Simultaneous detection of L-glutamate and nitric oxide from adherently growing cells at known distance using disk shaped dual electrodes.

An ex vivo system for simultaneous detection of nitric oxide (NO) and L-glutamate using integrated dual 250 microm platinum disk electrodes modified individually with suitable sensing chemistries has been developed. One of the sensors was coated with an electrocatalytic layer of Ni tetrasulfonate phthalocyanine tetrasodium salt (Ni-TSPc) covered by second layer of Nafion, which stabilises on the one hand the primary oxidation product NO(+) and prevents interferences from negatively charged compounds such as NO(2)(-). For glutamate determination, the second electrode was modified with a crosslinked redox hydrogel consisting of Os complex modified poly(vinylimidazol), glutamate oxidase and peroxidase. A manual x-y-z micromanipulator on top of an inverted optical microscope was used to position the dual electrode sensor at a defined distance of 5 microm from a cell population under visual control. C6 glioma cells were stimulated simultaneously with bradykinin or VEGF to release NO while KCl was used to invoke glutamate release. For evaluation of the glutamate sensors, in some experiments HN10 cells were used. To investigate the sensitivity and reliability of the system, several drugs were applied to the cells, e.g. Ca(2+)-channel inhibitors for testing Ca(2+)-dependence of the release of NO and glutamate, rotenone for inducing oxidative stress and glutamate antagonists for analysing glutamate release. With these drugs the NO and glutamate release was modulated in a similar way then expected from previously described systems or even in-vivo measurements. We therefore conclude that our system is suitable to analyse stress-induced mechanisms in cell lines.

Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes.

The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.

Cell-compatible array of three-dimensional tip electrodes for the detection of nitric oxide release.

An array of electrodes on which cells could be grown directly was fabricated using silicon anisotropic etching and a thick-photoresist process and employed for the detection of nitric oxide (NO) released from a population of adherently growing human umbilical vein endothelial cells (HUVEC). The electrodes are tip-shaped and are 40 microm high of which only the top 15 microm are exposed Pt-tips. After electrochemical induced modification of the exposed Pt tips using Ni phthalocyanine the individual addressable electrode tips were sensitive and selective for the detection of NO at an applied constant potential of 750 mV. The silicon nitride insulation of the lower part of the tip electrodes prevented the death of the cells upon the application of the working potential at which NO was detected. It also helped to avoid the perturbation of the integrity of the sensing chemistry imparted on the electrode surface that could have resulted from the contact of the adherently growing cells with the active electrode surface. The release of nitric oxide from HUVEC was successfully monitored with different numbers of tip electrodes simultaneously connected as combined working electrode.

Odorant-dependent generation of nitric oxide in Mammalian olfactory sensory neurons.

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis.

Dual microelectrodes for distance control and detection of nitric oxide from endothelial cells by means of scanning electrochemical microscope.

Dual Pt disk microelectrodes consisting of a 10-microm distance sensor and a 50-microm nitric oxide sensor were prepared. The 50-microm electrode was modified with Ni(4-N-tetramethyl)pyridyl porphyrin enclosed in the polymer network of a negatively charged electrodeposition paint. This paint prevented the dissolution of the otherwise soluble porphyrin in the aqueous test medium due to charge interactions. It also denied negatively charged ions in the analyte solution access to the electrode surface by electrostatic repulsion, thereby preventing interference from anions such as nitrite, nitrate, and ascorbate. With the aid of a scanning electrochemical microscope, it was possible to use the distance sensor by recording the negative feedback effect on the reduction of molecular oxygen to "guide" the nitric oxide sensor to various known distances from a layer of adherently growing human umbilical vein endothelial cells for the detection of nitric oxide released from the cells upon stimulation with bradykinin. The use of the distance sensor made it possible to preserve the integrity of the adherently growing cells concomitantly with the modified electrode by preventing the deterioration of the modifying layer during the distance adjustment step.

Functionalised electrode array for the detection of nitric oxide released by endothelial cells using different NO-sensing chemistries.

In a preliminary study aimed at developing strategies for the simultaneous detection of various biologically important molecules, a procedure is described that allows the electrochemical detection of nitric oxide (NO) released by a population of human umbilical vein endothelial cells (HUVEC) by using an array of electrodes comprising three individually addressable electrodes. Each electrode in the array was modified with a different NO-sensitive electrocatalyst, thereby demonstrating the possibility of modifying the individual electrodes in an array with different sensing chemistries. This study opens a doorway to the development of arrays of electrodes for the simultaneous detection of multiple analytes in a complex environment by suitably tailoring the sensitivity and selectivity of each electrode in the array to a specific analyte in the test medium.