ELECTROCHEMICAL CHARACTERIZATION OF CARMINIC ACID TOWARDS THE USE AS AN ELECTROCHEMICAL MOLECULAR BEACON FOR NUCLEIC ACID DETECTION by Santa Maria Gorbunova B.Sc., University of Brighton, 2011 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2015 © Santa Maria Gorbunova 2015 Abstract Worldwide, more than a million people die from tuberculosis (TB) every year. Although the disease is curable, treatment is complicated by multi-drug resistant and extensively drug-resistant TB strains. To detect TB and differentiate between its strains, a sensitive and specific point-of-care device is re- quired. Previous studies show that carminic acid (CA), an anthraquinone derivative, is suitable as an electrochemical molecular beacon due to the ability to switch on and off its electrochemical activity on its dimerization. Characterization of the electrochemical activity of CA at low concentrations (1 µM to 1 mM) over a range of pH values was performed using methods such as cyclic voltammetry, square wave voltammetry and Kouteck´y-Levich analysis on a rotating disk electrode. CA species of different protonation, which are predominant at pH 1.1, pH 4.1, pH 6.6 and pH 10.5, were examined in more detail. All measurements were carried out on a glassy carbon electrode in phosphate buffer solution electrolyte. It was found that CA undergoes a diffusion limited two proton two electron redox reaction with an overall peak potential shift of 61 mV per pH unit. Electrochemical measurements of the fully protonated CA resulted in additional current peaks that were assigned to an adsorption process of a CA reduction product. Generally, CA has faster electron transfer kinetics in more acidic environment and no elec- trochemical activity was observed for the fully deprotonated CA species at pH 10.5. While SWV could be used for quantitative analysis of CA for the concentrations up to 1 mM, its redox current signal was determined not to be concentration dependent at high measurement frequencies. These frequencies can also be adjusted to be more sensitive towards either the redox peak potentials with sharper peaks at low frequencies or the electron transfer kinetics based on kinetic dependent peak currents at high frequencies. The limit of detection for CA at pH 7.0 was found to be as low as 10 nM when measured using 200 Hz SWV. ii Preface The experimental work presented in this thesis is an original and unpublished work by the author, S. M. Gorbunova, and the development of the experimental procedures and data analyses were done in collaboration with the supervisor, D. Bizzotto. iii Table of Contents Abstract .................................................... ii Preface ..................................................... iii Table of Contents .............................................. iv List of Tables ................................................. vi List of Figures ................................................ vii Nomenclature .................................................x Acknowledgements ............................................. xiv 1 Introduction ...............................................1 1.1 Tuberculosis.............................................1 1.2 Carminic acid............................................3 1.3 Scope of the project........................................ 17 2 Electrochemistry Background .................................... 19 2.1 Theory................................................ 19 2.2 Methodology............................................. 26 3 Experimental ............................................... 37 3.1 Materials............................................... 37 3.2 Electrochemical cell........................................ 38 3.3 Instruments.............................................. 40 3.4 Measurements............................................ 40 iv Table of Contents 4 Results and Discussion ........................................ 41 4.1 Carminic acid electrochemistry at different pH......................... 41 4.2 Characterization of carminic acid electrochemical activity at low pH............ 48 4.3 Study of the electron transfer kinetics using an RDE..................... 50 5 Summary and Future Work ...................................... 59 References ................................................... 61 Appendices A Kinetic Analysis of Cobalt Hexammine ............................... 64 v List of Tables 3.1 Composition of electrolyte at the experimental pH values................... 38 3.2 List of the experimental values of CV sweep rates, SWV measurement frequencies and RDE rotations rates.......................................... 40 4.1 CA redox peak potentials and the estimated formal electrode potential (E0’) in different pH environments obtained from CV in 100 µM CA using a GC WE, sweep rate – 20 mV/s. 42 4.2 Summarized results of 100 µM CA kinetic parameters on a GC electrode......... 54 A.1 Data obtained from cobalt hexammine CV........................... 65 A.2 Summarized cobalt hexammine kinetic parameters obtained by different methods.... 68 vi List of Figures 1.1 Carminic acid structure with indicated deprotonation sites...................4 1.2 Fractions of CA species at different pH values..........................4 1.3 Classic polarography of 0.86 mM CA in 0.1 M LiClO4, scan rate: 4 mV/s.........5 1.4 SWV of CA modified graphite-polyester composite electrode in organic and aqueous so- lutions..................................................5 1.5 (a) Two electron two proton, (b) two electron one proton and (c) two electron reduction of quinone.................................................6 1.6 Half-wave potential and pH relationship of 2-hydroxy-1,4-benzoquinone..........7 1.7 A comparison of a 3 proton 2 electron and 2 proton 2 electron redox reaction of a hydrox- ylquinone................................................7 1.8 Two step quinone reduction steps.................................8 1.9 Pourbaix diagram for the disproportionation redox reaction of 2-AQMS...........9 1.10 Scheme of squares diagram of major quinone states..................... 10 1.11 Scheme of squares fitted for anthraquinones.......................... 10 1.12 A redox mechanism of a quinone................................. 11 1.13 CV of 2 mM 1,4-benzoquinone in DMSO with 0.2 M Bu4NPF6 at scan rate of 100 mV/s without addition of benzoic acid, with 0.03 M and 1 M benzoic acid............. 12 1.14 CV of 1 mM anthraquinone-2-sulfonic acid at scan rate of 100 mV/s on a gold electrode in aqueous buffered solutions of different pH.......................... 12 1.15 (a) Bound CA concentration dependence on DNA concentration. (b) Emitted fluorescence spectra of CA and CA - DNA complex............................... 14 1.16 Mechanism of CA as ECMB in a hairpin loop form....................... 15 1.17 Mechanism of CA as ECMB in a stem loop form........................ 15 1.18 CV of CA as an ECMB in its dimer form, as ECMB in its monomer form and in its free form (CA)............................................... 16 vii List of Figures 1.19 Differential pulse voltammetry of CA-ECMB with a different complementarity nucleic acid sequences............................................... 17 2.1 Schematic representation of electrical double layer....................... 20 2.2 Potential drop across the electrical double layer......................... 21 2.3 Energy barrier symmetry for different α values......................... 23 2.4 Example of a Tafel plot........................................ 24 2.5 Schematic representation of a cell as a circuit.......................... 26 2.6 The potential applied in linear sweep and the resulting charging current.......... 27 2.7 Applied potential and the resulting CV............................... 28 2.8 Potential steps in staircase CV................................... 30 2.9 Potential steps in DPV........................................ 30 2.10 Current response to the applied potential steps......................... 31 2.11 Example of DPV............................................ 31 2.12 Potential profile in SWV....................................... 32 2.13 Example of SWV........................................... 33 2.14 Mass transfer velocities and flows at the RDE.......................... 34 2.15 Example of current vs. potential measurement using RDE.................. 35 2.16 Example of a Kouteck´y-Levich plot................................ 36 3.1 Electrochemical cell setup..................................... 39 4.1 CV of 100 µM CA in different pH phosphate buffer solutions on GC WE (area 7.0 mm2), sweep rate of 20 mV/s........................................ 42 4.2 Peak potential and pH relationship of 100 µM CA in different pH phosphate buffer solutions on GC at a sweep rate of 20 mV/s................................. 43 4.3 CV of 100 µM CA in pH 6.6 phosphate buffer at different sweep rates on GC WE (area 7.0 mm2)................................................ 44 4.4 Peak current and pH relationship of 100 µM CA at different sweep rates on GC..... 44 4.5 Relationship between current and square root of sweep rate................ 45 4.6 CV of different concentration CA in pH 6.6 phosphate buffer on GC (area 7.0 mm2) with a sweep rate of 20 mV/s....................................... 46 4.7 Relationship between peak current (log) and concentration of CA (log).......... 46 viii List of Figures 4.8 Proposed CA