Flow Cytometric-Membrane Potential Detection of Sodium Channel Active Marine Toxins: Application to Ciguatoxins in Fish Muscle and Feasibility of Automating Saxitoxin Detection

Abstract

Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.

A novel and functional assay for pharmacological effects of marine toxins, saxitoxin and tetrodotoxin by cardiomyocyte-based impedance biosensor

“…5, it could be concluded that STX could significantly reduce the beating rate (P < 0.05, n = 6) in the concentration range (0.0007-1.62 M), while STX had no significant difference (P > 0.05, n = 6) in cell index and amplitude, which indicated that the beating rate of the biosensors was a sensitive parameter to STX. The results from our experiments correlated well with previous findings reported by other investigations, where different types of function-based assays were used [18,33,34].…”

Report of the Expert Meeting on Ciguatera Poisoning

“…This approach is promising as depolarization can be observed in minutes versus the overnight incubations typically used for the viability end point of the N2A-MTT assays. Flow cytometric detection (Manger et al, 2014) allows selection of the most responsive cells using electronic gating. Assays based on voltage-sensitive dyes (including N2A-Flow) have not been optimized or practised by other laboratories to the same extent as the N2A-MTT format, with the latter used extensively and refined over the past 25 years.…”

Potential Threats Posed by New or Emerging Marine Biotoxins in UK Waters and Examination of Detection Methodology Used in Their Control: Brevetoxins

“…However, with further experience in the method and with routine protocols in place for passaging the assay cells there could still be potential for further developments in this area, whilst the assay certainly remains an effective research tool. New flow cytometric assays using N2As in combination with voltage sensitive dyes are more rapid than the cytotoxicity assay, and respond to brevetoxins [ 105 ] but have not been applied to detecting brevetoxins in shellfish extracts. Similarly, a new fluorescence microscopy method involving the use of an alternative cell line to N2A cells, was developed for the assessment of cytotoxicity.…”