Current sensing devices designed to detect explosive vapours are bulky, expensive and in need of technological improvement. A wide variety of methods and an even wider range of physical chemistry issues are involved in the detection process. Consequently, new concepts and applications occur continually, making this an exciting field of electrochemistry; which is opening up a multitude of possibilities for fundamental research and applications.
This project involves a novel, genetically engineered nitroreductase containing a
sequence of six cysteine amino acids, enabling strong thiolate bonds to form on a gold electrode surface without the loss of enzyme activity. The cloned enzymes were shown to be active with a range of nitroaromatics and a nitro ester, namely 2-ethylhexyl nitrate, and afforded different rates of reaction for each analyte. Differences were also seen between different strains of nitroreductases. The optimum pH (pH 7.1) and temperature dependence ( < 40 ° C) for the enzymes were established along with their turnover numbers.
Evidence for the direct immobilisation of the enzymes, without the need for pretreatment of the surface with a self-assembled monolayer or a conducting polymer, was obtained by IR analysis, UV-visible spectroscopy, and cyclic voltammetry, illustrating that the cysteine tagged nitroreductases were successfully immobilised at the gold electrode surface. In addition, quartz crystal microbalance studies revealed that there are two stages to the absorption; the second stage was attributed to the structural reorganisation of surface
layers.
Experiments carried out with nitroreductases containing tags with various numbers of cysteines revealed that cys,2 obtained the greatest mass on the electrode. Hence, this was the modified nitroreductase chosen for the initial test of the amperometric sensor;
preliminary results demonstrate detection levels in the parts per trillion range, signifying tremendous promise towards an in situ sensor for the detection of explosives. The rate of the reduction proved to be proportional to the concentration of analytes in solution, and the system showed evidence of recovery after each sample, allowing successive samples to be taken. The potential applications associated with this novel, yet universally applicable
technique, for the immobilisation of enzymes is immeasurable.