Electrochemical detection of kinase by converting homogeneous analysis into heterogeneous assay through avidin-biotin interaction

In the classical heterogeneous electrochemical assay, phosphorylation of peptide substrate is usually performed on the solid-liquid surface. However, immobilization of probe on the solid surface may limit the interaction between the reaction site of probe and the active center of kinase due to the steric hindrance effect. In this work, we proposed a heterogeneous electrochemical method for kinase detection, in which the probe is immobilizationfree during the phosphorylation reaction. A biotinylated peptide was used as the kinase substrate. After phosphorylation, the biotinylated phosphopeptide was captured by the neutravidin (NA)-modified electrode through the avidin-biotin interaction. The phosphate groups on the electrode surface were then recognized by the conjugates preformed between biotinylated Phos-tag (TM) (Bio-tag-Phos) and ferrocene (Fc)-capped NA-modified gold nanoparticle (Fc-AuNP-NA). The method integrates the advantages of homogeneous reaction and heterogeneous detection with high simplicity, sensitivity and specificity. The strategy can be applied to design other heterogeneous biosensors without the immobilization of probe during the enzyme catalyzed reaction.