CAJ | 학술논문

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150 ms.
설계제작료용우단분자동역학실험적미류공혼합기,해혼합기용취이갑기규양완(PDMS)심편화석영재파편밀봉이성,구유저적형광배경,엄범적생물상용성,결합격광공취초현미경능구재비평형태하진행단분자형광탐측.아문설계적압력공제계통화진양류로방편이은정,보증료미류로중류형적장시간은정,종이실현료양품류속화류량적정준공제.저사기술특점보증료단분자탐측득도준학화고신조비적결과.이용단백질적탑축과정원쾌우혼합과정적특점,채용형광표기적금황색포도구균핵산매작위지시물,분변출단백질변성태적특정봉,병이용변성태적형광공진능량전체효솔수시간적변화표정출혼합기재괄합우단분자탐측조건하적혼합시간위150 ms.
We designed and built a microfluidic mixer based on the principle of hydrodynamic focusing govemed by Navier-Stokes equation for single-molecule kinetics experiments.The mixer is a cast of poly(dimethylsiloxane) (PDMS) sealed with transparent fused-silica coverglass,which results in low fluorescence background and broad biological compatibility and this enables single-molecule fluorescence detection under nonequilibrium conditions.The pressure regulated sample delivery system is convenient for loading a sample and allows for precise and stable flow velocity control.The combination of microfluidic mixer and single-molecule fluorescence resonance energy transfer (smFRET) allows us to measure the time course of the distribution of the smFRET efficiency in protein folding.We used the fact that denatured protein collapses much faster than the mixing process to characterize the mixing time using donor and acceptor dyes labeled staphylococcal nuclease (SNase) as an smFRET efficiency indicator.By monitoring the smFRET efficiency of denatured SNase during the course of mixing,we determined that the mixing time was 150 ms under conditions suitable for single-molecuie detection.