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设计制作了用于单分子动力学实验的微流控混合器,该混合器用聚二甲基硅氧烷(PDMS)芯片和石英载玻片密封而成,具有低的荧光背景,广泛的生物相容性,结合激光共聚焦显微镜能够在非平衡态下进行单分子荧光探测.我们设计的压力控制系统和进样流路方便而稳定,保证了微流路中流形的长时间稳定,从而实现了样品流速和流量的精准控制.这些技术特点保证了单分子探测得到准确和高信噪比的结果.利用蛋白质的塌缩过程远快于混合过程的特点,采用荧光标记的金黄色葡萄球菌核酸酶作为指示物,分辨出蛋白质变性态的特征峰,并利用变性态的荧光共振能量传递效率随时间的变化表征出混合器在适合于单分子探测条件下的混合时间为150ms.
A microfluidic mixer designed for single molecule dynamics experiments was designed and fabricated using a polydimethylsiloxane (PDMS) chip sealed with a quartz glass slide with low fluorescence background, a wide range of biological phases Capacitive, combined with confocal laser scanning microscopy in non-equilibrium single-molecule fluorescence detection.We designed the pressure control system and the injection flow path convenient and stable, to ensure that the microfluidic flow manifold long time stability, in order to achieve Accurate control of sample flow rate and flow rate.Their technical features ensure accurate and high signal-to-noise ratio results for single molecule detection.The use of protein collapsing process is much faster than the mixing process, using fluorescently labeled Staphylococcus aureus nuclease As an indicator, the characteristic peaks of protein denaturation were identified and the mixing time of the mixer under single molecule detection conditions was characterized by the change of the energy transfer efficiency of fluorescence resonance denaturation over time as 150 ms.