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  • UNIVERSIDADE DO ALGARVE

    Development of Biosensors

    for Molecular Analysis

    Joo Miguel Encarnao

    Doutoramento em Cincias Biotecnolgicas

    ( Especialidade de Biotecnologia Molecular)

    2007

  • UNIVERSIDADE DO ALGARVE

    Development of Biosensors

    for Molecular Analysis

    Joo Miguel Encarnao

    Doutoramento em Cincias Biotecnolgicas

    ( Especialidade de Biotecnologia Molecular)

    Tese orientada por:

    Professor Guilherme Ferreira

    Professor Peter Stallinga

    2007

  • Acknowledgments

    This dissertation represents the end of a long and hard process, but also the very fruitful and

    fascinating period of my PhD research.

    I am grateful to Prof. Guilherme Ferreira, my supervisor, for believing in me and convincing

    me to quit my job and embracing a fascinant world of knowledge, science and invaluable

    experience. Our scientific discussions helped planting the seeds of my research, crystallizing

    ideas and experimental results to a whole and harmonized picture.

    I would also like to express my deep gratitude to Prof. Peter Stallinga, my co-supervisor, to

    whom I owe much of my knowledge in how to be a passionate and credible scientist. Prof.

    Stallinga has literally raised me into the scientific world, guiding me slowly towards

    becoming an independent researcher.

    Special thanks to Prof. Henrique Gomes and Prof. Paulo Pinheiro with whom I learned a lot in

    their laboratories and collaborated in part of my work. Their theoretical insight helped clarify

    some of the underlying physical mechanisms and establish wider perspectives for interpreting

    my experimental results.

    1

  • I am grateful to all those who helped me to accomplish this work, like my colleagues Luis

    Rosa, Raul Baltazar, Luisa Pedro and Rogrio Rodrigues, and also Mr. Mrio Freitas for

    technical advises and help in constructing part of the experimental setups used in this work.

    I would like to acknowledge the Portuguese Science Foundation, an organism of the

    Portuguese Ministry of Science and Higher Education, for my PhD grant number

    SFHR/BD/12772/2003 and the funding to the projects POCTI/CTM/37719/2000 and

    POCI/BIO/61912/2004.

    Devoted to my family, specially my parents Jacinto and Suzel, my brother Carlos, my

    goddaughter Ins and my grandparents Antero and Marieta. They always knew I had the

    science bug in me.

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  • O homem no nasceu para trabalhar, mas para criar

    Professor Agostinho da Silva (1906-1996)

    3

  • Contents

    Resumo ...................................................................................................................................... 7

    Abstract ................................................................................................................................... 16

    List of Figures ......................................................................................................................... 18

    List of Tables........................................................................................................................... 28

    Introduction ............................................................................................................................ 29

    1.1. What is a Biosensor? ................................................................................................................................ 29 1.2. The piezoelectric effect ............................................................................................................................. 32 1.3. Quartz crystal microbalance- QCM ......................................................................................................... 33 1.4. Physical model of a quartz crystal piezoelectric sensor........................................................................... 37 1.5. QCM as a mass sensing tool .................................................................................................................... 40 1.6. Impedance analysis of QCM .................................................................................................................... 43 1.7. Deviations in the QCM response.............................................................................................................. 51 1.8. Thesis objectives and motivations ............................................................................................................ 55

    Experimental........................................................................................................................... 58

    2.1. Reagents.................................................................................................................................................... 58 2.2. Biologicals ................................................................................................................................................ 59

    2.2.1. Oligonucleotides............................................................................................................................... 59 2.2.2. Recombinant proteins ..................................................................................................................... 59 2.2.3. Other biologicals .............................................................................................................................. 60

    4

  • 2.3. Functionalization of gold nanoparticles with oligonucleotides .............................................................. 61 2.4. Quartz Crystal Sensors ............................................................................................................................. 61 2.5. QCM surface modification....................................................................................................................... 62

    2.5.1. Sensor activation with alkanethiols................................................................................................ 62 2.5.2. Sensor functionalization with proteins and biotin ........................................................................ 63 2.5.3. Sensor functionalization with oligonucleotides ............................................................................. 64

    2.6. Experimental set-up ................................................................................................................................. 65 2.7. Impedance Analysis.................................................................................................................................. 67 2.8. Electrochemical measurements ............................................................................................................... 68

    Instrumental setup of gold QCM sensors for real time molecular analysis ..................... 69

    3.1. System check- resonant frequency response ........................................................................................... 70 3.2. System check- impedance response ......................................................................................................... 72 3.3. Crystal cleaning and regeneration........................................................................................................... 78 3.4. Interferents ............................................................................................................................................... 81

    3.4.1. Temperature .................................................................................................................................... 82 3.4.2. Evaporation...................................................................................................................................... 82 3.4.3. Non-degassed fluids ......................................................................................................................... 85 3.4.4. Mechanical noise.............................................................................................................................. 86

    3.5. Conclusions .............................................................................................................................................. 89

    Influence of electrolytes in the QCM response: Discrimination and quantification of the

    interference to correct microgravimetric data .................................................................... 91

    4.1. Influence of charged species in the response of QCM sensors............................................................... 92 4.2. Effect of small increments of electrolytes ................................................................................................ 94 4.3. Piezoelectric resonators sense and respond to modifications of the diffusive electrical double layer . 100 4.4 Use of modified BVD data as corrective tool to evaluate and quantify DNA hybridization ................. 102 4.5. Conclusions ............................................................................................................................................ 106

    Electroacoustic impedance complemented piezoelectric quantitative analysis of

    molecular systems: self assembled monolayers and streptavidin-biotin binding........... 108

    5.1. Formation of a Self Assembled Monolayer (SAM) ............................................................................... 109 5.2- Piezoelectric detection and analysis of Streptavidin binding to Biotin................................................. 114 5.3- Conclusions ......................................................................................................................