Electroporation of single cells and tissues with an electrolyte-filled capillary.

We show how an electrolyte-filled capillary (EFC) coupled to a high-voltage power supply can be used as a versatile electroporation tool for the delivery of dyes, drugs, and biomolecules to the cytoplasm of single cells and cells in tissues. A large-voltage pulse applied across the EFC (fused silica, 30 cm long, 375-microm o.d., 30-microm i.d.) gives rise to a small electric field outside the terminus of the EFC, which causes pore formation in cell membranes and induces an electroosmotic flow of electrolyte. When the EFC contains cell-loading agents, then the electroosmotic flow delivers the agents at the site of pore formation. The combination of pore formation and delivery enables loading of materials into the cytoplasm. By patch-clamp and fluorescence microscopy, formation of pores was observed at estimated transmembrane voltages of <85 mV with half-maximum values around 206 mV. The electroporation protocol was demonstrated by introduction of fluorogenic dyes into single NG108-15 cells, cellular processes, and small populations of cells in organotypic hippocampal cultures. Preliminary results are shown in which this protocol was employed for in vivo electroporation of ventral mesencephalon in rat brains. The technique was also used to access organelle-based detection systems inside cells. As a demonstration, 1,4,5-inositoltriphosphate was added to the electrolyte and detected by intracellular organelles in electroporated cells.

Electroinjection of colloid particles and biopolymers into single unilamellar liposomes and cells for bioanalytical applications.

A combined electroporation and pressure-driven microinjection method for efficient loading of biopolymers and colloidal particles into single-cell-sized unilamellar liposomes was developed. Single liposomes were positioned between a approximately 2-microm tip diameter solute-filled glass micropipet, equipped with a Pt electrode, and a 5-microm-diameter carbon fiber electrode. A transient, 1-10 ms, rectangular waveform dc voltage pulse (10-40 V/cm) was applied between the electrodes, thus focusing the electric field over the liposome. Dielectric membrane breakdown induced by the applied voltage pulse caused the micropipet tip to enter the liposome and a small volume (typically 50-500 x 10(-15) L) of fluorescein, YOYO-intercalated T7-phage DNA, 100-nm-diameter unilamellar liposomes, or fluorescent latex spheres could be injected into the intraliposomal compartment. We also demonstrate initiation of a chemical intercalation reaction between T2-phage DNA and YOYO-1 by dual injection into a single giant unilamellar liposome. The method was also successfully applied for loading of single cultured cells.

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy.

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.

Nasal nitric oxide and its relationship to nasal symptoms, smoking and nasal nitrate.

Nitric oxide (NO) is produced in the nasal mucosa and in the paranasal sinuses. Increased nasal NO concentrations have been found in patients with asthma and/or rhinitis, and nasal NO has been suggested to be a marker of nasal inflammation. Measuring the stable end products of NO, nitrate and nitrite in nasal lavage fluid have been proposed as an indirect method for measuring NO concentration. The aim of this study was to measure nasal NO concentration, and to find out its relationship to nasal nitrate concentration and clinical parameters. 73 paper-mill workers were investigated with nasal and exhaled NO, nitrate in nasal lavage fluid and were given a respiratory questionnaire. Nasal air was sampled directly from a nasal mask and NO concentration was measured with a chemiluminescence analyser. Exhaled NO was measured with the subjects breathing tidal volumes and wearing nose clips. The nitric oxide metabolites were analysed as nitrate, after reduction of nitrite to nitrate. Smokers had lower nasal NO concentration (264 ppb) as compared to NO concentrations of 340 ppb among non-smokers (p = 0.02). There was no statistically significant relationship between nasal NO concentration and nitrate in nasal lavage fluid or nasal symptoms. Nasal NO concentration was significantly related to FVC (p = 0.047) and there was a relationship with borderline statistical significance (p = 0.06) to FEV1. In conclusion, we found no relationship between nitrate in nasal lavage and nasal NO, and neither of these were correlated to nasal symptoms or to nasal PIF. Nasal NO was significantly lower among smokers. Further controlled studies on subjects with rhinitis are needed, to evaluate the relation between nasal NO and nasal inflammation. In addition, there is also a need to develop methods for measuring nasal NO that minimise contamination from sinuses.