desarrollo de un multibiosensor de adn para el diagnóstico temprano de cáncer de mama

198 Pages·2008·5.12 MB·Spanish

DESARROLLO DE UN MULTIBIOSENSOR
DE ADN PARA EL DIAGNÓSTICO
TEMPRANO DE CÁNCER DE MAMA

Tesis para optar al grado de Doctora en Bioquímica

LAURA GARCÍA CARRASCOSA
Dirigida por Prof. Laura M. Lechuga
Tutor: Catedrático José M. Cuezva

Presentada ante el Departamento de Biología Molecular de la
Facultad de Ciencias de la Universidad Autónoma de Madrid

Llevada a cabo en el Instituto de Microelectrónica de Madrid del
Centro Nacional de Microelectrónica (IMM-CNM-CSIC) y en el
Centro de Investigación en Nanociencia y Nanotecnología
(CIN2:CSIC-ICN)

Madrid, Junio 2008

A mis padres

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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
Plasmon 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 quimisorption and
minimize fisisorption 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.
INDICE

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1. MOTIVACIÓN Y OBJETIVOS……………………………………………..5
2. ESTRUCTURA……………………………………………………………….9
3. INTRODUCCIÓN…………………………………………………………..11
3.1 Biosensores …………………………………………………………………11
3.1.1. INTRODUCCIÓN……………………………………………………11
3.1.2 TIPOS DE BIOSENSORES…………………………………………...14
3.1.3 BIOSENSORES ÓPTICOS……………………………………………20
3.1.3.1 Biosensor de resonancia de plasmón superficial………………...22
3.1.4 BIOSENSORES NANOMECÁNICOS……………………………….26
3.1.5 BIOSENSORES DE ADN…………………………………………….30
3.2 Inmovilización de biomoléculas sobre superficies biosensoras de oro …36
3.3 Detección precoz de cáncer de mama hereditario………………………..42
3.3.1. IMPORTANCIA DE LA GENÉTICA EN EL DESARROLLO DEL
CÁNCER………………………………………………………….…...42
3.3.1.1 Susceptibilidad genética y cáncer………………………….……..44
3.3.1.2 Tipos de mutaciones involucradas en cáncer……………………45
3.3.2 GENÉTICA DEL CÁNCER DE MAMA HEREDADO …………....48
3.3.2.1 Implicación de los genes BRCA1 y BRCA2……………………..49
3.3.2.2 El gen BRCA1………………………………………………….51
a) Estructura, expresión y función………………………………51
b) Mutaciones más frecuentes en el BRCA-1……………………53
3.3.2.3 Evaluación de riesgo genético en el cáncer de mama hereditario..54
3.3.3 TEST GENÉTICOS ACTUALES PARA LA DETECCIÓN DE
MUTACIONES EN LOS GENES BRCA1 Y BRCA2……………….58

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ÍNDICE
4. MATERIALES Y MÉTODOS………………………………………….63
4.1 Selección de secuencias de ADN y preparación de muestras reales de
pacientes con cáncer de mama heredado………………………………64
4.1.1 SECUENCIAS DE ADN DE CARÁCTER GENÉRICO……….64
4.1.2 SECUENCIAS DE ADN TIPO PCR PORTADORAS DE
MUTACIONES EN EL GEN BRCA-1…………………………65
4.2 Descripción de los sistemas biosensores empleados………………….69
4.2.1 BIOSENSOR DE RESONANCIA DE PLASMÓN SUPERFICIAL
(SPR)………………………………………………………….….69
4.2.2 BIOSENSOR NANOMECÁNICO……………………………...73
4.3 Técnicas ex-situ empleadas para apoyo a la caracterización de la
inmovilización e hibridación de ADN en biosensores……………….75
4.3.1 FLUORESCENCIA……………………………………………...75
4.3.2 MARCAJE RADIOACTIVO…………………………………….77
4.3.3 ESPECTROSCOPÍA FOTOELECTRÓNICA DE RAYOS X
(XPS)…………………………………………………………….77
4.4 Protocolos de inmovilización y hibridación……………………………79
4.4.1 BIOSENSOR SPR…………………………………………..……80
4.4.2 BIOSENSORES NANOMECÁNICOS………………………….81
4.4.3 TÉCNICAS EX-SITU …………………………………………....82

5 RESULTADOS……………………………………………………………83
5.1 Caracterización de los procesos de inmovilización e hibridación en
biosensores SPR y nanomecánicos………………….………………….83
5.1.1 CONDICIONES DE INMOVILIZACIÓN……………………..83
5.1.2 CONDICIONES DE HIBRIDACIÓN…………………………..96
5.1.3 OPTIMIZACIÓN DE LA DETECCIÓN POR MEJORA DE LA
ACCESIBILIDAD…………………………………………...…...98
5.2 Condiciones para la detección de mutaciones en SPR……………….105
5.3 Detección en SPR de mutaciones vinculadas al BRCA-1….......…….109
5.3.1 FORMATO DE DETECCIÓN DE UN ÚNICO ANALITO.....110
5.3.2 FORMATO DE DETECCIÓN MULTI-ANALITO................…112

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