Sensitive electrochemical biosensor combined with isothermal amplification for point-of-care COVID-19 tests

Abstract

We report an electrochemical biosensor combined with recombinase polymerase amplification (RPA) for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2. The electrochemical biosensor based on a multi-microelectrode array allows the detection of multiple target genes by differential pulse voltammetry. The RPA reaction involves hybridization of the RPA amplicon with thiol-modified primers immobilized on the working electrodes, which leads to a reduction of current density as amplicons accumulate. The assay results in shorter "sample-to-answer" times than conventional PCR without expensive thermo-cycling equipment. The limits of detection are about 0.972 fg/μL (RdRP gene) and 3.925 fg/μL (N gene), which are slightly lower than or comparable to that of RPA assay results obtained by gel electrophoresis without post-amplification purification. The combination of electrochemical biosensors and the RPA assay is a rapid, sensitive, and convenient platform that can be potentially used as a point-of-care test for the diagnosis of COVID-19.

Keywords: COVID-19; Coronavirus; Electrochemical detection method; Recombinase polymerase amplification; SARS-CoV-2.

Fig. 1

Electrochemical biosensor combined with isothermal amplification. Schematic of an electrochemical biosensor combined with recombinase polymerase amplification (RPA). The RPA reaction occurs on the working electrodes, and amplicon detection is quantified by differential pulse voltammetry (DPV).

Fig. 2

Characterization of the Ag/AgCl electrode. (a–b) Optical images (a) and cyclic voltammetry measurements (b) obtained from an Ag electrode after AgCl formation by immersing in a bleach solution for 30 s intervals. (c–e) X-ray photoelectron spectroscopy (XPS) spectra of Ag and AgCl electrodes: (c) survey spectrum, (d) Ag 3d 5/2 and Ag 3d 3/2 and (e) Cl 2p regions. (f–g) stability (f) and reproducibility (g) of the electrochemical biosensors by CV measurements.

Fig. 3

Feasibility verification of RPA in combination with electrochemical detection. The surface temperature of the Ag/AgCl electrode (a) and agarose gel electrophoresis analysis (b) for confirmation of the RPA assay. DPV signals obtained by on-chip RPA performed with specific target N gene (c) and RdRP gene (e). Change in the peak current for N gene (d) and RdRP gene (f) compared with an NTC and a blank control (***: p-value < 0.001.

Fig. 4

Reaction sensitivity of RPA coupled with electrochemical detection. The assay concentration ranged from 10³ to 10⁹ copies for 40 min under human body temperature. The variation in the peak current for each DPV curve measured during 40 min depending on different target concentrations: N gene (a) and RdRP gene (b) as well as for an NTC. DPV signals obtained at 20 min depending on template concentration: N gene (c) and RdRP gene (d). Inset shows the change in the peak current with respect to the target gene concentration.