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On-chip microfluidic production of cell-sized liposomes


In this protocol, we describe a recently developed on-chip microfluidic method to form monodisperse, cell-sized, unilamellar, and biocompatible liposomes with excellent encapsulation efficiency. Termed octanol-assisted liposome assembly (OLA), it resembles bubble-blowing on a microscopic scale. Hydrodynamic flow focusing of two immiscible fluid streams (an aqueous phase and a lipid-containing 1-octanol phase) by orthogonal outer aqueous streams gives rise to double-emulsion droplets. As the lipid bilayer assembles along the interface, each emulsion droplet quickly evolves into a liposome and a 1-octanol droplet. OLA has several advantages as compared with other on-chip techniques, such as a very fast liposome maturation time (a few minutes), a relatively straightforward and completely on-chip setup, and a biologically relevant liposome size range (5–20 μm). Owing to the entire approach being on-chip, OLA enables high-throughput liposome production (typical rate of tens of Hz) using low sample volumes (10 μl). For prolonged on-chip experimentation, liposomes are subsequently purified by removing the 1-octanol droplets. For device fabrication, a reusable silicon template is produced in a clean room facility using electron-beam lithography followed by dry reactive ion etching, which takes 3 h. The patterned silicon template is used to prepare polydimethylsiloxane (PDMS)-based microfluidic devices in the wet lab, followed by a crucial surface treatment; the whole process takes 2 d. Liposomes can be produced in 1 h and further manipulated, depending on the experimental setup. OLA offers an ideal microfluidic platform for diverse bottom-up biotechnology studies by enabling creation of synthetic cells, microreactors and bioactive cargo delivery systems, and also has potential as an analytical tool.

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Figure 1: Schematics showing OLA-based liposome production and purification.
Figure 2: Hydrophilic surface treatment of the microfluidic device.
Figure 3: Octanol-assisted liposome assembly.
Figure 4: On-chip purification of liposomes.


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We would like to acknowledge Y. Caspi, A. Birnie, F. Fanalista, K. Spoelstra, D. Hueting, M. van Doorn, M. Schaich, K. Al-Nahas, and S. Sachdev for their contributions to the development of the described protocol and valuable feedback. This work was supported by an NWO TOP-PUNT grant (no. 718014001), the Netherlands Organisation for Scientific Research (NWO/OCW) and a European Research Council Advanced Grant SynDiv (no. 669598).

Author information




S.D. performed the experiments. S.D. and C.D. wrote the paper.

Corresponding author

Correspondence to Cees Dekker.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Schematic flow diagram summarizing the key steps involved in the preparation of a master wafer and subsequent steps leading to the production of a microfluidic device.

(a) Master wafer is prepared in the clean room. A silicon wafer is spin-coated with a suitable negative resist and pre-baked. The wafer is then selectively exposed to e-beam to write the desired pattern. The wafer is further post-baked and chemically developed to remove the non-patterned resist. The developed wafer is appropriately etched, cleaned, and silanized to obtain a master wafer. (b) The microfluidic device is prepared in the wet lab. PDMS is poured over the master and cured by baking. The cured PDMs is then carefully peeled off and holes are punched at appropriate locations. The PDMS surface is then activated using plasma and bonded to a PDMS-coated, surface-activated glass slide. The bonded device is surface-treated to render it partially hydrophilic. The device is now ready to perform OLA. The master can be used multiple times.

Supplementary Figure 2 Various steps involved in the making of a microfluidic device.

(a) Master wafer enclosed within a well-like structure made up of aluminium foil. The design patterns are faintly visible on the wafer surface. (b) Cut-out PDMS block having a single OLA design (faint channels can be seen). Inlets, exit and separation hole are punched. (c) Glass slides (seen as faint outlines) immersed in a thin layer of PDMS, which is spread on a blank wafer. (d) Cured PDMS layer is peeled off to obtain PDMS-coated glass slides. The left-most glass slide is damaged severely during the peeling-off process and thus will not be used, while the down-most glass slide is broken only in the corner and can be used further. The remaining two glass slides are intact and in good condition. One glass slide has been removed showing a clean, reusable wafer surface. (e) A microfluidic device prepared by bonding the PDMS-coated glass slide to the design-containing PDMS block, right after the plasma treatment. The microfluidic channels are clearly visible. (f) Thicker (outer tube) and thinner (outer tube) tubes, placed in fittings, with the corresponding metal connectors inserted at one end.

Supplementary Figure 3 Production junction with bumpers.

Bright-field image of the production junction having bumper-like protrusions, in order to facilitate the surface treatment.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3. (PDF 579 kb)

Supplementary Data

.dxf file containing a standard design for OLA and subsequent liposome purification from 1-octanol droplets. (ZIP 91 kb)

Partial hydrophilic surface treatment of the microfluidic device.

1% (vol/vol) PVA solution is used. IA and LO channel (air) pressure: 50 mbar; OA channel (PVA) pressure: 120 mbar. (AVI 5817 kb)

Optimal double-emulsion droplet production.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 2960 kb)

Double-emulsion droplets with a prominent 1-octanol pocket flowing downstream.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 5173 kb)

Liposomes and budded-off 1-octanol droplets further downstream of the channel.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 1786 kb)

A top view across the separation hole, showing the separation process.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 9768 kb)

Optimal separation process, mainly yielding liposomes into the post-hole channel.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 383 kb)

Suboptimal production, yet double-emulsion droplets are still formed.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 9372 kb)

Suboptimal post-junction channel, showing aggregates of liposomes and 1-octanol droplets.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 474 kb)

Suboptimal purification process, showing unwanted 1-octanol aggregates entering the post-hole channel.

IA is 15% (vol/vol) glycerol; LO is 0.2% (wt/vol) lipids (DOPC/Rh-PE in a molar ratio of 1,000:1) in 1-octanol; OA is 15% (vol/vol) glycerol and 5% (wt/vol) P188. (AVI 434 kb)

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Deshpande, S., Dekker, C. On-chip microfluidic production of cell-sized liposomes. Nat Protoc 13, 856–874 (2018).

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