Highly Effective Protein Converting Strategy for Ultrasensitive Electrochemical Assay of Cystatin C

In this work, a highly effective protein converting strategy based on immunoreaction-induced DNA strand displacement and T7 Exonuclease (T7 Exo)-assisted protein cyclic enzymatic amplification for ultrasensitive detection of cystatin C was described. Herein, Au@Fe3O4 as magnetic separator was labeled by antibody 1-conjungated DNA (DNA1) and the DNA substrate of T7 Exo (DNA3) which initially hybridized with output DNA (S1) to form a stable S1/DNA1 duplex (S1/DNA3). Antibody 2 was labeled by competing DNA (DNA 2). In the presence of cystatin C, sandwich immunoreaction would induce proximity hybridization between DNA2 and DNA3 and thus displace S1 from the S1/DNA3 duplex with formation of a stable DNA2/DNA3 duplex, realizing the conversion of input target cystatin C into output S1. To enhance the conversion ratio, the DNA2/DNA3 duplex was then digested by T7 Exo with release of DNA2 which could act as competing DNA again to displace S1 from the S1/DNA3 duplex in adjacent locations and initiate another cleavage reaction. Through such a cyclic process, each input cystatin C could induce more than one output S1, enhancing detection sensitivity. A hairpin DNA modified electrode was used to capture the output S1, and then, a hybridization chain reaction is triggered on the biosensor surface. Then, thionine as electron mediator was embedded into the dsDNA polymers to produce a detection signal. The electrochemical biosensor exhibited a much wider linear range of 0.01 pg mL(-1) to 30 ng mL(-1) with low detection limit of 3 fg mL(-1). Moreover, this method introduced protein unrelated to nucleic acids into the realm of potential inputs for translation, which might create a new immunoassay method for sensitive detection of protein.