61st Annual Report on Research 2016

Reverse nanoemulsions (water-in-oil) are thermodynamically unstable while at the same time notoriously difficult to break. The small size of nanoemulsion droplets makes them difficult characterize without some assumptions regarding their dielectric and chemical properties.

In the first year of this PRF grant we used fluorimetry and DLS to characterize the pH and size of aqueous droplets in nanoemulsions. 

Considerable effort went into validating our use of fluorescein as a pH sensor, particularly since its fluorescence is sensitive to ionic strength as well as pH.  Fluorescein has dianionic, anionic, neutral, and cationic states, three of which are fluorescent. Using the ratio of fluorescence intensity at 550 nm and 515 nm, we generated calibration curves at fixed ionic strength between pH 1 and 10.  With care, we find that fluorescein is useable as a pH sensor between pH 2 and 8, quite a bit wider range than our earlier efforts.  This permitted us to show that the pH in attoliter volume droplets in perfluorinated oils is as low as 2 in the absence of surfactant.

In hydrocarbons the situation appears more complicated, probably due to surfactant impurities.  We found that the pH in nonane and squalane nanoemulsions, in the absence of added surfactant, were close to 6 and 7, respectively.  The addition of nonionic surfactants brought the pH down, exactly the opposite of the situation in perfluorinates.  Currently we believe this is due to surfactant impurities and we are now starting to purify all hydrocarbons using an aluminum oxide column. Our working theory predicts that the interior should be low pH in the absence of surfactant, particularly for the nonane droplets which were quite small (< 100 nm diameter).  Squalane droplets were substantially larger (> 500 nm diameter) which makes direct comparison difficult since the pH effect is thought to be due to sequestration of hydroxide ions at the low-dielectric surface and should therefore be sensitive to the volume-to-surface-area ratio.

Nanoemulsions are typically made by ultrasonication, which provides little control over droplet size.  In an attempt to control droplet size, we developed a protocol for making droplets using extrusion through a nucleopore membrane. We are in the process of characterizing these droplets for size and pH using DLS and fluorimetry.

Finally, in the last year we finished setting up a confocal microscope with the capability to do polarization anisotropy lifetime (PAL) measurements. Using burst-integrated fluorescence lifetime measurements, and a dye whose fluorescence lifetime is sensitive to pH, this apparatus will ultimately permit us to evaluate the pH in individual droplets. At the same time, PAL measurements allow us to investigate the rotational dynamics of the pH sensitive dye, which is important to validate our method: dyes that sense the droplet interior should have a fast (nanosecond) rotational coherence time, whereas dyes stuck at the interface will have a considerably longer rotational coherence time.

One undergraduate student and two graduate students received training under this grant in the last year. 

The senior most student (Sheema Rahmanseresht) was first author on the work that inspired this grant, in which we presented the first evidence that the pH of attoliter volume aqueous droplets was considerably lower than expected (Applied Physics Letters 106, 194107, 2015). Dr. Rahmanseresht also aligned and demonstrated the use of the confocal polarization anisotropy lifetime (PAL) apparatus and helped developed analysis techniques that we plan to use later in this project. Dr. Rahmanseresht received her PhD in May 2016. 

Kieran Ramos, the second graduate student, did an extensive calibration of fluorescein as a pH sensor and helped developed a Bayesian approach to data analysis.  He made extensive improvements to our fluorescence measurement protocol and modified the fluorimeter to accommodate the large dynamic range required for these measurements. He developed new techniques for making droplets using extrusion as described above.  Finally, Mr. Ramos supervised an undergraduate in the acquisition of DLS and fluorimetry data on droplets in hydrocarbons. He presented a contributed talk at the 2016 March Meeting of the APS titled “On the pH of Aqueous Attoliter-Volume Droplets.”

The undergraduate, Trevor Demille, is a senior physics major.  He has received extensive training in a variety of techniques relevant to this project and has written several reports outlining his progress.  He regularly prepares droplets with fluorescein, measures their size and mobility using DLS, measures fluorescence, and analyzes fluorescence data using methods developed by Ramos.  He has also become proficient in Matlab and Python while working on this grant.  Mr. Demille reported on his work at the Spring 2016 Meeting of the New England Section of the American Physical Society.