Faculty of Chemical Technology, University of Pardubice, Studentska 95, Pardubice CZ-53210, Czech Republic.
Faculty of Military Health Sciences, University of Defense, Trebesska 1575, Hradec Kralove CZ-50001, Czech Republic.
Sensors (Basel). 2017 Mar 24;17(4):676. doi: 10.3390/s17040676.
Magnetic particles (MPs) have been widely used in biological applications in recent years as a carrier for various molecules. Their big advantage is in repeated use of immobilized molecules including enzymes. Acetylcholinesterase (AChE) is an enzyme playing crucial role in neurotransmission and the enzyme is targeted by various molecules like Alzheimer's drugs, pesticides and warfare agents. In this work, an electrochemical biosensor having AChE immobilized onto MPs and stabilized through glutaraldehyde (GA) molecule was proposed for assay of the neurotoxic compounds. The prepared nanoparticles were modified by pure AChE and they were used for the measurement anti-Alzheimer's drug galantamine and carbamate pesticide carbofuran with limit of detection 1.5 µM and 20 nM, respectively. All measurements were carried out using screen-printed sensor with carbon working, silver reference, and carbon auxiliary electrode. Standard Ellman's assay was used for validation measurement of both inhibitors. Part of this work was the elimination of reversible inhibitors represented by galantamine from the active site of AChE. For this purpose, we used a lower pH to get the original activity of AChE after inhibition by galantamine. We also observed decarbamylation of the AChE-carbofuran adduct. Influence of organic solvents to AChE as well as repeatability of measurement with MPs with AChE was also established.
近年来，磁性颗粒 (MPs) 作为各种分子的载体，已在生物应用中得到广泛应用。它们的一个很大的优势在于可以重复使用固定化的分子，包括酶。乙酰胆碱酯酶 (AChE) 是一种在神经递质传递中起关键作用的酶，该酶是各种分子的靶标，如阿尔茨海默病药物、杀虫剂和战争制剂。在这项工作中，提出了一种将 AChE 固定在 MPs 上并通过戊二醛 (GA) 分子稳定的电化学生物传感器，用于测定神经毒性化合物。制备的纳米颗粒经纯 AChE 修饰，用于测定抗阿尔茨海默病药物加兰他敏和氨基甲酸酯类农药克百威，检测限分别为 1.5 µM 和 20 nM。所有测量均使用带有碳工作电极、银参比电极和碳辅助电极的丝网印刷传感器进行。标准的 Ellman 测定法用于验证两种抑制剂的测量。这项工作的一部分是从 AChE 的活性部位消除以加兰他敏为代表的可逆抑制剂。为此，我们使用较低的 pH 值，在加兰他敏抑制后获得 AChE 的原始活性。我们还观察到 AChE-克百威加合物的脱羰反应。还研究了有机溶剂对 AChE 的影响以及含有 AChE 的 MPs 的测量重复性。
