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Abstract
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Microfluidic chip systems are emerging as essential tools in biotechnology research [1]. These devices offer a cost-effective,
scalable, and user-friendly platform for the development of novel separation techniques [2]. In this study, two distinct
microfluidic chip architectures were utilized to establish a separation method for cyclotides. Compact microfluidic layouts are
advantageous in terms of speed, efficiency, and affordability [3]. The first chip incorporates three inlets and leverages an
applied electric current to facilitate separation based on molecular charge or mobility, enabling controlled and targeted
processing. In this configuration, the plant extract is introduced via the central inlet, while reagents are supplied through the
remaining channels. The second chip employs a membrane-based approach with two inlets and a PET membrane separating
the channels, allowing for passive diffusion-based separation without the need for external energy input. This design offers a
low-cost and straightforward solution suitable for small-scale laboratory work. In this configuration, a dilute acid solution—
either HCl or phosphoric acid—was introduced through the secondary inlet. Flow rates of 0.5 µL/min, 1 µL/min, and 2 µL/min
were employed during testing. To assess the method's performance, both chip types were tested using plant extract from Viola
ignobilis, collected in Iran’s West Azerbaijan region. The focus of the separation was cyclotides, small, highly stable cyclic
peptides with promising biomedical applications [4].
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