Extracting mycotoxins from edible vegetable oils by using green, ecofriendly deep eutectic solvents.

In this work, we developed an environmentally friendly liquid-liquid microextraction method using a natural deep eutectic solvent in combination with liquid chromatography for the simultaneous determination of four mycotoxins (deoxynivalenol, alternariol, ochratoxin A and zearalenone) in edible vegetable oils. A chemometric approach assessed the effect of the operational parameters on the mycotoxin extraction efficiency. The extracts were analyzed by HPLC coupled with a diode array and fluorescence detector. The optimum NADES composition resulted in the highest extraction recoveries, and it was applied to coextract the target mycotoxins in several types of edible vegetable oils without using hazardous solvents or requiring further clean-up. The limits of detection ranged from 0.07 to 300 µg kg-1, and recoveries were close to 100%, except for zearalenone (viz. 35%), with relative standard deviations below 9% in all cases. The proposed method was validated following the European Commission 2002/657/EC and 2006/401/EC.

Mycotoxin extraction from edible insects with natural deep eutectic solvents: a green alternative to conventional methods.

Edible insects are widely consumed in Africa, Asia, Oceania and Latin America, but less commonly so in Western countries. Since the turn of the millennium, however, entomophagy has aroused growing interest worldwide in response to the increasing scarcity of food resources. In fact, edible insects can be a source of high-quality protein, and also of fat, energy, minerals and vitamins. However, the lack of regulatory guidelines for microbiologically or chemically hazardous agents potentially present in these new foods (e.g., mycotoxins) may make their consumption unsafe. In this work, we developed an environmentally friendly analytical method using natural deep eutectic solvents (NADES or natural DES) in combination with ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of six mycotoxins of great concern owing to their toxic effects on humans and animals (namely, fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, ochratoxin A and mycophenolic acid) in insect-based food products. The target mycotoxins were co-extracted from cricket flour by using the optimum DES composition (namely, a mixture of choline chloride and urea, in a 1:2 mole ratio, containing 15% water which resulted in the highest extraction recoveries for all toxins). An experimental design method (Fractional Factorial Design (FFD) was used to examine the influence of the operational variables DES volume and water content, amount of sample, extraction time and extraction temperature on the extraction efficiency for each mycotoxin. Under optimum conditions, extraction recoveries were close to 100% except for fumonisin B2 (70%) and T-2 toxin (50%), with relative standard deviations (RSDs) below 13% in all cases. The proposed NADES-UHPLC-MS/MS method was validated in accordance with the European Commission 2002/657/EC and 2006/401/EC decisions, and used to determine the target compounds in cricket flour, silkworm pupae powder and black cricket powder.

Rapid determination of Alternaria mycotoxins in tomato samples by pressurised liquid extraction coupled to liquid chromatography with fluorescence detection.

A sensitive and reliable method using pressurised liquid extraction (PLE) followed by molecularly imprinted solid phase extraction (MISPE) and high performance liquid chromatography with fluorescence detection (HPLC-FLD) has been developed for the analysis of alternariol (AOH) and alternariol monomethyl ether (AME) in tomato samples. Influence of several extraction parameters that affect PLE efficiency were evaluated for the simultaneous extraction of both mycotoxins in the selected samples. AOH and AME were optimally extracted using MeOH/water (25:75, v/v) at 70°C as solvent, a pressure of 1000 psi and a single extraction cycle. The resulting PLE extracts were pre-concentrated by molecularly imprinted solid phase extraction (MISPE) cartridges followed of analysis by HPLC with fluorescence detection (λexc = 258, λem = 440 nm). The proposed method was applied to the analysis of AOH and AME in fortified tomato samples (20-72 µg· kg-1) with recoveries of 84-97% (RSD < 8%, n = 6) for AOH and 67-91% (RSD < 13%, n = 6) for AME. The detection limit for AOH and AME were 7 and 15 µg· kg-1, respectively. The ensuing PLE-MISPE-HPLC-FLD method was validated for the analysis of both mycotoxins in tomato samples in accordance with European Commission Decision 2002/657/EC.

Biosensing Based on Nanoparticles for Food Allergens Detection.

Food allergy is one of the major health threats for sensitized individuals all over the world and, over the years, the food industry has made significant efforts and investments to offer safe foods for allergic consumers. The analysis of the concentration of food allergen residues in processing equipment, in raw materials or in the final product, provides analytical information that can be used for risk assessment as well as to ensure that food-allergic consumers get accurate and useful information to make their food choices and purchasing decisions. The development of biosensors based on nanomaterials for applications in food analysis is a challenging area of growing interest in the last years. Research in this field requires the combined efforts of experts in very different areas including food chemistry, biotechnology or materials science. However, the outcome of such collaboration can be of significant impact on the food industry as well as for consumer’s safety. These nanobiosensing devices allow the rapid, selective, sensitive, cost-effective and, in some cases, in-field, online and real-time detection of a wide range of compounds, even in complex matrices. Moreover, they can also enable the design of novel allergen detection strategies. Herein we review the main advances in the use of nanoparticles for the development of biosensors and bioassays for allergen detection, in food samples, over the past few years. Research in this area is still in its infancy in comparison, for instance, to the application of nanobiosensors for clinical analysis. However, it will be of interest for the development of new technologies that reduce the gap between laboratory research and industrial applications.

Furfural Determination with Disposable Polymer Films and Smartphone-Based Colorimetry for Beer Freshness Assessment.

We have developed disposable color-changing polymeric films for quantification of furfural-a freshness indicator-in beer using a smartphone-based reader. The films are prepared by radical polymerization of 4-vinylaniline, as a furfural-sensitive indicator monomer, 2-hydroxymethyl methacrylate as a comonomer, and ethylene dimethyl methacrylate (EDMA) as a cross-linker. The sensing mechanism is based on the Stenhouse reaction in which aniline and furfural react in acidic media with the generation of a deep red cyanine derivative, absorbing at 537 nm, which is visible to the naked eye. The colorimetric response has been monitored using either a portable fiber-optic spectrophotometer or the built-in camera of a smartphone. Under the optimized conditions, a linear response to furfural in beer was obtained in the 39 to 500 µg L(-1) range, with a detection limit of 12 µg L(-1), thus improving the performance of other well-established colorimetric or chromatographic methods. The novel films are highly selective to furfural, and no cross-reactivity has been observed from other volatile compounds generated during beer aging. A smartphone application (app), developed for Android platforms, measures the RGB color coordinates of the sensing membranes after exposure to the analyte. Following data processing, the signals are converted into concentration values by preloaded calibration curves. The method has been applied to determination of furfural in pale lager beers with different storage times at room temperature. A linear correlation (r > 0.995) between the storage time and the furfural concentration in the samples has been confirmed; our results have been validated by HPLC with diode-array detection.

Development of magnetic molecularly imprinted polymers for selective extraction: determination of citrinin in rice samples by liquid chromatography with UV diode array detection.

In this work, we report the synthesis of novel magnetic molecularly imprinted polymers (m-MIPs) and their application to the selective extraction of the mycotoxin citrinin (CIT) from food samples. The polymers were prepared by surface imprinting of Fe3O4 nanoparticles, using 2-naphtholic acid (2-NA) as template molecule, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea and methacrylamide as functional monomers and ethyleneglycol dimethacrylate as cross-linker. The resulting material was characterized by transmission electron microscopy (TEM), and X-ray diffraction (XRD) and Fourier transform infrared spectroscopies (FT-IR). The polymers were used to develop a solid-phase extraction method (m-MISPE) for the selective recovery of CIT from rice extracts prior to its determination by HPLC with UV diode array detection. The method involves ultrasound-assisted extraction of the mycotoxin from rice samples with (7:3, v/v) methanol/water, followed by sample cleanup and preconcentration with m-MIP. The extraction (washing and elution) conditions were optimized and their optimal values found to provide CIT recoveries of 94-98 % with relative standard deviations (RSD) less than 3.4 % (n = 3) for preconcentrated sample extracts (5 mL) fortified with the analyte at concentrations over the range 25-100 µg kg(-1). Based on the results, the application of the m-MIPs facilitates the accurate and efficient determination of CIT in rice extracts.

Application of bacteriophages in sensor development.

Bacteriophage-based bioassays are a promising alternative to traditional antibody-based immunoassays. Bacteriophages, shortened to phages, can be easily conjugated or genetically engineered. Phages are robust, ubiquitous in nature, and harmless to humans. Notably, phages do not usually require inoculation and killing of animals; and thus, the production of phages is simple and economical. In recent years, phage-based biosensors have been developed featuring excellent robustness, sensitivity, and selectivity in combination with the ease of integration into transduction devices. This review provides a critical overview of phage-based bioassays and biosensors developed in the last few years using different interrogation methods such as colorimetric, enzymatic, fluorescence, surface plasmon resonance, quartz crystal microbalance, magnetoelastic, Raman, or electrochemical techniques.

Molecularly imprinted hydrogels as functional active packaging materials.

This paper describes the synthesis of novel molecularly imprinted hydrogels (MIHs) for the natural antioxidant ferulic acid (FA), and their application as packaging materials to prevent lipid oxidation of butter. A library of MIHs was synthesized using a synthetic surrogate of FA, 3-(4-hydroxy-3-methoxyphenyl)propionic acid (HFA), as template molecule, ethyleneglycol dimethacrylate (EDMA) as cross-linker, and 1-allylpiperazine (1-ALPP) or 2-(dimethylamino)ethyl methacrylate (DMAEMA), in combination with 2-hydroxyethyl methacrylate (HEMA) as functional monomers, at different molar concentrations. The DMAEMA/HEMA-based MIHs showed the greatest FA loading capacity, while the 1-ALLP/HEMA-based polymers exhibited the highest imprinting effect. During cold storage, FA-loaded MIHs protected butter from oxidation and led to TBARs values that were approximately half those of butter stored without protection and 25% less than those recorded for butter covered with hydrogels without FA, potentially extending the shelf life of butter. Active packaging is a new field of application for MIHs with great potential in the food industry.

Aluminum Nanoholes for Optical Biosensing.

Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that make them highly suitable for (bio)sensing applications. Either as efficient light scattering centers for surface plasmon excitation or metal-clad optical waveguides, they are able to form strongly localized optical fields that can effectively interact with biomolecules and/or nanoparticles on the nanoscale. As the metal of choice, aluminum exhibits good optical and electrical properties, is easy to manufacture and process and, unlike gold and silver, its low cost makes it very promising for commercial applications. However, aluminum has been scarcely used for biosensing purposes due to corrosion and pitting issues. In this short review, we show our recent achievements on aluminum nanohole platforms for (bio)sensing. These include a method to circumvent aluminum degradation--which has been successfully applied to the demonstration of aluminum nanohole array (NHA) immunosensors based on both, glass and polycarbonate compact discs supports--the use of aluminum nanoholes operating as optical waveguides for synthesizing submicron-sized molecularly imprinted polymers by local photopolymerization, and a technique for fabricating transferable aluminum NHAs onto flexible pressure-sensitive adhesive tapes, which could facilitate the development of a wearable technology based on aluminum NHAs.

Passivated aluminum nanohole arrays for label-free biosensing applications.

We report the fabrication and performance of a surface plasmon resonance aluminum nanohole array refractometric biosensor. An aluminum surface passivation treatment based on oxygen plasma is developed in order to circumvent the undesired effects of oxidation and corrosion usually found in aluminum-based biosensors. Immersion tests in deionized water and device simulations are used to evaluate the effectiveness of the passivation process. A label-free bioassay based on biotin analysis through biotin-functionalized dextran-lipase conjugates immobilized on the biosensor-passivated surface in aqueous media is performed as a proof of concept to demonstrate the suitability of these nanostructured aluminum films for biosensing.

Dextran-lipase conjugates as tools for low molecular weight ligand immobilization in microarray development.

The development of effective array biosensors relies heavily on careful control of the density of surface-immobilized ligands on the transducing platform. In this paper we describe the synthesis of new dextran-lipase conjugates for use in immobilizing low molecular weight haptens onto glass planar waveguides for immunosensor development. The conjugates were synthesized by immobilizing bacterial thermoalkalophilic lipases (Geobacillus thermocatenulatus lipase 2, BTL2) on agarose macroporous beads, followed by covalent coupling to dextran networks of variable molecular weight (1500-40000). The chimeras were immobilized via nonspecific hydrophobic interactions onto glass planar waveguides modified with 1,1,1,3,3,3-hexamethyldisilazane to obtain highly ordered and homogeneous molecular architectures as confirmed by atomic force microscopy. Microcystin LR (MCLR) was covalently bound to the dextran-BTL2 conjugates. The usefulness of this approach in immunosensor development was demonstrated by determining amounts of MCLR down to a few picograms per liter with an automated array biosensor and evanescent wave excitation for fluorescence measurements of attached DyLight649-labeled secondary antibody. Modifying BTL2 with dextrans of an increased molecular weight (>6000) provided surfaces with an increased loading capacity that was ascribed to the production of three-dimensional surfaces by the effect of analyte binding deep in the volume, leading to expanded dynamic ranges (0.09-136.56 ng L(-1)), lower limits of detection (0.007 ± 0.001 ng L(-1)), and lower IC50 values (4.4 ± 0.7 ng L(-1)). These results confirm the effectiveness of our approach for the development of high-performance biosensing platforms.

Synthesis, spectroscopic, and analyte-responsive behavior of a polymerizable naphthalimide-based carboxylate probe and molecularly imprinted polymers prepared thereof.

A naphthalimide-based fluorescent indicator monomer 1 for the integration into chromo- and fluorogenic molecularly imprinted polymers (MIPs) was synthesized and characterized. The monomer was equipped with a urea binding site to respond to carboxylate-containing guests with absorption and fluorescence changes, namely a bathochromic shift in absorption and fluorescence quenching. Detailed spectroscopic analyses of the title compound and various models revealed the signaling mechanism. Titration studies employing benzoate and Z-L-phenylalanine (Z-L-Phe) suggest that indicator monomers such as the title compound undergo a mixture of deprotonation and complex formation in the presence of benzoate but yield hydrogen-bonded complexes, which are desirable for the molecular imprinting process, with weakly basic guests like Z-l-Phe. Compound 1 could be successfully employed in the synthesis of monolithic and thin-film MIPs against Z-L-Phe, Z-L-glutamic acid, and penicillin G. Chromatographic assessment of the selectivity features of the monoliths revealed enantioselective discrimination and clear imprinting effects. Immobilized on glass coverslips, the thin-film MIPs of 1 displayed a clear signaling behavior with a pronounced enantioselective fluorescence quenching dependence and a promising discrimination against cross-analytes.

Multiresidue determination of ultratrace levels of fluoroquinolone antimicrobials in drinking and aquaculture water samples by automated online molecularly imprinted solid phase extraction and liquid chromatography.

The present work describes the development of a sensitive and highly selective innovative method for the simultaneous detection of six fluoroquinolone (FQ) antimicrobials (enrofloxacin, ciprofloxacin, norfloxacin, levofloxacin, danofloxacin, and sarafloxacin) in water samples. This detection is based on online solid phase extraction, coupled to liquid chromatography (LC), using for the first time tailor-made molecularly imprinted microspherical polymer particles prepared via precipitation polymerization. Various parameters affecting the extraction efficiency of the polymer have been optimized to reduce nonspecific interactions and to achieve selective uptake of the antibiotics from real samples. The method shows good recoveries ranging between 62% and 102% (V = 25 mL) for the different FQs tested and excellent interday and intraday precision with relative standard deviation (RSD) values between 2-5% and 2-6%, respectively. The detection limits were between 1-11 ng L(-1) (drinking water) and 1-12 ng L(-1) (fish farm water) when 25 mL samples were processed. The polymer showed selectivity for FQs containing a piperazine moiety whereas no retention was found for other antibiotics or nonrelated compounds. The method has been applied to the analysis of trace amounts of the FQs tested in drinking and fish farm water samples with excellent recoveries (>91%) and good precision (RSDs <5%).

An SPR biosensor for the detection of microcystins in drinking water.

A surface plasmon resonance (SPR) biosensor for the detection of microcystins (MCs) in drinking water has been developed. Several assay formats have been evaluated. The selected format is based on a competitive inhibition assay, in which microcystin-LR (MCLR) has been covalently immobilized onto the surface of an SPR chip functionalized with a self-assembled monolayer. The influence of several factors affecting sensor performance, such as the nature and concentration of the antibody, the composition of the carrier buffer, and the blocking and regeneration solutions, has been evaluated. The optimized SPR biosensor provides an IC(50) 0.67 ± 0.09 µg L(-1), a detection limit of 73 ± 8 ng L(-1), and a dynamic range from 0.2 to 2.0 µg L(-1) for MCLR. Cross-reactivity to other related MCs, such as microcystin-RR (88%) and microcystin-YR (94%), has also been measured. The SPR biosensor can perform four simultaneous determinations in 60 min, and each SPR chip can be reused for at least 40 assay-regeneration cycles without significant binding capacity loss. The biosensor has been successfully applied to the direct analysis of MCLR in drinking water samples, below the provisional guideline value of 1 µg L(-1) established by the World Health Organization for drinking water.

An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples.

Antibiotics are a class of pharmaceuticals that are of great interest due to the large volumes of these substances that are consumed in both human and veterinary medicine, and due to their status as the agents responsible for bacterial resistance. They can be present in foodstuffs and in environmental samples as multicomponent chemical mixtures that exhibit a wide range of mechanisms of action. Moreover, they can be transformed into different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher ecotoxicities and risks to human health than those of the individual compounds. Therefore, there is growing interest in the availability of multiclass methods for the analysis of antimicrobial mixtures in environmental and food samples at very low concentrations. Liquid chromatography (LC) has become the technique of choice for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS(2)). However, due to the complexity of the matrix, in most cases an extraction step for sample clean-up and preconcentration is required before analysis in order to achieve the required sensitivities. This paper reviews the most recent developments and applications of multiclass antimicrobial determination in environmental and food matrices, emphasizing the practical aspects of sample preparation for the simultaneous extraction of antimicrobials from the selected samples. Future trends in the application of LC-MS-based techniques to multiclass antibiotic analysis are also presented.

Microalgae fiber optic biosensors for herbicide monitoring using sol-gel technology.

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol-gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol-gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 microg L(-1)) and the broadest dynamic calibration range (19-860 microg L(-1)) with IC(50) 125+/-14 microg L(-1). Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.

Water-compatible molecularly imprinted polymer for the selective recognition of fluoroquinolone antibiotics in biological samples.

A novel water-compatible molecularly imprinted polymer (MIP), prepared with enrofloxacin (ENR) as the template, has been optimised for the selective extraction of fluoroquinolone antibiotics in aqueous media. The results of a morphological characterisation and selectivity tests of the polymer material for ENR and related derivatives are reported. High affinity for the piperazine-based fluoroquinolones marbofloxacin, ciprofloxacin, norfloxacin and ofloxacin was observed, whereas no retention was found for nonrelated antibiotics. Various parameters affecting the extraction efficiency of the polymer have been optimised to achieve selective extraction of the antibiotics from real samples and to reduce nonspecific interactions. These findings resulted in a MISPE/HPLC-FLD method allowing direct extraction of the analytes from aqueous samples with a selective wash using just 50% (v/v) organic solvent. The method showed excellent recoveries and precision when buffered urine samples fortified at five concentration levels (25-250 ng mL(-1) each) of marbofloxacin, ciprofloxacin, norfloxacin, enrofloxacin and sarafloxacin were tested (53-88%, RSD 1-10%, n = 3). Moreover, the biological matrix of the aqueous samples did not influence the preconcentration efficiency of the fluoroquinolones on the MIP cartridges; no significant differences were observed between the recovery rates of the antibiotics in buffer and urine samples. The detection limits of the whole process range between 1.9 and 34 ng mL(-1) when 5-mL urine samples are processed. The developed method has been successfully applied to preconcentration of norfloxacin in urine samples of a medicated patient, demonstrating the ability of the novel MIP for selective extraction of fluoroquinolones in urine samples.