Nanoelectrode ensembles as recognition platform for electrochemical immunosensors

Abstract

In this study we demonstrate the possibility to prepare highly sensitive nanostructured electrochemical immunosensors by immobilizing biorecognition elements on nanoelectrode ensembles (NEEs) prepared in track-etch polycarbonate membranes. The gold nanodisk electrodes act as electrochemical transducers while the surrounding polycarbonate binds the antibody-based biorecognition layer. The interaction between target protein and antibody is detected by suitable secondary antibodies labelled with a redox enzyme. A redox mediator, added to the sample solution, shuttles electrons from the nanoelectrodes to the biorecognition layer, so generating an electrocatalytic signal. This allows one to fully exploit the highly improved signal-to-background current ratio, typical of NEEs. In particular, the receptor protein HER2 was studied as the target analyte. HER2 detection allows the identification of breast cancer that can be treated with the monoclonal antibody trastuzumab. NEEs were functionalized with trastuzumab which interacts specifically with HER2. The biorecognition process was completed by adding a primary antibody and a secondary antibody labelled with horseradish peroxidase. Hydrogen peroxide was added to modulate the label electroactivity; methylene blue was the redox mediator generating voltammetric signals. NEEs functionalized with trastuzumab were tested to detect small amounts of HER2 in diluted cell lysates and tumour lysates.

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 (A_geom; area of nanodisks and polycarbonate), while the double-layer capacitive current (background) depends on the active area (A_act; 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 A_act 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.