Sven E. Eklund

Modification of the Cytosensor™ microphysiometer to simultaneously measure extracellular acidification and oxygen consumption rates

Abstract The Cytosensor™ microphysiometer has been modified by incorporating several platinum electrodes into the plunger head. The electrodes have been used to measure simultaneously the change in extracellular oxygen consumption rates and extracellular acidification rates during addition of various non-physiologic agents: 2-deoxy- d -glucose, sodium fluoride, paraoxon-ethyl, and antimycin A. Measurement of two parameters, oxygen and acidification, enables investigation of the aerobic and anaerobic metabolic consequences of these agents. Nafion membranes cast onto the platinum electrodes were useful in minimizing adsorption of cell-related interferents.

Metabolic Discrimination of Select List Agents by Monitoring Cellular Responses in a Multianalyte Microphysiometer

Harnessing the potential of cells as complex biosensors promises the potential to create sensitive and selective detectors for discrimination of biodefense agents. Here we present toxin detection and suggest discrimination using cells in a multianalyte microphysiometer (MMP) that is capable of simultaneously measuring flux changes in four extracellular analytes (acidification rate, glucose uptake, oxygen uptake, and lactate production) in real-time. Differential short-term cellular responses were observed between botulinum neurotoxin A and ricin toxin with neuroblastoma cells, alamethicin and anthrax protective antigen with RAW macrophages, and cholera toxin, muscarine, 2,4-dinitro-phenol, and NaF with CHO cells. These results and the post exposure dynamics and metabolic recovery observed in each case suggest the usefulness of cell-based detectors to discriminate between specific analytes and classes of compounds in a complex matrix, and furthermore to make metabolic inferences on the cellular effects of the agents. This may be particularly valuable for classifying unknown toxins.

Real-Time Cell Dynamics With a Multianalyte Physiometer

A technique for simultaneously measuring changes in extracellular glucose, lactate, and oxygen concentrations in conjunction with acidification rates on a Cytosensor Microphysiometer is described. Platinum electrodes are inserted into the standard Cytosensor plunger head and modified with enzymes and biocompatible polymeric films. The lactate and glucose oxidase enzymes catalyze the reaction of lactate and glucose. An end product of these catalyses, H2O2, is measured amperometrically. Extracellular oxygen is also measured amperometrically, while the acidification rate is measured potentiometrically by the Cytosensor. Useful information is obtained during the Cytosensor stop-flow cycles, which produce increasing or decreasing peaks, owing to the production of lactic and carbonic acid and consumption of glucose and oxygen by the cells. Fabrication of the modified sensor head and deposition of the electrode films is detailed, and the operation of the technique is described and illustrated by the simultaneous measurement of all four analytes during the addition of 20 mM fluoride to mouse fibro blast cells.

Determination of oxide in fluoride salts using an yttria-stabilized-zirconia oxygen pump.

An improved method over a previous technique has been developed to determine the ppm oxide concentration of fluoride salts. The oxide is released as oxygen gas by the reaction of the test salt with potassium bromotetrafluoride at 450 °C. The molecular oxygen released is then passed through a zirconia oxygen pump which selectively removes the oxygen. The current response is recorded as a chronoamperogram, from which the ppm oxide content of the salt can be obtained. Oxygen recovery from an yttrium oxide standard was better than 99%. The precision of analysis of FLINAK was better than 13% for samples containing 110-170 ppm oxide. The LOD was 12 μg of oxygen, and the analytical range was 120 ppm to >20% for a 0.1-g sample.