Single-use, Multichannel Microfluidic Chips for Capillary Electrophoresis

基于微芯片的毛细管电泳可以产生更快的分析时间，更低的试剂消耗，更容易的多路复用，更容易使用比CE在硅胶毛细管。 然而，通常使用的玻璃微芯片制造昂贵，需要大量的制造设施，并且可能不适合交叉污染是一个问题并且需要一次性装置的应用。 相比之下，塑料或聚合物微流体芯片可以用相对最少的设备用压印或模制技术制造，并且用热压印或注射模制技术制造，每个芯片几美分。 然而，聚合物微芯片中的激光诱导荧光检测提出了一些独特的挑战。 因为塑料基底比独立的二氧化硅毛细管基本上更荧光，所以需要空间选择性检测来隔离源自通道内的荧光信号，以便实现期望的灵敏度。 在过去，这需要一个共焦系统，通过光学元件的机械扫描实现多通道的测量。 我们已经开发并展示了一种新的方案，从多个微流控通道的灵敏，空间选择性和光谱分辨的激光诱导荧光检测，并将此方案应用于10 Hz的五色法医DNA分析在聚合物微流控装置。 自由空间488 nm激光激发用两个柱面透镜扩展成准直线，然后使用直径等于微通道间距的球面平凸透镜阵列分裂成多个聚焦光斑。 在每个激发光斑处，球透镜和光纤位于微通道下方。 通过使用高折射率球透镜和被定位成从通道获得聚焦光的直径显著较小的光纤来实现空间选择性。 检测光学器件可以自由地放置在每个通道附近，对通道布局和设计的限制最小。 光纤的另一端形成一维阵列，并指向成像光谱仪的入口狭缝。 在一个八通道配置的标准DNA碱基对梯子的分析显示出可比的灵敏度与使用商业共聚焦显微镜的单通道的测量获得。 在单个聚合物通道中，荧光素的检测限约为10 pM。 原型仪器是强大的，多功能的，只包含固定的光学部件，并有可能比竞争技术更便宜地实现。 平行检测的经济性和空间选择性的重要性使得该方法通常可用于具有多个微通道的聚合物基底中的分离。 虽然这项技术已经使用短串联重复DNA分离进行了评估，但该仪器可以很容易地用于大多数多色，多通道CE分析。 今年，我们已经组装了一个重复的仪器来解决这个问题，并致力于将制造聚合物微芯片的技术转移到我们在NIH的设施。 我们还致力于优化在不同聚合物基底中图案化和键合微芯片的配方，例如临床质量PMMA，聚碳酸酯和PDMS用于我们的设备，并在最大限度地减少蛋白质粘附到微通道壁上的方法上做了一些初步工作。 在接下来的一年里，我们计划使用这些技术和该仪器进行多重自由区CE免疫测定。

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. This year, 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, and have done some preliminary work on methods for minimizing protein adhesion to the microchannel walls. In the next year, we plan to use these techniques and this instrument for multiplexed free-zone CE immunoassays.