Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

Goto, Haruna; Kanai, Yuki; Yotsui, Arisa; Shimokihara, Shota; Shitara, Shunya; Oyobiki, Ryo; Fujiwara, Keigi; Watanabe, Takeshi; Einaga, Yasuaki; Matsumoto, Yoshinori; Miki, Norihisa; Doi, Nobuhide

doi:10.1039/c9lc01263j

Cited by 45 publications

(47 citation statements)

References 51 publications

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“…Goto et al ( 2020 ) applied a boron-doped diamond (BDD) electrode to measure the current change in 30-nl droplets for the DE of a NAD(P)-dependent oxidoreductase. As low as 1 μM of NADH could be detected, and a 3-fold higher activity of isocitrate dehydrogenase (IDH) mutant from Streptococcus mutants was screened ( Figure 3A ).…”

Section: Label-free Sortingmentioning

confidence: 99%

“… Label-free sorting methods based on electrochemical detection (A) [adapted with permission from Goto et al ( 2020 ) Copyright © Royal Society of Chemistry], mass spectrometry (B) [adapted with permission from Holland-Moritz et al ( 2020 ) Copyright © John Wiley and Sons, Inc.], Raman (C) [adapted with permission from Wang et al ( 2017 ) Copyright © American Chemical Society], and NMR (D) [adapted with permission from Davoodi et al ( 2020 ) Copyright © Royal Society of Chemistry]. …”

Section: Label-free Sortingmentioning

confidence: 99%

“…Highest frequency Minimal droplet size FADS 2.5 nM (Colin et al, 2015) 5 kHz (Neun et al, 2019) 2 pl (Colin et al, 2015) AADS 10 µM (Gielen et al, 2016) 100 Hz (Gielen et al, 2016) 100 pl (Maceiczyk et al, 2017) ECDS 1 µM (Goto et al, 2020) 10 Hz (Goto et al, 2020) 30 nl (Goto et al, give the absorbing dye WST-1 formazan. The sorting frequency could achieve ∼1 million droplets per hour.…”

Section: Sensitivitymentioning

confidence: 99%

“…The detection device on a microfluidic chip could also be coupled with a DEP sorting device, by converting electrochemical signal into digital signal and then into alternating current (AC) signal to turn on DEP sorter. Goto et al (2020) applied a boron-doped diamond (BDD) electrode to measure the current change in 30-nl droplets for the DE of a NAD(P)-dependent oxidoreductase. As low as 1 µM of NADH could be detected, and a 3-fold higher activity of isocitrate dehydrogenase (IDH) mutant from Streptococcus mutants was screened (Figure 3A).…”

Section: Electrochemical-based Droplet Sortingmentioning

confidence: 99%

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Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Zhang

et al. 2021

Front. Chem.

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Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE.

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Section: Label-free Sortingmentioning

confidence: 99%

Section: Label-free Sortingmentioning

confidence: 99%

Section: Sensitivitymentioning

confidence: 99%

Section: Electrochemical-based Droplet Sortingmentioning

confidence: 99%

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Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Zhang

et al. 2021

Front. Chem.

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“…Among them, microfluidic fluorescence-activated droplet sorting (FADS) was introduced to dramatically simplify the operations, improve sorting efficiency and flexibility, and reduce the cost of large library screening [18][19] . FADS has been successfully applied to screen lipase/esterase 20 , DNA/XNA polymerase 21 , cellulase 22 , and NAD(P)-dependent oxidoreductases 23 by incorporating highly sensitive and specific fluorescent enzymatic assays 24 . In this work, we developed a FADS-based approach for expediting the search of PETases and then validated its performance by benchmark PETases.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence-Activated Droplet Sorting of Polyethylene Terephthalate-degrading Enzymes

Qiao

Chen

et al. 2021

Preprint

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It is a great challenge to expand the spectrum of enzymes that can decompose synthetic plastics such as Polyethylene terephthalate (PET). However, a bottleneck remains due to the lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation of droplets encapsulating single cells, picoinjection of Fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with the method, including specificity and sensitivity for discriminating PETase from other enzymes, and optimized its performance and compatibility with environmental samples. The system was used to screen an environmental sample from a PET textile mill, successfully obtained PET-degrading species belonging to 9 different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate the FADS pipeline can be widely adopted to discover new PET-degrading microorganisms and enzymes from various environments, as well as directed evolution of PETases using synthetic biology.

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FACS‐Sortable Triple Emulsion Picoreactors for Screening Reactions in Biphasic Environments

Thompson,

Zhang,

Yang

et al. 2024

Adv Materials Inter

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Biphasic environments can enable successful chemical reactions where any single solvent results in poor substrate solubility or poor catalyst reactivity. For screening biphasic reactions at high throughput, a platform based on microfluidic double emulsions can use widely available FACS (Fluorescence Activated Cell Sorting) machines to screen millions of picoliter reactors in a few hours. However, encapsulating biphasic reactions within double emulsions to form FACS‐sortable droplet picoreactors requires optimized solvent phases and surfactants to produce triple emulsion droplets that are stable over multi‐hour assays and compatible with desired reaction conditions. This work demonstrates such FACS‐sortable triple emulsion picoreactors with a fluorocarbon shell and biphasic octanol‐in‐water core. First, surfactants are screened to stabilize octanol‐in‐water emulsions for the picoreactor core. With these optimized conditions, stable triple emulsion picoreactors (>70% of droplets survived to 24 hr), produced protein in the biphasic core via cell‐free protein synthesis are generated, and sorted these triple emulsions based on fluorescence using a commercial FACS sorter at >100 Hz with 75–80% of droplets recovered. Finally, an in‐droplet lipase assay with a fluorogenic resorufin substrate that partitions into octanol is demonstrated. These triple emulsion picoreactors have the potential for future screening bead‐encoded catalyst libraries, including enzymes such as lipases for biofuel production.

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Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

Abstract: We developed μTAS for screening of NAD(P)-dependent oxidoreductases whose activity can be detected with boron-doped diamond electrodes and whose genes can be recovered by dielectrophoretic sorting of nanoliter-size droplets.

Cited by 45 publications

References 51 publications

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Fluorescence-Activated Droplet Sorting of Polyethylene Terephthalate-degrading Enzymes

FACS‐Sortable Triple Emulsion Picoreactors for Screening Reactions in Biphasic Environments

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