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NanoMIPs that are prepared by solid phase synthesis have proven to be very versatile, but to date only limited attention has been paid to their use in solid phase extraction. Thus, since nanoMIPs show close similarities, in terms of binding behavior, to antibodies, it seems relevant to verify if it is possible to use them as mimics of the natural antibodies that are used in immunoextraction methods. As a proof-of-concept, we considered prepared nanoMIPs against fluoroquinolone ciprofloxacin. Several nanoMIPs were prepared in water with polymerization mixtures of different compositions. The polymer with the highest affinity towards ciprofloxacin was then grafted onto a solid support and used to set up a solid phase extraction–HPLC method with fluorescence detection, for the determination of fluoroquinolones in human urine. The method resulted in successful selection for the fluoroquinolone antibiotics, such that the nanoMIPs were suitable for direct extraction of the antibiotics from the urine samples at the µg mL−1 level. They required no preliminary treatment, except for a 1 + 9 (v/v) dilution with a buffer of pH 4.5 and they had good analyte recovery rates; up to 85% with precision in the range of 3 to 4.5%, without interference from the matrix. These experimental results demonstrate, for the first time, the feasibility of the use of nanoMIPs to develop solid phase extraction methods.

To accurately diagnose COVID-19 infection and its time-dependent progression, the rapid, sensitive, and noninvasive determination of immunoglobulins A specific to SARS-CoV-2 (IgA) in saliva and serum is needed to complement tests that detect immunoglobulins G and M. We have developed a dual optical/chemiluminescence format of a lateral flow immunoassay (LFIA) immunosensor for IgA in serum and saliva. A recombinant nucleocapsid antigen specifically captures SARS-CoV-2 antibodies in patient specimens. A labelled anti-human IgA reveals the bound IgA fraction. A dual colorimetric and chemiluminescence detection enables the affordable and ultrasensitive determination of IgA to SARS-CoV-2. Specifically, a simple smartphone-camera-based device measures the colour signal provided by nanogold-labelled anti-human IgA. For the ultrasensitive chemiluminescence transduction, we used a contact imaging portable device based on cooled CCD, and measured the light signal resulting from the reaction of the HRP-labelled anti-human IgA with a H2O2/luminol/enhancers substrate. A total of 25 serum and 9 saliva samples from infected and/or recovered individuals were analysed by the colorimetric LFIA, which was sensitive and reproducible enough for the semi-quantification of IgA in subjects with a strong serological response and in the early stage of COVID-19 infection. Switching to CL detection, the same immunosensor exhibited higher detection capability, revealing the presence of salivary IgA in infected individuals. For the patients included in the study (n = 4), the level of salivary IgA correlated with the time elapsed from diagnosis and with the severity of the disease. This IgA-LFIA immunosensor could be useful for noninvasively monitoring early immune responses to COVID-19 and for investigating the diagnostic/prognostic utility of salivary IgA in the context of large-scale screening to assess the efficacy of SARS-CoV-2 vaccines., Highlights • A LFIA for the point-of-care detection of IgA specific to SARS-CoV2 in serum and saliva was developed. • GNP and HRP-mediated optical/chemiluminescence signals were detected by a smartphone CMOS and a portable CCD, respectively. • The one-step colorimetric and two-step CL IgA-LFIA enabled the detection of anti-SARS-CoV-2 IgA in 15 min. • Salivary IgA were identified in four COVID-19 patients by the CL IgA-LFIA after two weeks from diagnosis.

Imprinted nanoparticles present several advantages respect to bulk imprinted materials, but, when prepared by traditional methods, their usefulness is limited as the approaches are costly or require complex optimization steps, while the purification from template molecules is challenging. An innovative approach is the solid-phase synthesis. It consists in the covalent immobilization of the template onto a solid support, the polymerization of nanoparticles around the template, the clean-up from unproductive components and the final release of the imprinted nanoparticles, which are free of template and demonstrate high affinity for the target molecule. Here we report the use of ciprofloxacin as immobilized template to evaluate the effect of different experimental conditions in the solid phase polymerization (template scaffolding, polymerization mixtures, polymerization medium) and different rebinding conditions (buffer pH) on the binding properties. The results confirm that the solid phase synthesis approach is a flexible approach, where the experimental conditions are decisive for the binding properties. The results show that this approach is a powerful technique to easily prepare nanoparticles fully compatible with the aqueous environment, with reduced non specific binding (≈104 Mol L−1), high equilibrium binding constants (105–107 Mol L−1) and fast association rate constants (≈106 Mol L−1 min−1), values which are comparable to those of natural antibodies.

Exposure to mycotoxins, which may contaminate food and feed commodities, represents a serious health risk for consumers. Ochratoxin A (OTA) is one of the most abundant and toxic mycotoxins, thus specific regulations for fixing its maximum admissible levels in foodstuff have been established. Lateral Flow ImmunoAssay (LFIA)-based devices have been proposed as screening tools to avoid OTA contamination along the whole food chain. We report a portable, user-friendly smartphone-based biosensor for the detection and quantification of OTA in wine and instant coffee, which combines the LFIA approach with chemiluminescence (CL) detection. The device employs the smartphone camera as a light detector and uses low-cost, disposable analytical cartridges containing the LFIA strip and all the necessary reagents. The analysis can be carried out at the point of need by non-specialized operators through simple manual operations. The biosensor allows OTA quantitative detection in wine and coffee samples up to 25 μg L−1 and with limits of detection of 0.3 and 0.1 μg L−1, respectively, which are below the European law-fixed limits. These results demonstrate that the developed device can be used for routine monitoring of OTA contamination, enabling rapid and reliable identification of positive samples requiring confirmatory analysis.

An innovative approach to imprinted nanoparticles (nanoMIPs) is represented by solid-phase synthesis. Since the polymeric chains grow over time and rearrange themselves around the template, the binding properties of nanoMIPs could depend on the polymerization time. Here we present an explorative study about the effect of different polymerization times on the binding properties of ciprofloxacin-imprinted nanoMIPs. The binding properties towards ciprofloxacin were studied by measuring the binding affinity constants (Keq) and the kinetic rate constants (kd, ka). Furthermore, selectivity and nonspecific binding were valued by measuring the rebinding of levofloxacin onto ciprofloxacin-imprinted nanoMIPs and ciprofloxacin onto diclofenac-imprinted nanoMIPs, respectively. The results show that different polymerization times produce nanoMIPs with different binding properties: short polymerization times (15 min) produced nanoMIPs with high binding affinity but low selectivity (Keq &gt, 107 mol L−1, α ≈ 1), medium polymerization times (30 min–2 h) produced nanoMIPs with high binding affinity and selectivity (Keq ≥ 106 mol L−1, α &lt, 1), and long polymerization times (&gt, 2 h) produced nanoMIPs with low binding affinity, fast dissociation kinetics and low selectivity (Keq ≤ 106 mol L−1, kdis &gt, 0.2 min−1, α ≈ 1). The results can be explained as the combined effect of rearrangement and progressive stiffening of the polymer chains around the template molecules.

The design, preparation and evaluation of molecularly imprinted polymers for roxarsone (4-hydroxy-3-nitrophenylarsonic acid), an organo-arsenic swine and poultry feed additive, using bi-substituted ureas and squaramide receptors as the functional monomers, are demonstrated. Pre-polymerisation studies of the template-monomer complexation performed by 1H NMR experiments show that squaramide-based monomers provide association equilibrium constant values higher than urea-based monomers. Equilibrium rebinding experiments in methanol show that two squaramide-based materials have good molecular recognition properties towards roxarsone, with high affinity (Keq = 16.85 × 103 L mol-1 and 14.65 × 103 L mol-1, respectively), high imprinting factors (4.73 and 3.64 respectively) and good selectivity towards two roxarsone-related compounds, acetarsone (3-acetamido-4-hydroxyphenylarsonic acid) and nitarsone (4-nitrophenylarsonic acid). Polymer MIP-SQ2 was successfully used to setup an experimental protocol for the direct solid phase extraction of roxarsone from surface water samples. The method gives clean HPLC traces, with recoveries between 91% and 95% at concentration levels of 5.0, 10, and 25 mg L-1. Sample preconcentration with good recoveries between 87% and 97%, are shown, confirming that it is possible to employ the developed materials to measure roxarsone down to 1 μg L-1 in water samples.

In this study, we report the simultaneous use of gold and silver nanoparticles to set a multicolor multiplex lateral flow immunoassay (xLFIA). Silver nanoparticles (AgNPs), spherical in shape and characterized by a brilliant yellow color, were obtained by a new viable one-step synthetic protocol. AgNPs were stable over time and acceptably robust to conditions used for fabricating LFIA devices. These AgNPs were employed as a colorimetric probe in combination with two different kinds of gold nanoparticles (AuNPs) to set a visual xLFIA for detecting allergens. Surface plasmon resonance peaks of probes (AgNPs, spherical and desert rose-like AuNPs) were centered at 420, 525, and 620 nm, respectively. Therefore, the xLFIA output was easily interpreted through a "yellow magenta cyan (YMC)" color code. The prospect of the YMC xLFIA was demonstrated by simultaneously detecting three major allergens in bakery products. Antibodies directed towards casein, ovalbumin, and hazelnut allergenic proteins were individually adsorbed onto metal nanoparticles to produce three differently colored specific probes. These were inserted in a LFIA comprising three lines, each responsive for one allergen. The trichromatic xLFIA was able to detect allergenic proteins at levels as low as 0.1 mg/l and enabled the easy identification of the allergens in commercial biscuits based on the color of the probes. Graphical Abstract.

The authors describe the preparation of desert rose-like gold nanoparticles (DR-GNPs) with a plasmon resonance band at 620 nm which gives them a blue color. They have a hydrodynamic diameter of ∼72 nm and were prepared by a seeding growth approach. The DR-GNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. These nonspherical GNPs were used as a label for the antibody in an immunochromatographic strip test (ICST). Despite their particular shape and the higher surface area compared to spherical gold nanoparticles, the DR-GNPs are useful blue labels for the GNP-based strip test. A multicolor ICST for aflatoxin B1 and fumonisins is described that employs both blue DR-GNPs and red spherical GNPs. It allows for simultaneous rapid determination of the two mycotoxins in maize flour, with visual cut-off levels of 2 μg⋅kg-1 for aflatoxin B1 and of 1000 μg⋅kg-1 for fumonisins.

Multiplex lateral flow immunoassay (LFIA) is largely used for point-of-care testing to detect different pathogens or biomarkers in a single device. The increasing demand for multitargeting diagnostics requires multi-informative single tests. In this study, we demonstrated three strategies to upgrade standard multiplex LFIA to multimodal capacity. As a proof-of-concept, we applied the strategies to the differential diagnosis of Human Immunodeficiency Virus (HIV) infection, a widespread pathogen, for which conventional multiplex LFIA testing is well-established. In the new two-parameter LFIA (x2LFIA), we exploited color encoding, in which the binding of multiple targets occurs in one reactive band and the color of the probe reveals which one is present in the sample. By combining the sequential alignment of several reactive zones along the membrane of the LFIA strip and gold nanoparticles and gold nanostars for the differential visualization, in this demonstration, the x2LFIA can furnish information on HIV serotype and stage of infection in a single device. Three immunosensors were designed. The use of bioreagents as the capturing ligand anchored onto the membrane or as the detection ligand labelled with gold nanomaterials affected the performance of the x2LFIA. Higher detectability was achieved by the format involving the HIV-specific antigens as capturing agent and labelled secondary bioligands (anti-human immunoglobulins M and protein G) as the probes.

We designed a versatile inorganic probe based on functionalized TiO2 nanoparticles for labeling immunoreagents and developing immunological tests. Similarly to enzymatic probes traditionally used in immunoassay, the TiO2 probe could be covalently linked to antigens or antibodies and exploited as the signal reporter in immunoassays. The TiO2 probe allowed revealing the rate of antigen-antibody complex formation by promoting the oxidation of a suitable chromogenic substrate with absorption in the visible. We demonstrated the suitability of the TiO2 probe as a label for immunoassay by coupling it to human serum albumin and developing a direct competitive assay to measure micro- and macro-albuminuria for diabetes diagnosis. The developed TiO2-based assay showed high sensitivity (detection limit 1.4 mg l−1), wide dynamic range (6-1270 mg l−1) and acceptable precision (within- and between-assay coefficient of variation %

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.

The biological activity of extracts from Ailanthus altissima is mainly due to the presence of ailanthone, a compound belonging to the quassinoid class. Recently, attention has been paid to its strong cytostatic activity. However, the extraction of ailanthone is based on very long and demanding procedures, which keep the price of the commercial product very high. Thus, the development of selective adsorbents for the purification of ailanthone from A. altissima leaves extracts could help in reduce the costs of production. In this work, we describe the rational design of a molecularly imprinted polymer selective for ailanthone based on the screening of a 96-members not-imprinted polymeric library to rapidly identify pre-polymerization mixtures able to generate MIPs with enhanced binding properties. A 4-vinylpyridine-co-trimethylolpropane trimethacrylate polymer showed high binding towards ailanthone. It was used to prepare an imprinted polymer with acetonitrile showed interesting binding affinity (Keq = 18.3 × 103 L mol−1), high imprinting factor (IF = 3.8) and fast binding kinetics (kass = 0.390 min−1, kdis = 0.021 mol L−1 min−1). The imprinted polymer was used to develop a successful purification protocol of extracts from Ailanthus altissima leaves. The purification was based on the combination of a preliminary clean-up of Soxhlet extracts onto a reverse phase-C18 cartridge and the subsequent isolation of ailanthone by a molecularly imprinted solid phase extraction. This approach allowed efficiently purifying the ailanthone contained in aqueous or methanolic Soxhlet extracts with high yields compared to the quantities reported in literature (water: 0.756 ± 0.027 mg g−1; methanol: 0.770 ± 0.030 mg g−1). Moreover, it allows processing sample volumes up to 15 mL without significant losses of the target compound.

The development of a new generation of ultra-sensitive sensors for analytical and bio-diagnostic devices requires a strong signal in front of very small quantity of analyte, often present in complex and interfering matrix. Taking advantage of peculiar plasmonic properties of nanoporous gold (NPG), a promising sensor for selective detection of Human Serum Albumin (HSA), a proof-of-concept bioanalyte, was prepared and tested in a conventional micro-Raman spectrometer using Surface Enhanced Raman Spectroscopy (SERS). NPG was synthesized by chemical de-alloying of an amorphous precursor, Au20Cu48Ag7Pd5Si20, starting from melt spun ribbons. A fully de-alloyed ribbon with ligaments of about 60 nm was obtained after 4 h of de-alloying at 70 °C and 10 M HNO3 + 0.5 M HF. This material is self-standing, mechanically resistant, and suitable for wide range of applications. At this stage, NPG is SERS-active toward several molecules, including pyridine, bi-pyridine, and rhodamine at very low concentration. In order to obtain the selective binding properties required to detect molecules in bio-diagnostic applications, immuno-functionalization of NPG was carried out by using an anti-HSA antibody (Ab-anti-HSA) covalently grafted on gold ligaments via the 4-aminothiophenol (4ATP) Raman probe, yielding the SERS-active nanohybrid NPG-4ATP-Ab. SERS signal recorded at increasing HSA concentrations was used for the acquisition of SERS maps and a chemometric regression model allowed to calibrate the sensor. In the best experimental conditions, we were able to get quantitative analysis of HSA at ultra-low concentrations (0.1 ng/l) with SERS. The methodology proposed in this paper has to be considered a major step toward a calibrated device for ultra-sensitive detection of biomolecules by SERS detection.

Carbon nitride nanoparticles (CNNPs) have been employed as fluorescent sensing tools owing to their unique features, e.g. low cost production, high stability in water and high photoluminescence quantum yield. Here, an easy and versatile synthetic approach was exploited to design fluorescent nanoparticles with surface functionalities suitable for covalent binding to bioligands. High hydrophilic, brightly fluorescent CNNPs, rich of superficial amines, were obtained from the thermal condensation of urea and lysine (CNNPLys) and by tuning the precursor ratio and the heating time. Structure and size of the functionalized nanoparticles were characterized through infrared (IR) spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). Their optical properties were studied by ultraviolet-visible (UV-Vis) and fluorescence spectroscopy. The superficial primary amino groups, furnished by the lysine co-precursor, enabled for covalently linking CNNPLys to model proteins. The CNNPLys-protein conjugates excited under UV irradiation emit in the 400–450 nm visible range (quantum yield 24%). The applicability of CNNPLys as novel fluorescent probes was demonstrated by a fluorescence quenching assay, in which gold nanoparticles (GNPs) were attached to Staphylococcal protein A and employed to quench the CNNPLys fluorescence by resonant energy transfer (FRET). The quenching occurred upon formation of the specific binding between the GNP-linked protein A and CNNPLys-tagged immunoglobulins, while the inhibition of the binding resulted in the recovery of CNNPLys luminescence. The synthetic strategy, based on combining a “conjugated polymer”-forming unit (urea) and a co-precursor able to provide the desired functional group (lysine), allows designing innovative materials for the development of new generation fluorescence biosensors in which easily functionalized fluorophores are needed.

Benomyl is a benzimidazol fungicide used against various crop pathogens. Although banned in many countries, it is still widely used worldwide and is listed in different monitoring programs among the substances to be monitored to assess human exposure to pesticide residues. The assessment of benomyl is mainly based on the analysis of the residues of its most important metabolite, carbendazim. Existing methods often lack of selectivity and display a limited performance because of the presence of co-extracted compounds. Molecularly imprinted polymers (MIPs) offer an alternative methodology, adsorbing preferentially those target molecules for which the polymers are specifically prepared. In this study, we optimized the synthesis of a polymer imprinted with benomyl. Tests of specificity recognition showed a good performance for carbendazim compared with other similar pesticides. The mean recovery of benomyl (measured as carbendazim) from water samples was estimated to be 90% for MIPs while with real soil samples collected in Morocco the recovery efficiency was 62%. Preliminary tests also suggest that this MIP can implement traditional SPE techniques for assessing benomyl residual concentrations in environmental samples.

The development of reliable molecularly imprinted sorbent assays is ensured by in-depth knowledge of the binding between the tracer, conventionally based on a template analogue conjugated to an enzyme, and the imprinted polymer used as a recognition element. To this end, the binding properties of a cortisol-3-(O-carboxymethyl)oxime-horseradish peroxidase conjugate to cortisol-imprinted microparticles previously adsorbed at the bottom of microplates were assessed. The effect of different blocking agents as well as of different percentages of Tween 20 in the working buffer were investigated in order to minimise the non-specific binding of the enzyme tracer to the adsorbed imprinted microparticles. The capability of the enzyme tracer to bind the imprinted solid phase and to compete with free cortisol was assessed by measuring the apparent equilibrium dissociation constant, KD (39.7 ± 13.5 pmol L−1), and the apparent binding site concentration, Bmax (21.7 ± 4.3 nmol L−1), whereas the IC50 value for the cortisol competition curve was found to be 5.32 ± 1.15 ng mL−1. Moreover, binding selectivity measured for several cortisol-related steroids confirmed the experimental results previously published for cortisol-imprinted polymers. A competitive assay for the determination of cortisol in human saliva was developed with a limit of detection of 1.02 ng mL−1, providing quantitative results comparable to those of a commercial ELISA.

A multiplex Lateral Flow Immunoassay was developed based on the use of a single Test line and multicolour gold nanoparticles (GNPs) as signal reporters. Red and blue GNPs were linked to antibodies directed towards two different analytes and included in a typical lateral flow immunoassay configuration, in which the Test line was formed by the mixture of two antigens. As a result of the immunoreactions occurring at the Test zone, diverse combinations of red and blue GNPs labels were captured. Therefore, the Test line assumed different colours depending on which – and how much – analyte is present in the sample. The multiplexing capability of the ‘colour-encoded assay’ is illustrated by the simultaneous detection of aflatoxin B1 (AFB1) and type-B fumonisins (FMs) in wheat and food products that made with wheat. Reproducible detection of AFB1 and FMs contamination in raw and processed food was achieved with visual cut-off levels at 1 ng mL−1 and 50 ng mL−1, respectively. The contaminant was identified based on the colour of the label according with a specific colour code. Furthermore, strips images were acquired by means of a common smartphone and analysed through RGB data analysis providing semi-quantitative detection of the two mycotoxins.

The diffusion of the legalization of cannabis for recreational, medicinal and nutraceutical uses requires the development of adequate analytical methods to assure the safety and security of such products. In particular, aflatoxins are considered to pose a major risk for the health of cannabis consumers. Among analytical methods that allows for adequate monitoring of food safety, immunoassays play a major role thanks to their cost-effectiveness, high-throughput capacity, simplicity and limited requirement for equipment and skilled operators. Therefore, a rapid and sensitive enzyme immunoassay has been adapted to measure the most hazardous aflatoxin B1 in cannabis products. The assay was acceptably accurate (recovery rate: 78&ndash, 136%), reproducible (intra- and inter-assay means coefficients of variation 11.8% and 13.8%, respectively), and sensitive (limit of detection and range of quantification: 0.35 ng mL&minus, 1 and 0.4&ndash, 2 ng mL&minus, 1, respectively corresponding to 7 ng g&minus, 1 and 8&ndash, 40 ng g-1 ng g&minus, 1 in the plant) and provided results which agreed with a HPLC-MS/MS method for the direct analysis of aflatoxin B1 in cannabis inflorescence and leaves. In addition, the carcinogenic aflatoxin B1 was detected in 50% of the cannabis products analyzed (14 samples collected from small retails) at levels exceeding those admitted by the European Union in commodities intended for direct human consumption, thus envisaging the need for effective surveillance of aflatoxin contamination in legal cannabis.

Rapid and sensitive competitive enzymatic immunoassays for measuring most relevant aflatoxins in eggs have been developed by synthesizing two hapten derivatives. Polyclonal antibodies raised against a hapten obtained from aflatoxin B1 (AFB1) were exploited to set an AFB1-selective assay, whereas antibodies obtained through immunising with a hapten derived from aflatoxin M1 (AFM1) allowed us to detect four principal aflatoxins (B1, G1, B2, and G2) and the most relevant AFB1 metabolite (AFM1) with detection limits in eggs of 0.3 μg kg−1 for AFB1, AFG1, and AFM1 and 3 μg kg−1 for AFB2 and AFG2, respectively. We also established a rapid and simple protocol for extracting aflatoxins from eggs by employing aqueous methanol (70%) followed by partitioning with hexane to remove fats. The whole analytical process is simple, very rapid (the extraction requires 14 min, and the assay is completed in 30 min) and proved to be accurate and precise enough (recoveries ranged from 84 to 100% and RSD% were within 20% for intra- and inter-assay experiments) to be proposed as a first level screening method for the monitoring of the occurrence of aflatoxins in egg.

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.

A lateral flow immunoassay (LFIA) was developed for the determination of fumonisin mycotoxins. The fluorescence of CdSe/ZnS quantum dots (QDs), observed at excitation/emission wavelengths of 365/525 nm, is suppressed by the addition of silver nanoparticles (SNPs) or gold nanoparticles (GNPs) because SNPs overlap the excitation bands of the QDs, and GNPs overlap the emission bands. The fluorescence of the QDs is recovered upon addition of fumonisins, allowing for the sensitive detection in “positive mode” of the target mycotoxin by monitoring the changes of the QDs fluorescence intensity. The SNPs are found to be the most effective quenchers, while the use of GNPs allows for an efficient recovery of fluorescence and can be employed in the LFIA. The method was successfully applied to the fluorometric determination of fumonisins in spiked maize flour samples. The visual detection limit is at the ng·mL−1 level. This is four times lower compared to the colorimetric LFIA based on the use of the conventional gold NPs.

Patulin is a water-soluble mycotoxin produced by several species of fungi. Governmental bodies have placed it under scrutiny for its potential negative health effects, and maximum residue limits are fixed in specific food matrices to protect consumers’ health. Confirmatory analysis of patulin in complex food matrices can be a difficult task, and sample clean-up treatments are frequently necessary before instrumental analyses. With the aim of simplifying the clean-up step, we prepared a 256-member combinatorial polymeric library based on 16 functional monomers, four cross-linkers and four different porogenic solvents. The library was screened for the binding towards patulin in different media (acetonitrile and citrate buffer at pH 3.2), with the goal of identifying polymer formulations with good binding properties towards the target compound. As a proof of concept, a methacrylic acid-co-pentaerithrytole tetraacrylate polymer prepared in chloroform was successfully used as a solid-phase extraction material for the clean-up and extraction of patulin from apple juice. Clean chromatographic patterns and acceptable recoveries were obtained for juice spiked with patulin at concentration levels of 25 (64 ± 12%), 50 (83 ± 5.6%) and 100 μg L−1 (76 ± 4.5%). The within-day and between-day reproducibility evaluated at a concentration level of 25 μg L−1 were 5.6 and 7.6%, respectively.

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.

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 the 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 an LF device for measuring ochratoxin A (OTA), thus permitting the attainment of a highly sensitive assessment of OTA contamination, with a sensitivity gain of more than 10-fold 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 microgram per liter level requested by the European legislation, as demonstrated by a highly results obtained through the quantitative silver-enhanced LFIA and a reference HPLC-FLD on 30 samples.

We have prepared molecularly imprinted beads with molecular recognition capability for target molecules containing the penicillanic acid substructure. They were prepared by (a) grafting mesoporous silica beads with 6-aminopenicillanic acid as the mimic template, (b) filling the pores with a polymerized mixture of methacrylic acid and trimethylolpropane trimethacrylate, and (c) removing the silica support with ammonium fluoride. The resulting imprinted beads showed good molecular recognition capability for various penicillanic species, while antibiotics such as cephalosporins or chloramphenicol were poorly recognized. The imprinted beads were used to extract penicillin V, nafcillin, oxacillin, cloxacillin and dicloxacillin from skimmed and deproteinized milk in the concentration range of 5–100 μg·L−1. The extracts were then analyzed by micellar electrokinetic chromatography by applying reverse polarity staking as an in-capillary preconcentration step, and this resulted in a fast and affordable method within the MRL levels, characterized by minimal pretreatment steps and recoveries of 64–90 %.

Salivary cortisol is regarded as a reliable parameter for the noninvasive assessment of the welfare of animals, because it is strictly related to stress levels. Several methods are available for salivary cortisol measurement in mammals, however rapid diagnostic test for detecting salivary cortisol are confined to humans. The availability of such non invasive diagnostic tools operable in situ would facilitate monitoring of animal welfare. The Cortisol stress™ test provides a simple and rapid tool to discriminate cortisol levels in canine saliva above or below 4ng/ml, which has been suggested as the cut-off value for distinguishing unstressed dogs from those experiencing stress. The test is based on a competitive immunochromatographic assay (ICT) using gold nanoparticles as probes, in which the color intensity of the Test line is inversely correlated to the salivary cortisol level. The qualitative result is obtained by the visual observation of the color formed on the Test line compared to that of the Control line We evaluated the accuracy of the test by determining salivary cortisol in 85 samples of canine saliva belonging to dogs with very variable age, sex, breed, and life history, and comparing the qualitative results to those obtained by a reference ELISA kit. Agreeing results were obtained through the two methods, and the ICT showed high diagnostic sensitivity, specificity and efficiency (100%, 98.4%, and 98.8%, respectively). Furthermore, we evaluated the precision of the test by an experimental design approach, which combines errors due to within-day and between-day variation with the biological variability, and demonstrated that the test could be reliably applied for correctly classifying canine samples, according to their salivary cortisol level. Moreover, we studied the shelf-life of the device in three experimental conditions. We confirmed the stability of the ICT at 4°C and 25°C for at least six months and observed similar results for an accelerated stability study conducted for 7days at 37°C, which suggest that the stability of ICT device could be estimated by the accelerated experiment alternatively to the real-time study.

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 © 2012 John Wiley & Sons, Ltd.

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.

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.

Molecular imprinting technology has been employed for the first time to prepare a specifically affinity chromatographic stationary phase for speciation purposes. Tributyltin has been chosen as the template molecule and the non-covalent approach has been applied. Three different polymerization methods have been evaluated: (i) a composite material, (ii) a polymer prepared via-Iniferter grafting; (iii) an emulsion polymer. Columns packed with different polymers have been evaluated by liquid chromatography (LC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). The chromatographic conditions as well as the analytical characteristics of the developed method are discussed in this paper. Our findings have shown formation of specific cavities in the grafted Iniferter as well as in the emulsion polymers with the latter achieving resolution of four organotin compounds. Detection limits are similar to those obtained with commercial, but not specific, stationary phases (6 pg for monobutyltin, MBT; 10 pg for both tributyltin, TBT, and triphenyltin, TPhT; and 20 pg for dibutyltin, DBT). The main advantage of this proposed stationary phase is that good recovery is obtained for all species, including MBT. Baseline resolution for TBT and TPhT has also been obtained. The high selectivity of this column prevents matrix interferences. The method has been validated by analyzing two biota reference materials (ERM-CE477 mussel tissue and T-38 oyster tissue).

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.

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).

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.

A novel and disposable cartridge for chemiluminescent (CL)-lateral flow immunoassay (LFIA) with integrated amorphous silicon (a-Si:H) photosensors array was developed and applied to quantitatively detect human serum albumin (HSA) in urine samples. The presented analytical method is based on an indirect competitive immunoassay using horseradish peroxidase (HRP) as a tracer, which is detected by adding the luminol/enhancer/hydrogen peroxide CL cocktail. The system comprises an array of a-Si:H photosensors deposited on a glass substrate, on which a PDMS cartridge that houses the LFIA strip and the reagents necessary for the CL immunoassay was optically coupled to obtain an integrated analytical device controlled by a portable read-out electronics. The method is simple and fast with a detection limit of 2.5mgL−1 for HSA in urine and a dynamic range up to 850mgL−1, which is suitable for measuring physiological levels of HSA in urine samples and their variation in different diseases (micro- and macroalbuminuria). The use of CL detection allowed accurate and objective analyte quantification in a dynamic range that extends from femtomoles to picomoles. The analytical performances of this integrated device were found to be comparable with those obtained using a charge-coupled device (CCD) as a reference off-chip detector. These results demonstrate that integrating the a-Si:H photosensors array with CL-LFIA technique provides compact, sensitive and low-cost systems for CL-based bioassays with a wide range of applications for in-field and point-of-care bioanalyses. [Figure not available: see fulltext.]

The assessment of the molecular recognition properties of a molecularly imprinted polymer (MIP) towards its target molecule requires the determination of its binding thermodynamic parameters through the measurement of the binding isotherm that governs the binding equilibrium. This measurement is usually performed assuming that the binding properties of a MIP are not affected by the simultaneous presence of two or more ligands of comparable affinity for the binding sites (full competitivity). The validity of this requirement is particularly critical when the selectivity of a MIP is evaluated. In fact, if conditions of full competitiveness fail to exist, the selectivity measured in the presence of one ligand at a time can be markedly different from the selectivity measured in the simultaneous presence of several different ligands. The goal of this work is to test whether the binding parameters of a MIP for its template molecule result are modified by the presence of a competitor of comparable affinity. As template and competitor molecules the herbicides 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) were chosen. The binding data obtained by partition experiments were fitted by using the general Freundlich–Langmuir competitive isotherm model for multiple ligands. The results show that the equilibrium constant (Keq) and the maximum binding site concentration (Bmax) calculated in both conditions are statistically indistinguishable. Thus, it can be concluded that the thermodynamics that govern the binding behavior of a MIP works in full competitive conditions.

A pyrimethanil-imprinted polymer (P1) was prepared by iniferter-mediated photografting a mixture of methacrylic acid and ethylene dimethacrylate onto homemade near-monodispersed chloromethylated polydivinylbenzene beads. The chromatographic behaviour of a column packed with these imprinted beads was compared with another column packed with irregular particles obtained by grinding a bulk pyrimethanil-imprinted polymer (P2). The comparison was made using the kinetic model of non-linear chromatography, studying the elution of the template and of two related substances, cyprodinil and mepanipyrim. Extension of the region of linearity, capacity factors for the template and the related substances, column selectivity, binding site heterogeneity, apparent affinity constant (K) and lumped kinetic association (ka) and dissociation rate constant (kd) were studied during a large interval of solute concentration, ranging between 1 and 2000 μg/ml. From the experimental results obtained, in the linearity region of solute concentration column selectivity and binding site heterogeneity remained essentially the same for the two columns, while column capacity (at 20 μg/ml, P1 = 23.1, P2 = 11.5), K (at 20 μg/ml, P1 = 8.3 × 106 M−1, P2 = 2.5 × 106 M−1) and ka (at 20 μg/ml, P1 = 3.5 μM−1 s−1, P2 = 0.47 μM−1 s−1) significantly increased and kd (at 20 μg/ml, P1 = 0.42 s−1, P2 = 0.67 s−1) decreased for the column packed with the imprinted beads. These results are consistent with an influence of the polymerisation method on the morphology of the resulting polymer and not on the molecular recognition properties due to the molecular imprinting process.

The molecular recognition properties of molecular imprinted polymers which bind the carbamate function were studied. Functional monomers potentially able to form non-covalent interactions with the model molecule N,O-dibenzylcarbamate were selected on the basis of a computational approach describing possible interactions between the template and a small library of vinylic monomers. These results were in accordance with N,O-dibenzylcarbamate batch-rebinding measurements performed on several miniMIPs prepared with the same library. From these preliminary results, four polymers were prepared by thermoinduced radical polymerization, using ethylene dimethacrylate as a cross-linker, chloroform (MIP1, MIP3) or acetonitrile (MIP2, MIP4) as porogens and methacrylic acid (MIP1, MIP2) or acrylamide (MIP3, MIP4) as functional monomers. Molecular recognition features of these materials were studied by high-performance liquid chromatography. In this manner selectivity was evaluated by considering the column retention of a library of sixteen structural analogues of dibenzylcarbamate, characterized by the transformation of the carbamate into a related function, or by the alteration of the molecular structure. The experimental results show that methacrylic acid is more efficient than acrylamide as a functional monomer (imprinting factors: MIP1 = 24.1, MIP2 = 25.6, MIP3 = 13.3, MIP4 = 2.44), and that chloroform enhances polymer selectivity. As regards structural motifs which conditionate the selectivity, the carbamate function strictly controls the presence/absence of molecular recognition, while shape and dimension of the substituents modulate the recognition itself. In particular, a marked recognition for analogs which were slightly bigger than the template was observed (N-benzyl-O-phenethylcarbamate: MIP1 α = 1.13, MIP2 α = 1.41, MIP3 α = 0.97; N-phenethyl-N-benzylcarbamate: MIP1 α = 1.61, MIP2 α = 1.17, MIP3 α = 0.81; N,O-diphenethylcarbamate: MIP1 α = 0.89, MIP2 α = 1.20, MIP3 α = 0.55).

The current opinion about molecular imprinted polymers (MIPs) is that their molecular recognition properties are due to the presence of nanocavities formed during a polymerization process developed in the presence of a template molecule. According to this principle, the shape of these nanocavities is complementary to that of the template and non-covalent interactions are established between the binding site and a single template molecule. Nevertheless, there are some experimental indications that the real molecular recognition mechanism involves clusters of template molecules being packed into the binding site. Recently, it has been proposed that template molecules covalently linked to the binding site can act as nucleation points, enhancing the formation of these molecular clusters. We have tested this hypothesis by studying the adsorption isotherms of polymers prepared by imprinting them with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Three different polymers were considered: P0, prepared without the template, P1, whose template was represented by 2,4,5-T molecules, and P2, whose template was 1/3 constituted by the polymerisable 2-(2,4,5-trichlorophenoxyacetoxy)-ethylmethacrylate (2,4,5-TEMA) and 2/3 by 2,4,5-T. The polymers were prepared by thermoinduced polymerization of template mixtures, 4-vinylpyridine and ethylene dimethacrylate. The crushed polymers were packed into HPLC columns and frontal chromatographic runs were performed by eluting the columns with a mobile phase containing variable amounts of 2,4,5-T. The experimental adsorption isotherms were fitted by using several isotherm models, and the Freundlich–Langmuir model was found to give the best fitting in terms of F -test. All the models considered showed a significant difference between the affinity constant values measured for the polymer P1 and P2, with a higher value for the polymer P2 (for Freundlich–Langmuir model: polymer P1, k =(2.00±0.43)×10 4 M −1 ; polymer P2, k =(1.93±0.0535)×10 5 M −1 ; ratio P2/P1, 9.65±2.09). Such experimental results support the hypothesis that a polymer prepared with a limited amount of template covalently attached to the binding site shows an increased affinity for the template itself.

In this paper we show that the carbodiimide-induced polymerisation of amino acid mixtures in aqueous medium and in presence of estradiol produces the mixtures of peptides with an average molecular weight of 2–6 kDa that are characterised by possessing molecular recognition properties towards estradiol. After the removal of the templating molecule, the binding properties of the peptide mixtures were studied using spectrophotometric and immunochemical methods. The experimental results show the presence of molecular recognition behaviour for all the peptide mixtures obtained by polymerisation in presence of estradiol, with affinity constant values between 0.44×10 9 and 6.6×10 9 M −1 , while the same mixtures obtained without estradiol show lower affinity constant values between 2.2×10 6 and 1.3×10 9 M −1 . The molecular recognition behaviour was found to be highly selective, as the binding constants of peptides towards the structural homologues testosterone and progesterone are lower than three orders of magnitude. Peptide fractions separated by ion-exchange chromatography show the same molecular recognition properties, with affinity constant values between 3.2×10 6 and 7.1×10 9 M −1 . Similarities and differences between this polymerisation technique and the molecular imprinting technique are briefly discussed.

An enzyme-linked immunosorbent assay (ELISA) was used for the determination of fenoxycarb in apple leaf samples. Single step extraction procedures with phosphate–citrate buffered solution containing different amounts of methanol were tested showing that a solvent percentage of 20% (v/v) was the best condition, with recoveries between 85 and 100% in the working range of 25–500 μg kg −1 and a negligible matrix effect. The low detection limit reached, 1 μg kg −1 against 50 μg kg −1 for the recommended liquid chromatographic method, makes the ELISA more suitable for determinations of the fenoxycarb residues in apple leaf samples. The reliability of the ELISA was evaluated by assaying the insecticide in spiked and contaminated samples by three different approaches: direct determination, standard addition method with a calibration graph, and the dilution test. The corresponding coefficients of variation were, respectively, 11, 22 and 27%. The direct determination on the (1+1) diluted apple leaf extract was used to measure the insecticide residues in samples collected in the north-eastern Italian regions of Veneto and Trentino-Alto Adige.

We have developed a simple and accurate biosensor based on a chemiluminescent (CL)-lateral flow immunoassay (LFIA) method integrated in a smartphone to quantitatively detect salivary cortisol. The biosensor is based on a direct competitive immunoassay using peroxidase-cortisol conjugate, detected by adding the chemiluminescent substrate luminol/enhancer/hydrogen peroxide. The smartphone camera is used as light detector, for image acquisition and data handling via a specific application. We 3D-printed simple accessories to adapt the smartphone. The system comprises a cartridge, which houses the LFIA strip, and a smartphone adaptor with a plano-convex lens and a cartridge-insertion slot. This provides a mini-darkbox and aligned optical interface between the camera and the LFIA membrane for acquiring CL signals. The method is simple and fast, with a detection limit of 0.3 ng/mL. It provides quantitative analysis in the range of 0.3-60 ng/mL, which is adequate for detecting salivary cortisol in the clinically accepted range. It could thus find application in the growing area of home-self-diagnostic device technology for clinical biomarker monitoring, overcoming the current difficulties in achieving sensitive and quantitative information with conventional systems taking the advantage of smartphone connectivity and the enhanced performance of the included camera.

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.

A multiplex chemiluminescent biosensor for simple, rapid and ultrasensitive on-site quantification of aflatoxin B1 and type B-fumonisins in maize samples has been developed. The biosensor integrates a multiplex indirect competitive lateral flow immunoassay (LFIA) based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a lensless "contact" imaging configuration. The developed assay requires a simple extraction of the analytes from maize flour samples followed by their detection with a 30 min assay time. The use of chemiluminescence detection allowed accurate and objective analytes quantification, enabling simultaneous detection of type B-fumonisins and aflatoxin B1 down to 6 mu g kg(-1) and 1.5 mu g kg(-1), respectively, thus fulfilling the standards imposed by the legislation of European Union. Maize flour samples spiked with both analytes were subjected to multiplex analysis obtaining recoveries ranging from 80 to 115% and the coefficient of variation below 20%. Finally, analysis of naturally contaminated maize samples resulted in a good agreement between CL-LFIA and a validated confirmatory HPLC-UV and commercial ELISA kit, obtaining recoveries in the range 88-120%. The proposed CL-LFIA protocol is rapid, inexpensive, easy-to-use, and fit for the purpose of rapid screening of mycotoxins in maize flour.

One of the goals of the new automated immunoassay analysers is to perform direct analysis in complex matrices thus overcoming traditional limits of homogeneous immunoassays: reduced sensitivity and ability to determine above all proteins and antibodies. The aim of this work was to demonstrate the feasibility of quantitative homogeneous immunoassay for small analytes in a complex matrix with a rapid and easy assay by exploiting an agglutination reaction. We studied the steroid hormone progesterone, and used new technology, the Copalistrade mark, which uses a special optical-sizing flow particle analysis and a semiconductor laser as a light source. We used polystyrene microbeads coated with the antigen as markers and put them in competition with the analyte in solution for the analytical antiserum. We synthesised and tested different conjugates of progesterone-bovine serum albumin, optimising all the experimental parameters. We performed a short pre-treatment of the serum and we obtained a detection limit of 0.011 ngcm(-3) and a working range between 0.026 and 43 ngcm(-3). We estimated human serum specimens and, with minor experimental revisions, the amounts of progesterone found agreed accurately with AutoDELFIAtrade mark analysis. The recoveries are good. All the experimental steps are easy, rapid and enable us to process many samples contemporarily.

The synthesis of polycarboxylated derivatives of β-cyclodextrin obtained by reacting maleic anhydride and suitable thiolic compounds is described. The derivatives were characterised by potentiometric analyses and 13C-NMR spectra. The products obtained show a great solubility in water, but also in ethanol and methanol and act as complexing agents for many bivalent metallic cations. These derivatives are probably biodegradable and non toxic.

A molecularly imprinted polymer which recognises the mycotoxin ochratoxin A was prepared using the mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L) -phenylalanine as a template. The polymer was obtained by dissolving the template, methacrylic acid and ethylendimethacrylate in chloroform and polymerising the mixture by thermal treatment at 60 °C. The monolith obtained was crushed, sieved to 30–90 μm and extensively washed till the template could no longer be found in the washing solution. The binding properties towards the template, ochratoxin A and several related molecules were measured by eluting with acetonitrile and chloroform a HPLC column packed with the imprinted polymer. The experimental results show that the polymer recognises not only the template well, but also the ochratoxin A. The specific molecular recognition effect is due to hydrogen bond interactions but in order to assure the full recognition effect adjunctive steric factors are necessary. The magnitude of these interactions can be controlled by the use of limited amounts of acetic acid in the mobile phase. From the measurement of the relative selectivity it was found that only the simultaneous presence of the carboxyl, the phenolic hydroxyl and certain peculiar substructures such as the chlorine atom assures the whole recognition of the template.

The equilibrium binding constants and the binding site concentrations of an immobilised rabbit antiserum towards progesterone-11α-hemisuccinate-bovine serum albumin were measured for different enzyme tracers. The enzyme tracers obtained by linking progesterone to horseradish peroxidase (HRP) through different spacer arm structures were: progesterone-11α-hemisuccinate-HRP (the homologous), progesterone-11α-hemiglutarate-HRP, progesterone-11α-hemiadipate-HRP, progesterone-11α-hemisebacate-HRP, progesterone-11α-hemisuccinamido-γ-aminobutyrate-HRP and progesterone-11α-hemisuccinamido-β-alaninate-HRP. If compared with the homologous enzyme tracer (provided with a five atom spacer arm and with an affinity of 1×10 9 M −1 ) the affinity of enzyme tracers with spacer arms of at most six atoms showed a 10-fold decrease whereas about a two-fold decrease was observed with spacer arms of seven and nine atoms. When an amido group which simulates the link between the carboxylic group of the progesterone derivative and the proteic lysine in the immunogen was inserted into 9 and 10 atom spacer arms in the very same position of the immunogen a sharp affinity increase was observed (1.2×10 9 M −1 for progesterone-11α-hemisuccinamido-β-alanilate-HRP and 1.5×10 9 M −1 for progesterone-11α-hemisuccinamido-γ-aminobutyrate-HRP). These results showed that in order to reach and exceed the homologous tracer affinity, a four or five atom spacer arm elongation must be coupled to a suitable amido group insertion. An identical trend was observed for the antibody binding site concentrations indicating the presence of two main classes of binding sites in the antiserum used.