Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis.
Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis.
复制标题
微流控碳黑聚二甲基硅氧烷装置，具有减少的紫外线背景荧光，可在单细胞分析中同时进行双色紫外线/可见激光诱导荧光检测。
作者：
Lukas Galla;Dominik Greif;J. Regtmeier;D. Anselmetti
在单细胞分析(SCA)中，正在研究的是不受总体平均或异质细胞群体影响的单个细胞特有的特性和不均匀的细胞反应。对于蛋白质组单细胞分析，超灵敏和可重复性的分离和检测技术是必不可少的。微流控器件与紫外激光诱导荧光(UV-LIF)检测相结合就是为了满足这些要求。在这里，我们报告了一种新的微流控芯片制造工艺，该工艺通过使用聚二甲基硅氧烷(PDMS)液滴和碳黑颜料(CBP)作为添加剂，将直接生产聚二甲基硅氧烷(PDMS)芯片与减少紫外光荧光背景(减少83%)相结合。CBP液滴被放置在检测点，而芯片的其余部分保持透明，确保对芯片的完全光学控制。我们系统地研究了在不同的紫外激光功率下，CBP和PDMS比值(从1：10到1：1000)的紫外背景荧光的关系。在CPP/PDMS比例为1：20时，可检测到100 NM色氨酸溶液(S/N = 3.5)，提供了86 NM(S/N = 3)的理论检出限。通过同时进行双色UV/VIS-LIF检测，我们成功地在30 S范围内实现了500 nm色氨酸(UV)和5 NM荧光素(VIS)混合物的电泳法分离。作为应用，我们还将双色LIF法应用于绿色荧光蛋白标记的单个果蝇(Sf9)昆虫细胞中蛋白质的分离。因此，只能在可见光谱范围内测量到一个单峰，该单峰可以与紫外线光谱中的一个单峰相关联。这标志着对标记蛋白γ-PKC的鉴定，并展望了未来对不止一种单一蛋白质的进一步可行鉴定。
In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensemble-averaging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N = 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N = 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFP-labeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein γ-PKC and envisions a further feasible identification of more than one single protein in the future.
使用金属蒸气 NeCu 激光器进行毛细管电泳中的紫外天然荧光检测。
影响因子：
7.4
作者：
Zhang,X;Sweedler,JV
通讯作者：
Sweedler,JV
影响因子：
7.4
作者：
Shadpour, Hamed;Hupert, Mateusz L.;Soper, Steven A.
通讯作者：
Soper, Steven A.