Faculty of Material Science and Chemical Engineering, Ningbo University, Ningbo 315211, China.
Faculty of Marine, Ningbo University, Ningbo 315211, China.
Biosensors (Basel). 2023 Mar 19;13(3):401. doi: 10.3390/bios13030401.
Both live and dead (S.T) are harmful to human health, but there are differences in pathological mechanism, dosage, and security. It is crucial to develop a rapid and simultaneous assay to distinguish and quantify live and dead S.T in foods. Herein, one dual-mode biosensor for simultaneous detection of live and dead S.T was fabricated based on two phage probes, using portable bioluminescence and fluorescent meter as detectors, respectively. Firstly, a magnetic phage capture probe (M-P1) and a phage signal tag (P2-S) labeled with SYTO 13 fluorescent dye were prepared, respectively. Both M-P1 and P2-S can specifically conjugate with S.T to form a magnetic sandwich complex. After magnetic separation, the isolated complex can emit a fluorescent signal under an excited 365 nm laser, which can reflect the total amount of S.T. Afterwards, the lysozyme was added to decompose the captured live S.T, which can release ATP and produce a bioluminescent signal corresponding to the live S.T amount. The dead S.T concentration can be deduced by the difference between total and live examples. The detection limit of 55 CFU/mL for total S.T and 9 CFU/mL for live ones was within 20 min. The assay was successfully employed in milk samples and prospectively for on-site screening of other dead and live bacteria, while changing the phages for the targets.
活的和死的（S.T）都对人体健康有害，但在病理机制、剂量和安全性方面存在差异。开发一种快速且同时测定食品中活的和死的 S.T 的方法至关重要。在此，我们基于两种噬菌体探针，使用便携式生物发光和荧光计分别作为检测器，构建了一种用于同时检测活的和死的 S.T 的双模式生物传感器。首先，制备了一种带有 SYTO 13 荧光染料标记的磁性噬菌体捕获探针（M-P1）和噬菌体信号标签（P2-S）。M-P1 和 P2-S 均可与 S.T 特异性结合，形成磁夹心复合物。磁分离后，分离的复合物在 365nm 激光激发下发出荧光信号，反映 S.T 的总量。随后，加入溶菌酶分解捕获的活 S.T，释放出与活 S.T 量相对应的 ATP 并产生生物发光信号。通过总样本和活样本之间的差异可以推断出死 S.T 的浓度。总 S.T 的检测限为 55CFU/mL，活 S.T 的检测限为 9CFU/mL，检测时间均在 20min 内。该方法成功应用于牛奶样品，并可用于现场筛选其他死菌和活菌，只需更换针对目标的噬菌体即可。
