School of Chemistry and Chemical Engineering, Guangdong Provincial Key Lab for Green Chemical Product Technology, South China University of Technology, Guangzhou 510640, P. R. China.
Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, P. R. China.
Nanoscale. 2020 Feb 14;12(6):3701-3714. doi: 10.1039/c9nr10123c. Epub 2020 Feb 5.
An acetylcholinesterase (AChE)-based electrochemical biosensor, as a promising alternative to detect organophosphates (OPs) and carbamate pesticides, has gained considerable attention in recent years, due to the advantages of simplicity, rapidity, reliability and low cost. The bio-activity of AChE immobilized on the surface and the direct electron transfer (DET) rate between an enzyme and an electrode directly determined the analytical performances of the AChE-based biosensor, and experimental studies have shown that the charged surfaces have a strong impact on the detectability of the AChE-based biosensor. Therefore, it is very important to reveal the behaviour of AChE in bulk solution and on charged surfaces at the molecular level. In this work, the adsorption orientation and conformation of AChE from Torpedo californica (TcAChE) on oppositely charged self-assembled monolayers (SAMs), COOH-SAM and NH-SAM with different surface charge densities, were investigated by parallel tempering Monte Carlo (PTMC) and all-atom molecular dynamics simulations (AAMD). Simulation results show that TcAChE could spontaneously and stably adsorb on two oppositely charged surfaces by the synergy of an electric dipole and charged residue patch, and opposite orientations were observed. The active-site gorge of TcAChE is oriented toward the surface with the "end-on" orientation and the active sites are close to the surface when it is adsorbed on the positively charged surface and the tunnel cost for the substrate is lower than that on the negatively charged surface and in bulk solution, while for TcAChE adsorbed on the negatively charged surface, the active site of TcAChE is far away from the surface and the active-site gorge is oriented toward the solution with a "back-on" orientation. It suggests that the positively charged surface could provide a better microenvironment for the efficient bio-catalytic reaction and quick DET between TcAChE and the electrode surface. Moreover, the RMSD, RMSF, dipole moment, gyration radius, eccentricity and superimposed structures show that only a slight conformational change occurred on the relatively flexible structure of TcAChE during simulations, and the native conformation is well preserved after adsorption. This work helps us better comprehend the adsorption mechanism of TcAChE on charged surfaces and might provide some guidelines for the development of new TcAChE-based amperometric biosensors for the detection of organophosphorus pesticides.
基于乙酰胆碱酯酶（AChE）的电化学生物传感器作为一种有前途的替代方法，用于检测有机磷（OP）和氨基甲酸酯类农药，近年来受到了相当大的关注，因为它具有简单、快速、可靠和低成本的优点。固定在表面上的 AChE 的生物活性以及酶与电极之间的直接电子转移（DET）速率直接决定了基于 AChE 的生物传感器的分析性能，实验研究表明，带电表面对基于 AChE 的生物传感器的检测能力有很强的影响。因此，揭示 AChE 在本体溶液和带电表面上的分子水平行为非常重要。在这项工作中，通过平行温度蒙特卡罗（PTMC）和全原子分子动力学模拟（AAMD）研究了来自加利福尼亚电鳐（TcAChE）的 AChE 在带相反电荷的自组装单层（SAM）COOH-SAM 和 NH-SAM 上的吸附取向和构象，这些 SAM 的表面电荷密度不同。模拟结果表明，TcAChE 可以通过电偶极子和带电残基斑块的协同作用自发且稳定地吸附在两个带相反电荷的表面上，并观察到相反的取向。TcAChE 的活性部位峡谷朝向表面，呈“端到端”取向，当它吸附在带正电荷的表面上时，活性位点靠近表面，并且底物的隧道成本低于带负电荷的表面和本体溶液，而对于吸附在带负电荷的表面上的 TcAChE，TcAChE 的活性位点远离表面，活性部位峡谷朝向溶液，呈“背向”取向。这表明带正电荷的表面可以为 TcAChE 与电极表面之间的高效生物催化反应和快速 DET 提供更好的微环境。此外，RMSD、RMSF、偶极矩、回转半径、偏心率和叠加结构表明，在模拟过程中，TcAChE 的相对灵活结构只发生了轻微的构象变化，并且在吸附后保持了天然构象。这项工作有助于我们更好地理解 TcAChE 在带电表面上的吸附机制，并可能为开发新的基于 TcAChE 的电流生物传感器用于检测有机磷农药提供一些指导。
