Addressing these challenges, Prof. Ki-Bum Lee and his team in the Department of Chemistry and Chemical Biology at Rutgers developed a CRISPR/Cas12a-based nucleic acid amplification-free biosensor by a surface-enhanced Raman spectroscopy (SERS)-assisted ultrasensitive detection system. As a proof-of-concept, the team focused on detecting multi-viral DNAs such as hepatitis B virus (HBV), human papillomavirus 16 (HPV-16), and HPV-18 [Figure 1].
To overcome the inherent drawback of the nucleic acid amplification process in CRISPR/Cas-based biosensing, the Raman signal was maximally enhanced by finely tuned homogeneous graphene oxide (GO)-triangle Au nanoflower array (GO-TANF). For the specific detection of viral DNAs, CRISPR RNA (crRNA) sequences were designed for binding on each target viral DNA and induced activation of Cas protein for trans-cleavage. Raman probe-functionalized Au nanoparticles (RAuNPs) were bound onto the surface of the GO-TANF via single-strand DNA (ssDNA). As a result, the developed sensing platform can measure a wide range of viral DNA concentrations (1 aM to 100 pM) without amplification. In addition, multiplexed detection of HBV, HPV-16, and HPV-18 was successfully done on a GO-TANF array.
In summary, our developed amplification-free CRISPR/Cas12-based biosensing system composed of GO-TANF and RAuNPs could provide a promising and versatile biosensing system for rapid, simple, precise, and ultrasensitive detection of viral nucleic acids. Besides, this system provides a novel strategy for enhancing SERS signal intensity and improving sensitivity for numerous biomarker detection, including viral DNA, genomic DNA, and cell-free DNA (cfDNA), and RNAs.
Publication: This work was recently published in ACS Nano, 2021, 15, 8, 13475-13485.
CORRESPONDENCE: Prof. Ki-Bum Lee (Rutgers University); Prof. Jeong-Woo Choi (Sogang University)
AUTHORS: Dr. Jin-Ha Choi, Dr. Letao Yang, Brian M. Conley, Dr. Jinho Yoon
KiBum Lee, Ph.D.
Jin-Ha Choi, Ph.D.
Letao Yang, Ph.D.
Brian M. Conley
Jinho Yoon, Ph.D.