Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk...

An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN)₆^4-), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core-shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.