It remains a huge challenge to integrate the sensitivity, stability, reproducibility, and anti-fouling ability of electrochemical biosensors for practical applications. Herein, we propose a self-assembled
electrode combining hexanethiol (HT),
poly-adenine (
poly-A), and cholesteryl-modified
DNA to meet this challenge. HT can tightly pack at the
electrode interface to form a hydrophobic self-assembled monolayer (SAM), effectively improving the stability and signal-to-noise ratio (SNR) of electrochemical detection. Cholesteryl-modified
DNA was immobilized at the
electrode through the hydrophobic interaction with HT to avoid the competition between the SAM and the
DNA probe on the
gold site. Thus, the assembly efficiency and uniformity of the
DNA probe as well as the detection reproducibility were increased remarkedly.
Poly-A was added on the HT assembled
electrode to occupy the unreacted sites of
gold to further enhance the anti-fouling ability. The combination of HT and
poly-A allows the
electrode to ensure favorable anti-fouling ability without sacrificing the detection performance. On this basis, we proposed a dual-signal amplification electrochemical biosensor for the detection of exosomal
microRNAs, which showed excellent sensitivity with a detection limit down to 1.46 aM. Importantly, this method has been successfully applied to detect exosomal microRNA-21 in cells and human serum samples, proving its potential utility in
cancer diagnosis.