Foundation for Applied Molecular Evolution, Alachua, Florida, United States of America.
Panamerican Biolabs LLC, Gainesville, Florida, United States of America.
PLoS One. 2022 Jan 20;17(1):e0252361. doi: 10.1371/journal.pone.0252361. eCollection 2022.
There is a rising interest in biotechnology for the compartmentalization of biochemical reactions in water droplets. Several applications, such as the widely used digital PCR, seek to encapsulate a single molecule in a droplet to be amplified. Directed evolution, another technology with growing popularity, seeks to replicate what happens in nature by encapsulating a single gene and the protein encoded by this gene, linking genotype with phenotype. Compartmentalizing reactions in droplets also allows the experimentalist to run millions of different reactions in parallel. Compartmentalization requires a fluid that is immiscible with water and a surfactant to stabilize the droplets. While there are fluids and surfactants on the market that have been used to accomplish encapsulation, there are reported concerns with these. Span® 80, for example, a commonly used surfactant, has contaminants that interfere with various biochemical reactions. Similarly, synthetic fluids distributed by the cosmetic industry allow some researchers to produce experimental results that can be published, but then other researchers fail to reproduce some of these protocols due to the unreliable nature of these products, which are not manufactured with the intent of being used in biotechnology. The most reliable fluids, immiscible with water and suitable for biochemical reactions, are fluorinated fluids. Fluorinated compounds have the peculiar characteristic of being immiscible with water while at the same time not mixing with hydrophobic molecules. This peculiar characteristic has made fluorinated fluids attractive because it seems to be the basis of their being biologically inert. However, commercially available fluorinated fluids have densities between 1.4 to 1.6 g/mL. The higher-than-water density of fluorinated oils complicates handling of the droplets since these would float on the fluid since the water droplets would be less dense. This can cause aggregation and coalescence of the droplets. Here, we report the synthesis, characterization, and use of fluorinated polysiloxane oils that have densities similar to the one of water at room temperature, and when mixed with non-ionic fluorinated surfactants, can produce droplets encapsulating biochemical reactions. We show how droplets in these emulsions can host many biological processes, including PCR, DNA origami, rolling circle amplification (RCA), and Taqman® assays. Some of these use unnatural DNA built from an Artificially Expanded Genetic Information System (AEGIS) with six nucleotide "letters".
人们对生物技术在水滴中进行生化反应分隔的兴趣日益浓厚。有几种应用，如广泛使用的数字 PCR，试图将单个分子包裹在一个液滴中进行扩增。另一种技术——定向进化，也越来越受欢迎，它试图通过包裹单个基因和该基因编码的蛋白质来复制自然界中发生的事情，将基因型与表型联系起来。在液滴中分隔反应也允许实验人员并行运行数百万种不同的反应。分隔反应需要一种与水不混溶的流体和一种表面活性剂来稳定液滴。虽然市场上有已经用于封装的流体和表面活性剂，但据报道这些产品存在一些问题。例如，Span® 80 是一种常用的表面活性剂，它的污染物会干扰各种生化反应。同样，化妆品行业分布的合成流体允许一些研究人员产生可以发表的实验结果，但由于这些产品不可靠，其他研究人员无法复制其中一些方案，这些产品并非为在生物技术中使用而制造。与水不混溶且适合生化反应的最可靠的流体是氟化流体。氟化化合物具有与水不混溶的奇特特性，同时也不会与疏水分子混合。这种奇特的特性使得氟化流体具有吸引力，因为它似乎是它们具有生物惰性的基础。然而，市售的氟化流体的密度在 1.4 到 1.6 g/mL 之间。氟化油的密度高于水，这使得处理液滴变得复杂，因为这些液滴由于密度较小而漂浮在流体上。这可能导致液滴的聚集和合并。在这里，我们报告了氟化聚硅氧烷油的合成、表征和使用，这些油在室温下的密度与水相似，并且当与非离子氟化表面活性剂混合时，可以产生包裹生化反应的液滴。我们展示了这些乳液中的液滴如何能够容纳许多生物过程，包括 PCR、DNA 折纸术、滚环扩增（RCA）和 Taqman®测定。其中一些使用由人工扩展遗传信息系统（AEGIS）构建的、由六个核苷酸“字母”组成的非天然 DNA。
