Elsevier

Biosensors and Bioelectronics

Volume 21, Issue 9, 15 March 2006, Pages 1727-1736
Biosensors and Bioelectronics

Electrochemical detection of DNA hybridization amplified by nanoparticles

https://doi.org/10.1016/j.bios.2005.08.011Get rights and content

Abstract

Detection of specific oligonucleotide (ODN) fragments has become an important field in many areas of biomedicine. We describe a novel ODN sensor based on electropolymerization of a conducting polymer (polypyrrole) in the presence of a sample containing ODN(s). The resulting trapped ODN(s) are then probed by addition of complimentary sequence ODN. By incorporating CdS nanoparticles with the probe, a significant improvement in sensor sensitivity was observed. Impedance spectroscopy suggested that optimal detection of hybridization occurred at frequencies ≥3000 Hz (for a 0.07 cm2 85 nm thick film). At these frequencies, the impedance signal was almost linear with the logarithm of ODN concentration in the range 3.7–370 nM with a detection limit of ∼1 nM ODN (for the sensor fabricated). Importantly, the sensor could be regenerated by removing hybridized ODN with NaOH suggesting possibility of the sensor re-use.

Introduction

The transcription of genes gives rise to messenger RNA (mRNA) which is, in turn, converted to proteins (such as enzymes) which give the cell its functional properties. As a result, detection and measurement of gene expression products is an important analytical tool with applications in areas that include biomedical research, clinical diagnosis and drug development. Most nucleic acid assays generally require sample labeling and complicated analysis procedures. The demand for faster (one-step) label-free gene detection has prompted extensive research into alternative methods that employ a range of readout modalities, including optical (Piunno et al., 1994, Isola et al., 1998), acoustic (Okahata et al., 1992, Caruso et al., 1997) and electronic (Immoos et al., 2004) methods.

Electrochemical DNA detection methods are attractive because they are amenable to direct electrical readout (Wang, 2002). A wide range of methods has been reported using, for example, metal complexes (Takenaka et al., 2000), organic redox indicators (Millan, 1993), enzymes (Carpini et al., 2004), nanoparticles (Wang et al., 2001, Wang et al., 2003, Cao et al., 2002) and nanotubes (Dai, 2004). A large variety of electrode materials have been investigated and several recent review articles summarize progress in this field (Palecek and Jelen, 2002, Kerman et al., 2004, Lucarelli et al., 2004).

Conducting polymers (CP) have been shown to be a versatile substrate for electrochemical DNA detection (Livache et al., 1995, Bidan et al., 2000, Korri-Youssoufi et al., 1997). The advantage of CPs over other electrodes (such as gold or carbon) resides in the perturbations in polymer chain conformation and/or electronic structure caused by the presence of the bioprobe/target conjugate leading to a change in macroscopic material properties (Livache et al., 2001, Gerard et al., 2002). These gene sensors can use covalent attachment of short complementary oligonucleotides (ODNs) to the substrate polymer chains. The change effected by hybridization can then be read out by, for example, cyclic voltammetry (Peng et al., 2005). Wang et al. (1999) described an alternative approach based on DNA hybridization between an unlabeled sample and ODN probes that were trapped in a polypyrrole film after acting as the sole counterion during electropolymerization. The latter approach is attractive since it simplifies sensor construction and does not require potentially lengthy synthesis of functionalized CP derivatives.

Nanoparticles are another class of materials that has been used recently for the construction of biosensors (Alivisatos, 2004, Sutherland, 2002). Commonly, these devices exploit the improved opto-electronic properties of nanoparticles (Murphy, 2002). Some optical sensing principles are based on changes in optical properties resulting from hybridization-dependent particle aggregation (e.g., Storhoff et al., 1998, Chakrabarti and Alexander, 2003). A sensor with opto-electronic readout generated detectable photocurrents in a DNA-cross-linked nanoparticle array (Willner et al., 2001). Very sensitive nanoparticle DNA sensors using purely electrical readout have also been realized (see Wang et al., 2003 for a review). Advantages of electrical detection include the inherent potential for miniaturization and system integration, as well as comparatively simple readout and low cost. The increased sensitivity of these sensors typically results from the use of electrochemical stripping (e.g., Wang et al., 2001) and/or magnetic and catalytic amplification that effectively concentrates nanoparticle–DNA complexes on the electrode. Another convenient electrical readout technique is electrochemical impedance spectroscopy (EIS) (Katz and Willner, 2003) which has been shown to be well suited to hybridization detection between covalently immobilized DNA-probe monolayers (e.g., Gheorghe and Guiseppi-Elie, 2003, Hang and Guiseppi-Elie, 2004). EIS has been used with a nanoparticle-based gene sensor by Xu et al. (2004) who attached DNA probes covalently to a gold electrode and detected impedance changes upon hybridization with nanoparticle-labeled sample DNA.

In this paper we have combined the use of CP substrates and the amplification afforded by semiconductor nanoparticles to construct novel DNA sensors. Sensitive electrical readout was provided by EIS and the sensors exhibited good specificity and mismatch discrimination. In our sensors the unlabeled sample DNA was trapped in a polypyrrole film during electropolymerization (resulting in comparatively simple sensor construction) and the film was then exposed to probe ODNs labeled with CdS nanoparticles. We demonstrate the signal amplification resulting from nanoparticle labeling and investigate the mechanisms by which impedance changes and amplification occur.

Section snippets

Reagents

Pyrrole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), mercaptoacetic acid and phosphate buffered saline pellets (PBS, pH 7.4) were obtained from Aldrich. Before use, pyrrole was distilled under vacuum. The other chemicals used were analytical grade or better. All reagents were used as supplied without further purification, unless otherwise stated.

Custom oligonucleotides were synthesized by Invitrogen Life Technologies Company. The sequences of ODN used in this work are as follows:

Immobilization of ODN target in polypyrrole

Wang and Jiang (2000) have demonstrated that ODNs can act as the sole dopant for polypyrrole films and showed that such films can be used to detect complementary sequences. We have used a similar method to prepare DNA sensor films based on polypyrrole. In the configuration investigated in this paper, pyrrole was electropolymerized in the presence of target ODNs to effectively trap the unlabeled sample in the polypyrrole film. The resulting sensors therefore allowed the detection of label-free

Conclusion

We have demonstrated that PPy films containing trapped DNA fragments used in combination with nanoparticle-labeled ODN probes can be used as effective gene sensors for unlabeled sample ODNs. An advantage of this sensor type resides in its simplified fabrication, since no functionalized pyrrole monomers need to be synthesized. Using quartz crystal microbalance experiments to study hybridization kinetics and efficiency in real-time, we showed that mass amplification can be achieved by labeling

Acknowledgements

The authors thank the Marsden Fund, the University of Auckland Vice-Chancellor's University Development Fund, the New Staff Research Fund and UniServices for financial support. The authors also thank Dr. Stephan Verdier for helpful discussion on AC impedance modeling.

References (41)

  • J. Wang et al.

    New label-free DNA recognition based on doping nucleic-acid probes within conducting polymer films

    Anal. Chim. Acta

    (1999)
  • J. Wang

    Electrochemical detection for microscale analytical systems: a review

    Talanta

    (2002)
  • P. Alivisatos

    The use of nanocrystals in biological detection

    Nat. Biotechnol.

    (2004)
  • G. Bidan et al.

    Electropolymerization as a versatile route for immobilizing biological species onto surfaces. Application to DNA biochips

    Appl. Biochem. Biotechnol.

    (2000)
  • Y.C. Cao et al.

    Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection

    Science

    (2002)
  • R.K. Chakrabarti et al.

    Nanocrystals modified with peptide nucleic acids (PNAs) for selective self-assembly and DNA detection

    J. Am. Chem. Soc.

    (2003)
  • P.H. Dai

    Aligned carbon nanotube–DNA electrochemical sensors

    Chem. Commun.

    (2004)
  • M. Dijksma et al.

    Effect of hexacyanoferrate (II/III) on self-assembled monolayers of thioctic acid and 11-mercaptoundecanoic acid on gold

    Langmuir

    (2002)
  • F. Caruso et al.

    Quartz crystal microbalance study of DNA immobilization and hybridization for nucleic acid sensor development

    Anal. Chem.

    (1997)
  • Cited by (0)

    View full text