Electrochemical detection of DNA hybridization amplified by nanoparticles
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:
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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)
- et al.
Oligonucleotide-modified screen-printed gold electrodes for enzyme-amplified sensing of nucleic acids
Biosens. Bioelectron.
(2004) - et al.
Application of conducting polymers to biosensors
Biosens. Bioelectron.
(2002) - et al.
Electrical frequency dependent characterization of DNA hybridization
Biosens. Bioelectron.
(2003) - et al.
Polypyrrole electrospotting for the construction of oligonucleotide arrays compatible with a surface plasmon resonance hybridization detection
Synth. Met.
(2001) - et al.
Biosensing effects in functionalized electroconducting conjugated polymer layers: addressable DNA matrix for the detection of gene mutations
Synth. Met.
(1995) - et al.
Carbon and gold electrodes as electrochemical transducers for DNA hybridization sensors
Biosens. Bioelectron.
(2004) - et al.
New materials for electrochemical sensing. V. Nanoparticles for DNA labeling
Trends Anal. Chem.
(2005) - et al.
Label-free electrochemical DNA sensor based on functionalised conducting copolymer
Biosens. Bioelectron.
(2005) - et al.
Fiber optic biosensor for fluorimetric detection of DNA hybridization
Anal. Chim. Acta
(1994) Quantum dots as luminescent probes in biological systems
Curr. Opin. Solid State Mater. Sci.
(2002)