College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA.
Sci Total Environ. 2024 Feb 20;912:168905. doi: 10.1016/j.scitotenv.2023.168905. Epub 2023 Nov 26.
In traditional CRISPR-based biosensors, the cleavage-induced signal generation is insufficient because only a signals is generated at a CRISPR-induced cleavage. Herein, we developed an improved CRISPR/Cas12a-based biosensor with an enlarged signal generation which integrated the hybridization chain reaction (HCR) and low-background Förster Resonance Energy Transfer (FRET) signal output mode. The HCR with nucleic acid self-assembly capability was used as a signal carrier to load more signaling molecules. To get the best signal amplification, three different fluorescence signal output modes (fluorescence recovery, FRET and low-background FRET) generated by two fluoresceins, FAM and Cy5, were fully investigated and compared. The results indicated that the low-background FRET signal output mode with the strictest signal generation conditions yielded the highest signal-to-noise ratio (S/N) (19.17) and the most obvious fluorescence color change (from red to yellow). In optimal conditions, the proposed biosensor was successfully applied for Salmonella Typhimurium (S. Typhimurium) detection with 6 h (including 4 h for sample pre-treatment) from the initial target processing to the final detection result. The qualitative sensitivity, reliant on color changes, was 10 CFU/mL. The quantitative sensitivity, calculated by the fluorescence value, were 1.62 × 10 CFU/mL, 3.72 × 10 CFU/mL, and 8.71 × 10 CFU/mL in buffer solution, S. Typhimurium-spiked milk samples, and S.Typhimurium-spiked chicken samples, respectively. The excellent detection performance of the proposed biosensor endowed its great application potential in food and environment safety monitoring.
在传统的基于 CRISPR 的生物传感器中，由于 CRISPR 诱导的切割仅产生一个信号，因此切割诱导的信号产生不足。在此，我们开发了一种改进的基于 CRISPR/Cas12a 的生物传感器，该传感器具有更大的信号产生能力，集成了杂交链反应（HCR）和低背景Förster 共振能量转移（FRET）信号输出模式。具有核酸自组装能力的 HCR 被用作信号载体，以加载更多的信号分子。为了获得最佳的信号放大效果，充分研究并比较了两种荧光素 FAM 和 Cy5 产生的三种不同的荧光信号输出模式（荧光恢复、FRET 和低背景 FRET）。结果表明，在最严格的信号产生条件下，低背景 FRET 信号输出模式产生的信号与噪声比（S/N）最高（19.17），荧光颜色变化最明显（从红色变为黄色）。在最佳条件下，该生物传感器成功应用于鼠伤寒沙门氏菌（S. Typhimurium）检测，从初始目标处理到最终检测结果，整个过程仅需 6 小时（包括 4 小时的样品预处理）。依赖于颜色变化的定性灵敏度为 10 CFU/mL。通过荧光值计算的定量灵敏度在缓冲溶液、添加 S. Typhimurium 的牛奶样品和添加 S.Typhimurium 的鸡肉样品中分别为 1.62×10 CFU/mL、3.72×10 CFU/mL 和 8.71×10 CFU/mL。该生物传感器具有出色的检测性能，使其在食品和环境安全监测方面具有巨大的应用潜力。
