Development of instrumentation to allow the detection of microorganisms using light scattering in combination with surface plasmon resonance.

This paper describes work carried out to develop a biosensor which allows two separate detection principles to operate simultaneously at the same surface. A prototype device was constructed that provided Kretschmann-configuration surface plasmon resonance (SPR) measurement of refractive index (RI) changes using an 820 nm LED light source, whilst a 635 nm diode laser was used to produce light scattering signals from bacterial spores. Both effects occurred at a gold-coated surface. The RI changes were measured conventionally from the side of the gold layer nearer to the light sources. The scattered light was imaged from the opposite face which was in contact with the aqueous sample. Specific detection of bacterial spores through the light scattering mode using antibody capture was investigated. The flow dynamics and interactions with the surface of individual spores were observed. A comparison with SPR for detection using the same antibody/antigen pair was made. Spore suspensions that were readily detectable by light scattering at 10(7) ml(-1) did not provide significant responses by SPR. The potential for future developments is discussed.

Specific assays for bacteria using phage mediated release of adenylate kinase.

A sensitive and rapid assay method for the specific detection of bacteria was developed using Escherichia coli and Salmonella newport as the test organisms. Bacteriophages were used to provide specific lysis of the bacteria and then the release of cell contents was measured by ATP bioluminescence. Increased sensitivity was obtained by focusing on the bacteria's adenylate kinase (AK) as the cell marker instead of ATP as conventionally used. Fewer than 10(3) E. coli cells could be readily detected in less than 1 h. Salmonella newport assays, although as sensitive, were slower and took up to 2 h. The effects of the culture medium, the phage, and the presence of non-specific bacteria were examined.

Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354.

We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position. Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.

Gene probe assays on a fibre-optic evanescent wave biosensor.

This report describes experiments to detect oligonucleotide hybridization at the surface of a fibre-optic evanescent wave biosensor. Conditions were optimized and the time course of hybridization reactions were found to be very rapid compared to conventional hybridization assays. Binding was easily reversed by heating and the sensor surface could be reused many times. Short (16- and 20-mer) oligonucleotides bound to the waveguide surface could be used to detect fluorescein-labelled complementary sequences at the nanomolar level. Polymerase chain reaction was used to generate a 204-base oligomer that was attached to the waveguide surface. Fluorescein-labelled oligos bound at either the proximal or distal ends of this probe were found to give similar outputs.