Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

Diffusion-based microfluidic PCR for "one-pot" analysis of cells.

Genetic analysis starting with cell samples often requires multi-step processing including cell lysis, DNA isolation/purification, and polymerase chain reaction (PCR) based assays. When conducted on a microfluidic platform, the compatibility among various steps often demands a complicated procedure and a complex device structure. Here we present a microfluidic device that permits a "one-pot" strategy for multi-step PCR analysis starting from cells. Taking advantage of the diffusivity difference, we replace the smaller molecules in the reaction chamber by diffusion while retaining DNA molecules inside. This simple scheme effectively removes reagents from the previous step to avoid interference and thus permits multi-step processing in the same reaction chamber. Our approach shows high efficiency for PCR and potential for a wide range of genetic analysis including assays based on single cells.

Focusing of mammalian cells under an ultrahigh pH gradient created by unidirectional electropulsation in a confined microchamber†Electronic supplementary information (ESI) available: Figures S1-S5 and videos S1-S2. See DOI: 10.1039/c4sc00319eClick here for additional data file.Click here for additional data file.Click here for additional data file.

The transport and manipulation of cells in microfluidic structures are often critically required in cellular analysis. Cells typically make consistent movement in a dc electric field in a single direction, due to their electrophoretic mobility or electroosmotic flow or the combination of the two. Here we demonstrate that mammalian cells focus to the middle of a closed microfluidic chamber under the application of unidirectional direct current pulses. With experimental and computational data, we show that under the pulses electrochemical reactions take place in the confined microscale space and create an ultrahigh and nonlinear pH gradient (∼2 orders of magnitude higher than the ones in protein isoelectric focusing) at the middle of the chamber. The varying local pH affects the cell surface charge and the electrophoretic mobility, leading to focusing in free solution. Our approach provides a new and simple method for focusing and concentrating mammalian cells at the microscale.

Thermal loading in flow-through electroporation microfluidic devices.

Thermal loading effects in flow-through electroporation microfluidic devices have been systematically investigated by using dye-based ratiometric luminescence thermometry. Fluorescence measurements have revealed the crucial role played by both the applied electric field and flow rate on the induced temperature increments at the electroporation sections of the devices. It has been found that Joule heating could raise the intra-channel temperature up to cytotoxic levels (>45 °C) only when conditions of low flow rates and high applied voltages are applied. Nevertheless, when flow rates and electric fields are set to those used in real electroporation experiments we have found that local heating is not larger than a few degrees, i.e. temperature is kept within the safe range (<32 °C). We also provide thermal images of electroporation devices from which the heat affected area can be elucidated. Experimental data have been found to be in excellent agreement with numerical simulations that have also revealed the presence of a non-homogeneous temperature distribution along the electroporation channel whose magnitude is critically dependent on both applied electric field and flow rate. Results included in this work will allow for full control over the electroporation conditions in flow-through microfluidic devices.

Characterizing osmotic lysis kinetics under microfluidic hydrodynamic focusing for erythrocyte fragility studies.

The biomechanics of erythrocytes, determined by the membrane integrity and cytoskeletal structure, provides critical information on diseases such as diabetes mellitus, myocardial infarction, hypertension, and sickle cell anemia. Here we demonstrate a simple microfluidic tool for examining erythrocyte fragility based on characterizing osmotic lysis kinetics. Hydrodynamic focusing is used for generating rapid dilution of the buffer and producing lysis of erythrocytes during their flow. The lysis kinetics are tracked by monitoring the release of intracellular contents from cells via recording the light intensity of erythrocytes at various locations in the channel. Such release profile reflects sensitively the changes in erythrocyte fragility induced by chemical, heating, and glucose treatment. Our tool provides a simple approach for probing red blood cell fragility in both basic research and clinical settings.