Sabine Borgmann

Electrochemical quantification of reactive oxygen and nitrogen: challenges and opportunities

AbstractReactive 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. FigureReactive oxygen and nitrogen species (ROS & RNS) are produced biological cells. An amperometric sensor is placed in close proximity. The recorded current I is used to determine fluxes of certain species.

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 NiII tetrakis(p‐nitrophenylporphyrin) on a 50‐μm diameter platinum disk electrode which was integrated, together with a 25‐μm 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.

Amperometric Enzyme Sensors based on Direct and Mediated Electron Transfer

Publisher Summary The simplest design of an amperometric biosensor is the direct measurement of either an enzymatically generated product or of an electron transfer (ET) mediator naturally involved in the biocatalytic process. For a principal understanding of the ET processes underlying the functionality of all amperometric biosensors, the theoretical background is used as it was developed for the elucidation of ET processes that play a vital role in a variety of biological reactions or in artificial “donor–acceptor system”. These donor–acceptor reactions can be very rapid even when the reactants are separated over longer distances. This chapter presents a brief summary of the Marcus-theory of ET and also focuses on the fabrication of suitable biosensor architectures facilitating electrochemical communication between the immobilized redox proteins and the electrode surface. The chosen immobilization technique and its impact on the biological recognition element affects, significantly, the overall biosensor performance and determines the selectivity, sensitivity, specificity, dynamic range, response time, and reliability of the biosensor. Several techniques for the immobilization of redox protein on electrode surfaces have been described in detail. In order to control the fabrication and later, function of an amperometric biosensor to its largest extent, the development of the so-called “reagentless biosensors” is of increasing importance. For the development of a reagentless biosensor, the most appropriate methods are the use of an enzyme with tightly bound redox centers and an ET pathway either by direct ET or via securely immobilized redox relays.

Robotic Systems for Combinatorial Electrochemistry

The scope of this chapter is to present the current state of the art in the field of combinatorial electrochemistry with the main focus on plate-based technologies. In particular, it is focused on the development and use of electrochemical robotic systems.