基于微流控混合器的连续流动态同步辐射圆二色谱发展
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摘要
基于微流控混合器,采用连续流探测方法,在北京同步辐射装置真空紫外光谱实验站发展了毫秒动态圆二色谱探测方法。石英微流控混合器采用深度离子刻蚀技术加工,通道深度44.5μm。混合器采用蛇形通道实现溶液的快速混合。通过荧光倒置显微镜,在模拟真实实验条件的高粘度溶液中,观察蛇形通道内溶液混合的荧光图像,进行混合效率测试。500μL·min~(-1)流量下,目前可实现4.5~270μs的时间尺度探测。利用微流控混合器进行动态探测,同步辐射紫外光必须聚焦,但由于聚焦透镜波长色散引起的焦点位移,导致圆二色谱发生畸变。通过精确测试不同波长对应焦点的相对位置,然后在圆二色谱扫描中实现波长和焦点位置精确的反馈控制,获得准确的圆二色谱。利用所发展的方法,测试了去折叠状态下的细胞色素c恢复折叠的动态同步辐射圆二色谱,在4.5μs处折叠恢复54%。这种方法将为生物大分子折叠动力学研究提供新的探测手段。
The setup of dynamic synchrotron radiation dichroism spectroscopy(SRCD)is discussed by using continuous flow probe with microfluidic mixer,developed at 4B8 ultraviolet vacuum spectroscopy beamline at Beijing Synchrotron Radiation Facility.The quartz microfluidic mixer was fabricated with deep ion etching with channel depth down to 44.5microns.The mixer is designed on serpentine configuration to reach the effective mixing.The mixing efficiency was evaluated based on fluorescence image of the mixing solution under practical high viscous solution condition.Dynamic SRCD measurement with mixer was validated after implementing focus position feedback,since wavelength dispersion of the focusing lens results in the changes of focus spot position during wavelength scan.The time range covers from 4.5to 270 ms at present under 500(L·min~(-1) flow rate within the wide observation channel.The performance of the time-resolved SRCD is demonstrated in snapshoting the folding of cytochrome c back to 54% at 4.5millisecond after completely mixing.
The setup of dynamic synchrotron radiation dichroism spectroscopy(SRCD)is discussed by using continuous flow probe with microfluidic mixer,developed at 4B8 ultraviolet vacuum spectroscopy beamline at Beijing Synchrotron Radiation Facility.The quartz microfluidic mixer was fabricated with deep ion etching with channel depth down to 44.5microns.The mixer is designed on serpentine configuration to reach the effective mixing.The mixing efficiency was evaluated based on fluorescence image of the mixing solution under practical high viscous solution condition.Dynamic SRCD measurement with mixer was validated after implementing focus position feedback,since wavelength dispersion of the focusing lens results in the changes of focus spot position during wavelength scan.The time range covers from 4.5to 270 ms at present under 500(L·min~(-1) flow rate within the wide observation channel.The performance of the time-resolved SRCD is demonstrated in snapshoting the folding of cytochrome c back to 54% at 4.5millisecond after completely mixing.
引文
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[5]LIN Bing-cheng,QIN Jian-hua(林炳承,秦建华).Microfluidic Chip(微流控芯片实验室).Beijing:Science Press(北京:科学出版社),2006.
[6]Jiang Liguo,Zeng Yan,Sun Qiqi.Anal.Chem.,2015,87(11):5589.
[7]Benedetta Marmiroli,Gianluca Grenci,Fernando Cacho-Nerin,2009,9:2063.
[8]Wu Ling,Lisa J Lapidus.Chem.,2013,85(10):4920.
[9]Avinash S Kane,Armin Hoffmann,Peter Baumgartel.Anal.Chem.,2008,80(24):9534.
[10]Tao Ye,Huang Yan,Gao Zhenghua.J.Synchrotron Rad.,2009,16:857.
[11]Martin B Ulmschneider,Jakob P Ulmschneider,Nina Schiller.Nature Communications,2014,5:4863.
[12]Lees J G,Smith B R,Wien F.Analytical Biochemistry,2004,332:285.
[13]Jay R Winkler.Current Opinion in Chemical Biology,2004,8:169.