Construction and optimization of an optical biosensor based on LSPR for the detection of Brettanomyces bruxellensis

The yeast Brettanomyces bruxellensis, is well-known in the oenological field for being the cause of significant defects in wine called “Brett character” that lead to consequent economic losses for the cellar. The methods used for the microbiological analysis base on the utilization of isolation media, and molecular techniques such as PCR, nested-PCR, RFLP, FISH and dot blot that use primers, endonucleases and labelling procedures. A new approach bases on the development of nanotechnology associated with Localized Surface Plasmon Resonance (LSPR) phenomenon which can reduce the time needed to obtain results. Nanometer scale DNA biosensors lead to several advantages such as short analysis time, minimum liquid handling and a multiple detection possibility. Moreover, the high sensitivity of LSPR have been utilized to fabricate a LSPR label-free optical biosensor in connection with gold nanoparticle substrate (Au NPs). The development of LSPR nanobiosensors for the rapid and sensitive detection of B. bruxellensis has been obtained by the deposition of gold nanoparticles onto a glass slide to create a surface for the immobilization of a thioled specific DNA probe (capture probe) used as bioreceptor for the target DNA molecules extracted from wine yeasts. Various protocols were designed to test different temperature and times for the immobilization of the capture probe. Moreover, several hybridization times were tested to optimize the hybridization between the complex capture probe and target DNA. Pure DNA from B. bruxellensis was used as positive control and pure DNA from Saccharomyces cerevisiae was used as negative control. A sensitivity of 10cells was obtained.