Fabricating a reversible and regenerable electrochemical biosensor for quantitative detection of antibody by using “triplex-stem” DNA molecular switch

A reversible and regenerable electrochemical biosensor is fabricated for quantitative detection of antibody based on “triplex-stem” molecular switches. A hairpin-shaped oligonucleotide (hairpin DNA) labeled with ferrocene (Fc) at the 3′-end is fixed on the gold electrode serving as a signal transduction probe. Its hairpin structure leads Fc close to the surface of gold electrode and produces a strong current signal (on-state). A single-strand oligonucleotide modified with two digoxin molecules on the two arm segments (capture DNA) interact with hairpin DNA with the help of Ag⁺ ions. The “triplex-stem” DNA forms, which separates Fc from the electrode and reduces the electrochemical signal (off-state). Binding of digoxin antibody to digoxin releases capture DNA from the hairpin DNA, creating an effective “off-on” current signal switch. The stability of the “triplex-stem” structure of hairpin/capture DNA is critical to the signal switch and the sensitivity of the method, which can be adjusted conveniently and efficiently by changing Ag⁺ concentrations. Based on the “off-on” current signal switch, this biosensor is used to detect digoxin antibody sensitively in blood serum. The linear range is 1.0–500 pg with a correlation coefficient of 0.996, and the detection limit is 0.4 pg. Also, this biosensor shows excellent reversibility and reproducibility, which are significant requirements for practical biosensor applications.