Yang Runlin, Xie Siying, Zhou Bin, Guo Mingming, Fan Jun, Su Fengli, Ji Zhirun, Chen Yue, Li Bingzhi
NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China.
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210023, China.
ACS Appl Mater Interfaces. 2024 Feb 28;16(8):9890-9899. doi: 10.1021/acsami.3c18732. Epub 2024 Feb 14.
CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.
基于CRISPR/Cas12a的生物传感技术正在迅速发展；然而，实现对Cas12a激活的灵敏且经济高效的报告仍然是一项挑战。作为回应，我们开发了一种无标记系统，该系统通过整合杂交链式反应（HCR）和DNA-铜纳米簇（DNA-CuNCs）能够对Cas12a激活进行后扩增。Cas12a的反式切割触发沉默的HCR，导致荧光DNA-CuNCs的原位组装，从而实现对Cas12a激活的开启式报告。无需预扩增，该检测方法能够检测低至5 fM的DNA。此外，当与预扩增相结合时，该系统实现了卓越的灵敏度，能够在人血清中检测到单拷贝的猴痘病毒（MPXV）质粒。在基于MPXV假病毒的验证测试中，所获得的结果与通过qPCR获得的结果一致，增强了该方法的稳健性。我们的研究首次尝试在HCR上操纵DNA-CuNC的形成，以实现对Cas12a的高灵敏且经济高效的报告，从而产生了一个用于生物安全应用的高效合成生物学驱动的传感平台。
