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  • br signal was obtained after Envision complex being poured


    signal was obtained after Envision complex being poured into the de- several experimental parameters such as the immobilization time of Ab1,
    tection solution (curve d). The reason was HRP contained in Envision Ab2 and the incubation time of HE4, h-BN@lucigenin@Ab2 bioconju-
    complex possessed intrinsically catalytic performance for the split of gates were studied in Fig. S4. It’s apparent that 2062-78-4 the ECL intensity declined
    H2O2 to output O2%−, just as Eq. (2), which participated in the lucigenin until the immobilization time of Ab1 up to 50 min and then tended to be
    ECL reaction. It was worth emphasizing that the ECL of lucigenin was stable (Fig. S4 A). Thus, 50 min was chose as the optimal immobilization
    initiated under alkaline condition, certifying the importance of OH− in time of Ab1. Similarly, the ECL signal dropped with the increasing im-
    ECL reaction. Therefore, the luminescence mechanism can be eluci- mobilization time of Ab2 and reached a platform at 30 min (Fig. S4B),
    dated by the following equations:
    thus the ideal immobilization time of Ab2 was 30 min. From Fig. S4C, the
    Luc ( OH)+ (9) ECL intensity decreased gradually as the incubation time increased, and a
    stable ECL intensity was obtained at 50 min, which was regarded as the
    Luc ( OH)• (10) optimal incubation time. Remarkably, the ECL signal from DBAE was
    descended and which from lucigenin was rose (Fig. S4D). Finally, two
    NMA* NMA hv (12) as the best incubation time of h-BN@lucigenin@Ab2 bioconjugates.
    3.6. Analytical performance of the ECL biosensor
    3.5. Optimization of experiment conditions
    Under the optimal conditions, two ECL signals from DBAE and luci-
    To ensure wonderful analytical performance in ECL detection, several
    genin were researched when different concentrations of HE4 was in-
    elements including the concentration of NiFe2O4 NTs, the adsorption cubated into the biosensor. Apparently, the ECL intensity of DBAE de-
    time of lucigenin and the concentration of NaOH were investigated. Seen clined while the ECL intensity of lucigenin increased with the increasing
    from Fig. S3 A, the highest ECL signal was realized at 1.0 mg/mL. concentrations of HE4, demonstrated in Fig. 5A. Fig. 5B indicated a good
    Therefoe, 1.0 mg/mL was identified as the optimal concentration of linear relationship between the ECL intensity ratio of lucigenin to DBAE
    NiFe2O4 NTs. Moreover, the adsorption time of lucigenin on h-BN was and the logarithm concentration of HE4 in the range of 10 fg/ml to
    reached a crest value at 3 h. Therefore, 3 h was selected as the optimum with a correlation coefficient of R = 0.991 (where I was the ECL in-
    adsorption time of lucigenin on h-BN. Meanwhile, hydroxide ions plays a tensity ratio of lucigenin to DBAE and C was the concentration of HE4).
    key role in the lucigenin ECL emission, as described at Fig. S3C, the ECL Moreover, the limit of detection (LOD) was low to 3.3 fg/ml (S/N = 3),
    intensity elevated gradually with the increasing concentration of NaOH. which was superior compared with other strategies listed in Table S1,
    Taking the reaction activity of biomolecules into consideration, 10 mM clarifying the excellent analytical performance of the proposed ratio-
    was selected
    the appropriate NaOH concentration. Additionally, metric biosensor for the determination of HE4.
    Table 1
    Recoveries tests of HE4 in serum samples (n = 3)a.
    Samples HE4 (pg/mL) Added (pg/mL) Found (pg/mL) Recovery (%)
    a n is the repetitive measurements number.
    3.7. Selectivity and stability of the proposed ECL biosensor
    To explore the selectivity of the biosensor, a contrast experiment was performed by employing 0.1 ng mL−1 AFP, CEA, IL-6, PSA as in-terferents. Seen from Fig. 5C, compared with the blank solution, almost no significant signal changes were obtained from those interferences,
    while the ratio of ECL intensity (ECLLucigenin/ECLDBAE) increased sharply in the presence of HE4, revealing the good selectivity towards HE4. As presented in Fig. 5D, the stability of the ECL ratiometric strategy was also evaluated under continuous cyclic potential scans for ten cycles at 1 pg/mL of HE4. Satisfyingly, the relative standard de-viation (RSD) of DBAE and lucigenin were 0.18% and 0.48%, respec-tively, indicating favourable stability of the biosensor.
    3.8. Analysis of practical samples
    For the purpose of examining the feasibility and validity of the ra-tiometric ECL strategy for HE4 analysis, the recovery experiments were carried out via a standard addition method. The results were shown in Table 1, after the serum samples were spiked with 1 pg mL−1, 100 pg mL−1 and 1000 pg mL−1 HE4, the recoveries were in the range of 89.8˜101%, demonstrating that the designed ratiometric ECL biosensor was capable of the quantitative detection of HE4 in real samples.