Microchip-based capillary electrophoresis could yield faster analysis times with lower reagent consumption, easier multiplexing, and greater ease of use than CE in silica capillaries. However, the glass microchips commonly used are expensive to manufacture, requiring extensive fabrication facilities, and can be ill-suited to applications for which cross-contamination is an issue and single-use devices are desired. In contrast, plastic, or polymeric microfluidic chips can be manufactured with imprinting or molding techniques with relatively minimal equipment, and manufactured with hot-embossing or injection molding techniques for pennies per chip. However, laser-induced fluorescence detection in polymeric microchips presents some unique challenges. Because the plastic substrate is substantially more fluorescent than freestanding silica capillaries, spatially selective detection is required to isolate the fluorescent signal originating from within the channel in order to achieve the desired sensitivity. In the past, this has required a confocal system, with the measurement of multiple channels achieved by mechanical scanning of the optical elements. We have developed and demonstrated a new scheme for sensitive, spatially selective and spectrally resolved laser-induced fluorescence detection from multiple microfluidic channels, and applied this scheme to 10 Hz five-color forensic DNA analysis in a polymeric microfluidic device. Free-space 488 nm laser excitation is spread into a collimated line with two cylindrical lenses and then split into multiple focused spots using an array of spherical plano-convex lenses with diameters equal to the microchannel spacing. At each excitation spot, a ball lens and an optical fiber is positioned underneath the microchannel. The spatial selectivity is achieved by using a high refractive index ball lens and a substantially smaller-diameter optical fiber positioned to obtain focused light from the channel. The detection optics can be freely positioned near each channel, placing minimal constraints on channel layout and design. The other ends of the optical fibers are formed into a 1-D array and directed onto the entrance slit of an imaging spectrograph. Analysis of standard DNA base-pair ladders in an eight-channel configuration shows comparable sensitivity to that obtained with measurements of a single channel using a commercial confocal microscope. The limit of detection is approximately 10pM for fluorescein in a single polymeric channel. The prototype instrument is robust, versatile, contains only fixed optical parts, and has the potential to be more cheaply implemented than competing technologies. The economies of parallel detection and the importance of spatial selectivity make this method generally useful for separations in polymeric substrates with multiple microchannels. Although this technology has been evaluated using short-tandem repeat DNA separations, the instrument can easily be used for most multi-color, multi-channel CE analyses. We have assembled a duplicate instrument in order to address this problem, and worked to transfer the technology for fabricating the polymer microchips to our facilities at NIH. We have also worked to optimize recipes for patterning and bonding microchips in different polymer substrates, such as clinical quality PMMA, polycarbonate, and PDMS for our equipment. This past year, there has been significant progress on several fronts. First, the fabrication of the PMMA channels was optimized, using a UV-ozone activation step prior to device bonding in lieu of the solvent assisted process used previously. This adjustment led to substantially higher device yield as well as greater reproducibility in channel cross section. Second, we developed a technique for coating the walls of the channels with a methyl cellulose, which substantially reduces interactions between the labeled peptides and the channel walls. Third, we worked to optimize buffer conditions for the separations. The most significant advance in this regard was the addition of betaine, which increased charge screening without adding substantially to the electrical conductivity of the buffer. This is particularly important for separations in plastic microchips, in which the lower thermal conductivity of the substrate gives rise to peak broadening from Joule heating at substantially lower dissipated power densities than in glass devices. As a result of these changes, the analyte peak widths were reduced by up to a factor of forty, and are now within a factor of three of the limit given by diffusional broadening of the injection plug. Using this laboratory-built setup, we have successfully separated nanogram-level quantities of several fluorescently labeled neuropeptides in less than two minutes. In addition, we explored the separation and labeling properties of a number of commercially available amine-reactive fluorophores, with the goal of finding two compatible dyes for multicolor detection. Using our prior experience in building a two-color laser-induced fluorescence detector for a capillary flow cell, we altered the microchip detector to measure the fluorescence excited by two laser wavelengths, and performed some proof-of-principle experiments on two-color detection of labeled peptides. Finally, we built a circuit to allow for more reproducible voltage control in these highly resistive channels, and began work on optimizing the injection conditions. In the next year, the primary goals are to further optimize separation conditions, quantify the reproducibility of the measurements, and move to an on-chip immunocapture step electrophoretic separation. Preliminary experiments using immunopurification to remove free dye from the labeled peptides are promising in this regard. We also hope to further develop the two-color detection scheme in order to measure recombinant standards along with the neuropeptides in the samples. The ability to simultaneously detect several internal standards along with the analytes of interest holds great potential for reducing analysis time and mitigating the effects of run-to-run variations in a capillary electrophoresis system. In addition to the detector for the microchip separations, our group had previously built two two-color laser-induced fluorescence detectors for capillary flow cells;one has been in use in a capillary electrophoresis system for some time. This year, the second two-color detector was incorporated into a nanoflow HPLC system. This detector is optimized for a square capillary flow cell with a 50 micrometer internal diameter, and has excitation wavelengths at 660 and 780 nm to reduce the contribution from sample autofluorescence. Sensitivity and spatial selectivity are achieved through the use of three high numerical aperture collimating lenses and a pinhole for each channel. The HPLC system and detector are currently in the process of characterization.
