Skip to main content
SpringerLink
Log in

Ficin-copper hybrid nanoflowers with enhanced peroxidase-like activity for colorimetric detection of biothiols

  • Original Paper
  • Published:
Microchimica Acta Aims and scope Submit manuscript

Abstract

The proteolytic enzyme ficin exhibits peroxidase-like activity but it is low and insufficient for real applications. Herein, we developed ficin-copper hybrid nanoflowers and demonstrated that they have significantly enhanced peroxidase-like activity of over 6-fold higher than that of free ficin, with one of the lowest Km and highest kcat values among all reported ficin-based peroxidase-like nanozymes. This was most likely caused by the synergistic catalysis of co-existing ficin and crystalline copper phosphate within nanoflower matrices having a large surface area. The nanoflowers were easily prepared by incubating ficin and copper sulfate at ambient temperature, causing coordination interactions between ficin’s amine/amide moieties and copper ions, followed by concomitant anisotropic growth of petals composed of copper phosphate crystals with ficin. When compared to free ficin and natural horseradish peroxidase, the resulting nanoflowers’ affinity toward H2O2 was greatly increased, yielding Km values of half and one-tenth, respectively, as well as noticeably improved stability. The nanoflowers were then applied to colorimetric determination of biological thiols (biothiols), such as cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), based on their inhibition of nanoflowers’ peroxidase-like activity, producing reduced color intensities as the concentration of biothiols increased. This strategy achieved highly sensitive colorimetric determinations of Cys, GSH, and Hcy after only 25-min incubation. Additionally, using this technique, biothiols in human serum were successfully determined with excellent precision, suggesting the potential application of this technology in clinical settings, particularly in point-of-care testing environments.

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

Data availability

Experimental data can be obtained from the corresponding author upon request.

References

  1. Lin J, Wang Q, Wang X, Zhu Y, Zhou X, Wei H (2020) Gold alloy-based nanozyme sensor arrays for biothiol detection. Analyst 145:3916–3921

    Article  CAS  Google Scholar 

  2. Mishanina TV, Libiad M, Banerjee R (2015) Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways. Nat Chem Biol 11:457–464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhang S, Ong CN, Shen HM (2004) Critical roles of intracellular thiols and calcium in parthenolide-induced apoptosis in human colorectal cancer cells. Cancer Lett 208:143–153

    Article  CAS  PubMed  Google Scholar 

  4. Hu Q, Yu C, Xia X, Zeng F, Wu S (2016) A fluorescent probe for simultaneous discrimination of GSH and Cys/Hcy in human serum samples via distinctly-separated emissions with independent excitations. Biosens Bioelectron 81:341–348

    Article  CAS  PubMed  Google Scholar 

  5. He L, Tao H, Koo S, Chen G, Sharma A, Chen Y, Lim IT, Cao QY, Kim JS (2018) Multifunctional fluorescent nanoprobe for sequential detections of Hg2+ ions and biothiols in live cells. ACS Appl Bio Mater 1:871–878

    Article  CAS  PubMed  Google Scholar 

  6. Lee HY, Choi YP, Kim S, Yoon T, Gou Z, Lee S, Swamy KMK, Kim G, Lee JY, Shin I, Yoon J (2014) Selective homocysteine turn-on fluorescent probes and their bioimaging applications. Chem Commun 50:6967–6969

    Article  CAS  Google Scholar 

  7. Liu J, Sun YQ, Zhang H, Huo Y, Shi Y, Guo W (2014) Simultaneous fluorescent imaging of Cys/Hcy and GSH from different emission channels. Chem Sci 5:3183–3188

    Article  CAS  Google Scholar 

  8. Townsend DM, Tew KD, Tapiero H (2003) The importance of glutathione in human disease. Biomed Pharmacother 57:145–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Patel BP, Rawal UM, Dave TK, Rawal RM, Shukla SN, Shah PM, Patel PS (2007) Lipid peroxidation, total antioxidant status, and total thiol levels predict overall survival in patients with oral squamous cell carcinoma. Integr Cancer Ther 6:365–372

    Article  CAS  PubMed  Google Scholar 

  10. Liu W, Chen J, Xu Z (2021) Fluorescent probes for biothiols based on metal complex. Coord Chem Rev 429:213638

    Article  CAS  Google Scholar 

  11. Chen X, Zhou Y, Peng X, Yoon J (2010) Fluorescent and colorimetric probes for detection of thiols. Chem Soc Rev 39(6):2120–2135

    Article  CAS  PubMed  Google Scholar 

  12. Zhu J, Dhimitruka I, Pei D (2004) 5-(2-Aminoethyl) dithio-2-nitrobenzoate as a more base-stable alternative to Ellman's reagent. Org Lett 6(21):3809–3812

    Article  CAS  PubMed  Google Scholar 

  13. Dai J, Ma C, Zhang P, Fu Y, Shen B (2020) Recent progress in the development of fluorescent probes for detection of biothiols. Dyes Pigments 177:108321

    Article  CAS  Google Scholar 

  14. Li ZJ, Zheng XJ, Zhang L, Liang RP, Li ZM, Qiu JD (2015) Label-free colorimetric detection of biothiols utilizing SAM and unmodified Au nanoparticles. Biosens Bioelectron 68:668–674

    Article  CAS  PubMed  Google Scholar 

  15. Mohammadi S, Khayatian G (2017) Colorimetric detection of biothiols based on aggregation of chitosan-stabilized silver nanoparticles. Spectrochim Acta A Mol Biomol Spectrosc 185:27–34

    Article  CAS  PubMed  Google Scholar 

  16. Li P, Lee SM, Kim HY, Kim S, Park S, Park KS, Park HG (2021) Colorimetric detection of individual biothiols by tailor made reactions with silver nanoprisms. Sci Rep 11:3937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mostafa IM, Liu H, Hanif S, Gilani MRHS, Guan Y, Xu G (2022) Synthesis of a novel electrochemical probe for the sensitive and selective detection of biothiols and its clinical applications. Anal Chem 94:6853–6859

    Article  CAS  PubMed  Google Scholar 

  18. Jin T, Li Y, Jing W, Li Y, Fan L, Li X (2020) Cobalt-based metal organic frameworks: a highly active oxidase-mimicking nanozyme for fluorescence “turn-on” assays of biothiol. Chem Commun 56:659–662

    Article  CAS  Google Scholar 

  19. Duan W, Qiu Z, Cao S, Guo Q, Huang J, Xing J, Lu X, Zeng J (2022) Pd–Fe3O4 Janus nanozyme with rational design for ultrasensitive colorimetric detection of biothiols. Biosens Bioelectron 196:113724

    Article  CAS  PubMed  Google Scholar 

  20. Le XA, Le TN, Kim MI (2021) Dual-functional peroxidase-copper phosphate hybrid nanoflowers for sensitive detection of biological thiols. Int J Mol Sci 23:366

    Article  PubMed  PubMed Central  Google Scholar 

  21. Bekhit AA, Hopkins DL, Geesink G, Bekhit AA, Franks P (2014) Exogenous proteases for meat tenderization. Crit Rev Food Sci Nutr 54:1012–1031

    Article  CAS  PubMed  Google Scholar 

  22. Salam SMA, Kagawa KI, Matsubara T, Kawashiro K (2008) Protease-catalyzed dipeptide synthesis from N-protected amino acid carbamoylmethyl esters and free amino acids in frozen aqueous solutions. Enzym Microb Technol 43:537–543

    Article  CAS  Google Scholar 

  23. Yang Y, Shen D, Long Y, Xie Z, Zheng H (2017) Intrinsic peroxidase-like activity of ficin. Sci Rep 7:43141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Huang Z, Yang Y, Long Y, Zheng H (2018) A colorimetric method for cysteine determination based on the peroxidase-like activity of ficin. Anal Methods 10:2676–2680

    Article  CAS  Google Scholar 

  25. Pan Y, Yang Y, Pang Y, Shi Y, Long Y, Zheng H (2018) Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid. Talanta 185:433–438

    Article  CAS  PubMed  Google Scholar 

  26. Zheng W, Shen D, Pan Y, Yi D, Long Y, Zheng H (2019) Enhancing the peroxidase-like activity of ficin by rational blocking thiol groups for colorimetric detection of biothiols. Talanta 204:833–839

    Article  CAS  PubMed  Google Scholar 

  27. Zheng W, Liu J, Yi D, Pan Y, Long Y, Zheng H (2020) Ficin encapsulated in mesoporous metal-organic frameworks with enhanced peroxidase-like activity and colorimetric detection of glucose. Spectrochim Acta A Mol Biomol Spectrosc 233:118195

    Article  CAS  PubMed  Google Scholar 

  28. Zhang M, Zhang Y, Yang C, Ma C, Tang J (2021) Enzyme-inorganic hybrid nanoflowers: classification, synthesis, functionalization and potential applications. Chem Eng J 415:129075

    Article  CAS  Google Scholar 

  29. Dadi S, Temur N, Gul OT, Yilmaz V, Ocsoy I (2023) In situ synthesis of horseradish peroxidase nanoflower@carbon nanotube hybrid nanobiocatalysts with greatly enhanced catalytic activity. Langmuir 39(13):4819–4828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Dadi S, Cardoso MH, Mandal AK, Franco OL, Ildiz N, Ocsoy I (2023) Natural molecule-incorporated magnetic organic-inorganic nanoflower: investigation of its dual fenton reaction-dependent enzyme-like catalytic activities with cyclic use. ChemistrySelect 8(13):e202300404

    Article  CAS  Google Scholar 

  31. Yilmoz SG, Demirbas A, Karaagac Z, Dadi S, Celik C, Yusufbeyoglu S, Ildiz N, Mandal AK, Cimen B, Ocsoy I (2022) Synthesis of taurine-Cu3(PO4)2 hybrid nanoflower and their peroxidase-mimic and antimicrobial properties. J Biotechnol 343:96–101

    Article  Google Scholar 

  32. Gul OT, Ocsoy I (2021) Co-enzymes based nanoflowers incorporated-magnetic carbon nanotubes: a new generation nanocatalyst for superior removal of cationic and anionic dyes with great repeated use. Environ Technol Innov 24:101992

    Article  CAS  Google Scholar 

  33. Gül OT, Ocsoy I (2021) Preparation of magnetic horseradish peroxidase-laccase nanoflower for rapid and efficient dye degradation with dual mechanism and cyclic use. Mater Lett 303:130501

    Article  Google Scholar 

  34. Dadi S, Celik C, Ocsoy I (2020) Gallic acid nanoflower immobilized membrane with peroxidase-like activity for m-cresol detection. Sci Rep 10:16765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Celik C, Tasdemir D, Demirbas A, Katı A, Gul OT, Cimen B, Ocsoy I (2018) Formation of functional nanobiocatalysts with a novel and encouraging immobilization approach and their versatile bioanalytical applications. RSC Adv 8(45):25298–25303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dang TV, Kim MI (2023) Diversified component incorporated hybrid nanoflowers: a versatile material for biosensing and biomedical applications. Korean J Chem Eng 40:302–310

    Article  CAS  Google Scholar 

  37. Ge J, Lei J, Zare RN (2012) Protein–inorganic hybrid nanoflowers. Nat Nanotechnol 7:428–432

    Article  CAS  PubMed  Google Scholar 

  38. Jiang X, Sun C, Guo Y, Nie G, Xu L (2015) Peroxidase-like activity of apoferritin paired gold clusters for glucose detection. Biosens Bioelectron 64:165–170

    Article  CAS  PubMed  Google Scholar 

  39. Carrilho E, Martinez AW, Whitesides GM (2009) Understanding wax printing: a simple micropatterning process for paper-based microfluidics. Anal Chem 81(16):7091–7095

    Article  CAS  PubMed  Google Scholar 

  40. Huang Y, Ran X, Lin Y, Ren J, Qu X (2015) Self-assembly of an organic–inorganic hybrid nanoflower as an efficient biomimetic catalyst for self-activated tandem reactions. Chem Commun 51:4386–4389

    Article  CAS  Google Scholar 

  41. Jiang W, Wang X, Yang J, Han H, Li Q, Tang J (2018) Lipase-inorganic hybrid nanoflower constructed through biomimetic mineralization: a new support for biodiesel synthesis. J Colloid Interface Sci 514:102–107

    Article  CAS  PubMed  Google Scholar 

  42. Dang TV, Heo NS, Cho HJ, Lee SM, Song MY, Kim HJ, Kim MI (2021) Colorimetric determination of phenolic compounds using peroxidase mimics based on biomolecule-free hybrid nanoflowers consisting of graphitic carbon nitride and copper. Microchim Acta 188:293

    Article  CAS  Google Scholar 

  43. Gonzalez DH, Kuang XM, Scott JA, Rocha GO, Paulson SE (2018) Terephthalate probe for hydroxyl radicals: yield of 2-hydroxyterephthalic acid and transition metal interference. Anal Lett 51:2488–2497

    Article  CAS  Google Scholar 

  44. Shang C, Wang Q, Tan H, Lu S, Wang S, Zhang Q, Gu L, Li J, Wang E, Guo S (2022) Defective PtRuTe as nanozyme with selectively enhanced peroxidase-like activity. JACS Au 2:2453–2459

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  46. Pan Y, Pang Y, Shi Y, Zheng W, Long Y, Huang Y, Zheng H (2019) One-pot synthesis of a composite consisting of the enzyme ficin and a zinc (II)-2-methylimidazole metal organic framework with enhanced peroxidase activity for colorimetric detection for glucose. Microchim Acta 186:1–8

    Article  CAS  Google Scholar 

  47. Li Z, Xia H, Li S, Pang J, Zhu W, Jiang Y (2017) In situ hybridization of enzymes and their metal–organic framework analogues with enhanced activity and stability by biomimetic mineralisation. Nanoscale 9:15298–15302

    Article  CAS  PubMed  Google Scholar 

  48. Turell L, Radi R, Alvarez B (2013) The thiol pool in human plasma: the central contribution of albumin to redox processes. Free Radic Biol Med 65:244–253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Buyukaslan H, Gulacti U, Gokdemir MT, Giden R, Celik H, Erel O, Dorterler EM (2019) Serum thiol levels and thiol/disulphide homeostasis in gunshot injuries. Eur J Trauma Emerg Surg 45:167–174

    Article  PubMed  Google Scholar 

Download references

Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT [NRF-2023R1A2C2007833]) and the Basic Science Research Program through the NRF funded by the Ministry of Education (Grant No. 2021R1A6A1A03038996). This research was also supported by the Gachon University research fund of 2021 (GCU-202110350001).

Author information

Authors and Affiliations

  1. Department of BioNano Technology, Gachon University, 1342 Seongnamdae-ro, Sujeong-gu, Seongnam, Gyeonggi, 13120, Republic of Korea

    Thinh Viet Dang, Jee Min Kim & Moon Il Kim

Authors
  1. Thinh Viet Dang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jee Min Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Moon Il Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.V.D.: conceptualization, methodology, investigation, validation, and writing—original draft. J.M.K.: investigation and validation. M.I.K.: conceptualization, supervision, writing—review and editing, funding acquisition, and project administration.

Corresponding author

Correspondence to Moon Il Kim.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s note

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

Supplementary Information

ESM 1

(DOCX 2820 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

Dang, T.V., Kim, J.M. & Kim, M.I. Ficin-copper hybrid nanoflowers with enhanced peroxidase-like activity for colorimetric detection of biothiols. Microchim Acta 190, 473 (2023). https://doi.org/10.1007/s00604-023-06070-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00604-023-06070-w

Keywords

Search

Navigation