Biopolym. Cell. 2024; 40(2):96-108.
Molecular and Cell Biotechnologies
Nanoparticles of prussian blue analogues as peroxidase mimetics for nanozyme – oxidase – based biosensors
1, 4Demkiv O. M., 1, 4Stasyuk N. Y., 2Grynchyshyn N. M., 3Klepach H. M., 1, 3Gonchar M. V.
  1. Institute of Cell Biology and Genetic Engineering, NAS of Ukraine
    148, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03143
  2. Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies of Lviv
    50, Pekarska Str., Lviv, Ukraine, 79010
  3. Drohobych Ivan Franko State Pedagogical University
    24, I. Franko Str., Drohobych, Ukraine, 82100
  4. Institute of Physical Chemistry, Polish Academy of Sciences
    44/52, Kasprzaka Str., Warsaw, Poland, 01‑224


Aim. This work aims to develop new H2O2-sensing elements and nanozyme-oxidase-based sensors for the detection of hydrogen peroxide, glucose, and galactose. Methods. Chemical synthesis. Assay of enzymatic activity. Scanning electron microscope. Results. Hexacyanoferrates of several transient metals were synthesized and tested for their nanozymes (HCF NZs) (certificial peroxidase) activity in solution. The best representative nanozymes (NZs) were used for the construction of H2O2-sensitive sensors and nanozyme-oxidase-based biosensors. The NZs-based sensors: nAuHCF/GE and nPtHCF/GE demonstrated higher sensitivity (7.5 and 9.4-fold) than the biosensor with natural peroxidase. The developed biosensors GOX/nPtHCF/GE and GaOx/nPtHCF/GE for the detection of glucose and galactose showed enhanced sensitivity: 900 and 540 A·M-1·m-2, respectively, broad linear range (0.02–0.2 mM). Along with \a broad linear range of detection these biosensors possess low limits of detection - 4.0 μM for glucose and 6.0 μM for galactose. Additionally, these biosensors exhibited improved stability when compared to the control. Conclusions. The novelty of the presented work is related to the synthesis of new peroxidase-like NZs and the evaluation of their functionality as the chemosensors on H2O2 and as the sensing components in the oxidase-based-biosensors for assay of glucose and galactose. The nPtHCF and nAuHCF NZs exhibit high sensitivity for the target analytes, a broad linear range, and satisfactory storage stability. The main advantages of the proposed nanozyme-oxidase-based biosensors are the simple architecture of the sensing layer and the ability to operate at low working electrode potentials.
Keywords: Prussian blue analogues, nanozymes, artificial peroxidase, glucose oxidase, galactose oxidase, amperometric biosensor


[1] Qi X, Tester RF. Fructose, galactose and glucose - In health and disease. Clin Nutr ESPEN. 2019; 33:18-28.
[2] Villena Gonzales W, Mobashsher AT, Abbosh A. The Progress of Glucose Monitoring-A Review of Invasive to Minimally and Non-Invasive Techniques, Devices and Sensors. Sensors (Basel). 2019; 19(4):800.
[3] Succoio M, Sacchettini R, Rossi A, Parenti G, Ruoppolo M. Galactosemia: Biochemistry, Molecular Genetics, Newborn Screening, and Treatment. Biomolecules. 2022; 12(7):968.
[4] Stasyuk N, Demkiv O, Gayda G, Zakalska O, Nogala W, Gonchar M. Amperometric biosensors based on alcohol oxidase and peroxidase-like nanozymes for ethanol determination. Mikrochim Acta. 2022; 189(12):474.
[5] Wijayanti SD, Tsvik L, Haltrich D. Recent Advances in Electrochemical Enzyme-Based Biosensors for Food and Beverage Analysis. Foods. 2023; 12(18):3355.
[6] Kanyong P, Krampa FD, Aniweh Y, Awandare GA. Enzyme-based amperometric galactose biosensors: a review. Mikrochim Acta. 2017; 184(10):3663-71.
[7] Liu Y, Yang L, Cui Y. Transdermal amperometric biosensors for continuous glucose monitoring in diabetes. Talanta. 2023; 253:124033.
[8] Attar F, Shahpar MG, Rasti B, Sharifi M, Saboury AA, Rezayat SM, Falahati M. Nanozymes with intrinsic peroxidase-like activities. J Mol Liq. 2019; 278:130-144.
[9] Komkova MA, Pasquarelli A, Andreev EA, Galushin AA, Karyakin AA. Prussian Blue modified boron-doped diamond interfaces for advanced H2O2 electrochemical sensors. Electrochim Acta. 2020; 339:135924.
[10] Puganova EA, Karyakin AA. New materials based on nanostructured Prussian blue for development of hydrogen peroxide sensors. Sens Actuators B Chem. 2005; 109:167-170.
[11] Stasyuk N, Smutok O, Demkiv O, Prokopiv T, Gayda G, Nisnevitch M, Gonchar M. Synthesis, Catalytic Properties and Application in Biosensorics of Nanozymes and Electronanocatalysts: A Review. Sensors (Basel). 2020; 20(16):4509.
[12] Qin L, Hu Y, Wei H. Nanozymes: Preparation and Characterization. In Nanotechnology for Electronics, Photonics, and Renewable Energy; Springer Science and Business Media LLC: Berlin/Heidelberg, Germany, 2020:pp. 79-101.
[13] Gayda GZ, Demkiv OM, Gurianov Y, Serkiz RY, Klepach HM, Gonchar MV, Nisnevitch M. "Green" Prussian Blue Analogues as Peroxidase Mimetics for Amperometric Sensing and Biosensing. Biosensors (Basel). 2021; 11(6):193.
[14] Demkiv O, Gayda G, Stasyuk N, Brahinetz O, Gonchar M, Nisnevitch M. Nanomaterials as Redox Mediators in Laccase-Based Amperometric Biosensors for Catechol Assay. Biosensors (Basel). 2022; 12(9):741.
[15] Violante-Mota F, Tellechea E, Moran JF, Sarath G, Arredondo-Peter R. Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants. Phytochemistry. 2010; 71(1):21-6.
[16] Maddinedi SB, Mandal BK. Peroxidase like activity of quinic acid stabilized copper oxide nanosheets. Austin J Anal Pharm Chem. 2014; 1,2:1008. ISSN:2381-8913.
[17] Bilalis P, Karagouni E, Toubanaki DK. Peroxidase-like activity of Fe3O4 nanoparticles and Fe3O4-graphene oxide nanohybrids: Effect of the amino- and carboxyl-surface modifications on H2O2 sensing. Appl Organomet Chem. 2022; 36(9):e6803.
[18] Pandey PC, Panday D, Pandey AK. Polyethylenimine mediated synthesis of copper-iron and nickel-iron hexacyanoferrate nanoparticles and their electroanalytical applications. J Electroanal Chem. 2016; 780:90-102.
[19] Daboss EV, Shcherbacheva EV, Karyakin AA. Simultaneous noninvasive monitoring of diabetes and hypoxia using core-shell nanozyme - oxidase enzyme biosensors. Sens Actuators B Chem. 2023; 380:133337.
[20] Figueiredo C, De Lacey AL, Pita M. Electrochemical studies of galactose oxidase. Electrochem Sci Adv. 2022; 2(5):e2100171.