Fundamentals and applications of a non-separation electrochemical enzyme immunoassay (NEEIA) system.

Abstract

The fundamental aspects of the recently developed NEELA system were explored along with ways to improve and expand the versatility of the technique. The overall usefulness of NEELA was enhanced by demonstrating that NEELA can be employed for competitive binding assays of small molecules (e.g., biotin, digoxin) in addition to sandwich type assays for proteins developed previously. Competitive assays were subsequently used to determine the relationship between assay performance and reagent binding affinity and to develop novel detection schemes for the NEELA system. It was found through a series of competitive assays towards digoxin, utilizing monoclonal antibodies with different affinities towards digoxin, that the NEELA system requires antibodies with K_A &ge; 1 x 10<super>10</super> M<super>-1</super> and becomes qualitative, at best, when K_A &le; 1 x 10<super>9</super> M<super>-1</super>. Additionally, it was demonstrated that labeling enzymes other than alkaline phosphatase (ALP) could be used in NEELA via competitive assays towards biotin employing beta-galactosidase (beta-GAL) and glucose oxidase (GOX) as labels. An enzyme channeling system with ALP as the label and immobilized tyrosinase completing the enzyme pair was also shown to be an effective detection scheme. The systems which used enzyme channeling and (beta-GAL) exhibit lower limits of detection (LODs) of approximately 10 nM towards biotin, while the GOX system had a LOD of 1 nM. Studies involving alternate enzyme labels combined with confocal microscopy experiments showed that it is unlikely that the pH gradient across the sensing membrane present in the NEELA system plays a crucial role in the spatial resolution of bound and unbound enzyme label. Indeed, the profile of pH vs. distance from the microporous nylon membrane surface with time shows that the gradient observed when using ALP as a label extends into the bulk of the solution. Results from competitive assays suggest that the specific activity of the enzyme label towards the substrate being employed is crucial for achieving low LODs with the NEELA system. It is also shown herein, that protein immobilized within the pores of the nylon membranes used for NEELA provides no significant contribution to the signal observed in a competitive assay for biotin. Additional studies comparing porous gold electrodes modified non-specifically to electrodes modified using activated thioctic acid in both competitive assays towards biotin and sandwich assays toward human chorionic gonadotropin, indicated that the use of thioctic: acid is not necessary to achieve good assay performance.