Ultrasensitive electrochemical detection of biotin using electrically addressable site-oriented antibody immobilization approach via aminophenyl boronic acid

A common approach towards developing immunoassays is to attach antibodies onto the surfaces of assay devices via a solid support. When directly adsorbed onto surfaces, however, antibodies generally adopt random orientations and therefore, often fail to exhibit their immunoaffinity. To preserve the antigen-binding activity of antibodies, there is an urgent need to develop specific and novel linking chemistries for attaching the antibodies to the solid surfaces in an oriented manner. In this paper, we report 2 alternative immobilization methods to enhance the orientation of antibodies onto screen-printed graphite electrodes (SPGEs). The first approach involves the deposition of gold nanoparticles (AuNPs) onto the SPGE and subsequent adsorption of monovalent half-antibody (monoAb) fragments of the anti-biotin antibody via Au-thiol bonds. For the second technique, we exploited the affinity of boronic acid towards sugar moieties by preparing a boronic acid-presenting SPGE surface to interact with the carbohydrate unit of this anti-biotin antibody. Using such approaches, we prepared an ultrasensitive electrochemical immunosensor, possessing a maximized epitope density, for the detection of biotin at concentrations as low as 0.19 pg. (C) 2010 Elsevier B.V. All rights reserved.