A real-time and label-free in vitro assay based on electric cell-substrate impedance sensing (ECIS) was established, validated, and compared to an end-point MTT assay within an experimental trial addressing the cytoprotective effects of 19 different flavonoids, flavonoid metabolites, and phenolic acids and their methyl esters on the HT-22 neuronal cell line, after induction of oxidative stress with tert-butyl hydroperoxide. Among the flavonoids under study, only those with a catechol unit and an additional 4-keto group provided cytoprotection. The presence of a 2,3-double bond was not a structural prerequisite for a neuroprotective effect. In the case of the phenolics, catechol substitution was the only structural requirement for activity. The flavonoids and other phenolics with a ferulic acid substitution or a single hydroxy group showed no activity. Electrochemical characterization of all compounds via square-wave voltammetry provided a rather specific correlation between cytoprotective activity and redox potential for the active flavonoids, but not for the active phenolics with a low molecular weight. Moreover this study was used to compare label-free ECIS recordings with results of the established MTT assay. Whereas the former provides time-resolved and thus entirely unbiased information on changes of cell morphology that are unequivocally associated with cell death, the latter requires predefined exposure times and a strict causality between metabolic activity and cell death. However, MTT assays are based on standard lab equipment and provide a more economic way to higher throughput.
High-resolution scanning electrochemical microscopy (SECM) in the constant-height mode was applied to image details of a nanostructured porous substrate as well as a confluent epithelial cell monolayer. Platinum disk ultramicroelectrodes (UMEs) with radii down to 100 nm were fabricated by sealing electrochemically etched Pt wires into soda-lime glass capillaries. A subsequent thermal treatment was used to improve the quality of electrode sealing. The size and shape of the SECM probes were adapted to the requirements of the inorganic and biological surfaces to enable high-resolution SECM imaging and to ensure stability and reliability during image acquisition. A mesoporous substrate (Si/SiO 2 ) with 800 nm rectangular pores was imaged with sub-mm resolution. In addition, confluent epithelial cell monolayers derived from kidney were investigated. Independent measurements with ferrocene methanol and hexamine ruthenium (III) chloride led to equivalent SECM results. High quality SECM images with well separated cells within the monolayer were obtained. Resolved regions of cell-cell contacts extending about 1-3 mm in lateral direction were imaged.
Electrochemically assisted injection (EAI) is a recently introduced injection concept for capillary electrophoresis (CE) enabling the separation of neutral analytes by means of electrochemical generation of charged species during the injection process. EAI‐CE is particularly attractive in combination with mass spectrometry (MS) leading to enhanced performance of MS detection. In this work, a fully automated EAI injection device was developed and applied to mechanistic and quantitative studies of the reduction of 4‐nitrotoluene (4‐NT). Three different carbon‐based screen‐printed electrodes (SPEs) were used including unmodified carbon, carbon nanofiber and reduced graphene oxide SPEs. Under acidic conditions the main products of 4‐NT reduction were 4‐hydroxylaminotoluene (4‐HAT) and 4‐aminotoluene (4‐AT). The EAI‐CE‐MS approach enabled the separation and selective determination of both compounds. On the basis of this methodical concept it was possible to study the formation of reduction products of 4‐NT in dependence on electrode potential and electrode material. In contrast to conventional electrochemical techniques like cyclic voltammetry EAI‐CE‐MS provides detailed information regarding changes of the product composition in dependence on various experimental conditions. Over the complete potential range studied the ratio of 4‐HAT/4‐AT was clearly different for the reduced graphene oxide SPE compared to the unmodified carbon and carbon fiber SPE. The construction of mass voltammograms added considerably to the information content of voltammetric experiments. Another aspect of this work was the reliable quantitative determination of 4‐NT by EAI‐CE‐MS using isotopically labeled 4‐NT as an internal standard. In this way the “classical” problem of changing response characteristics at solid electrodes could be eliminated based on the assumption that the target analyte and its isotopically labeled form behave in the same way. A corresponding protocol for quantitative EAI‐CE‐MS determinations of 4‐NT was elaborated and applied to standard solutions and spiked soil samples.
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