As described in this introduction, there is great value in producing hydrogel- encapsulated DIBs (eDIBs) which can contain DIB networks and communicate with an external aqueous environment via lipid bilayers. The hydrogel shell can provide with environmental compatibility and provide with robustness in comparison to previously described systems, such as multisomes and MCVs, allowing for the constructs to be fully freestanding and withstand mechanical handling. For this, the microfluidic methods used to produce W/O/W/O emulsions in the previous chapter will be adapted in order to controllably generate eDIBs in large numbers. The amphiphillic properties of lipids should allow for aqueous droplets to avoid coalescence, and thus forgo the use of surfactants as seen in section 2.3.2.2 and 2.3.2.3. Additionally, an external gelation method for alginate as described in section 3.2.2.2 should allow for the gelation of the alginate shell to occur after droplet formation, allowing for the continuous, in situ production of gelled eDIBs. Electrophysiology will be performed in order to demonstrate the formation of lipid bilayers between the internal compartments and the external environment of eDIBs, via capacitance measurements and also the incorporation of α-
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Hemolysin membrane proteins. Finally, the ability of eDIBs to survive in aqueous, air and oil environments will be demonstrated.
Figure 3.6 Diagram depicting the microfluidic approach employed to generate eDIBs. A T-
junction is employed to generate droplets of water in oil with lipid, which is then passed through a first coaxial droplet generator flowing alginate with dispersed CaCO3. The resulting flow is passed through a second coaxial droplet generator flowing oil with acetic acid, producing eDIBs. Gelation of the alginate phase occurs as the acetic acid from the oil phase partitions into the
alginate phase, lowering the pH and dissolving CaCO3 particles, which releases Ca2+ ions that
trigger gelation.
3.2 Methods
All chemicals have been purchased from Sigma-Aldrich UK unless stated otherwise. Methods pertaining the design of the microfluidic devices employed are described in section 2.4.1.
3.2.1 Preparation of fluids
Lipid in oil solution: Lyophilised 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti
Polar Lipids, USA) was dissolved in chloroform and dried down using nitrogen gas into a lipid film that adhered to the glass vial container. Once the lipid film was visibly dry and free of any chloroform, a 1:1 solution of hexadecane oil and silicone oil (AR20) was added to the vial to reach a lipid concentration of 5 mg mL-1. The solution is rested
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Alginate solution: 2% w/v low viscosity sodium alginate, 5 mg mL-1 calcium carbonate
nanoparticles and sodium chloride to make up a total ionic strength of 0.5 M are added to deionised water and stirred with use of a magnetic stirrer at 360 rpm for at least 3 hours, resulting in a homogeneously opaque and viscous solution.
Carrier oil solution: 0.5% v/v glacial acetic acid is added to light mineral oil (Sigma-
Aldrich product nº M8410) and stirred for at least 30 minutes until the solution appears clear and homogeneous.
Aqueous solution: Aqueous solutions used for droplet interface bilayers consisted of
50 mM dibasic sodium phosphate and sodium chloride to make up a total ionic strength of 0.5 M. 50 µM Sulphorhodamine B or 1 mM Lissamine Green are added for colour (resulting in a pink or blue colour, respectively).
α-Hemolysin: α-Hemolysin heptamers were prepared by Dr. Oliver Castell1 at a
concentration of ≈30 nM in a 10 mM imidazole, 0.5 M NaCl, 20 mM HEPES, pH 8 buffer. The solution was kept at -80ºC, and aliquots were diluted to ≈3 nM, and kept at 5ºC for electrophysiology experiments. These aliquots were used within a week of their preparation and kept on ice during use.
3.2.2 Microfluidics
A 3D-printed microfluidic device for the generation of triple emulsions via coaxial droplet generating geometries is employed as described in Chapter 2. 3 mL or 25 mL plastic, luer lock syringes (Fisher Scientific, UK) are filled with the fluids and air bubbles are expelled. The fluids are dispensed using perfusion syringe pumps (Legato™ series single and double syringe pumps, KD Scientific, USA).
Table 1 shows a typical regime of flow rates employed to generated eDIBs as well as notes regarding the operation of the microfluidic device.
128 Fluid Syringe Size Flow Rate (mL hr-1) Order of flow Notes Internal aqueous phase 3mL syringe 11.76 3rd
These fluids are pumped using the same syringe pump.
Prior to the production of eDIBs, these fluids should be pumped until the FEP tube that delivers these fluids into the first coaxial droplet generator is filled with a monodisperse regime of droplets of water in oil.
For the production of eDIBs, these fluids should be pumped only whence the device output FEP tube is filled with a monodisperse regime of alginate droplets in the carrier oil. Lipid-in-oil phase 3mL syringe Alginate phase 25mL syringe 250 1st
The fluid must not be kept in the syringe for longer than 30 minutes prior to use as the CaCO3 will
separate from the mixture.
Carrier oil phase
25mL
syringe 400 2
nd n/a
Table 1 Flow rates used per fluid in the microfluidic manufacture of eDIBs, plus additional
comments regarding the operation of the microfluidic device.
As the device is used, an accumulation of polymerised alginate may occur at the orifice of the glass capillary, obstructing the even flow of both the alginate and oil phase. A 100 mM EDTA solution is used to dissolve the adhered hydrogel, which is pumped through both the alginate and carrier oil inlets of the microfluidic device so that the affected areas are kept in this solution for at least 30 minutes. This can then be rinsed to get rid of the dissolved alginate and EDTA, and microfluidic operation resumed.
3.2.3 Electrophysiology
Electrophysiology was performed using custom electrodes connected to an Axopatch 200B with a 203BU headstage (Molecular Devices, USA). The electrode tips are coated in a droplet (pL volume range) of agarose, performed by briefly dipping the tip of
129 the electrode onto a glass coverslip containing 5 μL of a 7.5 mg mL-1 solution of low
melting point agarose in deionised water, which is kept at 90º on a heating block.
Electrophysiology recordings of bilayer capacitance and ion flux were made under applied potentials of a +/- 23 mV triangle wave at 1 Hz or a fixed potential of 10-50 mV, respectively. Data was recorded with the software WinEDR (University of Strathclyde). Electrophysiology traces were analogue filtered using the Axopatch 200B equipment (low-pass, 5 Khz) and digitally filtered post-acquisition with either a 100 Hz low-pass filter.
Droplet-droplet DIB electrophysiology is performed by pipetting two 0.2 µL droplets into a micromilled PMMA trough filled with a 1:1 mixture of hexadecane and silicone oil AR20 containing 5 mg mL-1 DPhPC lipid. The droplets are anchored onto the
electrodes. eDIB electrophysiology is performed by placing eDIBs in a petri dish submerged in mineral oil. The eDIB hydrogel shell is pierced to access the internal cores with the electrodes. The tip of the electrode containing a droplet of agarose is then inserted into the internal cores. Electrodes are mounted onto micromanipulators (Narishige, Japan).
3.2.3.1 Electrode preparation
A wire is prepared by stripping both sides of it (around 2 cm), exposing the internal wire filaments. A male crimp pin is soldered to one end and a female crimp pin to the other. The male crimp pin connects to the 203BU headstage whilst the female crimp pin connects to the Ag/AgCl electrode used for either droplet-droplet DIB or eDIB electrophysiology measurements.
Droplet-droplet Ag/AgCl electrodes are prepared from 3-6 cm of 0.1 mm diameter silver wire. The AgCl tip is prepared by lightly sanding the wire tip (around 2 mm) and exposing it to a solution of sodium hypochlorite for 30 minutes. The exposed tip will
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darken, which indicates that the silver wire tip has undergone chlorination. This silver wire can now be soldered to the female crimp pin to complete the electrode.
Electrodes used for eDIB measurements are prepared differently, as they must be able to pierce the alginate shell and also be shielded from it in order to avoid short circuits when probing the internal droplets. When electrophysiology is performed to probe the bilayers formed between internal eDIB cores and the alginate shell, only the electrode that pierces the internal cores needs to be prepared in this way.
Firstly, a glass capillary (1 mm ID, CM Scientific, UK) is pulled using a capillary puller, in order to produce a tapered and sharp capillary tip. The glass capillary is then cut from the non-tapered end so that the non-tapered part of the glass sheath is around 3 cm long. Ag/AgCl silver wire treated as described above is inserted into the glass capillary through the non-tapered side of the glass sheath so that it reaches the inside of the tapered side of the glass. Then, the tapered tip can be broken with aid of tweezers in order for the silver wire to exit through the tapered end of the glass. The electrode wire is then passed through the glass capillary so that roughly 1 mm of untreated wire is exposed from the tip (Figure 3.7a). Epoxy resin is applied to the non- tapered end of the glass capillary in order to fix the silver wire in place, and set aside for 20 minutes in order to dry.
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Figure 3.7 Preparation of electrodes for eDIB electrophysiology. a) Schematic depicting the stages in the
preparation of an eDIB electrode prior to the fixing of the electrode wire within the pulled glass capillary with epoxy resin. b) Schematic of the tip of the eDIB electrode at its final stage. c) Photograph showing the finished electrode, which has mostly been covered in parafilm in order to protect the fragile glass sheath around the silver wire.
Polydimethoxysilane (PDMS) (2-part Sylgard 184 elastomer, DOW Corning, UK) is prepared to seal the junction between the tapered side of the glass and the tip of the electrode (Figure 3.7b). The tip of the electrode is briefly dipped into the PDMS solution, which forms a droplet of PDMS between the glass capillary and the silver wire. The electrode is then heated in an oven at 120 ºC for 30 minutes to cure the PDMS. The process of dipping the tip in PDMS and curing the resulting droplet is repeated until only 0.75 mm of un-chlorinated silver wire is left exposed at the tip. Each subsequent repetition will grow onto the solid droplet of PDMS cured from the first instance which gradually reduces the exposed length of the wire. Once the desired length of 0.75 mm is reached, the excess chlorinated silver wire can be cut off using a scalpel, and the glass electrode soldered onto the female crimp pin. To finalise the process, parafilm is used to secure the glass electrode in place, especially at the junction between the crimp pin and the glass capillary where it is most fragile (Figure 3.7c).
a)
b)
c)
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A 7.5 mg ml-1 solution of low-melting point agarose in DI water was prepared and kept
at 90°C. The electrode tip was dipped in this solution prior to its use for electrophysiology, in order to encourage the wetting and hence the penetration of the electrode tip into water droplets contained within an eDIB.