The development of microfluidic technology and its application to biomedical assays has the potential to improve the quality and throughput of many widely used techniques. In particular, microchip-based capillary electrophoresis could yield faster analysis times with lower reagent consumption and greater ease of use than CE in silica capillaries. However, the glass microchips more commonly used are expensive to manufacture, 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 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. ? ? 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. In particular, we plan to explore the possibilities for multiplexed free-zone CE immunoassays within the next year. 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.? ? Our focus in this interagency project between the National Institute of Standards and Technology and the NIH has been the development of a robust, fiber optically coupled, laser induced fluorescence system. It offers both spectral dispersion and spatial selectively allowing sub-nanomolar concentration of fluorescent probes to be routinely detected in thermoplastic microfluidic devices. In summary, the overall key features of this interagency collaboration that differentiate this system from other microchannel electrophoresis systems include: inexpensive single-use thermoplastic microfluidic devices; unique and optimized polymeric sieving matrices; optimized surface activation and passivation coating tailored for thermoplastic devices; robust, multiplexed fluorescence excitation and emission optics; miniaturized, state-of-the-art components; low electrical power consumption; and no requirement for a specialized operational environment. The design should allow a facile transition to a field-deployable instrument while using economical consumable components and maintaining compatibility with existing laboratory instrumentation for fluidic handling and dispensing.
