A microfluidic platform for chemical stimulation and real time analysis of catecholamine secretion from neuroendocrine cells.

Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped "cell traps", each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion/repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.

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 Tyrode's 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.

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 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.

On-chip acidification rate measurements from single cardiac cells confined in sub-nanoliter volumes.

The metabolic activity of cells can be monitored by measuring the pH in the extracellular environment. Microfabrication and microfluidic technologies allow the sensor size and the extracellular volumes to be comparable to single cells. A glass substrate with thin film pH sensitive IrO( x ) electrodes was sealed to a replica-molded polydimethylsiloxane (PDMS) microfluidic network with integrated valves. The device, termed NanoPhysiometer, allows the trapping of single cardiac myocytes and the measurement of the pH in a detection volume of 0.36 nL. For wild-type (WT) single cardiac myocytes an acidification rate of 6.45 +/- 0.38 mpH/min was measured in comparison to 19.5 +/- 0.38 mpH/min for very long chain Acyl-CoA dehydrogenase (VLCAD) deficient mice in 0.8 mM of Ca(2+). VLCAD deficiency is a fatty acid oxidation disease leading to cardiomyopathy and arrhythmias. The acidification rate increased to 11.96 +/- 1.33 mpH/min for WT and to 32.0 +/- 4.64 mpH/min for VLCAD -/- in 1.8 mM of Ca(2+). The NanoPhysiometer concept can be extended to study ischemia/reperfusion injury or disorders of other biological systems to identify strategies for treatment and possible pharmacological targets.

Differential pH measurements of metabolic cellular activity in nl culture volumes using microfabricated iridium oxide electrodes.

In this paper we describe a new approach to measure pH differences in microfluidic devices and demonstrated acidification rate measurements in on-chip cell culture systems with nl wells. We use two miniaturized identical iridium oxide (IrOx) thin film electrodes (20 micromx400 microm), one as a quasi-reference electrode, the other as a sensing electrode, placed in two confluent compartments on chip. The IrOx electrodes were deposited onto microfabricated platinum (Pt) electrodes simultaneously using electrodeposition. Incorporating the electrodes into a microfluidic device allowed us to expose each electrode to a different solution with a pH difference of one pH unit maintaining a confluent connection between the electrodes. In this configuration, we obtained a reproducible voltage difference between the two IrOx thin film electrodes, which corresponds to the electrode sensitivities of -70 mV/pH at 22 degrees C. In order to measure the acidification rate of cells in nl cell culture volumes we placed one IrOx thin film electrode in the perfusion channel as a quasi-reference electrode and the other in the cell culture volume. We obtained an acidification rate of 0.19+/-0.02 pH/min for fibroblast cells using a stop flow protocol. These results show that we can use two identical miniaturized microfabricated IrOx electrodes to measure pH differences to monitor the metabolic activity of cell cultures on chip. Furthermore, our approach can also be applied in biosensor or bioanalytical applications.

Thin-film IrOx pH microelectrode for microfluidic-based microsystems.

Microsensors are valuable tools to monitor cell metabolism in cell culture volumes. The present research describes the fabrication and characterization of on-chip thin-film iridium oxide pH microsensors with dimensions of 20 microm x 20 microm and 20 microm x 40 microm suitable to be incorporated into nl volumes. IrOx thin films were formed on platinum microelectrodes by electrochemical deposition in galvanostatic mode. Anodically grown iridium oxide films showed a near super-Nernstian response with a slope of -77.6+/-2 mV/pH at 22 degrees C, and linear responses within the pH range of 4-11. Freshly deposited electrodes showed response times as low as 6s. Long-term studies showed a baseline drift of 2-3 mV/month, which could easily be compensated by calibration. This work demonstrated for the first time the use of planar IrOx pH microelectrodes to measure the acidification rate of CHO and fibroblast cells in an on chip cell culture volume of 25 nl with microfluidic control.