A microchip electrophoresis device with on-line microdialysis sampling and on-chip sample derivatization by naphthalene 2,3-dicarboxaldehyde/2-mercaptoethanol for amino acid and peptide analysis.

The integration of rapid on-chip sample derivatization employing naphthalene 2,3-dicarboxaldehyde and 2-mercaptoethanol (NDA/2ME) with an easily assembled microdialysis/microchip electrophoresis device was carried out. The microchip device consisted of a glass layer with etched microfluidic channels that was sealed with a layer of poly(dimethylsiloxane) (PDMS) via plasma oxidation. This simple sealing procedure alleviated the need for glass thermal bonding and allowed the device to be re-sealed in the event of blockages within the channels. The device was used for analysis of a mixture of amino acids and peptides derivatized on-chip with NDA/2ME for laser-induced fluorescence (LIF) detection. A 0.6 mM NDA/1.2 mM 2ME mixture was simply added into the buffer reservoir for dynamic on-column derivatization of sample mixtures introduced at a flow rate of 1.0 microl/min. Using this scheme, sample injection plugs were derivatized and separated simultaneously. Injections of ca. 12 fmol of 5 mM amino acid and peptide samples were conducted using the system. Finally, a three-component mixture of Arg, Gly-Pro, and Asp was sampled from a vial using microdialysis, derivatized, separated and detected with the system. The ultimate goal of this effort is the creation of a micro-total analysis system for high-temporal resolution monitoring of primary amines in biological systems.

Design and characterization of poly(dimethylsiloxane)-based valves for interfacing continuous-flow sampling to microchip electrophoresis.

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.

In-channel electrochemical detection for microchip capillary electrophoresis using an electrically isolated potentiostat.

A new electrode configuration for microchip capillary electrophoresis (CE) with electrochemical (EC) detection is described. This approach makes it possible to place the working electrode directly in the separation channel. The "in-channel" EC detection was accomplished without the use of a decoupler through the utilization of a specially designed, electrically isolated potentiostat. The effect of the working electrode position on the separation performance (in terms of plate height and peak skew) of poly(dimethylsiloxane)-based microchip CEEC devices was evaluated by comparing the more commonly used end-channel configuration with this new in-channel approach. Using catechol as the test analyte, it was found that in-channel EC detection decreased the total plate height by a factor of 4.6 and lowered the peak skew by a factor of 1.3. A similar trend was observed for the small, inorganic ion nitrite. Furthermore, a fluorescent and electrochemically active amino acid derivative was used to directly compare the separation performance of in-channel EC detection to that of a widely used laser-induced fluorescence (LIF) detection scheme. In this case, it was found that the plate height and peak skew for both detection schemes were essentially equal, and the separation performance of in-channel EC detection is comparable to LIF detection.

On-line coupling of microdialysis sampling with microchip-based capillary electrophoresis.

Microdialysis sampling is a technique that has been used for in vivo and in vitro monitoring of compounds of pharmaceutical, biomedical, and environmental interest. The coupling of a commercially available microdialysis probe to a microchip-based capillary electrophoresis (CE) system is described. A continuously flowing dialysate stream from a microdialysis probe was introduced into the microchip, and discrete injections were achieved using a valveless gating approach. The effect of the applied voltage and microdialysis flow rate on device performance was investigated. It was found that the peak area varied linearly with the applied voltage. Higher voltages led to lower peak response but faster separations. Perfusion flow rates of 0.8 and 1.0 microL/min were found to provide optimal performance. The on-line microdialysis/microchip CE system was used to monitor the hydrolysis of fluorescein mono-beta-d-galactopyranoside (FMG) by beta-d-galactosidase. A decrease of the FMG substrate with an increase in the fluorescein product was observed. The temporal resolution of the device, which is dependent on the CE separation time, was 30 s. To the best of our knowledge, this is the first reported coupling of a microdialysis sampling probe to a microchip capillary electrophoresis device.