Brevetoxins (BTXs) are highly toxic biotoxin and can cause human intoxication through food chain. The detection of brevetoxins is very difficult due to lack of optical and electrochemical (EC) signal. In this work, we developed an ultrasensitive capillary electrophoresis (CE) immunoassay and EC method for the determination of BTX-B by gold nanoparticles (AuNPs) assisted signal generation and sequential stacking concentration. The AuNPs were synthesized by sodium citrate reduction of HAuCl₄ in water. The AuNPs were conjugated with horseradish peroxidase (HRP) and antibody (Ab) to immobilize the HRP and Ab on the AuNPs surface with the molar ratio of HRP/Ab of 9/1. The Ab conjugated on the AuNPs surface incubated with limited amount of BTX-B in standard solution or shellfish samples to produce immunocomplex on the basis of the noncompetitive immunoreactions. Before sample injection, a NaOH plug with 10 cm height difference for 150 s was hydrodynamically injected into the separation capillary. After incubation for 40 min at room temperature, the immune sample was then electrokinetically injected into the capillary at 10 kV for 330 s. The positively charged analytes migrated rapidly into the capillary and were neutralized and stacked at the boundary between sample and NaOH plug, which led to the first preconcentration. After sample loading, the capillary inlet vial was changed to low-pH buffer solution, and H⁺ in the buffer solution moved rapidly into the capillary toward cathode across the neutral analytes zone. The neutralized analytes were positively charged again and the injected analytes were further condensed. Next, the formed immunocomplex, unbound HRP-Au-Ab probe and the excess HRP were separated by CE and sensitively detected by EC detection. AuNPs were used as carriers of HRP and Ab in order to carry out EC detection with the EC signals derived from catalytic reaction of the carried HRP to the H₂O₂/o-aminophenol system. Simultaneously, the EC signal was highly amplified by improving the HRP/Ab molar ratio on the surface of AuNPs. The proposed method by AuNPs assisted signal generation and on-line sequential concentration realized the sensitive and rapid determination of BTX-B in shellfish samples. In the range between 0.1 and 120 ng/mL, the assay allowed quantitative determination of BTX-B and the limit of detection (LOD) was 26 ng/L. The LOD was 365-fold lower than ELISA method. The amplified sensitivity was enhanced by high HRP/Ab ratio at AuNPs surface and sequential preconcentration. The proposed method provides a convenient and sensitive analytical approach for the determination of trace BTX in complex samples.

Key words: Brevetoxin B, capillary electrophoresis, gold nanoparticle, sequential stacking, immunoassay, electrochemical detection