Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel.
Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel.
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DOI：
10.1039/b416161k
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发表时间：
2005-02
期刊：
影响因子：
6.1
通讯作者：
S. Stavis;J. Edel;K. Samiee;H. Craighead
中科院分区：
文献类型：
--
作者：
S. Stavis;J. Edel;K. Samiee;H. Craighead
建立了一种用于检测和分析溶液中单分子的微流控光学系统。亚微米尺寸的流体通道被用来分离、检测和识别与有机荧光团共轭的单个量子点。这些通道是在500 nm平方截面的熔融二氧化硅中制备的。得到的约500 aL的焦体积减少了荧光背景，增加了单分子检测的信噪比。这些通道还可以快速检测穿过焦点体积的99%的量子点和有机荧光团。共轭物以2.3 kV cm（-1）的电场驱动通过通道，用单个波长为476 nm的激光激发，并用共聚焦显微镜检测。在光谱的绿色（500-590 nm）和红色（610-680 nm）区域同时采集荧光发射。由于量子点的发射光谱窄且对称，使信号抑制最小化。为了证明单分子结合的高效多色检测和表征，将Qdot 655链亲和素偶联物与Alexa Fluor 488分子结合并单独检测。光子计数直方图分析用于量化一致性检测和结合程度。荧光相关光谱法测定了结合和未结合物质的迁移率。亚微米尺寸的流体通道和量子点作为荧光标记的结合，实现了高效、快速的多路单分子检测和分析。
A microfluidic and optical system was created for the detection and analysis of single molecules in solution. Fluidic channels with submicrometer dimensions were used to isolate, detect and identify individual quantum dots conjugated with organic fluorophores. The channels were fabricated in fused silica with a 500 nm square cross section. The resulting focal volume of approximately 500 aL reduced fluorescent background and increased the signal to noise ratio of single molecule detection. The channels also enabled the rapid detection of 99% of quantum dots and organic fluorophores traversing the focal volume. Conjugates were driven through the channels electrokinetically at 2.3 kV cm(-1), excited with a single 476 nm wavelength laser and detected with a confocal microscope. Fluorescence emission was collected simultaneously from green (500-590 nm) and red (610-680 nm) regions of the spectrum. Signal rejection was minimized by the narrow and symmetric emission spectra of the quantum dots. To demonstrate efficient multicolor detection and characterization of single molecule binding, Qdot 655 Streptavidin Conjugates were bound to Alexa Fluor 488 molecules and individually detected. Photon counting histogram analysis was used to quantify coincident detection and degree of binding. Fluorescence correlation spectroscopy was used to measure the mobility of bound and unbound species. The union of fluidic channels with submicrometer dimensions and quantum dots as fluorescent labels resulted in efficient and rapid multiplexed single molecule detection and analysis.