Silica microspheres functionalized with the iminodiacetic acid/copper(II) complex as a peroxidase mimic for use in metal
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ORIGINAL PAPER
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 Burcu Gökçal 1 & Çiğdem Kip 1 & Dilek Şahinbaş 2 & Eda Çelik 1,2 & Ali Tuncel 1 Received: 6 July 2019 / Accepted: 16 December 2019 # Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract 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. Orthophenylenediamine 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. Keywords Protein assay . Nanozyme . Peroxidase-like activity . Hydrogen peroxide . Green fluorescent protein . SiO2 . Adsorption . Fluoresence . Iminodiacetic acid . Metal chelate complex
Introduction The nanostructures carrying transition metals are important family of nanozymes, particularly utilized for determination of various biomolecules. Copper-based nanozymes are an important class of enzyme-mimicking materials, which have been widely utilized in different assays [1–17]. Copperincorporated SBA-15 type silica, Cu-containing gels and Cu(II)-doped nanostructures were evaluated as nanozymes for determination of glucose, hydrogen peroxide and uric acid, Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-019-4087-0) contains supplementary material, which is available to authorized users. * Ali Tuncel [email protected] 1
Chemical Engineering Department, Hacettepe University, 06800 Ankara, Turkey
2
Institute of Science, Bioengineering Division, Hacettepe University, 06800 Ankara, Turkey
respectively [1–3]. Cu(II)-based metal-organic framework with peroxidase-like activity was also used for sensitive detection of hydrogen peroxide [4]. A chemiluminescence nanoprobe based on copper nanoclusters was developed based on the peroxidase-like activity for sensitive cholesterol detection [5]. A micro-RNA assay was developed using peroxidase-like activity of planar intercalated copper (II) complex [6]. Protein and tumor cell detection were performed by using the peroxidase-like activity
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