Parallel separations of oligonucleotides with optically gated sample introduction on multichannel microchips.

With the release of the human genome sequence, there has been increasing attention given to other genetic analyses, including the detection of genetic variations and fast sequencing of multiple samples for pharmacogenomics studies. Rapid injections of samples in multiplexed separation channels by optically gated sample introduction are shown here for DNA separation. Serial separations of four amino acids are shown in less than four seconds on a microchip with four multiplexed channels. Five short oligonucleotides have also been rapidly separated in 2% LPA with four channels using this technique. In addition, multiple unique samples have been simultaneously separated and five-base resolution has been demonstrated.

Rapid serial analysis of multiple oligonucleotide samples on a microchip using optically-gated injection.

Optically-gated injection of fluorescently-labeled DNA has been accomplished for the first time. Rapid, serial analysis of oligonucleotide ladders has been shown on a microchip using this injection technique. Separations of five- and six-component samples have been completed in 60 s or less with a capability to carry out serial injections of these samples every 15 s. The technique has been shown to have better than five base resolution for small oligonucleotides and excellent reproducibility in migration times (< or = 0.75% RSD). Currently, the limit of detection for the system is 0.23 microM. Additionally, multiple unique samples of DNA have been consecutively analyzed in a single separation lane using optical gating. Six consecutive injections of three different samples have been achieved with no sample carryover and a total analysis time of approximately 10 min. These results show the potential of optical gating as an alternative injection technique for high-throughput DNA applications, such as genotyping and monitoring dynamic processes.

Analysis of the stability of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde using high-performance liquid chromatography and mass spectrometry.

The stability of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) was investigated using a combination of high-performance liquid chromatography, solid-phase extraction, photodiode array spectrophotometric detection, and mass spectrometric (MS) characterization. The degradation of amino acid derivatives, generated using beta-mercaptoethanol as a nucleophile, was characterized under a variety of environmental influences, with a focus on understanding the degradation kinetics and identifying the degradation products. The predominant degradation product observed under most reaction conditions was the nonfluorescent lactam form of the originally fluorescent isoindole derivative. First, the time-dependent degradation of the isoindole derivative L-serine-NDA-beta-mercaptoethanol was found to follow pseudo-first order kinetics with a half-life of 2.0 min at pH 9.2 and room temperature. The isoindole derivative was observed to react further with methanol to form a more stable fluorescent methoxy-isoindole, shedding new light on the basis for enhanced stability of these derivatives in methanol. Tandem mass spectrometry (MS/MS) experiments were used to demonstrate unimolecular degradation of the protonated isoindole in the absence of solvent or atmosphere, suggesting an intramolecular reaction mechanism involving the hydroxyethylthio group. Finally, in photobleaching studies, NDA derivatives rapidly degraded into a variety of products within the first 2 min of photobleaching versus timed controls, with the predominant product being the lactam. These results suggest that the degradation pathway for NDA derivatives is similar to the previously reported pathway for o-phthalaldehyde derivatives and clearly identifies the reaction and degradation products under a variety of conditions.

Parallel analysis with optically gated sample introduction on a multichannel microchip.

As an alternative to the T-type injection on microchips, optically gated sample introduction previously has been demonstrated to provide fast, serial, and reproducible injections on a single-channel microchip. Here, the ability to perform high throughput, multichannel analysis with optically gated sample introduction is described using a voice coil actuator. The microchip is fixed on a stage, which moves back and forth via the voice coil actuator, scanning two laser beams across the channels on the microchip. For parallel analysis on a multichannel microchip, both the gating beam and the probe beam are scanned at 10 Hz to perform multiple injections and parallel detection. Simultaneous, fast separations of 4-choloro-7-nitrobenzofurazan (NBD)-labeled amino acids are demonstrated in multiple channels on a microchip. Serial separations of different samples in multiple channels are also reported. Optically gated sample introduction on multiple, parallel channels shows the potential for high-speed, high-throughput separations that are easily automated by using a single electronic shutter.

Dual fluorescence and electrochemical detection on an electrophoresis microchip.

Simultaneous amperometric and fluorescence detection in a microfabricated electrophoresis chip is reported. Detection limits of 448 nM and 1.52, 16, and 28 microM have been achieved for dopamine, catechol, NBD-arginine, and NBD-phenylalanine, respectively. These two orthogonal detection schemes allow analysis of a wider spectrum of compounds per separation, leading to higher throughput and enabling resolution of two neutral analytes, NBD-arginine and catechol. In addition, insight into the detection and separation mechanisms is realized. Differences in migration time and peak widths between the two detectors are compared, providing a better understanding of detector alignment. A common problem encountered in electrophoresis is run-to-run migration time irreproducibility for certain samples. This novel microchip dual detection system has been utilized to reduce this irreproducibility. An unknown sample is monitored with one detector while a standard (i.e., ladder) is added to the sample and monitored simultaneously with the other detector. This dual detection method is demonstrated to normalize unknown peak mobilities in a cerebral spinal fluid sample.