Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, Wageningen, 6708 WE, The Netherlands.
Lab Chip. 2018 Dec 18;19(1):79-86. doi: 10.1039/c8lc01175c.
Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height and a width of several micrometers confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observe DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level.
单分子检测方案提供了强大的手段，可以克服复杂样品中固有的静态和动态异质性。然而，以高时间和高分辨率探测生物分子相互作用和反应仍然具有挑战性，这通常需要表面固定化实体。在这里，我们介绍了玻璃纳米流控器件，用于通过基于相机的荧光显微镜对自由扩散的单个生物分子进行高通量检测。高度为 200nm 且宽度为数微米的纳米通道限制了生物分子的运动。通过在平行纳米通道阵列中采用压力驱动的流动，并通过跟踪荧光标记的 DNA 寡核苷酸的运动，我们可以以高通量观察构象变化。在具有纳米通道 T 形连接的器件几何形状中，我们通过连续混合反应物并触发化学反应来驱动稳态非平衡条件。我们使用该器件来探测 DNA 发夹的构象平衡，并实时连续观察 DNA 合成。我们的平台为在单分子水平上研究反应动力学提供了一种简单而稳健的方法。
