A one-step, separation-free amperometric enzyme immunosensor

A new one-step, separation-free, amperometric enzyme immunosensor is described. The sensor consists of an antibody electrode that is low cost, disposable, and operates without washing or separation steps. The immunosensor combines the following signal-amplification systems: enzyme-channeling immunoassay; accumulation of the redox mediators (I-2/I-); cyclic regeneration of an enzyme (peroxidase) substrate at the (polyethylenimine) polymer/electrode interface; and control of the hydrodynamic conditions at the interface of the antibody electrode. The immunological reactions were monitored electrochemically in situ, and the binding curves were directly visualized on a computer screen. The complete immunoassay can be performed in 5-20 min depending on the complexity of the immunological reactions. Model systems using rabbit IgG and human luteinizing hormone (hLH) in a 'sandwich' immunoassay revealed that the immunosensor can detect concentrations of hLH in human serum as low as 1 ng ml(-1).