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Analysis Kinetics of Domoic Acid (DA) by BLI (CAT#: STEM-MB-0282-CJ)

Introduction

Domoic acid (C15H7NO6, molecular weight 311.33) is a kind of small-molecular excitatory proline derivative and potent neurotoxin, which is mainly produced by the Pseudonitzschia genus. After being ingested by aquatic organisms, DA is enriched through the food chain, thus threatening human health. Importantly, the heat stability of DA makes its toxicity difficult to overcome through ordinary cooking methods. Domoic acid contains three carboxyl groups and a secondary amino group, and its form in solution is easily affected by pH. There are eight isomers of DA (A–H). These isomers may be the reaction products of DA exposed to ultraviolet light, rather than the natural products of algae. The main contaminant of marine and aquatic organisms is DA, which is the most toxic of all these isomers. There is a great similarity between the structures of DA, glutamic acid (Glu), and kainic acid (KA). However, the excitatory effect of DA on nerve cells is more than 100 times higher than Glu and 2–3 times higher than KA. Domoic acid is a stimulant that can directly or indirectly activate glutamate receptors, including the N-methyl-D-aspartate (NMDA) receptor, α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor, and kainate or kainic acid receptor. These receptors distribute in different regions of the central nervous system and are activated by DA under different physiological conditions. Domoic acid can cause damage to neurons in the hippocampus and neocortical neurons. It has been found that when the content of DA in shellfish reaches 40 mg/kg, it can cause poisoning, and 150 mg/kg leads to a risk of death. The acute reference dose (ARfD) of an adult is 30 μg DA/kg b.w. The European Union stipulated that the DA content should not exceed 20 mg/kg in shellfish, and the European Food Safety Authority (EFSA) recommended that the safe edible limit of DA be 4.5 mg/kg in shellfish. High toxicity, quick onset of symptoms, and the lack of suitable antidote bring great challenges to the detection and prevention of DA.




Principle

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.

Applications

Neurobiology/Neurodegeneration; Immunology/Inflammation; Toxicology

Procedure

1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.

Materials

• Equipment: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: DA and KA