Beijing National Laboratory for Molecular Sciences; Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing100190, P. R. China.
School of Chemical Sciences, University of Chinese Academy of Sciences, Beijing100049, P. R. China.
ACS Appl Bio Mater. 2022 Dec 19;5(12):5826-5831. doi: 10.1021/acsabm.2c00810. Epub 2022 Nov 28.
It is necessary to develop reliable chemiluminescence strategies for determination of intracellular adenosine triphosphate (ATP), which is vital in life science and clinical diagnosis. However, the current chemiluminescence methods based on firefly luciferase suffered from low delivery efficiency, unsatisfied targeting performance, and autohydrolysis in living biosystem. To circumvent these drawbacks, a thermoresponsive polymer nanocomposite modified with firefly luciferase and ATP aptamer (PFLNC@aptamer) was fabricated, which targeted ATP and determined the intracellular ATP levels via measuring the chemiluminescence signals at different temperatures. The PFLNC@aptamer exhibited capability for the enzymolysis efficiency regulation, increased 21.0% with temperature change from 37.0 to 25.0 °C. The ATP detection limit was 3.3 nM with a linear relationship from 10.0 nM to 0.1 mM. Moreover, the thermoresponsive nanocomposite could also effectively avoid the interference during delivering firefly luciferase into the living cells and effectively discriminate ATP via the immobilized ATP aptamer, which further confirmed its reliability for practical applications. It paves a specific avenue for effective intracellular ATP monitoring in fundamental and applied research.
有必要开发可靠的化学发光策略来测定细胞内三磷酸腺苷（ATP），这在生命科学和临床诊断中至关重要。然而，目前基于萤火虫荧光素酶的化学发光方法存在传递效率低、靶向性能差以及在活生物体系中自动水解等问题。为了克服这些缺点，制备了一种用萤火虫荧光素酶和 ATP 适体修饰的温敏聚合物纳米复合材料（PFLNC@aptamer），该复合材料通过在不同温度下测量化学发光信号来靶向 ATP 并测定细胞内 ATP 水平。PFLNC@aptamer 表现出酶解效率调节能力，当温度从 37.0 摄氏度降至 25.0 摄氏度时，酶解效率提高了 21.0%。ATP 的检测限为 3.3 nM，线性范围为 10.0 nM 至 0.1 mM。此外，温敏纳米复合材料还可以有效地避免在将萤火虫荧光素酶递送到活细胞过程中的干扰，并通过固定化的 ATP 适体有效地区分 ATP，这进一步证实了其在实际应用中的可靠性。它为基础和应用研究中有效的细胞内 ATP 监测开辟了一条特定的途径。
