Abstract of PhD Thesis

Investigation of redox polymers for enzymatic fuel cell and DNA sensor devices: synthesis, electrochemical characterisation and applications

Paul Kavanagh (2006) - National University of Ireland, Galway

This thesis is concerned with the synthesis of osmium-based redox complexes and redox polymers. Films of these redox polymers deposited on electrode surfaces are investigated for their application in biological fuel cells and DNA sensor devices.

Chapter 1 of this thesis serves as a general introduction to redox polymer modified electrodes and their application in sensor and biological fuel cell devices.

Chapters 2 and 3 are concerned with the synthesis and characterisation a range of osmium polypyridyl complexes and their conjugation to polymers. The preparation and characterisation of several non-commercially available polypyridyl ligands is also described in Chapter 2, including a high yielding synthesis of 4,4′-diamino-2,2′-bipyridine. The complexes were purified and characterised using a range of analytical techniques, including flash chromatography, CHN, 1H NMR, IR and UV-Vis spectroscopies, and mass spectrometry. Electrochemical characterisation was accomplished using cyclic voltammetry (CV). Redox potentials for these complexes ranged from −665 mV to 0 mV vs. Ag/AgCl. Chapter 3 describes the synthesis and electrochemical characterisation of redox polymers containing osmium polypyridyl metal sites. The electrochemical characteristics of films of the redox polymers on electrodes, were determined using CV, with redox potentials ranging from −0.115 V to 0.492 V. All polymer films displayed finite diffusion characteristics at low scan rates, with the onset of semi-infinite diffusion evident at higher scan rates (≥50 mV s-1). The design and study of a biological fuel cell consisting of a glucose oxidase-based anode and a laccase-based cathode using selected osmium-based redox polymers as mediators is also described in Chapter 3. The prototype biological fuel cell, under physiological conditions, yielded maximum power density of 16 µW/cm2 at a cell voltage of 0.25 V. At lower pH values maximum power density was 40 µW/cm2 at 0.4 V (pH 5.5) and 10 µW/cm2 at 0.3 V (pH 4.4).

Chapter 4 describes the production of stable and reproducible sensing surfaces that may be capable of detecting specific sequences of DNA. The co-immobilisation of redox polymer, [Os(2,2′-bipyridine)2(poly-vinylimidazole)10Cl]+/2+, and single-stranded DNA (coding for the ssrA gene of Listeria Monocytogenes­) at carbon electrodes yielded films that were insufficiently stable. Stability was improved by conjugation of the components to a pre-formed chemisorbed layer of cysteamine at a gold electrode surface. A peak height and peak area precision of 2.7 and 3.3 (% relative standard deviation) was obtained for a batch of these sensing surfaces (n = 8), with no change in surface stability over periods in excess of 1 hour.

The detection of DNA hybridisation using the sensing surfaces described in Chapter 4, is presented in Chapter 5. The initial approach involved attempts at detecting hybridisation through changes in the voltammetric response of the redox polymer film upon hybridisation. However, the film response upon interaction with complementary or non-complementary DNA under conditions of low ionic strength was as a result of non-specific electrostatic interactions between redox polymer and DNA. Increasing the salt concentration of the hybridisation buffer to 1 M NaCl eliminated this non-specific binding. However, no film response under conditions of high ionic strength was subsequently observed. Amplification of an electrochemical hybridisation signal using an enzyme-labeled target DNA sequence was therefore attempted. Hybridisation between the immobilised probe DNA and a biotin-conjugated target DNA sequence, followed by addition of a glucose oxidase-avidin conjugate, resulted in electrical contact between the enzyme and the mediating redox polymer film. A linear increase in current response with respect to concentration of target DNA in the range of 10-9 M to 10-6 M was observed, with a detection limit of ~1.4 fmol in a 7 µL droplet, corresponding to 0.2 nM of target DNA.  Regeneration of the sensing surface was possible by treatment with 0.25 M NaOH solution. After rehybridisation of the regenerated surface with the target DNA sequence, >95% of the current was recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface.

The main findings of the thesis are summarised in Chapter 6, and indicators to possible future research strands to further the concepts described in the thesis are presented.