Abstract
Tetrodotoxin (TTX) is a low molecular weight (~319 Da) neurotoxin found in a number of animal species, including pufferfish. Protection from toxin tainted food stuffs requires rapid, sensitive, and specific diagnostic tests. An emerging technique for the detection of both proteins and nucleic acids is Fluidic Force Discrimination (FFD) assays. This simple and rapid method typically uses a sandwich immunoassay format labeled with micrometer-diameter beads and has the novel capability of removing nonspecifically attached beads under controlled, fluidic conditions. This technique allows for near real-time, multiplexed analysis at levels of detection that exceed many of the conventional transduction methods (e.g., ELISAs). In addition, the large linear dynamic range afforded by FFD should decrease the need to perform multiple sample dilutions, a common challenge for food testing. By applying FFD assays to an inhibition immunoassay platform specific for TTX and transduction via low magnification microscopy, levels of detection of ~15 ng/mL and linear dynamic ranges of 4 to 5 orders of magnitude were achieved. The results from these studies on the first small molecule FFD assay, along with the impact to detection of seafood toxins, will be discussed in this manuscript.
Highlights
Tetrodotoxin (TTX), a low molecular weight neurotoxin, is found in a number of organisms including pufferfish, California newts, parrotfish, frogs of the genus Atelopus, blue-ringed octopus, starfish, angelfish, and xanthid crabs [1,2]
In the most basic form of these assays, an antibody is immobilized on the flow cell surface, the antigen is captured to the surface, and the bound complex is labeled with a secondary antibody
Fluidic Force Discrimination (FFD) is performed and the amount of beads bound to the surface quantified
Summary
Tetrodotoxin (TTX), a low molecular weight neurotoxin, is found in a number of organisms including pufferfish, California newts, parrotfish, frogs of the genus Atelopus, blue-ringed octopus, starfish, angelfish, and xanthid crabs [1,2]. The mouse bioassay does not test for a specific toxin, just the time it takes for a mouse to die following intraperitoneal injection of a sample In this assay, the time of death is proportional to toxicity; the dynamic range for this proportional relationship is small. By combining a modified inhibition immunoassay with the FFD technology, a rapid and sensitive detection platform was developed This proof of concept study indicated the ability of FFD to detect small molecules, TTX in this case, it showed that FFD can yield a large linear dynamic range of detection. The substrate preparation, immunoassay format, detection technique, and the results obtained from preliminary studies will be discussed
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