Progress 09/01/05 to 09/01/06
Outputs
Saxitoxin (STX), tetrodotoxin (TTX) and their congeners are low-molecular weight neurotoxins, which can selectively block voltage-sensitive Na+ ion channels with high affinity. In this work, custom-built multi-channel surface plasmon resonance (SPR) biosensors are applied to the detection of STX, TTX and their congeners using antibody- or receptor-based assays. Antibody-Based Detection: A novel sensing surface was developed by immobilizing TTX via formaldehyde chemistry on a gold film covered with a mixed self-assembled monolayer (SAM) consisting of amine and hydroxyl terminated oligo-ethylene glycol (OEG) alkanethiol. The SAM composition and TTX immobilization chemistry were optimized to maximize specific binding while minimizing nonspecific binding. An inhibition assay was used to establish calibration curves for different antibody concentrations. The lowest detection limit of 0.3 ng/ml was achieved, which is better than that from ELISA. Furthermore, it was
demonstrated that one can easily regenerate the sensor surface using 50 mM NaOH. This work is presented in a publication submitted to Sensors and Actuators B Chemical. We are extending this method to the detection of STX and TTX in both clinically and food relevant complex matrices, including blood, urine, and puffer fish extracts. Receptor-Based Detection: Direct measurement of the STX and TTX toxicity of a sample is currently realized using a radio-labeled toxin and rat brain membrane preparations containing the Na+ gated ion channels. We are developing alternatives to the radio-labeled STX receptor binding assay which are also based on the ion channels purified from animals. The first approach is a competition assay using SPR with a protein-toxin conjugate; we have immobilized the purified Na+ gated ion channels from rat brain membranes onto a SPR-active gold film and have tested several STX and TTX conjugates. Results show that the biological activity of the conjugate is dependent
on the tether length. A biotin-(OEG)n-TTX conjugate with varying length OEG tethers is being produced to test this hypothesis. For the second approach a competition assay using SPR with conotoxins is being developed, conotoxins have been shown to compete with STX and TTX for the same site I binding in Na+ gated ion channels, and dissociation kinetics are much slower. Thus, conotoxins can be used to replace the protein-OEG-TTX conjugates in the competition assay. In this case, synaptosome prepared from electric eel electroplax is needed. We have obtained the eel electroplax and are currently purifying voltage-sensitive Na+ ion channels for this assay. The advantage of using conotoxins as compared to the TTX conjugate is that conotoxins are much larger (~3000 Da), enabling direct detection with SPR. In addition, we are producing and characterizing fluorescently tagged TTX conjugates for fluorescence-based detection to replace radio-labeled detection. We have developed methods for
purifying the toxin conjugates by HPLC.
Impacts
Fast, reliable, and quantitative methods are needed to both identify food with toxic levels of saxitoxin (STX) and tetrodotoxin (TTX) type molecules and to study how these toxins are produced and transferred through the food web, ultimately to human consumers. In this work, a surface plasmon resonance (SPR) biosensor is applied to the detection of STX, TTX and their congeners using antibody- or receptor-based assays. SPR sensors are fast, reliable, label-free, and quantitative tools which are suitable for this purpose. Studying the sources and vectors for toxin accumulation in food will enable regulation and identification of both existing and emerging toxin sources.
Publications
- Taylor, A.D., Ladd, J., Etheridge, S., Deeds, J., Hall, S., Jiang, S. 2006. Quantitative Detection of Tetrodotoxin (TTX) by a Surface Plasmon Resonance (SPR) Sensor. Sensors and Actuators B: Chemical. (submitted).
|