We report on an efficient ultrasound based technique for lysing Escherichia coli and Pichia pastoris with oscillating cavitation bubbles in an integrated microfluidic system. The system consists of a meandering microfluidic channel and four piezoelectric transducers mounted on a glass substrate, with the ultrasound exposure and gas pressure regulated by an automatic control system. Controlled lysis of bacterial and yeast cells expressing green fluorescence protein (GFP) is studied with high-speed photography and fluorescence microscopy, and quantified with real-time polymerase chain reaction (qRT-PCR) and fluorescence intensity. The effectiveness of cell lysis correlates with the duration of ultrasound exposure. Complete lysis can be achieved within one second of ultrasound exposure with a temperature increase of less than 3.3 °C. The rod-shaped E. coli bacteria are disrupted into small fragments in less than 0.4 seconds, while the more robust elliptical P. pastoris yeast cells require around 1.0 second for complete lysis. Fluorescence intensity measurements and qRT-PCR analysis show that functionality of GFP and genomic DNA for downstream analytical assays is maintained.

Chemistry. It incorporates referee's comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document.

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult-to-transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B-cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP-positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP-positive cells, respectively. Here, we present sonoporation as a feasible non-viral strategy for stable and highly efficient heterologous transfection of recalcitrant B-cell lines. This is the first demonstration of a non-viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B-cell lines.

In this study, two immiscible liquids in a microfluidics channel has been successfully emulsified by acoustic cavitation bubbles. These bubbles are generated by the attached piezo transducers which are driven to oscillate at resonant frequency of the system (about 100 kHz) [1, 2]. The bubbles oscillate and induce strong mixing in the microchamber. They induce the rupture of the liquid thin layer along the bubble surface due to the high shear stress and fast liquid jetting at the interface. Also, they cause the big droplets to fragment into small droplets. Both water-in-oil and oil-in-water emulsions with viscosity ratio up to 1000 have been produced using this method without the application of surfactant. The system is highly efficient as submicron monodisperse emulsions (especially for water-in-oil emulsion) could be created within milliseconds. It is found that with a longer ultrasound exposure, the size of the droplets in the emulsions decreases, and the uniformity of the emulsion increases. Reference: [1] Tandiono, SW Ohl et al., “Creation of cavitation activity in a microfluidics device through acoustically driven capillary waves,” Lab Chip 10, 1848–1855 (2010). [2] Tandiono, SW Ohl et al., “Sonochemistry and sonoluminescence in microfluidics,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5996–5998 (2011).

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