Washing-Free Electrochemical Detection of Amplified Double-Stranded DNAs Using a Zinc Finger Protein

Recombinase polymerase amplification (RPA) has been combined with electrochemical detection for simple and rapid point-of-care testing. However, there are two major hindrances to this simple and rapid testing: (i) washing or purification steps are required to remove unbound labeled probes and interfering species in the sample; (ii) it is difficult to quantify double-stranded DNA (dsDNA) electrochemically by using biospecific affinity binding without dsDNA denaturation. In the present study, we describe a wash-free and rapid electrochemical method to detect RPA-amplified dsDNAs using a zinc finger protein, Zif268. Electrochemical detection is achieved using proximity-dependent electron mediation of ferrocenemethanol between a glucose-oxidase (GOx) label and an electrode, which differentiates the specifically electrode-bound and -unbound labels without a washing or purification step. RPA-amplified dsDNA containing a biotin-terminated forward primer is specifically bound to a neutravidin-modified electrode, and GOx-conjugated Zif268 is specifically bound to the dsDNA. The whole detection is performed within 17 min (15 min for the RPA reaction and <2 min for the electrochemical measurement). Electrochemical detection is carried out without an additional incubation period, because the specific binding between Zif268 and the dsDNA occurs during the RPA reaction. The detection method could discriminate between target template DNA of Piscirickettsia salmonis and nontarget DNAs (random sequence and calf thymus DNA). The detection limit for the target DNA is approximately 300 copies in 13.2 μL, indicating that the detection method is ultrasensitive. We believe that the method could offer a promising solution for simple and rapid point-of-care testing.