Aptamer–17β‐estradiol–antibody sandwich ELISA for determination of gynecological endocrine function

17β‐Estradiol‐E2 (17β‐E2) is a steroid hormone that plays a major role in the reproductive endocrine system and is involved in various processes, such as pregnancy, fertility, and menopause. In this study, the performance of an enzyme‐linked immunosorbent assay (ELISA) for 17β‐E2 quantification was enhanced by using a gold nanoparticle (GNP)‐conjugated aptamer. An anti‐17β‐E2–aptamer–GNP antibody was immobilized on an amine‐modified ELISA surface. Then, 17β‐E2 was allowed to interact with and be sandwiched by antibodies. Aptamer–GNP conjugation was confirmed by colorimetric assays via the naked eye and UV–visible light spectroscopy. The detection limit based on a signal‐to‐noise ratio (S/N) of 3 was estimated to be 1.5 nM (400 pg/mL), and the linear range was 1.5–50 nM. Control experiments (without 17β‐E2/with a complementary aptamer sequence/with a nonimmune antibody) confirmed the specific detection of 17β‐E2. Moreover, 17β‐E2 spiking of human serum did not interrupt the interaction between 17β‐E2 and its antibody and aptamer. Thus, the developed ELISA can be used as an alternate assay for quantification of 17β‐E2 and assessment of endocrine‐related gynecological problems. Sandwich ELISA for determination of gynecological endocrine function.