Skip to main content
SpringerLink
Log in

Quadruple signal amplification strategy based on hybridization chain reaction and an immunoelectrode modified with graphene sheets, a hemin/G-quadruplex DNAzyme concatamer, and alcohol dehydrogenase: ultrasensitive determination of influenza virus subtype H7N9

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

Abstract

We report on a new amplification strategy for use in an immunoassay for influenza virus subtype H7N9. Graphene sheets were first placed on a glassy carbon electrode (GCE), and gold nanoparticles were then electrodeposited as a support for a layer of alcohol dehydrogenase (ADH) in a sol–gel containing thiol groups. Protein A was used to properly orientate immobilized antibody against H7N9 on the sol–gel, and this is shown to result in strongly improved specificity of the antigen-antibody binding. Thus, a sensitive and specific immunosensor was obtained in which a quadruple signal amplification strategy is employed, viz. (a) via the use of graphene sheets, (b) via a hybridization chain reaction, (c) the use of hemin/G-quadruplex DNAzyme concatamers, and (d) the use of ADH. The hemin/G-quadruplex is a typical DNAzyme, which simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme. The hybridization chain reaction-based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system. Sandwich immunoreactions occurred between the capture antibody on the electrode and the secondary antibody labeled with MWCNTs. Positively charged Methylene Blue (MB) was then used as an intercalator to detect the DNAzyme concatamer formed. The differential pulse voltammetric signals for MB are related to the concentration of H7N9 in the range from 8 to 60 pg · mL−1, and the detection limit is 0.81 pg · mL−1 (at an S/N ratio of 3). This immunoassay is very sensitive, specific and robust.

An electrochemical sandwich immunosensor has been developed for sensitive and specific detection of influenza virus subtype H7N9. Protein A was used to properly orientate antibody. The hybridization chain reaction based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Bimbo LM, Denisova OV, Mäkilä E et al (2013) Inhibition of influenza A virus infection in vitro by saliphenylhalamide-loaded porous silicon nanoparticles. ACS Nano 7(8):6884–6893

    Article  CAS  Google Scholar 

  2. Shen F, Wang J, Xu Z et al (2012) Rapid flu diagnosis using silicon nanowire sensor. Nano Lett 12(7):3722–3730

    Article  CAS  Google Scholar 

  3. Chen Y, Liang W, Yang S et al (2013) Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet 381(9881):1916–1925

    Article  CAS  Google Scholar 

  4. Wei J, Zheng L, Lv X et al (2014) Analysis of influenza viruses receptor specificity using glycan-functionalized gold nanoparticles. ACS Nano 8(5):4600–4607

    Article  CAS  Google Scholar 

  5. Peng D, Hu S, Hua Y et al (2007) Comparison of a new gold-immunochromatographic assay for the detection of antibodies against avian influenza virus with hemagglutination inhibition and agar gel immunodiffusion assays. Vet Immunol Immunopathol 117(1–2):17–25

    Article  CAS  Google Scholar 

  6. Bamrungsap S, Apiwat C, Chantima W et al (2013) Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles. Microchim Acta 181(1–2):223–230

    Google Scholar 

  7. Xu D, Liu L, Guan J et al (2013) Label-free microcantilever-based immunosensors for highly sensitive determination of avian influenza virus H9. Microchim Acta 181(3–4):403–410

    Google Scholar 

  8. Moreno A, Lelli D, Brocchi E et al (2013) Monoclonal antibody-based ELISA for detection of antibodies against H5 avian influenza viruses. J Virol Methods 187(2):424–430

    Article  CAS  Google Scholar 

  9. Uyeki TM, Nguyen DC, Rowe T et al (2012) Seroprevalence of antibodies to avian influenza A (H5) and A (H9) viruses among market poultry workers, Hanoi, Vietnam, 2001. PLoS One 7(8), e43948

    Article  CAS  Google Scholar 

  10. Ellis JS, Zambon MC (2002) Molecular diagnosis of influenza. Rev Med Virol 12(6):375–389

    Article  CAS  Google Scholar 

  11. Yanik AA, Huang M, Kamohara O et al (2010) An optofluidic nanoplasmonic biosensor for direct detection of live viruses from biological media. Nano Lett 10(12):4962–4969

    Article  CAS  Google Scholar 

  12. Yang J, Wen W, Zhang X, Wang S (2015) Electrochemical immunosensor for the prostate specific antigen detection based on carbon nanotube and gold nanoparticle amplification strategy. Microchimica Acta

  13. Bodelón G, Mourdikoudis S, Yate L et al (2014) Nickel Nanopar ticle-doped paper as a bioactive scaffold for targeted and robust immobilization of functional proteins. ACS Nano 8(6):6221–6231

    Article  Google Scholar 

  14. Tsai WC, Pai PJR (2009) Surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin B. Microchim Acta 166(1–2):115–122

    Article  CAS  Google Scholar 

  15. De M, Ghosh PS, Rotello VM (2008) Applications of Nanoparticles in Biology. Adv Mater 20(22):4225–4241

    Article  CAS  Google Scholar 

  16. Xia C, Li Y, Yuan G, Guo Y, Yu C (2015) Immunoassay for serum amyloid A using a glassy carbon electrode modified with carboxy-polypyrrole, multiwalled carbon nanotubes, ionic liquid and chitosan. Microchim Acta

  17. Yi H, Xu W, Yuan Y et al (2014) A pseudo triple-enzyme cascade amplified aptasensor for thrombin detection based on hemin/G-quadruplex as signal label. Biosens Bioelectron 54:415–420

    Article  CAS  Google Scholar 

  18. Liu L, Jiang S, Wang L, Zhang Z, Xie G (2014) Direct detection of microRNA-126 at a femtomolar level using a glassy carbon electrode modified with chitosan, graphene sheets, and a poly(amidoamine) dendrimer composite with gold and silver nanoclusters. Microchim Acta 182(1–2):77–84

    Google Scholar 

  19. Huang KJ, Li J, Liu Y et al (2012) Disposable immunoass ay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate. Microchim Acta 177:419–426

    Article  CAS  Google Scholar 

  20. Ran XQ, Yuan R, Chai YQ et al (2010) A sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/DNA/Thi/nano-Au modified glassy carbon electrode. Colloids Surf B 79(2):421–426

    Article  CAS  Google Scholar 

  21. Pérez-López B, Merkoçi A (2012) Carbon nanotubes and graphene in analytical sciences. Microchim Acta 179(1–2):1–16

    Article  Google Scholar 

  22. Yi H, Xu W, Yuan Y et al (2013) A sensitive electrochemical aptasensor for thrombin detection based on exonuclease-catalyzed target recycling and enzyme-catalysis. Biosens Bioelectron 47:368–372

    Article  CAS  Google Scholar 

  23. Peng K, Zhao H, Yuan Y, Yuan R, Wu X (2014) Mediator-free triple-enzyme cascade electrocatalytic aptasensor with exonuclease-assisted target recycling and hybridization chain reaction amplification. Biosens Bioelectron 55:366–371

    Article  CAS  Google Scholar 

  24. Zhang Y, Li B, Jin Y (2011) Label-free fluorescent detection of thrombin using G-quadruplex-based DNAzyme as sensing platform. Analyst 136(16):3268–3273

    Article  CAS  Google Scholar 

  25. Chen Y, Yang M, Xiang Y et al (2014) Binding-induced autonomous disassembly of aptamer-DNAzyme supersandwich nanostructures for sensitive electrochemiluminescence turn-on detection of ochratoxin A. Nanoscale 6(2):1099–1104

    Article  CAS  Google Scholar 

  26. Fu X, Huang R, Wang J, Chang B (2013) Sensitive electrochemical immunoassay of a biomarker based on biotin-avidin conjugated DNAzyme concatamer with signal tagging. RSC Adv 3(32):13451–13456

    Article  CAS  Google Scholar 

  27. Yuan Y, Chai Y, Yuan R et al (2013) An ultrasensitive electrochemical aptasensor with autonomous assembly of hemin-G-quadruplex DNAzyme nanowires for pseudo triple-enzyme cascade electrocatalytic amplification. Chem Commun 49(66):7328–7330

    Article  CAS  Google Scholar 

  28. Zhou J, Xu M, Tang D et al (2012) Nanogold-based bio-bar codes for label-free immunosensing of proteins coupling with an in situ DNA-based hybridization chain reaction. Chem Commun 48(100):12207–12209

    Article  CAS  Google Scholar 

  29. Zhang J, Chai Y, Yuan R et al (2013) A novel electrochemical aptasensor for thrombin detection based on the hybridization chain reaction with hemin/G-quadruplex DNAzyme-signal amplification. Analyst 138(16):4558–4564

    Article  CAS  Google Scholar 

  30. Jena BK, Raj CR (2006) Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles. Anal Chem 78:6332–6339

    Article  CAS  Google Scholar 

  31. Song C, Xie G, Wang L et al (2014) DNA-based hybridization chain reaction for an ultrasensitive cancer marker EBNA-1 electrochemical immunosensor. Biosens Bioelectron 58:68–74

    Article  CAS  Google Scholar 

  32. Wan Y, Su Y, Zhu X et al (2013) Development of electrochemical immunosensors towards point of care diagnostics. Biosens Bioelectron 47:1–11

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No.81171415) and the Education Commission of Chongqing Municipality (No. KJ1400211).

Author information

Authors and Affiliations

  1. Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Juan Yang, Chao Song, Lingzhi Liu, Xiaoying Jing, Guoming Xie & Hua Xiang

  2. Department of Chemistry, Tsinghua University, Beijing, 100084, People’s Republic of China

    Yu Xiang

Authors
  1. Juan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chao Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lingzhi Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoying Jing
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guoming Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hua Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Guoming Xie or Hua Xiang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 11427 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, J., Xiang, Y., Song, C. et al. Quadruple signal amplification strategy based on hybridization chain reaction and an immunoelectrode modified with graphene sheets, a hemin/G-quadruplex DNAzyme concatamer, and alcohol dehydrogenase: ultrasensitive determination of influenza virus subtype H7N9. Microchim Acta 182, 2377–2385 (2015). https://doi.org/10.1007/s00604-015-1583-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00604-015-1583-8

Keywords

Search

Navigation