ABSTRACT
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.
Subject(s)
Biosensing Techniques/methods , Enzymes, Immobilized/chemistry , Glucose Oxidase/chemistry , Glucose/analysis , Lactic Acid/analysis , Mixed Function Oxygenases/chemistry , Animals , Cell Line, Tumor , Cells, Cultured , Electrochemistry/methods , Fibroblasts/chemistry , Fibroblasts/metabolism , Glucose/metabolism , Glucose Oxidase/metabolism , Humans , Lactic Acid/metabolism , Mice , Microelectrodes , Microscopy/methods , Mixed Function Oxygenases/metabolism , Oxidation-Reduction , Platinum/chemistryABSTRACT
Voltage-gated biological ion channels were simulated by insertion of the peptaibol antibiotic alamethicin into reconstituted phosphatidylcholine bilayer lipid membranes (BLMs). Scanning electrochemical microscopy (SECM) was utilized to probe initial BLM resistivity, the insertion of alamethicin pores, and mass transport across the membrane. Acquired SECM images show the spatial location of inserted pore bundles, the verification of voltage control over the pore conformational state (open/closed), and variations in passive mass transport corresponding to different topographical areas of the BLM. SECM images were also used to evaluate overall BLM integrity prior to insertion as well as transport (flux in open state) and leakage (flux in closed state) currents following insertion.