Ultra-sensitive detection of norovirus using a three-in-one CRISPR platform based on a DNA hydrogel and composite functional nanomaterials

The ultrasensitive sensor with three optical response mechanisms was proposed for the determination of trace amounts of norovirus using a 3-in-1 GCSNAs (a gap-containing spherical nucleic acid nanoparticles) probe. A simple and highly sensitive three-mode biosensor with Raman, colorimetric, and fluorescence functions was proposed and implemented using the GCSNAs probe and a DNA hydrogel for norovirus detection. When the virus exists, the trans-cleavage activity of CRISPR-Cas12a was activated by double-stranded dsDNA (dsDNA) generated by reverse transcription and recombinase polymerase isothermal amplification (RT-RPA) to degrade the DNA hydrogel/GCSNA composition and release the three-in-one (3-in-1) probe-GCSNA, realising the triple ultrasensitive detection of norovirus. The colorimetric sensing mode allows for semi-quantitative on-site detection, which is visible to the naked eye and the quantitative detection can be achieved by conducting grayscale analysis using the "Colour Grab" function of a smartphone. This new triple sensor achieved the successful quantification of norovirus at concentrations as low as the femtomolar scale with an excellent selectivity and accuracy. Considering the colorimetric properties of rolling circle amplification (RCA)-based DNA hydrogels and GCSNAs, the proposed method has a broad application prospect in virus on-site detection in food. It should be applicable for virus detection in a wide range of fields such, as environmental analysis, medical diagnosis, and food safety. It is anticipated that this mechanism will open new avenues for the development of multimodal analyses and multifunctional sensing platforms for various applications. We anticipate that this sensing mechanism will open up a new way for the development of food safety detection.