Tunable Synthesis of Encapsulated Microbubbles by Coupled Electrophoretic Stabilization and Electrochemical Inflation

Microbubbles are immensely important targeted therapeutic delivery vehicles and imaging contrast agents. But they are also incredibly challenging to manufacture and store owing to extreme internal pressures generated by the high surface curvature at these small size scales. Here we show how these limitations can be overcome by exploiting an interplay between microscale electrokinetics and electrochemistry that simultaneously confines macromolecular encapsulants near an electrode surface while inflating them with gases evolved from electrochemical reactions localized there. Stabilized microbubbles incorporating a remarkably broad range of morphologies can be produced by manipulating these fundamental processes. We demonstrate this versatility by generating dense clouds of small microbubbles that enable label-free detection of virtually any charged macromolecule, and by synthesizing larger discrete encapsulated microbubbles of tunable size. The inherent instability of microbubbles—that is, gasfilled bubbles in the 1–50 mm size range—results from the enormous Laplace pressures imposed by their highly curved interfacial topologies. Macromolecular encapsulants (e.g., amphiphilic surfactants, lipids) are often employed as stabilization agents to counter these extreme conditions by bolstering the interfacial barrier against gas outflow. The resulting stabilized microbubbles offer unique properties that have long been appreciated in the context of affinity separations (i.e., colloidal gas apherons), and more recently as contrast agents for ultrasound imaging, as well as convenient platforms for tethering chemical compounds (receptors, antibodies, etc.) to enable highly specific in vivo targeting (either to precisely deliver drug payloads or to locally destroy surrounding tissue when ruptured by an applied ultrasonic field). But despite these exciting applications, conventional batch-scale approaches used to produce encapsulated microbubbles are often relatively crude, based on mechanical agitation to entrain surrounding gas. Incredible progress has been made toward development of improved strategies at both the macro-scale (e.g., sonication and emulsification) and micro-scale (e.g., flow focusing and T-junctions), but a critical need still exists for approaches offering greater robustness to tune microbubble sizes and properties, as well as providing scalability from large batches to single-dose amounts. We have developed an electrokinetically actuated microbubble synthesis method that addresses these needs by establishing localized zones inside a microchannel network where an encapsulant can become enriched to a very high concentration while simultaneously experiencing infusion with electrochemically generated gases. The cornerstone of our approach is a microfluidic system incorporating arrays of individually addressable electrodes (patterned along the microchannel floor) that make it possible to transport charged macromolecules from the bulk solution to an electrode surface where they become compacted into an ultra-thin film (Figure 1a). This design enables very small potentials (1–3 V) to produce high electric fields (hundreds of Vcm ) because of the close inter-electrode spacings (tens to hundreds of mm) achievable within the array. The compaction process is illustrated by observing the behavior of fluorescently labeled DNA in response to a 1 VDC potential applied across neighboring electrodes. The negatively charged DNA contained between active electrodes migrates toward the anode and experiences local enrichment, yielding a fluorescent zone at the electrode surface (Figure 1b). Remarkably, we discovered that this compacted film can also be clearly seen under ordinary white light even when the DNA sample is unlabeled, with no excitation or emission filters in the optical path (Figure 1c). An anomalous highly reflective zone is formed at the electrode surface that is most clearly evident under oblique illumination, becoming visible within 10–20 s and completely dissipating within 5–10 s after the voltage is switched off (Figure 1d–g). This unexpected phenomenon can be widely applied, making it possible to detect a variety of unlabeled macromolecules including proteins (Figure 1h–j) and small-molecule cyclodextrans (Figure 1k). The effect can be tuned to localize compaction at either the anode or cathode by adjusting the buffer pH relative to the sample s isoelectric point (Figure 1 i). To determine the morphology of the compacted macromolecular films, we repeated the above mentioned DNA capture experiments under transmitted illumination using optically transparent thin gold electrode arrays. Observing the process under polarized light reveals that dense stabilized microbubble clouds are embedded within the enriched DNA [*] Y.-W. Huang, F. A. Shaikh, Prof. V. M. Ugaz Artie McFerrin Department of Chemical Engineering Texas A&M University, 3122 TAMU College Station, TX 77843 (USA) Fax: (+1)979-458-1002 E-mail: ugaz@tamu.edu [**] This work was supported in part by the Camille & Henry Dreyfus Foundation and the US National Science Foundation (NSF) under grant CTS-0554108. We thank Prof. Gyula Vigh for graciously providing cyclodextran samples, as well as for many helpful discussions. We also thank the staff in the Materials Characterization Facility (MCF) and the Microscopy& Imaging Center (MIC) at Texas A&M. V.M.U. gratefully acknowledges support from the K. R. Hall Professorship at Texas A&M.