Single-use, Multichannel Microfluidic Chips for Capillary Electrophoresis

基于微芯片的毛细管电泳法比毛细管电泳法分析速度快，试剂消耗少，更易于多路传输，更易于在硅胶毛细管中使用。然而，通常使用的玻璃微芯片制造成本高，需要广泛的制造设施，并且不适合交叉污染问题和需要一次性设备的应用。相比之下，塑料或聚合物微流控芯片可以用相对最少的设备用压印或成型技术制造，并用热压或注射成型技术制造，每个芯片只需几分钱。然而，聚合物微芯片中的激光诱导荧光检测面临着一些独特的挑战。由于塑料衬底比独立的二氧化硅毛细管具有更强的荧光，因此需要进行空间选择性检测来隔离源自通道内的荧光信号，以实现所需的灵敏度。在过去，这需要共焦系统，通过光学元件的机械扫描来实现对多个通道的测量。 我们开发和论证了一种从多个微流控通道进行灵敏、空间选择性和光谱分辨的激光诱导荧光检测的新方案，并将该方案应用于聚合物微流控装置中的10赫兹五色法医DNA分析。利用直径等于微通道间距的球面平凸透镜阵列，将自由空间的488 nm激光扩展成一条带有两个柱面透镜的准直线，然后分裂成多个聚焦光斑。在每个激发点，球透镜和光纤位于微通道的下方。通过使用高折射率球透镜和定位成从通道获得聚焦光的直径小得多的光纤来实现空间选择性。探测光学器件可以自由地放置在每个通道附近，对通道布局和设计施加最小的限制。光纤的另一端形成一维阵列，定向到成像光谱仪的入射狭缝上。对八通道配置的标准DNA碱基对梯形图的分析表明，与使用商用共聚焦显微镜对单通道进行测量所获得的灵敏度相当。在单个聚合物通道中，荧光素的检测下限约为10 pm。原型仪器坚固耐用，通用性强，只包含固定的光学部件，并且有可能比竞争对手的技术更便宜地实施。平行检测的经济性和空间选择性的重要性使该方法通常适用于具有多个微通道的聚合物基质的分离。 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颜色、多通道的CE分析。为了解决这个问题，我们已经组装了一个重复的仪器，并努力将制造聚合物微芯片的技术转移到我们在NIH的设施。我们还致力于在不同的聚合物衬底上优化微芯片的图案化和粘合配方，例如用于我们设备的临床质量PMMA、聚碳酸酯和PDMS。我们利用实验室自制的激光诱导荧光检测仪实现了游离染料和几种蛋白质的分离，但壁层与蛋白质之间的相互作用限制了最小峰宽。目前，我们的努力集中在优化器件制造的成品率，以及减少我们希望分析的神经肽与通道壁之间的相互作用。明年的目标是在微芯片电泳分离之前进入免疫捕获步骤，同时检测脑脊液中的多种神经肽。

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. We have achieved separation of free dye and several proteins with laser-induced fluorescence detection using our laboratory-built instrument, but the interaction between the wall and the proteins limits the minimum peak width. Currently, our efforts are focused on optimizing yield in the device fabrication, and reducing the interaction between the neuropeptides we wish to analyze and the channel walls. In the next year, the goal is to move to an immunocapture step before the microchip electrophoresis separation, for simultaneous detection of multiple neuropeptides in cerebrospinal fluid.