National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, People's Republic of China.
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China.
Biosens Bioelectron. 2023 Jun 1;229:115229. doi: 10.1016/j.bios.2023.115229. Epub 2023 Mar 15.
Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes (Au, Ag, Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218 fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics.
对多种感兴趣的分析物进行敏感定量对于临床诊断具有重要意义。CRISPR Cas 平台为提高基于核酸的诊断的特异性、灵敏度和速度提供了一种策略，而其多重分析能力仍然有限且具有挑战性。在此，我们开发了一种基于 DNA 四面体（DTN）的新型生物传感器，该传感器基于空间分离的 CRISPR Cas 自我扩增策略和多种金属纳米粒子标记，并结合电感耦合等离子体质谱（ICP-MS）检测，以提高该平台用于 HPV-DNA（HPV-16、HPV-18 和 HPV-52）多重检测的灵敏度和可行性。鉴于靶 DNA 诱导 Cas12a/crRNA 双链体的强大转切割活性，并且周围相应的单链 DNA（ssDNA）接头被切割成短片段，无法将金属纳米粒子探针（Au、Ag、Pt）键合到 DTN 修饰的磁性珠探针（MBs-DTN）上，导致 ICP-MS 信号明显变化。值得注意的是，与 ssDNA 功能化 MBs 相比，我们的系统通过使用 MBs-DTN 获得了更高的信噪比，通过调节 MBs 表面的探针进一步放大了信号。正如预期的那样，通过该提出的策略，可以以低至 218 fM 的检测限检测 HPV-DNA，并以高准确度和特异性进行一次测试的多重分析。此外，我们证明了可以检测宫颈拭子样本中的 HPV-DNA，与 DNA 测序结果具有高度一致性。我们相信，这项工作为设计基于 CRISPR 的多重检测系统提供了有前途的选择，以实现高灵敏度、良好特异性和临床分子诊断。
