Imaging of ATP membrane transport with dual micro-disk electrodes and scanning electrochemical microscopy.

Extracellular adenosine-5'-triphosphate (ATP) is involved in a variety of relevant regulatory mechanisms at a cellular level and has therefore been focus of extensive research. One of the major challenges associated with measuring this key regulatory analyte is the ability to detect and localize extracellular ATP with sufficient spatial and temporal resolution in physiological environments. In this study, scanning electrochemical microscopy (SECM) utilizing an amperometric micro-biosensor based on co-immobilization of the enzymes glucose oxidase and hexokinase is applied for imaging ATP transport through a porous polycarbonate membrane under physiologically relevant conditions. The enzymatic biosensor operates on competitive consumption of the substrate glucose between the immobilized enzymes glucose oxidase and hexokinase involving ATP as a co-substrate. Quantitative determination of the ATP concentration is based on a linear correlation between the glucose consumption and the ATP level. Integration of the amperometric ATP micro-biosensor into a dual micro-disk electrode configuration is achieved by immobilizing the enzymes at one of the micro-disk electrodes while the second disk serves as an unmodified amperometric probe for controlled positioning of the micro-biosensor in close proximity to the sample surface enabling quantification of the obtained current signal.

Numerical simulation of scanning electrochemical microscopy experiments with frame-shaped integrated atomic force microscopy--SECM probes using the boundary element method.

Integrated submicroelectrodes for combined AFM-SECM measurements are characterized with numerical simulations using the boundary element method. SECM approach curves and SECM images are calculated and analyzed for a model substrate containing pronounced topographical and electrochemical features. The theoretically calculated image has been compared to the experimental data and shows excellent quantitative agreement. Hence, the applicability of integrated AFM-SECM electrodes for combined electrochemical and topographical imaging and a profound theoretical description including quantification of the obtained results are demonstrated.

Nanoelectrodes integrated in atomic force microscopy cantilevers for imaging of in situ enzyme activity.

For investigation of laterally resolved information on biological activity, techniques for simultaneously obtaining complementary information correlated in time and space are required. In this context, recent developments in scanning probe microscopy are aimed at information on the sample topography and simultaneously on the physical and chemical properties at the nanometer scale. With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy with AFM is demonstrated. This instrumentation enables simultaneous imaging of topography and obtainment of laterally resolved electrochemical information in AFM tapping mode. Hence, topographical and electrochemical information on soft surfaces (e.g., biological species) and polymers can be obtained. The functionality of tip-integrated electrodes is demonstrated by simultaneous electrochemical and topographical studies of an enzyme-modified micropattern.

Analysis of corrosion processes at the surface of diamond-like carbon protected zinc selenide waveguides.

A detailed surface analytical study on the corrosion behavior of unprotected and diamond-like carbon (DLC)-coated mid-infrared (MIR) waveguides used in remote sensing applications at strongly oxidizing conditions is presented. High-quality DLC films, with a thickness of 100 nm serving as MIR-transparent corrosion barrier, have been produced at the surface of zinc selenide (ZnSe) attenuated total reflection waveguides via pulsed laser deposition techniques. IR microscopy and atomic force microscopy are applied to investigate the chemical inertness of DLC-based membranes against aqueous solutions of hydrogen peroxide. These stability studies show that uncoated ZnSe waveguides are subject to severe chemical surface modifications, while DLC-protected waveguides maintain their optical properties and chemical integrity. In situ studies on the corrosion behavior by a recently developed approach combining scanning electrochemical microscopy (SECM) with Au/Hg amalgam ultramicroelectrodes in a scanning stripping voltammetry experiment provides additional insight into the mechanisms of the corrosion process. It is demonstrated that the combination of surface analytical techniques and, in particular, the innovative application of SECM with amalgam electrodes provides superior information on corrosion processes at the surface of optical waveguides. This detailed study confirms the efficiency of protective DLC coatings deposited onto IR-transparent optical waveguides, rendering this novel concept ideal for sensing applications in harsh environments.

Amperometric ATP biosensor based on polymer entrapped enzymes.

A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1).