Departamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 14040-90, Brazil.
SEPARARE Núcleo de Pesquisa Em Cromatografia, Departamento de Química, Universidade Federal de São Carlos, Caixa Postal 676, São Carlos, 13565-905, Brazil.
Anal Chim Acta. 2019 Sep 23;1072:81-86. doi: 10.1016/j.aca.2019.04.057. Epub 2019 Apr 26.
This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.
本工作描述了一种新的基于固定化酶反应器（ICERs）的同时在线双平行酶分析方法，具有质谱检测功能。这项工作的新颖之处在于，只需单次进样，在不到 6 分钟的时间内即可同时筛选两种不同的酶。该系统由两个固定化毛细管酶反应器（ICERs）组成。更具体地说，ICERs 包含两种不同的酶，它们平行容纳，并放置在液相色谱（LC）系统和质谱仪（MS）之间。该系统可适用于其他类型的具有不同载体的酶反应器。系统中的所有元件均通过两个 10 端口/双位置切换阀进行接口连接。不同的管尺寸允许我们在同一次分析中独立监测每种酶的活性。选择乙酰胆碱酯酶（AChE）和丁酰胆碱酯酶（BChE）生物反应器作为概念验证。使用乙酰胆碱（ACh）作为底物；在有和没有标准胆碱酯酶抑制剂加兰他敏的情况下，监测其质子化酶解产物离子胆碱的面积[M+H]+m/z 104.0。该方法被证明是一种快速、同时、独立的无标记双酶抑制剂分析的有趣工具。
