Rapid identification of bacterial spores is of fundamental importance in diagnostics and bio-defence. The concept of a reflection
hologram as the interactive element in a biosensor presents a novel approach to the detection of pathogenic organisms, in which the holographic element provides both the analyte-responsive matrix and the optical interrogation and reporting transducer. This thesis describes the development and characterisation of novel holographic sensors for the detection of
Bacillus species spore
germination and subsequent vegetative growth. Divalent metal ion-sensitive
holograms containing a methacrylated analogue of nitrilotriacetic acid (NTA) as the chelating monomer were successfully used to monitor Ca2+ ions released during
B. subtilis spore germination in real-time, which was within minutes of sample addition. Similarly, pH-sensitive holograms comprising methacrylic acid (MAA) as the ionisable functional monomer were shown to monitor the pH change associated with early vegetative metabolism following germination of
B. megaterium spores. Casein and starch–based holographic matrices, prepared by co-polymerisation of the appropriate substrate with acrylamide, were used to detect exo-enzymes released during various stages of
B. megaterium and
B. subtilis vegetative cell growth. Developments have also been made towards a spore-specific sensor through exploitation of small, acid-soluble spore proteins (SASPs) in the holographic matrix. These holograms were shown to act as substrates for recombinant germination protease (GPRS) activated by calcium dipicolinate (Ca2+-DPA), demonstrating proof-of principle of an enzyme-linked holographic sensor for potential signal amplification.To provide detection selectivity, integration of these sensors into a ‘capture/culture’ device is proposed, in which spores are captured using specific anti-spore antibodies prior to holographic detection. Given this intention, germination of antibody-captured
B. subtilis spores was demonstrated and revealed that the kinetics of spore germination were unaffected by antibody-binding. For the purpose of reducing detection limits, the effect of inoculum size on germination kinetics was also investigated. This revealed little evidence for density-dependent behaviour, suggesting that the detection of low spore numbers within reasonable time-frames (with respect to germination kinetics) is achievable, given adequate sensitivity of the sensor system.This study demonstrates that a range of holographic sensors, when used in combination and interfaced with an initial antibody-capture stage for specificity, provide a novel, comprehensive means for the detection of viable bacterial spores.