Tesis doctoral de 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.
Datos académicos de la tesis doctoral «Desarrollo de un multibiosensor de adn para diagnóstico temprano de cáncer de mama«
- Título de la tesis: Desarrollo de un multibiosensor de adn para diagnóstico temprano de cáncer de mama
- Autor: Laura García Carrascosa
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 17/07/2008
Dirección y tribunal
- Director de la tesis
- Laura Lechuga Gomez
- Tribunal
- Presidente del tribunal: isabel Correas hornero
- césar Fernández sánchez (vocal)
- arben Merkoí§i (vocal)
- Antonio Bernard miana (vocal)