基于液滴微流控技术的高通量纤维素酶筛选系统。
A high-throughput cellulase screening system based on droplet microfluidics.
机构信息
Department of Physics, School of Engineering and Applied Sciences, Harvard University , Cambridge, Massachusetts 02138, USA.
出版信息
Biomicrofluidics. 2014 Jul 2;8(4):041102. doi: 10.1063/1.4886771. eCollection 2014 Jul.
Abstract
A new ultra-high-throughput screening assay for the detection of cellulase activity was developed based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, we have achieved up to 300-fold enrichments of the active population of cells and greater than 90% purity after just one sorting round. In addition, we proved that we can sort the cellulase-expressing cells from mixtures containing less than 1% active cells.
摘要
一种新的基于微流控分选的超高通量筛选检测纤维素酶活性的方法被开发出来。纤维素酶活性是通过一系列偶联酶检测的，这些酶会导致荧光产物的形成，而这种荧光产物可以在芯片上检测到。使用这种方法，我们在仅仅一轮分选后，就实现了活性细胞群体的 300 倍富集，纯度大于 90%。此外，我们证明了我们可以从含有不到 1%活性细胞的混合物中分拣出表达纤维素酶的细胞。
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2
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