A new gene detection platform based on surface-enhanced Raman spectroscopy (SERS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system is developed, which is applied for virus gene detections. CRISPR/Cas12a activated by CRISPR ribonucleic acid (crRNA) can recognize and lock the complementary target double-stranded deoxyribonucleic acid (dsDNA), followed by the cis -cleavage on the dsDNA and the trans -cleavage on the nearby linker single-stranded DNA (ssDNA). Target-induced trans -ssDNA cleavage leads to a failure in an aggregation behavior of the designed SERS probe pair that was bridged by the linker ssDNA, which causes the SERS intensity reduced relative to the control trial that gives much stronger SERS due to the severe aggregation state of SERS probes caused by intact linker ssDNA. This sensing method achieves the highly sensitive detections of human papillomavirus (HPV) genes in serum and pseudovirus within 40 min. The detection sensitivity of this CRISPR/Cas-SERS platform can reach pM. Owing to the flexible designs of the DNA probe and crRNA, this CRISPR/Cas-SERS sensing system is a universal gene detection tool, which can be applied for the in-vitro diagnosis field and point-of-care testing.
Lihua Chen,Mingchao Liu,Yan Tang,Chuangfu Chen,Xingxing Wang,Zhiqiang Hu
Gold magnetic particles as a new carrier of disease diagnosis probes have attracted wide attention but encountered a bottleneck. That is, the interfacial properties of gold magnetic particles are susceptible to the influence of nonspecific biological molecules in actual diagnostic samples. Here, a novel nanoparticle made by the covalent attachment of polyethyleneimine and hyperbranched polyether polyol onto the gold shell surface of a magnetic bead demonstrated not only low fouling properties but also excellent stability in a variety of external environments, especially in complex biological systems. Most importantly, in its application as the probe for sensitive and selective fluorescence detection of high-risk human papillomavirus (HPV) genotypes 18, 16 in buffer, even in 100% serum, a good linear correlation with the concentration of HPV18/16 target DNA ranging from 5 nM to 1 μM was shown with the low detection limits. To our knowledge, this is one of the few successful examples of direct application of magnetic beads to the detection of disease markers in whole serum, suggesting that this material has good commercial potential and value.
危险品化学品经营许可证（带存储）