An ultrasensitive electrochemical DNA sensing strategy free from pre-immobilization via G-quadruplex based homogenous proximity hybridization

Motivated by the desire for simple, rapid and highly sensitive DNA detection, we presented a signal-on electrochemical DNA (E-DNA) sensing strategy utilizing cooperative proximity hybridization based on a G-quadruplex (G4) probe labeled with the SH, which could specifically hybridize with its target DNA in homogenous solution. In the presence of target DNA, proximity hybridization was triggered to form a Y-shaped complex and the SH was released from G4 probe stem, companied by chemisorption on the electrode surface through Au-S binding when applied a positive potential, which brought Fc labeled on the signal probe close to the electrode surface. Thus, electrochemical signal dramatically increased, ensuring the highly sensitive “signal-on” assay. Such an E-DNA sensing strategy allows for ultrasensitive DNA detection with a detection limit as low as 2.82 × 10−15 M and a wide linear response from 1.0 × 10−9 to 1.0 × 10−15 M. In addition, the powerful discriminating ability of the mismatched DNA from the perfect matched target DNA was also demonstrated. More importantly, this homogenous proximity hybridization strategy could expand to colorimetric assay by incorporating G4 probe with hemin to form DNAzyme, which could effectively catalyze ABTS to generate a visual color change. Taking the joint advantages of G4 stem-loop probe and homogenous proximity hybridization, this sensing strategy exhibits greatly enhanced sensitivity and excellent specificity, making it a promising strategy for point-of-care testing. [Display omitted] •Utilizing coorperative proximity hybridization and potential-assisted Au-S chemisorption technique.•A G4 stem-loop probe was designed as the recognition probe.•The thiol was locked in G4 stem, avoiding the non-specific adsorption of recognition probe on the electrode surface.•The E-DNA sensing strategy allows for ultrasensitive DNA detection with a detection limit as low as 2.82 fM.•Single base mismatch can be effectively differentiated.