Glucose and lactate biosensors for scanning electrochemical microscopy imaging of single live cells.

We have developed glucose and lactate ultramicroelectrode (UME) biosensors based on glucose oxidase and lactate oxidase (with enzymes immobilized onto Pt UMEs by either electropolymerization or casting) for scanning electrochemical microscopy (SECM) and have determined their sensitivity to glucose and lactate, respectively. The results of our evaluations reveal different advantages for sensors constructed by each method: improved sensitivity and shorter manufacturing time for hand-casting, and increased reproducibility for electropolymerization. We have acquired amperometric approach curves (ACs) for each type of manufactured biosensor UME, and these ACs can be used as a means of positioning the UME above a substrate at a known distance. We have used the glucose biosensor UMEs to record profiles of glucose uptake above individual fibroblasts. Likewise, we have employed the lactate biosensor UMEs for recording the lactate production above single cancer cells with the SECM. We also show that oxygen respiration profiles for single cancer cells do not mimic cell topography, but are rather more convoluted, with a higher respiration activity observed at the points where the cell touches the Petri dish. These UME biosensors, along with the application of others already described in the literature, could prove to be powerful tools for mapping metabolic analytes, such as glucose, lactate, and oxygen, in single cancer cells.

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.