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High-throughput microfluidic chip with silica gel‐C18 channels for cyclotide separation

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Abstract

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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Acknowledgements

Thanks to the Turkish Scientific and Technological Council (TÜBİTAK) for their support under the grant numbers of 119N608, 20AG003 and 20AG031. Thanks to the Iranian Ministry of Science Research and Technology (MSRT), University of Tabriz, and Azarbaijan Shahid Madani University for their support under the grant number of IRTU-99-24-800. Thanks to the Scientific Research Projects (BAP- the priority areas project (ONAP) for their support under the grant number of TOA-2022-2307 of Eskisehir Osmangazi University.

Funding

- Turkish Scientific and Technological Council (TÜBİTAK) under the grant number of 119N608, and TUBİTAK 1004- Regenerative and Restorative Medicine Research and Applications) under the grant numbers of 20AG003 and 20AG031, and Scientific Research Projects (BAP- the priority areas project (ONAP) under the grant number of TOA-2022-2307 of Eskisehir Osmangazi University.

- Iranian Ministry of Science Research and Technology (MSRT) under the grant number of IRTU-99-24-800.

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Authors and Affiliations

  1. Cellular Therapy and Stem Cell Production Application and Research Center (ESTEM), Eskişehir Osmangazi University, Eskişehir, Turkey

    Aliakbar Ebrahimi, Hamed Ghorbanpoor & Huseyin Avci

  2. Department of Biomedical Engineering, Ankara Yildirim Beyazit University, Ankara, Turkey

    Aliakbar Ebrahimi, Reza Didarian & Fatma Dogan Guzel

  3. Department of Metallurgical and Materials Engineering, Eskişehir Osmangazi University, Eskişehir, Turkey

    Reza Didarian & Huseyin Avci

  4. Department of Biomedical Engineering, Eskişehir Osmangazi University, Eskişehir, Turkey

    Hamed Ghorbanpoor

  5. Department of Chemistry, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran

    Hossein Hashempour

  6. Translational Medicine Research and Clinical Center (TATUM), Eskişehir Osmangazi University, Eskişehir, Turkey

    Huseyin Avci

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  1. Aliakbar Ebrahimi
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AE: investigation, conceptualization, methodology, formal analysis, writing original draft — review and editing. RD: formal analysis, data curation, writing — original draft. HG: software, writing original draft — review and editing. FDG: project administration, conceptualization, funding acquisition, writing — review and editing. HH: design the project, project administration, methodology, formal analysis, writing — review and editing. HA: design the project, supervision, conceptualization, funding acquisition, project administration, writing — review and editing.

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Ebrahimi, A., Didarian, R., Ghorbanpoor, H. et al. High-throughput microfluidic chip with silica gel‐C18 channels for cyclotide separation. Anal Bioanal Chem 415, 6873–6883 (2023). https://doi.org/10.1007/s00216-023-04966-3

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