Ultrasensitive CRISPR/Cas12a-Driven SERS Biosensor for On-Site Nucleic Acid Detection and Its Application to Milk Authenticity Testing

Abstract

An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN) 6 4− ), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core−shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.

CRISPR‐Cas‐based detection for food safety problems: Current status, challenges, and opportunities

“…Additionally, a range of platforms have been developed by leveraging the advantages of CRISPR‐Cas with nucleic acid amplification technologies such as PCR (Ma, Peng, et al., 2021), RAA (Li, Ye, Chen, Xiang, et al., 2021), RPA (Chen et al., 2020; Zhuang et al., 2022), LAMP (Li, Chen, et al., 2022), multiple cross‐displacement amplification (Zhu et al., 2021), strand displacement amplification (Wang, Liu, et al., 2019; Zhou et al., 2018), rolling circle amplification (Qing et al., 2021), hybridization chain reaction (Liu et al., 2022), exponential amplification method (Huang et al., 2018; Tian et al., 2020), nicking enzyme‐assisted amplification (Bai et al., 2022), and so on. The CRISPR‐Cas biosensors are usually based on fluorescence signal readouts; other signal readouts such as colorimetry (Li, Zheng, et al., 2021), electrochemistry (Qing et al., 2021), lateral flow assay (Marsic et al., 2021), photothermal effect (Ma, Peng, et al., 2021), portable personal glucose meter (Liu, Hu, et al., 2021), surface‐enhanced Raman scattering (SERS) assay (Kim, Lee, Seo, et al., 2020; Pan et al., 2022), gas bubble signal (Silva et al., 2021), microfluidic paper‐based analytical device (μPAD) (Zhuang et al., 2022), hydrogel‐integrated paper‐based analytical device (μReaCH‐PAD) (Huang, Ni, et al., 2021), coupling biolayer interferometry (Qiao, Liu, et al., 2021), luminescence resonance energy transfer (Lin et al., 2022), and so on are established for visual and rapid biosensing applications.…”

Innovative Application of SERS in Food Quality and Safety: A Brief Review of Recent Trends

“…The authenticity of food is another important research subject [ 56 ]. The adulteration of food products with cheaper materials for economic gain can pose serious health threats to consumers [ 57 ].…”

“…The adulteration of food products with cheaper materials for economic gain can pose serious health threats to consumers [ 57 ]. As vibrational spectroscopic techniques, Raman/SERS have the potential to fulfill the industrial need for food quality and authenticity analyses [ 56 ]. Two strategies to judge authenticity include: (1) combination with chemometrics to achieve the identification and quantification of food samples; (2) combination with specific target DNA-modified SERS substrates with a target-responsive Raman dye that can recognize the target ( Table 7 ) [ 58 , 59 ].…”

Sensitive Detection of a Single-Nucleotide Polymorphism in Foodborne Pathogens Using CRISPR/Cas12a-Signaling ARMS-PCR

“…Owing to the specific recognition from guide RNA (gRNA) and the multipleturnover trans-cleavage, integrating the CRISPR/Cas12a system into ARMS-PCR may enable high sensitivity with simpler equipment and operations. 27,28 Herein, we constructed a CRISPR/Cas12a-signaling ARMS-PCR assay, termed cARMS, to identify fluoroquinoloneresistant Salmonella with a single-nucleotide mutation. Owing to the high specificity of the ARMS method and the CRISPR/ Cas12a system, as well as the high amplification efficiency of the PCR method, the cARMS assay yielded single-nucleotide resolution and high SNP detection sensitivity (down to yielded a detection limit lower than that of gel electrophoresis (detection limit, ∼5%) and equivalent to that of qPCR (detection limit, ∼0.5%).…”