Journal of Nanoparticle Research

, 15:1934 | Cite as

Preparation of cholesterol oxidase nanoparticles and their application in amperometric determination of cholesterol

  • Sheetal Chawla
  • Rachna Rawal
  • Sonia
  • Ramrati
  • C. S. PundirEmail author
Research Paper


The nanoparticle (NP) aggregates of commercial cholesterol oxidase (ChOx) were prepared by desolvation method. The formation and characterization of ChOxNP aggregates were studied by transmission electron microscopy and scanning electron microscopy. NP aggregates were more stable, active and had a higher shelf life than that of free enzyme. An amperometric cholesterol biosensor was constructed by immobilizing ChOxNPs onto Au electrode. The biosensor showed optimum response within 8 s at pH 6.0 and 35 °C, when polarized at +0.27 V versus Ag/AgCl. The biosensor possesses high sensitivity and measures cholesterol concentrations as low as 1.56 mg/dl. The working linear range was 12.5–700 mg/dl for cholesterol. The biosensor was evaluated and employed for measurement of total cholesterol in human serum. The enzyme electrode lost 50 % of its initial activity during its regular use for 180 times over a period of 90 days when stored in 0.1 M sodium phosphate buffer, pH 7.0 at 4 °C.


Cholesterol oxidase nanoparticles Cholesterol biosensor Au electrode Serum cholesterol 


  1. Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC (1974) Enzymatic determination of serum total cholesterol. Chin Chem 20(4):470–475Google Scholar
  2. Arvind SSJ, Baby TT, Arockiadoss T, Rakhi RB, Ramprabhu S (2011) Cholesterol biosensor based on gold nanoparticles decorated functionalized graphene nanoplates. Thin solid filmsGoogle Scholar
  3. Bhatia D, Suman, Pundir CS (2005) Preparation of a reusable enzyme strip for determination of serum cholesterol. Ind J Biotechnol 4:471–475Google Scholar
  4. Boller T, Meier C, Menzler S (2002) EUPERGIT oxirane acrylic beads: how to make enzymes fit for biocatalysis. Org Process Res Dev 6:509–519CrossRefGoogle Scholar
  5. Brahim S, Narinesingh D, Guiseppi-Elie A (2001) Amperometric determination of cholesterol in serum using a biosensor of cholesterol oxidase contained within a polypyrrole-hydrogel membrane. Anal Chim Acta 448:27–36CrossRefGoogle Scholar
  6. Cao L, Van-Rantwijk F, Sheldon RA (2000) Cross-linked enzymes aggregates: a simple and effective method for the immobilization of penicillin acylase. Org Lett 2:1361–1364CrossRefGoogle Scholar
  7. Cao L, Van-Langen L, Sheldon RA (2003) Immobilized enzymes: carrier bound or carrier free. Curr Opin Biotechnol 14:387–394CrossRefGoogle Scholar
  8. Chauhan N, Narang J, Pundir CS (2010) Amperometric determination of serum cholesterol with pencil graphite rod. Am J Anal Chem 2:41–46CrossRefGoogle Scholar
  9. Jianping L, Peng T, Peng Y (2006) A cholesterol biosensors based on entrapment of cholesterol oxidase in a silicic sol–gel matrix at a prussian blue modified. Electroanalysis 15:1031–1033Google Scholar
  10. Kundu N, Yadav S, Pundir CS (2012) Preparation and characterization of glucose oxidase nanoparticles and their application in dissolve oxygen metric determination of serum glucose. J Nanosci Nanotechnol 12:1–7CrossRefGoogle Scholar
  11. Li YF, Liu ZM, Liu YL, Yang YH, Shen GL, Yu RQ (2006) A mediator-free phenol biosensor based on immobilizing tyrosinase to ZnO nanoparticles. Anal Biochem 349:33–40CrossRefGoogle Scholar
  12. Liu G, Lin Y, Ostatna V, Wang J (2005) Enzyme nanoparticles-based electronic biosensor. Chem Commun 27:3481–3483CrossRefGoogle Scholar
  13. Nien PC, Chen PY, Ho KC (2009) Fabricating an amperometric cholesterol biosensor by a covalent linkage between poly(3-thiopheneacetic acid) and cholesterol oxidase. Sensors 9:1794–1806CrossRefGoogle Scholar
  14. Norouzi P, Rafiei-Sarmazdeh Z, Faridbod F, Adibi M, Ganjali MR (2010) Er3+ carbon paste electrode based on new nano-composite. Int J Electrochem Soc 5:367–376Google Scholar
  15. Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG, Van-Rantwijk F (2004) Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 87:754–762CrossRefGoogle Scholar
  16. Shah S, Sharma A, Gupta MN (2006) Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder. Anal Biochem 351:207–213CrossRefGoogle Scholar
  17. Sheldon RA (2011) Cross-linked enzyme aggregates as industrial biocatalysts. Org Process Res Dev 15:213–223CrossRefGoogle Scholar
  18. Suman, Pundir CS (2003) Co-immobilization of cholesterol esterase, cholesterol oxidase and peroxidase onto alkylamine glass beads for measurement of total cholesterol in serum. Curr Appl Phys 3:129–133CrossRefGoogle Scholar
  19. Umar A, Rahman MM, Vaseem M, Hahn YB (2009) Ultra-sensitive cholesterol biosensor based on low-temperature grown ZnO nanoparticles. Electrochem Commun 11:118–121CrossRefGoogle Scholar
  20. Van-Langon LM, Selassa RP, Van-Rantwijk F, Sheldon RA (2005) Cross-linked aggregates of R-oxynitrilase: a stable, recyclable biocatalyst for enantioselective hydrocyanation. Org Lett 7:327–329CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Sheetal Chawla
    • 1
  • Rachna Rawal
    • 1
  • Sonia
    • 1
  • Ramrati
    • 1
  • C. S. Pundir
    • 1
    Email author
  1. 1.Department of BiochemistryM. D. UniversityRohtakIndia

Personalised recommendations