[show abstract][hide abstract] ABSTRACT: 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 0.19pg.
[show abstract][hide abstract] ABSTRACT: This work describes the development of a cost-effective, easy-to-use, portable immunoanalytical platform technology with sufficient sensitivity for use in the detection of physiologically important targets. Biotin, also known as vitamin H, was selected as the model analyte. The detecting system employs biotin-tagged, potassium ferrocyanide-encapsulated liposomes as the signal amplifier and PAH (poly allylamine hydrochloride)-modified, nanosized-Au particles assembled screen-printed electrode (nanoAu-SPE) as the working electrode. The diagnostic procedures are based on selective immunoanalytical recognitions and sensitive electrochemical detection. The model analyte biotin was determined based on a "competitive-type" immunoassay in which competition occurs between the analyte biotin and potassium ferrocyanide-encapsulated, biotin-tagged liposomes for a limited number of anti-biotin antibody binding sites, which were immobilized on the PAH/nanoAu/SPE surface. The nanostructured Au SPE surface was covalently bonded to the PAH layer, which subsequently interacted with anti-biotin antibodies. The ferrocyanide released from ruptured bound-liposomes was finally measured using square-wave voltammetry. The calibration curve for biotin had a linear range of 10(-11)-10(-2) M, covering nine orders of magnitude. The detection limit of this immunodetecting system was as low as 9.1 pg of biotin (equivalent to 4.5/microL of 8.3 x 10(-9) M).
Journal of Nanoscience and Nanotechnology 05/2009; 9(4):2324-9. · 1.15 Impact Factor