Enzyme electrodes to monitor glucose consumption of single cardiac myocytes in sub-nanoliter volumes.
Monitoring the metabolic activity of cells in automated culture systems is one of the key features of micro-total-analysis-systems. We have developed a microfluidic device that allows us to trap single cardiac myocytes (SCMs) in sub-nanoliter volumes and incorporate amperometric glucose-sensing electrodes with working areas of 0.002 mm(2) to measure the glucose consumption of SCM. The miniaturized planar glucose electrodes were fabricated by spin coating platinum electrodes on glass substrates with a glutaraldehyde/enzyme solution and a protective Nafion membrane. The glucose electrodes demonstrate a high enzymatic activity characterized by an apparent Michaelis-Menten constant of 7.52+/-0.18 mM and a sensitivity of approximately 33.8 and approximately 13.2 mA/Mcm(2) at glucose concentration from 0-6 to 6-20 mM in Tyrode's solution, respectively. The response time of the glucose electrodes was between 5 and 15s, and the sensitivity of the electrodes did not degrade over a period of 8 weeks. A replica molded polydimethylsiloxane microfluidic device with a sub-nanoliter sensing volume was sealed to the glass substrate and aligned with the glucose microelectrodes. SCM can be trapped in the sensing volume above the glucose electrodes to measure the glucose consumption over time. The average glucose consumption of SCM was 0.211+/-0.097 mM/min (n=7) in Tyrode's solution with 5 mM of glucose.
Ges
Igor A
IA
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235-1631, USA.
Baudenbacher
Franz
F
D015374:Biosensing Techniques / Q000295:instrumentation*; D018929:Cell Culture Techniques / Q000295:instrumentation; D002469:Cell Separation / Q000295:instrumentation*; D004563:Electrochemistry / Q000295:instrumentation*; D004566:Electrodes; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005947:Glucose / Q000378:metabolism*; D005949:Glucose Oxidase / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D032383:Myocytes, Cardiac / Q000502:physiology*; D036103:Nanotechnology / Q000295:instrumentation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005949:Glucose Oxidase; D005947:Glucose