Microchimica Acta

, 185:114 | Cite as

Immobilization of horseradish peroxidase on amino-functionalized carbon dots for the sensitive detection of hydrogen peroxide

  • Ya Su
  • Xuan Zhou
  • Yumei LongEmail author
  • Weifeng LiEmail author
Original Paper


The authors describe the preparation of amino-functionalized carbon dots (NH2-CDs) via a one-step hydrothermal process using silver nitrate and chitosan as the precursors. The NH2-CDs have a fairly consistent size distribution with an average size of 2.8 ± 0.5 nm. This is attributed to the introduction of Ag(I) both as a catalyst and a precipitant. The NH2-CDs are highly crystalline. Their surface carries amino groups and carboxy groups which is confirmed by transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy. Horseradish peroxidase (HRP) was immobilized in the NH2-CDs and then placed on a glassy carbon electrode (GCE). Spectroscopic and electrochemical analyses evidenced the stability and good bioactivity of the immobilized HRP. This reveals that NH2-CD is a desirable matrix for enzyme immobilization. The modified GCE exhibits enhanced electro-catalytic activity towards hydrogen peroxide (H2O2) reduction as compared to that of plain CDs. The effects of pH value and loading on the performances of the modified GCEs were studied. Under optimized conditions, the biosensor has a linear response in the 5 to 590 nM H2O2 concentration range, with a 1.8 nM defection limit (at an S/N ratio of 3). The sensor is stable, reproducible and selective. Finally, the sensor was applied to determine H2O2 in real samples, and satisfactory recoveries were achieved.

Graphical abstract

Amino-functionalized carbon dots (NH2-CDs) can provide more active sites and a friendly microenvironment for horseradish peroxidase (HRP) immobilization. Thus, a novel sensitive H2O2 biosensor has been developed.


Hydrothermal method Electro-catalytic reduction Glassy carbon electrode Cyclic voltammetry Amperometric response Biosensor H2O2 Analytical property 



This work is supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Project of Scientific and Technologic Infrastructure of Suzhou (SZS201207).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2017_2629_MOESM1_ESM.doc (3.3 mb)
ESM 1 (DOC 3382 kb)


  1. 1.
    Song HY, Ni YN, Kokot S (2013) A glassy carbon electrode modified with poly(anthranilic acid), poly(diphenylamine sulfonate) and CuO nano-particles for the sensitive determination of hydrogen peroxide. Microchim Acta 180:1263–1270. CrossRefGoogle Scholar
  2. 2.
    Jia NQ, Lian Q, Wang ZY, Shen HB (2009) A hydrogen peroxide biosensor based on direct electrochemistry of hemoglobin incorporated in PEO–PPO–PEO triblock copolymer film. Sensors Actuators B Chem 137:230–234. CrossRefGoogle Scholar
  3. 3.
    Li M, Gao H, Wang X, Wang Y, Qi H, Zhang C (2017) A fluorinedoped tin oxide electrode modified with gold nanoparticles for electrochemiluminescent determination of hydrogen peroxide released by living cells. Microchim Acta 184:603–610. CrossRefGoogle Scholar
  4. 4.
    Fusco G, Bollella P, Mazzei F, Favero G, Antiochia R, Tortolini C (2016) Catalase-baed modified graphite electrode for hydrogen peroxide detection in different beverages. J Anal Methods Chem 2016:8174913. CrossRefGoogle Scholar
  5. 5.
    Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzym Microb Technol 39:235–251. CrossRefGoogle Scholar
  6. 6.
    Castillo J, Gáspár S, Leth S, Niculescu M, Mortari A, Bontidean I, Soukharev V, Dorneanu SA, Ryabov AD, Csöregi E (2004) Biosensors for life quality: design, development and applications. Sensors Actuators B Chem 102:179–194. CrossRefGoogle Scholar
  7. 7.
    Liu YD, Liu XH, Guo ZP, ZG H, Xue ZG, XQ L (2017) Horseradish peroxidase supported on porous graphene as a novel sensing platform for detection of hydrogen peroxide in living cells sensitively. Biosens Bioelectron 87:101–107. CrossRefGoogle Scholar
  8. 8.
    Thenmozhi K, Narayanan SS (2017) Horseradish peroxidase and toluidine blue covalently immobilized leak-free sol-gel composite biosensor for hydrogen peroxide. Mater Sci Eng C 70:223–230. CrossRefGoogle Scholar
  9. 9.
    Wu C, Liu Z, Sun HH, Wang X, Xu P (2016) Selective determination of phenols and aromatic amines based on horseradish peroxidase-nanoporous gold co-catalytic strategy. Biosens Bioelectron 79:843–849. CrossRefGoogle Scholar
  10. 10.
    Mao CJ, Chen XB, Niu HL, Song JM, Zhang SY, Cui RJ (2012) A novel enzymatic hydrogen peroxide biosensor based on Ag/C nanocables. Biosens Bioelectron 31:544–547. CrossRefGoogle Scholar
  11. 11.
    Zhao XJ, Mai ZB, Kang XH, Zou XY (2008) Direct electrochemistry and electrocatalysis of horseradish peroxidase based on clay-chitosan-gold nanoparticle nanocomposite. Biosens Bioelectron 23:1032–1038. CrossRefGoogle Scholar
  12. 12.
    XY X, Ray R, YL G, Ploehn HJ, Gearheart L, Raker K, Scrivens WA (2004) Electrophoretic analysis and purification of fluorescent single-wall carbon nanotube fragments. J Am Chem Soc 126:12736–12737. CrossRefGoogle Scholar
  13. 13.
    Pumera M, Sánchez S, Ichinose I, Tang J (2007) Electrochemical nanobiosensors. Sensors Actuators B Chem 123:1195–1205. CrossRefGoogle Scholar
  14. 14.
    Willner I, Baron R, Willner B (2007) Integrated nanoparticle-biomolecule systems for biosensing and bioelectronics. Biosens Bioelectron 22:1841–1852. CrossRefGoogle Scholar
  15. 15.
    Bollela P, Fusco G, Tortolini C, Sanzò G, Favero G, Gorton L, Antiochia R (2017) Beyond graphene:electrochemical sensors and biosensors for biomarkers detection. Biosens Bioelectron 89:152–166. CrossRefGoogle Scholar
  16. 16.
    Mazzei F, Favero G, Bollella P, Tortolini C, Mannina L (2015) Recent trends in electrochemical nanobiosensors for environmental analysis. Int J Environ Health 7:267–291. CrossRefGoogle Scholar
  17. 17.
    Peng ZL, Han X, Li SH, Al-Youbi AO, Bashammakh AB, El-Shahawi MS, Leblanc RM (2017) Carbon dots: biomacromolecule interaction, bioimaging and nanomedicine. Coord Chem Rev 343:256–277. CrossRefGoogle Scholar
  18. 18.
    Lim SY, Shen W, Gao ZQCZ (2015) Carbo quantum dots and their applications. Chem Soc Rev 44:362–381. CrossRefGoogle Scholar
  19. 19.
    Cao L, Wang X, Meziani MJ, FS L, Wang HF, Luo PG, Lin Y, Harruff BA, Veca LM, Murray D, Xie SY, Sun YP (2007) Carbon dots for multiphoton bioimaging. J Am Chem Soc 129:11318–11319. CrossRefGoogle Scholar
  20. 20.
    Jin HL, Huang HH, He YH, Feng X, Wang S, Dai LM, Wang JC (2015) Graphene quantum dots supported by grapheme nanoribbons with ultrahigh electrocatalytic performance for oxygen reduction. J Am Chem Soc 137:7588–7591. CrossRefGoogle Scholar
  21. 21.
    Ray SC, Saha A, Jana NR, Sarkar R (2009) J Phys Chem C 113:18546–18551CrossRefGoogle Scholar
  22. 22.
    Park Y, Yoo J, Lim B, Kwon W, Rhee SW (2016) Improving the functionality of carbon nanodots: doping and surface functionalization. J Mater Chem A 4:18546–18551. Google Scholar
  23. 23.
    Sun XC, Lei Y (2017) Fluorescent carbon dots and their sensing applications. Trends Anal Chem 89:163–180. CrossRefGoogle Scholar
  24. 24.
    Ding CQ, Zhu AW, Tian Y (2014) Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging. Acc Chem Res 47:20–30. CrossRefGoogle Scholar
  25. 25.
    Baker SN, Baker GA (2010) Luminescent carbon nanodots: emergent nanolights. Angew Chem Int Ed 49:6726–6744. CrossRefGoogle Scholar
  26. 26.
    Gowthaman NSK, Sinduja B, Karthikeyan R, Rubini K, John SA (2017) Fabrication of nitrogen-doped carbon dots for screening the purine metabolic disorder in human fluids. Biosens Bioelectron 94:30–38. CrossRefGoogle Scholar
  27. 27.
    Wang YL, Wang ZC, Rui YP, Li MG (2015) Horseradish peroxidase immobilization on carbon nanodots/CoFe layered double hydroxides: direct electrochemistry and hydrogen peroxide sensing. Biosens Bioelectron 64:57–62. CrossRefGoogle Scholar
  28. 28.
    Li Y, Zhao Y, Cheng H, Hu Y, Shi G, Dai L, Qu L (2012) Nitrogen-doped graphene quantum dots with oxygen-rich functional groups. J Am Chem Soc 134:15–18. CrossRefGoogle Scholar
  29. 29.
    Yang YH, Cui JH, Zheng MT, CF H, Tan SZ, Xiao Y, Yang Q, Liu YL (2012) One-step synthesis of amino-functionalized fluorescent carbon nanoparticles by hydrothermal carbonization of chitosan. Chem Commun 48:380–382. CrossRefGoogle Scholar
  30. 30.
    Chowdhury D, Gogoi N, Majumdar G (2012) Fluorescent carbon dots obtained from chitosan gel. RSC Adv 2:12156–12159. CrossRefGoogle Scholar
  31. 31.
    Mao SX, Long YM, Li WF, YF T, Deng AP (2013) Core-shell structured Ag@C for direct electrochemistry and hydrogen peroxide biosensor applications. Biosens Bioelectron 48:258–262. CrossRefGoogle Scholar
  32. 32.
    Wang J, Wang CF, Chen S (2012) Amphiphilic egg-derived carbon dots: rapid plasma fabrication, pyrolysis process, and multicolor printing patterns. Angew Chem Int Ed 51:9297–9301. CrossRefGoogle Scholar
  33. 33.
    Wang SF, Chen T, Zhang ZL, Shen XC, ZX L, Pang DW, Wong KY (2005) Direct electrochemistry and electrocatalysis of heme proteins entrapped in agarose hydrogel films in room-temperature ionic liquids. Langmuir 21:9260–9266. CrossRefGoogle Scholar
  34. 34.
    Adams S, Higgins AM, Jones RAL (2002) Surface-mediated folding and misfolding of proteins at lipid/water interfaces. Langmuir 18:4854–4861. CrossRefGoogle Scholar
  35. 35.
    Kang XH, Wang J, Wu H, Aksay IA, Liu J, Lin YH (2009) Glucose oxidase-graphene-chitosan modified electrode for direct electrochemistry and glucose sensing. Biosens Bioelectron 25:901–905. CrossRefGoogle Scholar
  36. 36.
    Ren XL, Meng XW, Chen D, Tang FQ, Jiao J (2005) Using silver nanoparticle to enhance current response of biosensor. Biosens Bioelectron 21:433–437. CrossRefGoogle Scholar
  37. 37.
    Ko S, Takahashi Y, Fujita H, Tatsuma T, Sakoda A, Komori K (2012) Peroxidase-modified cupstacked carbon nanofiber networks for electrochemical biosensing with adjustable dynamic range. RSC Adv 2:1444–1449. CrossRefGoogle Scholar
  38. 38.
    Harbury HA (1957) Oxidation-reduction potentials of horseradish peroxidase. J Biol Chem 225:1009–1024Google Scholar
  39. 39.
    Dai HX, Lv WJ, Zuo XW, Zhu Q, Pan CJ, Niu XY, Liu JJ, Chen HL, Chen XG (2017) A novel biosensor based on boronic acid functionalized metal-orgnic frameworks for the determination of hydrogen peroxide released from living cells. Biosens Bioelectron 95:131–137. CrossRefGoogle Scholar
  40. 40.
    Ren QQ, Wu J, Zhang WC, Wang C, Qin X, Liu GC, Li ZX, Yu Y (2017) Real-time in vitro detection of cellular H2O2 under camptothecin stress using horseradish peroxidase, ionic liquid, and carbon nanotube-modified carbon fiber ultramicroelectrode. Sensors Actuators B Chem 245:615–621. CrossRefGoogle Scholar
  41. 41.
    Liu H, Guo K, Duan CY, Chen XJ, Zhu ZF (2016) A novel biosensor based on the direct electrochemistry of horseradish peroxidase immobilized in the three dimensional flower-like Bi2WO6 microspheres. Sci Eng C 64:243–248. CrossRefGoogle Scholar
  42. 42.
    CM Y, Wang L, Li WB, Zhu C, Bao N, HY G (2015) Detection of cellular H2O2 in living cells based on horseradish peroxidase at the interface of Au nanoparticles decorated grapheme oxide. Sensors Actuators B Chem 211:17–24. CrossRefGoogle Scholar
  43. 43.
    Song HY, Ni YN, Kokot S (2014) Investigations of an electrochemical platform based on the layered MoS2-graphene and horseradish peroxidase nanocomposite for direct electrochemistry and electrocatalysis. Biosens Bioelectron 56:137–143. CrossRefGoogle Scholar
  44. 44.
    Kaçar C, Dalkiran B, Erden PE, Kiliç E (2014) An amperometric hydrogen peroxide biosensor based on Co3O4 nanoparticles and multiwalled carbon nanotube modified glassy carbon electrode. Appl Surf Sci 311:139–146. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Chemistry, Chemical engineering and Materials ScienceSoochow UniversitySuzhouPeople’s Republic of China
  2. 2.The Key Lab of Health Chemistry and Molecular Diagnosis of SuzhouSuzhouPeople’s Republic of China
  3. 3.State Key Laboratory of Chemo/Biosensing and ChemometricsHunan UniversityChangshaPeople’s Republic of China

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