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A direct, label-free immunosensor was designed for the rapid detection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions using quartz crystal microbalance with dissipation (QCM-D) as transduction method. The sensing layer including the anti-SEA antibody was constructed by chemisorption of a self-assembled monolayer of cysteamine on the gold electrodes placed over the quartz crystal sensor followed by activation of the surface amino groups with the rigid homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDITC) and covalent linking of binding protein (protein A or protein G). Four anti-SEA antibodies (two of which from commercial source) have been selected to set up the most sensitive detection device. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1000 or 2000 ngml(-1) with a detection limit of 20 ngml(-1). The total time for analysis was 15 min. Using a sandwich type assay, the response was ca. twice higher and consequently the lowest measurable concentration dropped down to 7 ngml(-1) for a total assay time of 25 min., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) are a family of contaminants originating from the incomplete combustion of organic compounds, present in the urban air, water, soil and also foodstuff. Due to their recognised carcinogenicity, their concentration in urban air is routinely monitored and governmental instances have set rules as regards their maximum allowable concentration. Benzo[a]pyrene (BaP) is one of the PAHs, highly toxic, whose concentration correlates well with the total PAHs contents in environmental samples; its detection by immunosensors has been rarely described because of its low molecular weight and poor solubility in water. In this paper, we report the design and testing of optical and piezoelectric immunosensors for the determination of BaP by using Polarisation-Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS) or Quartz Crystal Microbalance with dissipation measurements (QCM-D) as transduction techniques, and a comparison of their analytical performances. We found relevant to set up immunosensors based on a direct or indirect competitive format. BaP was detected by PM-IRRAS with both immunosensor formats with similar sensitivity, ca. 5 μM. Flow-injection piezoelectric transduction in the indirect competitive format set up led to a slightly better sensitivity than that reached by PM-IRRAS. No general conclusion about the most efficient set up could be drawn from comparing the sensitivities, but the reported data illustrate the potential of PM-IRRAS as well as flow-injection QCM-D as efficient label-free transduction techniques., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Two antibody immobilisation procedures were compared to set up an immunosensor for goat anti-rabbit immunoglobulin (anti-rIgG), i.e. rIgG covalently bound or immobilised via affinity to protein A (PrA). In both cases, the first layer of protein was covalently bound to a mixed self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) and mercaptohexanol (C6OH) on a gold surface. The elaboration of the sensitive surfaces, as well as their selectivity and sensitivity were studied step by step by polarization modulation-reflection absorption infra-red spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM) with impedance measurement. QCM measurements showed that the viscoelastic properties of the antibody layer were markedly modified during the antigen recognition when the antibody was bound by affinity to PrA. The specific detection of antigen within a complex medium was assessed by PM-RAIRS thanks to the grafting of cobalt-carbonyl probes. Affinity constants between the immobilised rIgG and the anti-rIgG were determined from PM-RAIRS analysis.

We present a DNA biosensor based on self-assembled monolayers (SAMs) of thiol-derivatized peptide nucleic acid (PNA) molecules adsorbed on gold surfaces. Previous works have shown that PNA molecules at an optimal concentration can be self-assembled with their molecular axes normal to the surface. In such structural configuration BioSAMs of PNAs maintain their capability for recognizing complementary DNA. We describe the combined use of PM-RAIRS and synchrotron radiation XPS for the detection and spectroscopic characterization of PNA-DNA hybridization process on gold surfaces. RAIRS and XPS are powerful techniques for surface characterization and molecular detection, which do not require a fluorescence labeling of the target. We present a characterization of the spectroscopic IR and XPS features, some of them associated to the phosphate groups of the DNA backbone, as an unambiguous signature of the PNA-DNA heteroduplex formation. The N(1s) XPS core level peak after DNA hybridization is decomposed in curves components, and every component assigned to different chemical species. Therefore, the results obtained by means of two complementary structural characterization techniques encourage the use of PNA-based biosensors for the detection of DNA molecules on natural samples.

Immunosensors, based on the immobilization of a model rabbit antibody on mixed self-assembled monolayers and Protein A as a linking agent on gold transducers, were elaborated and characterized at each step by modulated polarization-infrared spectroscopy (PM-IRRAS) and occasionally by atomic force microscopy (AFM) and quartz crystal microbalance (QCM). By testing two different mixed SAMs comprising 11-mercaptoundecanoic acid (MUA), together with either decanethiol (C9CH3) or mercaptohexanol (C6OH), the role of the chemical composition and structure of the antibody attachment layer upon the sensor performance was demonstrated.