A ratiometric electrochemiluminescent immunoassay for calcitonin by using N-(aminobutyl)-N-(ethylisoluminol) and graphite-like carbon nitride

A ratiometric electrochemiluminescent (ECL) assay is described for the determination of the calcium(II) regulator calcitonin (CT). The method is making use of (a) graphite-like carbon nitride (g-C 3 N 4 ) as the cathodic luminophore, (b) N-(aminobutyl)-N-(ethylisoluminol) (ABEI) as the anodic luminophore, and (c) peroxodisulfate and dissolved oxygen as coreactants. The luminous potential of g-C 3 N 4 and ABEI can be well distinguished because of their different luminescent properties. Energy transfer between g-C 3 N 4 and ABEI is not observed, and the coreactants peroxodisulate and oxygen do not interfere with each other. Au nanoparticles were functionalized with g-C 3 N 4 and placed on the electrode to serve as a matrix for immobilization of primary antibody (Ab 1 ). In the presence of CT, it will bind to the electrode. Then secondary antibody (Ab 2 ) modified with polyaniline (PANI) and ABEI is incubated onto the electrode. With the increase in the concentration of CT, the blue ECL of g-C 3 N 4 is quenched by PANI, while the blue luminescence of ABEI is enhanced. This enables ratiometric detection of calcitonin by ratioing the internsities at 460 and 475 nm. Response is linear in the 0.1~40 pg·mL −1 CT concentration range, and the limit of detection is 23 fg·mL −1 . The method breaks the limitation of common ECL ratiometric strategy, namely, two luminophores often share the common coreactant. Schematic representation of an immunoassay where polyaniline (PANI) in a BSA-Ab 2 -ABEI-Au@PANI composite quenches the cathodic signal of a graphitic carbon nitride (Au-g-C 3 N 4 ) modified with gold nanoparticles (Au), while N-(aminobutyl)-N-(ethylisolumino) (ABEI) in the BSA-Ab 2 -ABEI-Au@PANI composit produces an anodic signal that enables quantitation of calcitonin.