Single-use, Multichannel Microfluidic Chips for Capillar

微流控技术的发展及其在生物医学检测中的应用，有可能提高许多广泛使用的技术的质量和产量。特别是，在硅胶毛细管中，基于微芯片的毛细管电泳比毛细管电泳法分析速度更快，试剂消耗更少，更易于使用。然而，更常用的玻璃微芯片制造成本较高，而且可能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。 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。特别是，我们计划在明年探索多种自由区CE免疫分析的可能性。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。 在美国国家标准与技术研究院和美国国立卫生研究院之间的这个跨部门项目中，我们的重点一直是开发一种强大的、光纤耦合的激光诱导荧光系统。它同时提供光谱色散和空间选择性，允许在热塑性微流控设备中常规检测亚纳摩尔浓度的荧光探针。总而言之，这一跨机构合作使该系统有别于其他微通道电泳系统的总体主要特点包括：廉价的一次性热塑性微流控设备；独特和优化的聚合物筛选基质；为热塑性设备量身定做的优化的表面活化和钝化涂层；坚固耐用的多路荧光激发和发射光学系统；小型化、尖端组件；低功耗；以及对专业操作环境的无要求。设计应允许方便地过渡到可现场部署的仪器，同时使用经济的消耗性部件，并保持与现有实验室仪器的兼容性，用于流体处理和分配。

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.