使用多色荧光检测液滴微流控光纤
Summary
Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.
Abstract
Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.
Introduction
液滴的微流体通过在大量悬浮在载体油1水性液滴的compartmentalizing实验提供用于高通量生物学的平台。液滴已被用于应用程序作为不同的单细胞分析2中 ，数字聚合酶链反应（PCR）3，和酶进化4。荧光测定法检测的液滴的微流体的标准模式下，作为其亮信号和快速响应时间与在千赫速率检测子纳升液滴体积兼容。许多应用需要荧光检测用于同时至少两种颜色。例如，我们的实验室通常进行的PCR活化使用一个检测信道为一个化验结果液滴排序的实验中，并使用二次背景染料，使测定阴性液滴可数5。
液滴微流体典型检测站BAsed的上表面荧光显微镜，并需要复杂的光操作计划，以从自由空间激光引入激励光成显微镜被聚焦在样品上。荧光从液滴射出后，所发射的荧光的光进行滤波，使得每个检测信道利用中心波长频带设为一光电倍增管（PMT）。落射荧光显微镜基于光学检测系统，由于其费用，复杂性提供了一个进入门槛，并要求维修。光纤提供的装置构造的简化和健壮检测方案，由于纤维可以手动插入微流体装置，除去基于镜面光路由的需要，并且允许光路用光纤连接器相连接。
在本文中，我们描述了一个紧凑的模块化方案的组装和验证，利用光纤的阵列进行多色荧光检测DA单光电探测器6。光纤被耦合到单独的激光和被插入垂直于一个L形的流动通道以规则的空间偏移。的荧光收集纤维取向平行于激励区域，并连接到一个单独的PMT。因为液滴穿过在不同的时间的激光束，由光电倍增管记录的数据显示了一个时间偏移，允许用户向液滴由每个不同的激光束激发后发出的荧光区分。这个时间偏移消除了需要发射的光分离成使用一系列分色镜和带通滤波器的分离的光电倍增管。以验证检测器的效能，我们定量液滴种群包封不同颜色和浓度的染料的荧光。该系统的灵敏度进行了研究为单色荧光检测，并示出了以检测与浓度液滴向下至0.1nM，一个200×灵敏度曝光的能力ovement相比，在文献7日报道最近的基于光纤的方法为。
Protocol
Representative Results
Discussion
光纤检测需要的光纤相对于流体通道的取向。因为我们的器件利用具有多层光刻制造导槽，以相对于彼此的掩模放置是非常重要的。如果光纤导槽太靠近流体通道，对于流体泄漏的可能性;如果导向通道位于太远或错位，由检测纤维收集的荧光信号可以显著减少。适当的对准可以通过设计对准标记，例如同心圆入口罩相片图案形成过程中共同定位来辅助。此外，光纤的手动插入装置是具有打破装置…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by DARPA grant number 84389.01.44908, an NSF CAREER award (DBI-1253293), an NIH exploratory/developmental research grant (CA195709), and NIH New Innovator Awards (HD080351, DP2-AR068129-01), and a New Directions grant from the UCSF resource allocation program.
Materials
| Photomasks | CadArt Servcies | ||
| 3" silicon wafers, P type, virgin test grade | University Wafers | 447 | |
| SU-8 3035 | Microchem | Y311074 | |
| SU-8 2050 | Microchem | Y111072 | |
| Sylgard 184 silicone elastomer kit | Krayden | 4019862 | |
| 1 ml syringes | BD | 309628 | |
| 10 ml syringes | BD | 309604 | |
| 27 gaugue needles | BD | 305109 | |
| PE 2 polyethylene tubing | Scientific Commodities, Inc. | B31695-PE/2 | |
| Novec 7500 | Fisher Scientific | 98-0212-2928-5 | Commonly knowns as HFE 7500 |
| Ionic Krytox Surfactant | Synthesis instructions in ref #10 | ||
| Dextran- conjugated cascade blue dye | Life Technologies | D-1976 | |
| Fluorescein sodium salt | Sigma | 28803 | |
| Quad bandpass filter | Semrock | FF01-446/510/581/703-25 | |
| PMT | Thorlabs | PMM02 | |
| Fiber port | Thorlabs | PAFA-X-4-A | |
| lens tube | Thorlabs | SM1L05 | |
| Patch cable with 200 um core / 225 um cladding optical fiber with one stripped end and one FC/PC connector | Thorlabs | Custom | |
| Patch cable with 105 um core / 125 um cladding optical fiber with one stripped end and one FC/PC connector | Thorlabs | Custom | |
| 125 um fiber stripping tool | Thorlabs | T08S13 | |
| 225 um fiber stripping tool | Thorlabs | T10S13 | |
| laser fiber adapter | OptoEngine | FC/PC Adapter | |
| 405 nm CW laser at 50 mW | OptoEngine | MDL-III-405 | Distributor for CNI lasers |
| 473 nm CW laser at 50 mW | OptoEngine | MLL-FN-473-50 |
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