Christian Holtze

 

Non-Ionic Fluorosurfactants for Biological and Chemical Applications inside Aqueous and Organic Emulsion droplets

Emulsions—dispersions of two immiscible fluids—have many applications in industry and everyday life. These range from paints to crop protection. They are also increasingly attractive for analytical applications. Traditional emulsions consist of water and hydrocarbon oil. However, this combination does not allow for a wide variety of applications. It is therefore of advantage to replace one of these liquids with a third class of liquids that are immiscible with both, water and hydrocarbon oils / organic solvents: the liquids of choice are fluorocarbon oils. The application of fluorocarbon oils as a continuous emulsion phase offers a number of advantages over the conventional water-in-oil (w/o) and oil-in-water (o/w) emulsions. For example, fluorocarbon oils provide an excellent barrier between individual emulsion droplets that consist of water, hydrocarbon oils or hydrocarbon solvents. Therefore, these droplets may be regarded as perfectly isolated entities, in which chemical or biological processes may occur independently.

Emulsions are inherently unstable. They have to be stabilized with appropriate surfactants to prevent the coalescence of droplets. So far there are no non-ionic fluorosurfactants available that would be suited for the stabilization of water-in-fluorocarbon (w/f) and oil-in-fluorocarbon (o/f)) emulsions. However, such surfactants are crucial for exciting new applications. We will present two examples from our current research for both, w/f and o/f emulsions.

Given the potential commercial value of this new class of surfactants, we filed a patent on it.

(1) New Surfactants for Biological Applications in Microfluidics

Biological Systems in Small Droplets

Emulsions may be produced with great control in microfluidic devices. Our group has much expertise in using hydrodynamic flow focusing for producing water-in-oil emulsions (water droplets in a continuous oil phase). These so-called inverse emulsions are of interest to various biological applications: cells may be incorporated in water-droplets (Katie Humphrey, Amy Rowat) and biomolecules, such as DNA or proteins, may be investigated in droplets, as it is done in in-vitro-compartmentalization (Jeremy Agresti).

Figure 1: Biological applications may involve the incorporation of cells (left), DNA, RNA, proteins, and other biological materials as well as substrates (right) in aqueous emulsion drolets dispersed in a continuous oil phase.

Emulsions with a Continuous Fluorocarbon Phase

While the biological systems work in water, the continuous phase may be chosen from a range of chemicals that are immiscible with water. Fluorocarbon oils seem to be ideally suited, as they (a) do not allow for cross-contamination, as they (b) are compatible with PDMS and as they (c) provide an efficient transport of oxygen to cells inside the droplets.

Surfactants for these systems have to fulfill two requirements: First, they have to keep two colliding droplets from coalescing. This can be done by steric stabilization and it sets criteria for choosing the part of the surfactant that is soluble in the fluorocarbon oil. Secondly, biological compounds, such as DNA, may be rendered inactive upon adsorption to the interfaces. Therefore, the water-soluble part of the surfactant must be made to prevent their adsorption. However, appropriate surfactants are not available commercially.

Synthesis of Fluorosurfactants

Commercial surfactants for water-in-fluorocarbon emulsions carry charges on their hydrophilic moiety. Charges, however, interact strongly with proteins and DNA resulting in an unfolding of the three dimensional structure and inactivity of these compounds. Non-ionic surfactants for stabilizing water-in-fluorocarbon emulsions are not available. Therefore, we chose to synthesize a new class of fluorosurfactants. It consists of oligomeric fluorocarbon blocks for steric stabilization and hydrophilic PEO-blocks, as this polymer is known to keep biomolecules from adsorbing to interfaces. We investigated various ways to couple these two blocks chemically.

Having found a working coupling reaction, a further challenge was the geometry of the surfactant. In order to form a dense layer of surfactant molecules, its “shape” must be tuned to fit around the water droplet. This can be achieved by tuning the relative chain lengths or the morphology of the surfactant, e. g. by synthesizing a double-tail surfactant.

 

Figure 2: left: The surfactant geometry of a surfactant is crucial to emulsion stability. It may be tuned by changing the relative chain length of the two blocks constituting the surfactant and by tuning the morphology of the surfactant (single or double tail surfactant). right: A surfactant layer assembles at the interface of an emulsion droplet. The geometry in this case is suitable for colloidal stabilization.

Results

Indeed, one product of the coupling reaction actually stabilized emulsions for a long time, which means that the blocks are chosen well, the chemical bond is sufficiently stable and its geometry is suitable. Samples, in which the PEO-blocks were chosen to be too big, did not stabilize the droplets against coalescence for a reasonable time.

Figure 3: Comparison of two emulsions stabilized with the same amount of chemically equivalent surfactants with different relative block sizes: The upper image shows a surfactant of suitable geometry, the lower one exhibits pronounced coalescence due to its wrong geometry.

Biocompatibility could be shown through in-vitro translation yielding a fluorescent dye. The new surfactant allows for translation inside the droplets, while the blind experiment does not show fluorescence.

Figure 4: Biological experiments inside aqueous droplets dispersed in fluorocarbon oils: An in-vitro translation mix has been incorporated in both w/f emulsions. A fluorescent product will only be yielded, if adsorption of DNA to the droplet interface is prevented by an appropriate surfactant system. Emulsions stabilized with the surfactant described above show pronounced fluorescence (right), while a blind sample stabilized with a commercial anionic surfactant (left) exhibits no fluorescence.

Summarizing, we found appropriate moieties, developed a coupling reaction and tuned the geometry for a new class of non-ionic fluorosurfactants for w/f emulsions: These surfactants stabilize aqueous droplets against coalescence and at the same time are capable of preventing the adsorption of biological material to the interfaces.

(2) Cross-Linked Polyurethane Latices through Suspension Polymerization in Fluorocarbon Oils

Stabilization of o/f Emulsions

The above surfactants contain polyethyleneglycol (PEG) moieties as “inner” blocks. Given that PEG is soluble in most of the common organic solvents (CH₂Cl₂, THF, Acetone, MeOH, EtOH, DMF, DMSO, etc.) and given that these solvents are immiscible with fluorocarbon oils as well, our new surfactants should not only be suited for stabilizing water droplets in fluorocarbon oils, but they should also work for droplets of organic solvents. The advantage of o/f emulsions is that emulsion droplets of organic solvents may now be made in entirely water-free systems. In order to show the value of o/f emulsions for classical organic chemistry, we chose an industrially important example that involves water-sensitive isocyanates: the production of polyurethane latices.

Latices and Polyurethanes

Organic emulsion droplets in a continuous fluorocarbon phase may be used as precursors for cross-linked polyurethane latex particles. The availability of surfactants for o/f emulsions allows for suspension polymerization in a water-free environment.

A latex is a dispersion of solid polymer particles in a liquid. Latices are important components of paints and coatings; they are also used in the production of latex gloves. Latices may be made from emulsion droplets containing the precursors of a latex particle.

Figure 5: Surfactant-stabilized droplets of a liquid latex precursor shall be converted to solid polymer latex particles.

Latices of polyurethanes are hard to make. Polyurethanes are produced through the polyaddition of diisocyanates and diols. Diisocyanates are water-sensitive. Hydrolysis yields carbon dioxide, which is exploited in the production of polyurethane foams for insulating construction materials. For coatings, however, the excellent mechanical properties of polyurethanes are desired and the side-reaction with water has to be suppressed. Hence, polymerization has to take place in the absence of water.

 

Figure 6: Polyurethanes (PU) are the products of the polyaddition reaction of diisocyanates and diols. Isocyanates are water-sensitive chemicals that react to the amines, liberating CO₂ (foaming reaction). If PU-foams are not desired, polymerization has to be carried out in a water-free environment.

The Production of Polyurethane Latices

Because of the water-sensitivity of isocyanates, up to date industry produces polyurethane latices in a fairly complicated process: First, a polymer solution is produced in bulk. This is then emulsified in a continuous water-phase. Finally, latex particles are obtained after the evaporation of the solvent. For cross-linked particles an additional cross-linking step is required.

Figure 7: The current procedure for the production of polyurethane latices is a three step process: Synthesis of a polyurethane solution in bulk, emulsification, and solvent evaporation. A fourth step would be required for the production of cross-linked polyurethane latex particles.

Suspension polymerization, on the other hand, would be much simpler: The polymer precursor is emulsified and reacts in the droplets, yielding polymer latex particles. Those are 1:1 copies of the emulsion droplets.

Figure 8: Suspension polymerization would be a much simpler way to polyurethane latices, however, it relies of the availability of a water-free environment: Emulsification of the polymer precursor will yield droplets, in which polymerization will take place yielding the solid latex particles after a 1:1 conversion. Even cross-linked particles may be obtained without an additional step. No solvents are required for this process (“green chemistry”).

However, if this process is to be applied, water may not be used as a continuous phase because it would degrade the diisocyanates. Fluorocarbon oils as the continuous emulsion phase provide at the same time a water-free emulsion system and the required immiscibility with the polyurethane precursors.

Sufficient colloidal stabilization of the emulsion droplets against coalescence during the latex production and later on of the latex particles against aggregation is crucial to the success of the comparatively simple approach of suspension polymerization. This stabilization may be provided by the non-ionic fluorosurfactants described above.

Results

Using the PEG-block-fluorocarbon copolymer fluorosurfactants (s. a.) with a geometry that was tuned to suit the specific system, we have been able to synthesize polyurethane latex particles through suspension polymerization in a continuous fluorocarbon phase.

Figure 9: Polyurethane latex made by suspension polymerization (emulsification in a PDMS-device). 1,6-diisocyanatohexane and PEG of 200 g/mol molecular weight were used as reactants.

Moreover, as shown at the example of a shaken emulsion in Figure 10, even cross-linked polyurethane particles have been obtained in a one-step process.

 

Figure 10: Cross-linked polyurethane latex made by suspension polymerization (emulsification by simple shaking). 1,6-diisocyanatohexane and PEG 200 g/mol were applied with polycaprolactone triol as cross-linker.

We showed that our new class of fluorosurfactants is suited for the stabilization of not only aqueous, but also organic emulsion droplets in fluorocarbon oils. This enables us to carry out water sensitive reactions inside the droplets: We presented a new way to produce polyurethane latices through simple suspension polymerization. Even cross-linked particles may be produced in a one-step process. Our emulsification techniques in microfluidic PDMS devices are applicable to this system due to the chemical compatibility of the polyurethane precursors with PDMS rubbers. These techniques will yield highly monodisperse particles. In the same way, also polyester particles may be made. Beside these types of polymerization, many other water-sensitive chemical reactions may be carried out in hydrocarbon-in-fluorocarbon emulsions. Their promises apply not only to synthetic but also to analytical applications, including single molecule investigations inside emulsion droplets.