Dendronized fluorosurfactants for highly stable emulsions  

We design surfactants that form robust microdroplets with improved high stability (in PCR thermal cycling) and resistance to inter-droplet transfer (of fluorescent dye salts, drugs, etc) using dendritic oligo-glycerol-based surfactants with a high degree of inter-and intramolecular hydrogen bonding.

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Fluorosurfactant-stabilized microfluidic droplets are widely used as pico- to nanoliter volume reactors in chemistry and biology. However, current surfactants cannot completely prevent the inter-droplet transfer of small organic molecules encapsulated or produced inside the droplets. In addition, the microdroplets typically coalesce at temperatures higher than 80 °C. Therefore, the use of droplet-based platforms for ultrahigh-throughput combination drug screening and polymerase chain reaction (PCR)-based rare mutation detection has been limited. Here, we provide insights into designing surfactants that form robust microdroplets with improved stability and resistance to inter-droplet transfer. We used a panel of dendritic oligo-glycerol-based surfactants to demonstrate that a high degree of inter-and intramolecular hydrogen bonding, as well as the dendritic architecture, contribute to high droplet stability in PCR thermal cycling and minimize the inter-droplet transfer of the water-soluble fluorescent dye sodium fluorescein salt and the drug doxycycline. (Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules. Nat Commun 10, 4546 (2019). https://doi.org/10.1038/s41467-019-12462-5)

Fig 1 Synthesis of fluorosurfactants with dendritic glycerol head groups. PFPE tails of two different fluoro-chain lengths (M, L) were conjugated to the mono-glycerol (G) polar head (upper drawing, light blue) to create surfactants M-G and L-G; PFPE tails of three different molecular weights (H, M, L) were conjugated to dendritic tri-glycerol (dTG) polar head to create surfactants H-dTG, M-dTG, and L-dTG (lower drawing, blue). These surfactants orient at the interface (deep gray) of water (light red) and fluorinated oil (gray) to stabilize picolitre-volume (pL) water-in-oil (W/O) emulsion droplets, as depicted in the picture
Fig 2 Polar head group geometry dictates droplet stability. a Polydimethylsiloxane (PDMS) based microfluidic devices are used to prepare microdroplets containing PCR reagents. 

Fig 2 Polar head group geometry dictates droplet stability. b The three tri-glycerol (dTG)-based surfactants (H-dTG, M-dTG, L-dTG), the two mono-glycerol (G)-based surfactants (M-G, L-G), and the PEG600-based fluorosurfactant, PEG-PFPE2, were used to create droplets for stability testing (chemical structures in b). c Droplets produced using the tri-glycerol, the mono-glycerol, and the PEG600-based surfactants showed no merging during >24 h incubation at 4 °C (c showing pre-PCR droplets). d After 35 cycles of PCR, L-dTG-stabilized droplets showed almost no merging. In contrast, droplets generated using mono-glycerol (G)-based surfactants (M-G, L-G) merged completely during the PCR. Droplets stabilized with PEG-PFPE2 surfactant showed substantial merging (c depicting post-PCR droplets). Scale bar, 100 µm
Fig 3 Influence of dense hydrogen bond network on inter-droplet diffusion. a For each surfactant, we used parallel drop maker to create a mixture comprising equal amounts of PBS-only-droplets and PBS + sodium fluorescein salt-containing droplets. Confocal fluorescent imaging of droplets after 72 h incubation at 37 °C is shown in top panel. b We incubated these mixtures, took fluorescence images at the indicated time points, and then performed quantitative analysis of the fluorescence intensity of the 10 randomly selected PBS-only droplets. Box-plot demonstrates that dye was almost completely retained in droplets stabilized by M-dTG, while some transfer was detected in M-G droplets. PEG-PFPE2 surfactant-stabilized droplets showed substantial transfer (left to right). The box plots represent the median (center line), the interquartile range (box) and the non-outlier range (whisker). 
Fig 3 Influence of dense hydrogen bond network on inter-droplet diffusion. c A model representing the plausible inter- and intramolecular hydrogen bonding from M-dTG, M-G, and PEG-PFPE2 surfactants (left to right). The lines denote hydrogen bonding at the interface of oil and water. For PEG-PFPE2 surfactant, as there is no hydrogen bond donor, there is no inter- and intramolecular hydrogen bond present. Scale bars = 100 µm; a.u. = arbitrary units. Source data of Fig. 3b (middle panel) are provided as a Source Data file
Fig 4 Cell-based reporter system to test inter-droplet drug diffusion. a HEK 293 cells stably transfected with a doxycycline responsive GFP reporter construct (reporter cells) show no GFP expression in the absence of drugs and become highly fluorescent after 48 h incubation in the presence of 500 nM DOX-solution. b Homogeneous population of DOX-containing droplets and Reporter-cell-containing droplets was generated using a parallel drop maker that creates an equal number of droplets from two independent aqueous streams. 
Fig 4 Cell-based reporter system to test inter-droplet drug diffusion. c Cell-loading into droplets roughly follows Poisson predictions. We imaged 533 droplets immediately after cell encapsulation to determine cell occupancy. We used an input density of five cells per droplet volume and found that 83% of the droplets contained one or more cells. Therefore, with cells at this concentration, the parallel drop maker generates a mixed droplet population in which 42% droplets contain one or more cells, 50% contain only doxycycline, and 8% are empty. d Box-plot of droplet size distribution after 24, 48, and 72 h incubation at 37 °C demonstrates that the mean average droplet diameter remained constant (~75 µm) over the time course (n = 204–226 droplets for each time point). The box plots represent the median (center line), the interquartile range (box) and mean ± 1.5 x s.d. (whisker). e Viability of cells cultured in wells or in droplets. As a positive control we cultured cells at standard concentration in culture wells (CW–SC) (1 × 106 cells/ml). We used M-dTG and PEG-PFPE2 surfactants to generate cell encapsulated droplets and incubated droplets at 37 °C for the indicated times. We isolated cells from the droplets to perform live/dead assay using Calcein AM and Ethidium Homodimer-1 dyes based live/dead cell viability assay kit (Invitrogen). We counted ~500–2000 cells to estimate the cell survival rate at the indicated time points. Data are presented as mean ± s.d., n = 4 images from distinct areas. Source data of c–e are provided as a Source Data file
Fig 5 DOX-inducible GFP-reporter cells to quantify drug transfer. We used the parallel drop maker to generate homogenous mixtures in which 50% of the droplets contained DOX (at the indicated concentrations), 42% contained ≥1 cell, and 8% were empty. We incubated droplets at 37 °C for the indicated times, isolated the cells from the droplets, and quantified the GFP+ cells using flow cytometer. GFP intensity is plotted against forward scatter-area (FSC-A). For the positive control in bulk, we cultured the DOX-GFP-HEK 293 cells with DOX in six well plates using standard cell culture methods. For the Positive Control (Droplet), all droplets, including those with cells, contained DOX at the indicated concentration

Related reading:https://orcid.org/0000-0001-7114-1095  https://www.nature.com/articles/s41467-019-12462-5  https://www.nature.com/articles/s41578-020-00247-y  https://www.nature.com/articles/s41467-021-21436-5  

Other microfluidics studies:https://doi.org/10.1021/acsnano.0c02701

https://doi.org/10.1016/j.bios.2019.111597 https://doi.org/10.2144/btn-2019-0134

Dr. Xingcai Zhang, Harvard/MIT Research Fellow; Science Writer/Editorial (Advisory) Board Member for Springer Nature, Elsevier, Materials Today, Royal Society of Chemistry, Wiley; Nature Nano Ambassador with 5 STEM degrees/strong background in sustainable Nature-derived/inspired/mimetic materials for biomed/sensing/catalysis/energy/environment applications, with around 100 high-impact journal publications in Nature Reviews Materials (featured cover paper), etc.  https://scholar.google.com/citations?hl=en&user=2vDraMoAAAAJ&view_op=list_works&sortby=pubdate

Contact: Dr. Xingcai Zhang xingcai@mit.edu  chemmike1984@gmail.com +1-2253041387 wechat:drtea1

Xingcai Zhang

Harvard/MIT Research Fellow, Harvard University

Sustainable Nature-inspired/dervied/mimetic materials, nanomaterials, biomedicine, lab-on-chip, 2D/C/porous/polymer materials, tea. Dr. Xingcai Zhang, Harvard/MIT Research Fellow; Science Writer/Editorial (Advisory) Board Member for Springer Nature, Elsevier, Materials Today, Royal Society of Chemistry, Wiley; Nature Nano Ambassador with 5 STEM degrees/strong background in sustainable Nature-derived/inspired/mimetic materials for biomed/sensing/catalysis/energy/environment applications, with around 100 high-impact journal publications in Nature Reviews Materials (featured cover paper), etc. https://scholar.google.com/citations?hl=en&user=2vDraMoAAAAJ&view_op=list_works&sortby=pubdate https://orcid.org/0000-0001-7114-1095 Contact: xingcai@mit.edu +1-2253041387 wechat:drtea1