In the current paradigm about molecular imprinting, the imprinted binding sites exist as a consequence of the polymerization process around templates, and the properties of non-imprinted polymers (NIPs) has been largely overlooked. Thus, nothing can be affirmed a priori on the binding properties of NIPs. We propose an alternative view where the imprinting effect is due to the presence of a template molecule which enhances the pre-existing binding properties of a polymer. If a NIP shows no binding properties towards a target molecule the corresponding imprinted polymer (MIP) will show a weak imprinting effect. On the other hand, if a NIP shows binding properties towards 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 towards 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 constant measured for the NIP-and the MIP-library, 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.

Silver nucleation on gold has been exploited for signal amplification and has found application in several qualitative and quantitative bio-sensing techniques, thanks to the simplicity of the method and the high sensitivity achieved. Very recently, this technique has been tentatively applied to improve performance of gold-based immunoassays. In this work, the exploitation of the signal amplification due to silver deposition on gold nanoparticles has been first applied to a competitive lateral flow immunoassay (LFIA). The signal enhancement due to silver allowed us to strongly reduce the amount of the competitor and of specific antibodies employed to build a LF device for measuring ochratoxin A (OTA), thus determining the attainment of the high sensitive assessment of OTA contamination, with a sensitive gain of more than 10-folds compared to the gold-based LFIA that used the same immunoreagents and to all previously reported LFIA for measuring OTA. In addition, a less sensitive "quantitative"-LFIA could be established, by suitably tuning competitor and antibody amounts, which was characterized by reproducible and accurate OTA determinations (RSD% 6-12%, recovery% 82-117%). The quantitative system allowed a reliable OTA quantification in wines and grape musts at the μg/l level requested by the European legislation, as demonstrated by agreeing results obtained through the "quantitative" silver enhanced-LFIA and a reference HPLC-FLD on 30 samples.

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.

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An extraction method based on molecularly imprinted polymer prepared through a mimic template approach was used for the determination of ochratoxin A in 17 red wines from different geographical regions of Italy. Sample loading (wine sample diluted 1:1 with 1% v/v aqueous solution of PEG 8000), washing (2 mL water/acetonitrile 4:1 v/v), and elution (2 mL of acetonitrile/acetic acid 98:2 v/v) conditions allowed the optimization of the extraction method, capable of preconcentrating ochratoxin A below the maximum permitted level of 2 ng/mL. Under optimized conditions, recoveries of ochratoxin A from spiked samples ranged from 88 to 102% with sample volumes up to 20 mL. The HPLC determination by fluorescence detection allowed limits of detection and quantification, respectively, of 0.075 and 0.225 ng/mL. Sample extractions by an immunoaffinity protocol showed the method to be comparable, demonstrating the potential of the imprinting approach to substitute for the current immunoaffinity method.