Tandiono

Microbubble-mediated sonoporation for highly efficient transfection of recalcitrant human B- cell lines.

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.

Intense cavitation in microfluidics for bio-technology applications

This study reports the use of intense ultrasonic cavitation in the confinement of a microfluidics channel [1], and the applications that has been developed for the past 4 years [2]–[5]. The cavitation bubbles are created at the gas-water interface due to strong capillary waves which are generated when the system is driven at its natural frequency (around 100 kHz) [1]. These bubbles oscillate and collapse within the channel. The bubbles are useful for sonochemistry and the generation of sonoluminescence [2]. When we add bacteria (Escherichia coli), and yeasts (Pichia pastoris) into the microfluidics channels, the oscillating and collapsing bubbles stretch and lyse these cells [3]. In another application, human red blood cells are added to a microchamber. Cell stretching and rapture are observed when a laser generated cavitation bubble expands and collapses next to the cell [4]. A numerical model of a liquid pocket surrounded by a membrane with surface tension which was placed next to an oscillating bubble ...

Surfactant-free emulsification in microfluidics using strongly oscillating bubbles

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:...

Cavitation in confined spaces

Cavitation phenomena in real world are typically confined by one or more boundaries. Confining cavitation in small channels allows to study their interaction with cells, the formation of emulsions, and even sonochemical reactions in far greater detail as it would be possible in the bulk. However, it was expected that boundary layers will hinder bubble collapse more and more as the structure sizes are reduced. In this presentation the channel size is reduced even further, thus from microfluidic to nanofluidic channels. In microfluidic channels cavitation bubbles are generated with focused laser pulses and with acoustic waves. Acoustic cavitation in micrometer sized allows the formation of homogeneous emulsions, rapid rupture of cells (yeats and bacterias), and the dispersion of nanoparticles. While laser induced cavitation bubbles allow the study of bubble dynamics and bubble interaction in nanofluidic channels. In particular we will present experimental results on the dynamics of single bubbles and bubble...

Cell lysis using acoustic cavitation bubbles in microfluidics

Analysis of intracellular contents, such as proteins and nucleic acids, in a micro-scale system is gaining its importance in biomedical research. However, an efficient cell lysis needs to be achieved before the analysis can be carried out. The standard lysis methods in microfluidics, for example: by means of chemicals, thermal, or electrical lysis, suffer from the undesirable temperature increase and cross-contamination, which may lead to the denaturation of proteins or interfere with subsequent assays. Here, we present a technique to mechanically lyse microbial cells using acoustically driven cavitation bubbles in a polydimethylsiloxane based microfluidic channel attached on a glass slide. The cavitation bubbles are created by exciting gas-liquid interfaces in the microchannel into nonlinear interface instability with ultrasonic vibrations. The strongly oscillating bubbles create regions with intense mixing and high shear stress, which can deform and rupture the nearby cells. Escherichia coli (bacteria) and Pichia pastoris (yeast) cells are completely lysed in less than 0.4 seconds and 1.0 second, respectively. The temperature increase of the samples during the ultrasound exposures is less than 3.3 °C. Fluorescence intensity measurements and real-time polymerase chain reaction (qRT-PCR) analysis suggest that the functionality of the harvested protein and genomic DNA is maintained.

Study of nano/micro jets generated by laser-induced bubbles in thin films

The paper presents a liquid jet in thin films (height varies from micro to nanometers) and the study of its dynamics under the effects of the thin films dimension and viscosity. Furthermore, the penetrating jet velocity is investigated in terms of the laser energy and the distance between the laser focus and the targeted gas bubble surface. In the microchannel, strong shear stress ruptures part of the gas bubble and Rayleigh-Plateau instability further shattered it into small bubbles. On the other hand, in the nanochannel, the nanojets accelerate liquids with thickness of hundreds of nanometers.

Ultrasonic bubbles in microfluidics for red blood cells, bacteria, and yeast lysis

A custom microfluidic ultrasound device has been designed and implemented to efficiently create oscillating ultrasonic bubbles. These bubbles interact with the red blood cells, bacteria (Escherichia coli), and yeast (Pichia pastoris). Observations using high speed photography show that the red blood cells were strongly stretched. A numerical model using the Boundary Element Method is used to simulate the oscillating bubbles interaction with a nearby elastic pocket (a red blood cell model). Complex dynamics are discussed. Our ultrasonic microfluidic device is efficient in lysing E. coli and yeast cells for the harvesting of active intracellular content. Complete lysis of E. coli takes only 0.4 seconds while it takes about 1 second for the yeast cell. Temperature rise is minimal. We perform flourescent intensity measurement and qRT-PCR (real-time polymerase chain reaction) to show that the functional integrity of the proteins and DNA is preserved.

Acoustics cavitation in microfluidics for sonoluminescence and sonochemistry

Strong ultrasound is applied to a microfluidic channel to generate nonlinear surface waves which entrap bubbles at the gas–liquid interface to form oscillating bubbles. The ultrasound is generated by the piezoelectric transducer on the side of polydimethylsiloxane microchannel. The microchannel is attached to a glass slide through plasma bonding, while the transducer is glued by epoxy for strong coupling. The high speed photography shows that continuous cavitation clusters are formed within the microchannel as gas is injected. As they collapse rapidly, they are able to produce very intense concentration of energy that is able to emit light. This phenomenon is known as sonoluminescence. Previously, sonoluminescence is achieved via a single bubble or multiple bubbles in a bulk liquid. The authors report a realization of sonoluminescence in a microfluidic device. The same oscillating bubbles can also be used as micro-labs. They can trigger chemical reactions that require high temperature and pressure. We ach...