Target-triggered cascade signal amplification for sensitive electrochemical detection of SARS-CoV-2 with clinical application

Abstract

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the outbreak of the 2019 coronavirus (COVID-19) disease, which greatly challenges the global economy and health. Simple and sensitive diagnosis of COVID-19 at the early stage is important to prevent the spread of pandemics. Herein, we have proposed a target-triggered cascade signal amplification in this work for sensitive analysis of SARS-CoV-2 RNA. Specifically, the presence of SARS-CoV-2 RNA can trigger the catalytic hairpin assembly to generate plenty of DNA duplexes with free 3'-OH termini, which can be recognized and catalyzed by the terminal deoxynucleotidyl transferase (TdT) to generate long strand DNA. The prolonged DNA can absorb substantial Ru(NH₃)₆³⁺ molecules via electrostatic interaction and produce an enhanced current response. The incorporation of catalytic hairpin assembly and TdT-mediated polymerization effectively lowers the detection limit to 45 fM, with a wide linear range from 0.1 pM to 3000 pM. Moreover, the proposed strategy possesses excellent selectivity to distinguish target RNA with single-base mismatched, three-base mismatched, and random sequences. Notably, the proposed electrochemical biosensor can be applied to analyze targets in complex circumstances containing 10% saliva, which implies its high stability and anti-interference. Moreover, the proposed strategy has been successfully applied to SARS CoV-2 RNA detection in clinical samples and may have the potential to be cultivated as an effective tool for COVID-19 diagnosis.

Keywords: COVID-19; Cascade signal amplification; Electrochemical biosensor; SARS-CoV-2 RNA.

Graphical abstract

Scheme 1

Schematic diagram of the target-triggered signal amplification for sensitive electrochemical detection of SARS-CoV-2 RNA.

Fig. 1

(A) EIS measurements corresponding to the stepwise treatment of Au electrode. (a) bare Au electrode, (b) MCH/HP1/Au, (c) (HP2+target)/MCH/HP1/Au, (d) (TdT + HP2+target)/MCH/HP1/Au. (B) DPV signals under different reaction conditions: (a) TdT/MCH/HP1/Au incubated with Ru(NH₃)₆³⁺, (b) (HP2+target)/MCH/HP1/Au incubated with Ru(NH₃)₆³⁺, (c) (TdT + HP2+target)/MCH/HP1/Au incubated with Ru(NH₃)₆³⁺. The concentration of the target RNA is 1 nM.

Fig. 2

The quantitative analysis for the detection of SARS-CoV-2 RNA. (A) The DPV signals corresponding to SARS-CoV-2 RNA with different concentrations. a–i: 0, 0.1, 1, 5, 10, 100, 500, 1000, 3000 pM, respectively. (B) Linear relationship between the DPV signals and the logarithm of SARS-CoV-2 RNA concentration. Error bars indicate standard deviations (n = 3).

Fig. 3

(A) Selectivity of the proposed biosensor. SM represents for single-base mismatched sequence, TM represents for three-base mismatched sequence. The concentration of the analyte is 500 pM. (B) Analytical performance in Tris-HCl and 10% saliva without or with SARS-CoV-2. The concentration of the target is 1000 pM. Error bars indicate standard deviations (n = 3).

Fig. 4

(A) DPV responses of the clinical specimens collected from healthy group (HG) and patient group (PG). (B) Scatterplot of the clinical specimens of HG and PG. (***, p < 0.001). Error bars indicate standard deviations (n = 3).