Microfluidic Chips and Multicolor Detectors 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。 我们已经开发并展示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。自由空间的488 nm激光通过两个柱面透镜传播到一条准直线上，然后利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。在每个激发点，球透镜和光纤组合被放置在微通道的下方。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。去年，我们开始在器件键合前使用紫外光臭氧激活步骤，取代了以前使用的溶剂辅助过程。这种调整导致了更高的器件成品率以及更大的沟道横截面重现性。今年，我们开始使用一种粘合方法，在粘合过程中使用牺牲材料来保护通道，我们希望这一变化将进一步提高器件成品率和粘合强度，从而能够使用更激进的化学方法来处理通道壁，以最大限度地减少非特异性相互作用。目前，我们正在用甲基纤维素处理通道的壁，这大大减少了标记多肽与通道壁之间的相互作用，但随着时间的推移，涂层会有一些降解。 今年，我们还继续优化分离的缓冲条件。与玻璃芯片相比，塑料微芯片中的分离最重要的附加限制是保持整体沟道电导率较低，因为基板的较低热导率可能会在比玻璃器件低得多的耗散功率密度下引起焦耳加热引起的峰值展宽。由于我们的工作，分析物的峰宽度减少了高达40倍，现在是注射塞扩散加宽所给出的极限的3倍以内。 使用我们实验室建立的装置，我们在不到两分钟的时间内成功地分离出了纳克级的几种荧光标记神经肽。此外，我们已经开始了旨在实施芯片上免疫捕获步骤的实验，然后再进行电泳分离。使用荧光显微镜验证表面化学的成功以及使用与释放捕获的神经肽所需的类似缓冲条件的分离的初步实验结果是有希望的。

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 previously 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 via 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 optical fiber combination 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 transferred 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. Last year, we started 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. This year, we started using a bonding method that employs a sacrificial material to protect the channels during the bonding process, a change that we hope will further improve device yield and bond strength, enabling the use of more aggressive chemistries for treating the channel walls to minimize non-specific interactions. Currently, we are treating the walls of the channels with methyl cellulose, which substantially reduces interactions between the labeled peptides and the channel walls, but there is some degradation of the coating over time. This year, we have also continued to optimize buffer conditions for the separations. The most important additional constraint for separations in plastic microchips, as opposed to glass ones, is to keep the overall channel conductivity low, as the lower thermal conductivity of the substrate can give rise to peak broadening from Joule heating at substantially lower dissipated power densities than in glass devices. As a result of our work, 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 our laboratory-built setup, we have successfully separated nanogram-level quantities of several fluorescently labeled neuropeptides in less than two minutes. In addition, we have begun experiments aimed at implementing an on-chip immunocapture step prior to electrophoretic separation. The results of preliminary experiments using fluorescent microscopy to verify the success of the surface chemistry, and separations using similar buffer conditions as needed for release of the capture neuropeptides, are promising.