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.

Lateral-flow immunoassays for mycotoxins and phycotoxins: a review.

Natural 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.

A lateral flow immunoassay for the rapid detection of ochratoxin A in wine and grape must.

A one-step lateral flow immunoassay was developed for semiquantitatively detecting ochratoxin A (OTA) in wines and grape musts. Matrix-matched calibration curves carried out in blank wines showed a detection limit of 1 µg L(-1) and IC(50) of 3.2 µg L(-1). Relative standard deviations for intra- and interday precision were in the 20-40% range. A simple treatment of samples, which only included dilution with sodium bicarbonate and polyethylene glycol (4% w/v) for red and white wines and the further addition of ethanol (12% v/v) for grape musts, was established. The developed assay allowed OTA detection in 5 min and proved to be accurate and sensitive enough to allow the correct attribution of samples as compliant or noncompliant according to EU legislation. Results agreeing with those of a reference chromatographic method were obtained on 38 wines and 16 musts. Although some lateral flow devices aimed at detecting OTA have been previously described, this is the first assay capable of measuring the toxin in wine and grape must, which represent a major source of OTA dietary intake. Analytical performances of the method are comparable to or better than previously reported assays showed. In addition, the assay, including sample treatments, is extremely simple and rapid and can be effectively regarded as a one-step assay usable virtually anywhere.

An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization.

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.

A rational route to the development of a competitive capillary electrophoresis immunoassay: assessment of the variables affecting the performances of a competitive capillary electrophoresis immunoassay for human serum albumin.

Affinity capillary electrophoresis is a powerful analytical tool to extract quantitative information about the binding properties of different interacting systems. The use of LIF detection makes the technique suitable for screening strong binding interactions. The non-equilibrium electrophoretic separations of pre-equilibrated mixtures of ligand and receptor are generally used for such strong molecular interactions allowing the assessment of capillary electrophoresis immunoassays, mostly in competitive formats. As the analytical performances of the assay strongly depend on the preservation of the binding properties during the separation, a rational route to assay development has to be followed to get the best conditions. The paper describes the steps followed to set-up a competitive immunoassay for human serum albumin (HSA) by using a labeled protein (HSA-FITC) and an anti-HSA polyclonal antiserum. A labeling degree of around 1 of the HSA-FITC conjugates is needed to get narrow electrophoretic peak while the titration curve is used to define the optimal antiserum dilution. An antiserum-labeled protein affinity constant of 1.34×10(7) M(-1) was measures in the selected separation conditions. Furthermore, in order to maximize the assay competition between the labeled and unlabelled HSA a short pre-incubation step of the antiserum with the unlabelled HSA (the analyte) was introduced to promote a sharp increase in assay sensitivity.

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 experiment's 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.

Chemiluminescence-based biosensor for fumonisins quantitative detection in maize samples.

A compact portable chemiluminescent biosensor for simple, rapid, and ultrasensitive on-site quantification of fumonisins (fumonisin B1+fumonisin B2) in maize has been developed. The biosensor integrates a competitive lateral flow immunoassay based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a contact imaging configuration. The use of chemiluminescence detection allowed accurate and objective analyte quantification, rather than qualitative or semi-quantitative information usually obtained employing conventional lateral flow immunoassays based on colloidal gold labeling. A limit of detection of 2.5 µgL(-1) for fumonisins was achieved, with an analytical working range of 2.5-500 µgL(-1) (corresponding to 25-5000 µgkg(-1) in maize flour samples, according to the extraction procedure). Total assay time was 25 min, including sample preparation. A simple and convenient extraction procedure, performed by suspending the sample in a buffered solution and rapidly heating to eliminate endogenous peroxidase enzyme activity was employed for maize flour samples analysis, obtaining recoveries in the range 90-115%, when compared with LC-MS/MS analysis. The chemiluminescence immunochromatography-based biosensor is a rapid, low cost portable test suitable for point-of-use applications.

Binding behaviour of molecularly imprinted polymers prepared by a hierarchical approach in mesoporous silica beads of varying porosity.

One of the most interesting methods for preparing molecularly imprinted polymers with controlled morphology consists in filling the pores of silica beads with an imprinting mixture, polymerizing it and dissolving the support, leaving porous imprinted beads that are the "negative image" of the silica beads. The main advantage of such an approach consists in the easy preparation of spherical imprinted polymeric particles with narrow diameter and pore size distribution, particularly indicated for chromatographic applications. In this approach it has been shown that the resulting morphology of polymeric beads depends essentially on the porosity and surface properties of the silica beads that act as microreactors for the thermopolymerization process. Anyway, it is not yet clear if the porosity of the silica beads influences the binding properties of the resulting imprinted beads. In this paper, we report the effect of different porosities of the starting mesoporous silica beads on the resulting binding properties of imprinted polymers with molecular recognition properties towards the fungicide carbendazim. The morphological properties of the imprinted beads prepared through this hierarchical approach were measured by nitrogen adsorption porosimetry and compared with a reference imprinted material prepared by bulk polymerization. The chromatographic behaviour of HPLC columns packed with the imprinted materials were examined by eluting increasing amounts of carbendazim and extracting the binding parameters through a peak profiling approach. The experimental results obtained show that the resulting binding properties of the imprinted beads are strongly affected by the polymerization approach used but not by the initial porosity of the silica beads, with the sole exception of the binding site density, which appears to be inversely proportional to them.

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, r²= 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.

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.

Molecularly imprinted polymers for corticosteroids: analysis of binding selectivity.

In this work we prepared a library of cortisol-imprinted polymers via a sequential approach by combining 10 different functional monomers, 7 cross-linkers and 5 porogen solvents. The best combinations of functional monomers, cross-linkers and porogen solvents in terms of cortisol binding were used to prepare three imprinted polymers--polyacrylamide-co-ethylene dimethacrylate (porogen: chloroform), poly-4-vinylpyridine-co-ethylene dimethacrylate (porogen: chloroform) and polyacrylamide-co-ethylene dimethacrylate (porogen: acetonitrile)--whose selectivity towards 10 synthetic corticosteroids and 4 natural steroidal hormones was tested. The experimental results obtained show how different combinations of functional monomers, cross-linkers and porogen solvents produce cortisol-imprinted polymers with very different selectivity patterns, and that a careful optimization of the pre-polymerization mixtures makes it possible to increase the number of target steroids recognized by the resulting imprinted polymer. Moreover, through the use of a Free-Wilson analysis of the binding selectivity, it has been possible to obtain insights on the steroidal structural motifs able to increase or decrease the molecular recognition of corticosteroids by the imprinted polymers.

Mycotoxins in food and feed: Extraction, analysis and emerging technologies for rapid and on-field detection.

Mycotoxins are toxic compounds produced by various moulds, which can affect a variety of crops. Due to their high toxicity and wide diffusion, mycotoxins constitute a severe risk for human health, therefore maximum tolerance levels in food and feed products have been set up all over the world and analytical controls are mandatory for many commodities. Despite validated analytical methods for assessing mycotoxin contamination already existing, a number of papers describing new methods of extraction, identification or measurement appear daily in literature. Nevertheless, the extraction and determination of such contaminants in food and feed is a topic of constant and increasing interest, as also attested by the number of related patents which have been applied for in the last few years. Scientific papers dealing with mycotoxin occurrence, potential risk and determination have recently been reviewed. Nevertheless, the objective of this review is to focus on patent activity rather than on the scientific breakthroughs on this subject. Therefore, the most recent patents regarding the whole analytical protocol to measure mycotoxins in food, starting from 2006 to date are presented and discussed. The possibility of a technology transfer for the various innovations presented is also discussed as are future developments in the field.

Molecularly imprinted polymer/cryogel composites for solid-phase extraction of bisphenol A from river water and wine.

Superporous monolithic hydrogels (cryogel monoliths) are elastic, sponge-like materials that can be prepared in an aqueous medium through a cryotropic gelation technique. These monoliths show interesting properties for the development of high-throughput solid-phase extraction supports to treat large volumes of aqueous samples. In this work, a cryogel-supported molecularly imprinted solid-phase extraction approach for the endocrine disruptor bisphenol A (BPA) from river water and wine samples is presented. An imprinted polymer with molecular recognition properties for BPA was prepared in acetonitrile by thermal polymerization of a mixture of 4,4'-dihydroxy-2,2-diphenyl-1,1,1,3,3,3-trifluoropropane as a mimic template of BPA, 4-vinylpyridine and trimethylolpropane trimethacrylate in a molar ratio of 1 + 6 + 6. Fine imprinted particles (<10 microm) were embedded in a poly-acrylamide-co-N,N'-methylenbisacrylamide cryogel obtained by ammonium persulfate-induced cryopolymerization at -18 degrees C. The resulting monolithic gel was evaluated for its use as a sorbent support in an off-line solid-phase extraction approach to recover BPA from dilute aqueous samples with minimum pre-loading work-up. The optimized extraction protocol resulted in a reliable MISPE method suitable to selectively extract and preconcentrate BPA from river water and red wine samples, demonstrating the practical feasibility of cryogel-trapped imprinted polymers as solid-phase extraction materials.

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.

Development of enzyme-linked immunosorbent assays for Sudan dyes in chilli powder, ketchup and egg yolk.

This study aimed at developing sensitive competitive enzyme-linked immunosorbent assays (ELISAs) for the banned Sudan dyes using polyclonal antibodies. Three different formats were developed and characterized in terms of sensitivity, selectivity and rapidity. A competitive indirect ELISA was developed, which showed an IC(50) of 3.8 microg l(-1). Two competitive direct ELISAs were also developed, in which the antibody was added before or simultaneously with the other reagents; the first showed an IC(50) of 8.3 microg l(-1) and the latter showed an IC(50) of 4.9 microg l(-1). Nevertheless, considering dilution of extracts which is needed to offset matrix interference, the limits of detection of the three formats were substantially the same (10 microg kg(-1)). The antibodies in all three test formats were able to recognize Sudan I and partially Sudan II, III and IV; no cross-reactivity was observed with the five edible dyes. Twenty food samples, including chilli powder, paprika, ketchup, and egg, were extracted by a simple sample preparation and very limited dilution. Extracts were analyzed by the developed competitive direct ELISA with the simultaneous addition of reagents. A good correlation was observed (y = 1.19 x-10.0, r(2) = 0.991, n = 20) when the data was compared with that obtained through a conventional HPLC method.

Homogeneous immunoassay based on gold nanoparticles and visible absorption detection.

A sensitive homogeneous immunoassay, using human serum albumin (HSA) as a model analyte coupled with simple visible absorption detection, has been developed. The new assay is based on the use of gold nanoparticles functionalized with the target protein, which compete with the analyte for the binding of a specific polyclonal antibody. The binding of antibodies to the functionalized nanoparticles determines a shift of the visible absorption maximum of the gold colloid, and quantification of the analyte could be obtained as the competitive inhibition of the binding of antibodies to the nanoparticles. The proposed immunoassay has been optimized and successfully applied to measuring HSA in human urine samples, in which results agreed well with those obtained by a nephelometric reference method.

Synthetic peptides as artificial receptors towards proteins from genetically modified organisms.

The aim of this work was the preparation of peptide ligands with good affinity and selectivity towards proteins from genetically modified organisms, namely neomycin phosphotransferase II (Npt II) and the endotoxin Cry1A. A 12 x 12 combinatorial solid phase synthesis in aqueous medium was performed to prepare peptide libraries. From this library, two dipeptides with binding properties towards the chosen ligands (Pro-Lys for Npt II, K(eq) 7.59 x 10(4)M(-1); Trp-Gln for Cry 1A, K(eq) 4.35 x 10(4)M(-1)) were selected as scaffolds for the synthesis of new tetrapeptide libraries. The equilibrium constants of the newly selected tetrapeptides increased slightly respect to the dipeptides (Pro-Lys-His-Phe for Npt II, K(eq) 7.88 x 10(4)M(-1); Trp-Gln-Ala-Phe for Cry 1A, K(eq) 5.65 x 10(4)M(-1)), but selectivity towards other proteins (wheat gliadins, bovine gamma-globulins, bovine serum albumin and chicken ovalbumin) became higher. It was demonstrated that selected tetrapeptides recognised well the ligands also in presence of very complex mixtures of potentially interfering proteins, such as whole cell lysates. This approach can be considered as a general method to obtain tailor-made reagents with antibody-like binding properties towards biomacromolecules.

Aptamers and molecularly imprinted polymers as artificial biomimetic receptors in affinity capillary electrophoresis and electrochromatography.

Artificial biomimetic receptors, such as aptamers and molecular-imprinted polymers, show antibody-like properties which are due to molecular recognition phenomena characterized by high affinity and selectivity. These binding features have made them suitable in all those application fields in which selective recognition is required. Thus, it is not surprising that they are finding applications in affinity CE as well. Recently, a variety of ACE methods have shown themselves to be suitable tools to provide a detailed quantitative characterization of the thermodynamic and kinetic aspects of binding. At the same time, affinity CE can exploit the peculiarities of these binding interactions to set up CE-based analytical tools for the separation and the determination of specific target molecules in microscale formats. This review will provide a detailed description of affinity CE methods recently reported in the literature and related to these topics.

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).

Binding properties of a monoclonal antibody against the Cry1Ab from Bacillus Thuringensis for the development of a capillary electrophoresis competitive immunoassay.

Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable-(5.0 +/- 1.2) x 10(6) M(-1) and (9.06 +/- 5.7) x 10(6) M(-1)-reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 mug L(-1)). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.