Silica microspheres functionalized with the iminodiacetic acid/copper(II) complex as a peroxidase mimic for use in metal affinity chromatography-based colorimetric determination of histidine-tagged proteins


Monodisperse porous silica microspheres were functionalized with the iminodiacetic acid/copper(II) complex and then evaluated as a group-specific peroxidase-mimicking nanozyme for colorimetric determination of histidine-tagged (His-tagged) proteins. The green fluorescent protein (GFP) was selected as a typical His-tagged protein. The specificity for GFP and the peroxidase-like activity for the selected substrate were obtained by immobilizing the complex on the porous microspheres. The modified microspheres were also evaluated as a group specific immobilized metal affinity chromatography (IMAC) sorbent for the purification of GFP from Escherichia coli extract. The peroxidase-like activity of the microspheres was inhibited by the GFP adsorbed onto the microspheres due to the interaction of His-tagged protein with the immobilized Cu(II) complex. Ortho-phenylenediamine is used as a substrate for the enzyme mimic. The photometric response (measured at 416 nm) is linear in the 9.0–92 μg·mL−1 GFP concentration range in E. coli lysate. The limit of detection is 6.9 μg·mL−1.

Schematic representation of metal affinity chromatography-based colorimetric determination of histidine-tagged proteins using silica microspheres functionalized with iminodiacteic acid/copper (II) complex as a peroxidase mimic.

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This research was supported by Hacettepe University Scientific Research Projects Coordination Unit under contract numbered as FBA-2019-17337. Special thanks are extended to Turkish Academy of Sciences (TUBA) for the research support provided to Dr. Ali Tuncel as a full-member. Council of Higher Education of Turkey is acknowledged for providing the CoHE-100/2000 Doctoral Scholarship to Dilek Şahinbaş.

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Gökçal, B., Kip, Ç., Şahinbaş, D. et al. Silica microspheres functionalized with the iminodiacetic acid/copper(II) complex as a peroxidase mimic for use in metal affinity chromatography-based colorimetric determination of histidine-tagged proteins. Microchim Acta 187, 121 (2020).

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  • Protein assay
  • Nanozyme
  • Peroxidase-like activity
  • Hydrogen peroxide
  • Green fluorescent protein
  • SiO2
  • Adsorption
  • Fluoresence
  • Iminodiacetic acid
  • Metal chelate complex