Clustered Regularly Interspaced Short Palindromic Repeats-Mediated Amplification-Free Detection of Viral DNAs Using Surface-Enhanced Raman Spectroscopy-Active Nanoarray

Abstract

Nucleic acid biomarkers have been widely used to detect various viral-associated diseases, including the recent pandemic COVID-19. The CRISPR-Cas-based trans-activating phenomenon has shown excellent potential for developing sensitive and selective detection of nucleic acids. However, the nucleic acid amplification steps are typically required when sensitive and selective monitoring of the target nucleic acid is needed. To overcome the aforementioned challenges, we developed a CRISPR-Cas12a-based nucleic acid amplification-free biosensor by a surface-enhanced Raman spectroscopy (SERS)-assisted ultrasensitive detection system. We integrated the activated CRISPR-Cas12a by viral DNA with a Raman-sensitive system composed of ssDNAimmobilized Raman probe-functionalized Au nanoparticles (RAuNPs) on the graphene oxide (GO)/triangle Au nanoflower array. Using this CRISPR-based Raman-sensitive system improved the detection sensitivity of the multiviral DNAs such as hepatitis B virus (HBV), human papillomavirus 16 (HPV-16), and HPV-18 with an extremely low detection limit and vast detection range from 1 aM to 100 pM without the amplification steps. We suggest that this ultrasensitive amplification-free detection system for nucleic acids can be widely applied to the precise and early diagnosis of viral infections, cancers, and several genetic diseases.

Applications of surface‐enhanced Raman spectroscopy in environmental detection

“…There are a wide variety of SERS methods that may be used for virus detection, but currently few have been applied to detect environmental samples. Promising avenues for future research include magnetic nanoparticles (MagSERS) in conjunction with immunoassays and digital microfluidics, 220,221 as well as amplification‐free Class 2 CRISPR/Cas systems 223,224,226–228 . For the detection of bacteria with SERS, there are a number of available methods, and many have already been applied to environmental safety research.…”

“…Recently, several articles have achieved SERS detection of a variety of samples using amplification‐free CRISPR, demonstrating that CRISPR/Cas is a promising technology for SERS detection in the field. Amplification‐free CRISPR has been used to detect HIV‐1 dsDNA (LOD = 0.3 fM), 223 hepatitis B virus, HPV‐16, and HPV‐18 DNA (LOD = 1 aM), 224 SARS‐CoV‐2 RNA (LOD = 1 fM), 226 SARS‐CoV‐2 N‐gene (LOD = 5 fM) 227 and SARS‐CoV‐2 RNA in clinical samples (LOD = 100 copies/µL) 228 . There are multiple ways to bypass the amplification step while still attaining low levels of detection, such as coupling CRISPR with a lateral flow assay, 223 performing detection on a microchamber platform 227 and using multiple different guide RNAs 227,228 .…”

PrimedSherlock: A tool for rapid design of highly specific CRISPR-Cas12 crRNAs

“…By performing off-target analysis of crRNA sequences, we can control for user-provided primer design independent of off-target enzyme activation. Further, the shift to diagnostic assays directly from sample without amplification is highly debated [33, 34, 35]. By maintaining this redundancy in our analysis, users should be able to adapt the toolset for CRISPR-Cas assays independent of sample amplification and account for the risks associated with multiplexed crRNA approaches.…”

Prospects of Surface-Enhanced Raman Spectroscopy for Biomarker Monitoring toward Precision Medicine

“…CRISPR-Cas12a activation causes the release of the SERS probes from graphene oxide-Au nanoflower arrays, resulting in a decrease of the SERS signal. Reproduced with permission from ref ( 236 ). Copyright ACS 2021.…”

“…Viral DNA could be detected by this methodology at extremely low concentrations, down to 1 aM, within only 20 min. 236 The combination of SERS and CRISPR systems has not only been tested for viral DNA detection, but also for the detection of multidrug-resistant bacteria. 237 …”