Ultrahigh‐Throughput Detection of Enzymatic Alcohol Dehydrogenase Activity in Microfluidic Droplets with a Direct Fluorogenic Assay

Abstract

The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for > 6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an > 800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.

Sub-single-turnover quantification of enzyme catalysis at ultrahigh throughput via a versatile NAD(P)H coupled assay in microdroplets

“…However, the individual populations could still be distinguished (Figure 3A) with only 3% overlap. This stands in contrast to the NAD(P)H probe resorufin -fluorescence microscopy revealed near complete equilibration of previously empty and full droplets in only 6 hours [54] , suggesting at least a 64-fold better retention of the uncaged fluorophore. However, leakage of product was not the only consideration, as all active components of a successful cascade should be retained within each droplet to prevent cross-talk.…”

“…Leakage is a well-documented challenge in microfluidic assay development, preventing the use of the commonly large and aromatic probes for all but the fastest reactions [48][49][50][51][52] . This poses a hitherto unsolved challenge for fluorogenic NAD(P)H quantification as resorufin, the sole published fluorogenic probe for NAD(P)H detection [49,53] , is hydrophobic and consequently only compatible with very short reaction times in microfluidic droplets [54] . Previous work has shown that adding charged groups to fluorophores [52] and specifically coumarins [12,55] mitigates droplet leakage and improves aqueous solubility.…”

A Coupled Ketoreductase‐Diaphorase Assay for the Detection of Polyethylene Terephthalate‐Hydrolyzing Activity

“…After sufficient incubation for PET hydrolysis, the reaction mix proposed in this work was added to the droplets by picoinjection [37,38] or droplet fusion, [39] following a short incubation to reveal the presence of hydrolysis products before proceeding to on-chip droplet sorting and recovery of the DNA from the droplets by PCR (Figure S9). Although resorufin was not the optimal fluorophore for ultrahigh-throughput screening due to its low retention in w/o droplets compared to the less "leaky" fluorescein [40] or pyranine molecules, [41] workflows in which resorufin was the fluorescent product of an enzymatic cascade with incubation times compatible with the proposed assay herein were described. [42,43] Furthermore, modified resazurin substrates with improved retention in droplets are available [44,45] and their use would be particularly suitable for this application.…”

Multifunctional Droplets Formed by Interfacially Self-Assembled Fluorinated Magnetic Nanoparticles for Biocompatible Single Cell Culture and Magnet-Driven Manipulation

“…Droplet microfluidics − is an incredible technology due to its ability to compartmentalize samples and reagents in picoliter to nanoliter droplets with a highly reproducible volume. Droplets working as microreactors can be further manipulated including mixing, sorting, splitting, coalescing, and trapping, which are basic elements for integration on a chip. − These manipulations make it possible to explore multiple aspects in directed enzyme evolution, − addressing cellular heterogeneity, − identification of rare cells in microbial communities, , recognition of antibiotic resistance genes, and biomarker discovery for diseases. , In the last 20 years, various methods for active droplet manipulation have been developed to deflect and recover the droplet of interest, , including electric control, , acoustic control, , magnetic control, − pneumatic control, , and so on. Except for magnetic control, these methods need a complex setup with high costs and a sorting chip requiring sophisticated fabrication for droplets manipulation with a significant accuracy, sensitivity, and response time. ,, …”