Electrochimica Acta 53 (2007) 803–810 Development of a membrane-based electrochemical immunosensor Guiwan Koh, Shuchi Agarwal, Pui-Sze Cheow, Chee-Seng Toh ∗ Department of Chemistry, Faculty of Science, National University of Singapore, Singapore 117543, Singapore Received 31 May 2007; received in revised form 23 July 2007; accepted 25 July 2007 Available online 31 July 2007 Abstract A membrane-based electrochemical immunosensor sensitive towards proteins is described, based on nanoporous alumina-modified platinum wire electrodes. The sensing mechanism depends on the blocking of pore channels when the protein antigen molecules bind to antibody molecules attached to the channel walls, impeding the diffusion of redox probe, ferrocenemethanol, towards the sensing platinum wire overlaid by the nanoporous alumina film. The antibody and antigen used in this work were anti-glucose oxidase and glucose oxidase, respectively. The immunosensor showed a low limit of detection of 100 ng L -1 antigen concentration and was selective towards glucose oxidase protein in the presence of another protein, glucothione S-transferase. © 2007 Elsevier Ltd. All rights reserved. Keywords: Alumina; Immunosensor; Glucose oxidase; Nanostructure; Immunoglobulin 1. Introduction Development of immunosensors with capability for rapid, sensitive and selective detection of infectious diseases, contin- ues to be an important subject for research and development [1–4]. It is equally desirable to have fast response sensing capa- bility towards other analytes, including proteins, DNAs and haptens in environmental studies, pharmaceutical applications and biomedical diagnostics, as long as their complementary binding immunoglobulins can be produced. Current methods used by in vitro immunoassay such as ELISA incorporates immunoglobulins or antigens tagged with markers into appro- priate biorecognition materials and coupled to a transducer such as optical [5], fluorescence [6] or electrochemical sensor [7–9]. Typically, these techniques give a linear response of 10 gL -1 to 150 mg L -1 and detection limit of 10 gL -1 [10]. Herein, we describe a method for measuring the amount of protein antigen based on monitoring the magnitude of diffu- sion limited faradiac current of a redox probe diffusing within narrow channels of a nanoporous alumina matrix. Nanoporous alumina is a highly regular, rigid and dense porous material with nominal pore sizes ranging from 10 to 200 nm pore density of about 1 × 10 10 pores cm -2 [11,12] and is chemically and ther- ∗ Corresponding author. Tel.: +65 6516 3887; fax: +65 6779 1691. E-mail address: chmtohcs@nus.edu.sg (C.-S. Toh). mally stable [13]. These features are relatively easy to achieve and inexpensive by comparison to conventional lithographic techniques. We reason that these same features could be used to trap specific-binding antibody such as immunoglobulin G within the confined spaces of the vertical channels within an alumina matrix. In addition, these immunoglobulin G coated channels could function as diffusion paths for a redox probe, ferrocenemethanol, chosen for its neutral charge and electro- chemically reversible behaviour. Fig. 1 shows the basic design of the biosensor which explains its scheme of operation. A layer of aluminum (ca. 400 nm thick) was sputtered onto a home-made platinum wire electrode tip and anodized to alumina using a pipette anodization method, which yields barrier-free alumina [14]. A sub-monolayer or monolayer of immunoglob- ulin G was then immobilized along the nano-channel walls of the porous alumina, followed by immobilization of bovine serum albumin (BSA) to block the unspecific adsorption sites. The alumina-modified platinum wire electrode was subse- quently used for antigen detection, in the presence of the redox probe. Upon binding the complementary antigen to the immunoglobulin G, formation of the antigen–antibody (Ag–Ab) complexes blocked the approach of ferrocenemethanol towards the exposed platinum surface beneath the porous alumina layer. Differential pulse voltammetry (DPV) was employed to mon- itor the faradiac current limited by the diffusion rate of the redox probe diffusing towards the underlying platinum elec- trode. 0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.07.055