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  • _______________________________________________________________________

    Electrochemical Biosensor: Multistep

    functionalization of thiolated ssDNA on gold-coated

    microcantilever

    Jorge Dulanto Carbajal

    Department of Phisics

    McGill University

    Montréal, Québec

    September, 2011

    A Thesis submitted to

    McGill University

    In partial fulfillment of the requirements for the degree of

    Master in Science

    © Jorge Dulanto Carbajal, 2011

  • Acknowledgements

    This thesis is dedicated to my mother Ana, my father Jorge, my brother Renzo

    and his family who always supported me during this challenge.

    I would like to thank my supervisor Peter Grütter who gave me this amazing

    opportunity to learn and to live Physics. Thanks also goes to Yoshihiko Nagai ‘Tigre’

    who taught me almost everything I know about this project. I would also like to thank

    James Hedberg for his help in the proof-reading of this thesis.

    Thanks to all my friends in the Physics department who always gave me energy

    to complete this wonderful period of my life.

  • Table of Contents

    Abstract.............................................................................................................. i

    Résumé............................................................................................................... ii

    1 Introduction....................................................................................................... 1

    1.1 Motivation.................................................................................................. 1

    1.2 Micromechanical cantilever-based sensor.................................................. 2

    2 Instrumentation and Methods...........................................................................7

    2.1 Electrochemical setup..................................................................................7

    2.1.1 Electrodes…..…. .................................................................................8

    2.1.1.1 Reference electrode.....................................................................8

    2.1.1.2 The counter electrode ................................................................ .8

    2.1.1.3 The working electrode .................................................................9

    2.1.2 Electrochemical cell…….....................................................................10

    2.1.3 Apparatus specifications .................................................................... 12

    2.2 Electrochemical Techniques……………………....................................... 13

    2.2.1 Cyclic Voltammetry............................................................................ 13

    2.2.2 Square wave potential (Chronoamperometry).................................... 16

    2.2.3 Double layer structure…………......................................................... 17

    2.2.4 Potential of zero charge....................................................................... 20

    2.3 Optical deflection sensor………................................................................ 21

    2.3.1 Microcantilever……………………………........................................ 21

    2.3.2 Surface stress calculation.......………... .............................................. 22

    3 Organothiol-monolayers and surface stress on gold surfaces………….......25

    3.1 Organothiol SAM formation……………………….....................................26

    3.1.1 Structure of thiol functional group.......................................................27

    3.2 Surface stress………………………………….............................................28

    3.2.1 Origin of surface stress…………………………………...…………28

    3.2.2 Surface stress at adsorbate-covered solids..........................................29

    3.2.3 Cantilever bending method……..........................................................30

  • 4 Characterization of DNA functionalization with electrochemical protocols.......................................................................................................... 33

    4.1 Experimental procedure….......................................................................... 33

    4.2 Response of DNA under electrochemical stimulation…............................ 37

    4.2.1 Response to square wave potential vs. Ag/AgCl…………............... 39

    4.2.2 Fluorescence quenching experiment………………………............... 40

    4.3 DNA single step functionalization protocol…............................................43

    4.4 DNA multistep functionalization protocol…...........................................44

    4.4.1 Characterization of DNA multistep characterization protocol……...…45

    4.4.2 Surface stress change evolution using multistep functionalization

    protocol……………………………………………………………...…48

    4.4.3 Shape evolution of stress gram using DNA multistep

    functionalization......................................................................................50

    4.4.4 Comparison of sensitivity between cyclic voltammograms and stress

    grams...................................................................................................... 55

    5 Conclusions and outlook……........................................................................ 59

    5.1 Conclusions............................................................................................ 59

    5.2 Outlook……………..……….................................................................60

    Appendix A: Desalting method for thiolated-DNA sample………………...... 63

    Appendix B: Electrochemical cell design and upgrade..................................... 65

    Appendix C: Dipoles moments on macromolecules………………...………....71

    References…………................................................................................................75

  • i

    Abstract:

    Bio-chemical sensors are an emerging and vibrant area of research. The use of

    micromechanical cantilevers is relatively new as biomechanical recognition detectors.

    Reactions on a gold coated and chemically functionalized surface produce a mechanical

    deflection of the cantilever which is used as the input signal of the detector. Within the

    area of biosensors, DNA-sensors have a wide range of applications such as DNA

    hybridization detectors, DNA mismatch sequence detectors and protein detectors.

    We designed and built a microcantilever sensor system which allows for control

    and characterization of surface conditions. This includes controlled functionalization

    which can be a dominant factor in signal generation and reproducibility in these

    systems. Additionally, we developed a multistep functionalization protocol which

    consists of a sequence of short incubations and characterizations of thiolated ssDNA on

    a gold-coated cantilever.

    Multistep functionalization is a new protocol that is used to control the ssDNA

    surface density on a gold-coated cantilever. Repeatable responses and feasible

    biosensors are obtained using this protocol.

  • ii

    Résumé:

    Les capteurs biochimiques sont un domaine émergent et dynamique de la

    recherche. L’utilisation de cantilevers micromécaniques est relativement récente en tant

    que détecteur par reconnaissance biomécanique. Des réactions sur la surface recouverte

    d’or et fonctionnalisée chimiquement produisent une déviation mécanique du cantilever

    qui constitue la source de mesure du détecteur. Dans le domaine des biocapteurs, les

    capteurs d’ADN ont un large champ d’application tel que les détecteurs d’hybridation

    d’ADN, les détecteurs de mésappariement d’ADN et les aptamères (détecteurs de

    protéines).

    Nous avons conçu et construit un système de capteur microcantilever permettant

    le contrôle et la caractérisation des conditions de surface. Une fonctionnalisation

    contrôlée est un facteur dominant pour la génération de signal et de la reproductibilité

    dans ces systèmes. Afin d’atteindre cet objectif, nous avons développé le protocole de

    fonctionnalisation en plusieurs étapes qui consiste en une séquence d’incubations de

    courte durée (5min chacune) et de caractérisations d’ADN simple brin thiolé sur le

    cantilever recouvert d’or.

    La fonctionnalisation en plusieurs étapes est un nouveau protocole qui contrô

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