One-step enzyme kinetics measurement in 3D printed microfluidics devices based on a high-performance single vibrating sharp-tip mixer.
One-step enzyme kinetics measurement in 3D printed microfluidics devices based on a high-performance single vibrating sharp-tip mixer.
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DOI：
10.1016/j.aca.2021.338677
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发表时间：
2021-08-08
影响因子：
6.2
通讯作者：
Li P
中科院分区：
文献类型：
--
作者：
Li X;He Z;Li C;Li P
测量酶动力学对于理解许多生物过程和改进生物传感和工业应用是非常重要的。测量酶动力学的常规方法需要制备一系列具有不同底物浓度的溶液，并使用这些溶液测量随时间的信号响应，导致繁琐的样品制备步骤、高试剂/样品消耗以及难以研究快速酶动力学。在这里，我们报告了一种使用3D打印微流体设备测量酶动力学的一步测定法，该方法消除了制备和处理多种溶液的步骤，从而大大简化了整个工作流程。该分析通过高效的振动尖端混合方法实现，该方法可以在最短的混合长度（~300 μm）和时间内混合多股流体（低至3 ms），工作流速范围从1.5 μL/min到750 μL/min。由于混合器的高性能，在一次实验运行中，通过简单地调节从三个入口装载的试剂的流速来进行具有不同底物浓度的一系列实验。在此体系中测定了辣根过氧化物酶（HRP）催化H_2O_2与氨酰红反应的米氏动力学。米氏常数的计算值与文献值和常规分析方法的计算值一致。该方法具有制作简单、操作简便、分析速度快、功耗低（1.4 ~ 45.0mW）、时间分辨率高等优点，将极大地促进酶动力学的测量，并为优化酶生物传感实验和探测许多生化过程提供了巨大的潜力。
Measuring enzyme kinetics is of great importance to understand many biological processes and improve biosensing and industrial applications. Conventional methods of measuring enzyme kinetics require to prepare a series of solutions with different substrate concentrations and measure the signal response over time with these solutions, leading to tedious sample preparation steps, high reagents/sample consumption, and difficulties in studying fast enzyme kinetics. Here we report a one-step assay to measure enzyme kinetics using a 3D-printed microfluidic device, which eliminates the steps of preparing and handling multiple solutions thereby simplifying the whole workflow significantly. The assay is enabled by a highly efficient vibrating sharp-tip mixing method that can mix multiple streams of fluids with minimal mixing length (~300 μm) and time (as low as 3 ms), and a wide range of working flow rates from 1.5 μL/min to 750 μL/min. Owing to the high performance of the mixer, a series of experiments with different substrate concentrations are performed by simply adjusting the flow rates of reagents loaded from three inlets in one experiment run. The Michaelis-Menten kinetics of the horseradish peroxidase (HRP)-catalyzed reaction between H2O2 and amplex red is measured in this system. The calculated Michaelis constant is consistent with the values from literature and conventional analysis methods. Due to the simplicity in fabrication and operation, rapid analysis, low power consumption (1.4 – 45.0 mW), and high temporal resolution, this method will significantly facilitate enzyme kinetics measurement, and offers great potential for optimizing enzyme based biosensing experiments and probing many biochemical processes.
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DOI：
10.1038/nrm4041
发表时间：
2015-09
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
Duncombe TA;Tentori AM;Herr AE
通讯作者：
Herr AE
影响因子：
6.1
作者：
Ahmed, Daniel;Mao, Xiaole;Huang, Tony Jun
通讯作者：
Huang, Tony Jun
影响因子：
2.8
作者：
Ahmed, Daniel;Mao, Xiaole;Huang, Tony Jun
通讯作者：
Huang, Tony Jun
影响因子：
7.4
作者：
Bui, Minh-Phuong Ngoc;Li, Cheng Ai;Seong, Gi Hun
通讯作者：
Seong, Gi Hun
影响因子：
15.1
作者：
Huang, Po-Hsun;Zhao, Shuaiguo;Huang, Tony Jun
通讯作者：
Huang, Tony Jun