A ratiometric electrochemiluminescence method using a single luminophore of porous g-C3N4 for the ultrasensitive determination of alpha fetoprotein.

In this work, we report a simple ratiometric electrochemiluminescence (ECL) method for ultra-sensitive immunoanalysis. A glassy carbon electrode (GCE) was modified by a mixture of porous g-C3N4 nanosheets and carbon nanotubes (CNTs). Secondary antibodies were labeled using CuS nanoparticles as the tags. After immune recognition, CuS nanoparticles in the immunocomplex were dissolved as Cu2+, which can quench the ECL of g-C3N4. The amount of Cu2+ was determined to quantify the concentration of the target antigen. To enhance the sensitivity, Cu2+ ions were firstly enriched and reduced to Cu on the surface of GCE/CNTs-g-C3N4, and the cathodic ECL of g-C3N4 was measured as the reference signal in the ratiometric ECL measurements. After applying a potential of 0.6 V (vs. Ag/AgCl) for 6 s, Cu was dissolved as Cu2+, which can quench the ECL of g-C3N4 with much higher efficiency because the freshly dissolved Cu2+ ions were distributed mainly within the Helmholtz layer of GCE/CNTs-g-C3N4. By using the ECL intensity ratio of GCE/CNTs-g-C3N4 (Cu2+) to GCE/CNTs-g-C3N4 (Cu) measured under the potentiostatic model as the signal indictor, the ratiometric ECL method was used to detect a biomarker of alpha fetoprotein with the limit of detection of 0.1 fg mL-1. It was shown that the influence of the difference in electrode modification and ECL measurement conditions on the determination of Cu2+ is suppressed greatly in the ratiometric ECL method. The combination of ratiometric ECL with electrochemical enrichment and biometallization is a useful strategy to enhance the sensitivity and reproducibility in immunoanalysis.