Amplified terminal protection assay of small molecule/protein interactions via a highly characteristic solid-state Ag/AgCl process

In this work, we describe a new sensitive strategy for electrochemical detection of protein via small molecule/protein interactions. This assay is based on a terminal protection mechanism that small molecule-linked single-stranded DNA (ssDNA) is protected against hydrolysis by exonuclease I when the target protein is captured by the corresponding small molecule recognition element. Positively charged gold nanoparticles (AuNPs) are attached to the termini-protected and negatively charged ssDNA through electrostatic interactions. Subsequent AuNP-catalyzed silver enhancement followed by a highly characteristic and sensitive solid-state Ag/AgCl process is introduced to the sensing platform to amplify the signal output. By combining the amplification ability resulting from the silver deposition on the surface-captured AuNPs with the inherent high sensitivity of the electrochemical solid-state Ag/AgCl process, our method expands its range to the detection of macromolecules that bind to specific small molecules and enables low picomolar detection of protein. As a model of biotin/streptavidin interaction, a detection limit of 10 pM for streptavidin is readily achieved with desirable sensitivity and specificity, which indicates that the terminal protection assay coupled with the electrochemical solid-state Ag/AgCl process can offer a promising platform for the determination of various of types of proteins or small molecule-protein interactions.

► Small molecule/protein interaction is interrogated by using a terminal protection strategy.
► The association of protein with small molecule-linked ssDNA protects ssDNA from digesting by exonuclease I.
► Small molecule/protein interaction is amplified by a highly characteristic solid-state Ag/AgCl electrochemical process.
► The proposed method offers selective and sensitive detection of protein down to the low picomolar level.