A dual fragment triggered DNA ladder nanostructure based on logic gate and dispersion-to-localization catalytic hairpin assembly for efficient fluorescence assay of SARS-CoV-2 and H1N1.

DNA nanotechnology has been widely utilized in the construction of various functional nanostructures. However, most DNA nanostructures have the shortcomings of low response rate and serious background leakage. Herein, we proposed the conception of AND logic gate cascaded dispersion-to-localization catalytic hairpin assembly (AND gate-DLCHA) for the fabrication of novel DNA ladder nanostructures. In our design, the entropy-driven AND logic gate can precisely recognize two fragments of the target nucleic acid sequences. After AND logic gate activation by target nucleic acids, dispersion-to-localization catalytic hairpin assembly was initiated. Consequently, tremendous DNA ladder nanostructures were generated and the response signal was rapidly enhanced, which can be used for rapid and amplied detection of nucleic acids. Taking advantage of the sensitivity and specificity of AND gate-DLCHA strategy, the fluorescence sensors were established and successfully applied in ultrasensitive assay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (H1N1) within 45 min with the limit of detection (LOD) as low as 66 copies mL-1 (SARS-CoV-2) and 33 copies mL-1 (H1N1), which showed perspectives in pathogen identification and biomedical application. The high selectivity and reliability of established sensors was attributed to the dual-fragment analysis. Meanwhile, the sensors possessed minimal leakage and greatly enhanced signal to background (S/B) ratio owing to substrate transduction from dispersion into colocalization. This rationally developed logic gate cascaded dispersion-to-localization catalytic hairpin assembly strategy presented a new approach for the development of DNA nanostructures.

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

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.