Sensitive fluorescence biosensor for SARS-CoV-2 nucleocapsid protein detection in cold-chain food products based on DNA circuit and g-CNQDs@Zn-MOF

Abstract

SARS-CoV-2 isolation from cold-chain food products confirms the possibility of outbreaks through cold-chain food products. RNA extraction combined with RT-PCR is the primary method currently utilized for the detection of SARS-CoV-2. However, the requirement of hours of analytical time and the high price of RT-PCR hinder its worldwide implementation in food supervision. Here, we report a fluorescence biosensor for detection of SARS-CoV-2 N protein. The fluorescence biosensor was fabricated by aptamer-based conformational entropy-driven circuit where molecular beacon strands were labeled with graphitic carbon nitrides quantum dots@Zn-metal-organic framework (g-CNQDs@Zn-MOF) and Dabcyl. The detection of the N protein was achieved via swabbing followed by competitive assay using a fixed amount of N-48 aptamers in the analytical system. A fluorescence emission spectrum was employed for the detection. The detection limit of our fluorescence biosensor was 1.0 pg/mL for SARS-CoV-2 N protein, indicating very excellent sensitivity. The fluorescence biosensor did not exhibit significant cross-reactivity with other N proteins. Finally, the biosensor was successfully applied for the detection of SARS-CoV-2 N protein in actual cold-chain food products showing same excellent accuracy as RT-PCR method. Thus, our fluorescence biosensor is a promising analytical tool for rapid and sensitive detection of SARS-CoV-2 N protein.

Keywords: Aptamer; Cold-chain food product; Conformational entropy-driven circuit; G-CNQDs@Zn-MOF; SARS-CoV-2.

Scheme 1

Detection of N protein with the proposed fluorescent biosensor based on DNA circuit and g-CNQDs@Zn-MOF. Abbreviation: aptamer of N protein (N-48), catalyst strand (C), fuel strand (F), three-stranded substrate complex (S), displacing output (OP), signal strands (SB), linker strand (LB), waste complex (W) and molecular beacon (MB).

Fig. 1

Scan electron micrographs of (A) g-CNQDs, (B)Zn-MOF, (C&D) g-CNQDs@Zn-MOF.

Fig. 2

Fluorescence emission spectra of g-CNQDs, Zn-MOF and g-CNQDs@Zn-MOF.

Fig. 3

Polyacrylamide gel electropherograms of different reaction systems: the toehold region ‾5 and toehold region ‾3 were (A) 6 nt/4 nt, (B) 5 nt/5 nt, and (C) 4 nt/6 nt; the concentration ratios of F strand to substrate probe were 0.5:1 (lanes 10–12), 1:1 (lanes 7–9), 1.5:1 (lane 4–6), and 2:1 (lane 1–3); lanes 1, 4, 7, and 10 were control groups, lane 13 was C, lane 14 was S. Abbreviation: three-stranded substrate complex (S), catalyst strand (C), fuel strand (F) output strand (OB), signal strand (SB),linker strand (LB), and waste complex (W).

Fig. 4

(A) The PL intensities of conformational entropy-driven circuit system with MB1 strand or MB2 strand; Structures of (B) MB1 strand and (C) MB2 strand. The error bars represent the standard deviations of three independent experiment. Abbreviation: molecular beacon (MB).

Fig. 5

(A) Fluorescence emission spectra overlay for detection of SARS-CoV-2 N protein in the linear range of 5.0 pg/mL∼1.0 × 10³ pg/mL; the calibration plot for the concentration of N protein and PL intensity acquired by the fluorescence biosensor; (B) Selectivity evaluation of the proposed biosensor for the detection of N protein of SARS-CoV-2 against other four kinds of proteins (the concentration of all proteins used in the experiment was 100 pg/mL). The error bars represent the standard deviations of three independent experiment.