Plasma stencilling methods for cell patterning.

In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS-PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability.

Electrochemical quantification of reactive oxygen and nitrogen: challenges and opportunities.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a crucial role in chemical signaling processes of biological cells. Electrochemistry is one of the rare methods able to directly detect these species. ROS and RNS can be monitored in the local microenvironment of cells in real time at the site where the actual signaling takes place. This review presents recent advances made with amperometric electrochemical techniques. Existing challenges for the quantification of ROS and RNS in biological systems are discussed to promote the development of innovative and reliable cell-based assays.

Lipid and phospholipid profiling of biological samples using MALDI Fourier transform mass spectrometry.

Here we describe a study of the feasibility of lipid and phospholipid (PL) profiling using matrix assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) for two different applications. In this work PL profiles of different mammalian tissues as well as those of whole cell organisms were examined. In particular, comparative analysis of lipid and PL profiles of tissues from mice fed different diets was done and, in another application, MALDI FTMS was used to analyze PL profiles of genetically modified Saccharomyces cerevisiae. Computational sorting of the observed ions was done in order to group the lipid and PL ions from complex MALDI spectra. The PL profiles of liver tissues from mice fed different diets showed a cross correlation coefficient of 0.2580, indicating significant dissimilarity, and revealed more than 30 significantly different peaks at the 99.9% confidence level. Histogram plots derived from the spectra of wild type and genetically modified yeast resulted in a cross correlation coefficient 0.8941 showing greater similarity, but still revealing a number of significantly different peaks. Based on these results, it appears possible to use MALDI FTMS to identify PLs as potential biomarkers for metabolic processes in whole cells and tissues.

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes.

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants K(M)(app) and maximum currents I(max)(app) were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0-0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04 mM.

Characterizing the phospholipid profiles in mammalian tissues by MALDI FTMS.

Discussed here is an analytical method for profiling lipids and phospholipids directly from mammalian tissues excised from Mus musculus (house mouse). Biochemical analysis was accomplished through the use of matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry, where whole tissue sections of mouse brain, heart, and liver were investigated. Lipid and phospholipid ions create complex MALDI mass spectra containing multiple ions with different m/z values corresponding to the same fundamental chemical species. When a computational sorting approach is used to group these ions, the standard deviation for observed relative chemical abundance can be reduced to 6.02%. Relative standard deviations of 10% are commonly accepted for standard chromatographic phospholipid analyses. Average mass measurement accuracy for 232 spectra representing three tissue types from 12 specimens was calculated to be 0.0053 Da. Further it is observed, that the data and the analysis between all the animals have near-identical phospholipid contents in their brain, heart, and liver tissues, respectively. In addition to the need to accurately measure relative abundances of phospholipid species, it is essential to have adequate mass resolution for complete and accurate overall analysis. It is reasonable to make mass composition assignments with spectral resolving power greater than 8000. However, results from the present study reveal 14 instances (C12 carbon isotope) of multiple m/z ions having the same nominal value that require greater resolution in order that overlap will not occur. Spectra measured here have an average resolving power of 12 000. It is established that high mass resolution and mass accuracy coupled with MALDI ionization provide for rapid and accurate phospholipid analysis of mammalian tissue sections.

Electrochemical high-content screening of nitric oxide release from endothelial cells.

Release of nitric oxide (NO) is of high importance for regulating endothelial cell functions during vasodilatation, vascular remodeling, and angiogenesis. Thus, a direct and reliable real-time method for NO detection that takes into account time-dependent variations of the NO concentration in the complex reaction within the diffusion zone above the cells is vital for obtaining information about the role of NO in intracellular endothelial signal transduction and its impact on the surrounding cells. In this study, the time course of vascular endothelial growth factor E (VEGF-E) stimulated NO release from transformed human umbilical vein endothelial cells (T-HUVEC) was investigated by means of metalloporphyrin-based NO sensors employed in an electrochemical robotic system. The NO sensor was obtained by electrochemically induced deposition of Ni(II) tetrakis(p-nitrophenylporphyrin) on a 50-microm diameter platinum disk electrode which was integrated, together with a 25-microm diameter platinum disk, in a double-barrel electrode arrangement. The second electrode was used as a guidance sensor for the automatic and highly reproducible positioning of the NO sensor at a known distance from a layer of adherently growing cells by using z-approach curves in the negative feedback mode of scanning electrochemical microscopy (SECM). The electrochemical robotic system allows the fully automated detection of NO with high sensitivity and selectivity to be performed in real time within 96-well microtiter plates. A functional cell assay was established to allow the standardized detection of NO released upon stimulation from T-HUVEC with a sensor positioned at a known distance above the endothelial cells. The overall system was evaluated by automatic detection of NO release from T-HUVEC upon stimulation with VEGF-E after incubation with a variety of drugs that are known to act on different sites in the complex signal-transduction pathway that finally invokes NO release.