Micro-electrochemistry for enzyme and cellular transport studies

Miklós Gratzl

Department of Biomedical Engineering

&

Department of Physiology and Biophysics

Case Western Reserve University

Cleveland , OH 44106, USA

Biomedical measurements based on principles of micro-electrochemistry are increasingly used in research as well as in clinical settings. In this talk emerging applications in three areas will be discussed. Activities of certain enzymes can be used as markers for different clinical conditions. Such information is also required in basic research involving small laboratory animals. In both application areas it is desirable, and often necessary to use small quantities of specimen. This problem is addressed here using principles of electrochemistry to realize reagentless pH-stating in microliter volumes of biological samples. Current injection via a Pt mini-electrode generates H⁺ or OH^- ions at a rate that is sufficient to maintain pH in the sample during the studied bio-catalytic reaction that may consume or generate OH^- or H⁺. In this arrangement enzyme activity directly equals the amount of current required for pH-stating. Results obtained with a total electrochemical pH-stat for 20 microliter biomedical specimens will be discussed. Epithelial cells cover internal organs in tight monolayers that ensure directional mass transport in or out of the organ. Healthy epithelia are crucial to maintain proper organ function. Malfunctioning epithelia cause many diseases including many cancers and genetic disorders. Despite of their importance relatively little direct information is available on ionic secretion from epithelial monolayers and co-secretion of mucus. In this study we adapted ion-selective electrodes and a novel mathematical approach to deconvolution to gain insight in how Cl^-, K⁺ and Ca²⁺ are co-secreted with mucins from a model cell line. In spite of many decades of intense basic and clinical research the ultimate failure rate of cancer chemotherapy is still high. This is mainly due to poor selectivity of the available drugs, and inherent or acquired multidrug resistance (MDR) of cancer cells. Significant progress is being made in solving the former problem but new experimental approaches are required to successfully address clinical MDR. We have used carbon fiber based microvoltammetry to monitor variations in extracellular concentration of doxorubicin (DOX), a clinically important cancer drug at sparse monolayers and at single cancer cells. These experiments are complemented with the assessment of uptake dependent efflux by employing a Diffusional Microburet (DMB) to deliver DOX into single cancer cells under a confocal fluorescence microscope. To make such cellular transport studies more comprehensive and controlled, a BioMEMS platform has also been devised and tested. Results from these studies have shed new light on drug transport at cancer cells and its contribution to MDR.

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