Doctor of Philosophy
Department of Chemistry
Minett, Andrew Ian, Electrochemical detection of microorganisms using conducting polymers, Doctor of Philosophy thesis, Department of Chemistry, University of Wollongong, 2000. http://ro.uow.edu.au/theses/1141
The rapid detection of microorganisms in the food, pharmaceutical, clinical medicine, environmental and agricultural industries is of the utmost importance. Unfortunately, the best current technology has been able to offer is detection times of several days for quantitative results. The development of conducting polymer films has led to an increase in research activity involving the use of these materials for the design and application of sensors. Their ability to undergo electrochemical switching between oxidised and reduced states, which is correlated with the movement of anions and cations, has seen conducting polymers successfully used to produce electrochemical responses for particular species of interest. Conducting polymers can also be used as a matrix for the immobilisation of biological components such as enzymes, proteins or antibodies. The immobilisation of these components adds specificity to the sensors. It the combination of a biological component, immobilised onto a suitable transducer, that is the basis of the expanding field of biosensor research.
Recent successes in biosensor research have resulted in reduced detection times of the order of minutes or hours. The main disadvantage of these techniques however, has been the inability to successfully detect small viable numbers (<104 microorganisms/ml (mo/ml) in these short periods of time.
The focus of this work was the development of a technique suitable for the rapid and quantitative detection of a particular microorganism, Listeria monocytogenes. Chapter 3 investigated the incorporation of an antibody into a conducting polymer matrix. A comparison of various immobilisation techniques including physical entrapment, co-immobilisation and covalent attachment of the biological component was undertaken. It was found that the covalent attachment method provided films with more reproducibility and sensitivity compared to those prepared by the other methods.
In Chapter 4, an investigation into signal generation is described. Conventional electrochemistry and the comparison of two different resistometric methods were investigated. It has been suggested that resistance measurements offer greater sensitivity than amperometric or potentiometric detection methods, however this was found not to be true in this instance. A lack of reproducibility and a high detection limit was the limiting factor in this work, so an alternative signal generation method was considered. Conventional mediators were trialled to enhance the bioelectrochemical transfer of electrons from the microorganism to the transducer. This latter part of this chapter investigates the ability of ferricyanide, toluidine blue, uniblue and two anthraquinones to mediate the microorganism-electrode interaction. Toluidine blue was the only mediator tested where significant changes upon the addition of Listeria were observed. These changes were not proportional to concentration, making this technique non-ideal.
In Chapter 5, a successful approach to the mediation of the bioelectrochemical process is described. This research has shown a water-soluble conducting polymer (polymethoxyaniline sulfonate (PMAS)) to be successful in mediating the electron transfer from a microorganism (Listeria monocytogenes) to a glassy carbon substrate. Graphical representation of the change in current, at a potential of +0.8V, observed after the addition of the Listeria, showed a linear relationship between peak current and microorganism concentration. The detection limit of this technique has been determined to be between lOmo/ml and lOOmo/ml and a detection time in the order of a few minutes (<15mins).
The changes recorded in the electrochemistry can also be observed using UV-vis spectroscopy. Addition of the microorganism to the parent polymer results in changes absorbance being observed. Fractionating the polymer into eight separate molecular weight fractions between 1700 and 21000amu gave spectra with different absorbance values resulting from the addition of the Listeria to each individual fraction.
The uniqueness of the PMAS response has been confirmed by studies using another water-soluble conducting polymer, poly-pyrrole butane sulfonate. This water soluble polypyrrole showed no responses to the addition of the microorganisms using either electrochemistry or spectroscopy. It is suggested that the high sensitivity of this technique can be attributed to the unique nature of the PMAS. It is the interaction the microorganism and the conveyance of the electron processes along the polymer backbone that allows small viable numbers to be reproducibly detected.