Low-concentration 17β-estradiol (E2) in environmental media (µg/L or even lower) is one of the strongest endocrine disruptors in environmental estrogens. A quantitative analysis of E2 in the environment lacks sufficient sensitivity and accuracy. To fill the gap in existing environmental estrogen detection technology, electrochemiluminescence (ECL) and immunosensor techniques were combined to construct a nano-ZrO₂ (ZrO₂NPs)-based ECL immunosensor (ECLI). The potential of the immunosensor for detecting trace E2 was evaluated.

First, ZrO₂NPs were prepared by a sol–gel method, and polyaniline (PANI)-coated gold nanoparticles (AuNPs) (PANI@AuNPs) were prepared using a gold nano seed coating method. Thereafter, ZrO₂NPs were introduced onto the surface of a glass electrode (GCE), and sheep-derived E2 polyclonal antibody (Ab) was immobilized on the electrode to prepare a ZrO₂NPs-based ECLI. Finally, the ECLI was immersed in the E2 test solution to capture E2, followed by the introduction of E2-labeled PANI@AuNPs (PANI@AuNPs-BSA-E2) to bind with Ab that was not pre-bound to E2. During this process, the electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and ECL response characteristics of the GCE surface at different stages of incubation were observed. The influence of the following factors on the ECL intensity of the ECLI during E2 detection was simultaneously investigated: substrate concentration, buffer solution environment (pH and co-reactant concentration), concentration of ZrO₂NP suspension, potential scanning rate for ECL detection, Ab and E2 interaction time, and interaction time for Ab not bound to E2 with PANI@AuNPs-BSA-E2. E2 in the samples was quantitatively detected based on the relationship between the ECL intensity and E2 concentration. Finally, the stability, selectivity, regenerability, and applicability of the ECLI sensor for E2 detection in natural water and vegetable juice were investigated through 10 consecutive scans. Experiments were carried out by immersing the sensor in the sample for 1–12 days; replacing the target antigen E2 with diethylstilbestrol, estriol, estrone, dichlorodiphenyltrichloroethane, ethinylestradiol, or bisphenol A; adding E2 standard solution; regenerating the sensor via microwave desorption; and immersing it in natural water and vegetable juice samples containing a certain concentration of E2.

The resistance of the multi-level sensor with the surface-modified GCE gradually increased. The ECL signal originated from bonding between Ab and E2 loaded on the surface of the ZrO₂NPs. The optimal conditions for E2 detection by the ECLI were as follows: pH 7.0 buffer system; K₂S₂O₈ (co-reactant) concentration of 100 mmol/L; ZrO₂NPs suspension concentration of 2.5 g/L; Ab and E2; interaction time of 2.5 h for Ab not bound to E2 with PANI@AuNPs-BSA-E2; and potential scanning rate of 0.1 V/s. Under the optimal detection conditions, the ECL intensity of the ECLI exhibited a good linear response to the E2 concentration, with a correlation coefficient (R²) of 0.998 6 and a detection limit of 0.011 µg/L. After 10 consecutive scans and 12 days of storage, the ECL performance of the ECLI still reached ≥98% of its original level. The constructed ECLI was not affected by environmental endocrine disruptors. In natural water and vegetable juice, the recovery rate of E2 was in the range of 99.3%–104.0% and 98.7%–103.0%, respectively, indicating the satisfactory detection ability of the sensor. Regeneration using the ultrasound method indicated that ECLI had good regeneration and reusability.

The constructed ZrO₂NPs-based ECLI sensor achieved quantitative detection of trace amounts of E2 in environmental samples, with good stability and selectivity, affording a wide detection range, low detection limit, and high sensitivity.