An ultrasensitive sandwich electrochemical multiplex immunosensor designed for simultaneous detection of cancer biomarkers CEA and YES1 was developed. The immunosensor was created by integrating gold nano clusters with thiolated protein G, glutaraldehyde, CEA and YES1 antibodies, bovine serum albumin (BSA), and Horseradish peroxidase (HRP) functionalized gold nanoparticles on a glassy carbon electrode. Gold nanoparticles (AuNPs) were produced using Mangifera indica leaves extract and characterized through UV Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and Dynamic light scattering (DLS), with an average size range of 8 40nm.

Differential pulse voltammetry (DPV) measurements identified distinct oxidation signals at −0.18V and + 0.53V, corresponding to YES1 and CEA, respectively. The immunosensor demonstrated excellent sensitivity with detection limits of 0.0022 ng/mL for YES1 and 0.0034 ng/mL for CEA within a linear range of 0.1 50 ng/ mL. Comparative analysis of human plasma samples using this immunosensor and ELISA indicated relative error values ranging from 4.66% to 6.59% for CEA and 7.83% to 1.75% for YES1. The immunosensor displayed stability over two weeks and minimal cross reactivity with interfering compounds in human plasma. This innovative approach utilizing bio synthesized gold nanoparticles and protein G coupled gold clusters holds significant promise for early diagnosis of circulating cancer biomarkers in clinical applications