An electrochemical biosensor for sensitive analysis of the SARS-CoV-2 RNA

Abstract

The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is continuously worsening globally, herein we have proposed an electrochemical biosensor for the sensitive monitoring of SARS-CoV-2 RNA. The presence of target RNA firstly triggers the catalytic hairpin assembly circuit and then initiates terminal deoxynucleotidyl transferase-mediated DNA polymerization. Consequently, a large number of long single-stranded DNA products can be produced, and these negatively charged DNA products will bind a massive of positively charged electroactive molecular of Ru(NH₃)₆³⁺ due to the electrostatic adsorption. Therefore, significantly amplified electrochemical signals can be generated for sensitive analysis of SARS-CoV-2 RNA in the range of 0.1-1000 pM with the detection limit as low as 26 fM. Besides the excellent distinguishing ability for SARS-CoV-2 RNA against single-base mismatched RNA, the proposed biosensor can also be successfully applied to complex matrices, as well as clinical patient samples with high stability, which shows great prospects of clinical application.

Keywords: COVID-19; Catalytic hairpin assembly; SARS-CoV-2; TdT-mediated DNA polymerization.

Scheme 1

Principle of the proposed electrochemical biosensor for sensitive analysis of SARS-CoV-2 RNA.

Fig. 1

(A) EIS characterizations corresponding to different treatments of the electrode: (a) AuE, (b) MCH/HP/AuE, (c) (HP1 + HP2 + target RNA)/MCH/HP/AuE, and (d) (HP1 + HP2 + target RNA+TdT)/MCH/HP/AuE. (B) DPV responses under different conditions: (a) (TdT + Ru(NH₃)₆³⁺) MCH/HP/AuE, (b) (HP1 + HP2 + TdT + Ru(NH₃)₆³⁺)/MCH/HP/AuE, (c) (HP1 + HP2 + target RNA + TdT + Ru(NH₃)₆³⁺)/MCH/HP/AuE.

Fig. 2

(A) DPV currents of the biosensor with various concentrations of the target RNA. (B) The linear relationship between the value of peak current and the logarithm of the target RNA concentrations. Error bars: SD, n = 3.

Fig. 3

Selective study of the proposed biosensor for the target RNA, single-base mismatched (SM) RNA, two-base mismatched (TM) RNA and random sequence at the concentrations of 100 pM and 1 pM, respectively. Error bars: SD, n = 3.

Fig. 4

Current responses for different concentrations of the target RNA in buffer, serum and saliva. Error bars: SD, n = 3.

Fig. 5

Clinical sample analysis of the SARS-COV-2 RNA. (A) DPV responses and (B) scattered dot plots of SARS-COV-2 RNA from healthy samples (HS) and patient samples (PS). The statistical significances are calculated by t-test (**, p < 0.01). Error bars: SD, n = 3.