Igor A. Ges

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.

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 Tyrodes 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 Tyrodes solution with 5 mM of glucose.

Electrochemical detection of catecholamine release using planar iridium oxide electrodes in nanoliter microfluidic cell culture volumes

Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20 μm×300 μm) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0-400 μM, with a sensitivity of 23.1±0.5 mA/M mm(2). The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50mM) containing Tyrodes solution at a flow rate of 1 nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ~52 μM. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells.

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α‐demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad‐spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.