Construction and interrogation of enzyme microarrays using scanning electrochemical microscopy - optimisation of adsorption and determination of enzymatic activity.
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.
Cranfield Health, Cranfield University, Bedford, UK.
William S
WS
Roberts
Frank
F
Davis
Stuart D
SD
Collyer
Séamus P J
SP
Higson
D000327:Adsorption; D016227:Benzoquinones / Q000032:analysis; D004800:Enzymes, Immobilized / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D008839:Microelectrodes; D008855:Microscopy, Electron, Scanning; D040081:Protein Array Analysis; D013379:Substrate Specificity
D016227:Benzoquinones; D004800:Enzymes, Immobilized; C004532:quinone; D006735:Horseradish Peroxidase