Skip to main content

Advertisement

SpringerLink
Log in

Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the conjugates.

DNA sensing platform based on an immobilized ssDNA capture probe and a hybrid ssDNA-dsDNA detection probe that specifically hybridize with the ssDNA target. The hybrid ssDNA-dsDNA detection probe also provides the binding sites for the enzyme-DNA binding protein conjugates (HRP-scCro and GOx-dHP) that generate the colorimetric signal

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Jain KK. Nanodiagnostics: application of nanotechnology in molecular diagnostics. Expert Rev Mol Diagn. 2014;3:153–61.

    Article  Google Scholar 

  2. Shields D, Dhabale S. 2014. https://www.alliedmarketresearch.com/DNA-diagnostics-market. Accessed 10 Oct 2016.

  3. Kiechle FL. Molecular pathology and infectious diseases. In: Grody WW, Nakaruma RM, Strom CM, Kiechle FL, editors. Molecular diagnostics: techniques and applications for the clinical laboratory. London: Academic Press; 2010. p. 99–106.

    Chapter  Google Scholar 

  4. Pieretti M. Signal amplification methods in molecular diagnostics. In: Grody WW, Nakaruma RM, Strom CM, Kiechle FL, editors. Molecular diagnostics: techniques and applications for the clinical laboratory. London: Academic Press; 2010. p. 15–9.

    Chapter  Google Scholar 

  5. Aktas GB, Skouridou V, Masip L. Novel signal amplification approach for HRP-based colorimetric genosensors using DNA binding protein tags. Biosens Bioelectron. 2015;74:1005–10.

    Article  CAS  Google Scholar 

  6. Ariga K, Ji Q, Mori T, Naito M, Yamauchi Y, Abe H, Hill JP. Enzyme nanoarchitectonics: organization and device application. Chem Soc Rev. 2013;42:6322–45.

    Article  CAS  Google Scholar 

  7. Riccardi CM, Mistri D, Hart O, Anuganti M, Lin Y, Kasi RM, Kumar CV. Covalent interlocking of glucose oxidase and peroxidase in the voids of paper: enzyme–polymer “spider webs”. Chem Commun. 2016;52:2593–6.

    Article  CAS  Google Scholar 

  8. Küchler A, Yoshimoto M, Luginbühl S, Mavelli F, Walde P. Enzymatic reactions in confined environments. Nat Nanotechnol. 2016;11:409–20.

    Article  Google Scholar 

  9. Wu D, Ren X, Hu L, Fan D, Zheng Y, Wei Q. Electrochemical aptasensor for the detection of adenosine by using PdCu@MWCNTs-supported bienzymes as labels. Biosens Bioelectron. 2015;74:391–7.

    Article  CAS  Google Scholar 

  10. Lai YH, Lee CC, King CC, Chuang MC, Ho JAA. Exploitation of stem-loop DNA as a dual-input gene sensing platform: extension to subtyping of influenza A viruses. Chem Sci. 2014;5:4082–90.

    Article  CAS  Google Scholar 

  11. Xin L, Zhou C, Yang Z, Liu D. Regulation of an enzyme cascade reaction by a DNA machine. Small. 2013;9:3088–91.

    Article  CAS  Google Scholar 

  12. Civit L, Fragoso A, Hölters S, Dürst M, O’Sullivan CK. Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples. Anal Chim Acta. 2012;715:93–8.

    Article  CAS  Google Scholar 

  13. Gao F, Courjean O, Mano N. An improved glucose/O2 membrane-less biofuel cell through glucose oxidase purification. Biosens Bioelectron. 2009;25:356–61.

    Article  CAS  Google Scholar 

  14. Hudson JM, Fried MG. Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter. J Mol Biol. 1990;214:381–96.

    Article  CAS  Google Scholar 

  15. Kalodimos CG, Folkers GE, Boelens R, Kaptein R. Strong DNA binding by covalently linked dimeric Lac headpiece: evidence for the crucial role of the hinge helices. Proc Natl Acad Sci U S A. 2001;98:6039–44.

    Article  CAS  Google Scholar 

  16. Spronk CA, Folkers GE, Noordman AM, Wechselberger R, van den Brink N, Boelens, Kaptein R. Hinge-helix formation and DNA bending in various lac repressor-operator complexes. EMBO J. 1999;18(22):6472–80.

    Article  CAS  Google Scholar 

  17. Kohen A, Jonsson T, Klinman JP. Effect of protein glycosylation on catalysis: changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction. Biochemistry. 1997;36:2603–11.

    Article  CAS  Google Scholar 

  18. Munteanu MG, Vlahovicek K, Parthasaraty S, Simon I, Pongor S. Rod models of DNA: sequence-dependent anisotropic elastic modelling of local bending phenomena. Trends Biochem Sci. 1998;23:341–6.

    Article  CAS  Google Scholar 

  19. Manera M, Miro M, Estela JM, Cerdá V. A multisyringe flow injection system with immobilized glucose oxidase based on homogeneous chemiluminescence detection. Anal Chim Acta. 2004;508:23–30.

    Article  CAS  Google Scholar 

  20. Wilner OI, Weizmann Y, Gill R, Lioubashevski O, Freeman R, Willner I. Enzyme cascades activated on topologically programmed DNA scaffolds. Nat Nanotechnol. 2009;4:249–54.

    Article  CAS  Google Scholar 

  21. Freeman R, Sharon E, Teller C, Wilner I. Control of biocatalytic transformations by programmed DNA assemblies. Chem Eur J. 2010;16:3690–8.

    Article  CAS  Google Scholar 

  22. Frías IAM, Avelino KYPS, Silva RR, Andrade CAS, Oliveira MDL. Trends in biosensors for HPV: identification and diagnosis. J Sens. 2015; doi:10.1155/2015/913640.

    Google Scholar 

  23. Ortiz M, Torréns M, Alakulppi N, Strömbom L, Fragoso A, O’Sullivan CK. Amperometric supramolecular genosensor self-assembled on cyclodextrin-modified surfaces. Electrochem Commun. 2011;13:578–81.

    Article  CAS  Google Scholar 

  24. Bartolome JP, Echegoyen L, Fragoso A. Reactive carbon nano-onion modified glassy carbon surfaces as DNA sensors for human papillomavirus oncogene detection with enhanced sensitivity. Anal Chem. 2015;87:6744–51.

    Article  CAS  Google Scholar 

  25. Zhao Z, Tapec-Dytioco R, Tan W. Unltrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles. J Am Chem Soc. 2003;125:11474–5.

    Article  CAS  Google Scholar 

  26. Riccò R, Meneghello A, Enrichi F. Signal enhancement in DNA microarray using dye doped silica nanoparticles: application to human papilloma virus (HPV) detection. Biosens Bioelectron. 2011;26:2761–5.

    Article  Google Scholar 

  27. Lee CC, Liao YC, Lai YH, Chuang MC. Recognition of dual targets by a molecular beacon-based sensor: subtyping of influenza A virus. Anal Chem. 2015;87:5410–6.

    Article  CAS  Google Scholar 

  28. Li H, Rothberg L. Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc Nat Ac Sci USA. 2004;101:14036–9.

    Article  CAS  Google Scholar 

  29. Li J, Song S, Liu X, Wang L, Pan D, Huang Q, Zhao Y. Enzyme-based multi-component optical nanoprobes for sequence-specific detection of DNA hybridization. Adv Mater. 2008;20:497–500.

    Article  CAS  Google Scholar 

  30. Ma C, Wang W, Mulchandani A, Shi C. A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification. Anal Biochem. 2014;457:19–23.

    Article  CAS  Google Scholar 

  31. Lu S, Hu T, Wang S, Sun J, Yang X. Ultra-sensitive colorimetric assay system based on the hybridization chain reaction-triggered enzyme cascade amplification. ACS Appl Mater Interfaces. 2017;9:167–75.

    Article  CAS  Google Scholar 

  32. Sun J, Ge J, Liu W, Lan M, Zhang H, Wang P, Wang Y, Niu Z. Multi-enzyme co-embedded organic-inorganic hybrid nanoflowers: synthesis and application as a colorimetric sensor. Nano. 2014;6:255–62.

    CAS  Google Scholar 

  33. Fu J, Liu M, Liu Y, Woodbury NW, Yan H. Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J Am Chem Soc. 2012;134:5516–9.

    Article  CAS  Google Scholar 

  34. Müller J, Niemeyer CM. DNA-directed assembly of artificial multienzyme complexes. Biochem Biophys Res Commun. 2008;377:62–7.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported financially by the FP7-PEOPLE-2011-CIG DeCoDeB project grant awarded to LM. GBA acknowledges the Universitat Rovira i Virgili for the doctoral fellowship. The authors thank Dr. Gert Folkers (Utrecht University, Netherlands) for the generous gift of the construct containing the LacI headpiece domain gene.

Author information

Authors and Affiliations

  1. Bioengineering and Bioelectrochemistry Group, Interfibio, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain

    Gülsen Betül Aktas, Vasso Skouridou & Lluis Masip

Authors
  1. Gülsen Betül Aktas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vasso Skouridou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lluis Masip
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lluis Masip.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(PDF 569 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aktas, G.B., Skouridou, V. & Masip, L. Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures. Anal Bioanal Chem 409, 3623–3632 (2017). https://doi.org/10.1007/s00216-017-0304-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-017-0304-z

Keywords

Search

Navigation