Novel solution-phase immunoassays for molecular analysis of tumor markers

Molecular forms of serum prostate-specific antigens (PSA-free, PSA-total, PSA-complex) were conjugated with an N-hydroxysuccinimide ester of a ruthenium(II) tris-bipyridine (Ru(bpy)32+–NHS) which provided both electrochemiluminescence (ECL) and phosphorescence. ECL intensity of labeled PSA, produced upon oxidation of Ru(bpy)32+ in a solution containing tri-n-propylamine, was proportional to the concentration of PSA and inversely proportional to its mass and size of PSA. ECL intensity of the PSA-free antigen decreased upon binding with its monoclonal antibody (MAB) due to the increased mass and size of the conjugated pair and the decrease in diffusion coefficient. The binding affinity of PSA-free antigen with its MAB at 1 × 1010 M−1 was determined. However, no binding of PSA-free antigen with the MAB of PSA-complex was observed. A similar approach was applied to study labeled PSA-complex indicating affinity of PSA-complex to its MAB at 3 × 109 M−1 and a step-wise binding process with PSA-free MAB. Thus, this solution-phase quantitative ECL immunoassay allowed measurement of the affinity of serum PSAs with their MABs and screening of PSAs based upon their affinity to MABs. Unlike other immunoassays, this immunoassay demonstrated one-step rapid analysis while simultaneously eliminating immobilization, separation and washing steps and detected PSA at a level of 1.7 pg mL−1, which is 1000-fold more sensitive than current PSA immunoassays. Furthermore, single-molecule (SM) phosphorescence microscopy was developed to detect single serum PSA-free and PSA-complex molecules in solution with no use of antibody showing that PSA-free molecules diffused faster than PSA-complex molecules in solution. This finding is consistent with ECL measurements and implies the possibility of screening individual analytes in a complex mixture using their distinct SM diffusion distance. This is the first report describing the detection of single protein molecules labeled with a metal-complex using phosphorescence microscopy and also the screening of serum tumor markers using ECL and SM phosphorescence solution-phase assays.