Cyclic accumulation of nanoparticles: A new strategy for electrochemical immunoassay based on the reversible reaction between dethiobiotin and avidin

A new method based on cyclic accumulation of gold nanoparticles has been proposed for determinating human immunoglobulin G (IgG) by anodic stripping voltammetry. The dissociation reaction between dethiobiotin and avidin in the presence of biotin provides efficient means for the cyclic accumulation of gold nanoparticles used for the final analytical quantification. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG is immobilized on the wells of microtiter plates. The human IgG analyte is first captured by the primary antibody and then sandwiched by secondary antibody labeled with dethiobiotin. Avidin-Au solution was introduced to react with dethiobiotinylated antibody followed by adding of bioitin solution to wash it down. The dissociation of avidin-Au from dethiobiotinylated antibody was caused by displacing function of biotin to dethiobiotin. Making use of the ability of dethiobiotinylated antibody keeping its activity after the association/dissociation reaction, the alternatively treatment with avidin-Au and biotin solutions could realize cyclic accumulation of gold nanoparticles for ASV quantification. The anodic peak current increases gradually with the increasing accumulation cycles. Five cycles of accumulation are sufficient for the assay. The experiment conducted with a blank solution without addition of the analyte showed an extremely low background even with the number of accumulation cycles reached 10. The low background of the proposed method is a distinguished advantage providing a possibility for determination of at least 0.1 ng/ml human IgG. The RSD of the method is 9.57% for eight times determination of 1 ng/ml human IgG under the same conditions.