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.