Ultrafast microfluidic mixers for investigating early folding


Ultrafast microfluidic mixers for investigating early folding
Session Topic: Microfluidic Control
Presentation Type: Oral
The function of protein or nucleic acid strongly depends on its structure. Folding kinetic
investigation helps to reveal the mechanism of how the molecule folds from the random
coil into the final functional structure, which has highly been focused in chemical and
biological science. Biomacromolecular folding usually occurs within a time scale of
microseconds in their early stage[1-3]. Conventional stopped-flow technique is widely
used for investigating the kinetics of molecular reaction. However, the temporal
resolution of approximately 1 ms of the stopped-flow method limits its applications to
characterize the folding kinetics of microsecond or even sub-microsecond levels. In this
presentation, we proposed several ultrafast mixing approaches with microfluidic chip
for interrogating the early folding kinetics of nucleic acids and proteins. For example,
we demonstrated a “crossed microfluidic beam” device as shown in Fig.1 capable of
completely mixing solutions within 0.6 µs, enabling for the first time the observation of
cytochrome c folding beyond its folding speed limit (~1 µs). This novel approach led to
the discovery of a complex initial folding process hidden in the “burst” phase previously
reported. Instead of a single exponential process, sequential events of non-exponential
and exponential processes were discovered as shown in Fig.2.
[1] Editorial, Science 309 (2005), 78-102.
[2] Ying Li, Chao Liu, Xiaojun Feng, Youzhi Xu, Bi-Feng Liu, Anal. Chem. 86 (2014)
[3] Ying Li, Youzhi Xu, Xiaojun Feng, and Bi-Feng Liu, Anal. Chem. 84 (2012)
Figure 1 | Fabrication of the microfluidic mixer. (a) Design of the arrow-shaped
microfluidic mixer. (b) Schematic assembly of the micromixer with a custom-built
PMMA holder. (c) Size comparison of the microchip assembly with a US one-cent coin.
Figure 2 Investigating the folding kinetics of cyt c. (a) The folding kinetics of cyt c
monitored by fluorescence changes after pH jumps from 2.0 to 2.0, 4.0, 5.5 and 6.5. (b)
Four phases during the collapse of cyt c at pH 6.5. Phase III and IV were fitted with
single exponential functions. Error bars, relative standard deviations of 6 experiments.

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