Matthew K. Hulvey

Detecting thiols in a microchip device using micromolded carbon ink electrodes modified with cobalt phthalocyanine

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.

Separation and detection of peroxynitrite using microchip electrophoresis with amperometric detection.

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 ONOO(-) is made difficult by its short half-life under physiological conditions (approximately 1 s). Here we report a method for the separation and detection of ONOO(-) from other electroactive species utilizing a microchip electrophoresis device incorporating an amperometric detection scheme. Microchip electrophoresis permits shorter separation times (approximately 25 s for ONOO(-)) and higher temporal resolution than conventional capillary electrophoresis (several minutes). This faster analysis allows ONOO(-) to be detected before substantial degradation occurs, and the increased temporal resolution permits more accurate tracking of dynamic changes in chemical systems.

Amperometric detection in microchip electrophoresis devices: effect of electrode material and alignment on analytical performance.

The fabrication and evaluation of different electrode materials and electrode alignments for microchip electrophoresis with electrochemical detection is described. The influences of electrode material, both metal and carbon‐based, on sensitivity and 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, and catechol (CAT) as test analytes, it was found that pyrolyzed photoresist electrodes with end‐channel alignment yielded the lowest LOD (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 CAT in comparison to end‐channel alignment.

In‐channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat

The combination of microchip electrophoresis 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 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 (as a buffer modifier) for the separation of nitrite (NO  2− ), glutathione, ascorbic acid, and tyrosine. A half‐wave potential shift of approximately negative 500 mV was observed for NO  2− and H2O2 standards in the in‐channel configuration compared to end‐channel. Higher separation efficiencies were observed for both NO  2− 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.

A microchip-based endothelium mimic utilizing open reservoirs for cell immobilization and integrated carbon ink microelectrodes for detection

This paper describes the fabrication and characterization of a microfluidic device that utilizes a reservoir-based approach for endothelial cell immobilization and integrated embedded carbon ink microelectrodes for the amperometric detection of extracellular nitric oxide (NO) release. The design utilizes a buffer channel to continuously introduce buffer or a plug of stimulant to the reservoir as well as a separate sampling channel that constantly withdraws buffer from the reservoir and over the microelectrode. A steel pin is used for both the fluidic connection to the sampling channel and to provide a quasi-reference electrode for the carbon ink microelectrode. Characterization of the device was performed using NO standards produced from a NONOate salt. Finally, NO release from a layer of immobilized endothelial cells was monitored and quantified using the system. This system holds promise as a means to electrochemically detect extracellular NO release from endothelial cells in either an array of reservoirs or concurrently with fluorescence-based intracellular NO measurements.

Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection

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′,7′-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

Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers

The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2(-)) in cell lysates. There was a 2.5- to 4-fold increase in NO2(-) production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2(-) inside a single unstimulated macrophage cell was estimated to be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions.

Fabrication and evaluation of a 3-dimensional microchip device where carbon microelectrodes individually address channels in the separate fluidic layers

A fabrication method that results in a 3-dimensional fluidic device containing poly(dimethylsiloxane) (PDMS) -embedded microelectrodes that individually address each layer is described. The two electrode-containing layers and the polycarbonate membrane are reversibly sealed together, eliminating the need for plasma oxidation during device assembly, while enabling simultaneous amperometric detection in membrane-separated fluidic channels. The electrodes were characterized using microchip-based flow analysis. It was found that PDMS-embedded electrodes have a limit of detection (400 nM for catechol) that is 5-fold lower than that reported for microchip-based flow analysis with similar electrodes in a hybrid PDMS-glass device. The selectivity of the carbon ink microelectrodes can be tuned by a simplified modification procedure; this was demonstrated by the selective detection of nitric oxide over possible interferents. Finally, the ability to monitor processes occurring in separate layers of a 3-dimensional device was shown by the simultaneous detection of catechol on either side of the polycarbonate membrane. The electrode response in each fluidic channel was found to be linear as a function of concentration and the transport between layers could be controlled by varying the linear velocities of each fluidic channel. The ability to fabricate and operate this type of 3-dimensional device will be useful for the development of cell-based in vivo mimics that involve the transport of molecular messengers and/or pharmaceuticals across layers of immobilized cells.