1DROPLET STABILITY BY USING
DSURF FLUOROSURFACTANT
INTRODUCTION
Biomillenia’s microbiome-on-a-chip technology maintains a much higher diversity ofliving microbes in millions of parallel culture vessels than with standard microbiology
methods. It uses its proprietary droplet microfluidics platform to create bacterial cellbanks, evaluate microorganisms for desired phenotypes and to find new routes toprevent and treat dysbiosis. Dysbiosis of the human microbiome is a research area ofincreasing interest, especially in cancer immunotherapy[1,2], skin diseases[3,4], and in general
in inflammatory diseases[5,6]. Droplet-microfluidics with its miniaturized culture vessels
and its inherent high throughout was proven to be an excellent tool for microbiome
research. Most of the microfluidic droplet techniques require biocompatible surfactants
to keep the droplets stable. By forming a monolayer at the water-oil interphase theamphiphilic molecules prevent droplet coalescence even when droplets are in directcontact with each other. However, the droplet stability can be affected by growth ofbacteria in droplets. Especially complex bacterial communities extracted from natural
samples can pose a risk to the emulsion quality, due to various secreted metabolites,
as well as different growth rates and morphologies exhibited by the various species.
Additionally, some bacterial species derived from natural samples are particularly
sensitive to the growth micro-environment. Microfluidic surfactants might be one factor
to be considered when establishing the micro-environment for the growth of naturalbacteria. Hence, the optimal surfactant is providing droplet stability also during longterm incubation while being biocompatible for a wide spectrum of bacterial species.
Here, the performance of three commonly used surfactants are compared at threedifferent concentrations. The droplet stability over time and the droplet occupation rates
were determined by encapsulating a microbial community derived from human skin.

Biomillenia is a team of experts in microfluidics, microbiology, molecular biology andbioinformatics who are passionate about harnessing smart microbes to restore thebody’s healthy balance and help prevent lifestyle diseases.

For additional information, contact Fluigent by email : contact@fluigent.com or consult our website : www.fluigent.com
More information on Biomillenia’s microbiome-on-a-chip technology, please consult : www.biomillenia.com

2APPLICATION NOTE
MATERIAL AND METHODS
Reagents
Novec HFE7500 (3M) containing 5%, 2% or 0.5% (w/w) concentration of Competitor 1
surfactant, Competitor 2 surfactant, or dSurf (Fluigent), respectively.
Microbe sample
A microbial sample collected from skin of healthy volunteers was used for droplet
generation. A general microbial skin sample, as opposed to a single strain like E. coli, wasused here because a bacterial community consisting of many species better represents
the experimental condition for microbiological research. The microbial skin sample wasprepared according to Biomillenia’s proprietary standard sample preparation workflow
and was suspended in a standard medium for skin microbes containing 0.5% (v/v)
TWEEN80. TWEEN80 is a lipophilic molecule in the aqueous phase that interfers withthe droplet stabilizing characteristics of the surfactants.
Droplet generation
The droplets were prepared on Biomillenia’s proprietary microfluidic platform withPDMS chips. The two liquid phases were controlled during droplet generation by theuse of high precision pressure pumps (Fluigent, Flow EZ). Droplets were generated at
frequencies of 7-10 kHz. The droplet volume was set to 20 pL. Droplets were collectedand incubated in custom made vessels at 37 °C allowing fot the storage of the droplets
without exposing them to a direct gas interface.
Droplet observation
From the droplet collection vessel, a small
number of droplets were sampled to checkfor droplet stability and bacterial occupation
at 0, 1, 3 and 7 days of incubation. For imaging
by microscopy, droplets were spread onto amonolayer surrounded by the respective oilsurfactant combination.

A		B		CRESULTS
Droplet generation
During droplet generation we found that all
three tested surfactants at all concentrations
provided an immediate stabilization of
aqueous droplets. For all tested surfactants
similar pressure settings for the continuous oilphase and the aqueous phase could be usedthroughout the droplet generation process,resulting in droplet generation frequenciesof 7-10 kHz and monodispersed dropletpopulations with an average volume of ~20 pL(Fig. 1).
We did not observe any droplet coalescence inthe exit channel of the chip, in which droplets
were having direct contact to each other.

Figure 1. Droplet generation with dSURF (A), Competitor 1(B) and Competor 2 (C). Immediate droplet stability during
droplet generation was observed at the flow focusing unit
for all tested concentrations of 0.5% of reagent

3

APPLICATION NOTE
Droplet stability and bacteria occupancy
The collected droplets were spread in Biomillenia’s proprietary observation chip for best
imaging of microbial occupation in droplets after 0, 1, 3 and 7 days incubation at 37 °C.
As shown in Fig. 2, a monolayer of droplets surrounded by the respective oil-surfactant
combination was imaged microscopically.
For each tested surfactant the concentration of 0.5% sufficient to ensure the stabilityof droplets over long term periods. The extent of droplet coalescence is highest forCompetitor 1 after one day while for dSURF and Competitor 2 the instability becomesvisible only later and to a lesser extent. Surfactant concentrations of 2% and higher
result in stable populations over 7 days at 37 °C for dSURF and Competitor 2 while forCompetitor 1 a concentration of 5% is required to reach similar results. (Fig. 2).
0.5%A1

B1

C1

A2

B2

C2

A3

B3

C32%5%Figure 2. Images of droplets with dSURF–0.5% (A1), dSURF–2% (A2), dSURF–5% (A3), with Competitor 1–0.5% (B1), Competitor 1–2% (B2), Competitor 1–5% (B3), and with Competitor 2–0.5% (C1), Competitor 2–2% (C2), Competitor 2–5% (C3) after 7 days incubation.

4APPLICATION NOTE
For droplets of the expected size,
we determined the occupation withmicrobial cultures by image analysis
after at least one day of incubation
for the droplet images shown above.
Droplets containing more than threecells were categorized as occupied.
We analysed at least 1000 dropletsin total for each droplet population.
Since the aqueous cell suspension wasprepared under the same conditions
for each droplet population a similar
droplet occupation was expected. Theemulsion failure for Competitor 1 at theconcentration of 0.5% prevented thedetermination of the occupation rate.

dSURF–0.5%
dSURF–2%
dSURF–5%
Competitor2–0.5%
Competitor2–2%
Competitor2–5%
Competitor1–0.5%
Competitor1–2%
Competitor1–5%

Figure 3. Droplet occupation frequency over time. The occupation was determined by image analysis from microscopy images of droplets at eachsampling time point. The emulsion failure for Competitor 1 at the concentration of 0.5 % prevented the determination of the occupation rate.

CONCLUSION

The stability of microfluidic emulsions strongly depends on the content of droplets
and its interplay with the surfactant used. Hydrophobic compounds in the aqueous
phase, along with microbial growth of various species present far from ideal
conditions for microfluidic droplets, but exemplify well the experimental challenges
encountered when microfluidic techniques are truly applied in microbiology.
Hence, surfactants are needed that help to accommodate those complex sample
characteristics.
Within the described experimental setting dSURF performed best among thetested surfactants in terms of maitaining droplet stability and biocompatibility. The
required surfactant concentration for keeping the droplets with microbial growthstable over long term incubation at 37 °C was 2% (w/w) for each of them, compared
to 5% (w/w) necessary for Competitor 1. Using lower surfactant concentrations is
not only of advantage for financial reasons but is also a way to prevent inter-droplet
transport of molecules from the aqueous phase.[7]In addition to improved droplet stability, a trend in the droplet occupation ratewith bacterial cultures is also detectable. As the aqueous phase for each droplet
population was prepared with the same amount of cells, a similar occupation
rate can be expected assuming no differences in biocompatibility for the threeused surfactants. dSURF seems to have a higher occupation rate which could beindicative of better biocompatibility.
In conclusion, dSURF is a new surfactant for droplet microfluidics in the market
which performs very well compared to widely established microfluidic surfactants
and is highly suitable for complex biological applications.

5

APPLICATION NOTEREFERENCES[1] Nature Reviews Drug Discovery, volume 18, page 569–572 (2019)[2] Cancer Letters, volume 447, page 41–47 (2019)[3] Nature Reviews Microbiology, volume 16, page 143–155 (2018)[4] Microbiome, volume 7, article number 95 (2019)[5] Nature Reviews Microbiology, volume 17, page 497–511 (2019)[6] Cell, volume 178, page 1041–1056, (2019)[7] Nature Communications, volume 7, article number 10392 (2016)CONTACTSFLUIGENT SAS,BIOMILLENIA SAS,

Francewww.fluigent.com

Francewww.biomillenia.com

67 Avenue de Fontainebleau
Okabé Bureaux94270 Le Kremlin Bicêtre102 Avenue Gaston Roussel
c/o BIOCITECH, Building Lavoisier93230 Romainville

6APPENDIX
A			B			CFigure S1. Droplet generation with dSURF (top left), Competitor
1 (top right) and Competor 2 (bottom right). Immediate droplet
stability during droplet generation was observed at the flow focusing unit for all tested concentrations of 0.5% (A), 2% (B) and5% (C) of reagent

A			B			CA			B			C

7APPENDIX
00.5%

13

72%5%Figure S2-A. Droplet images for dSURF (A) for all three concentrations after the indicated number of days of incubation

0.5%01372%

5%

Figure S2-B. Droplet images for Competitor 1 for all three concentrations after the indicated number of days of incubation

8APPENDIX
00.5%

13

72%

5%

Figure S2-C. Droplet images for Competitor 2 for all three concentrations after the indicated number of days of incubation