A CRISPR/Cas12a-Assisted SERS Nanosensor for Highly Sensitive Detection of HPV DNA
Jianqing Ye, Yongshi Shen, Zhizhong Lin, Luyun Xu, Lingna Wang, Xueliang Lin, Baoxing Huang, Zhiqing Ma, Zongyang Yu, Duo Lin, Wenjuan Chen, Shangyuan Feng
Abstract
The lack of timely and effective screening and diagnosis is a major contributing factor to the high mortality rate of cervical cancer in low-income countries and resource-limited regions. Therefore, the development of a rapid, sensitive, and easily deployable diagnostic tool for HPV DNA is of critical importance. In this study, we present a novel high-sensitivity and high-specificity detection method for HPV16 and HPV18 by integrating the CRISPR/Cas12a system with surface-enhanced Raman scattering (SERS) technology. This method leverages the trans-cleavage activity of the CRISPR/Cas12a system, which cleaves biotin-modified spherical nucleic acids (Biotin-SNA) in the presence of target DNA, releasing free Biotin-DNA. The released Biotin-DNA preferentially binds to streptavidin-modified magnetic beads (SAV-MB), reducing the capture of Biotin-SNA by SAV-MB and thereby significantly enhancing detection sensitivity. This method offers the potential for point-of-care diagnostics as it operates efficiently at 37 °C without the need for thermal cycling. Using standard DNA samples, we demonstrated that this biosensor achieved detection limits as low as 209 copies/μL and 444 copies/μL within 95 min. When combined with recombinase polymerase amplification (RPA), the sensor demonstrated enhanced sensitivity, enabling detection of target DNA at concentrations as low as 1 copy/μL within approximately 50 min. Furthermore, validation with clinical samples confirmed the feasibility and practical applicability of this method. This novel SERS-based sensor offers a new and effective tool in the prevention and detection of cervical cancer.