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 ssDNA-immobilized 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.
Keywords: CRISPR-Cas12a; amplification-free detection; graphene oxide (GO); surface-enhanced Raman spectroscopy (SERS); triangle Au nanoflower; viral DNA.