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: 7967891
• 负责人: Nicole Y Morgan
• 金额: $ 11.23万
• 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/7967891
• 关键词: Address; Amines; Base Pairing; Betaine; Biological Assay; Blood capillaries; Buffers; Caliber; Capillary Electrophoresis; Cells; Charge; Clinical; Color; Consumption; DNA; DNA analysis; Detection; Development; Devices; Dyes; Electric Conductivity; Elements; Equipment; Fluorescein; Fluoresceins; Fluorescence; Forensic Medicine; Glass; Goals; Heating; High Pressure Liquid Chromatography; Image; Immunoassay; Injection of therapeutic agent; Intention; Label; Laboratories; Lasers; Light; Measurement; Measures; Mechanics; Methods; Methylcellulose; Microchip Electrophoresis; Microfluidic Microchips; Microfluidics; Microscope; Molds; Neuropeptides; Optics; Ozone; Pattern; Peptides; Plastics; Polymers; Polymethyl Methacrylate; Positioning Attribute; Process; Property; Reagent; Recipe; Recombinants; Refractive Indices; Reproducibility; Research Personnel; Running; Sampling; Scanning; Scheme; Screening procedure; Short Tandem Repeat; Signal Transduction; Silicon Dioxide; Solvents; Spottings; System; Techniques; Technology; Technology Transfer; Thermal Conductivity; Time; United States National Institutes of Health; Variant; Width; Work; base; capillary; color detection; density; design; detector; experience; fluorophore; imprint; improved; insight; instrument; interest; lens; microchip; optical fiber; polycarbonate; prototype; research study; voltage

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. This past year, there has been significant progress on several fronts. First, the fabrication of the PMMA channels was optimized, 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. Second, we developed a technique for coating the walls of the channels with a methyl cellulose, which substantially reduces interactions between the labeled peptides and the channel walls. Third, we worked to optimize buffer conditions for the separations. The most significant advance in this regard was the addition of betaine, which increased charge screening without adding substantially to the electrical conductivity of the buffer. This is particularly important for separations in plastic microchips, in which the lower thermal conductivity of the substrate gives rise to peak broadening from Joule heating at substantially lower dissipated power densities than in glass devices. As a result of these changes, 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 this laboratory-built setup, we have successfully separated nanogram-level quantities of several fluorescently labeled neuropeptides in less than two minutes. In addition, we explored the separation and labeling properties of a number of commercially available amine-reactive fluorophores, with the goal of finding two compatible dyes for multicolor detection. Using our prior experience in building a two-color laser-induced fluorescence detector for a capillary flow cell, we altered the microchip detector to measure the fluorescence excited by two laser wavelengths, and performed some proof-of-principle experiments on two-color detection of labeled peptides. Finally, we built a circuit to allow for more reproducible voltage control in these highly resistive channels, and began work on optimizing the injection conditions. In the next year, the primary goals are to further optimize separation conditions, quantify the reproducibility of the measurements, and move to an on-chip immunocapture step electrophoretic separation. Preliminary experiments using immunopurification to remove free dye from the labeled peptides are promising in this regard. We also hope to further develop the two-color detection scheme in order to measure recombinant standards along with the neuropeptides in the samples. The ability to simultaneously detect several internal standards along with the analytes of interest holds great potential for reducing analysis time and mitigating the effects of run-to-run variations in a capillary electrophoresis system. In addition to the detector for the microchip separations, our group had previously built two two-color laser-induced fluorescence detectors for capillary flow cells; one has been in use in a capillary electrophoresis system for some time. This year, the second two-color detector was incorporated into a nanoflow HPLC system. This detector is optimized for a square capillary flow cell with a 50 micrometer internal diameter, and has excitation wavelengths at 660 and 780 nm to reduce the contribution from sample autofluorescence. Sensitivity and spatial selectivity are achieved through the use of three high numerical aperture collimating lenses and a pinhole for each channel. The HPLC system and detector are currently in the process of characterization.

与硅胶毛细管中的毛细管电泳相比，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、更容易的多重检测和更易于使用。然而，常用的玻璃微芯片制造成本昂贵，需要大量的制造设施，并且可能不适合存在交叉污染问题且需要一次性设备的应用。相比之下，塑料或聚合物微流体芯片可以通过压印或模制技术以相对最少的设备制造，并通过热压花或注射模制技术制造，每个芯片成本低廉。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。由于塑料基板比独立式二氧化硅毛细管荧光性更强，因此需要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。过去，这需要共焦系统，通过光学元件的机械扫描来实现多通道的测量。 我们开发并演示了一种新方案，用于从多个微流体通道进行灵敏、空间选择性和光谱分辨的激光诱导荧光检测，并将该方案应用于聚合物微流体装置中的 10 Hz 五色法医 DNA 分析。自由空间 488 nm 激光激发通过两个柱面透镜传播成一条准直线，然后使用直径等于微通道间距的球面平凸透镜阵列分成多个聚焦点。在每个激发点，球透镜和光纤位于微通道下方。空间选择性是通过使用高折射率球透镜和直径小得多的光纤来实现的，该光纤被定位以获得来自通道的聚焦光。检测光学器件可以自由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引导到成像光谱仪的入口狭缝上。对八通道配置中的标准 DNA 碱基对梯的分析显示出与使用商用共聚焦显微镜测量单通道所获得的灵敏度相当的灵敏度。单个聚合物通道中荧光素的检测限约为 10pM。该原型仪器坚固耐用、用途广泛，仅包含固定光学部件，并且有可能比竞争技术更便宜地实施。并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质的分离。 尽管该技术已使用短串联重复 DNA 分离进行了评估，但该仪器可轻松用于大多数多色、多通道 CE 分析。为了解决这个问题，我们组装了一个重复的仪器，并致力于将制造聚合物微芯片的技术转移到我们在 NIH 的设施中。我们还致力于优化在不同聚合物基材上图案化和粘合微芯片的配方，例如用于我们设备的临床质量 PMMA、聚碳酸酯和 PDMS。去年，在几个方面都取得了重大进展。 首先，优化了 PMMA 通道的制造，在器件键合之前使用紫外线臭氧激活步骤代替之前使用的溶剂辅助工艺。 这种调整导致器件产量显着提高，通道横截面的再现性也更高。 其次，我们开发了一种用甲基纤维素涂覆通道壁的技术，这大大减少了标记肽和通道壁之间的相互作用。 第三，我们致力于优化分离的缓冲条件。 这方面最显着的进步是添加了甜菜碱，它增加了电荷屏蔽，而不会显着增加缓冲液的电导率。 这对于塑料微芯片的分离尤其重要，其中基板的较低热导率导致焦耳加热导致峰展宽，且耗散功率密度比玻璃器件低得多。 这些变化的结果是，分析物峰宽减少了多达四十倍，现在处于进样塞扩散展宽限制的三倍以内。 使用这种实验室构建的装置，我们在不到两分钟的时间内成功分离了纳克级数量的几种荧光标记的神经肽。 此外，我们还探索了许多市售胺反应性荧光团的分离和标记特性，目的是找到两种用于多色检测的兼容染料。 利用我们之前为毛细管流通池构建双色激光诱导荧光检测器的经验，我们改变了微芯片检测器以测量由两个激光波长激发的荧光，并对标记肽的双色检测进行了一些原理验证实验。 最后，我们构建了一个电路，以便在这些高电阻通道中实现更可重复的电压控制，并开始优化注入条件。 明年的主要目标是进一步优化分离条件，量化测量的再现性，并转向芯片上免疫捕获步骤电泳分离。 在这方面，使用免疫纯化去除标记肽中游离染料的初步实验是有希望的。 我们还希望进一步开发双色检测方案，以便测量重组标准品以及样品中的神经肽。 同时检测多种内标以及感兴趣的分析物的能力对于减少分析时间和减轻毛细管电泳系统中运行间差异的影响具有巨大的潜力。 除了用于微芯片分离的检测器外，我们小组之前还构建了两个用于毛细管流通池的双色激光诱导荧光检测器；其中一种已经在毛细管电泳系统中使用了一段时间。 今年，第二个双色检测器被纳入纳流 HPLC 系统。 该检测器针对内径为 50 微米的方形毛细管流通池进行了优化，激发波长为 660 和 780 nm，以减少样品自发荧光的影响。 通过使用三个高数值孔径准直透镜和每个通道的针孔来实现灵敏度和空间选择性。 HPLC 系统和检测器目前正在进行表征过程。