Synthesis and application of nanozymes in immunoassays


In a modern laboratory diagnostics, the main method of quantitative analysis of biomarkers is enzyme-linked immunosorbent assay (ELISA). Enzymes such as horseradish peroxidase and alkaline phosphatase convert colorless substrates into colored products, which makes them effective labels in ELISA. Nanozymes are nanomaterials possessing enzyme-like activity, though mechanism of action of nanozymes and enzymes can be different. Nanozymes mimicking horseradish peroxidase (transition metal nanoparticles, carbon nanomaterials) offer some advantages in comparison with natural enzyme:

  • Better physical-chemical stability

  • Tunable activity

  • Cheaper synthesis


We develop methods of nanozyme synthesis, size tuning, and functionalization. The most interesting for us are Prussian blue nanoparticles, platinum nanoparticles, and iron oxide nanoparticles. Prussian blue is a blue pigment with a formula AFeIII[FeII(CN)6]·xH2O (where A is Na, K, NH4) or FeIII4[FeII(CN)6]3·xH2O. Prussian blue nanoparticles are usually prepared by the double precoursor method (e.g. co-precipitation of FeCl3+K4[Fe(CN)6]) or single precursor method (e.g. hydrothermal decomposition of K4[Fe(CN)6] or K3[Fe(CN)6]). Recently, prof. Arkady Karyakin's group discovered that Prussian blue nanoparticles prepared by the reduction of FeCl3/K3[Fe(CN)6] mixture have increased peroxidase-like activity (10.1021/jacs.8b05223). They called these nanoparticles 'artificial peroxidase'. We synthesized 'artificial peroxidase' nanoparticles ranging in size from 90 to 300 nm. Size of nanoparticles can be controled by the addition of chelating agents like citric or oxalic acids.


Prussian blue nanoparticles synthesized by citric-acid assisted method were used as catalytic labels in immunoassay of prostate specific antigen (PSA) and antibodies against tetanus toxoid. For this, gelatin A layer was formed around nanoparticles. Gelatin molecules were cross-linked with glutaraldehyde, then, anti-PSA antibodies or Streptococcal protein G were covalently attached to free aldehyde groups of the gelatin coat. Gelatin endows Prussian blue nanoparticles with excellent colloidal stability at different pH and high ionic strength. Antibodies provide specific recognition of target in the course of analysis.


In priciple, we demonstrated that Prussian blue nanozymes are prospective labels for colorimetric assays, however they do not outperform natural peroxidase yet. Their catalytic activity, long-term stability, and fucntionalization routes need to be improved. Our current reresearch aims:

  1. Synthesis of 'artificial peroxidase' nanoparticles smaller than 90 nm for application in lateral flow and immunofiltration (flow through) assay

  2. Large-scale synthesis of Prussian blue nanoparticles

  3. Enhancement of nanozyme performance by the optimization of substrate solution composition



Khramtsov, P.; Kropaneva, M.; Minin, A.; Bochkova, M.; Timganova, V.; Maximov, A.; Puzik, A.; Zamorina, S.; Rayev, M. Prussian Blue Nanozymes with Enhanced Catalytic Activity: Size Tuning and Application in ELISA-like Immunoassay. Nanomaterials 2022, 12, 1630.