Resumen de Desarrollo de un multibiosesor de adn para el diagnostico temprano de cancer de mama
Laura García Carrascosa
This Thesis describes the development of a new methodology based on%&/label-free biosensing to detect multiple mutations within a gene. As proof of%&/concept it has been chosen the BRCA1 gene, which is related to early onset%&/of inherited breast cancer.%&/Two different biosensing technologies, Nanomechanical and Surface%&/Plasmón Resonance (SPR) biosensors have been evaluated as an alternative%&/tool to routine analytical methods for the detection of DNA point mutations.%&/For setting up an optimised biosensor method for this type of%&/diagnostics, the following issues have been addressed:%&/- The enhancement of DNA immobilization onto gold surfaces at both biosensing%&/platforms: It has been used the well-known method of thiol self assembly%&/manolayers to immobilize DNA sequences in a controlled%&/way. Three different thiol groups have been evaluated to test DNA%&/linking to the gold surface in order to maximize chemisorption and%&/minimize phisysorption phenomena, leading to highly dense DNA%&/receptor monolayers.%&/- The enhancement of hybridization onto DNA monolayers surfaces at both%&/biosensing platforms: Different strategies, as the use of lateral and%&/vertical spacers, have been tested to control the self-assembly method%&/and to improve target accessibility, leading to higher hybridization%&/efficiency.%&/- Evaluation of both biosensing platforms for DNA detection: Detection of 12%&/and 25 mer DNA sequences have been tried. Only SPR biosensor%&/were able to detect hybridization and to discriminate a single%&/mismatch within a sequence. Nanomechanical biosensors that use a%&/single microcantilever as transducer were unable to differentiate%&/between a fully complementary sequence and a non-complementary%&/one. For the nanomechanical biosensor a reference cantilever must%&/be used in order to compensate other events not related to%&/hybridization which could hide the detection of the specific%&/hybridization. On the other hand, SPR biosensors were able to detect%&/12 mer and 25 complementary sequences with a 10 nM and 100 nM%&/limit of detection, respectively. In addition, a clear discrimination of a%&/single mismatch was demonstrated. Therefore this biosensing%&/method was chosen for setting up a multianalyte label-free detection%&/format.%&/- Set-up of a multi-analyte detection format able to discriminate a single%&/mismatched in PCR like products: A methodology of DNA%&/immobilization of multiple receptor sequences, based on the%&/simultaneous co-inmobilization of two and four different sequences%&/related to BRCA1 gene have been tried. Sequential detection of%&/several PCR like target products using the same DNA monolayer was%&/demonstrated, addressing a detection limit at the nM range (50 nM).%&/The main goal of this Thesis has been the demonstration of the ability of%&/the SPR biosensor system for DNA detection and discrimination of single%&/mismatches through a multiplex format. The multiplex detection format has%&/never been described before. The establishment of this methodology, as well as%&/its ability to address a limit of detection in the nM range, sets a landmark in the%&/direct and label-free detection of DNA by biosensor devices. This allocates the%&/biosensor technology as a competitive and complementary tool for DNA%&/analysis.
