Development of electrogenerated chemiluminescence-based enzyme linked immunosorbent assay for sub-pM detection

This paper reports the development and characterization of a highly sensitive enzyme linked immunosorbent assay realized by the electrogenerated chemiluminescence (ECL) detection of a thiol monolayer formed by an enzyme labeled antibody. We used two monoclonal anti tumor necrosis factor-alpha (TNF-alpha) antibodies for a sandwich immunoassay. One was a capture antibody, and the other was a detection antibody labeled with an enzyme via an avidin-biotin interaction. Acetylcholinesterase was used as the labeling enzyme to convert acetylthiocholine to thiocholine. Then the thiocholine was collected on a gold electrode surface by gold-thiol binding. A bright and distinctive emission was observed at 1150 mV (vs Ag-AgCl) on the gold electrode with a thiocholine monolayer as a coreactant in the presence of tris(2,2'-bipyridyl)ruthenium complex. This method can greatly enhance the immunoassay signal since a large number of coreactant molecules can be generated by the enzymatic reaction, which is advantageous compared with a previously reported ECL based immunoassay that directly labels the detection antibody with a coreactant or luminophore. In addition, a surface accumulated coreactant is superior to the previously reported coreactant system in a bulk solution, because ECL emission occurs only very close to an electrode surface. As a result, high sensitivity and a low detection limit of 0.2 pM (3.4 pg/mL) TNF-alpha were achieved with excellent reproducibility by optimizing the conditions for the immuno-reaction, thiocholine accumulation, and ECL generation.