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Abstract
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Over the past two decades, microfluidic-based separations have been used for the purification, isolation, and separation of
biomolecules to overcome difficulties encountered by conventional chromatography-based methods including high cost,
long processing times, sample volumes, and low separation efficiency. Cyclotides, or cyclic peptides used by some plant
families as defense agents, have attracted the interest of scientists because of their biological activities varying from antimicrobial to anticancer properties. The separation process has a critical impact in terms of obtaining pure cyclotides for drug
development strategies. Here, for the first time, a mimic of the high-performance liquid chromatography (HPLC) on microfluidic chip strategy was used to separate the cyclotides. In this regard, silica gel-C18 was synthesized and characterized by
Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) and then filled inside
the microchannel to prepare an HPLC C18 column-like structure inside the microchannel. Cyclotide extract was obtained
from Viola ignobilis by a low voltage electric field extraction method and characterized by HPLC and matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF). The extract that contained vigno 1, 2, 3, 4, 5, and varv A cyclotides
was added to the microchannel where distilled water was used as a mobile phase with 1 µL/min flow rate and then samples
were collected in 2-min intervals until 10 min. Results show that cyclotides can be successfully separated from each other
and collected from the microchannel at different periods of time. These findings demonstrate that the use of microfluidic
channels has a high impact on the separation of cyclotides as a rapid, cost-effective, and simple method and the device can
find widespread applications in drug discovery research.
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