Skip to main content
SpringerLink
Log in

Bioconjugation of nanozyme and natural enzyme for ultrasensitive detection of cholesterol

  • Original Paper
  • Published:
Analytical Sciences Aims and scope Submit manuscript

Abstract

When nanozymes are used in biological analysis, higher activity can improve the detection sensitivity, and better selectivity can eliminate other interference. To improve the specificity and sensitivity, we fabricated an innovative bioconjugated nanozyme with natural enzyme (BNNZ), in which natural ChOx was immobilized onto histidine-modified Fe3O4 (His–Fe3O4) with hydrophilic poly(ethylene glycol) (PEG) as a linker. ChOx could specifically catalyze the oxidation of cholesterol to generate H2O2 molecule, and then the newly formed H2O2 oxidized the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into blue ox-TMB by peroxidase-like His–Fe3O4. According to the above cascade reaction, the BNNZ-based colorimetric strategy was proposed for the detection of cholesterol. Wherein, natural enzymes specifically catalyzed substrates, which endowed BNNZ with excellent specificity for target molecules; meanwhile, the introduction of histidine on His–Fe3O4 effectively increased the peroxidase-like activity of BNNZ, which provided a guarantee for sensitivity. Furthermore, BNNZ after reaction could be rapidly separated by an external magnetic field without interfering with colorimetric quantitative detection. The proposed strategy exhibited excellent sensitivity with limit of detection of 0.446 μM and was successfully used for the detection of cholesterol in spiked human serum sample with recovery and relative standard deviation in the range of 97.9–103.5% and 2.5–4.0%, respectively. This work indicates that the bioconjugation of nanozyme and natural enzyme may be a universal strategy for synthesis of high-performance enzyme–nanozyme systems, and the new-type BNNZ will be widely used in biological detection and disease treatment.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. R. Zhang, X. Yan, K. Fan, Nanozymes inspired by natural enzymes. Acc. Mater. Res. 2(7), 534–547 (2021)

    Article  CAS  Google Scholar 

  2. Y. Huang, J. Ren, X. Qu, Nanozymes: classification, catalytic mechanisms, activity regulation, and applications. Chem. Rev. 119(6), 4357–4412 (2019)

    Article  CAS  PubMed  Google Scholar 

  3. M. Liang, X. Yan, Nanozymes: from new concepts, mechanisms, and standards to applications. Acc. Chem. Res. 52(8), 2190–2200 (2019)

    Article  CAS  PubMed  Google Scholar 

  4. H. Jin, D. Ye, L. Shen, R. Fu, Y. Tang, J.C.Y. Jung, H. Zhao, J. Zhang, Perspective for single atom nanozymes based sensors: advanced materials, sensing mechanism, selectivity regulation, and applications. Anal. Chem. 94(3), 1499–1509 (2022)

    Article  CAS  PubMed  Google Scholar 

  5. B. Das, J.L. Franco, N. Logan, P. Balasubramanian, M.I. Kim, C. Cao, Nanozymes in point-of-care diagnosis: an emerging futuristic approach for biosensing. Nano-Micro Lett. 13(1), 193 (2021)

    Article  CAS  Google Scholar 

  6. M. Wei, J. Lee, F. Xia, P. Lin, X. Hu, F. Li, D. Ling, Chemical design of nanozymes for biomedical applications. Acta Biomater. 126, 15–30 (2021)

    Article  CAS  PubMed  Google Scholar 

  7. L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang, J. Feng, D. Yang, S. Perrett, X. Yan, Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2(9), 577–583 (2007)

    Article  CAS  PubMed  Google Scholar 

  8. Y. Hu, H. Cheng, X. Zhao, J. Wu, F. Muhammad, S. Lin, J. He, L. Zhou, C. Zhang, Y. Deng, P. Wang, Z. Zhou, S. Nie, H. Wei, Surface-enhanced Raman scattering active gold nanoparticles with enzyme-mimicking activities for measuring glucose and lactate in living tissues. ACS Nano 11(6), 5558–5566 (2017)

    Article  CAS  PubMed  Google Scholar 

  9. X. Wang, F. Wen, L. He, J. Su, P. Jiang, D. He, Engineering porous Co–Mn oxide nanosheets with abundant oxygen vacancy as an efficient oxidase-like mimic for heparin colorimetric sensing. Anal. Chim. Acta 1198, 339564 (2022)

    Article  CAS  PubMed  Google Scholar 

  10. X. Meng, D. Li, L. Chen, H. He, Q. Wang, C. Hong, J. He, X. Gao, Y. Yang, B. Jiang, G. Nie, X. Yan, L. Gao, K. Fan, High-performance self-cascade pyrite nanozymes for apoptosis–ferroptosis synergistic tumor therapy. ACS Nano 15(3), 5735–5751 (2021)

    Article  CAS  PubMed  Google Scholar 

  11. N. Wang, J. Shi, Y. Liu, W. Sun, X. Su, Constructing bifunctional metal–organic framework based nanozymes with fluorescence and oxidase activity for the dual-channel detection of butyrylcholinesterase. Anal. Chim. Acta 1205, 339717 (2022)

    Article  CAS  PubMed  Google Scholar 

  12. H. Sun, A. Zhao, N. Gao, K. Li, J. Ren, X. Qu, Deciphering a nanocarbon-based artificial peroxidase: chemical identification of the catalytically active and substrate-binding sites on graphene quantum dots. Angew. Chem. Int. Ed. 54(24), 7176–7180 (2015)

    Article  CAS  Google Scholar 

  13. T. Zhan, J. Kang, X. Li, L. Pan, G. Li, W. Hou, NiFe layered double hydroxide nanosheets as an efficiently mimic enzyme for colorimetric determination of glucose and H2O2. Sens. Actuators B Chem. 255, 2635–2642 (2018)

    Article  CAS  Google Scholar 

  14. R. Cai, D. Yang, S. Peng, X. Chen, Y. Huang, Y. Liu, W. Hou, S. Yang, Z. Liu, W. Tan, Single nanoparticle to 3D supercage: framing for an artificial enzyme system. J. Am. Chem. Soc. 137(43), 13957–13963 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. R. Cai, D. Yang, K. Lin, T.S. Cao, Y. Lyv, K. Chen, Y. Yang, J. Ge, L. Xia, G. Christou, Y. Zhao, Z. Chen, W. Tan, 3D halos assembled from Fe3O4/Au NPs with enhanced catalytic and optical properties. Nanoscale 11(43), 20968–20976 (2019)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. A.A. Vernekar, T. Das, S. Ghosh, G. Mugesh, A remarkably efficient MnFe2O4-based oxidase nanozyme. Chem. Asian J. 11(1), 72–76 (2016)

    Article  CAS  PubMed  Google Scholar 

  18. Z. Chen, J.J. Yin, Y.T. Zhou, Y. Zhang, L. Song, M. Song, S. Hu, N. Gu, Dual enzyme-like activities of iron oxide nanoparticles and their implication for diminishing cytotoxicity. ACS Nano 6(5), 4001–4012 (2012)

    Article  CAS  PubMed  Google Scholar 

  19. M.L. Ye, Y. Zhu, Y. Lu, L. Gan, Y. Zhang, Y.G. Zhao, Magnetic nanomaterials with unique nanozymes-like characteristics for colorimetric sensors: a review. Talanta 230, 122299 (2021)

    Article  CAS  PubMed  Google Scholar 

  20. S. Li, Y. Zhang, Q. Wang, A. Lin, H. Wei, Nanozyme-enabled analytical chemistry. Anal. Chem. 94(1), 312–323 (2022)

    Article  CAS  PubMed  Google Scholar 

  21. M.S. Kim, J. Lee, H.S. Kim, A. Cho, K.H. Shim, T.N. Le, S.S.A. An, J.W. Han, M.I. Kim, J. Lee, Heme cofactor-resembling Fe–N single site embedded graphene as nanozymes to selectively detect H2O2 with high sensitivity. Adv. Funct. Mater. 30(1), 1905410 (2020)

    Article  CAS  Google Scholar 

  22. W. Wu, Q. Wang, J. Chen, L. Huang, H. Zhang, K. Rong, S. Dong, Biomimetic design for enhancing the peroxidase mimicking activity of hemin. Nanoscale 11(26), 12603–12609 (2019)

    Article  CAS  PubMed  Google Scholar 

  23. K. Fan, H. Wang, J. Xi, Q. Liu, X. Meng, D. Duan, L. Gao, X. Yan, Optimization of Fe3O4 nanozyme activity via single amino acid modification mimicking an enzyme active site. Chem. Commun. 53(2), 424–427 (2017)

    Article  CAS  Google Scholar 

  24. L. Dewangan, J. Korram, I. Karbhal, R. Nagwanshi, M.L. Satnami, N-doped carbon quantum dot-MnO2 nanowire FRET pairs: detection of cholesterol, glutathione, acetylcholinesterase, and chlorpyrifos. ACS Appl. Nano Mater. 4(12), 13612–13624 (2021)

    Article  CAS  Google Scholar 

  25. C. Hong, L. Chen, C. Wu, D. Yang, J. Dai, Z. Huang, R. Cai, W. Tan, Green synthesis of Au@WSe2 hybrid nanostructures with the enhanced peroxidase-like activity for sensitive colorimetric detection of glucose. Nano Res. 15(2), 1587–1592 (2022)

    Article  CAS  Google Scholar 

  26. T.L. Steck, Y. Lange, Cell cholesterol homeostasis: mediation by active cholesterol. Trends Cell Biol. 20(11), 680–687 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. J. Zhang, Q. Liu, Cholesterol metabolism and homeostasis in the brain. Protein Cell 6(4), 254–264 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. G. Llaverias, C. Danilo, I. Mercier, K. Daumer, F. Capozza, T.M. Williams, F. Sotgia, M.P. Lisanti, P.G. Frank, Role of cholesterol in the development and progression of breast cancer. Am. J. Pathol. 178(1), 402–412 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. F.J. Alenghat, A.M. Davis, Management of blood cholesterol. JAMA 321(8), 800–801 (2019)

    Article  PubMed  PubMed Central  Google Scholar 

  30. F.R. Maxfield, I. Tabas, Role of cholesterol and lipid organization in disease. Nature 438(7068), 612–621 (2005)

    Article  CAS  PubMed  Google Scholar 

  31. V. Narwal, R. Deswal, B. Batra, V. Kalra, R. Hooda, M. Sharma, J.S. Rana, Cholesterol biosensors: a review. Steroids 143, 6–17 (2019)

    Article  CAS  PubMed  Google Scholar 

  32. L. Dewangan, J. Korram, I. Karbhal, R. Nagwanshi, V.K. Jena, M.L. Satnami, A colorimetric nanoprobe based on enzyme-immobilized silver nanoparticles for the efficient detection of cholesterol. RSC Adv. 9(72), 42085–42095 (2019)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. M. Chung, Y. Jang, M. Kim, Convenient colorimetric detection of cholesterol using multi-enzyme Co-incorporated organic-inorganic hybrid nanoflowers. J. Nanosci. Nanotechnol. 18(9), 6555–6561 (2018)

    Article  CAS  PubMed  Google Scholar 

  34. C. Hong, X. Zhang, C. Wu, Q. Chen, H. Yang, D. Yang, Z. Huang, R. Cai, W. Tan, On-site colorimetric detection of cholesterol based on polypyrrole nanoparticles. ACS Appl. Mater. Interfaces 12(49), 54426–54432 (2020)

    Article  CAS  PubMed  Google Scholar 

  35. Q. Wu, L. He, Z.W. Jiang, Y. Li, Z.M. Cao, C.Z. Huang, Y.F. Li, CuO nanoparticles derived from metal-organic gel with excellent electrocatalytic and peroxidase-mimicking activities for glucose and cholesterol detection. Biosens. Bioelectron. 145, 111704 (2019)

    Article  CAS  PubMed  Google Scholar 

  36. L. Zhao, Z. Wu, G. Liu, H. Lu, Y. Gao, F. Liu, C. Wang, J. Cui, G. Lu, High-activity Mo, S co-doped carbon quantum dot nanozyme-based cascade colorimetric biosensor for sensitive detection of cholesterol. J. Mater. Chem. B 7(44), 7042–7051 (2019)

    Article  CAS  PubMed  Google Scholar 

  37. J. Li, T. Liu, R.A. Dahlgren, H. Ye, Q. Wang, Y. Ding, M. Gao, X. Wang, H. Wang, N, S-co-doped carbon/Co1-xS nanocomposite with dual-enzyme activities for a smartphone-based colorimetric assay of total cholesterol in human serum. Anal. Chim. Acta 1204, 339703 (2022)

    Article  CAS  PubMed  Google Scholar 

  38. G.I. Berglund, G.H. Carlsson, A.T. Smith, H. Szöke, A. Henriksen, J. Hajdu, The catalytic pathway of horseradish peroxidase at high resolution. Nature 417(6887), 463–468 (2002)

    Article  CAS  PubMed  Google Scholar 

  39. Z. Mu, S. Wu, J. Guo, M. Zhao, Y. Wang, Dual mechanism enhanced peroxidase-like activity of iron–nickel bimetal–organic framework nanozyme and its application for biosensing. ACS Sustain. Chem. Eng. 10(9), 2984–2993 (2022)

    Article  CAS  Google Scholar 

  40. M.A. Voinov, J.O.S. Pagán, E. Morrison, T.I. Smirnova, A.I. Smirnov, Surface-mediated production of hydroxyl radicals as a mechanism of iron oxide nanoparticle biotoxicity. J. Am. Chem. Soc. 133(1), 35–41 (2011)

    Article  CAS  PubMed  Google Scholar 

  41. Q. Liu, A. Zhang, R. Wang, Q. Zhang, D. Cui, A review on metal- and metal oxide-based nanozymes: properties, mechanisms, and applications. Nano-Micro Lett. 13(1), 154 (2021)

    Article  CAS  Google Scholar 

  42. C. Zhang, C. Chen, D. Zhao, G. Kang, F. Liu, F. Yang, Y. Lu, J. Sun, Multienzyme cascades based on highly efficient metal–nitrogen–carbon nanozymes for construction of versatile bioassays. Anal. Chem. 94(8), 3485–3493 (2022)

    Article  CAS  PubMed  Google Scholar 

  43. A. Menotti, M. Lanti, A. Zanchetti, G. Botta, M. Laurenzi, O. Terradura-Vagnarelli, M. Mancini, The role of HDL cholesterol in metabolic syndrome predicting cardiovascular events. The Gubbio population study. Nutr. Metab. Cardiovas. 21(5), 315–322 (2011)

    Article  CAS  Google Scholar 

  44. S. Qu, Z. Li, Q. Jia, Detection of purine metabolite uric acid with picolinic-acid-functionalized metal–organic frameworks. ACS Appl. Mater. Interfaces 11(37), 34196–34202 (2019)

    Article  CAS  PubMed  Google Scholar 

  45. I. Kim, Y.I. Kim, S.W. Lee, H.G. Jung, G. Lee, D.S. Yoon, Highly permselective uric acid detection using kidney cell membrane-functionalized enzymatic biosensors. Biosens. Bioelectron. 190, 113411 (2021)

    Article  CAS  PubMed  Google Scholar 

  46. Y. Li, S. Li, M. Bao, L. Zhang, C. Carraro, R. Maboudian, A. Liu, W. Wei, Y. Zhang, S. Liu, Pd nanoclusters confined in ZIF-8 matrixes for fluorescent detection of glucose and cholesterol. ACS Appl. Nano Mater. 4(9), 9132–9142 (2021)

    Article  CAS  Google Scholar 

  47. Y. Huang, Y. Gu, X. Liu, T. Deng, S. Dai, J. Qu, G. Yang, L. Qu, Reusable ring-like Fe3O4/Au nanozymes with enhanced peroxidase-like activities for colorimetric-SERS dual-mode sensing of biomolecules in human blood. Biosens. Bioelectron. 209, 114253 (2022)

    Article  CAS  PubMed  Google Scholar 

  48. J. Hassanzadeh, A. Khataee, Ultrasensitive chemiluminescent biosensor for the detection of cholesterol based on synergetic peroxidase-like activity of MoS2 and graphene quantum dots. Talanta 178, 992–1000 (2018)

    Article  CAS  PubMed  Google Scholar 

  49. V. Román-Pizarro, M. Ramírez-Gutiérrez, A. Gómez-Hens, J.M. Fernández-Romero, Usefulness of magnetically-controlled MNPs-enzymes microreactors for the fluorimetric determination of total cholesterol in serum. Talanta 208, 120426 (2020)

    Article  PubMed  Google Scholar 

  50. H.C. Chang, J.A. Ho, Gold nanocluster-assisted fluorescent detection for hydrogen peroxide and cholesterol based on the inner filter effect of gold nanoparticles. Anal. Chem. 87(20), 10362–10367 (2015)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant No. 82172829, 21605114).

Funding

National Natural Science Foundation of China, 82172829, Hong-Tao Zhao, 21605114, Xian-Hua Wang.

Author information

Authors and Affiliations

  1. Pharmaceutical Department of the Second Hospital of Tianjin Medical University, Tianjin, 300211, China

    Hong-Tao Zhao & Zhe Wang

  2. Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, Building B for School of Pharmacy, Tianjin Medical University, 22 Qixiangtai Road, Heping District, Tianjin, 300072, China

    Jin-Ye Lang, Chen-Chen Bai & Xian-Hua Wang

  3. Department of Pharmacy of Tianjin Chest Hospital, Tianjin, 300350, China

    Zhan-Song Hu

Authors
  1. Hong-Tao Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jin-Ye Lang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhe Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhan-Song Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chen-Chen Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xian-Hua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xian-Hua Wang.

Ethics declarations

Conflict of interest

The authors declare that they no conflict of interest.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 5466 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, HT., Lang, JY., Wang, Z. et al. Bioconjugation of nanozyme and natural enzyme for ultrasensitive detection of cholesterol. ANAL. SCI. 39, 503–515 (2023). https://doi.org/10.1007/s44211-022-00258-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s44211-022-00258-5

Keywords

Search

Navigation