Bio-functionalization of nanostructured electrodes for biosensing applications
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Bio-functionalization refers to the modification of surfaces using biomolecules such as peptides, enzymes, DNA, aptamers, antibodies, and viruses etc. Bio-functionalization of electrode surfaces could impart new properties that enable their use in a specific application such as biosensing. This dissertation mainly focused on two types of bio-functionalization of nanostructured electrodes for electrochemical sensing applications. The first was based on enzymes for oxygen sensing applications and the second was based on viruses for bacterial cell isolation and detection applications. In the first application of oxygen sensing, laccase enzyme derived from two different sources, namely Trametes versicolor (fungi) and Bacillus FNT (thermophilic bacterium) were used for electrode bio-functionalization. Metal oxides were first functionalized with laccase from Trametes versicolor (TVL). However, the resulting electrodes exhibited less than ideal stability for electrochemical sensing applications. The limitation was overcome by using a thermophilic bacterium laccase Bacillus sp. FNT (BL). Multiwall carbon nanotubes (MWCNT) modified electrode was functionalized with Bacillus FNT laccase. The resulting electrodes exhibited excellent electrochemical stability and high enzyme activity compared to TVL functionalized electrodes for oxygen reduction bio-electrocatalysis. A detailed electro-kinetic study was also conducted using BL functionalized electrodes for oxygen reduction reaction. In the second application, new methods were developed for functionalizing electrode surfaces with viruses for whole bacterial cell biosensor applications. Carbon nanotube modified electrode surfaces were functionalized with bacteriophages using a charge-directed, electric-field induced immobilization method developed specifically for myoviruses. The T2 phages functionalized electrodes were established for the capture and electrochemical detection of Escherichia coli B. In order to achieve simultaneous detection of foodborne pathogens from real food matrix, a bacteria separation method was developed by bio-functionalization of magnetic particles using bacteriophages. P100 phages were used to functionalize magnetic particles for selective isolation and enrichment of Listeria monocytogenes from complex media. The work was complemented by a demonstration of simultaneous L. monocytogenes enrichment and detection using an electrochemical biosensor. To enable preferred orientation of P100 on the magnetic particles, the bacteriophage was genetically engineered to express biotin on the capsid of the phage, which could be crosslinked to the streptavidin present on the magnetic particle surface.