One-step enzyme kinetics measurement in 3D printed microfluidics devices based on a high-performance single vibrating sharp-tip mixer.

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 HO 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.