Dual-Mode Electrochemical Immunoassay for Insulin Based on Cu₇S₄-Au as a Double Signal Indicator

The detection of insulin by electrochemical (EC) immunoassay is desirable but highly challenged due to the obstacle of improving its accuracy, especially in a single-response system. In this work, based on Cu₇S₄-Au as a dual signal indicator, we fabricated a dual-mode electrochemical immunoassay for insulin. Especially, Cu₇S₄ presents a strong differential pulse voltammetry (DPV) signal for the electron transfer between Cu²⁺ and Cu⁺, without the addition of K₃[Fe(CN)₆] or other electron transfer mediators. Furthermore, Cu₇S₄ displays high sensitivity and high electrocatalytic activity toward the reduction of H₂O₂ through chronoamperometry (CA). The introduction of Au nanoparticles can not only link on the surface of Cu₇S₄ by the chemical bond of Au-SH, but also connect the second antibody (Ab₂) by the chemical bond of Au-N. Due to the superior electroconductivity of Au nanoparticles and the synergistic effect between the Au nanoparticles and Cu₇S₄, a high sensitivity is achieved by means of DPV and CA. To improve the loading capacity of antibodies, nanofiber polyaniline covalently grafted graphene (GS-PANI) linked with Au nanoparticles (GS-PANI-Au) as the matrix material was prepared. Based on Cu₇S₄-Au as a double signal indicator, the developed EC immunoassay for insulin exhibits a wide linear response for insulin detection in the range from 0.1 pg/mL to 50 ng/mL, with a low detection limit of 35.8 and 12.4 fg/mL through DPV and CA modes, respectively. Furthermore, the immunosensor displays an excellent analytical capability for insulin and promises application in quantitative detection of other disease markers in clinical diagnosis.