Cristina Giovannoli

Molecularly imprinted solid-phase extraction sorbent for the clean-up of chlorinated phenoxyacids from aqueous samples.

A molecularly imprinted polymer (MIP) was synthesized using the herbicide 2,4,5-trichlorophenoxyacetic acid as a template, 4-vinylpyridine as an interacting monomer, ethylendimethacrylate as a cross-linker and a methanol-water mixture as a porogen. The binding properties and the selectivity of the polymer towards the template were investigated by frontal and zonal liquid chromatography. The polymer was used as a solid-phase extraction material for the clean-up of the template molecule and some related herbicides (2,4-dichlorophenoxyacetic acid, fenoprop, dichlorprop) from river water samples at a concentration level of ng/ml with quantitative recoveries comparable with those obtained with a traditional C18 reversed-phase column when analyzed by capillary electrophoresis. The results obtained show that the MIP-based approach to the solid-phase extraction is comparable with the more traditional solid-phase extraction with C18 reversed-phase columns in terms of recovery, but it is superior in terms of sample clean-up.

Lateral-flow immunoassays for mycotoxins and phycotoxins: a review

AbstractNatural toxin (for example mycotoxin and phycotoxin) contamination of food is of safety and economic concern, so much effort is devoted to the development of screening methods which enable the toxins to be continuously and widely monitored in food and feed. More generally speaking, rapid and non-instrumental assays for detection of a variety of food contaminants are generating ever-increasing scientific and technological interest because they enable high-throughput, economical, on-site monitoring of such contaminants. Among rapid methods for first-level screening of food contaminants, lateral-flow immunoassay (LFIA), also named immunochromatographic assay or immune-gold colloid immunoassay, has recently attracted scientific and industrial interest because of its attractive property of enabling very rapid, one-step, in-situ analysis. This review focuses on new aspects of the development and optimization of lateral-flow devices for mycotoxin and phycotoxin detection, including strategies for management of matrix interference and, particularly, for investigation of the improvements achieved by signal-enhancing strategies or by application of non-gold nanoparticle signal reporters. Figure 1Competitive lateral flow immunoassay for myco- or phycotoxin: the Test zone is formed by adsorbing a conjugate of the target compound (toxin); Control zone is formed by anti-species antibodies (white), reporters are specific (anti-toxin antibodies, black) and non-specific (grey) antibodies labelled with gold nanoparticles (GNP). Focalization of GNP-labelled antibodies determines a visible/detectable colour appearance on both the Test and Control lines, which can be related to analyte amount in a liquid sample.

A connection between the binding properties of imprinted and nonimprinted polymers: a change of perspective in molecular imprinting.

In the current paradigm for molecular imprinting, the imprinted binding sites exist as a consequence of the polymerization process around templates, and the properties of nonimprinted polymers (NIPs) have largely been overlooked. Thus, nothing can be affirmed a priori concerning the binding properties of NIPs. We propose an alternative view where the imprinting effect is due to the presence of a template molecule that enhances the pre-existing binding properties of a polymer. If a NIP shows no binding properties toward a target molecule, the corresponding imprinted polymer (MIP) will show a weak imprinting effect. On the other hand, if a NIP shows binding properties toward a target molecule, the corresponding MIP will show a significant imprinting effect. To verify this hypothesis, we prepared a 96-member combinatorial polymeric library in the absence of any template molecule. This library was screened for several potential ligands, and with no exceptions, the composition of the best-binding NIP produced a MIP with excellent binding properties, whereas a low-binding NIP formulation produced a MIP with comparable low binding. To validate these results, the binding properties toward naproxen and ibuprofen were measured for two combinatorial libraries of polymers prepared in the presence (MIP library) and the absence (NIP library) of the template molecule. The experiments results showed a correlation between the apparent affinity constants measured for the NIP and MIP libraries, confirming the proposed hypothesis. Moreover, for closely related molecules, it was shown that binding selectivity is an emergent property derived from the imprinting process and not a property of NIPs.

Development and application of solvent-free extraction for the detection of aflatoxin M1 in dairy products by enzyme immunoassay.

The official methods for the quantification of aflatoxin M1 in dairy products (cheese and yogurt) include extraction into dichloromethane or chloroform, evaporation of the solvent, partitioning of the reconstituted residue with hexane, and subsequent analysis. To secure a rapid and inexpensive screen for aflatoxin M1 contamination, a sensitive competitive ELISA, using a rabbit polyclonal antibody, was developed for measuring aflatoxin M1 in milk and used in a comparative study for measuring the extraction efficiency of aflatoxin M1 in aqueous or organic solvent buffers using yogurt samples. An aqueous sodium citrate solution was found to be suitable for extracting aflatoxin M1, thus eliminating the need for organic solvents. The citrate extraction proved to be efficient (recovery ranged from 70 to 124%) in fortified samples of very different kinds of dairy products, including yogurt and six types of cheese. Fourteen yogurt and cheese samples were extracted with citrate solution and analyzed by ELISA. A good correlation was observed (y=0.95x-0.59, r2=0.98) when the data were compared with those obtained through the official method, across a wide range of aflatoxin M1 contaminations (10-200 ng/kg).

Determination of banned Sudan dyes in food samples by molecularly imprinted solid phase extraction-high performance liquid chromatography.

A method for molecularly imprinted SPE of banned Sudan azo-dyes from food samples was investigated. The molecularly imprinted polymer was obtained by suspension polymerization using 1-(4-chlorophenyl)azonaphthalen-2-ol as the mimic template. The molecular recognition properties of imprinted beads were evaluated for use as a SPE sorbent, in order to develop a selective extraction protocol for the Sudan class of dyes. The optimized extraction protocol resulted in a reliable molecularly imprinted SPE (MISPE) method suitable for HPLC analysis. It was selective for the main analyte, Sudan I, and the related azo-dyes Sudan II, III, IV, Sudan Red B, and Sudan Red 7B, while the permitted azo-dyes Allura Red AC, Neococcin, and Sunset Yellow FCF were not extracted. The method was tested for Sudan I, II, III, and IV in five different food samples (hot chilli pepper, hot chilli tomato sauce, sausage, tomato sauce, and hard boiled egg yolk) at three concentration levels (15, 100, and 300 microg/g). It demonstrated itself to be insensitive to the presence of different complex matrices, precise, accurate, and with good recovery rates (85-101%). The LOD and LOQ were satisfactory for most analytical determinations.

Binding properties of 2,4,5-trichlorophenoxyacetic acid-imprinted polymers prepared with different molar ratios between template and functional monomer.

Several molecularly-imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared with a molar ratio between the functional monomer and the template molecule in the pre-polymerisation mixture set between 1+2 and 20+1. The functional monomer used was 4-vinylpyridine (4-VP), the cross-linker was ethylene dimethacrylate, and the porogenic solvent was a mixture of methanol-water 3+1 (v/v). The polymers obtained were grinded, sieved and packed in 100 mm x3.9 mm HPLC columns. The effects of the mobile phase composition were evaluated by eluting the columns with acetonitrile-water mixtures. The results obtained indicate that column capacity, selectivity factor and the imprinting effect are controlled by ion-pair and hydrophobic interactions between the analyte and the stationary phase. In the full range of ratios considered, column capacity, selectivity factor and imprinting effect are inversely proportional to the molar ratio between the template molecule and the functional monomer.

Development and application of a quantitative lateral flow immunoassay for fumonisins in maize

A quantitative lateral flow immunoassay for measuring fumonisins in maize was developed. Strip preparation and assay parameters were optimized to obtain a dipstick usable outside the laboratory with different samples, and which shows performance comparable with that of other screening methods, as confirmed by the intra- and the inter-day precision of data (RSD 5-16%). Quantification was obtained by an external calibration curve, which can be stored and used for measurements made with strips of the same batch in different days and at varying temperatures (22-37°C). Limit of detection (120 μgL(-1)) and dynamic range (200-5000 μgL(-1)) allow the direct assessment of fumonisin contamination at all levels of regulatory relevance. Twenty-seven maize samples were analyzed after a simple sample preparation which avoids the use of organic solvent. Linear correlation was observed (y=1.071x-0.2, r(2)=0.990) when data was compared with that obtained through a reference LC-MS/MS method, across a wide range of fumonisin contamination.

Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk.

A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M1 (AFM1) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM1 contamination at nanograms per litre level (LOD 20 ng L(-1), IC50 99 ng L(-1)), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained. Direct detection of the target toxin in milk could be obtained by acquiring images of the strips and correlating intensities of the coloured lines with analyte concentrations. The one-step assay can be completed in 17 min, including a very simple and rapid sample preparation, which allowed the application of the assay to milk samples which differ in fat and protein contents. Although imprecise (mean RSD about 30%), the method proved to be accurate and sensitive enough to allow the correct attribution of sample as compliant or non-compliant according to EU legislation in force. Agreeing results to those of a reference ELISA were obtained on 40 milk samples by matrix-matched calibration in pasteurized milk.

Chromatographic characterization of molecularly imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid

Two polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared by utilising the technique of the non-covalent molecular imprinting polymerisation in an aqueous medium. The polymers obtained were packed in HPLC columns and the effects of the mobile phase composition on the retention of the imprinting molecule and the selectivity of the stationary phases towards several analogous structures were studied by liquid chromatography. The columns showed a good level of selectivity towards the template and strictly related molecules. It was found that the molecular recognition mechanism acting on the columns was dependent on a combination of ion pair and hydrophobic interactions.

Development of a quantitative lateral flow immunoassay for the detection of aflatoxins in maize

An immunoassay-based lateral flow device for the quantitative determination of four major aflatoxins in maize has been developed. The one-step assay has performance comparably with that of other screening methods, as confirmed by the intra- and the inter-day precision of the data (RSD 10–22%), and can be completed in 10 min. Quantification was obtained by acquiring images of the strip and correlating intensities of the coloured lines with analyte concentration by means of a stored calibration curve carried out by diluting aflatoxins in the extract from a blank maize sample. Limit of detection (1 µg kg–1) and dynamic range (2–40 µg kg−1) allows the direct assessment of aflatoxin contamination in maize at all levels of regulatory relevance. All reagents are immobilized on the lateral flow device. In addition, very simple sample preparation, using an aqueous buffered solution, has been demonstrated to allow the quantitative extraction of aflatoxins. Twenty-five maize samples were extracted with the aqueous medium and analyzed by the developed assay. A good correlation was observed (y = 0.97x + 0.07, r 2 = 0.980) when data was compared with that obtained through an official method. The developed method is reliable, rapid and allows for application outside the laboratory as a point-of-use test for screening purposes.