Karlsson Anders, Sott Kristin, Markström Martin, Davidson Max, Konkoli Zoran, Orwar Owe
Department of Chemistry, Göteborg University, SE-412 96 Göteborg, Sweden.
J Phys Chem B. 2005 Feb 3;109(4):1609-17. doi: 10.1021/jp0459716.
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 Lambda 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.
我们提出了一种技术，可在三维表面活性剂网络所不可或缺的微米级仿生囊泡（10⁻¹²至10⁻¹⁵升）内引发涉及少量反应物的化学反应。这些网络的形状处于动态控制之下，能够随意实现两种或多种反应物的转移与混合。具体而言，通过显微注射将两个由纳米管相连的囊泡分别填充反应物（底物和酶）。起初，囊泡相距甚远，由于纳米管直径狭窄且轴向延伸较长，在相关实验时间尺度上，分离囊泡内的物质之间的任何扩散混合都会受到阻碍。为引发反应，将囊泡拉近，纳米管被囊泡消耗，在临界距离时，纳米管 - 囊泡连接点扩张，导致形成一个球形反应器，从而使内容物混合。我们使用一个模型酶促反应来演示这一概念，该反应产生一种荧光产物（碱性磷酸酶对荧光素二磷酸进行两步水解），通过荧光漂白恢复（FRAP）荧光显微镜协议测量产物形成随时间的变化。通过将酶活性与大量测量结果进行比较，可以确定囊泡内的酶浓度。对于限制在反应囊泡内的酶分子少至约15个的系统，也能够追踪反应。为描述这些实验，我们使用一个简单的扩散控制反应模型，并采用生存概率方法求解。与实验的一致性是定性的，但该模型能很好地描述趋势。结果表明，该模型正确预测了：（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。引入数值校正因子Λ以确保实验与理论之间达成半定量一致。结果表明，这个数值因子对囊泡半径和酶的数量的依赖较弱，因此仅在一次校准测量中确定该因子一次就足够了。
