Glutathione detection in human serum using gold nanoparticle decorated, monodisperse porous silica microspheres in the magnetic form.

A nanozyme for glutathione (GSH) detection in a broad concentration range was synthesized. GSH is usually detected up to an upper limit of 100 µM using current noble metal nanozymes due to the sharp decrease in the colorimetric response with the increasing GSH concentration. Strong inhibition of colorimetric reactions by GSH adsorbed onto noble metal based nanozymes in the form of non-porous, nanoscale particulate materials dispersed in an aqueous medium is the reason for the sharp decrease in the colorimetric response. In the present study, a new magnetic nanozyme synthesized by immobilization of Au nanoparticles (Au NPs) on magnetic, monodisperse porous silica microspheres (>5 µm) obtained by a "staged-shape templating sol-gel protocol" exhibited peroxidase-like activity up to a GSH concentration of 5000 µM. A more controlled linear decrease in the peroxidase-like activity with a lower slope with respect to that of similar nanozymes was observed with the increasing GSH concentration. The proposed design allowed the GSH detection in a broader concentration range depending on the adsorption of GSH onto the Au NPs immobilized on magnetic, monodisperse porous silica microspheres. A calibration plot allowing the detection of GSH in a broad concentration range up to 3300 µM was obtained using the magnetic nanozyme. The GSH concentration was also determined in human serum by elevating the upper detection range and adjusting the sensitivity of detection via controlling the nanozyme concentration.

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. Graphical abstractSchematic 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.

Aggregation-resistant nanozyme containing accessible magnetite nanoparticles immobilized in monodisperse-porous silica microspheres for colorimetric assay of human genomic DNA.

Bridging-induced aggregation of individual nanozyme particles by long-chain biomacromolecules causes loss of peroxidase mimetic activity for various nanozymes. When a common nanozyme (Fe3O4 nanoparticles) was used for colorimetric assay of a long-chain biomacromolecule, human genomic DNA (hgDNA, 14.000 kDa, containing 22 kilobases), peroxidase-like activity was absent owing to the irreversible aggregation of Fe3O4 nanoparticles in the presence of hgDNA. We synthesized an aggregation-resistant nanozyme containing Fe3O4 nanoparticles immobilized in 5.1 µm monodisperse-porous silica microspheres. Fe3O4 nanoparticles were immobilized in a form accessible to the substrate and large biomolecules in the solution within the monodispersed silica microspheres including both mesopores and macropores. An appreciable and stable spectrophotometric response was obtained, originating from the satisfactorily high peroxidase-like activity of the synthesized nanozyme. No aggregation was observed in the aqueous dispersion of the nanozyme in the presence of hgDNA. Large particle size, low particle number density, high surface area, and the presence of macropores were evaluated as factors contributing to the adsorption of hgDNA chains onto the synthesized nanozyme without interparticle bridge formation between the individual microspheres causing aggregation. Here, for the first time a colorimetric assay was developed based on the enhancement of peroxidase-mimetic activity of non-aggregated porous silica microspheres to determine hgDNA concentration up to 300 ng/µL. hgDNA could be also isolated with 47% yield and an equilibrium hgDNA adsorption of 19.000 ng/mg, using the same nanozyme. Hence, a material acting as a nanozyme for colorimetric determination of hgDNA was also evaluated as a magnetic, solid phase extraction sorbent for the first time.