Microfluidic Chips and Multicolor Detectors for Capillary Electrophoresis

用于毛细管电泳的微流控芯片和多色检测器

• 批准号: 8158001
• 负责人: Nicole Y Morgan
• 金额: $ 20.32万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158001
• 关键词: Capillary Electrophoresis; Microfluidics; detector

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.

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。 我们以前已经开发并证明了一种新的方案，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激光激发通过两个柱面透镜传播成准直线，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。在每个激发点处，球透镜和光纤组合位于微通道下方。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。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。我们还致力于优化在不同聚合物基板上图案化和键合微芯片的配方，例如临床质量的PMMA、聚碳酸酯和PDMS。去年，我们开始在器件键合之前使用紫外线臭氧活化步骤，以代替之前使用的溶剂辅助工艺。这种调整导致显著更高的器件产率以及沟道横截面的更大再现性。今年，我们开始使用一种键合方法，该方法在键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的产量和键合强度，从而能够使用更具侵略性的化学物质来处理通道壁，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壁，这大大减少了标记肽和通道壁之间的相互作用，但随着时间的推移，涂层会发生一些降解。 今年，我们还继续优化分离的缓冲条件。与玻璃微芯片相反，塑料微芯片中分离的最重要的附加约束是保持整体通道电导率低，因为基板的较低热导率可以在比玻璃器件低得多的耗散功率密度下引起焦耳加热的峰展宽。作为我们工作的结果，分析物峰宽减少了高达四十倍，现在在注射塞扩散加宽极限的三倍以内。 使用我们的实验室构建的设置，我们已经成功地分离了纳克级数量的几个荧光标记的神经肽在不到两分钟。此外，我们已经开始了旨在在电泳分离之前实施芯片上免疫捕获步骤的实验。 初步实验的结果，使用荧光显微镜，以验证成功的表面化学，和分离使用类似的缓冲液条件所需的释放的捕获神经肽，是有前途的。