monomer and cross-linker formulation for polymer synthesis. The MIP prepared using enoxacin (ENOX) as the template - a mixture of methacrylic acid (MAA) and trifluoromethacrylic acid (TFMAA) as functional monomers and ethylene glycol dimethacrylate (EDMA) as the cross-linker - showed superior FQ recognition properties than the rest of the materials generated. MIP spherical particles were prepared using silica beads as sacrificial scaffolds. The polymers were packed in solid phase extraction (SPE) cartridges. The optimized MISPE-HPLC method allows the extraction of the antimicrobials from aqueous samples followed by a selective washing with acetonitrile/water (0.005% TFA, pH=3.0), 20:80 (v/v) and elution with 5% trifluoroacetic acid in methanol. Optimum MISPE conditions led to recoveries of the target FQs in chicken muscle samples ranging between 68 and 102% and precisions in the 3-4% range (RSD, n=18). The method has been validated according to European Union Decision 2002/657/EC, in terms of linearity, accuracy, precision, selectivity, decision limit (CCα) and detection capability (CCß) by HPLC-FLD and HPLC-MS/MS. The limits of detection were improved using HPLC-MS/MS analysis and ranged between 0.2 and 2.7µgkg(-1) (S/N=3) for all the FQs tested.

Immuno-like assays and biomimetic microchips.

Biomimetic assays with molecularly imprinted polymers (MIPs) are bound to be an alternative to the traditional immuno-analytical methods based on antibodies. This is due to the unique combination of advantages displayed by the artificial materials including the absence of animal inoculation and sacrifice, unnecessary hapten conjugation to a carrier protein for stimulated production, the possibility of manufacturing MIPs against toxic substances, excellent physicochemical stability, reusability, ease of storage, and recognition in organic media. If the selectivity and affinity of MIPs are increased, many more immuno-like assays will be developed using radioactive, enzymatic, colorimetric, fluorescent, chemiluminescent, or electrochemical interrogation methods. This chapter provides a comprehensive comparison between the bio- and biomimetic entities and their usage.

Automated portable array biosensor for multisample microcystin analysis in freshwater samples.

An automated array biosensor based on evanescent-wave excitation has been developed for the detection of microcystins (MCs) in freshwater samples. The sensing surface consisted of microcystin-leucine-arginine (MCLR) covalently immobilized onto a planar waveguide (microscope slide). The binding of anti-MCLR monoclonal antibodies, spiked in the sample, to the immobilized MCLR was competitively inhibited by MCLR in solution and the amount of antibody bound to the patterned antigens was revealed using Cy5-labeled rabbit anti-mouse IgG. Surface chemistry has been optimized to improve biosensor performance in terms of sensitivity, regeneration ability and to avoid non specific binding for further application to environmental monitoring. The optimized biosensor assay presents an IC(50) value of 0.34 ± 0.01 µg/L, a detection limit of 16 ± 3 ng/L and a dynamic range from 0.06 to 1.5 µg/L MCLR, improving the performance of previously reported devices. Cross-reactivity to other related MCs, such as microcystin-RR (MCRR, 90%), microcystin-RR desmethylated (dm-MCRR, 95%) and microcystin-YR (MCYR, 91%), was also evaluated. The automated microarray can assay up to six different samples in parallel, with a total analysis time of about 60 min. The sensing surface was regenerated with 50mM NaOH and each chip was reused for, at least, 15 assay-regeneration cycles without significant binding capacity loss. The immunosensor has been successfully applied to the direct analysis of MCs in surface water samples and the results were in close agreement with those provided by LC-MS/MS.

Molecularly imprinted polymer diffraction grating as label-free optical bio(mimetic)sensor.

Micropatterned molecularly imprinted polymer (MIP) transmissive 2D diffraction gratings (DGs) are fabricated and evaluated as label-free antibiotic bio(mimetic)sensors. Polymeric gratings are prepared by using microtransfer molding based on SiO(2)/Si molds. The morphology of the MIP gratings is studied by optical and atomic force microscopes. MIP 2D-DGs exhibit 2D optical diffraction patterns, and measurement of changes in diffraction efficiency is used as sensor response. The refractive index of the micropatterned MIP material was estimated, via solvent index matching experiments, to be 1.486. Immersion of a MIP 2D-DG in different solutions of target-antibiotic enrofloxacin leads to significant variations in diffraction efficiency, demonstrating target-molecule detection. On the other hand, no significant response is observed for both control experiments: MIP grating exposed to a non-retained analyte and an equivalent non-imprinted polymer grating exposed to the target analyte, showing highly specific antibiotic label-free optical recognition.

Multiresidue determination of fluoroquinolone antimicrobials in baby foods by liquid chromatography.

According to the current EU legislation, the presence of antimicrobial residues in baby foods is forbidden. Nevertheless, there is a lack of analytical methods to determine veterinary antimicrobials in baby foods and support the zero tolerance policy for this type of foods. This paper describes a simple method based on molecularly imprinted solid phase extraction (MISPE) and liquid chromatography with fluorescence detection (LC-FLD) for the determination of residues of fluoroquinolones (FQs) in baby foods. The method involves sample extraction with a solution of o-phosphoric acid (50mM, pH 3.0)/ACN (20:80, v/v) and further clean-up by loading the extracts onto MIP cartridges. Optimum MISPE conditions led to recoveries of the target FQs in the range of 92-106%, with RSDs <8%. Method validation has been performed according to Commission Decision 2002/657/EC, in terms of linearity, accuracy, precision, selectivity, decision limit (CCα) and detection capability (CCß). The proposed method has been successfully applied to the analysis of baby foods of different compositions bought in local supermarkets and pharmacies. The results did not show the presence of residual amounts of FQs in the analysed samples above the method's decision limits (CCα between 5 and 151µgkg(-1)).

Development of a microalgal PAM test method for Cu(II) in waters: comparison of using spectrofluorometry.

Test methods are needed to monitor Cu concentrations in reservoirs and water supplies. Dictyosphaerium chlorelloides (Chlorophyta) cells were immobilized in a silicate sol-gel and the toxic effects of Cu(II) were examined using different techniques: fluorescence measurements (using a spectrofluorometer with an optic fiber coupled to a flow cell or a 96-well-plate reader) or by Pulse Amplitude Modulation (PAM) parameters using a portable instrument and the pulse saturation method. Fm' and qN were the most sensitive indicator parameters when performing Cu analysis in water. D. chlorelloides PAM biosensor presented a detection limit of 0.6 mg l(-1) for Cu(II), within the limits to establish if Cu concentrations exceeded regulatory levels. Moreover, a 1.9 mg Cu l(-1) (30 microM) resistant strain of the D. chlorelloides microalgae was produced in order to obtain more selectivity on the metal determination.

Optimization of a pressurized liquid extraction method by experimental design methodologies for the determination of fluoroquinolone residues in infant foods by liquid chromatography.

In the present study, we have developed a method based on pressurized liquid extraction (PLE) and liquid chromatography with fluorescence detection (LC-FLD) for the determination of residues of fluoroquinolones (FQs) in infant food products. PLE extraction has been optimized by the application of experimental design methodologies. Initially, a fractional factorial design (FFD) was used to screen the significance of four extraction parameters: solvent composition, temperature, pressure and number of cycles. The most significant factors, identified by ANOVA analysis, were the solvent composition, temperature and pressure, which were further optimized with the aid of a face centred design (FCD) and the desirability function. The optimized operating PLE conditions were as follows: ACN/o-phosphoric acid 50mM pH 3.0 (80:20, v/v), 80 degrees C, 2000psi and three extraction cycles of 5min. Under these conditions, recoveries of the target FQs varied between 69% and 107% with RSDs below 9%. The whole method was validated according to the Commission Decision 2002/657/EC guidelines. The proposed method has been successfully applied to the analysis of different infant food products bought in local supermarkets and pharmacies. The results showed the presence of residues of enrofloxacin in a non-compliant baby food sample corresponding to a chicken-based formulation, which were also confirmed and quantified by LC-MS/MS analysis.

Quantitative determination of penicillin V and amoxicillin in feed samples by pressurised liquid extraction and liquid chromatography with ultraviolet detection.

A rapid and simple method is proposed for the routine determination of amoxicillin (AMOX) and penicillin V (PENV) in swine feedingstuffs. The method is based on pressurised liquid extraction (PLE) followed by high performance liquid chromatography with ultraviolet detection (PLE-HPLC-UV) for antibiotic analysis. Parameters affecting PLE procedure, such as temperature, solvent composition, number of extraction cycles and sample cell size, were evaluated in order to achieve the highest extraction efficiency. The optimised method employed 11mL extraction cells, acetonitrile-water mixtures (25:75, v/v) for AMOX and (50:50, v/v) for PENV, as extraction solvent, 102.07atm of extraction pressure, 50 degrees C of extraction temperature, 5min of static time and 60% flush volume of the cell size. Extracts were filtered and directly analysed by HPLC-DAD/UV without further clean-up. Mean recovery rates for feed samples fortified with 200-500mgkg(-1) of both antibiotics were 86% for AMOX (RSD< or =6%) and 95% for PENV (RSD< or =3%). The method was successfully applied to the analysis of a commercial medicated swine feedingstuff, and the results were in good agreement with those obtained using mechanical shaking or ultrasonic extraction combined with solid phase extraction (UE-SPE), previously applied in the literature for feed analysis. The extraction efficiencies were evaluated by statistical comparison (analysis of variance, ANOVA-single factor) of the results obtained using the different extraction methods. Compared to the alternative techniques, PLE offers several practical advantages: easy to perform, fast, savings in solvent volume and in time, all steps are fully automated and further clean-up is not necessary for penicillin analysis.

Development and validation of a solid-phase extraction method coupled to liquid chromatography with fluorescence detection for the determination of fluoroquinolone residues in powdered infant formulae. Application to the analysis of samples from the Spanish and Latin American market.

This paper describes a new method for the effective extraction, clean-up and chromatographic analysis of residues of four fluoroquinolones (ciprofloxacin, enrofloxacin, danofloxacin and sarafloxacin) in powdered infant formulae and follow-on preparations. Samples were reconstituted following the manufacturer's recommendations and treated with trichloroacetic acid in methanol 10% (w/v) for deproteinization. Two solid-phase extraction cartridges have been evaluated for sample clean-up and preconcentration, Strata Screen A and Strata X and the later provided the best recoveries for all the analytes tested. Chromatographic analysis has been carried out using a polar endcapped column (AQUA C(18)) and fluorescence detection, with lomefloxacin (LOME) as internal standard. Method validation has been performed according to European Commission Decision 2002/657/EC criteria, in terms of linearity, recovery, precision, specificity, decision limit (CC(alpha)) and detection capability (CC(beta)). Typical recoveries ranged between 70 and 110% at levels below and above the maximum residue limits of the target analytes in bovine milk, with an excellent intralab reproducibility (RSDs<7%). Matrix effects did not significantly affect method accuracy, as evidenced by analyzing different brands of milk. The method has been successfully applied to the analysis of 100 samples of infant and follow-on formulae of the Spanish and Latin American market, using LC-MS/MS as confirmatory technique.

Development of a new sample pretreatment procedure based on pressurized liquid extraction for the determination of fluoroquinolone residues in table eggs.

A new method for the simultaneous determination of three fluoroquinolones (FQs) enrofloxacin (ENRO) ciprofloxacin (CIPRO) and sarafloxacin (SARA) in table eggs has been developed, applying pressurized liquid extraction (PLE) and liquid chromatography (LC) with fluorescence detection (LC-FLD). The influence of several extraction parameters (e.g. solvent mixture, temperature and extraction time) on FQs extraction efficiency and coextracted matrix interferents was evaluated using fortified control eggs and matrix matched standard curves. The results showed that FQs extraction efficiency depends mainly on solvent composition and the optimum extraction mixture was found to be phosphate 50mM, pH 3.0/acetonitrile (50:50, v/v). The optimized procedure employed 50% flush volume, 5min of static time and three extraction cycles at 70 degrees C and 1500psi. Method validation was performed according to the guidelines of the Directive 96/23/EC, using control egg samples, fortified with the target FQs in the range 50-1000ngg(-1) and applying the optimized extraction conditions on three different days, providing recoveries between 67-90% with RSDs lower than 11% in all cases. The decision limit (CCalpha) and detection capability (CCbeta) of the analytical method were found to be within the range 17-24ngg(-1) and 30-41ngg(-1), respectively. The method was successfully applied to the determination of ENRO and its metabolite CIPRO in incurred egg samples from ENRO-treated hens and LC-MS has been used and for confirmatory purposes.

Molecularly imprinted polymers with a streamlined mimic for zearalenone analysis.

Molecularly imprinted polymers (MIPs) with selective recognition properties for zearalenone (ZON), an estrogenic mycotoxin, and structurally related compounds have been prepared using the non-covalent imprinting approach. A rationally designed ZON analogue, cyclododecyl 2,4-dihydroxybenzoate (CDHB), that exhibits resemblance to ZON in terms of size, shape and functionality has been synthesized and used as template for MIP preparation instead of the natural toxin. Several functional monomers have been evaluated to maximize the interactions with the template molecule during the polymerization process. The polymer material prepared with 1-allylpiperazine (1-ALPP) as functional monomer, trimethyl trimethacrylate (TRIM) as cross-linker and acetonitrile as porogen (in a 1:4:20 molar ratio) displayed superior binding capacities than any other of the MIPs tested. Selectivity of this material for ZON and structurally related and non-related compounds has been evaluated using it as stationary phase in liquid chromatography. Our results demonstrate that the imprinted polymer shows significant affinity in the porogenic solvent for the template mimic (CDHB) as well as for the ZON and other related target metabolites in food samples, dramatically improving the performance of previously reported MIPs for ZON recognition. Therefore, MIPs can be an excellent alternative for clean-up and preconcentration of the mycotoxin in contaminated food samples.

Multiresidue determination of fluoroquinolones in milk by column liquid chromatography with fluorescence and ultraviolet absorbance detection.

Column liquid chromatography with fluorescence (FLD) and UV-diode array detection (UV-DAD) was used for the simultaneous determination of ciprofloxacin (CIPRO), enrofloxacin (ENRO), marbofloxacin (MARBO), danofloxacin (DANO) and sarafloxacin (SARA) residues in milk, using norfloxacin (NOR) as internal standard. Two solid-phase extraction (SPE) cartridges, were evaluated for sample clean-up and preconcentration, Strata X, based on a modified styrene-divinylbenzene polymer, and Strata Screen A, a mixed anion exchanger/C8 reversed-phase sorbent. The fluoroquinolones (FQs) were separated on a polar endcapped column (AQUA C18). The recoveries for raw milk spiked with the antibiotics at three concentrations close to the maximum residue limit (MRL), were 80-103% for ENRO, CIPRO and DANO, with relative standard deviations (R.S.D.) lower than 6.6%. SARA recoveries were 70% (R.S.D. = 7%) and values in the order of 95% (R.S.D. = 1.5%) were obtained for MARBO at the MRL level. The quantification limits ranged from 2.4 to l0 ng ml(-1) and are below the MRL established for these drugs by the European Union. The method was successfully applied to the analysis of ENRO and its metabolite CIPRO in an incurred milk sample.

Fiber-optic luminescent sensors with composite oxygen-sensitive layers and anti-biofouling coatings.

Anti-biofouling polymers containing phosphorylcholine (PC)-substituted methacrylate units have been prepared by copolymerization with dodecyl methacrylate and used to coat luminescent oxygen sensors. Nanometer-sized coatings of such materials are shown to reduce significantly the adhesion of marine bacteria (more than 70%) and thrombocytes (more than 90%) to the surface of tris-(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)-doped silicone layers. A thorough analytical characterization of both the PC-coated and the uncoated dyed films has demonstrated that the anti-biofouling layers do not alter dramatically the performance of the fiber-optic oxygen sensors in aqueous media and are mechanically stable for more than one year of continuous immersion. The slope of the linear calibration plots in the 0-8 mg L(-1) oxygen concentration range (ca. 1.0 L mg(-1)) decreases 8-11% after applying the 50-nm protective layer with no change in the sensor precision (1.1-1.9% RSD, n = 6). The response time of the 200-microm O2-sensitive layers (1.5-6 min) increases up to 2-fold, depending on the nature of the PC polymer used, but the temperature effect on the sensor response (0.020 L mg(-1) degrees C(-1)) remains essentially unchanged. Oxygen detection limits as low as 0.04 mg L(-1) have been measured with the coated optodes. The novel biofouling-resistant optosensors have been successfully validated against a commercial oxygen electrode and are shown to respond faster than the electrochemical device for large oxygen concentration changes. The biomimetic coatings will be particularly useful for drift-free long-term operation of environmental optosensors and in vivo fiber-optic oxygen analyzers.

Application of an antibody biochip for p53 detection and cancer diagnosis.

Detection of the p53 tumor suppressor gene is important in early cancer diagnostics because alterations in the gene have been associated with carcinogenic manifestations in several tissue types in humans. We have developed an antibody-based detection instrument, the biochip, to detect the presence of the anti-p53 antibody in human serum. The design of this highly integrated detector system is based on miniaturized phototransistors having multiple optical sensing elements, amplifiers, discriminators, and logic circuitry on an IC board. The system utilizes laser excitation and fluorescence signals to detect complex formation between the p53 monoclonal antibody and the p53 antigen. Recognition antibodies are immobilized on a nylon membrane platform and incubated in solutions containing antigens labeled with Cy5, a fluorescent cyanine dye. Subsequently, this membrane is placed on the detection platform of the biochip and fluorescence signal is induced using a 632.8-nm He-Ne laser. Using this immuno-biochip, we have been able to detect binding of the p53 monoclonal antibody to the human p53 cancer protein in biological matrices. The performance of the integrated phototransistors and amplifier circuits of the biochip, previously evaluated through measurement of the signal output response for various concentrations of fluorescein-labeled molecules, have illustrated the linearity of the microchip necessary for quantitative analysis. The design of this biochip permits sensitive, selective and direct measurements of a variety of antigen-antibody formations at very low concentrations. Furthermore, the acquisitions of the qualitative and quantitative results are accomplished rapidly, in about 15 min. These features demonstrate the potential of this antibody-based biochip for simple, rapid and early biomedical diagnostics of cancer.

Antibody-based biosensor for breast cancer with ultrasonic regeneration.

We describe a novel method and instrumental setup for regenerating antibodies immobilized on a fiberoptic probe of an immunosensor using ultrasonic irradiation with broadband imaging transducers. The instrumental setup and irradiation conditions for antibody regeneration using ultrasound are described. The results of the measurements with antibody against breast cancer antigen illustrate the effectiveness and potential of the regenerable immunosensor. A 65% removal of the antigens bound to the Mab immobilized on the fiber surface is attained after ultrasound regeneration.

Fiber optic monitoring of carbamate pesticides using porous glass with covalently bound chlorophenol red.

An optical fiber biosensor for the determination of the pesticides propoxur (Baygon) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described. A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes. The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG-immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle. In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l(-1) and from 0.03 to 0.50 mg l(-1), respectively. The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng). Reproducibility, stability and interference studies of the optical device are reported. The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed.