Fabio Di Nardo

A simple and compact smartphone accessory for quantitative chemiluminescence-based lateral flow immunoassay for salivary cortisol detection

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.

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.

Multicolor immunochromatographic strip test based on gold nanoparticles for the determination of aflatoxin B1 and fumonisins

AbstractThe 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. Graphical abstractBlue desert rose-like gold nanoparticles (DR-GNPs) were synthesized, characterized and applied as label for the ImmunoChromatographic Strip Test (ICST) technique, in which red spherical GNPs (s-GNPs) are usually employed. The combined use of the blue DR-GNP and red s-GNPs allowed developing of an intuitive multicolor ICST for the simultaneous detection of aflatoxin B1 and fumonisins in maize flour.

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.

Miniaturized Biosensors to Preserve and Monitor Cultural Heritage: from Medical to Conservation Diagnosis

The point-of-care testing concept has been exploited to design and develop portable and cheap bioanalytical systems that can be used on-site by conservators. These systems employ lateral flow immunoassays to simultaneously detect two proteins (ovalbumin and collagen) in artworks. For an in-depth study on the application of these portable biosensors, both chemiluminescent and colorimetric detections were developed and compared in terms of sensitivity and feasibility. The chemiluminescent system displayed the best analytical performance (that is, two orders of magnitude lower limits of detection than the colorimetric system). To simplify its use, a disposable cartridge was designed ad hoc for this specific application. These results highlight the enormous potential of these inexpensive, easy-to-use, and minimally invasive diagnostic tools for conservators in the cultural heritage field.

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.

Screening of a Combinatorial Library of Organic Polymers for the Solid-Phase Extraction of Patulin from Apple Juice

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.