Fluorescence and Luminescence Techniques
For the purposes of endotoxin detection, mutant firefly luciferase shows
potential in becoming a fast and reliable detection method
95,96. Experiments have been performed using a mutated
version of the firefly luciferase that are able to quickly and precisely
identify solutions containing endotoxins 96. The
mutated form of North American luciferase used in the experiment
generates a luminescence 10 times as intense as the standard, wild-type
luciferase 97. The enzyme was dissolved later in a
TMAT buffer solution. In short, endotoxin is detected by Factor C,
previously discussed in the LAL test section. This activates the
protein, which cascades to activate Factor B, which in turn activates a
pro-clotting enzyme, then a clotting enzyme. The resulting protein
contains Luciferin, a precursor to the light-emitting Amino-Luciferin.
When this activated form of the fluorophore is created, the detectable
light is emitted. The results of the experiment show a distinct increase
in luminescence intensity as endotoxin concentration increases. The
lowest endotoxin concentration recorded was 0.0001 EU/mL, or 1 x
10-5 ng/mL, while the researchers report a detection
limit of this mutant-type luciferase bioluminescence technique was
0.0005 EU/ml, or 5 x 10-5 ng/ml 23.
Another important factor to mention is that this detection limit was
reported in 15 minutes. This required time is blistering in comparison
to the LAL gel-clot techniques estimated required time of 138 minutes to
nearly 1.5 hours 98. However, using firefly luciferase
returns to the issue of animal testing that can be avoided using a
recombinant version of the mutated luciferase 99.
Experiments have been performed using a peptide biosensor and attached
fluorescent probes, fluorescein-maleimide (F5M), and
tetramethylrhodamine-5-malemide (TMR5M) 73. Recently,
a fluorophore BODIPY
((4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s -Indacene)
with excitation and emission wavelengths of 485/20 and 528/20 nm were
used to quantify presence and removal of endotoxin from biological
solutions (Figure 5 ) 100-103. BODIPY dye
which is a lipid biomarker, in presence of endotoxin quenches due to
endotoxin binding to its surface signaling endotoxin contamination
100,101,104. Endotoxin detection studies have been
conducted using Alexa Fluor-labeled fluorescent endotoxin with
excitation and emission wavelengths of 490 and 525 nm
105. In this study, C-18 acyl chain modified
Fe3O4/Au/Fe3O4
nanoflowers were used for simultaneous capture and detection of
endotoxins from water samples 105. The lowest
endotoxin detection limit using this technique was 1 ng/ml
105.