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Acoustically-levitated drop reactor (ldr) employable for kinetics measurements of biochemical networks

机译：声悬浮液滴反应器（ldr）可用于生化网络的动力学测量

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摘要

Accurate measurement of enzyme kinetics is an essential part of understanding the mechanisms of biochemical reactions. The typical means of studying such systems use stirred cuvettes, stopped-flow apparatus, microfluidic systems, or other small sample containers. These reaction-kinetics measurements reactors are usually made of quartz, glass, or an easily-moldable polymer such as polydimethylsiloxane. For studying enzyme kinetics or free radical chemistry, using small volumes with high concentrations is desirable, but interactions of biochemical moieties with these materials can be problematic. The main obstacle is that, as the system volume shrinks, the ratio of surface area to volume increases. As a result, the importance of adsorption can increase, and proteins sticking to walls, biofilms grow on walls, and radicals annihilating on walls can become significant. Thus, under these circumstances, typical microfluidic systems are incompatible with the demands of accurate study of many biochemical systems.Over the past five years, acoustic levitation has become a technique uniquely suited to studying biochemical reactions. Acoustic levitation offers several advantages over typical reaction systems, including small sample volume (and mass), the prevention of chemical contamination between drops and external objects, rapid mixing within the drop as a result of circulation driven by the levitating ultrasound, and freedom from wall interactions. We have developed an Acoustically-Levitated Drop Reactor (LDR) to study enzyme-catalyzed reaction kinetics related to free-radical and oxidative stress chemistry. Microliter-scale droplet generation, reactant introduction, maintenance, and fluid removal are all important aspects in conducting reactions in a levitated drop. Therefore, we developed a three-capillary bundle system to address these needs. Since the capillary system is used to introduce drops to the levitation cavity, our LDR system is not 100% wall-less, but the ratio of solid surface area to drops surface area is less than 20%, which is low compared to all the other reaction systems. The largest interface seen by reactants is a liquid/gas interface. Herein, I report kinetic measurements for both luminol chemiluminescence and the reaction of pyruvate with nicotinamide adenine dinucleotide, catalyzed by lactate dehydrogenase in a levitated drop. Observations of the chemiluminescence experiment showed second-order kinetics were detected within experimental error prior to deviations seen after 10 s. For the enzyme-catalyzed reaction kinetic measurements, it was found that the KM for lactate dehydrogenase matched literature values to better than one standard deviation of the literature experiment. Also studied were the effects of laser exposure on the stability of Myeloperoxidase at the liquid/gas interface. It was found that although myeloperoxidase degrades over several hours in buffer, there is no indication that the degradation is due to either denaturation at the free surface or to two-photon photolysis. Based on our accomplishments, we demonstrate the feasibility of using a levitated drop as a microreactor. The reactions discussed utilized in-house developed fiber optic and laser-based optical detection systems to monitor reaction kinetics inside a levitated drop via chemiluminescence and fluorescence. Outgrowths of these accomplishments will hopefully lead to the use of additional detection systems (including electrochemical and mass spectrometry) with the LDR to study a wide variety of biochemical reaction kinetics.
机译：酶动力学的准确测量是理解生化反应机理的重要组成部分。研究此类系统的典型方法是使用比色皿，停止流动的仪器，微流体系统或其他小的样品容器。这些反应动力学测量反应器通常由石英，玻璃或易成型的聚合物（例如聚二甲基硅氧烷）制成。为了研究酶动力学或自由基化学，需要使用小体积高浓度的试剂，但是生化部分与这些材料的相互作用可能会出现问题。主要障碍在于，随着系统体积的缩小，表面积与体积之比增加。结果，吸附的重要性会增加，蛋白质粘附在壁上，生物膜会在壁上生长，自由基消灭在壁上会变得很重要。因此，在这种情况下，典型的微流体系统不符合对许多生化系统进行精确研究的要求。在过去的五年中，声悬浮已成为一种独特的适合研究生化反应的技术。与典型的反应系统相比，声悬浮具有几个优势，包括较小的样品量（和质量），防止液滴与外部物体之间的化学污染，由于悬浮超声驱动的循环而导致液滴在液滴内快速混合以及不受壁的影响互动。我们已经开发了一种声悬浮反应器（LDR），以研究与自由基和氧化应激化学有关的酶催化反应动力学。微升级液滴的产生，反应物的引入，维持和流体的去除都是在悬浮液滴中进行反应的重要方面。因此，我们开发了一种三毛细管束系统来满足这些需求。由于使用毛细管系统将液滴引入悬浮腔，因此我们的LDR系统并非没有100％无壁，但固体表面积与液滴表面积的比率小于20％，与所有其他比率相比较低反应系统。反应物看到的最大界面是液体/气体界面。在这里，我报道了在悬浮液中乳酸脱氢酶催化的鲁米诺化学发光以及丙酮酸与烟酰胺腺嘌呤二核苷酸反应的动力学测量。化学发光实验的观察结果表明，在10秒后出现偏差之前，在实验误差范围内检测到了二级动力学。对于酶催化的反应动力学测量，发现乳酸脱氢酶的KM与文献值相匹配，好于文献实验的一个标准差。还研究了激光暴露对髓过氧化物酶在液/气界面处稳定性的影响。已经发现，尽管髓过氧化物酶在缓冲液中降解数小时，但没有迹象表明降解是由于自由表面的变性或双光子光解引起的。基于我们的成就，我们证明了使用悬浮液滴作为微反应器的可行性。讨论的反应利用内部开发的基于光纤和激光的光学检测系统通过化学发光和荧光监测悬浮液滴内部的反应动力学。这些成就的产生将有望导致LDR使用其他检测系统（包括电化学和质谱）来研究各种生化反应动力学。

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    Pierre Zakiah N.;

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  • 年度 2011
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  • 正文语种 {"code":"en","name":"English","id":9}
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