Controlled initiation of enzymatic reactions in micrometer-sized biomimetic compartments
Controlled initiation of enzymatic reactions in micrometer-sized biomimetic compartments
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DOI：
10.1021/jp0459716
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发表时间：
2005-02-03
影响因子：
3.3
通讯作者：
Orwar, O
中科院分区：
文献类型：
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作者：
Karlsson, A;Sott, K;Orwar, O
我们提出了一种技术，引发化学反应，涉及几个反应物内的微米级仿生囊泡（10（-12）至10（-15）L）集成到三维表面活性剂网络。这些网络的形状处于动态控制之下，允许两种或多种反应物随意转移和混合。具体而言，两个纳米管连接的囊泡填充反应物（底物和酶，分别）通过显微注射。最初，囊泡相距很远，并且由于纳米管的窄直径和长轴向延伸，分离的囊泡的内容物之间的任何扩散混合（在相关的实验时间尺度上）受到阻碍。为了引发反应，使囊泡靠近在一起，纳米管被囊泡消耗，并且在临界距离处，纳米管-囊泡连接处扩张，导致形成一个球形反应器，并因此混合内容物。我们证明的概念，使用模型酶促反应，产生荧光产物（碱性磷酸酶的荧光素二磷酸的两步水解），其中产品的形成作为时间的函数，使用FRAP荧光显微镜协议进行测量。通过将酶活性与体积测量值进行比较，可以确定囊泡内的酶浓度。对于具有仅约15个酶分子限制在反应器囊泡中的系统，可以进行反应。为了描述实验，我们使用一个简单的扩散控制的反应模型，并使用生存概率的方法来解决它。与实验的协议是定性的，但该模型描述的趋势。结果表明，该模型正确地预测了（i）几秒钟后的单指数衰减，以及（ii）底物衰减常数取决于酶的数量和反应容器的几何形状。数值修正因子A的引入，以确保实验和理论之间的半定量协议。结果表明，这个数值因子弱依赖于囊泡半径和酶的数量，因此它是足够的，以确定这个因素只有一次在一个单一的校准测量。
We present a technique to initiate chemical reactions involving few reactants inside micrometer-scale biomimetic vesicles (10(-12) to 10(-15) L) integral to three-dimensional surfactant networks. The shape of these networks is under dynamic control, allowing for transfer and mixing of two or several reactants at will. Specifically, two nanotube-connected vesicles were filled with reactants (substrate and enzyme, respectively) by microinjection. Initially, the vesicles are far apart and any diffusive mixing (on relevant experimental time scales) between the contents of the separated vesicles is hindered because of the narrow diameter and long axial extension of the nanotube. To initiate a reaction, the vesicles were brought close together, the nanotube was consumed by the vesicles and at a critical distance, the nanotube-vesicle junctions were dilated leading to formation of one spherical reactor, and hence mixing of the contents. We demonstrate the concept using a model enzymatic reaction, which yields a fluorescent product (two-step hydrolysis of fluorescein diphosphate by alkaline phosphatase), where product formation was measured as a function of time using a FRAP fluorescence microscopy protocol. By comparing the enzymatic activity with bulk measurements, the enzyme concentration inside the vesicle could be determined. Reactions could be followed for systems having as few as approximately 15 enzyme molecules confined to a reactor vesicle. To describe the experiments we use a simple diffusion-controlled reaction model and solve it using a survival probability approach. The agreement with experiment is qualitative, but the model describes the trends well. It is shown that the model correctly predicts (i) single-exponential decay after a few seconds, and (ii) that the substrate decay constant depends on the number of enzymes and geometry of reaction container. The numerical correction factor A is introduced in order to ensure semiquantitative agreement between experiment and theory. It was shown that this numerical factor depends weakly on vesicle radius and number of enzymes, thus it is sufficient to determine this factor only once in a single calibration measurement.