In this paper, we report a new technique to pattern carbon microelectrodes for use in microfluidics. This technique, termed micromolding of carbon inks, uses poly(dimethylsiloxane)(PDMS) microchannels to define the size of the microelectrode. First, PDMS microchannels of the approximate dimensions desired for the microelectrode are made by soft lithography. The PDMS is then reversibly sealed to a substrate and the microchannels are filled with carbon ink. After a heating step the PDMS mold is removed, leaving a carbon microelectrode with a size slightly smaller than the original PDMS microchannel. The resulting microelectrode (27 microm wide and 6 microm in height) can be reversibly sealed to a PDMS-based flow channel. Fluorescence microscopy showed that no leakage occurred around the chip/electrode seal, even up to flow rates of 10 microL min(-1). The electrode was characterized by microchip-based flow injection analysis. Injections of catechol in Hank's Balanced Salt Solution (pH 7.4), showed a linear response from 2 mM to 10 microM (r(2)= 0.995), with a sensitivity of 56.5 pA microM(-1) and an estimated limit of detection of 2 microM (0.27 picomole, S/N=3). Reproducibility of the electrode response was shown by repeated injections (n= 10) of a 500 microM catechol solution, resulting in a RSD of 4.6%. Finally, selectivity was demonstrated by coating the microelectrode with Nafion, a perfluoronated cation exchange polymer. Dopamine exhibited a response at the modified microelectrode while ascorbic acid was rejected by the Nafion-coating. These electrodes provide inexpensive detectors for microfluidic applications while also being viable alternatives to use of other carbon microelectrode materials, such as carbon fibers. Furthermore, the manner in which the microelectrodes are produced will be of interest to researchers who do not have access to state of the art microfabrication facilities.

A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 microg ml(-1)) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 microm width x 6 microm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 microM) from being transported to the electrode surface while nitric oxide (NO, 10 microM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 microM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.

A novel method is described for measuring the deformability of red blood cells (RBCs) in tubing whose diameters approximate forces encountered in vivo. Here, RBCs from rabbits are loaded into a 50 cm section of 75 microm id microbore tubing and connected to a syringe pump. This section of tubing is then connected to a 15 cm section of 25 microm id tubing. As buffer is pumped through the flow system, the RBCs are evacuated from both sections of tubing. However, the inability of the RBCs to move freely through the 25 mirom id section of tubing results in a buildup of cells at the inlet of this portion of tubing. The continued force output by the syringe pump results in a deformation of the RBCs until all of the cells are eventually evacuated from the flow system. It was found that a measurement of the time required to reach half of the maximum pressure (1/2 P(max)) may be used as an indicator of the RBC deformability. For a given sample, a simple buffer results in less time to reach 1/2 P(max) (6.9 +/- 0.2 s) than deformable RBCs (21.6 +/- 0.8 s). To verify that the increased amount of time to reach 1/2 P(max) is indeed due to the RBCs, various hematocrits of an RBC sample were investigated and, as expected, it was found that a 12% RBC hematocrit had a higher 1/2 P(max) value (26.0 s +/- 2.2 s) when compared to a 7% hematocrit (19.1 +/- 0.3 s). In addition, RBCs chemically stiffened with glutaraldehyde were shown to be 25% less deformable than normal RBCs. Finally, a study was performed to examine the relationship between RBC deformability and ATP release and it was found that ATP release increased as a function of RBC deformability. This method greatly simplifies deformability measurements, employing only a syringe pump and microbore tubing, and may lead to a more complete understanding of the physiological significance of erythrocyte deformability.

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Tailoring the interfacial region of composite modified electrodes has been a topic of discussion for over a decade. This research examines the electrochemical effects of the formation of unique interfacial regions in surface-modified glass microsphere/polyvinylpyridine composite modified electrodes and surface-modified glass microsphere/polystyrene sulfonate composite modified electrodes. The surfaces of the glass microspheres are modified with different organic functional groups by binding organosilanes to the surfaces of the glass microspheres through a siloxane linkage. This research showed that surface-modified glass microspheres can alter the electrochemical flux through both surface-modified glass microsphere/polyvinylpyridine composites and surface-modified glass microsphere/polystyrene sulfonate composites of hydroquinone, Ru(bpy)3 2+, and ferricyanide. It was also shown that the polymer itself plays a crucial role in the formation and the properties of the interfacial region. The interfacial region was imaged using fluorescence microscopy, and the microscopy showed that a highly concentrating interfacial region is formed for all of the surface-modified glass microsphere/polymer composites studied regardless of whether there is an electrochemical effect. Further studies with smaller particles are necessary to obtain a large enough interfacial region to be useful for sensor development.