Short communicationNanoelectrode ensembles as recognition platform for electrochemical immunosensors
Introduction
Nanoelectrode ensembles (NEEs) are new nanoelectrochemical tools very useful for electroanalysis and sensors (Menon and Martin, 1995, Ugo et al., 2002). They are prepared by electroless deposition of gold within the pores of track-etch polycarbonate membranes. A NEE is made by a very large number of very small ultramicroelectrodes confined in a rather small space, with a density ≥108 electrodes/cm2. NEEs can exhibit distinct voltammetric response regimes depending on the scan rate or distance between the nanoelectrode elements. The total overlap regime is commonly observed at ensembles of nanodisk electrodes prepared from commercial track-etched membranes. Under these conditions, the faradaic current (signal) is proportional to the geometric area (Ageom; area of nanodisks and polycarbonate), while the double-layer capacitive current (background) depends on the active area (Aact; area of the nanodisks). Therefore, NEEs are characterized by detection limits two to three orders of magnitude lower than regular electrodes (Ugo et al., 1996, Brunetti et al., 2000).
In typical schemes used for electrochemical biosensors, a biorecognition layer is immobilized directly on the electrode surface and the signal is produced by exchange of electrons with the underlying electrode; this was applied also to arrays of nanoelectrodes (Lin et al., 2004, Li et al., 2003, Lapierre-Devlin et al., 2005, Delvaux et al., 2005). However, for extremely miniaturized electrodes, such as NEEs, the amount of biomolecule immobilized on the nanoelectrodes can be too small to furnish useful signals (De Leo et al., 2007a). In order to increase the electrode area available for the immobilization, the template membrane can be etched (Yu et al., 2003, Lapierre-Devlin et al., 2005, Krishnamoorthy and Zoski, 2005) to obtain ensembles of gold nanofibers. However, this causes the increase of capacitive current and lowering of signal-to-background current ratios (De Leo et al., 2007a).
This prompted us to explore a different approach, using the template membrane of the ensemble and not the nanoelectrodes to immobilize the biorecognition elements. In such a design no increase in Aact is required and voltammetric signals should be produced at the highest signal/background current ratio.
On the basis of its popularity in classical ELISA tests as well as in advanced electrochemical immunoassays (Yu et al., 2006) we chose horseradish peroxidase (HRP) as the enzyme label, using methylene blue (MB) as the redox mediator (in the solution phase) to shuttle electrons from the nanoelectrodes to the label. We tested this approach with an extremely actual issue that is the determination of the expression levels of the HER2 receptor and the binding activity of its agonist trastuzumab (or Herceptin®), a drug used in the adjuvant therapy of the breast cancer (Molina et al., 2001). The possibility to detect HER2 is extremely important for the identification of cancers that can be treated with Herceptin®, providing a good opportunity to define the so-called personalized therapies.
Section snippets
Electrochemical apparatus
All electroanalytical measurements were carried out at room temperature (22 ± 1 °C) with a CH660A potentiostat, using a three-electrode single-compartment cell equipped with a platinum counter electrode and an Ag/AgCl (KCl-saturated) reference electrode.
Chemicals
All chemicals used were reagent grade and used without further purification. HRP type VI, 298 purpurogallin units/mg solid, was from Sigma. Purified water was obtained using a Milli-Ro plus Milli-Q (Millipore) system.
Sensors
NEEs were prepared by template
Results and discussion
Preliminary tests in solution showed that the best mediator suitable to shuttle electron from NEE to HRP is MB.
The dotted line cyclic voltammetry (CV) in Fig. 1, shows a well-resolved reduction peak recorded at a NEE, relevant to the reversible reduction (Ye and Baldwin, 1988):MB + 2e + H+ ⇆ LBwhere LB is the leuco (reduced) form of MB.
MB signals are significantly better resolved from background currents at a NEE than at a conventional Au-electrode, under the same experimental conditions (not shown).
Conclusions
This is, up to now, the first report demonstrating the usefulness of NEE as detection platforms for immunosensors where the high signal/background ratio typical of nanoelectrodes is preserved. This is particularly attractive for detection of trace proteins such as HER2. T-NEEs detect the target protein in diluted samples, where traditional immunochemical methods, such as Western blotting, fail. Specialized studies for quantifying the analytical performances of T-NEEs (detection limit, dynamic
Acknowledgments
We thank Prof. Giuseppe Firrao (University of Udine) for helpful discussion and MUR (Rome), PRIN 2006, for financial support.
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