A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detection

A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detection

Yin, B. H.; Zhang, Q.; Xia, X. Y.; Li, C. Q.; Ho, W. K. H.; Yan, J. X.; Huang, Y. Y.; Wu, H. L.; Wang, P. ; Yi, C. Q.; Hao, J. H.; Wang, J. F.; Chen, H. L.; Wong, S. H. D.; Yang, M^*

Theranostics, 2022, 12, 5914-5930

? https://doi.org/10.7150/thno.75816

Abstract. 

Background: CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection.

Methods: To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising “on-off” nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their separation from the disassembled nanostructures to suppress the background for improving detection sensitivity.

Results: As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM.

Conclusion: Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors.