Holographic sensors for the detection of bacterial spores.
Holographic sensors for the detection of Bacillus species spore germination and vegetative growth are described. Reflection holograms were fabricated using a diffusion method for the distribution of ultra-fine silver bromide grains into pre-formed polymer films, followed by holographic recording using a frequency doubled Nd:YAG (532 nm) laser. Changes in holographic replay wavelength or diffraction intensity were used to characterise the swelling behaviour or structural integrity of a range of holographic matrices in response to various extracellular products of bacterial spore germination and vegetative metabolism. 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; the holographic response manifested as a 16 nm blue-shift in diffraction wavelength over the progress of germination. Similarly, pH-sensitive holograms comprising methacrylic acid (MAA) as the ionisable monomer were responsive to changes in pH associated with early vegetative metabolism following germination of B. megaterium spores; a visually perceptible blue-shift in holographic replay wavelength of 75 nm was observed. Casein and starch-based holographic matrices, prepared by co-polymerisation of the appropriate substrate with acrylamide, were used to detect exo-enzymes released during later stages of B. megaterium and B. subtilis vegetative cell growth; holographic responses of both matrices were visible as a reduction in diffraction intensity due to progressive fringe disruption caused by enzymatic cleavage. The combined monitoring of various germination and growth events using the range of aforementioned holographic sensors provides a novel, comprehensive means for the detection of viable bacterial spores.
Bhatta
D
D
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom.
Christie
G
G
Madrigal-González
B
B
Blyth
J
J
Lowe
C R
CR
D020106:Acrylamide / Q000378:metabolism; D001412:Bacillus subtilis / Q000254:growth & development / Q000378:metabolism*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002364:Caseins / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D006696:Holography / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D013171:Spores, Bacterial / Q000254:growth & development / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D000516:alpha-Amylases / Q000378:metabolism