Signal-on electrochemical immunoassay for APE1 using ionic liquid doped Au nanoparticle/graphene as a nanocarrier and alkaline phosphatase as enhancer

Abstract

In this paper, the Au nanoparticles decorated graphene nanosheets (AuNPs/Gr) were prepared as nanocarriers using ionic liquid (IL) as linker reagent. Then the alkaline phosphatase (ALP) and the ferrocene tagged detection antibodies (Fc-Ab2) were loaded on the IL doped AuNPs/Gr as a trace label for ultrasensitive measurements of human apurinic/apyrimidinic endonuclease 1 (APE1), which is a multifunctional protein in the DNA base excision repair pathway relating to various types of cancer. Several labeling protocols were investigated for the determination of the APE1 protein concentration and improved analytical features were obtained with the proposed carriers of IL doped AuNPs/Gr which were labeled with Fc-Ab2 and ALP (ALP/Fc-Ab2/AuNPs/IL/Gr). The reason may be that the IL doped AuNPs/Gr carriers (AuNPs/IL/Gr) could not only enhance the immobilized amount of ALP and Fc-Ab2, but also promote the electron transfer rate. Thus, through the specific recognition of antigen–antibody, numerous ALP/Fc-Ab2/AuNPs/IL/Gr, which are captured onto every single immunocomplex, could further catalyze the ascorbic acid 2-phosphate (AA-p) reaction to amplify the electrochemical signal. Transmission electron microscopy (TEM) images of the AuNPs/IL/Gr nanocomposites revealed the formation of a functionalized surface network structure. The resulting immunosensor exhibited a linear response to APE1 in the concentration range of 0.1–80 pg mL⁻¹ with a detection limit of 0.04 pg mL⁻¹, indicating potential applications in clinical diagnostics.