Browsing by Author "Roberts, William St John"

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    Construction and interrogation of enzyme microarrays using scanning electrochemical microscopy - optimisation of adsorption and determination of enzymatic activity.
    (Royal Society of Chemistry, 2011-12-21T00:00:00Z) Roberts, William St John; Davis, Frank; Collyer, Stuart D.; Higson, Seamus P. J.
    Scanning electrochemical microscopy (SECM) has been used to image and study the catalytic activity of horseradish peroxidase (HRP) immobilised in a patterned fashion onto glass slides. Microarrays of HRP islands could be deposited on amino-modified glass slides using glutaraldehyde crosslinking combined with the SECM being used as a micro-deposition device. The enzymatic activity of the immobilised enzyme on the surface was in the presence of its substrate observed to give rise to substantial positive feedback between the slide and the SECM microelectrode tip. Conversely when either blank slides - or slides coated with HRP which had been subsequently thermally denatured were utilised, these showed negative feedback effects. Various conditions such as enzyme concentration, incubation time and substrate concentration were systematically varied to optimise sensitivity. Regular arrays of HRP could be assembled and when imaged, displayed lower limits of detection of 1.2 × 10(-12) mol ml(-1) of benzoquinone.
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    Scanning electrochemical microscopy for the interrogation of biologically modified surfaces
    (Cranfield University, 2007-01) Roberts, William St John; Higson, Seamus P. J.; Johnson, G.; Lonsdale, D.; Griffiths, J.; Smart, Palie
    This thesis describes two novel applications of scanning electrochemical microscopy (SECM) to biological systems. The first involves the characterisation of a novel, impedance based genomic DNA biosensor - previously developed within the group. SECM in feedback mode was used to interrogate a DNA-polyelectrolyte film to determine whether the changes observed by impedance were detectable by SECM. Using the SECM micropositioning device to pattern a carbon ink substrate, a dotted array of polyethylenimine (PEI) and single stranded DNA (ssDNA) was fabricated. Using hexamine ruthenium chloride as the redox couple, the array was then interrogated by a SECM area scan before and following exposure to complementary and non-complementary DNA. Upon the exposure of the DNA/PEI array to complementary DNA, the feedback current over the functionalised region was observed to increase, whereas on exposure of the array to non-complementary DNA, an increase in feedback current was also observed - but to a lesser degree. The second SECM application described involves the use of SECM to detect protein expression in cells. Using an established immunochemical protocol, the transmembrane protein, CD44, expressed by cultured RT112 cells was labelled via a primary/secondary antibody complex to horseradish peroxidase. Using hydrogen peroxide and hydroquinone, the activity of the HRP label was subsequently detected by SECM in feedback mode. The microelectrode tip was biased at a potential of -0.4V, a potential sufficient for the reduction of benzoquinone - the redox active product of the HRP catalysed reaction. The work presented represents the first application of SECM to detecting protein expression in cells and effectively demonstrates the promise this technique holds for immunochemical applications. An analysis of Uniscan’s innovation network is also presented, which provides a valuable insight into the management of such resources and how they may be orchestrated to extract maximal innovative value for all parties involved in a collaborative relationship.