Coupling of background reduction with rolling circle amplification for highly sensitive protein detection via terminal protection of small molecule-linked DNA.

In this work, by coupling background current reduction with rolling circle amplification (RCA), we describe the development of an ultrasensitive electrochemical sensing method for protein detection based on a small molecule-linked DNA terminal protection strategy. Our detection platform employs a typical streptavidin (STV)-biotin interaction system. Biotin-linked single-stranded DNA (SH-ssDNA-biotin) is self-assembled on a gold electrode to capture the target protein, STV. The binding of STV with the biotin small molecule recognition element protects the SH-ssDNA-biotin against hydrolysis by exonuclease I (Exo I), while the unbound SH-ssDNA-biotin is effectively hydrolyzed and removed from the electrode surface. The bound STV further interacts with long, RCA-amplified biotin DNAs to facilitate the adsorption of numerous electroactive reporters, hexaammineruthenium(III) chloride (RuHex) via electrostatic interactions, which results in significantly amplified signals for the quantitative determination of STV. Moreover, the removal of the unbound SH-ssDNA-biotin probes from the sensing electrode obviates the accumulation of RuHex and leads to a highly minimized background current. The simultaneous RCA signal amplification and background current reduction is expected to significantly enhance the signal-to-noise ratio and to achieve ultrahigh sensitivity. The results reveal that the developed strategy provides a low detection limit of 0.4 pM with high selectivity.