Ultra-High-Throughput Absorbance-Activated Droplet Sorting for Enzyme Screening at Kilohertz Frequencies

Droplet microfluidics
is a valuable method to “beat the
odds” in high throughput screening campaigns such as directed
evolution, where valuable hits are infrequent and large library sizes
are required. Absorbance-based sorting expands the range of enzyme
families that can be subjected to droplet screening by expanding possible
assays beyond fluorescence detection. However, absorbance-activated
droplet sorting (AADS) is currently ∼10-fold slower than typical
fluorescence-activated droplet sorting (FADS), meaning that, in comparison,
a larger portion of sequence space is inaccessible due to throughput
constraints. Here we improve AADS to reach kHz sorting speeds in an
order of magnitude increase over previous designs, with close-to-ideal
sorting accuracy. This is achieved by a combination of (i) the use
of refractive index matching oil that improves signal quality by removal
of side scattering (increasing the sensitivity of absorbance measurements);
(ii) a sorting algorithm capable of sorting at this increased frequency
with an Arduino Due; and (iii) a chip design that transmits product
detection better into sorting decisions without false positives, namely
a single-layered inlet to space droplets further apart and injections
of “bias oil” providing a fluidic barrier preventing
droplets from entering the incorrect sorting channel. The updated
ultra-high-throughput absorbance-activated droplet sorter increases
the effective sensitivity of absorbance measurements through better
signal quality at a speed that matches the more established fluorescence-activated
sorting devices.

微滴微流控技术是一种极具价值的策略，用以克服高通量筛选活动中的种种挑战，如定向进化等，在这些活动中，有价值的发现较为罕见，且需构建庞大的文库。基于吸光度的排序技术拓宽了可供微滴筛选的酶家族范围，其原理在于将可能的检测方法扩展至荧光检测之外。然而，吸光度激活的微滴排序（AADS）目前的速度约为典型荧光激活的微滴排序（FADS）的1/10，这意味着由于通量限制，相较于FADS，有更大比例的序列空间无法触及。在本研究中，我们改进了AADS，使其达到千赫兹的排序速度，相较于先前设计实现了数量级的提升，并保持了近乎理想的排序精度。这一成就得益于以下几方面的结合：（一）采用折射率匹配油，通过消除侧向散射来提升信号质量（从而增加吸光度测量的灵敏度）；（二）一种算法，能够在提高的频率下使用Arduino Due进行排序；（三）一种芯片设计，它能更有效地将产物检测信息传递至排序决策，避免假阳性，具体设计包括单层入口以增加空间微滴之间的距离，以及注入“偏移油”形成流体屏障，防止微滴进入错误的排序通道。该更新的超高通量吸光度激活微滴排序器，通过在速度上与更成熟的荧光激活排序设备相匹配的同时，改善了信号质量，从而提高了吸光度测量的有效灵敏度。