C3N4 nanosheet-supported Prussian Blue nanoparticles as a peroxidase mimic: colorimetric enzymatic determination of lactate


Prussian Blue nanoparticles were deposited on g-C3N4 nanosheets. The resulting nanocomposite possesses peroxidase-like (POx) activity and can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine at room temperature in the presence of H2O2. This leads to formation of a blue product with an absorption maximum at 650 nm. The formation of the Prussian Blue nanoparticles on the g-C3N4 nanosheets, and the POx-like activity of the composite were characterized in detail. The POx mimic was used for determination of L-lactic acid via detection of H2O2 that is produced by the enzyme lactate oxidase (LOx). The assay has a linear range that extends from 5 to 100 μM, and the detection limit is 2.2 μM. The method was successfully applied to the determination of L-lactic acid in spiked human serum.

Ultra-small Prussian Blue (PB) nanoparticles were used to modify g-C3N4 nanosheets, and their peroxidase-like activity was explored for detection of L-lactic acid. LOx represent L-lactate oxidase, and TMB represents 3,3′,5,5′-tetramethylbenzidine.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Scheme 1
Fig. 7


  1. 1.

    Lu Y, Yeung N, Sieracki N, Marshall NM (2009) Design of functional metalloproteins. Nature 460:855

    CAS  Article  Google Scholar 

  2. 2.

    Bonar-Law RP, Sanders JKM (1995) Polyol recognition by a steroid-capped porphyrin. Enhancement and modulation of misfit guest binding by added water or methanol. J Am Chem Soc 117:259–271

    CAS  Article  Google Scholar 

  3. 3.

    Royer GP, Klotz IM (1969) Enhanced rates due to Apolar interactions between polymer and substrate. J Am Chem Soc 91:5885–5886

    CAS  Article  Google Scholar 

  4. 4.

    Zhang X, Xu H, Dong Z, Wang Y, Liu J, Shen J (2004) Highly efficient dendrimer-based mimic of glutathione peroxidase. J Am Chem Soc 126(34):10556–10557

    CAS  Article  Google Scholar 

  5. 5.

    Aiba Y, Sumaoka J, Komiyama M (2011) Artificial DNA cutters for DNA manipulation and genome engineering. Chem Soc Rev 40(12):5657–5668

    CAS  Article  Google Scholar 

  6. 6.

    Wei H, Wang E (2013) Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem Soc Rev 42(14):6060–6093

    CAS  Article  Google Scholar 

  7. 7.

    Huang L, Zhu W, Zhang W, Chen K, Wang J, Wang R, Yang Q, Hu N, Suo Y, Wang J (2018) Layered vanadium(IV) disulfide nanosheets as a peroxidase-like nanozyme for colorimetric detection of glucose. Microchim Acta 185(1):7

    Article  Google Scholar 

  8. 8.

    Wei H, Wang E (2008) Fe3O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2O2 and glucose detection. Anal Chem 80(6):2250–2254

    CAS  Article  Google Scholar 

  9. 9.

    Chen W, Chen J, Liu A-L, Wang L-M, Li G-W, Lin X-H (2011) Peroxidase-like activity of cupric oxide nanoparticle. ChemCatChem 3(7):1151–1154

    CAS  Article  Google Scholar 

  10. 10.

    Tian Z, Li J, Zhang Z, Gao W, Zhou X, Qu Y (2015) Highly sensitive and robust peroxidase-like activity of porous nanorods of ceria and their application for breast cancer detection. Biomaterials 59:116–124

    CAS  Article  Google Scholar 

  11. 11.

    Nasir M, Nawaz MH, Yaqub M, Hayat A, Rahim A (2017) An overview on enzyme-mimicking nanomaterials for use in electrochemical and optical assays. Microchim Acta 184:323–342

    CAS  Article  Google Scholar 

  12. 12.

    Wu G-W, He S-B, Peng H-P, Deng H-H, Liu A-L, Lin X-H, Xia X-H, Chen W (2014) Citrate-capped platinum nanoparticle as a smart probe for ultrasensitive mercury sensing. Anal Chem 86(21):10955–10960

    CAS  Article  Google Scholar 

  13. 13.

    Shi W, Wang Q, Long Y, Cheng Z, Chen S, Zheng H, Huang Y (2011) Carbon nanodots as peroxidase mimetics and their applications to glucose detection. Chem Commun 47(23):6695–6697

    CAS  Article  Google Scholar 

  14. 14.

    Qu F, Li T, Yang M (2011) Colorimetric platform for visual detection of cancer biomarker based on intrinsic peroxidase activity of graphene oxide. Biosens Bioelectron 26(9):3927–3931

    CAS  Article  Google Scholar 

  15. 15.

    Lin T, Zhong L, Wang J, Guo L, Wu H, Guo Q, Fu F, Chen G (2014) Graphite-like carbon nitrides as peroxidase mimetics and their applications to glucose detection. Biosens Bioelectron 59:89–93

    CAS  Article  Google Scholar 

  16. 16.

    Shi W, Zhang X, He S, Huang Y (2011) CoFe2O4 magnetic nanoparticles as a peroxidase mimic mediated chemiluminescence for hydrogen peroxide and glucose. Chem Commun 47(38):10785–10787

    CAS  Article  Google Scholar 

  17. 17.

    Zhou D, Zeng K, Yang M (2019) Gold nanoparticle-loaded hollow Prussian blue nanoparticles with peroxidase-like activity for colorimetric determination of L-lactic acid. Microchim Acta 186(2):121

    Article  Google Scholar 

  18. 18.

    Darabdhara G, Boruah PK, Das MR (2019) Colorimetric determination of glucose in solution and via the use of a paper strip by exploiting the peroxidase and oxidase mimicking activity of bimetallic cu-Pd nanoparticles deposited on reduced graphene oxide, graphitic carbon nitride, or MoS2 nanosheets. Microchim Acta 186(1):102

    Article  Google Scholar 

  19. 19.

    Ramalingam M, Ponnusamy VK, Sangilimuthu SN (2019) A nanocomposite consisting of porous graphitic carbon nitride nanosheets and oxidized multiwalled carbon nanotubes for simultaneous stripping voltammetric determination of cadmium(II), mercury(II), lead(II) and zinc(II). Microchim Acta 186(2):69

    Article  Google Scholar 

  20. 20.

    Wang Z, Dong K, Liu Z, Zhang Y, Chen Z, Sun H, Ren J, Qu X (2017) Activation of biologically relevant levels of reactive oxygen species by au/g-C3N4 hybrid nanozyme for bacteria killing and wound disinfection. Biomaterials 113:145–157

    CAS  Article  Google Scholar 

  21. 21.

    Karyakin AA, Karyakina EE (1999) Prussian blue-based `artificial peroxidase' as a transducer for hydrogen peroxide detection. Application to biosensors. Sensor Actuat B Chem 57(1):268–273

    CAS  Article  Google Scholar 

  22. 22.

    Komkova MA, Karyakina EE, Karyakin AA (2018) Catalytically synthesized Prussian blue nanoparticles defeating natural enzyme peroxidase. J Am Chem Soc 140(36):11302–11307

    CAS  Article  Google Scholar 

  23. 23.

    Cao L, Liu Y, Zhang B, Lu L (2010) In situ controllable growth of Prussian blue Nanocubes on reduced graphene oxide: facile synthesis and their application as enhanced Nanoelectrocatalyst for H2O2 reduction. ACS App Mater Inter 2(8):2339–2346

    CAS  Article  Google Scholar 

  24. 24.

    Zhang X-Z, Zhou Y, Zhang W, Zhang Y, Gu N (2016) Polystyrene@au@prussian blue nanocomposites with enzyme-like activity and their application in glucose detection. Colloid Surf A: Physicochem Engineer 490:291–299

    CAS  Article  Google Scholar 

  25. 25.

    Singh S, Tripathi P, Kumar N, Nara S (2017) Colorimetric sensing of malathion using palladium-gold bimetallic nanozyme. Biosens Bioelectron 92:280–286

    CAS  Article  Google Scholar 

  26. 26.

    Zhang Y, Thomas A, Antonietti M, Wang X (2009) Activation of carbon nitride solids by protonation: morphology changes, enhanced ionic conductivity, and photoconduction experiments. J Am Chem Soc 131:50–51

    CAS  Article  Google Scholar 

  27. 27.

    Wang X, Maeda K, Thomas A, Takanabe K, Xin G, Carlsson JM, Domen K, Antonietti M (2008) A metal-free polymeric photocatalyst for hydrogen production from water under visible light. Nat Mater 8:76

    Article  Google Scholar 

  28. 28.

    Dong F, Wu L, Sun Y, Fu M, Wu Z, Lee SC (2011) Efficient synthesis of polymeric g-C3N4 layered materials as novel efficient visible light driven photocatalysts. J Mater Chem 21(39):15171–15174

    CAS  Article  Google Scholar 

  29. 29.

    Dong H, Guo X, Yang C, Ouyang Z (2018) Synthesis of g-C3N4 by different precursors under burning explosion effect and its photocatalytic degradation for tylosin. Appl Catalysis B Environ 230:65–76

    CAS  Article  Google Scholar 

  30. 30.

    Zhang X-Q, Gong S-W, Zhang Y, Yang T, Wang C-Y, Gu N (2010) Prussian blue modified iron oxide magnetic nanoparticles and their high peroxidase-like activity. J Mater Chem 20(24):5110–5116

    CAS  Article  Google Scholar 

  31. 31.

    Yang H, Sun L, Zhai J, Li H, Zhao Y, Yu H (2014) In situ controllable synthesis of magnetic Prussian blue/graphene oxide nanocomposites for removal of radioactive cesium in water. J Mater Chem A 2(2):326–332

    CAS  Article  Google Scholar 

  32. 32.

    Wang N, Han Z, Fan H, Ai S (2015) Copper nanoparticles modified graphitic carbon nitride nanosheets as a peroxidase mimetic for glucose detection. RSC Adv 5(111):91302–91307

    CAS  Article  Google Scholar 

  33. 33.

    Zeng K, Yang M, Liu Y-N, Rasooly A (2018) Dual function hollow structured mesoporous Prussian blue mesocrystals for glucose biosensors. Anal Methods 10(32):3951–3957

    CAS  Article  Google Scholar 

  34. 34.

    Wang K, Xu J-J, Chen H-Y (2006) Biocomposite of cobalt phthalocyanine and lactate oxidase for lactate biosensing with MnO2 nanoparticles as an eliminator of ascorbic acid interference. Sensor Actuat B Chem 114(2):1052–1058

    CAS  Article  Google Scholar 

  35. 35.

    Jena BK, Raj CR (2006) Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles. Anal Chem 78(18):6332–6339

    CAS  Article  Google Scholar 

  36. 36.

    Shen X, Zhang G, Zhang D (2012) A new Fluorometric turn-on detection of l-lactic acid based on the Cascade enzymatic and chemical reactions and the abnormal fluorescent behavior of Silole. Org Lett 14(7):1744–1747

    CAS  Article  Google Scholar 

Download references


The authors thank the support of this work by the National Natural Science Foundation of China (Grant No.21575165).

Author information



Corresponding author

Correspondence to Minghui Yang.

Ethics declarations

Conflict of interest

The author(s) declare that they have no competing interests. All experiments were in accordance with the guidelines of the National Institute of Health, China, and approved by the Institutional Ethical Committee (IEC) of the Second Xiangya hospital that attached to Central South University. We also obtained informed consent for any experimentation with human serum samples.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material


(DOC 142 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhou, D., Wang, C., Luo, J. et al. C3N4 nanosheet-supported Prussian Blue nanoparticles as a peroxidase mimic: colorimetric enzymatic determination of lactate. Microchim Acta 186, 735 (2019). https://doi.org/10.1007/s00604-019-3834-6

Download citation


  • g-C3N4 nanosheet
  • Prussian Blue
  • Enzyme mimic
  • Colorimetry
  • L-lactate oxidase