An ultrasensitive electrochemiluminescence immunosensor for alpha-fetoprotein based on a poly(aniline-luminol)/graphene oxide nanocomposite

  • Lu Wei
  • Yanhui Zhang
  • Nurguzal Eziz
  • Yaru Yang
  • Guixin LiEmail author
  • Ming Guan
Research Paper


An ultrasensitive electrochemiluminescence (ECL) immunosensor for alpha-fetoprotein (AFP) detection based on a poly(aniline-luminol)/graphene oxide nanocomposite was developed. The nanocomposite, which was prepared using a fast and simple chemical oxidation strategy for the first time, showed excellent ECL performance. This outstanding ECL performance is due to the formation of poly(aniline-luminol) on the graphene oxide (GO) surface and the excellent electron-transfer properties of GO. Moreover, the poly(aniline-luminol)/graphene oxide nanocomposite has abundant amino groups at its surface, making it a good platform for biomacromolecule labeling. Using the nanocomposite, a novel ECL immunosensor for the determination of AFP was successfully developed. Anti-AFP was immobilized on the surface of a poly(aniline-luminol)/graphene oxide nanocomposite-modified electrode using a glutaraldehyde crosslinking method to form the ECL immunosensor. The AFP was then captured at the modified electrode surface through an antigen–antibody immunoreaction. When the AFP was captured by its antibody, the ECL intensity decreased. This ECL immunosensor for the detection of AFP exhibited a linear range of 1.7 × 10−12 to 1.7 × 10−8 mg mL−1 and a detection limit of 5 × 10−13 mg mL−1, indicating high sensitivity and linearity across a wide concentration range. Furthermore, the immunosensor was successfully applied to determine AFP in a real-world human serum sample.

Graphical abstract

A new poly(aniline-luminol)/graphene oxide nanocomposite was prepared using a fast and simple strategy for the first time, and an ultrasensitive electrochemiluminescence (ECL) immunosensor for alpha-fetoprotein (AFP) detection based on this nanocomposite was developed


Alpha-fetoprotein Electrochemiluminescence Immunosensor Chemical oxidation Poly(aniline-luminol)/graphene oxide nanocomposite 



This research was financially supported by the National Natural Science Foundation of China (no. 21665025), the Outstanding Young Science and Technology Personnel Training Project of the Xinjiang Uyghur Autonomous Region (no. QN2016YX0306), and Key Projects of the Engineering Research Center of Electrochemical Technology and Application of Xinjiang Normal Universitly (XJNUGCZX122017A01); all of this support is gratefully acknowledged.

Compliance with ethical standards

Ethics approval and consent to participate

All procedures performed in this study involving human participants were in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments, and were approved by the Ethical Committee of Xinjiang Normal University. All blood samples were from healthy persons, and informed consent was obtained from all individual participants included in the study.

Human and animal rights

No violation of human or animal rights occurred during this investigation.

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary material

216_2019_1897_MOESM1_ESM.pdf (3 mb)
ESM 1 (PDF 2.96 mb)


  1. 1.
    Liu XX, Song XD, Dong ZY, Meng XT, Chen YP, Yang L. Photonic crystal fiber-based immunosensor for high-performance detection of alpha fetoprotein. Biosens Bioelectron. 2017;91:431–5.CrossRefGoogle Scholar
  2. 2.
    Xu R, Jiang YD, Xia L, Zhang TX, Xu L, Zhang S, et al. A sensitive photoelectrochemical biosensor for AFP detection based on ZnO inverse opal electrodes with signal amplification of CdS-QDs. Biosens Bioelectron. 2015;74:411–7.CrossRefGoogle Scholar
  3. 3.
    Yuan YL, Li SS, Xue YW, Liang JT, Cui LJ, Li QB, et al. A Fe3O4@Au-based pseudo-homogeneous electrochemical immunosensor for AFP measurement using AFP antibody-GNPs-HRP as detection probe. Anal Biochem. 2017;534:56–63.Google Scholar
  4. 4.
    Sun JS, Tian DZ, Guo QJ, Zhang L, Jiang WT, Yang MH. A label-free electrochemical immunosensor for the detection of cancer biomarker α-fetoprotein (AFP) based on hydroxyapatite induced redox current. Anal Methods. 2016;8:7319–23.CrossRefGoogle Scholar
  5. 5.
    Liang JM, Wang J, Zhang LW, Wang SJ, Yao CP, Zhang ZX. Conductometric immunoassay of alpha-fetoprotein in sera of liver cancer patients using bienzyme-functionalized nanometer-sized silica beads. Analyst. 2019;144:265–73.CrossRefGoogle Scholar
  6. 6.
    Jiao L, Mu ZG, Zhu CZ, Wei Q, Li H, Du D. Graphene loaded bimetallic Au@Pt nanodendrites enhancing ultrasensitive electrochemical immunoassay of AFP. Sensors Actuators B. 2016;231:513–9.CrossRefGoogle Scholar
  7. 7.
    Yu ZZ, Cai GN, Ren RR, Tang DP. A new enzyme immunoassay for alpha-fetoprotein in a separate setup coupling an aluminium/Prussian blue-based self-powered electrochromic display with a digital multimeter readout. Analyst. 2018;143:2992–6.CrossRefGoogle Scholar
  8. 8.
    Tsai HY, Li SY, Fuh CB. Magnetofluorescent nanocomposites and quantum dots used for optimal application in magnetic fluorescence-linked immunoassay. Anal Bioanal Chem. 2018;410:1923–9.CrossRefGoogle Scholar
  9. 9.
    Chen JX, Zhao GC. A novel signal-on photoelectrochemical immunosensor for detection of alpha-fetoprotein by in situ releasing electron donor. Biosens Bioelectron. 2017;98:155–60.CrossRefGoogle Scholar
  10. 10.
    Shen HY, Wang XQ, Wang YY, Qu F. Immunoassay for alpha fetal protein in serum based on quartz capillary materials by gray level analysis. Chin J Anal Chem. 2017;45:83–8.Google Scholar
  11. 11.
    Babamiri B, Hallaj R, Salimi A, Akhtari K. Potential-resolved electrochemiluminescence immunoassay for simultaneous determination of CEA and AFP tumor markers using dendritic nanoclusters and Fe3O4@SiO2 nanoparticles. Microchim Acta. 2017;184:3613–23.CrossRefGoogle Scholar
  12. 12.
    Wang JR, Guo XL, Li HJ, Jin YC, Chen LS, Kang Q. A signal-on electrochemiluminescence immunosensor for detecting alpha fetoprotein using gold nanoparticle-graphite-like carbon nitride nanocomposite as signal probe. Int J Electrochem Sci. 2017;12:9784–97.CrossRefGoogle Scholar
  13. 13.
    Zhang SP, Zang LL, Zhang XZ, Dai H, Xu GF, Zhang QR, et al. Signal-on electrochemiluminescent immunosensor based on poly(amidoamine) dendrimer functionalized carbon nanodots amplification for ultrasensitive detection of α-fetoprotein. Electrochim Acta. 2016;196:67–74.CrossRefGoogle Scholar
  14. 14.
    Xiao LJ, Chai YQ, Wang HJ, Yuan R. Electrochemiluminescence immunosensor using poly(l-histidine)-protected glucose dehydrogenase on Pt/Au bimetallic nanoparticles to generate an in situ co-reactant. Analyst. 2014;139:4044–50.Google Scholar
  15. 15.
    Zheng HL, Yi H, Lin W, Dai H, Hong ZS, Lin YY, et al. A dual-amplified electrochemiluminescence immunosensor constructed on dual-roles of rutile TiO2 mesocrystals for ultrasensitive zearalenone detection. Electrochim Acta. 2018;260:847–54.CrossRefGoogle Scholar
  16. 16.
    Guo ZY, Sha YH, Hu YF, Yu ZQ, Tao YY, Wu YJ, et al. Faraday cage-type electrochemiluminescence immunosensor for ultrasensitive detection of Vibrio vulnificus based on multi-functionalized graphene oxide. Anal Bioanal Chem. 2016;408:7203–11.Google Scholar
  17. 17.
    Gao HF, Wen LK, Wu YH, Fu ZF, Wu G. An ultrasensitive label-free electrochemiluminescent immunosensor for measuring Cry1Ab level and genetically modified crops content. Biosens Bioelectron. 2017;97:122–7.CrossRefGoogle Scholar
  18. 18.
    Pur MRK, Hosseini M, Faridbod F, Dzefuli AS, Ganjali MR. A novel solid-state electrochemiluminescence sensor for detection of cytochrome c based on ceria nanoparticles decorated with reduced graphene oxide nanocomposite. Anal Bioanal Chem. 2016;408:7193–202.CrossRefGoogle Scholar
  19. 19.
    Liu C, Wei XH. Tu YF. Development of a reagentless electrochemiluminescent electrode for flow injection analysis using copolymerised luminol/aniline on nano-TiO2 functionalised indium-tin oxide glass. Talanta. 2013;111:156–62.CrossRefGoogle Scholar
  20. 20.
    Ferreira V, Cascalheira AC, Abrantes LM. Electrochemical preparation and characterisation of poly(luminol-aniline) films. Thin Solid Films. 2008;516:3996–4001.Google Scholar
  21. 21.
    Wei XH, Xiao CB, Liu C, Wang K, Tu YF. The sensitized solid-phase electrochemiluminescence of electrodeposited poly-luminol/aniline on AuAg/TiO2 nanohybrid functionalized electrode for flow injection analysis. Electroanalysis. 2014;26:807–14.CrossRefGoogle Scholar
  22. 22.
    Wang YJ, Li GX, Zheng XW. Electrochemiluminescence performance of poly(aniline-luminol) composite nanowires synthesized by chemical oxidation. Chin J Anal Chem. 2015;43:141–5.Google Scholar
  23. 23.
    Xiao CB, Zhao Q, Tu YF. An innovative route to prepare the all-solid-state electrochemiluminescent electrode—using the nano-rods of luminol/aniline copolymer. Electrochim Acta. 2014;147:791–6.Google Scholar
  24. 24.
    Zheng J, Ma XF, He XC, Gao MJ, Li G. Preparation, characterizations, and its potential applications of PANi/graphene oxide nanocomposite. Procedia Eng. 2012;27:1478–87.Google Scholar
  25. 25.
    Binh Phan T, Thanh Luong T, Mai TX, Thanh Thuy Mai T. Effect of nanostructured graphene oxide on electrochemical activity of its composite with polyaniline titanium dioxide. Adv Nat Sci-Nanosci. 2016;7:1–5.Google Scholar
  26. 26.
    Mohamed MA, Atty SA, Merey HA, Fattah TA, Foster CW, Banks CE. Titanium nanoparticles (TiO2)/graphene oxide nanosheets (GO): an electrochemical sensing platform for the sensitive and simultaneous determination of benzocaine in the presence of antipyrine. Analyst. 2017;142:3674–9.CrossRefGoogle Scholar
  27. 27.
    Li J, Liu S, Yu JH, Lian WJ, Cui M, Xu W, et al. Electrochemical immunosensor based on graphene-polyaniline composites and carboxylated graphene oxide for estradiol detection. Sensors Actuators B. 2013;188:99–105.CrossRefGoogle Scholar
  28. 28.
    Wu HP, Lin SL, Chen CX, Liang W, Liu XY, Yang HX. A new ZnO/rGO/polyaniline ternary nanocomposite as photocatalyst with improved photocatalytic activity. Mater Res Bull. 2016;83:434–41.CrossRefGoogle Scholar
  29. 29.
    Li M, Yin WC, Han XL, Chang XQ. Hierarchical nanocomposites of polyaniline scales coated on graphene oxide sheets for enhanced supercapacitors. J Solid State Electrochem. 2016;20:1941–8.CrossRefGoogle Scholar
  30. 30.
    Vellaichamy B, Periakaruppan P, Ponnaiah SK. A new in-situ synthesized ternary CuNPs-PANI-GO nano composite for selective detection of carcinogenic hydrazine. Sensors Actuators B. 2017;245:156–65.CrossRefGoogle Scholar
  31. 31.
    Wang JD, Wang XY, Tang HS, Gao ZH, He SQ, Li J, et al. Ultrasensitive electrochemical detection of tumor cells based on multiple layer CdS quantum dots-functionalized polystyrene microspheres and graphene oxide-polyaniline composite. Biosens Bioelectron. 2017;100:1–7.CrossRefGoogle Scholar
  32. 32.
    Gao K, Sun Z, Pan BG, Qiao XW, Hong CL. Electrochemical immunosensor for alpha-fetoprotein based on Prussian blue-carbon nanotube@polydopamine. Micro Nano Lett. 2018;13:58–62.Google Scholar
  33. 33.
    Li YK, Feng T, Deng SM, Wang XF, Xie MX. Development of magnetic and fluorescent immune sensors for the detection of alpha fetoprotein. Spectrosc Spectr Anal. 2017;37:1667–72.Google Scholar
  34. 34.
    Kuila T, Bose S, Mishra AK, Khanra P, Kim NH, Lee JH. Chemical functionalization of graphene and its applications. Prog Mater Sci. 2012;57:1061–105.CrossRefGoogle Scholar
  35. 35.
    Han ZL, Li F, Shu JN, Gao LF, Liu XY, Cui H. Acridinium ester functionalized carbon nanomaterials: general synthesis strategy and outstanding chemiluminescence. ACS Appl Mater Interfaces. 2016;8:17454–60.CrossRefGoogle Scholar
  36. 36.
    Wang ZY, Guo WY, Di JW, Tu YF. The studies of electrochemical polymerization of luminol and the electrochemiluminescence properties on indium-tin oxide glass electrode. Chin J Anal Chem. 2005;33:763–6.Google Scholar
  37. 37.
    He S, Liang JM, Hu HL, Yuan JN, He JB, Ni ZF. Preparation of graphene oxide/chitosan layer-by-layer self-assembled composite film and its application in environmental protection. Mater Rev. 2016;30:1–5.Google Scholar
  38. 38.
    Yu M, Liu PR, Sun YJ, Liu JH, An JW, Li SM. Fabrication and characterization of graphene-Ag nanoparticles composites. J Inorg Mater. 2012;27:89–94.Google Scholar
  39. 39.
    Wang LL, Xing RG, Zhang BW, Hou Y. Preparation and electrochemical properties of functionalized graphene/polyaniline composite electrode materials. Acta Phys-Chim Sin. 2014;30:1659–66.Google Scholar
  40. 40.
    Jain M, Annapoorni S. Raman study of polyaniline nanofibers prepared by interfacial polymerization. Synth Met. 2010;160:1727–32.CrossRefGoogle Scholar
  41. 41.
    Al-Mashat L, Shin K, Kalantar-Zadeh K, Plessis JD, Han SH, Kojima RW, et al. Graphene/polyaniline nanocomposite for hydrogen sensing. J Phys Chem C. 2010;114:16168–73.CrossRefGoogle Scholar
  42. 42.
    Li GX, Lian JL, Zheng XW, Cao J. Electrogenerated chemiluminescence biosensor for glucose based on poly(luminol-aniline) nanowires composite modified electrode. Biosens Bioelectron. 2010;26:643–8.CrossRefGoogle Scholar
  43. 43.
    Haapakka KE, Kankare JJ. The mechanism of the electrogenerated chemiluminescence of luminol in aqueous alkaline solution. Anal Chim Acta. 1982;138:263–75.CrossRefGoogle Scholar
  44. 44.
    Chang YB, Feng YJ, Yang XL, He Q. The preparation of immunosensor modified by Pt-Ni alloy nanotubes. J Anal Sci. 2016;32:529–32.CrossRefGoogle Scholar
  45. 45.
    Yang SH, Zhang FF, Wang ZH, Liang QL. A graphene oxide-based label-free electrochemical aptasensor for the detection of alpha-fetoprotein. Biosens Bioelectron. 2018;112:186–92.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Lu Wei
    • 1
  • Yanhui Zhang
    • 1
  • Nurguzal Eziz
    • 2
  • Yaru Yang
    • 1
  • Guixin Li
    • 1
    Email author
  • Ming Guan
    • 1
  1. 1.Engineering Research Center of Electrochemical Technology and Application, School of Chemistry and Chemical EngineeringXinjiang Normal UniversityUrumqiChina
  2. 2.School of ScienceHotan Teachers CollegeHotanChina

Personalised recommendations