Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran 14115-175, Iran.
Biosensors (Basel). 2022 Oct 24;12(11):918. doi: 10.3390/bios12110918.
A bioluminescence-based assay for ATP can measure cell viability. Higher ATP concentration indicates a higher number of living cells. Thus, it is necessary to design an ATP sensor that is low-cost and easy to use. Gold nanoparticles provide excellent biocompatibility for enzyme immobilization. We investigated the effect of luciferase proximity with citrate-coated gold, silver, and gold-silver core-shell nanoparticles, gold nanorods, and BSA-Au nanoclusters. The effect of metal nanoparticles on the activity of luciferases was recorded by the luminescence assay, which was 3-5 times higher than free enzyme. The results showed that the signal stability in presence of nanoparticles improved and was reliable up to 6 h for analytes measurements. It has been suggested that energy is mutually transferred from luciferase bioluminescence spectra to metal nanoparticle surface plasmons. In addition, we herein report the 27-base DNA aptamer for adenosine-5'-triphosphate (ATP) as a suitable probe for the ATP biosensor based on firefly luciferase activity and AuNPs. Due to ATP application in the firefly luciferase reaction, the increase in luciferase activity and improved detection limits may indicate more stability or accessibility of ATP in the presence of nanoparticles. The bioluminescence intensity increased with the ATP concentration up to 600 µM with a detection limit of 5 µM for ATP.
基于生物发光的 ATP 分析可用于测量细胞活力。较高的 ATP 浓度表明有更多的活细胞。因此，有必要设计一种低成本、易于使用的 ATP 传感器。金纳米粒子为酶固定提供了极好的生物相容性。我们研究了荧光素酶与柠檬酸包覆的金、银和金-银核壳纳米粒子、金纳米棒和 BSA-Au 纳米团簇的接近程度的影响。金属纳米粒子对荧光素酶活性的影响通过发光分析进行了记录，其活性比游离酶高 3-5 倍。结果表明，在纳米粒子存在下，信号稳定性提高，分析物测量的可靠性长达 6 小时。据认为，能量从荧光素酶生物发光光谱相互转移到金属纳米粒子表面等离激元。此外，我们在此报告了用于腺苷 5'-三磷酸 (ATP) 的 27 碱基 DNA 适体，作为基于萤火虫荧光素酶活性和 AuNPs 的 ATP 生物传感器的合适探针。由于 ATP 在萤火虫荧光素酶反应中的应用，荧光素酶活性的增加和检测限的提高可能表明在纳米粒子存在下 ATP 更稳定或更易接近。生物发光强度随 ATP 浓度的增加而增加，ATP 的检测限为 5µM。
