Towards the development of a single-step immunosensor based on an electrochemical screen-printed electrode strip coupled with immunomagnetic beads

AbstractThis work investigates the behaviour of two alternative systems that model the crucial event involved in any ELISA test, i.e. the molecular recognition between an antigen and its specific antibody on a solid phase, and its measurement. Each approach is devised with the goal of making possible a single-step, separation and wash-free amperometric magneto-immunosensor. Magnetic particles (MBs) are used as support for the immobilization of rabbit IgGs that are recognized by the specific anti-rabbit IgG-HRP. The assay protocol is based on the use of a series of small “reservoirs” containing phosphate buffer, hydroquinone, anti-rabbit IgG-HRP and an appropriate amount of MB-rabbit IgG. After a brief incubation, the content of each “reservoir” is transferred to one of the wells of a 8-well magnetized-screen-printed electrode strip. The resulting MB-IgG-anti-IgG-HRP chain, is then concentrated on the working electrode surface for electrochemical measurement. Two different approaches to monitor this immunological reaction are investigated. The first one is based on the enzyme-channeling principle (ECP) and involves the use of a second enzyme, glucose oxidase (GOD), immobilized on the working electrode previously modified with Prussian Blue. Since the H2O2 produced by GOD is the co-substrate of the HRP enzyme, glucose is added into the well and the current, generated by the residual H2O2, is measured. The second, more direct, approach is performed without exploiting ECP (no GOD enzyme), by adding H2O2 into the well and measuring the current generated by the HRP product on a pristine screen-printed electrode. Both approaches yielded a typical sigmoidal binding curve, illustrating the discrimination between the signal produced by the immuno-bound HRP concentrated on the electrode surface, and the background signal due to HRP in the bulk solution. FigureSchematic representation of the single-step immunoassay: in the upper part, the content of the ‘reservoirs’ (containing MB-rabbit IgG, TPi, HQ and various concentration levels of anti-rabbit IgG-HRP) are transferred to an 8 well/sensor strip coupled with an special magnetic support which can draw the IMBs to the electrode surface; the lower part shows the two electrochemical approaches proposed to monitor the immunological reaction