Goldner Lori S, Jofre Ana M, Tang Jianyong
Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA.
Methods Enzymol. 2010;472:61-88. doi: 10.1016/S0076-6879(10)72015-2.
We describe a method for molecular confinement and single-fluorophore sensitive measurement in aqueous nanodroplets in oil. The sequestration of individual molecules in droplets has become a useful tool in genomics and molecular evolution. Similarly, the use of single fluorophores, or pairs of fluorophores, to study biomolecular interactions and structural dynamics is now common. Most often these single-fluorophore sensitive measurements are performed on molecules that are surface attached. Confinement via surface attachment permits molecules to be located and studied for a prolonged period of time. For molecules that denature on surfaces, for interactions that are transient or out-of-equilibrium, or to observe the dynamic equilibrium of freely diffusing reagents, surface attachment may not be an option. In these cases, droplet confinement presents an alternative method for molecular confinement. Here, we describe this method as used in single-fluorophore sensitive measurement and discuss its advantages, limitations, and future prospects.
我们描述了一种在油包水纳米液滴中进行分子限制和单荧光团灵敏测量的方法。将单个分子隔离在液滴中已成为基因组学和分子进化中的一种有用工具。同样，使用单个荧光团或荧光团对来研究生物分子相互作用和结构动力学现在也很常见。这些单荧光团灵敏测量大多是在表面附着的分子上进行的。通过表面附着进行限制可使分子被定位并长时间研究。对于在表面变性的分子、短暂或非平衡的相互作用，或者为了观察自由扩散试剂的动态平衡，表面附着可能不是一个选择。在这些情况下，液滴限制为分子限制提供了一种替代方法。在此，我们描述这种用于单荧光团灵敏测量的方法，并讨论其优点、局限性和未来前景。
