Thomas Erichsen

Quinohemoprotein alcohol dehydrogenase-based reagentless amperometric biosensor for ethanol monitoring during wine fermentation

This paper describes the development and optimization of an amperometric biosensor for monitoring ethanol in beverages. The biosensor is constructed by cross-linking a quinoprotein alcohol dehydrogenase (QH-ADH) to an Os-complex-modified poly(vinylimidazole) redox polymer using poly(ethylene glycol) diglycidyl ether. The optimum biosensor configuration was evaluated by changing the ratio between enzyme, redox polymer, and cross-linker using conventional graphite rods as basis electrodes. The optimized sensor showed a sensitivity of 0.336+/-0.025 A M-1 cm(2) for ethanol and a detection limit (calculated as three times the signal-to-noise ratio) of 1 muM. This biosensor configuration was further evaluated in a conventional flow-injection system and the applicability for the determination of ethanol in diverse wine samples could be successfully demonstrated. Adaptation of this sensor configuration to screen-printed (SP) electrodes allowed their integration into an automated sequential-injection analyzer and the successful on-line monitoring of ethanol during wine fermentation processes. (Less)

Combinatorial microelectrochemistry: Development and evaluation of an electrochemical robotic system

An electrochemical robotic system using standard microtiter plates as reaction wells for potentiostatic and galvanostatic electrosynthesis and high-throughput electroanalysis was conceived and realized using stepmotor driven positioning stages in combination with a flexible software. Electrode bundles specifically adapted to the experimental needs are accurately positioned in the wells of a microtiter plate followed by the automatic performance of sequences of electrosynthetic or electroanalytical techniques. The system allows us to work under inert-gas atmosphere, in aqueous and organic solvents, and to add or remove solutions by means of integrated syringe pumps. A specifically developed script language permits the user to perform very complex experimental sequences in the different wells of the microtiter plate. The hardware and software features of the developed electrochemical robotic system, the design of suitable electrode arrangements for electrosynthesis and electroanalytical techniques, as well ...

4D shearforce-based constant-distance mode scanning electrochemical microscopy.

4D shearforce-based constant-distance mode scanning electrochemical microscopy (4D SF/CD-SECM) is designed to assess SECM tip currents at several but constant distances to the sample topography at each point of the x,y-scanning grid. The distance dependent signal is achieved by a shearforce interaction between the in-resonance vibrating SECM tip and the sample surface. A 4D SF/CD-SECM measuring cycle at each grid point involves a shearforce controlled SECM tip z-approach to a point of closest distance and subsequent stepwise tip retractions. At the point of closest approach and during the retraction steps, pairs of tip current (I) and position are acquired for various distances above the sample surface. Such a sequence provides x,y,I maps, that can be compiled and displayed for each selected data acquisition distance. Thus, multiple SECM images are obtained at known and constant distances above the sample topography. 4D SF/CD-SECM supports distance-controlled tip operation while continuous scanning of the SECM tip in the shear-force distance is avoided. In this way, constant-distance mode SECM imaging can be performed at user-defined, large tip-to-sample distances. The feasibility and the potential of the proposed 4D SF/CD-SECM imaging is demonstrated using on the one hand amperometric feedback mode imaging of a Pt band electrode array and on the other hand the visualization of the diffusion zone of a redox active species above a microelectrode in a generator/collector arrangement.

Reagentless biosensors based on co-entrapment of a soluble redox polymer and an enzyme within an electrochemically deposited polymer film

A novel biosensor architecture, which is based on the combination of a manual and a non-manual deposition technique for sensor components on the electrode surface is reported. A water-soluble Os-poly(vinyl-imidazole) redox hydrogel is deposited on a graphite electrode by drop-coating (i.e. manually) followed by the electrochemically-induced deposition of an enzyme-containing non-conducting polymer film. The local polymer deposition is initiated by electrochemical generation of H(3)O(+) exclusively at the electrode surface causing a pH-shift to be established in the diffusion zone around the electrode (i.e. non-manually). This pH-shift leads to the protonation of a dissolved polyanionic polymer which in consequence changes significantly its solubility and is hence precipitating on the electrode surface. In the presence of a suitable enzyme, such as quinohemoprotein alcohol dehydrogenase (QH-ADH), the polymer precipitation leads to an entrapment of the redox enzyme within the polymer film. Simultaneously, the water-soluble Os-poly(vinyl-imidazole) redox hydrogel, which is slowly dissolving from the electrode surface after addition of the electrolyte, is co-entrapped within the precipitating polymer layer. This provides the pre-requisite for an efficient electron-transfer pathway from the redox enzyme via the polymer-bound redox centres to the electrode surface. The sensor preparation protocol has been optimised aiming on a high mediator concentration in the polymer film and an effective electron transfer.

Frequency-Dependent Alternating-Current Scanning Electrochemical Microscopy (4D AC-SECM) for Local Visualisation of Corrosion Sites

For a better understanding of the initiation of localised corrosion, there is a need for analytical tools that are capable of imaging corrosion pits and precursor sites with high spatial resolution and sensitivity. The lateral electrochemical contrast in alternating-current scanning electrochemical microscopy (AC-SECM) has been found to be highly dependent on the frequency of the applied alternating voltage. In order to be able to obtain data with optimum contrast and high resolution, the AC frequency is swept in a full spectrum at each point in space instead of performing spatially resolved measurements at one fixed perturbation frequency. In doing so, four-dimensional data sets are acquired (4D AC-SECM). Here, we describe the instrument set-up and modus operandi, along with the first results from the imaging of corroding surfaces. Corrosion precursor sites and local defects in protective organic coatings, as well as an actively corroding pit on 304 stainless steel, have been successfully visualised. Since the lateral electrochemical contrast in these images varies with the perturbation frequency, the proposed approach constitutes an indispensable tool for obtaining optimum electrochemical contrast.

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.

Imaging Biocatalytic Activity of Enzyme−Polymer Spots by Means of Combined Scanning Electrochemical Microscopy/Electrogenerated Chemiluminescence

The purpose of this study was to develop a scanning electrochemical microscopy (SECM) and scanning electrogenerated chemiluminescence (SECL) setup to visualize the localized enzymatic activity using glucose oxidase as a model. Combination of SECM and electrogenerated chemiluminescence (ECL) was made possible by integrating a photomultiplier tube (PMT) within a SECM setup which is mounted on top of an inverted microscope. An enzyme-polymer spot formed on a glass slide and placed on top of the entrance window of the PMT was used as a model sample to evaluate the potential of the combined SECM/ECL setup. Hydrogen peroxide, which was locally generated by the glucose oxidase (GOx)-catalyzed reaction, reacted with oxidized luminol which was simultaneously electrochemically generated at the positioned SECM electrode tip. By using the phase-sensitive lock-in amplifier, the potential applied to the SECM tip was sinusoidally swept to invoke an associated oscillation of the ECL. Thus, sensitivity of SECL could be substantially enhanced. Images of the local immobilized enzyme activity obtained both by ECL and generator/collector (GC) mode of SECM were compared to elucidate the pathway in which the SECM and SECL signals are generated.

Robotic sequential analysis of a library of metalloporphyrins as electrocatalysts for voltammetric nitric oxide sensors

A library of 83 metalloporphyrins with varying substitution pattern at the meso-position of the porphyrins and different central metal ions in the core region has been synthesized in small quantities using a parallel synthesis strategy. By means of a specially designed electrochemical robotic device integrating a 96-well microtitre plate and an easily movable assembly of working, counter and reference electrodes, the different porphyrins were automatically applied in sequence to an in-well electrochemical preparation and testing of NO sensors. Screening the entire compound collection suggested initial considerations concerning the influence of varied functionalities of the metalloporphyrins on their electrocatalytic properties for the oxidation of NO and helped to identify the quality of the investigated catalyst candidates. As compared to manually performed quality tests, the proposed strategy of automation has advantages in convenience, rapidity and especially reproducibility avoiding any inaccuracies introduced by manually performing all steps of the complex sensor formation and testing sequence.

On-Line Analysis of Mercury by Sequential Injection Stripping Analysis (SISA) Using a Chemically Modified Electrode

A highly flexible, automatic sequential-injection stripping analysis (SISA) system has been developed and applied for monitoring the levels of mercury in aqueous solutions using a chemically modified electrode. The preparation of the modified electrode comprises spin coating of an ethanolic solution of poly(4-vinylpyridine) and Kryptofix-222 onto a glassy carbon electrode (GCE) followed by cross-linking the polymer. The analysis is based on the anodic stripping voltammetry of mercury using differential pulse voltammetry. A sequence of 36 operations was needed to complete a full cycle of cleaning, calibration and analysis.

Constant-distance mode SECM as a tool to visualize local electrocatalytic activity of oxygen reduction catalysts.

Summary Multidimensional shearforce-based constant-distance mode scanning electrochemical microscopy (4D SF/CD-SECM) was utilized for the investigation of the activity distribution of oxygen reduction catalysts. Carbon-supported Pt model catalyst powders have been immobilized in recessed microelectrodes and compared to a spot preparation technique. Microcavities serve as platform for the binder-free catalyst sample preparation exhibiting beneficial properties for constant-distance mode SECM imaging concerning modified surface area and catalyst loading. The integration of the redox competition mode of SECM into the detection scheme of the 4D SF/CD mode is demonstrated for specifically adapting high-resolution SECM experiments to powder-based catalyst preparations.