NANOSTRUCTURED BIOSENSORS FOR THE DETECTION OF FOODBORNE PATHOGENS AND TOXINS
Creators: name: MURA, STEFANIA
Publisher: name: Universit� degli Studi di Milano
Year: 2010

description: New, fast, low cost and simple methods were developed to provide tools to detect a possible contamination of foodborne pathogens or environmental toxins, avoiding the diffusion of dangerous food. New biosensors were created using nanostructured thin films deposited on silicon wafers as substrates. In detail mesoporous materials (pores of 2-40 nm) of SiO2, TiO2, HfO2, ZrO2 were synthesized via ?evaporation induced self assembly?, and the behaviour of these materials was studied at different temperature and in PBS solution. Mesoporous titania thin films have been identified as the materials with the best properties and therefore were used for the development of the final devices. First of all a biosensor for the detection of dioxins was developed. Titania films were functionalized with APTES, an organic linker, and three peptides selective for Dioxins (TCDD) were linked to the amino-terminal groups. The peptides were bonded to a fluorescent marker, FITC, and the fluorescence variations were studied after the absorption of TCDD on the films. In this way it was possible to obtain a chip able to detect pM levels of TCDD. The second part of the work was based on the detection of pathogens with FTIR, using mesoporous titania as substrate for the development of the biosensor. This time titania thin films were functionalized with specific antibodies for the detection of E.coli O157:H7. The absorption of antibodies was carried out directly on the substrates and on the substrates functionalized with APTES and GA (Glutaraldehyde) used as cross linker. With the functionalized films were reached detection limits of 1x102 CFU/ ml of E.coli. These results obtained with our devices have been competitive with the existing bioassay methods but with advantages compared to the old techniques as low cost, simplicity, speed of analysis, and possibility to use on-field. This methods can be effective for a screening of samples but, for more specific analysis, a better sensibility is required. To reach lower detection limits was used another vibrational technique, Raman spectroscopy, complementary to FTIR, taking advantage of new properties due to the SERS effect. This technique is based on the detection of analytes adsorbed, or in close proximity, of a special resonant surface of a noble metal (Ag). Starting from our materials (mesoporous titania thin films) were grown Ag0 nanoparticles into the pores of titania films that worked as a mold for directing the size and the distribution of nanoparticles, avoiding nanoparticle dispersion and aggregation. The features were monitored to obtain a material able to detect analytes in very low concentration and the effectiveness of these materials was studied dipping titania-AgNPs thin films in Rhodamine B isothiocyanate (RhBITC) and Cytochrome C (CytC) solutions. The results obtained from raman measures revealed that films with the analyte (TiO2 films without AgNPs) showed a Raman spectrum visible until a concentration of 1x10-3M; while with our new substrates (TiO2 films with AgNPs) it was possible to discriminate the spectrum of RhITC and Cytocrome C until a concentration of 1x10-16 M, obtaining an analytical enhancement factor AEF of 1x1013. This is an excellent result that can be applied to the detection of different contaminants because each one has a characteristic fingerprint and can be discriminated with this technique with or without the use of labels. In this way will be possible to use these substrates for the detection of contaminants in very low concentration, until a single molecule level, opening the route to new ways of detection, simple, fast, low cost; achieving the objective of our work.