Department of Physics, Chalmers University of Technology, Gothenburg, Sweden.
Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Lab Chip. 2017 Feb 28;17(5):830-841. doi: 10.1039/c6lc01302c.
We present a nanofluidic device for fluorescence-based detection and characterization of small lipid vesicles on a single particle basis. The device works like a nano flow cytometer where individual vesicles are visualized by fluorescence microscopy while passing through parallel nanochannels in a pressure-driven flow. An experiment requires less than 20 μl sample volume to quantify both the vesicle content and the fluorescence signals emitted by individual vesicles. We show that the device can be used to accurately count the number of fluorescent synthetic lipid vesicles down to a vesicle concentration of 170 fM. We also show that the size-distribution of the vesicles can be resolved from their fluorescence intensity distribution after calibration. We demonstrate the applicability of the assay in two different examples. In the first, we use the nanofluidic device to determine the particle concentration in a sample containing cell-derived extracellular vesicles labelled with a lipophilic dye. In the second, we demonstrate that dual-color detection can be used to probe peptide binding to synthetic lipid vesicles; we identify a positive membrane-curvature sensing behavior of an arginine enriched version of the Antennapedia homeodomain peptide penetratin. Altogether, these results illustrate the potential of this nanofluidic-based methodology for characterization and quantification of small biological vesicles and their interactors without ensemble averaging. The device is therefore likely to find use as a quantitative analytical tool in a variety of fields ranging from diagnostics to fundamental biology research. Moreover, our results have potential to facilitate further development of automated lab-on-a-chip devices for vesicle analysis.
我们提出了一种基于纳米流控的设备，用于在单个颗粒基础上基于荧光检测和表征小脂质体。该设备的工作原理类似于纳米流式细胞仪，其中单个囊泡在压力驱动的流动中通过平行纳米通道时通过荧光显微镜可视化。实验需要少于 20 μl 的样品体积即可定量测量囊泡含量和单个囊泡发射的荧光信号。我们表明，该设备可用于准确计数荧光合成脂质体的数量，直至囊泡浓度低至 170 fM。我们还表明，通过校准后可以从囊泡的荧光强度分布中分辨出它们的大小分布。我们在两个不同的示例中证明了该测定的适用性。在第一个示例中，我们使用纳米流控设备来确定含有用亲脂染料标记的细胞衍生细胞外囊泡的样品中的颗粒浓度。在第二个示例中，我们证明了双色检测可用于探测肽与合成脂质体的结合；我们确定富含精氨酸的 Antennapedia 同源域肽 penetratin 的正膜曲率感应行为。总之，这些结果说明了这种基于纳米流控的方法在不进行总体平均的情况下对小生物囊泡及其相互作用物进行表征和定量的潜力。因此，该设备有望成为从诊断到基础生物学研究等各种领域的定量分析工具。此外，我们的结果有可能促进用于囊泡分析的自动化片上实验室设备的进一步发展。
