期刊
THERANOSTICS
卷 12, 期 13, 页码 5914-5930出版社
IVYSPRING INT PUBL
DOI: 10.7150/thno.75816
关键词
gold nanoparticles; magnetic manipulation; surface-enhanced Raman spectroscopy; CRISPR-Cas12a; nucleic acid detection
资金
- Shenzhen-Hong Kong-Macao Science and Technology Plan Project [SGDX2020110309260000]
- Research Grant Council (RGC) of Hong Kong [PolyU C5110-20G]
- Department of Biomedical Engineering [0033912]
- Start-up Fund for RAPs under the Strategic Hiring Scheme [0035876]
- Hong Kong Polytechnic University (PolyU, University Grant Council)
- RGC of the Hong Kong General Research Grant [PolyU 15217621]
- Hong Kong Polytechnic University Internal Fund [1-ZVVQ]
- University Research Facility in Life Sciences of PolyU
This study overcomes the steric hindrance of nanomaterials to CRISPR-Cas12a by designing specific DNA/RNA hairpins, and constructs a nucleic acid biosensor with high sensitivity and selectivity.
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.
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