Simultaneous multiplexed stripping voltammetric monitoring of marine toxins in seafood based on distinguishable metal nanocluster-labeled molecular tags.

Marine toxins from microscopic algae can accumulate through the food chain and cause various neurological and gastrointestinal illnesses for human health. Herein, we designed a new ultrasensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of brevetoxin B (BTX-2) and dinophysistoxin-1 (DTX-1) in seafood using distinguishable metal nanocluster-labeled molecular tags as traces on bifunctionalized magnetic capture probes. To construct such a bifunctionalized probe, monoclonal mouse anti-BTX-2 (mAb(1)) and anti-DTX-1 (mAb(2)) antibodies were co-immobilized on a magnetic bead (MB-mAb(1,2)). The distinguishable metal nanoclusters including cadmium nanoclusters (CdNC) and copper nanoclusters (CuNC) were synthesized using the artificial peptides with amino acid sequence CCCYYY, which were used as distinguishable signal tags for the label of the corresponding bovine serum albumin-BTX-2 and bovine serum albumin-DTX-1 conjugates. A competitive-type immunoassay format was adopted for the online simultaneous monitoring of BTX-2 and DTX-1 on a homemade flow-through magnetic detection cell. The assay was based on the stripping voltammetric behaviors of the labeled CdNC and CuNC at the various peak potentials in pH 2.5 HCl containing 0.01 M KCl using square wave anodic stripping voltammetry (SWASV). Under optimal conditions, the multiplexed immunoassays enabled simultaneous detection of BTX-2 and DTX-1 in a single run with wide working ranges of 0.005-5 ng mL(-1) for two marine toxins. The limit of detection (LOD) and limit of quantification (LOQ) were 1.8 and 6.0 pg mL(-1) for BTX-2, while those for DTX-1 were 2.2 and 7.3 pg mL(-1), respectively. No non-specific adsorption and electrochemical cross-talk between neighboring sites were observed during a series of procedures to detect target analytes. The covalent conjugation of biomolecules onto the nanoclusters and magnetic beads resulted in good repeatability and intermediate precision down to 9.5%. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of 12 spiked samples, including Sinonovacula constricta , Musculista senhousia , and Tegillarca granosa , between the multiplexed immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for analysis of BTX-2 and DTX-1.