Integration of multiple components in polystyrene-based microfluidic devices part II: cellular analysis.

In Part II of this series describing the use of polystyrene (PS) devices for microfluidic-based cellular assays: various cellular types and detection strategies are employed to determine three fundamental assays often associated with cells. Specifically, using either integrated electrochemical sensing or optical measurements with a standard multi-well plate reader, cellular uptake, production, or release of important cellular analytes are determined on a PS-based device. One experiment involved the fluorescence measurement of nitric oxide (NO) produced within an endothelial cell line following stimulation with ATP. The result was a four-fold increase in NO production (as compared to a control), with this receptor-based mechanism of NO production verifying the maintenance of cell receptors following immobilization onto the PS substrate. The ability to monitor cellular uptake was also demonstrated by optical determination of Ca(2+) into endothelial cells following stimulation with the Ca(2+) ionophore A20317. The result was a significant increase (42%) in the calcium uptake in the presence of the ionophore, as compared to a control (17%) (p < 0.05). Finally, the release of catecholamines from a dopaminergic cell line (PC 12 cells) was electrochemically monitored, with the electrodes being embedded into the PS-based device. The PC 12 cells had better adherence on the PS devices, as compared to use of PDMS. Potassium-stimulation resulted in the release of 114 ± 11 µM catecholamines, a significant increase (p < 0.05) over the release from cells that had been exposed to an inhibitor (reserpine, 20 ± 2 µM of catecholamines). The ability to successfully measure multiple analytes, generated in different means from various cells under investigation, suggests that PS may be a useful material for microfluidic device fabrication, especially considering the enhanced cell adhesion to PS, its enhanced rigidity/amenability to automation, and its ability to enable a wider range of analytes to be investigated, even analytes with a high degree of hydrophobicity.

Integration of multiple components in polystyrene-based microfluidic devices part I: fabrication and characterization.

In Part I of a two-part series, we describe a simple and inexpensive approach to fabricate polystyrene devices that is based upon melting polystyrene (from either a Petri dish or powder form) against PDMS molds or around electrode materials. The ability to incorporate microchannels in polystyrene and integrate the resulting device with standard laboratory equipment such as an optical plate reader for analyte readout and pipets for fluid propulsion is first described. A simple approach for sample and reagent delivery to the device channels using a standard, multi-channel micropipette and a PDMS-based injection block is detailed. Integration of the microfluidic device with these off-chip functions (sample delivery and readout) enables high-throughput screens and analyses. An approach to fabricate polystyrene-based devices with embedded electrodes is also demonstrated, thereby enabling the integration of microchip electrophoresis with electrochemical detection through the use of a palladium electrode (for a decoupler) and carbon-fiber bundle (for detection). The device was sealed against a PDMS-based microchannel and used for the electrophoretic separation and amperometric detection of dopamine, epinephrine, catechol, and 3,4-dihydroxyphenylacetic acid. Finally, these devices were compared against PDMS-based microchips in terms of their optical transparency and absorption of an anti-platelet drug, clopidogrel. Part I of this series lays the foundation for Part II, where these devices were utilized for various on-chip cellular analysis.

Microfluidic transendothelial electrical resistance measurement device that enables blood flow and postgrowth experiments.

Transendothelial electronic resistance (TEER) measurements are performed across a cell layer immobilized on a microfluidic device that also enables the cell layer to interact with a flowing stream of red blood cells (RBCs). A bipolar pulsed square wave potential is applied across a monolayer of bovine pulmonary artery endothelial cells, and the resulting current response is measured and integrated. The overall impedance of the cell layer provides an indicator of cell layer integrity. After cell seeding on the device, a decrease in TEER signal from 22.3 ± 1.6 µC to 3.5 ± 0.4 µC (corresponding to a resistance of 40.9 ± 2.9 Ω·cm(2) to 259.1 ± 27.4 Ω·cm(2)) was observed after 8 h of cell growth. Intracellular nitric oxide (NO) production by the immobilized endothelial cells that had reached confluence was 34% higher than those cells that had not reached confluence, as indicated by the integrated TEER system. Importantly, this NO production by the confluent endothelium was stimulated by ATP released from RBCs flowing under the endothelial cells. In this construct, the described microfluidic device enables both a TEER-based evaluation of cell layer integrity and molecularly communicated interactions of these cells with a flowing stream of blood components.

Direct plate-reader measurement of nitric oxide released from hypoxic erythrocytes flowing through a microfluidic device.

The ability to perform a fluorescence-based quantitative determination of a biologically important analyte directly released from mammalian cells using a standard microtiter plate reader to measure wells integrated into a microfluidic device is reported. Specifically, the amount of nitric oxide (NO) released from flowing erythrocytes (ERYs) exposed to a hypoxic buffer is measured using a fluorescein-based probe. The ERYs are pumped through channels in one layer of the poly(dimethylsiloxane) (PDMS) device; as these cells release NO, it flows through a porous polycarbonate membrane to the probe. The device is then placed into a standard microtiter plate reader for measurement, with the entire calibration and analyte determination occurring simultaneously. Using this method, NO release from hypoxic ERYs was determined to be 6.9 +/- 1.8 microM, a significantly increased value in comparison to that from normoxic ERYs of 0.60 +/- 0.04 microM (p < 0.001, n = 4 rabbits). Furthermore, the reproducibility (reported as a %RSD) of measuring fluorescence standards was 3.5%. Detection limits, dynamic range, and optimal membrane pore diameters are also reported. This device enables the use of a standard high-throughput tool (the plate reader) to measure analytes in a microfluidic device, the ability to improve the quantitative determination of a relatively unstable molecule (NO), and the incorporation of a flow component and blood constituent into a system that can be combined with microtiter plate technology.