Rolling circle amplification based on signal-enhanced electrochemical DNA sensor for ultrasensitive transcription factor detection

An electrochemical biosensor based on specific protein-DNA binding and rolling circle amplification (RCA) signal enlargement was developed for transcription factor (TF) detection. The TF biosensor was constructed by three oligo-DNA probes. Probe 1 (P1) was immobilized on gold electrode. Probe 2 (P2) hybridized with P1 to form a partially complementary double-stranded DNA . The complementary part contained a TF binding sequence, while the non-complementary part of P2 integrated a RCA primer sequence. Probe 3 (P3) can displace P2 by hybridizing with P1 to produce a completely complementary double-stranded DNA. When TF protein was introduced, a stable TF-DNA complex would form and the displacement reaction mentioned above was hindered, which made the RCA possible. Finally, the electrochemical redox probe methylene blue (MB) was bound to the RCA product and offered the available detection signal. Using nuclear factor kappa B (NF-κB) p65 as a model target, the fabricated electrochemical biosensor exhibited a wide linear region and a low detection limit.