A reagent-free binding assay for cholera toxin based on sequestration electrochemistry using lactose-functionalized magnetic microbeads.

We established a reagent-free one-step electrochemical binding assay for cholera toxin (CT) using magnetic microbeads functionalized with pyrroloquinoline quinone (PQQ) as an electroactive compound and lactose as the ligand for CT. The detection principle in this assay is based on "sequestration electrochemistry", i.e., the binding of CT to the lactose portion immobilized on the magnetic microbead surface causes a decrease in the electrochemical response of PQQ, which is also immobilized by binding with lactose on the microbead surface. The voltammetric response of the PQQ-modified magnetic microbeads was measured using a magnetic carbon paste rotating-disk electrode, onto which the functionalized magnetic microbeads were adsorbed by rotating the electrode at 1000 rpm for 60 s. The response of the functionalized microbeads decreased with increasing the CT concentration. The specificity of the assay was confirmed by adding bovine g-globulin, which has a similar isoelectric point as CT, to the solution containing the functionalized microbeads. No decrease in the electrochemical response was observed in this case. After magnetic preconcentration, the limit of detection for CT was 58.8 ng ml-1. This simple and sensitive approach provides a rapid platform for CT detection and highlights the potential of sequestration electrochemistry for toxin monitoring in complex samples.