Laura Bonel

An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer

Ochratoxin A (OTA) is one of the most important mycotoxin contaminants of foods, particularly cereals and cereal products, with strict low regulatory levels (of ppb) in many countries worldwide. An electrochemical competitive aptamer-based biosensor for OTA is described. Paramagnetic microparticle beads (MBs) were functionalized with an aptamer specific to OTA, and were allowed to compete with a solution of the mycotoxin conjugated to the enzyme horseradish peroxidase (OTA-HRP) and free OTA. After separation and washing steps helped with magnetic separations, the modified MBs were localized on disposable screen-printed carbon electrodes (SPCEs) under a magnetic field, and the product of the enzymatic reaction with the substrate was detected with differential-pulse voltammetry. In addition to magnetic separation assays, other competitive schemes (direct/indirect aptasensors performed on the SPCEs surface or using gold nanoparticles functionalized with the aptamer) were preliminary tested, optimized and compared. The magnetic aptasensor showed a linear response to OTA in the range 0.78-8.74 ng mL(-1) and a limit of detection of 0.07±0.01 ng mL(-1), and was accurately applied to extracts of certified and spiked wheat samples with an RSD lower than about 8%.

Electrochemical affinity biosensors for detection of mycotoxins: A review

This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago.

Ochratoxin A nanostructured electrochemical immunosensors based on polyclonal antibodies and gold nanoparticles coupled to the antigen

Competitive electrochemical immunosensors, based on disposable screen-printed electrodes (SPCEs), have been developed for the determination of the mycotoxin ochratoxin A (OTA). Two indirect immunoassays schemes were assessed using polyclonal antibodies and through the physical adsorption of OTA conjugated to albumin from bovine serum (OTA-BSA) and newly synthesized OTA-BSA bound to gold nanoparticles (OTA-BSA-AuNPs) onto the working electrode surface. After the competition step, detection was facilitated by a secondary aIgG antibody labelled with alkaline phosphatase and differential-pulse voltammetry using α-naphtyl-phosphate as substrate. The performance of the optimized immunosensors in terms of sensitivity, reproducibility and selectivity was studied. The linear working range of the described biosensors ranged between 0.9 and 9.0 ng mL−1 for the OTA-BSA based immunosensor and between 0.3 and 8.5 ng mL−1 for the gold nanostructured immunosensor, with a limit of detection (LOD) equal to 0.86 ng mL−1 (RSD = 10.6%) and 0.20 ng mL−1 (RSD = 8.0%) of OTA, respectively. The nanostructured immunosensor was applied to a certified wheat standard and a non-contaminated wheat material spiked with OTA, obtaining recoveries from about 104 ± 0.07% to 107 ± 0.08%. The influence of the wheat matrix on the analytical performance of the immunoassay was also studied.

Improved electrochemical competitive immunosensor for ochratoxin A with a biotinylated monoclonal antibody capture probe and colloidal gold nanostructuring

We report a sensitive electrochemical immunosensor for Ochratoxin A (OTA), which is a frequent mycotoxin contaminant in cereals and other kinds of agricultural commodities, based on a biotinylated monoclonal antibody against OTA (mAbOTA–bi) and the avidin–biotin coupling of the tracer extravidin–horseradish peroxidase (ea–HRP). The analytical performance has been improved with respect to a previously developed immunosensor based on a polyclonal antibody against OTA and a secondary aIgG antibody labeled with alkaline phosphatase as tracer. The immunosensor relies on indirect competitive assay format after the passive physical adsorption of the antigen conjugated to bovine serum albumin (OTA–BSA) or bound to gold nanoparticles (OTA–BSA–AuNPs), and the screen-printed technology for voltammetric (DPV) measurements. The new design is simpler (only one capture probe), requires less time (only one incubation time with antibody), and shows an increased slope at the linear range of the calibration plot due to higher affinity of the monoclonal antibody compared to the polyclonal one. The newly designed immunosensor has a linear dynamic range of 0.15 to 9.94 ng mL−1 of OTA (R ≥ 0.9900), lower detection limit (0.10 ng mL−1 OTA), and a variability between assays of about 10%. The immunosensor was validated with certified wheat samples after extraction of OTA in acetonitrile : water (6/4) (v/v), and allows the determination of OTA in concentration levels well below those permitted in cereals under European Union recommendations (3 ng g−1).

Rapid determination of recent cocaine use with magnetic particles-based enzyme immunoassays in serum, saliva, and urine fluids

Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h).

Molecularly Imprinted On-Line Solid-Phase Extraction Coupled with Fluorescence Detection for the Determination of Ochratoxin A in Wheat Samples

An automatic solid-phase extraction (SPE) system coupled with a fluorescence detector was developed for the sensitive determination of Ochratoxin A (OTA) in wheat at the ng mL−1 levels. Cross-linked molecularly imprinted polymers (MIPs) were synthesized as sorbents by a non-covalent approach using N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L)-phenylalanine (OTAm) as a mimic template, methacrylic acid as functional monomer, and ethylendimethacrylate as the cross-linker in chloroform porogenic solvent, polymerizing the mixture by thermal treatment at 60°C. These MIP polymers showed excellent affinity and specificity in the recognition of natural OTA mycotoxin compared to the results obtained with nonimprinted polymers. The polymers were slurry packed into a SPE column (V∼1.7 mL) and used in a SPE flowing system coupled to on-line molecular fluorescence detection (named MISPE-FLD) for the OTA determination. Methanol (MeOH), MeOH:H2O (1:9) + 1% acetic acid (AcOH), and MeOH:tributylamine (99:1) were tested as desorbing solvents. A dynamic binding capacity of 118 ± 9 ng of OTA (n = 3) was calculated for 45 mg of dry MIP particles. The described instrument demonstrated the cleanup of the matrix by the imprinted column and the simultaneous determination of OTA in wheat samples in the range 3–18 ng mL−1(LOD 1.2 ng mL−1 of OTA). The recoveries of reference wheat flour materials spiked with OTA were about 93 ± 9%. The MISPE-FLD instrument can be used for concentrations below the maximum levels of the EC regulatory limits for OTA in unprocessed cereals (5.0 µg kg−1) (EC Commission Regulation 2006).

A validated multi-channel electrochemical immunoassay for rapid fumonisin B1 determination in cereal samples

Fumonisin mycotoxins are natural contaminants of cereals mostly found in maize samples. Owing to their acute and chronic toxicity, very strict regulations have been imposed on the levels of fumonisin mycotoxins in cereal and cereal-based foods worldwide. We report a rapid direct competitive multi-channel electrochemical immunoassay for fumonisin B1 (FB1) determination in cereal and cereal-based foods. Monoclonal antibodies against FB1 were immobilized on the surface of modified magnetic beads (2.8 μm diameter), and the transduction was performed on an 8× screen-printed array allowing the multi-channel electrochemical detection of eight samples in parallel (amperometric detection, E = −0.35 V). The FB1 electrochemical immunoassay has a limit of detection of 0.58 ± 0.05 μg L−1 and EC50 = 4.34 ± 0.15 μg L−1, for FB1 concentration in the range of up to 54 μg L−1. The precision (within-day, between-days) was in the range of about 7–13% RSD. The optimized method (extraction and the electrochemical immunoassay determination) was successfully validated with maize certified reference materials at various levels of concentrations, and with an official AOAC liquid chromatography with fluorescence detection (HPLC-FLD) method (2001.04). We also compared our results with a commercial enzyme-linked immunosorbent analysis (ELISA) kit. Cross-reactivities with other mycotoxins (ochratoxins and deoxynivalenol) were less than 10%. The lifetime of immobilized antibodies was about 28 days. The reported FB1 multi-channel electrochemical immunoassay is a rapid and reliable alternative to HPLC methods.