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

An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed foron-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-cleavageability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In thepresence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PBnanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPsin the microplate was triggered, producing massive ferricyanide anions (Fe(CN)64-), which could exhibit a unique characteristicRaman peak that was located in thebiological Raman-silent region. By mixing the alkali-treated solution with the SERS substrate,Au@Ag core-shell NP, the concentration of the target DNA wasfinally 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 224aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor wassuccessfully applied to milk authenticity detection.

Automated summary

This study proposes an ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a for on-site nucleic acid detection. The biosensor achieves precise target DNA recognition and signal amplification through specific base pairing and efficient trans-cleavage capability of CRISPR/Cas12a. The strategy exhibits an ultralow detection limit and is successfully applied to milk authenticity detection.