Horseradish peroxidase-screen printed biosensors for determination of Ochratoxin A

This work summarizes the manufacturing procedure of Horseradish peroxidase (HRP) based biosensors for the determination of the mycotoxin Ochratoxin A (OTA). The biosensors have been fabricated using the single technology of screen-printing. That is to say, an HRP containing ink has been directly screen-printed onto carbon electrodes, which offers a higher rapidity and simplicity in the manufacturing process of biosensors for OTA determination. The formal redox potential of the Fe(III/II) moiety of HRP has been used to demonstrate the effective loading of enzyme into the ink. The chronoamperometric oxidation current registered has been successfully related to the concentration of OTA in solution from different samples, including beer ones. Under the optimum conditions of the experimental variables, precision in terms of reproducibility and repeatability has been calculated in the concentration range from 23.85 to 203.28 nM. A relative standard deviation for the slopes of 10% (n = 4) was obtained for reproducibility. In the case of repeatability, the biosensor retained a 30% of the initial sensitivity after the third calibration. The average capability of detection for 0.05% probabilities of false positive and negative was 26.77 ± 3.61 nM (α = 0.05 and β = 0.05, n = 3).     

Supramolecular solvent-based microextraction of ochratoxin A in raw wheat prior to liquid chromatography-fluorescence determination

A supramolecular solvent made up of reverse micelles of decanoic acid, dispersed in a continuous phase of THF:water, was proposed for the simple, fast and efficient microextraction of OTA in wheat prior to liquid chromatography-fluorescence determination. The method involved the stirring of 300 mg-wheat subsamples (particle size 50 μm) and 350 μL of supramolecular solvent for 15 min, subsequent centrifugation for 15 min and the direct quantitation of OTA in the extract, previous 5.7-fold dilution with ethanol/water/acetic acid (49.5/49.5/1), against solvent-based calibration curves. No clean-up of the extracts or solvent evaporation was needed. Interactions between the supramolecular solvent and major matrix components in the wheat (i.e. carbohydrates, lipids and proteins) were investigated. The reverse micelles in the extractant induced gluten flocculation but only in the coacervation region of lower analytical interest (i.e. at percentages of THF above 11%). The quantitation of OTA was interference-free. Representativity of the 300 mg-wheat subsamples was proved by analysing a reference material. OTA recoveries in wheat ranged between 84% and 95% and the precision of the method, expressed as relative standard deviation, was 2%. The quantitation limit of the method was 1.5 μg kg⁻¹ and was below the threshold limit established for OTA in raw cereals by EU directives (5.0 μg kg⁻¹). The method developed was validated by using a certified reference material and it was successfully applied to the determination of OTA in different wheat varieties from crops harvested in the South of Spain. OTA was not detected in any of the analysed samples. This method allows quick and simple microextraction of OTA with minimal solvent consumption, while delivering accurate and precise data.     

Modified magnetic nanoparticles in an electrochemical method for the ochratoxin A determination in Vitis vinifera red grapes tissues

This work described the development and characterization of an electrochemical method using square wave voltammetry (SWV) combined with the use of modified magnetic nanoparticles (MNPs), which had shown a rapid and sensitive determination of ochratoxin A (OTA) in wine grapes (Cabernet Sauvignon, Malbec and Syrah) post-harvest tissues. The wine grapes were inoculated with Aspergillus ochraceus to obtain OTA in artificially infected samples. The OTA was directly determined using square-wave voltammetry. The current obtained is directly proportional to the concentration of OTA present in the samples. This method has been used for OTA determination in wine grapes and it was validated against a commercial ELISA test kit. The limits of detection calculated for electrochemical detection and the ELISA were 0.02 and 1.9 μg kg⁻¹, respectively and the coefficients of variation for accuracy and precision dates were below 5.5%. This method promises to be suitable for the detection and quantification of OTA in apparently healthy fruits post-harvest for assuring safety and quality of food as well as consumer's health.     

An electrochemical immunosensor for ochratoxin A determination in wines based on a monoclonal antibody and paramagnetic microbeads

We report a direct competitive immunosensor for the rapid determination of ochratoxin A (OTA) in wine samples. Magnetic beads (1 ± 0.5 μm diameter) covered with streptavidin were functionalized with a monoclonal antibody against OTA, and then left to incubate in a solution of tracer (ochratoxin conjugated to the enzyme peroxidase) and a range of OTA concentrations (10(-4) to 1,000 ng mL(-1)). After washing and separation steps helped with a magnetic field, a volume of the dispersion was put on screen-printed electrodes under a magnet, and after adding the substrate the p-benzoquinone generated enzymatically was detected by differential-pulse voltammetry. Wine samples (2 mL) were easily prepared simply by adjusting to pH = 7.5 with diluted NaOH and by adding polyvinylpyrrolidone for complexing polyphenols, without any other clean-up or preconcentration steps. The limit of detection for detecting OTA in wines was of 0.11 ± 0.01 ng L(-1), well below the permitted content of the mycotoxin by the European Union (<2 ng mL(-1)). Spiked wines were subjected to immunosensor calibrations to study the matrix effects. OTA concentrations measured with the immunosensor were compared with those obtained by high-performance liquid chromatography coupled to fluorescence detection (AOAC official method 2001.01). The OTA levels from two red wines of "Campo de Borja", Spain, ranged from about 0.027 to 0.033 ng mL(-1) of OTA.     

Electrochemical immunosensor for rapid and sensitive determination of estradiol

This work describes the preparation of an electrochemical immunosensor for estradiol based on the surface modification of a screen printed carbon electrode with grafted p-aminobenzoic acid followed by covalent binding of streptavidin (Strept) and immobilization of biotinylated anti-estradiol (anti-estradiol-Biotin). The hormone determination was performed by applying a competitive immunoassay with peroxidase-labelled estradiol (HRP–estradiol) and measurement of the amperometric response at −200 mV using hydroquinone (HQ) as redox mediator. The calibration curve for estradiol exhibited a linear range between 1 and 250 pg mL⁻¹ (r = 0.990) and a detection limit of 0.77 pg mL⁻¹ was achieved. Cross-reactivity studies with other hormones related with estradiol at physiological concentration levels revealed the practical specificity of the developed method for estradiol. A good reproducibility, with RSD = 5.9% (n = 8) was also observed. The operating stability of a single bioelectrode modified with anti-estradiol-Biotin-Strept was nine days when it was stored at 8 °C under humid conditions between measurements. The developed immunosensor was applied to the analysis of certified serum and spiked urine samples with good results.     

A high-performance liquid-chromatographic method for the determination of ochratoxin a in human plasma

Summary A high-performance liquid-chromatographic method is described for the quantitative determination of the mycotoxin ochratoxin A (OTA) in human plasma. The assay involves extraction with chloroform and sodium bicarbonate then HPLC with fluorescence detection. The method was validated in terms of selectivity, recovery, linearity, precision (within-day and between-day variability), accuracy, detection and quantification limits, and the stability of OTA in plasma and treated samples. The limit of detection was 0.4 ng mL⁻¹ of OTA in methanol, corresponding to 0.52 ng ml⁻¹ OTA in plasma. This assay was successfully applied for the determination of OTA levels in human plasma.     

Ionic liquid-based miniature electrochemical sensors for the voltammetric determination of catecholamines

Screen-printed electrodes modified with carbon paste that consisted of graphite powder dispersed in ionic liquids (IL) were used for the electrochemical determination of dopamine, adrenaline and dobutamine in aqueous solutions by means of cyclic voltammetry. The IL plays a dual role in modifying compositions, acting both as a binder and chemical modifier (ion-exchanger); ion-exchange analyte pre-concentration increases analytical signal and improves the sensitivity. Calibration graphs are linear in concentration range 3.9 × 10⁻⁶ to 1.0 × 10⁻⁴ M (dopamine), 2.9 × 10⁻⁷ to 1.0 × 10⁻⁴ M (adrenaline) and 1.7 × 10⁻⁷ to 1.0 × 10⁻⁴ M (dobutamine); detection limits are (1.2 ± 0.1) × 10⁻⁶, (1.3 ± 0.1) × 10⁻⁷ and (5.3 ± 0.1) × 10⁻⁸ M, respectively. Using an additive of Co (III) tetrakis-(tert-butyl)-phthalocyanine leads to the increase of signal and lowering detection limit. Some practical advises concerning both the sensor design and selectivity of catecholamine determination are provided.     

Comparison of methods for the determination of ochratoxin A in wine

Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS-MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC-MS-MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.     

Determination of ochratoxin A content in wheat bread samples collected from the Algarve and Bragança regions, Portugal: Winter 2007

Ochratoxin A (OTA) is a mycotoxin produced by fungi such as Penicillium verrucosum and Aspergillium spp. and has been found to have a variety of potentially deadly toxic effects. The favoured substrate for fungal growth and OTA production appears to be cereals and flour-based products, including bread. Due to the dietary relevance of bread for the Portuguese population, it is imperative that its OTA content remains well within safe quantities. As such, bread samples collected from commercial surfaces across the Algarve region and from the city of Bragança during the winter of 2007 were tested for OTA through extraction with immunoaffinity columns and quantification by liquid chromatography coupled with fluorescence detection. Although OTA content was found to be above the limit of quantification in approximately 60% and 50% of the analysed samples from Algarve and Bragança, respectively, all samples were found to be compliant with European Commission. OTA content reached maximums of 0.49 ng/g in Algarve and 0.43 ng/g in Bragança, and was thus below the maximum limit established by European legislation for bread of 3 ng/g. The results of the present study put the estimated daily intake of OTA from bread at approximately 0.26 ng/kg bw/day in Algarve and 0.38 ng/kg bw/day in Bragança, circa 1.5% and 2.0% of the TDI established by either the EFSA or the FAO/WHO, or over 4.5% and 6.5% if we consider the FAO/WHO advised bread consumption of 250 g/day. These results seem to suggest that, in these two Portuguese regions, OTA contamination is well under control and unlikely to represent a threat to consumer health.     

A normal phase HPTLC method for the quantitative determination of ochratoxin A in rice

Summary A previously published [1] liquid chromatographic method for the determination of ochratoxin A in corn, barley and kidney has been modified for application to parboiled rice with quantification by high performance thin layer chromatography (HPTLC). The method has been validated by spiking uncontaminated extracts of rice with ochratoxin A over the range 0 to 198 g kg^–1. The proposed method has some significant advantages over the current AOAC method [2], especially for determining low levels of ochratoxin A in parboiled rice.     

Ultrasensitive one-step rapid detection of ochratoxin A by the folding-based electrochemical aptasensor

A one-step electrochemical aptasensor using the thiol- and methylene blue- (MB-) dual-labeled aptamer modified gold electrode for determination of ochratoxin A (OTA) was presented in this research. The aptamer against OTA was covalently immobilized on the surface of the electrode by the self-assembly effect and used as recognition probes for OTA detection by the binding induced folding of the aptamer. Under the optimal conditions, the developed electrochemical aptasensor demonstrated a wide linear range from 0.1 pg mL⁻¹ to 1000 pg mL⁻¹ with the limit of detection (LOD) of 0.095 pg mL⁻¹, which was an extraordinary sensitivity compared with other common methods for OTA detection. Moreover, as a practical application, this proposed electrochemical aptasensor was used to monitor the OTA level in red wine samples without any special pretreatment and with satisfactory results obtained. Study results showed that this electrochemical aptasensor could be a potential useful platform for on-site OTA measurement in real complex samples.     

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase.In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection.Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.     

Aptamer-targeted magnetic nanospheres as a solid-phase extraction sorbent for determination of ochratoxin A in food samples

A sorbent based on the aptamer for ochratoxin A was immobilized onto magnetic nanospheres (MNS) and used to develop a magnetic solid-phase extraction procedure to clean up food samples in conjunction with high-performance liquid chromatography separation and fluorescence detection. Specific retention of ochratoxin A by the sorbent was demonstrated, and the capacity of the MNS-aptamer sorbent was determined. The efficacy of this new approach was successfully evaluated through comparison with solid-phase extraction on commercial C18 cartridge. Several different food samples fortified in the range of with 2.5-50 μg/kg yielded mean recoveries from 67% to 90%, respectively. Finally, this oligosorbent was applied to the selective extraction of ochratoxin A from unfortified food samples.     

Recent developments in the field of screen-printed electrodes and their related applications

The development of analytical methods that respond to the growing need to perform rapid ‘in situ’ analyses shows disposable screen-printed electrodes (SPEs) as an alternative to the traditional electrodes. This review presents recent developments in the electrochemical application of disposable screen-printed sensors, according to the types of materials used to modify the working electrode. Therefore, unmodified SPE, film-modified SPE, enzyme-modified SPE and antigen/antibody-modified SPE are described. Applications are included where available.     

CYP450 biosensors based on screen-printed carbon electrodes for the determination of cocaine

A new electrochemical method has been described and characterized for the determination of cocaine using screen-printed biosensors. The enzyme cytochrome P450 was covalently attached to screen-printed carbon electrodes. Experimental design methodology has been performed to optimize the pH and the applied potential, both variables that have an influence on the chronoamperometric determination of the drug. This method showed a reproducibility of 3.56% (n = 4) related to the slopes of the calibration curves performed in the range from 19 up to 166 nM. It has been probed the used of this kind of biosensors in the determination of cocaine in street samples, with an average capability of detection of 23.05 ± 3.53 nM (n = 3, α = β = 0.05).     

Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine

Gel-based and membrane-based flow-through immunoassay formats were investigated for rapid ochratoxin A (OTA) detection in red wine. The flow-through set-up consisted of an antibody containing gel or membrane placed at the bottom of a standard solid-phase extraction column (i.e. the flow-through column), combined with a clean-up column. Different clean-up methods were studied for red wine clarification and purification. The optimal method consisted of passing wine, diluted with an aqueous solution containing 1% polyethylene glycol (PEG 6000) and 5% sodium hydrogencarbonate, through strong anion exchange (SAX) silica. An immunoassay for OTA detection in red wine was optimized and a cut-off level at 2 μg L⁻¹ according to EU legislation was achieved with both formats. A more significant colour difference between blank and spiked samples was observed for the gel-based assay making this superior to the membrane-based assay. The proposed rapid gel-based test was compared with a standard immunoaffinity column - high-performance liquid chromatography - fluorescent detection (IAC-HPLC-FLD) method and a good correlation of the results was obtained for naturally contaminated wine samples.     

Validation of a liquid chromatography method for the simultaneous quantification of ochratoxin A and its analogues in red wines

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the simultaneous quantification of ochratoxin A (OTA) and its analogues (ochratoxin B (OTB), ochratoxin C (OTC) and methyl ochratoxin A (MeOTA)) in red wine at trace levels is described. Before their analysis by HPLC-FLD, ochratoxins were extracted and purified with immunoaffinity columns from 50 mL of red wine at pH 7.2. Validation of the analytical method was based on the following parameters: selectivity, linearity, robustness, limits of detection and quantification, precision (within-day and between-day variability), recovery and stability. The limits of detection (LOD) in red wine were established at 0.16, 0.32, 0.27 and 0.17 ng L(-1) for OTA, OTB, MeOTA and OTC, respectively. The limit of quantification (LOQ) was established as 0.50 ng L(-1) for all of the ochratoxins. The LOD and LOQ obtained are the lowest found for OTA in the reference literature up to now. Recovery values were 93.5, 81.7, 76.0 and 73.4% for OTA, OTB, MeOTA and OTC, respectively. For the first time, this validated method permits the investigation of the co-occurrence of ochratoxins A, B, C and methyl ochratoxin A in 20 red wine samples from Spain.     

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA–BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL⁻¹, and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74–110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL⁻¹ for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R ² = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples.     

Occurrence of ochratoxin A in Turkish wines

A total of 95 wine samples including 34 white, 10 rosé and 51 red wines originating from four different Turkish areas were analysed for ochratoxin A (OTA). An analytical method based on immunoaffinity column (IAC) for clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD) was used to determine OTA in wines. The limit of detection (LOD) was estimated as 0.006 ng ml^− 1 for white wine and 0.010 ng ml^− 1 for rosé and red wines. The limit of quantification (LOQ) was estimated as 0.020 ng ml^− 1 in white wine and 0.030 ng ml^− 1 in rosé and red wines. Recovery experiments were carried out with spiked samples in the range 0.1–1 ng ml^− 1 of OTA. The average OTA recoveries from spiked white wine samples varied from 79.43% to 85.07%; while the mean recoveries for rosé and red wine samples were in the range of 77.48–83.96% and 76.61–83.55%, respectively. OTA was detected in 82 (86%) wine samples at levels of < 0.006–0.815 ng ml^− 1, which were below the maximum allowable limit established by the European Community. The mean OTA concentration in red wines was slightly higher than in white and rosé wines. Furthermore, our data indicate that the geographic region of origin has strong influence on OTA level for white, rosé and red wines: wines originating from Thrace (n = 44, mean = 0.158 ng ml^− 1) and Aegean (n = 28, mean = 0.060 ng ml^− 1) regions of Turkey were more contaminated with OTA compared with wines originating from central (n = 15, mean = 0.027 ng ml⁻¹) and east Anatolia (n = 8, mean = 0.027 ng ml^− 1) areas. This study showed that the occurrence of OTA in Turkish wines is high, but at levels that probably leads to a non-significant human exposure to OTA by consumption of wines.     

Occurrence of ochratoxin A in bread consumed in Morocco

One hundred samples of commercial bread purchased from January to October (2006) from retail bakeshops in five different cities (Rabat, Témara, Salé, Casablanca and Meknès) in Morocco were surveyed for the presence of ochratoxin A (OTA) using liquid chromatography coupled to fluorescence detection. The identification of OTA in positive bread samples was confirmed by methyl ester derivatization. Analytical results showed that forty eight (48%) samples were positive with OTA greater than the limit of quantification (LOQ = 0.051 ng/g). Levels of OTA in positive samples ranged between 0.14 and 149 ng/g. The average contamination of bread samples with OTA was 13 ± 1.5 ng/g. The highest frequency of positive samples (61.5%) and the most contaminated sample (149 ng/g) were found in bread commercialized in the Casablanca area. Twenty six of the positive samples exceeded the maximum level of 3 ng/g set by European regulations for OTA in cereals and derivatives. Based in the results presented in this study, the estimated daily intake of OTA from bread was 126 ng/kg bw/day. The present paper is the first ever drafted on the natural occurrence of OTA in bread consumed in Morocco. Data on the daily intake of OTA by the Moroccan population are also estimated for the first time.