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Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

ABSTRACT: In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K(m) and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K(m) and k(cat) can then be obtained from the reaction rate curves for different concentrations of substrate while holding the enzyme concentration constant. We studied the enzymatic reaction for ?-galactosidase and its substrate (resorufin-?-D-galactopyranoside) and found K(m) and k(cat) to be 333 ± 130 ?M and 64 ± 8 s(-1), respectively, which are in agreement with published data. Our approach is valuable for studying the kinetics of high-speed enzymatic reactions and other chemical reactions.

SUBMITTER: Xie Y

PROVIDER: S-EPMC3991781 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

Xie Yuliang Y Ahmed Daniel D Lapsley Michael Ian MI Lin Sz-Chin Steven SC Nawaz Ahmad Ahsan AA Wang Lin L Huang Tony Jun TJ

Analytical chemistry 20120814 17

In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K(m) and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K(m) and k(cat) can then be obtained from the reaction rate ...[more]

PMID: 22880882

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Project description:Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustical, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency ultrasound (7.44MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5MHz) ultrasound pulse (duration 13.3 or 40?s) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d=0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106±0.032?m (n=70) for acoustic pressure of 1.5MPa (duration 13.3?s), and increased to 0.171±0.030?m (n=125) for acoustic pressure of 1.7MPa and to 0.182±0.052?m (n=112) for a pulse duration of 40?s (1.5MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.

Dataset Information

Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

Publications

Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

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