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

Bohan Yin; Qin Zhang; Xinyue Xia; Chuanqi Li; Willis Kwun Hei Ho; Jiaxiang Yan; Yingying Huang; Honglian Wu; Pui Wang; Changqing Yi; Jianhua Hao; Jianfang Wang; Honglin Chen; Siu Hong Dexter Wong; Mo Yang

doi:10.7150/thno.75816

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

Theranostics. 2022 Aug 8;12(13):5914-5930. doi: 10.7150/thno.75816. eCollection 2022.

Authors

Bohan Yin¹, Qin Zhang¹, Xinyue Xia², Chuanqi Li¹, Willis Kwun Hei Ho¹, Jiaxiang Yan¹, Yingying Huang¹, Honglian Wu¹, Pui Wang³, Changqing Yi⁴, Jianhua Hao⁵, Jianfang Wang², Honglin Chen³, Siu Hong Dexter Wong^{1

6}, Mo Yang¹

Affiliations

¹ Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong 999077, China.
² Department of Physics, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China.
³ Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong 999077, China.
⁴ Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of Biomedical Engineering, Sun Yat-Sen University, Guangzhou, 510006, P. R. China.
⁵ Department of Applied Physics, The Hong Kong Polytechnic University, Kowloon, Hong Kong 999077, China.
⁶ Research Institute for Sports Science and Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong 999077, China.

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.

Keywords: CRISPR-Cas12a; gold nanoparticles; magnetic manipulation; nucleic acid detection; surface-enhanced Raman spectroscopy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics
DNA / chemistry
Gold / chemistry
Metal Nanoparticles* / chemistry
Nucleic Acids*
RNA

Substances

Nucleic Acids
RNA
Gold
DNA