Enzyme-coated microelectrodes to monitor lactate production in a nanoliter microfluidic cell culture device.
Monitoring the degree of anaerobic respiration of cells in high density microscale culture systems is an enabling key technology and essential for cell-based biosensors. We have fabricated and incorporated miniature amperometric lactate sensing electrodes with working areas from 3 to 5×10(-2) mm2 into a microfluidic-based microscale cell culture system to measure the lactate production rate of fibroblasts in nanoliter volumes. Planar thin film platinum electrode arrays on glass substrates were spin coated with lactate oxidase and a protective Nafion layer. The lactate electrodes had a high enzymatic activity described by a Michaelis-Menten constant of 2.6±0.1 mM, a linear response in the range 0.01-2.5 mM and a sensitivity of 7.3×10(-2) mA/mM cm2. A replica-molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. We trapped fibroblasts in the cell culture volume and measured the lactate production rate using a stop-flow protocol. The average lactate production rate was 0.011±0.0049 mM/min. The lactate production was suppressed with the addition of 2-deoxy-D-glucose, which binds to hexokinase. The blocking of hexokinase prevents the generation of pyruvate, the intermittent substrate required for lactate production even in the presence of glucose.
Ges
Igor A
IA
Department of Biomedical Engineering, Vanderbilt University, 5824 Stevenson Center, VU Station B 351631, Nashville, TN 37235-1631, USA.
Baudenbacher
Franz
F
D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation*; D018929:Cell Culture Techniques / Q000295:instrumentation; D002460:Cell Line; D003217:Conductometry / Q000295:instrumentation*; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005347:Fibroblasts / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D008839:Microelectrodes; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D006899:Mixed Function Oxygenases / Q000737:chemistry*
D004800:Enzymes, Immobilized; D019344:Lactic Acid; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase