Article pubs.acs.org/ac
First Report of a Direct Surface Plasmon Resonance Immunosensor for a Small Molecule Seafood Toxin Betsy Jean Yakes,*,† Kelsey M. Kanyuck,‡ and Stacey L. DeGrasse† †
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5100 Paint Branch Parkway, College Park, Maryland 20740, United States ‡ Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, Maryland 20742, United States ABSTRACT: Tetrodotoxin (TTX), a small molecular weight neurotoxin, is responsible for poisoning events that traditionally occur from consumption of contaminated puffer fish. Recent studies have shown a growing number of foods contaminated with TTX and a larger number of waters and associated countries where the toxin may occur. The apparent expanding prevalence of TTX supports a growing need for screening assays that can be used to detect potentially harmful food. In the past few years, surface plasmon resonance (SPR) biosensors have been developed for rapid, robust detection of TTX; however, these assays focus on detection of unbound antibody from an inhibition reaction with the toxin. This manuscript introduces the first direct immunoassay for a seafood toxin, specifically TTX. Major advantages of this assay compared to indirect assays include increased speed of analysis, decreased use of biological reagents, and improved confidence in the detection of the toxin, along with the ability to characterize the antibody/toxin interaction. The analytical method introduced in this paper could be applied to other seafood toxins, as well as to a wide range of low molecular weight targets.
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in the United States. Examples of these events include illness due to consumption of imported f ugu;12 illegally imported, misbranded puffer fish;13 and individual consumption of imported puffer fish.14 Other poisonings have occurred in Europe on the Israeli Coast of the Eastern Mediterranean from Lessepsian immigrant puffer fish15 and from TTX contaminated trumpet shells from waters off Portugal.16 Furthermore, consumable species, including gastropods, containing TTX have been found in water off the coast of Portugal17 and in puffer fish in the Aegean Sea of Greece,18 both of which indicate a potential growing risk of illnesses from tetrodotoxin poisoning. In order to minimize risk of poisoning events, the United States Food and Drug Administration (FDA) provides guidance to field personnel via an Import Alert on all species of puffer fish and fishery products with puffer fish as well as strict guidance on the preparation process and importation of puffer fish19 as a means to control hazardous product from entering the country. Even with these guidelines, there is a need for rapid, sensitive, and accurate detection methods for tetrodotoxin in order to identify potentially toxic food as well as to confirm the toxin associated with potential outbreaks. Detection methods for TTX include traditional bio/ analytical techniques such as high-performance liquid chroma-
etrodotoxin (TTX) is a low molecular weight (319 Da) toxin with multiple analogs that is putatively produced by bacteria.1 This potent neurotoxin can accumulate in certain marine vectors2 and, upon consumption of contaminated food, cause illness in humans and other animals. TTX poisoning is through toxin binding to site 1 on voltage-gated sodium channels3 and is generally characterized by numbness of the lips and tongue, tingling in the face and extremities, nausea/ vomiting, diarrhea, shortness of breath and, in extreme cases, death in affected individuals. This toxin, deemed the “quintessential natural toxin”,4 has a lethal dose for 50% of the population (LD50) in mammals of 2−10 μg/kg intravenously5 and has earned a position on the U.S. Department of Health and Human Services (HHS) Select Agents and Toxins list.6 TTX poisonings mainly have been associated with consumption of Indo-Pacific Ocean puffer fish (i.e., f ugu); however, poisonings have also occurred from puffer fish obtained from the Atlantic Ocean, Gulf of Mexico, and Gulf of California.7 TTX has also been found in other vectors including xanthid crabs,8 marine gastropods,9 and goby fish.10 In addition to waters traditionally known to harbor TTXcontaining organisms, there is concern that rising water temperatures and increasing marine life diversity may allow this toxin to emerge in nontraditional areas.11 Further, an increasing globalization of food stuffs could lead to poisoning events. For example, while illnesses most commonly occur in Asia, specifically Japan where puffer fish is native and f ugu is considered a delicacy,2 poisonings have also recently occurred © XXXX American Chemical Society
Received: June 20, 2014 Accepted: August 13, 2014
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dx.doi.org/10.1021/ac502271y | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Article
tography (HPLC) with fluorescence detection,20 liquid chromatography with mass spectrometry (LC-MS),21 mouse bioassay,22 receptor binding assays,23 and enzyme-linked immunosorbent assays (ELISAs).24 Additionally, emerging sensor methods have been developed to improve upon existing techniques in the areas of speed of screening samples, reliability, specificity for TTX, and decreased use of animals/ biological reagents.25 Previously designed surface plasmon resonance (SPR) biosensors have been shown to be capable of detecting low concentrations of TTX in puffer fish extracts, spiked milk, and spiked apple juice,26,27 and a single-laboratory validated SPR assay for TTX in gastropod extracts has also been established.11 While these SPR biosensors offer advantages such as reduced use of animals/biological reagents and improved screening over traditional detection of TTX, an assay that directly detects the toxin itself (rather than unbound antibody from an inhibition reaction) could be of benefit for increased speed of analysis, decreased use of biological reagents, improved confidence in the detection of the toxin, and improved ability to characterize the antibody/toxin interaction. Direct assays for small molecules (
