Label-free electrochemical immunoassay for ultrasensitive detection of norethindrone

Abstract

A label-free electrochemical immunoassay based on the graphene sheet (GS)–carboxymethyl chitosan (CMC)–thionine (TH) matrix (GS–CMC–TH matrix) for the sensitive determination of norethindrone antigen has been prepared. GS has excellent electrochemical activity and the synergistic effect between GS and TH in the CMC matrix was investigated. The anti-norethindrone was immobilized onto the GS–CMC–TH matrix modified glassy carbon electrode (GC) through covalent binding. The stable thin film formed with the GS–CMC–TH matrix on the surface of GC indicated high electroactivity, and it was found that the electroactivity of TH was greatly improved in the presence of GS due to the synergistic effect between GS and TH. After anti-norethindrone was conjugated onto the film of GS and TH through N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, the modified electrode could be used as an amperometric immunoassay for the detection of norethindrone antigen. The specific antibody-antigen immunocomplex formed on the electrode resulted in the decrease of amperometric signal of the electrode. The amperometric signal decreased linearly with norethindrone antigen concentration in the range of 0.01–4.2 ng/cm3 with a detection limit of 2.01 pg/cm3. The immunoassay had the advantages of high sensitivity, good selectivity and stability, and was applied to the analysis of norethindrone antigen in aquatic product with satisfactory results. .