Potential-resolved electrochemiluminescent immunoassay based on dual co-reactants regulation

Multi-signal-based self-calibrating biosensors have become a research focus due to their superior accuracy and sensitivity in recent years. Herein, the potential-resolved differential ECL immunoassay based on dual co-reactants regulation was developed. Meso-tetra(4-carboxyphenyl)porphyrin (TCPP) functionalized zirconium dioxide (ZrO₂) composites (TCPP-ZrO₂) was first synthesized using TCPP as the luminophore and ZrO₂ as the enhancer and stabilizer. The organic-inorganic hybrid structure of TCPP-ZrO₂ limits the intramolecular motion of TCPP and accelerates the production of reactive radicals of co-reactants, thus exhibiting excellent ECL intensity and stability. Then, the potential-resolved ECL properties of TCPP-ZrO₂ regulated only by two co-reactants were explored. On this basis, a differential ECL immunosensor was constructed for the sensitive and accurate determination of heart-fatty acid binding protein (hFABP) using 2-(dibutylamino)ethanol modified gold nanoparticles (DBAE@Au) as labels (ECL-1, +1.3 V) and ECL-2 (-1.3 V) triggered by potassium persulfate (K₂S₂O₈) as the internal reference. In the presence of hFABP, DBAE@Au was captured by the sensing interface to generate ECL-1 and consume K₂S₂O₈, leading to a significant decrease of ECL-2. According to the intensity difference between ECL-2 and ECL-1, the detection of hFABP was achieved with a low detection limit and a wide linear range. The proposed differential ECL immunosensor has been applied in testing human serum samples with satisfactory results, demonstrating its promising applications for clinical diagnosis.