Bujnowicz Łukasz, Pietras Rafał, Sarewicz Marcin, Osyczka Artur
Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
HardwareX. 2023 Mar 1;14:e00409. doi: 10.1016/j.ohx.2023.e00409. eCollection 2023 Jun.
Experiments based on fast reagent mixing and observation of reaction progress are considered a powerful tool for investigating the kinetics of chemical and enzymatic reactions. Various spectroscopic methods are used in monitoring the reaction progress, which require different sample preparation methods. Stopped-flow is the most widespread method, where the reaction in the liquid phase is observed by optical absorption spectroscopy. Albeit less popular, the freeze-quench method is also used, in which the reaction is rapidly stopped by freezing the sample at a given time point after the reaction onset. The frozen droplets of the sample are collected and measured at low temperatures in the solid state. Currently, many commercial solutions are available for freeze-quench or stopped-flow experiments, but despite the high price of the devices, most of these do not allow combining both these methods in a single experiment. This study presents a relatively simple solution that combines both these methods, thus making a complete study of chemical or enzymatic reactions possible. Besides, the presented solution enables sequential double mixing of reagents, which is generally problematic and cannot be done using commercial instruments.
基于快速试剂混合和反应进程观察的实验被认为是研究化学和酶促反应动力学的有力工具。监测反应进程会用到各种光谱方法，这需要不同的样品制备方法。停流法是最广泛使用的方法，通过光学吸收光谱观察液相中的反应。尽管冷冻淬灭法不太常用，但也会被使用，即在反应开始后的给定时间点通过冷冻样品迅速终止反应。收集样品的冷冻液滴并在低温下以固态进行测量。目前，有许多商业解决方案可用于冷冻淬灭或停流实验，但尽管这些设备价格高昂，其中大多数不允许在单个实验中同时结合这两种方法。本研究提出了一种相对简单的解决方案，它结合了这两种方法，从而使对化学或酶促反应的完整研究成为可能。此外，所提出的解决方案能够实现试剂的顺序双重混合，这通常存在问题且无法使用商业仪器完成。
