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

Abstract

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.

Recombinase Polymerase Amplification-Based Biosensors for Rapid Zoonoses Screening

“…However, a certain degree of nonspecific amplification is generated at the affinity-modified electrode, with centrifugal filtration required to remove the electroactive dithiothreitol, which adds a reaction step. 87 In addition, a reagent-free DNA sensor enhanced by RPA was developed using a combination of ferrocene and reporter probe-modified AuNPs and modified primers (Figure 5b). RPA was performed using primer binding sites and C3 spacer-modified primers to produce amplicons with single-stranded tails at both ends.…”

A No‐washing Point‐of‐Care Electrochemical Biosensor Based on CuS Nanoparticles for Rapid and Sensitive Detection of Neuron‐specific Enolase

“…Therefore, the design of the no-washing biosensor happens to meet the needs of rapid and convenient detection [3,9], avoiding sample contamination and detection inaccuracy caused by washing in the complex system. Compared with conventional biosensors, no-washing biosensors are more suitable for point-of-care testing (POCT) [10][11][12][13].…”

Electrochemical Detection of Genomic DNA Utilizing Recombinase Polymerase Amplification and Stem-Loop Probe

“…By having the redox tag bound to the probe, the sample matrix can be analyzed without any modification. There have been some recent successful attempts to electrochemically detect RPA-amplified DNA. − These methods, however, all require multiple complex steps: the work by Lau et al, for example, requires gold nanoparticles, magnetic beads, heating, and wash steps, and the work by Sanchez-Salcedo et al requires multiple washing steps with different buffers. In order to create a point-of-care diagnostic device, having as few steps as possible is preferable as it cuts down on reagents, costs, and complexity.…”