Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

By J. Agresti, E. Antipov, Adam R. Abate, K. Ahn, A. Rowat, Jean-Christophe Baret, M. Márquez, Alexander M. Klibanov, A. Griffiths, David A. Weitz. Proceedings of the National Academy of Sciences of the United States of America, 2010

1,102 citations recorded by the source corpus.

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼107. After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼108 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Subfields: Biomedical Engineering, Electrical and Electronic Engineering

View Paper: https://www.semanticscholar.org/paper/e2c758239142e7171cc36edc05e37c8392691150

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Citations in the corpus

Automating drug discovery (2017, background): “Droplets are especially suitable for performing enzyme-controlled processes 146,147 and may contain cells for probing drug effects in continuous flow 148 .”
Droplet barcoding for single cell transcriptomics applied to embryonic stem cells (2015, positive): “Cells can be compartmentalized into droplets and assayed for different bio-molecules (Mazutis et al. 2013), their genes amplified (Eastburn et al. 2013) and droplets sorted at high-throughput rates (Agresti et al. 2010).”
Advances in Microfluidic PCR for Point-of-Care Infectious Disease Diagnostics (2011, methodology): “…methods (Huebner et al. , 2008, Rane et al. , 2010, Teh et al. , 2008), and fusion or sorting of multiple droplets by applying localized electric field, droplet surface modification, and variation of channel geometries (Agresti et al. , 2010, Chabert and Viovy, 2008, Mazutis et al. , 2009b).”