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

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.

A Multilayered Mesoporous Gold Nanoarchitecture for Ultraeffective Near‐Infrared Light‐Controlled Chemo/Photothermal Therapy for Cancer Guided by SERS Imaging

“…To examine the SERS property of pAS@AuNCs, we measured the Raman signal and selected the peak of MBA at 1078 cm −1 , which corresponds to its ring stretch vibration mode, [ 41,42 ] as the characteristic peak (MBA‐peak). For SERS intensity comparison, we included two other groups: 40 nm AuNP (Au 40 NP, with a similar size to the core of ASNP) and ASNP, both of which were modified with mSiO 2 shell (denoted as Au 40 @mSiO 2 NP and AS@mSiO 2 NP, respectively) ( Figure a; Figures S1 and S7, Supporting Information).…”

“…To examine the SERS property of pAS@AuNCs, we measured the Raman signal and selected the peak of MBA at 1078 cm −1 , which corresponds to its ring stretch vibration mode, [41,42] as the characteristic peak (MBA-peak). For SERS intensity comparison, we included two other groups: 40 nm and c) colors of Au 13 NPs, ASNPs, AS@mSiO 2 NPs, and pAS@AuNCs suspended in nanopure water with surface plasmon resonance (SPR) peaks at 518, 700, 734, 806 nm, respectively.…”

Research progress of CRISPR/Cas systems in nucleic acid detection of infectious diseases

“…The Raman signal is weak compared to the system without target DNA. Because steric hindrance of the modified nanomaterials at both ends of ssDNA limits the cleavage of ssDNA by the CRISPR/Cas12a system, Yin et al [79] designed a SERS probe and a DNA/RNA chimeric hairpin probe to replace direct cleavage of ssDNA by the bulky CRISPR/Cas12a system with a small volume of RNA. AuNPs-DNA1 was used as the core of the SERS probes, and multiple AuNPs-DNA2 coated with 4-mercaptobenzoic acid were linked by complementary DNA pair DNA1/ DNA2.…”

Current advance of CRISPR/Cas-based SERS technology

“…60 (G) Schematic diagram of a CRISPR/ Cas12a integrated SERS nanoplatform with a chimeric DNA/RNA hairpin guidance for nucleic acid detection. 61 (H) Schematic diagram of CRISPR/ Cas12a-based core-shell Au@PB@Au NPs SERS substrate for detection of AFB1. 62 Choi et al 57 proposed a CRISPR/Cas12a-assisted SERS analysis system that is also a nucleic acid amplification-free biosensor (Fig.…”

“…Plasmonic nanosatellites are often found in homogeneous system detection and exhibit excellent SERS sensing performance because they support strong plasmonic coupling between the metal core and its satellite particles. Yin et al 61 recently proposed a CRISPR/Cas12a integrated SERS nanoplatform for nucleic acid detection based on a plasmonic satellite nanoconjugate. Interestingly, they adopted a chimeric DNA/RNA hairpin for gene amplification, significantly improving the detection sensitivity (Fig.…”