This work describes the fabrication and evaluation of a poly(dimethyl)siloxane (PDMS)-based device that enables the discrete injection of a sample plug from a continuous-flow stream into a microchannel for subsequent analysis by electrophoresis. Devices were fabricated by aligning valving and flow channel layers followed by plasma sealing the combined layers onto a glass plate that contained fittings for the introduction of liquid sample and nitrogen gas. The design incorporates a reduced-volume pneumatic valve that actuates (on the order of hundreds of milliseconds) to allow analyte from a continuously flowing sampling channel to be injected into a separation channel for electrophoresis. The injector design was optimized to include a pushback channel to flush away stagnant sample associated with the injector dead volume. The effect of the valve actuation time, the pushback voltage, and the sampling stream flow rate on the performance of the device was characterized. Using the optimized design and an injection frequency of 0.64 Hz showed that the injection process is reproducible (RSD of 1.77%, n = 15). Concentration change experiments using fluorescein as the analyte showed that the device could achieve a lag time as small as 14 s. Finally, to demonstrate the potential uses of this device, the microchip was coupled to a microdialysis probe to monitor a concentration change and sample a fluorescein dye mixture.

Here, we show that an array of endothelial cells, addressable by an underlying microfluidic network of channels containing red blood cells, can be employed as an in vitro model of in vivo circulation to monitor cellular communication between different cell types in the drug discovery process.

Nitric oxide (NO) is a biologically important short-lived reactive species that has been shown to be involved in a large number of physiological processes. The production of NO is substantially increased in immune and other cell types through the upregulation of inducible nitric oxide synthase (iNOS) caused by exposure to stimulating agents such as lipopolysaccharide (LPS). NO production in cells is most frequently measured via fluorescence microscopy using diaminofluorescein-based probes. Capillary electrophoresis with laser-induced fluorescence detection has been used previously to separate and quantitate the fluorescence derivatives of NO from potential interferences in single neurons. In this paper, microchip electrophoresis (ME) coupled to laser-induced fluorescence (LIF) detection is evaluated as a method for measurement of the NO production by Jurkat cells under control and stimulating conditions. ME is ideal for such analyses due to its fast and efficient separations, low volume requirements, and ultimate compatibility with single cell chemical cytometry systems. In these studies, 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein diacetate (DAF-FM DA) was employed for the detection of NO, and 6-carboxyfluorescein diacetate (6-CFDA) was employed as an internal standard. Jurkat cells were stimulated using lipopolysaccharide (LPS) to produce NO, and bulk cell analysis was accomplished using ME-LIF. Stimulated cells exhibited an approximately 2.5-fold increase in intracellular NO production compared to the native cells. A NO standard prepared using diethylamine NONOate (DEA/NO) salt was used to construct a calibration curve for quantitation of NO in cell lysate. Using this calibration curve, the average intracellular NO concentrations for LPS-stimulated and native Jurkat cells were calculated to be 1.5 mM and 0.6 mM, respectively.

The combination of microchip electrophoresis (ME) with amperometric detection leads to a number of analytical challenges that are associated with isolating the detector from the high voltages used for the separation. While methods such as end-channel alignment and the use of decouplers have been employed, they have limitations. A less common method has been to utilize an electrically isolated potentiostat. This approach allows placement of the working electrode directly in the separation channel without using a decoupler. This paper explores the use of microchip electrophoresis and electrochemical detection (ME-EC) with an electrically isolated potentiostat for the separation and in-channel detection of several biologically important anions. The separation employed negative polarity voltages and tetradecyltrimethylammonium bromide (TTAB, as a buffer modifier) for the separation of nitrite (NO2-), glutathione (GSH), ascorbic acid (AA), and tyrosine (Tyr). A half-wave potential (E½) shift of approximately negative 500 mV was observed for NO2- and H2O2 standards in the in-channel configuration compared to end channel. Higher separation efficiencies were observed for both NO2- and H2O2 with the in-channel detection configuration. The limits of detection were approximately two-fold lower and the sensitivity was approximately two-fold higher for in-channel detection of nitrite when compared to end-channel. The application of this microfluidic device for the separation and detection of biomarkers related to oxidative stress is described.

Peroxynitrite (ONOO -) is a highly reactive species implicated in the pathology of several cardiovascular and neurodegenerative diseases. It is generated in vivo by the diffusion-limited reaction of nitric oxide (NO • ) and superoxide anion ( • O 2 -) and is known to be produced during periods of inflammation. Detection of ONOOis made difficult by its short half-life under physiological conditions (∼1 s). Here we report a method for the separation and detection of ONOOfrom other electroactive species utilizing a microchip electrophoresis device incorporating an amperometric detection scheme. Microchip electrophoresis permits shorter separation times (∼25 s for ONOO -) and higher temporal resolution than conventional capillary electrophoresis (several minutes). This faster analysis allows ONOOto be detected before substantial degradation occurs, and the increased temporal resolution permits more accurate tracking of dynamic changes in chemical systems.Oxidative stress and nitration are thought to be involved in the pathology of several cardiovascular diseases including stroke, myocardial infarction, hypertension, atherosclerosis, and chronic heart failure, as well as the neurodegenerative diseases multiple sclerosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. 1 Peroxynitrite (ONOO -) is a highly reactive, oxidizing species formed in vivo from the reaction of nitric oxide (NO • ) with the superoxide anion. The reaction between these two species occurs at a diffusion-limited rate (6 × 10 9 M -1 s -1 ) 2 that can supersede the scavenging of superoxide by superoxide dismutase (2 × 10 9 M -1 s -1 ). 1 In situations such as the proinflammatory state, peroxynitrite can be produced faster than it can be scavenged, resulting in oxidative insult.

The fabrication and evaluation of different electrode materials and electrode alignments for microchip electrophoresis with electrochemical (EC) detection is described. The influences of electrode material, both metal and carbon-based, on sensitivity and limits of detection (LOD) were examined. In addition, the effects of working electrode alignment on analytical performance (in terms of peak shape, resolution, sensitivity, and LOD) were directly compared. Using dopamine (DA), norepinephrine (NE), and catechol (CAT) as test analytes, it was found that pyrolyzed photoresist electrodes with end-channel alignment yielded the lowest limit of detection (35 nM for DA). In addition to being easier to implement, end-channel alignment also offered better analytical performance than off-channel alignment for the detection of all three analytes. In-channel electrode alignment resulted in a 3.6-fold reduction in peak skew and reduced peak tailing by a factor of 2.1 for catechol in comparison to end-channel alignment.

A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a twofold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.

This paper describes the fabrication and evaluation of a chemically modified carbon ink microelectrode to detect thiols of biological interest. The detection of thiols, such as homocysteine and cysteine, is necessary to monitor various disease states. The biological implications of these thiols generate the need for miniaturized detection systems that enable portable monitoring as well as quantitative results. In this work, we utilize a microchip device that incorporates a micromolded carbon ink electrode modified with cobalt phthalocyanine to detect thiols. Cobalt phthalocyanine (CoPC) is an electrocatalyst that lowers the potential needed for the oxidation of thiols. The CoPC/carbon ink composition was optimized for the micromolding method and the resulting microelectrode was characterized with microchip-based flow injection analysis. It was found that CoPC lowers the overpotential for thiols but, as compared to direct amperometric detection, a pulsed detection scheme was needed to constantly regenerate the electrocatalyst surface, leading to improved peak reproducibility and limits of detection. Using the pulsed method, cysteine exhibited a linear response between 10-250 microM (r(2) = 0.9991) with a limit of detection (S/N = 3) of 7.5 microM, while homocysteine exhibited a linear response between 10-500 microM (r(2) = 0.9967) with a limit of detection of 6.9 microM. Finally, to demonstrate the ability to measure thiols in a biological sample using a microchip device, the CoPC-modified microelectrode was utilized for the detection of cysteine in the presence of rabbit erythrocytes.