We have demonstrated the electrochemistry of a redox active species, Fe(CN)(6)(4-), flowing in a nitrocellulose membrane incorporated into two plates with a pole array and electrodes, and applied it by measuring the activity of an enzyme, glucose oxidase (GOx), captured by immunoreaction to develop an electrochemical immunochromatographic format. The flow in the membrane between the flat surfaces with electrodes is unstable because the solution seeps into the surfaces by capillary force and the substrate support and electrode materials have different wettabilities. Thus, electrochemical responses obtained were also unstable. we prepared the small pole array and incorporated a microelectrode in a pole to diminish the total contact area used to support the membrane. The solution flowed uniformly and stably in the membrane supported on the plates with the pole array, and a steady-state oxidation current of Fe(CN)(6)(4-) was observed by amperometry. The mouse IgG used as a model target was captured in the antibody immobilization area prepared in the membrane. Subsequent flowing of GOx to conjugate the antibody allowed the labeling of the captured target by the formation of a sandwich-type immunocomplex. Fe(CN)(6)(4-) produced through the enzyme reaction in the presence of glucose flowed downstream to give an electrochemical response with the electrode arranged 2 mm from the antibody immobilization area. Electrochemical detection of redox species flowing in the membrane indicated the possibility for developing a single-step immunochromatographic assay format with electrochemical quantitation. (c) 2012 Elsevier Ltd. All rights reserved.