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Medcalf, E. J.

Publications and source records attributed to Medcalf, E. J..

2 recordsLinked to original sources

Sorting of droplets at kHz rates using absorbance activated acoustic sorting

Droplet microfluidics allows one to address the ever-increasing demand to screen large libraries of biological samples. Absorbance spectroscopy complements the golden standard of fluorescence detection by label free target identification and providing more quantifiable data. However, this is limited by speed and sensitivity. In this paper we increase the speed of sorting by including acoustofluidics, achieving sorting rates of target droplets of 1 kHz. We improved the devices design for detection of absorbance using fibre-based interrogation of samples with integrated lenses in the microfluidic PDMS device for focusing and collimation of light. This optical improvement reduces the scattering and refraction artefacts, improving the signal quality and sensitivity. The novel design allows us to overcome limitations based on dielectrophoresis sorting, such as droplet size dependency, material and dielectric properties of samples. Our acoustic activated absorbance sorter removes the need for offset dyes or matching oils and sorts about a magnitude faster than current absorbance sorter.

biophysics↗

Ultrahigh-throughput Absorbance Activated Droplet Sorting (UHT-AADS) 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 landscape of 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 reaching 4 kHz 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 ultrahigh-throughput absorbance activated droplet sorter (UHT-AADS) increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/507731v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1c81dc3org.highwire.dtl.DTLVardef@119fe88org.highwire.dtl.DTLVardef@195c06borg.highwire.dtl.DTLVardef@8bce56_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗