Controlled Cavitation in Microfluidic Systems

We report on cavitation in confined microscopic environments which are commonly called microfluidic or lab-on-a-chip systems. The cavitation bubble is created by focusing a pulsed laser into these structures filled with a light-absorbing liquid. At the center of a 20 µm thick and 1 mm wide channel, pancake-shaped bubbles expand and collapse radially. The bubble dynamics compares with a two-dimensional Rayleigh model and a planar flow field during the bubble collapse is measured. When the bubble is created close to a wall a liquid jet is focused towards the wall, resembling the jetting phenomenon in axisymmetry. The jet flow creates two counter-rotating vortices which stir the liquid at high velocities. For more complex geometries, e.g., triangle- and square-shaped structures, the number of liquid jets recorded correlates with the number of boundaries close t

Single cell electroporation using microfluidic devices

Electroporation is a powerful technique to increase the permeability of cell membranes and subsequently introduce foreign materials into cells. Pores are created in the cell membrane upon application of an electric fi eld (kV/cm). Most applications employ bulk electroporation, at the scale of 1 mL of cells (ca. one million cells). However, recent progresses have shown the interest to miniaturize the technique to a single cell. Single cell electroporation is achieved either using microelectrodes which are placed in close vicinity to one cell, or in a microfl uidic format. We focus here on this second approach, where individual cells are trapped in micrometer-size structures within a microchip, exposed in situ to a high electric fi eld and loaded with either a dye (proof-of-principle experiments) or a plasmid. Specifi cally, we present one device that includes an array of independent electroporation sites for customized and successive poration of nine cells. The different steps of the single cell electroporation protocol are detailed including cell sample preparation, cell trapping, actual cell poration and on-chip detection of pore formation. Electroporation is illustrated here with the transport of dyes through the plasma membrane, the transfection of cells with GFP-encoding plasmids, and the study of the ERK1 signaling pathway using a GFP–ERK1 protein construct expressed by the cells after their transfection with the corresponding plasmid. This last example highlights the power of microfl uidics with the implementation of various steps of a process (cell poration, culture, imaging) performed at the single cell level, on a single device

Determination of the electroporation onset of bilayer lipid membranes as a novel approach to establish ternary phase diagrams: example of the L-α-PC/SM/cholesterol system

The lipid matrix of cell membranes contains phospholipids belonging to two main classes, glycero- and sphingolipids, as well as cholesterol. This matrix can exist in different phases, liquid disordered (l(d)), liquid ordered (l(o)) and possibly solid (s(o)), or even a combination of these. The precise phase composition of a membrane depends on its molecular content and more specifically on the presence and amount of cholesterol. This in turn dictates the membrane properties. In this work, the resistance of membranes to the process of electroporation is studied and related to the membrane phase composition. Specifically, the threshold voltage for electroporation is measured (V-th) when DC pulses with increasing amplitude are applied to membranes prepared from various mixtures of a glycerolipid (Heart PC (L-alpha-PC)), a sphingolipid (Egg SM (SM)) and cholesterol (Ch), introduced in various ratios. Binary mixtures (L-alpha-PC/Ch, L-alpha-PC/SM, SM/Ch) and L-alpha-PC/SM/Ch ternary mixtures are successively employed. For all binary and ternary systems, dramatic changes in V-th are measured as a function of the membrane molecular composition, and the variation patterns of V-th are successfully correlated with the membrane phase composition. Interestingly, the measure of the electroporation onset can be employed as a novel methodology to establish ternary phase diagrams, and this is illustrated with the L-alpha-PC/SM/cholesterol ternary system

Parallel probing of drug uptake of single cancer cells on a microfluidic device

Drug resistance is frequently developing during treatment of cancer patients. Intracellular drug uptake is one of the important characteristics to understand mechanism of drug resistance. However, the heterogeneity of cancer cells requires the investigation of drug uptake at the single cell level. Here, we developed a microfluidic device for parallel probing of drug uptake. We combined a v-type valve and peristaltic pumping to select individual cells from a pool of prostate cancer cells (PC3) and place them successively in separate cell chambers in which they were exposed to the drug. Six different concentrations of doxorubicin, a naturally fluorescent anti-cancer drug, were created in loop-shaped reactors and exposed to the cell in closed 2 nL volume chambers. Monitoring every single cell over time in 18 parallel chambers revealed increased intracellular fluorescence intensity according to the dose of doxorubicin, as well as nuclear localization of the fluorescent drug after 2 h of incubation. The herein proposed technology demonstrated a first series of proof of concept experiments and it shows high potential to use for probing drug sensitivity of single cancer cell

Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function