Skip to main content
SpringerLink
Log in

Probe accessibility effects on the performance of electrochemical biosensors employing DNA monolayers

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

Abstract

Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets.

Here we demonstrate that the density of the DNA probe monolayer is a crucial determinant of the final signalling of DNA-bases sensors used for the detection of proteins, enzymes, and other macromolecules.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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
Fig. 6

Similar content being viewed by others

References

  1. Katz E, Willner I (2003) Probing biomolecular interactions at conductive and semiconductive surfaces by impedance spectroscopy: Routes to impedimetric immunosensors, DNA-sensors and enzyme biosensors. Electroanalysis 15(11):913–947

    Article  CAS  Google Scholar 

  2. Liu J, Cao Z, Lu Y (2009) Functional nucleic acid sensors. Chem Rev 109(5):1948–1998

    Article  CAS  Google Scholar 

  3. Wang J (2000) From DNA biosensors to gene chips. Nucleic Acids Res 28(16):3011–3016

    Article  CAS  Google Scholar 

  4. Ricci F, Volpe G, Micheli L, Palleschi G (2007) A review on novel developments and applications of immunosensors in food analysis. Anal Chim Acta 605(2):111–129

    Article  CAS  Google Scholar 

  5. Drummond TG, Hill MG, Barton JK (2003) Electrochemical DNA sensors. Nat Biotechnol 21(10):1192–1199

    Article  CAS  Google Scholar 

  6. Wang J (2002) Electrochemical nucleic acid biosensors. Anal Chim Acta 469(1):63–71

    Article  CAS  Google Scholar 

  7. Homola J (2003) Present and future of surface plasmon resonance biosensors. Anal Bioanal Chem 377(3):528–539

    Article  CAS  Google Scholar 

  8. Lequin RM (2005) Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 51(12):2415–2418

    Article  CAS  Google Scholar 

  9. Ricci F (2008) Plaxco KW (2008) E-DNA sensors for convenient, label-free electrochemical detection of hybridization. Microchim Acta 163(3–4):149–155

    Article  CAS  Google Scholar 

  10. Cosnier S (1999) Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. Biosens Bioelectron 14(5):443–456

    Article  CAS  Google Scholar 

  11. Gooding JJ, Mearns F, Yang W, Liu J (2003) Self-assembled monolayers into the 21st century: Recent advances and applications. Electroanalysis 15(2):81–96

    Article  CAS  Google Scholar 

  12. Fan C, Plaxco KW, Heeger AJ (2003) Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc Natl Acad Sci U S A 100(16):9134–9137

    Article  CAS  Google Scholar 

  13. Kelley SO, Boon EM, Barton JK, Jackson NM, Hill MG (1999) Single-base mismatch detection based on charge transduction through DNA. Nucleic Acids Res 27(24):4830–4837

    Article  CAS  Google Scholar 

  14. Tosar JP, Brañas G, Laíz J (2010) Electrochemical DNA hybridization sensors applied to real and complex biological samples. Biosens Bioelectron 26(4):1205–1217

    Article  CAS  Google Scholar 

  15. Park J, Park S (2009) DNA hybridization sensors based on electrochemical impedance spectroscopy as a detection tool. Sensors 9(12):9513–9532

    Article  CAS  Google Scholar 

  16. Vercoutere W, Akeson M (2002) Biosensors for DNA sequence detection. Curr Opin Chem Biol 6(6):816–822

    Article  CAS  Google Scholar 

  17. Jayasena SD (1999) Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clin Chem 45(9):1628–1650

    CAS  Google Scholar 

  18. Willner I, Zayats M (2007) Electronic aptamer-based sensors. Ang Chemie Int Ed 46(34):6408–6418

    Article  CAS  Google Scholar 

  19. Xiao Y, Rowe AA, Plaxco KW (2007) Electrochemical detection of parts-per-billion lead via an electrode-bound DNAzyme assembly. J Am Chem Soc 129(2):262–263

    Article  CAS  Google Scholar 

  20. Xiao Y, Piorek BD, Plaxco KW, Heeger AJ (2005) A reagentless signal-on architecture for electronic, aptamer-based sensors via target-induced strand displacement. J Am Chem Soc 127(51):17990–17991

    Article  CAS  Google Scholar 

  21. Xiao Y, Lubin AA, Heeger AJ, Plaxco KW (2005) Label-free electronic detection of thrombin in blood serum by using an aptamer-based sensor. Ang Chemie Int Ed 44(34):5456–5459

    Article  CAS  Google Scholar 

  22. Radi A, Acero Sánchez JL, Baldrich E, O'Sullivan CK (2006) Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor. J Am Chem Soc 128(1):117–124

    Article  CAS  Google Scholar 

  23. Strehlitz B, Nikolaus N, Stoltenburg R (2008) Protein detection with aptamer biosensors. Sensors 8(7):4296–4307

    Article  CAS  Google Scholar 

  24. Qureshi A, Gurbuz Y, Kallempudi S, Niazi JH (2010) Label-free RNA aptamer-based capacitive biosensor for the detection of C-reactive protein. Phys Chem Chem Phys 12(32):9176–9182

    Article  CAS  Google Scholar 

  25. Ricci F, Bonham AJ, Mason AC, Reich NO, Plaxco KW (2009) Reagentless, electrochemical approach for the specific detection of double- and single-stranded DNA binding proteins. Anal Chem 81(4):1608–1614

    Article  CAS  Google Scholar 

  26. Ricci F, Adornetto G, Moscone D, Plaxco KW, Palleschi G (2010) Quantitative, reagentless, single-step electrochemical detection of anti-DNA antibodies directly in blood serum. Chem Comm 46(10):1742–1744

    Article  CAS  Google Scholar 

  27. Wang J, Onoshima D, Aki M, Okamoto Y, Kaji N, Tokeshi M et al (2011) Label-free detection of DNA-binding proteins based on microfluidic solid-state molecular beacon sensor. Anal Chem 83(9):3528–3532

    Article  CAS  Google Scholar 

  28. Cash KJ, Ricci F, Plaxco KW (2009) An electrochemical sensor for the detection of protein–small molecule interactions directly in serum and other complex matrices. J Am Chem Soc 131(20):6955–6857

    Article  CAS  Google Scholar 

  29. Cash KJ, Ricci F, Plaxco KW (2009) A general electrochemical method for label-free screening of protein–small molecule interactions. Chem Commun 41:6222–6224

    Article  Google Scholar 

  30. Rant U, Pringsheim E, Kaiser W, Arinaga K, Knezevic J, Tornow M, Fujita S, Yokoyama N, Abstreiter G (2009) Detection and size analysis of proteins with switchable DNA layers. Nano Lett 9(4):1290–1295

    Article  CAS  Google Scholar 

  31. Tort N, Salvador JP, Eritja R, Poch M, Martınez E, Samitier J, Marco MP (2009) Fluorescence site-encoded DNA addressable hapten microarray for anabolic androgenic steroids.Trends. Anal Chem 28(6):718–728

    CAS  Google Scholar 

  32. Chen S, Phillips MF, Cerrina F, Smith LM (2009) Controlling oligonucleotide surface density in light-directed DNA array fabrication. Langmuir 25(11):6570–6575

    Article  CAS  Google Scholar 

  33. Kjällman THM, Peng H, Soeller C, Travas-Sejdic J (2008) Effect of probe density and hybridization temperature on the response of an electrochemical hairpin-DNA sensor. Anal Chem 80(24):9460–9466

    Article  Google Scholar 

  34. White RJ, Phares N, Lubin AA, Xiao Y, Plaxco KW (2008) Optimization of electrochemical aptamer-based sensors via optimization of probe packing density and surface chemistry. Langmuir 24(18):10513–10518

    Article  CAS  Google Scholar 

  35. Ricci F, Lai RY, Heeger AJ, Plaxco KW, Sumner JJ (2007) Effect of molecular crowding on the response of an electrochemical DNA sensor. Langmuir 23(12):6827–6834

    Article  CAS  Google Scholar 

  36. Peterson AW, Heaton RJ, Georgiadis RM (2001) The effect of surface probe density on DNA hybridization. Nucleic Acids Res 29(24):5163–5168

    Article  CAS  Google Scholar 

  37. Lee C, Gong P, Harbers GM, Grainger DW, Castner DG, Gamble LJ (2006) Surface coverage and structure of mixed DNA/Alkylthiol monolayers on gold. Characterization by XPS, NEXAFS, and fluorescence intensity measurements. Anal Chem 78(10):3316–3325

    Article  CAS  Google Scholar 

  38. Liao TH, Salnikow J, Moore S, Stein WH (1973) Bovine pancreatic deoxyribonuclease A. isolation of cyanogen bromide peptides; complete covalent structure of the polypeptide chain. J Biol Chem 248(4):1489–1495

    CAS  Google Scholar 

  39. Xiao Y, Lai RY, Plaxco KW (2007) Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat Protoc 2(11):2875–2880

    Article  CAS  Google Scholar 

  40. Phares N, White RJ, Plaxco KW (2009) Improving the stability and sensing of electrochemical biosensors by employing trithiol-anchoring groups in a six-carbon self-assembled monolayer. Anal Chem 81(3):1095–1100

    Article  CAS  Google Scholar 

  41. O'Connor SD, Olsen GT, Creager SE (1999) Nernstian electron source model for the ac voltammetric response of a reversible surface redox reaction using large-amplitude ac voltages. J Electroanal Chem 466(2):197–202

    Article  Google Scholar 

  42. Creager SE, Wooster TT (1998) A new way of using ac voltammetry to study redox kinetics in electroactive monolayers. Anal Chem 70(20):4257–4263

    Article  CAS  Google Scholar 

  43. Sumner JJ, Weber KS, Hockett LA, Creager SE (2000) Long-range heterogeneous electron transfer between ferrocene and gold mediated by n-alkane and W-aikyl-carboxamide bridges. J Phys Chem B 104(31):7449–7454

    Article  CAS  Google Scholar 

  44. Lubin AA, Plaxco KW (2010) Folding-based electrochemical biosensors: The case for responsive nucleic acid architectures. Acc Chem Res 43(4):496–505

    Article  CAS  Google Scholar 

  45. Castronovo M, Radovic S, Grunwald C, Casalis L, Morgante M, Scoles G (2008) Control of steric hindrance on restriction enzyme reactions with surface-bound DNA nanostructures. Nano Lett 8(12):4140–4145

    Article  CAS  Google Scholar 

  46. Castronovo M, Lucesoli A, Parisse P, Kurnikova A, Malhotra A, Grassi M et al (2011) Two-dimensional enzyme diffusion in laterally confined DNA monolayers. Nature Comm 2(1)

  47. Palanisamy R, Connolly AR, Trau M (2010) Considerations of solid-phase DNA amplification. Bioconjug Chem 21(4):690–695

    Article  CAS  Google Scholar 

  48. McCalla SE, Luryi AL, Tripathi A (2009) Steric effects and mass-transfer limitations surrounding amplification reactions on immobilized long and clinically relevant DNA templates. Langmuir 25(11):6168–6175

    Article  CAS  Google Scholar 

  49. Bar M, Bar-Ziv RH (2009) Spatially resolved DNA brushes on a chip: Gene activation by enzymatic cascade. Nano Lett 9(12):4462–4466

    Article  CAS  Google Scholar 

  50. Ricci F, Zari N, Caprio F, Recine S, Amine A, Moscone D et al (2009) Surface chemistry effects on the performance of an electrochemical DNA sensor. Bioelectron 76(1–2):208–213

    CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge members of our research groups for helpful discussions and comments on the manuscript. This work was supported by the Italian Ministry of University and Research (MIUR) through the project FIRB “Futuro in Ricerca”.

Author information

Authors and Affiliations

  1. Dipartimento di Scienze e Tecnologie Chimiche, University of Rome, Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy

    Vanessa Biagiotti, Alessandro Porchetta, Sara Desiderati, Giuseppe Palleschi & Francesco Ricci

  2. Consorzio Interuniversitario Biostrutture e Biosistemi “INBB”, Viale Medaglie d’Oro 305, 00136, Rome, Italy

    Giuseppe Palleschi & Francesco Ricci

  3. Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA, 93106, USA

    Kevin W. Plaxco

Authors
  1. Vanessa Biagiotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alessandro Porchetta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sara Desiderati
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kevin W. Plaxco
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Giuseppe Palleschi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Francesco Ricci
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francesco Ricci.

Additional information

Vanessa Biagiotti and Alessandro Porchetta contributed equally to this work.

Published in the 10th Anniversary Issue.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Biagiotti, V., Porchetta, A., Desiderati, S. et al. Probe accessibility effects on the performance of electrochemical biosensors employing DNA monolayers. Anal Bioanal Chem 402, 413–421 (2012). https://doi.org/10.1007/s00216-011-5361-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5361-0

Keywords

Search

Navigation