Scanning electrochemical microscopy for the characterisation of surfaces modified with biological molecules

Abstract

This thesis describes a novel fabrication procedure for microelectrodes to be used with the scanning electrochemical microscope (SECM), the characterisation of a variety of novel impedance based immunosensors, and the characterisation of a novel oligonucleotide biosensor. The thesis firstly describes the development of a protocol for the fabrication of reproducible microelectrodes characterised to identify suitability in use with the SECM. The thesis then describes the use of SECM in feedback mode to interrogate a variety of antibody-polyelectrolyte films determining whether the changes observed by impedance were detectable by SECM. A screen printed carbon ink surface was patterned with an array of biotinylated polyethyleneimine (PEI) which was exposed to Neutravidin and then the biotinylated antibody of interest. Using ferrocenecarboxylic acid as the redox couple, the array was interrogated by SECM, scanning before and following exposure to a series of concentrations of the complementary antigen and a non-complementary antigen. Upon the exposure of the PEI/Neutravidin/biotinylated antibody array to the antigen, the feedback current over the functionalised region was observed to change. The change observed increased as the concentration of the antigen exposed to the array was increased showing linear correlation. On exposure of the array to a non-complementary antigen, only a small change in the feedback current was observed. NSE, PSA, Ciprofloxacin and NTx were all investigated with limits of detection of 0.5 pg ml-1, 1 pg ml-1, 0.1 ng ml-1 and 1 nM respectively. Finally using a similar method as employed above, SECM was utilised in the detection of binding events of short oligonucleotides. Once again scans were conducted before and after exposure to both complementary and non-complementary oligonucleotide sequences and by subtraction absolute changes in feedback current were determined. On exposure to the complementary oligonucleotide sequence a change in feedback was observed when the array was exposed to the non-complementary oligonucleotide sequence, as with the antibody/antigen array, only a small change in the feedback current was observed.