Microchimica Acta

, 174:97 | Cite as

Biocompatible and label-free amperometric immunosensor for hepatitis B surface antigen using a sensing film composed of poly(allylamine)-branched ferrocene and gold nanoparticles

Original Paper


An amperometric immunosensor has been developed for sensitive determination of hepatitis B surface antigen as a model protein. A glassy carbon electrode was modified with an assembly of positively charged poly(allylamine)-branched ferrocene (PAA-Fc) and negatively charged gold nanoparticles (Au NPs). The formation of PAA-Fc effectively avoids the leakage of Fc, retains its electrochemical activity, and enhances the conductivity of the composite. The adsorption of Au NPs onto the PAA-Fc matrix provides sites for the immobilization of the antigen and a favorable micro-environment to maintain its activity. The morphologies and electrochemistry of the sensing film were investigated via scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Factors influencing the performance of the immunosensor were studied in detail. The concentration of the antigen can be quantitated (by measuring the decrease of the amperometric response resulting from the specific binding between antigen and antibody) in the range between 0.1 and 150 ng mL–1, with a detection limit of 40 pg mL–1 (S/N = 3). The method is economical, efficient, and potentially attractive for clinical immunoassays.


A novel and sensitive amperometric immunosensor based on the assembly of biocompatible positively charged poly(allylamine)-branched ferrocene and negatively charged Au nanoparticles onto a glassy carbon electrode has been developed for sensitive determination of hepatitis B surface antigen as a model protein.


Amperometric immunosensor Poly(allylamine) Ferrocene Gold nanoparticles Hepatitis B surface antigen 

Supplementary material

604_2011_585_MOESM1_ESM.doc (96 kb)
ESM 1(DOC 95 kb)


  1. 1.
    Gitlin N (1997) Hepatitis B: diagnosis, prevention, and treatment. Clin Chem 43:1500–1506Google Scholar
  2. 2.
    Satoh K, Iwata-Takakura A, Yoshikawa A, Gotanda Y, Tanaka T, Yamaguchi T, Mizoguchi H (2008) A new method of concentrating hepatitis B virus (HBV) DNA and HBV surface antigen: an application of the method to the detection of occult HBV infection. Vox Sang 95:174–180CrossRefGoogle Scholar
  3. 3.
    Lee HS, Han CJ, Kim CY (1993) Predominant etiologic association of hepatitis C virus with hepatocellular carcinoma compared with hepatitis B virus in elderly patients in a hepatitis B-endemic area. Cancer 72:2564–2567CrossRefGoogle Scholar
  4. 4.
    Zhang S, Zou J, Yu F (2008) Investigation of voltammetric enzyme-linked immunoassay based on a new system of HAP-H2O2-HRP. Talanta 76:122–127CrossRefGoogle Scholar
  5. 5.
    Siitari H, Hemmila I, Soini E, Lovgren T, Koistinen V (1983) Detection of hepatitis B surface antigen using time-resolved fluoroimmunoassay. Nature 301:258–260CrossRefGoogle Scholar
  6. 6.
    Chen D, Kaplan LA (2006) Performance of a new-generation chemiluminescent assay for hepatitis B surface antigen. Clin Chem 52:1592–1598CrossRefGoogle Scholar
  7. 7.
    Wu J, Fu Z, Yan F, Ju H (2007) Biomedical and clinical applications of immunoassays and immunosensors for tumor markers. TrAC Trends Anal Chem 26:679–688CrossRefGoogle Scholar
  8. 8.
    Liu ZY, Yuan R, Chai YQ, Zhuo Y, Hong CL, Yang X (2008) Highly sensitive, reagentless amperometric immunosensor based on a novel redox-active organic-inorganic composite film. Sens Actuators B 134:625–631CrossRefGoogle Scholar
  9. 9.
    Aguilar ZP, Vandaveer WR, Fritsch I (2002) Self-contained microelectrochemical immunoassay for small volumes using mouse IgG as a model system. Anal Chem 74:3321–3329CrossRefGoogle Scholar
  10. 10.
    Yuan R, Zhang L, Li Q, Chai Y, Cao S (2005) A label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of Japanese B encephalitis vaccine. Anal Chim Acta 531:1–5CrossRefGoogle Scholar
  11. 11.
    Berggren C, Johansson G (1997) Capacitance measurements of antibody-antigen interactions in a flow system. Anal Chem 69:3651–3657CrossRefGoogle Scholar
  12. 12.
    Yeo J, Park JY, Bae WJ, Lee YS, Kim BH, Cho Y, Park SM (2009) Label-free electrochemical detection of the p53 core domain protein on its antibody immobilized electrode. Anal Chem 81:4770–4777CrossRefGoogle Scholar
  13. 13.
    Chua JH, Chee RE, Agarwal A, Wong SM, Zhang GJ (2009) Label-free electrical detection of cardiac biomarker with complementary metal-oxide semiconductor-compatible silicon nanowire sensor arrays. Anal Chem 81:6266–6271CrossRefGoogle Scholar
  14. 14.
    Zhang J, Lei JP, Xu CL, Ding L, Ju HX (2010) Carbon nanohorn sensitized electrochemical immunosensor for rapid detection of microcystin-LR. Anal Chem 82:1117–1122CrossRefGoogle Scholar
  15. 15.
    Zhao G, Zhan X (2010) Facile preparation of disposable immunosensor for Shigella flexneri based on multi-wall carbon nanotubes/chitosan composite. Electrochim Acta 55:2466–2471CrossRefGoogle Scholar
  16. 16.
    Santandreu M, Alegret S, Fàbregas E (1999) Determination of [beta]-HCG using amperometric immunosensors based on a conducting immunocomposite. Anal Chim Acta 396:181–188CrossRefGoogle Scholar
  17. 17.
    Huang KJ, Sun JY, Xu CX, Niu DJ, Xie WZ (2010) A disposable immunosensor based on gold colloid modified chitosan nanoparticles-entrapped carbon paste electrode. Microchim Acta 168:51–58CrossRefGoogle Scholar
  18. 18.
    Zhang LY, Liu Y, Chen T (2009) Label-free amperometric immunosensor based on antibody immobilized on a positively charged gold nanoparticle/L-cysteine-modified gold electrode. Microchim Acta 164:161–166CrossRefGoogle Scholar
  19. 19.
    Li N, Yuan R, Chai Y, Chen S, An H, Li W (2007) New antibody immobilization strategy based on gold nanoparticles and Azure I/multi-walled carbon nanotube composite membranes for an amperometric enzyme immunosensor. J Phys Chem C 111:8443–8450CrossRefGoogle Scholar
  20. 20.
    Yuan Y, Yuan R, Chai Y, Zhuo Y, Shi Y, He X, Miao X (2007) A reagentless amperometric immunosensor for alpha-fetoprotein based on gold nanoparticles/TiO2 colloids/prussian blue modified platinum electrode. Electroanalysis 19:1402–1410CrossRefGoogle Scholar
  21. 21.
    Lv P, Min LG, Yuan R, Chai YQ, Chen SH (2010) A novel immunosensor for carcinoembryonic antigen based on poly(diallyldimethylammonium chloride) protected prussian blue nanoparticles and double-layer nanometer-sized gold particles. Microchim Acta 171:297–304CrossRefGoogle Scholar
  22. 22.
    Das J, Jo K, Lee JW, Yang H (2007) Electrochemical immunosensor using p-aminophenol redox cycling by hydrazine combined with a low background current. Anal Chem 79:2790–2796CrossRefGoogle Scholar
  23. 23.
    Tripathi VS, Kandimalla VB, Ju HX (2006) Amperometric biosensor for hydrogen peroxide based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite. Biosens Bioelectron 21:1529–1535CrossRefGoogle Scholar
  24. 24.
    Qiu JD, Wang R, Liang RP, Xia XH (2009) Electrochemically deposited nanocomposite film of CS-Fc/Au NPs/GOx for glucose biosensor application. Biosens Bioelectron 24:2920–2925CrossRefGoogle Scholar
  25. 25.
    Liu D, Liu H, Hu N (2010) pH-controllable bioelectrocatalysis of glucose by glucose oxidase loaded in weak polyelectrolyte layer-by-layer films with ferrocene derivative as mediator. Electrochim Acta 55:6426–6432CrossRefGoogle Scholar
  26. 26.
    Zhang SX, Fu YQ, Sun CQ (2003) Fabrication of poly(allylamine)ferrocene monolayer based on electrostatic interaction and its electrocatalytic oxidation of ascorbic acid. Electroanalysis 15:739–746CrossRefGoogle Scholar
  27. 27.
    Deng L, Liu Y, Yang G, Shang L, Wen D, Wang F, Xu Z, Dong S (2007) Molecular “wiring” glucose oxidase in supramolecular architecture. Biomacromolecules 8:2063–2071CrossRefGoogle Scholar
  28. 28.
    Ho JA, Chang HC, Shih NY, Wu LC, Chang YF, Chen CC, Chou C (2010) Diagnostic detection of human lung cancer-associated antigen using a gold nanoparticle-based electrochemical immunosensor. Anal Chem 82:5944–5950CrossRefGoogle Scholar
  29. 29.
    Yuan YR, Yuan R, Chai YQ, Zhuo Y, Miao XM (2009) Electrochemical amperometric immunoassay for carcinoembryonic antigen based on bi-layer nano-Au and nickel hexacyanoferrates nanoparticles modified glassy carbon electrode. J Electroanal Chem 626:6–13CrossRefGoogle Scholar
  30. 30.
    Cui RJ, Huang HP, Yin ZZ, Gao D, Zhu JJ (2008) Horseradish peroxidase-functionalized gold nanoparticle label for amplified immunoanalysis based on gold nanoparticles/carbon nanotubes hybrids modified biosensor. Biosens Bioelectron 23:1666–1673CrossRefGoogle Scholar
  31. 31.
    Wang F, Hu SS (2009) Electrochemical sensors based on metal and semiconductor nanoparticles. Microchim Acta 165:1–22CrossRefGoogle Scholar
  32. 32.
    Hodak J, Etchenique R, Calvo EJ, Singhal K, Bartlett PN (1997) Layer-by-layer self-assembly of glucose oxidase with a poly(allylamine)ferrocene redox mediator. Langmuir 13:2708–2716CrossRefGoogle Scholar
  33. 33.
    Qiu JD, Peng HZ, Liang RP, Li J, Xia XH (2007) Synthesis, characterization, and immobilization of prussian blue-modified Au nanoparticles: application to electrocatalytic reduction of H2O2. Langmuir 23:2133–2137CrossRefGoogle Scholar
  34. 34.
    Siebrands T, Giersig M, Mulvaney P, Fischer CH (1993) Steric exclusion chromatography of nanometer-sized gold particles. Langmuir 9:2297–2300CrossRefGoogle Scholar
  35. 35.
    Hu MH, Yamaguchi Y, Okubo T (2005) Self-assembly of water-dispersed gold nanoparticles stabilized by a thiolated glycol derivative. J Nanopart Res 7:187–193CrossRefGoogle Scholar
  36. 36.
    Zhou YL, Li YZ (2004) Studies of interaction between poly(allylamine hydrochloride) and double helix DNA by spectral methods. Biophys Chem 107:273–281CrossRefGoogle Scholar
  37. 37.
    Park MK, Deng SX, Advincula RC (2004) pH-sensitive bipolar ion-permselective ultrathin films. J Am Chem Soc 126:13723–13731CrossRefGoogle Scholar
  38. 38.
    Burke SE, Barrett CJ (2004) pH-dependent loading and release behavior of small hydrophilic molecules in weak polyelectrolyte multilayer films. Macromolecules 37:5375–5384CrossRefGoogle Scholar
  39. 39.
    Huang HZ, Yang XR (2003) Chitosan mediated assembly of gold nanoparticles multilayer. Colloids Surf A 226:77–86CrossRefGoogle Scholar
  40. 40.
    Chen J, Yan F, Du D, Wu J, Ju HX (2006) Electrochemical immunoassay of human chorionic gonadotrophin based on its immobilization in gold nanoparticles-chitosan membrane. Electroanalysis 18:670–676CrossRefGoogle Scholar
  41. 41.
    Wu Y, Zheng JW, Li Z, Zhao YR, Zhang Y (2009) A novel reagentless amperometric immunosensor based on gold nanoparticles/TMB/Nafion-modified electrode. Biosens Bioelectron 24:1389–1393CrossRefGoogle Scholar
  42. 42.
    Zhuo Y, Yuan R, Chai YQ, Tang DP, Zhang Y, Wang N, Li XL, Zhu Q (2005) A reagentless amperometric immunosensor based on gold nanoparticles/thionine/Nafion-membrane-modified gold electrode for determination of alpha-1-fetoprotein. Electrochem Commun 7:355–360CrossRefGoogle Scholar
  43. 43.
    Zhuo Y, Yuan PX, Yuan R, Chai YQ, Hong CL (2008) Nanostructured conductive material containing ferrocenyl for reagentless amperometric immunosensors. Biomaterials 29:1501–1508CrossRefGoogle Scholar
  44. 44.
    Zhou L, Yuan R, Chai YQ (2007) On-off PVC membrane based potentiometric immunosensor for label-free detection of alpha-fetoprotein. Electroanalysis 19:1131–1138CrossRefGoogle Scholar
  45. 45.
    Qiu JD, Liang RP, Wang R, Fan LX, Chen YW, Xia XH (2009) A label-free amperometric immunosensor based on biocompatible conductive redox chitosan-ferrocene/gold nanoparticles matrix. Biosens Bioelectron 25:852–857CrossRefGoogle Scholar
  46. 46.
    Lee HJ, Namkoong K, Cho EC, Ko C, Park JC, Lee SS (2009) Surface acoustic wave immunosensor for real-time detection of hepatitis B surface antibodies in whole blood samples. Biosens Bioelectron 24:3120–3125CrossRefGoogle Scholar
  47. 47.
    Liang RP, Peng HZ, Qiu JD (2008) Fabrication, characterization, and application of potentiometric immunosensor based on biocompatible and controllable three-dimensional porous chitosan membranes. J Colloid Interface Sci 320:125–131CrossRefGoogle Scholar
  48. 48.
    Yuan R, Tang DP, Chai YQ, Zhong X, Liu Y, Dai JY (2004) Ultrasensitive potentiometric immunosensor based on SA and OCA techniques for immobilization of HBsAb with colloidal Au and polyvinyl butyral as matrixes. Langmuir 20:7240–7245CrossRefGoogle Scholar
  49. 49.
    Hanaee H, Ghourchian H, Ziaee AA (2007) Nanoparticle-based electrochemical detection of hepatitis B virus using stripping chronopotentiometry. Anal Biochem 370:195–200CrossRefGoogle Scholar
  50. 50.
    Tang DY, Xia BY (2008) Electrochemical immune bioassay for the antigen–antibody interaction based on Fe(CN)64−/3− and AuCl4 ions-derivated biomimetic interface. Ionics 14:329–334CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Department of Chemistry and Institute for Advanced StudyNanchang UniversityNanchangPeople’s Republic of China

Personalised recommendations