Surface-enhanced Raman scattering (SERS)-based biosensors are promising tools for virus nucleic acid detection. However, it remains challenging for SERS-based biosensors using a sandwiching strategy to detect long-chain nucleic acids such as nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because the extension of the coupling distance (CD) between the two tethered metallic nanostructures weakens electric field and SERS signals. Herein, we report a magnetic-responsive substrate consisting of heteoronanostructures that controls the CD for ultrasensitive and highly selective detection of the N gene of SARS-CoV-2. Significantly, our findings show that this platform reversibly shortens the CD and enhances SERS signals with a 10-fold increase in the detection limit from 1 fM to 100 aM, compared to those without magnetic modulation. The optical simulation that emulates the CD shortening process confirms the CD-dependent electric field strength and further supports the experimental results. Our study provides new insights into designing a stimuli-responsive SERS-based platform with tunable hot spots for long-chain nucleic acid detection.
Various
mycotoxins widely co-exist in agro-products, and their
combined effects cause toxicity and potential carcinogenicity to humans
and animals. In this work, we developed an economical and sensitive
quantum dots (QDs)/QD microbead (QDs/QB)-based multiplex immunochromatographic
assay (mICA) for the rapid detection of fumonisin B1 (FB1), zearalenone (ZEN), and ochratoxin A (OTA) without the building-up
process of mycotoxin conjugates. QDs and QBs were selected as fluorescent
reporters and conjugated with antimycotoxin monoclonal antibodies
for improving sensitivity. Furthermore, phage-displayed FB1, ZEN, and OTA mimotope peptide-based soluble and monovalent fusions
to maltose-binding protein (MBP) were applied onto the test line of
the mICA as the mimetic coating antigen. Under the optimized conditions,
the visual detection limits (vLODs) of peptide–MBP-based mICA
could be obtained as 0.25 ng/mL for FB1, 3.0 ng/mL for
ZEN, and 0.5 ng/mL for OTA within 10 min. The results for spiked real
sample detection indicate good accuracy, reproducibility, and practicability.
In addition, the proposed mICA was comparable with ultraperformance
liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)
in terms of reliability in detecting FB1, ZEN, and OTA
using natural samples. From the point of promoting commercial production,
these time-saving and low-cost peptide–MBP antigens applied
in ICA might provide promising potential for promoting productivity
and decreasing the cost of production.
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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