Acoustic sorting of microfluidic droplets at kHz rates using optical absorbance

Richter, Esther S; Link, Andreas; McGrath, John S.; Sparrow, Raymond; Gantz, Maximilian; Medcalf, Elliot J; Hollfelder, Florian; Franke, Thomas

doi:10.1039/d2lc00871h

Cited by 14 publications

(9 citation statements)

References 31 publications

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“…Also, since the sorting uses Arduino-like microcontrollers, improvement in hardware will result in increased computation speed while retaining the accessibility of being inexpensive and open-source. Complementary work by Richter et al has shown an increased sorting throughput and removal of droplet trace artifacts by using a combination of surface acoustic waves and microlenses in the form of an optical air cavity. We envision that a further upgrade to AADS designs could incorporate design improvements from both studies.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2023

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“…Then the droplet is forced into the collection channel. 120,121 In addition, a membrane valve can also be used to sort the droplets. The valve is pressurized to squeeze the waste channel and increase its resistance.…”

Section: Droplet Manipulationmentioning

confidence: 99%

Development and future of droplet microfluidics

Nan,

Zhang,

Weitz

et al. 2024

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“…Attempts to increase the sensitivity and sensitivity of absorbance sorting have been made: (i) Duncombe et al introduced UVADS (UV–Vis Spectra Activated Droplet Sorter) in a channel design with increased path length (by installing a right-angled turn at the detection interface) and by recording entire spectra (200–1050 nm) as unique signatures in UV–Vis Spectra-Activated Droplet Sorting. (ii) Richter et al . have shown kHz sorting throughput in a model separation based on removal of droplet trace artifacts by using a combination of surface acoustic waves and microlenses in the form of an optical air cavity.…”

Section: Detection and Sortingmentioning

confidence: 99%

“…128 Attempts to increase the sensitivity and sensitivity of absorbance sorting have been made: (i) Duncombe et al 206 introduced UVADS (UV−Vis Spectra Activated Droplet Sorter) in a channel design with increased path length (by installing a rightangled turn at the detection interface) and by recording entire spectra (200−1050 nm) as unique signatures in UV−Vis Spectra-Activated Droplet Sorting. (ii) Richter et al 207 have shown kHz sorting throughput in a model separation based on removal of droplet trace artifacts by using a combination of surface acoustic waves and microlenses in the form of an optical air cavity. (iii) Medcalf et al 208 overcame the scattering caused by droplet edges in an improved microfluidic design (i.e., with a single-layered inlet leading to enabling more even spacing), refractive index matching, and faster sorting algorithms (compared to ref 128), so sorting around 1 kHz became possible.…”

Section: Detection and Sortingmentioning

confidence: 99%

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

et al. 2023

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Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number of candidates tested. Water-in-oil emulsion droplets can replace the classical test tube, to provide in vitro compartments as an alternative screening format, containing genotype and phenotype and enabling a readout of function. The scale-down to micrometer droplet diameters and picoliter volumes brings about a >10 7 -fold volume reduction compared to 96-well-plate screening. Droplets made in automated microfluidic devices can be integrated into modular workflows to set up multistep screening protocols involving various detection modes to sort >10 7 variants a day with kHz frequencies. The repertoire of assays available for droplet screening covers all seven enzyme commission (EC) number classes, setting the stage for widespread use of droplet microfluidics in everyday biochemical experiments. We review the practicalities of adapting droplet screening for enzyme discovery and for detailed kinetic characterization. These new ways of working will not just accelerate discovery experiments currently limited by screening capacity but profoundly change the paradigms we can probe. By interfacing the results of ultrahigh-throughput droplet screening with next-generation sequencing and deep learning, strategies for directed evolution can be implemented, examined, and evaluated. CONTENTSAA 11.6.2. Combining High-Throughput Selections with High-Throughput Analysis AB 12. Conclusions

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Acoustic sorting of microfluidic droplets at kHz rates using optical absorbance

Abstract: Droplet microfluidics allows one to address the ever-increasing demand to screen large libraries of biological samples. Absorbance spectroscopy complements the established fluorescence detection by alternative target identification and providing quantifiable...

Cited by 14 publications

References 31 publications

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

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

Development and future of droplet microfluidics

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

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