Passive Droplet Microfluidic Platform for High-Throughput Screening of Microbial Proteolytic Activity

Overview

Journal Anal Chem

Specialty Chemistry

Date 2024 Sep 25

PMID 39320273

Authors

Luca Potenza

Lukasz Kozon

Lukasz Drewniak

Tomasz S Kaminski

Affiliations

Soon will be listed here.

Abstract

Traditional bacterial isolation methods are often costly, have limited throughput, and may not accurately reflect the true microbial community composition. Consequently, identifying rare or slow-growing taxa becomes challenging. Over the past decade, a new approach has been proposed to replace traditional flasks or multiwell plates with ultrahigh-throughput droplet microfluidic screening assays. In this study, we present a novel passive droplet-based method designed for isolating proteolytic microorganisms, which are crucial in various biotechnology industries. Following the encapsulation of single cells in gelatin microgel compartments and their subsequent clonal cultivation, microcultures are passively sorted at high throughput based on the deformability of droplets. Our novel chip design offers a 50-fold improvement in throughput compared to a previously developed deformability-based droplet sorter. This method expands an array of droplet-based microbial enrichment assays and significantly reduces the time and resources required to isolate proteolytic bacteria strains.

References

Dhar M, Lam J, Walser T, Dubinett S, Rettig M, Di Carlo D . Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay. Proc Natl Acad Sci U S A. 2018; 115(40):9986-9991. PMC: 6176626. DOI: 10.1073/pnas.1803884115. View

Morris L, Evans J, Marchesi J . A robust plate assay for detection of extracellular microbial protease activity in metagenomic screens and pure cultures. J Microbiol Methods. 2012; 91(1):144-6. DOI: 10.1016/j.mimet.2012.08.006. View

Gray C, Barker S, Dhariwal M, Sullivan J . Assay of the high-alkaline proteinase from alkalophilic bacillus PB92 using a chromogenic tripeptide substrate. Biotechnol Bioeng. 1985; 27(12):1717-20. DOI: 10.1002/bit.260271213. View

Wu W, Zhang S, Zhang T, Mu Y . Immobilized Droplet Arrays in Thermosetting Oil for Dynamic Proteolytic Assays of Single Cells. ACS Appl Mater Interfaces. 2021; 13(5):6081-6090. DOI: 10.1021/acsami.0c21696. View

Mahler L, Niehs S, Martin K, Weber T, Scherlach K, Hertweck C . Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities. Elife. 2021; 10. PMC: 8081529. DOI: 10.7554/eLife.64774. View

Ng E, Miller M, Jing T, Chen C . Single cell multiplexed assay for proteolytic activity using droplet microfluidics. Biosens Bioelectron. 2016; 81:408-414. DOI: 10.1016/j.bios.2016.03.002. View

Watterson W, Tanyeri M, Watson A, Cham C, Shan Y, Chang E . Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes. Elife. 2020; 9. PMC: 7351490. DOI: 10.7554/eLife.56998. View

Zengler K, Toledo G, Rappe M, Elkins J, Mathur E, Short J . Cultivating the uncultured. Proc Natl Acad Sci U S A. 2002; 99(24):15681-6. PMC: 137776. DOI: 10.1073/pnas.252630999. View

Saito K, Ota Y, Tourlousse D, Matsukura S, Fujitani H, Morita M . Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort. Sci Rep. 2021; 11(1):9506. PMC: 8096817. DOI: 10.1038/s41598-021-88974-2. View

10.

Morellon-Sterling R, El-Siar H, Tavano O, Berenguer-Murcia A, Fernandez-Lafuente R . Ficin: A protease extract with relevance in biotechnology and biocatalysis. Int J Biol Macromol. 2020; 162:394-404. DOI: 10.1016/j.ijbiomac.2020.06.144. View

11.

Kasana R, Salwan R, Yadav S . Microbial proteases: detection, production, and genetic improvement. Crit Rev Microbiol. 2011; 37(3):262-76. DOI: 10.3109/1040841X.2011.577029. View

12.

Nosho K, Yasuhara K, Ikehata Y, Mii T, Ishige T, Yajima S . Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation. Microbiology (Reading). 2018; 164(9):1122-1132. PMC: 6230765. DOI: 10.1099/mic.0.000673. View

13.

Staskiewicz K, Dabrowska-Zawada M, Kozon L, Olszewska Z, Drewniak L, Kaminski T . Droplet microfluidic system for high throughput and passive selection of bacteria producing biosurfactants. Lab Chip. 2024; 24(7):1947-1956. DOI: 10.1039/d3lc00656e. View

14.

Zurek P, Hours R, Schell U, Pushpanath A, Hollfelder F . Growth amplification in ultrahigh-throughput microdroplet screening increases sensitivity of clonal enzyme assays and minimizes phenotypic variation. Lab Chip. 2020; 21(1):163-173. DOI: 10.1039/d0lc00830c. View

15.

Hatch A, Patel A, Beer N, Lee A . Passive droplet sorting using viscoelastic flow focusing. Lab Chip. 2013; 13(7):1308-15. DOI: 10.1039/c2lc41160a. View

16.

Craik C, Page M, Madison E . Proteases as therapeutics. Biochem J. 2011; 435(1):1-16. PMC: 4503466. DOI: 10.1042/BJ20100965. View

17.

CHARNEY J, TOMARELLI R . A colorimetric method for the determination of the proteolytic activity of duodenal juice. J Biol Chem. 2010; 171(2):501-5. View

18.

Neun S, Kaminski T, Hollfelder F . Single-cell activity screening in microfluidic droplets. Methods Enzymol. 2019; 628:95-112. DOI: 10.1016/bs.mie.2019.07.009. View

19.

Baret J, Miller O, Taly V, Ryckelynck M, El-Harrak A, Frenz L . Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. Lab Chip. 2009; 9(13):1850-8. DOI: 10.1039/b902504a. View

20.

Najah M, Calbrix R, Mahendra-Wijaya I, Beneyton T, Griffiths A, Drevelle A . Droplet-based microfluidics platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms. Chem Biol. 2014; 21(12):1722-32. DOI: 10.1016/j.chembiol.2014.10.020. View