An electrochemical aptasensor with N protein binding aptamer-complementary oligonucleotide as probe for ultra-sensitive detection of COVID-19

Abstract

The emergence of the COVID-19 epidemic has affected the lives of hundreds of millions of people globally. There is no doubt that the development of fast and sensitive detection methods is crucial while the worldwide effective vaccination programs are miles away from actualization. In this study, we have reported an electrochemical N protein aptamer sensor with complementary oligonucleotide as probe for the specific detection of COVID-19. The electrochemical aptasensor was prepared by fixing the double-stranded DNA hybrid obtained by the hybridization of N protein aptamer and its Fc-labeled complementary strand on the surface of a gold electrode. After incubation with the target, the aptamer dissociated from the labeled complementary DNA oligonucleotide hybrid to preferentially bind with N protein in the solution. The concentration of N protein was measured by detecting the changes in electrochemical current signals induced by the conformational transformation of the complementary DNA oligonucleotide left on the electrode surface. The sensor had a linear relationship between the logarithm of the N protein concentration from 10 fM to 100 nM (ΔI_p = 0.098 log C_{N protein}/fM - 0.08433, R² = 0.99), and the detection limitation was 1 fM (S/N = 3). The electrochemical aptamer sensor was applied to test the spiked concentrations of throat swabs and blood samples from three volunteers, and the obtained results proved that the sensor has great potentials for the early detection of COVID-19 in patients.

Keywords: Electrochemical aptasensor; N protein detection; Rapid COVID-19 diagnosis; Regenerable aptamer sensor.

Scheme 1

Schematic diagram of the electrochemical aptasensor based on N protein-binding aptamer and Fc labeled complementary DNA oligonucleotide as probe. (A) Fc-labeled N protein aptamer conformation switching to a hairpin loop structure (before target incubation) and the change in conformation (after incubation with target). The change in the current signal is related to the Fc-electrode surface distance. (B) Incubation of ds-DNA hybrid (aptamer + Fc labeled complementary DNA oligonucleotide) with N protein. High affinity aptamer dissociated from the complementary DNA oligonucleotide to bind to the N protein, resulting in a stronger current signal change (related to the Fc-complementary DNA oligonucleotide structure switching to a hairpin loop) than strategy A.

Fig. 1

(A) Agarose gel electrophoresis showing aptamer (lane 1), complementary DNA oligonucleotide (lane 2), and ds-DNA hybrid (lane 3). (B) CD spectra of aptamer (black curve), ds-DNA hybrid before (red curve) and after incubation with the target (blue curve) for 60 min at 25 °C. Inset: CD spectrum of N protein alone.

Fig. 2

(A) Differential pulse voltammetry (DPV) response signals recorded in Tris buffer. (B) The relationship between the logarithm of N protein concentration and ΔI_p value. The error bars indicate the standard deviations after three measurements.

Fig. 3

Differential pulse voltammetry signals of several interferents in throat swabs (A) and human blood samples (B).

Fig. 4

(A) DPV values at different concentrations of N protein added to the throat swabs. (B) Determination of different concentrations N protein in blood samples.