The Crystal Optimizer: Kinetic Control of Protein Crystallization

描述(由申请人提供)：蛋白质结晶仍然是确定蛋白质结构的目标的瓶颈。一种被称为晶体优化器(XOpt)的微流控装置已经被开发出来，用于优化蛋白质晶体成核和生长的动力学路径，一旦通过其他筛选方法确定了引导条件。该装置驱动数百滴候选蛋白质溶液中的每一滴在不同的持续时间内达到不同程度的过饱和度，然后将过饱和度降低到较低的值。其概念是找到两步动力学路径，首先在适当的时间内熄灭液滴，使少数晶体成核，然后将过饱和降低到抑制额外晶体成核的水平，但仍过饱和到足以使晶体缓慢生长。除了控制浓度，XOpt还测量作为时间函数的溶质和蛋白质浓度，因此可以获得关于样品经过的实际动力学路径的定量信息，并在随后的几轮优化中利用这些信息来设计改进的动力学路径。增加一个简单的温度阶段，在芯片上产生温度梯度，用来寻找最优动力学路径的最佳温度。 有两个关键点使Crystal Optimizer具有创新性和实用性。首先，蛋白质结晶是一个激活的、非平衡的过程，而XOpt是唯一的微流体设备，旨在以可控和可逆的方式系统地优化这一物理特性。其次，XOpt使用简单，制造成本低廉。

DESCRIPTION (provided by applicant): Protein crystallization remains a bottleneck in the goal to determine the protein's structure. A microfluidic device denoted the Crystal Optimizer (XOpt) has been developed to optimize the kinetic pathway for the nucleation and growth of protein crystals once the lead conditions have been identified by other screening methods. The device drives each of hundreds of drops of the candidate protein solution to different degrees of supersaturation for variable durations and then reduces the supersaturation to a low value. The concept is to find the two-step kinetic pathway that firstly quenches the drop for the right amount of time to nucleate just a few crystals and secondly reduces the supersaturation to a level that suppresses nucleation of additional crystals, but is still supersaturated enough for the crystals to grow slowly. Besides controlling concentration, the XOpt measures the solute and protein concentrations as a function of time so quantitative information on the actual kinetic pathway traversed by the samples can be obtained and exploited to design improved kinetic pathways in subsequent rounds of optimization. Addition of a simple temperature stage that generates a thermal gradient across the chip is used to find the optimal temperature of the optimal kinetic path. Two key points make the Crystal Optimizer innovative and useful. First, protein crystallization is an activated, non-equilibrium process and the XOpt is the sole microfluidic device designed to systematically optimize this physical feature in a controlled and reversible manner. Second, the XOpt is simple to use and inexpensive to manufacture.