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  • with a correlation coefficient of R where I


    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 FLAG tag Peptide 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.
    4. Conclusions
    In summary, a NiFe2O4 NTs catalyzing oxygen evolution reaction promoted ratiometric ECL biosensor for the sensing of HE4 was de-veloped based on DBAE and lucigenin as the new ECL emitter unit for the first time. In this protocol, NiFe2O4 NTs with superior properties not only served as a matrix to load large amounts of Envision-Ab1 complex, but catalyzed the OER process to yield oxygen, leading to a significantly promoted ECL response of DBAE. Interestingly, Envision complex an-chored with substantial HRP could accelerate the decomposition of H2O2 to release O2%−, which enhanced the two ECL reaction of DBAE and lucigenin. Additionally, the h-BN nanosheets was revealed to be an effective probe scaffold for the first time in this strategy, which achieved the high loading of lucigenin and maintained the intense and stable ECL signal of lucigenin for its strong adsorption capability and excellent resistance to corrosion. Therefore, by virtue of the above features, a highly sensitive ratiometric ECL strategy was designed for the sensitive determination of HE4, which holds great promise in the early ovarian cancer monitoring and clinical diagnosis.
    Appendix A. Supplementary data
    [9] T. Ma, M. Zhang, Y. Wan, Y. Cui, L. Ma, Preparation of an acridinium ester-labeled antibody and its application in goldmag nanoparticle-based, ultrasensitive chemi-luminescence immunoassay for the detection of human epididymis protein 4, Micromachines 8 (2017).