Cinzia Passini

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.

Determination of Ochratoxin A in Italian Red Wines by Molecularly Imprinted Solid Phase Extraction and HPLC Analysis

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.

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.

A fluorescent immunochromatographic strip test using Quantum Dots for fumonisins detection.

A fluorescent immunochromatographic strip test (ICST) based on the use of Quantum Dots (QD) was developed and applied to detect fumonisins in maize samples. A limit of detection for fumonisin B1 of 2.8 µg L(-1) was achieved, with an analytical working range of 3-350 µg L(-1), corresponding to 30-3500 µg kg(-1) in maize flour samples, according with the extraction procedure. The time required to perform the analysis was 22 min, including sample preparation. Recovery values in the range from 91.4% to 105.4% with coefficients of variation not exceeding 5% were obtained for fortified and naturally contaminated maize flour samples. To evaluate the possible improvements due to the use of QD for ICST technology, we performed a direct comparison of the proposed QD-ICST to a gold nanoparticles- and a chemiluminescent-ICST previously developed for fumonisins detection, in which the same immunoreagents were employed.

Peptide-based affinity media for solid-phase extraction of Ochratoxin A from wine samples: Effect of the solid support on binding properties

A suitable sample clean up is a key point in the development of an analytical method. Peptide-based affinity media have recently gained attention in the selective extraction of defined target analytes from complex samples. In this paper we investigated the thermodynamic and kinetic binding properties of different stationary phases (Amberlite IRC-50, Lewatit CNP105, Toyopearl CM-650 M, porous silica gel beads and micrometric glass beads) functionalized with a hexapeptide sequence binding the Ochratoxin A. The highest values of the equilibrium binding constant (Keq) and binding site concentration (Bmax) were obtained for Lewatit CNP105 (Keq: 98.1×10(6) M(-1), Bmax: 30.8 μmol/g), followed by Toyopearl and micrometric glass beads, whereas the worst performances were obtained with Amberlite IRC-50 and porous silica gel beads. Also kinetic performances show the same trend. These results highlight that the surface chemical nature has a key role in the binding properties of solid supports used as affinity media for the selective extraction of well-defined target molecules. Finally, Lewatit CNP105 was compared with Amberlite IRC-50 as solid support in SPE extraction of OTA from 14 wine samples fortified with OTA at 2 and 4 μg l(-1) levels. The extracts were analyzed by HPLC with fluorescence detection (λexc 333 nm, λem 460 nm) showing no significant matrix effects, with a LOD and LOQ of 0.45 and 1.5 μgl(-1), respectively, and good recoveries between 71% and 108% for Amberlite IRC-50 and 91% and 101% for Lewatit CNP105. While both supports showed a statistically comparable extraction accuracy, a statistically significant difference was found in terms of extraction precision, confirming that the solid phase based on Lewatit CNP105 performs better than the solid phase based on Amberlite IRC-50.

Comparison of binding behavior for molecularly imprinted polymers prepared by hierarchical imprinting or Pickering emulsion polymerization

The aim of this study was the evaluation of the binding performances and selectivity of molecularly imprinted beads prepared toward several penicillins (i) by hierarchical bulk polymerization in the pores of template-grafted silica microbeads (hMIPs) and (ii) by Pickering emulsion polymerization in the presence of template-decorated silica nanobeads (pMIPs). 6-Aminopenicillanic acid was chosen as the common fragmental mimic template. Both approaches produced micron-sized polymeric beads with good recognition properties toward the target ligands whereas the selectivity pattern appeared quite different. The polymer prepared by the Pickering emulsion approach showed binding properties similar to imprinted beads prepared by hierarchical approach. Equilibrium binding constants changed their values from 0.1-0.2 × 10(6) (hMIPs) to 0.2-0.6 × 10(6) M(-1) (pMIPs), while the binding site densities changed from 3.7-4.8 (hMIPs) to 0.3-0.55 μmol/g (pMIPs). Compared to the hierarchical polymerization, Pickering emulsion polymerization represents a more practical approach when a template mimic needs to be used.

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. Copyright

A broad-selective enzyme immunoassay for non-invasive stress assessment in African penguins (Spheniscus demersus) held in captivity

We applied a direct competitive immunoassay for measuring corticosterone and glucocorticoid metabolites in faeces (FGMs) as a non-invasive tool for monitoring the stress response of African penguins (Spheniscus demersus) held in captivity in a zoological facility. The developed assay, validated in-house, proved to be rapid (the test could be completed in 90 minutes), sensitive (LOD for corticosterone 0.2 μg l−1, dynamic range 0.75–75 μg l−1) and broad-selective, as it cross-reacted with the major corticosteroids, thus allowing the detection of excreted FGMs resulting from a biological stressor. Matrix interference, due to components of faecal samples, was overcome by diluting sample extracts (1 + 4 or 1 + 9, depending on the sample). The assay enabled us to investigate the response to stress in five animals – three adult males and two adult females – over a period of 30 hours, and to identify the peak of FGM production as being 7–10 hours after the stressful event.

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.

Affinity Capillary Electrochromatography of Molecularly Imprinted Thin Layers Grafted onto Silica Capillaries Using a Surface-Bound Azo-Initiator and Living Polymerization

Molecularly imprinted thin layers were prepared in silica capillaries by using two different surface polymerization strategies, the first using 4,4′-azobis(4-cyanovaleric acid) as a surface-coupled radical initiator, and the second, S-carboxypropyl-S’-benzyltrithiocarbonate as a reversible addition-fragmentation chain transfer (RAFT) agent in combination with 2,2′-azobisisobutyronitrile as a free radical initiator. The ability to generate imprinted thin layers was tested on two different polymerization systems: (i) a 4-vinylpyridine/ethylene dimethacrylate (4VP-EDMA) in methanol-water solution with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a template; and (ii) methacrylic acid/ethylene dimethacrylate (MAA-EDMA) in a chloroform solution with warfarin as the template molecule. The binding properties of the imprinted capillaries were studied and compared with those of the corresponding non-imprinted polymer coated capillaries by injecting the template molecule and by measuring its migration times relative to a neutral and non-retained marker. The role of running buffer hydrophobicity on recognition was investigated by studying the influence of varying buffer acetonitrile concentration. The 2,4,5-T-imprinted capillary showed molecular recognition based on a reversed phase mechanism, with a decrease of the template recognition in the presence of higher acetonitrile content; whereas warfarin-imprinted capillaries showed a bell-shaped trend upon varying the acetonitrile percentage, illustrating different mechanisms underlying imprinted polymer-ligand recognition. Importantly, the results demonstrated the validity of affinity capillary electrochromatography (CEC) to screen the binding properties of imprinted layers.