br Preparation of NiFe O NTs Envision Ab
2.5. Preparation of NiFe2O4 NTs/Envision-Ab1 hybrids
In brief, 80 μL 4.2 μg/mL Ab1 was activated with 40 μL EDC/NHS (molar ratio 4:1) for 40 min at 4 °C. Then the above solution was mixed with 80 μL of Envision complex and reacted for 50 min to realize the amide reaction between Envision complex and Ab1, resulting in the formation of Envision-Ab1 complex. Subsequently, 200 μL Envision-Ab1 complex was added into 200 μL of 1 mg/mL NiFe2O4 NTs under stirring and kept for 4 h at room temperature to finish the self-assembly of Envision-Ab1 complex. After removing excess Envision-Ab1 complex, the NiFe2O4 NTs /Envision-Ab1 complex was obtained and stored at 4 °C for further use after the process of centrifugation and washing for several times.
2.6. Preparation of [email protected]@Ab2 bioconjugates
Simply, 200 μL h-BN (5 mg/mL) was utilized to adsorb lucigenin (200 μL, 1 mM) under stirring for 3 h. After removing the unabsorbed lucigenin, 80 μL AP (10 mM) was assembled to the [email protected] composites via the π-π staking reaction between the h-BN and AP. Then with the help of GLD, 80 μL Ab2 was bound to the above amino func-tionalized hybrids under stirring at room temperature. Afterwards, 1.0% BSA was used to block the nonspecific bind sites. After centrifu-ging and washing with ultrapure water, the [email protected]@Ab2 bioconjugates were obtained and stored at 4 °C for further use.
2.7. Synthetic process of the ratiometric strategy
Prior to modification, the GCE (Ф = 3 mm) was polished with 0.3 and 0.05 μm alumina slurry to acquired a bright surface and then wa-shed thoroughly with alcohol and water. As schematized in Scheme 1, after drying at room temperature, 3 μL of NiFe2O4 NTs/Envision-Ab1 hybrids solution was directly dropped on the clean electrode surface. After drying under infrared lamp, 3 μL of BSA (1%) was spread on the electrode to block nonspecific VH-298 for 30 min at 4 °C. Fol-lowing that, the modified electrode was incubated with 3 μL different concentrations of HE4 standards for 50 min at 4 °C. Ultimately, 3 μL of [email protected]@Ab2 bioconjugates were decorated on the electrode and incubated for 40 min at 4 °C to construct the ratiometric ECL bio-sensor.
2.8. The analysis of practical samples and standard addition method
First, the fresh blood was obtained from the first affiliated hospital of Fujian medical university. Before assay, 5 mL blood sample was Sensors & Actuators: B. Chemical 288 (2019) 80–87
centrifuged at the rate of 3000 r/min for 15 min. Afterwards, the su-pernate was diluted 10 times and applied the ratiometric ECL biosensor to realize the HE4 analysis in serum sample. Additionally, the standard addition method was performed by adding the same volume of HE4 standard solution with different concentrations of 1 pg mL−1, 100 pg mL−1 and 1000 pg mL−1 into the serum sample, respectively, and analyzed by the proposed ratiometric ECL strategy.
3. Results and discussion
3.1. Characterization of different materials
Initially, the typical transmission electron microscopy (TEM) were performed to illustrate the morphology and structure of NiFe2O4 NTs. Seen clearly from Fig. 1A, large amounts of slightly curved one-di-mensional nanomaterial with hollow morphology at the end of them revealed the nanotube structure of NiFe2O4 NTs. Meanwhile, the XRD pattern was further conduced to confirm the successful formation of NiFe2O4 NTs. As shown in Fig. 1B, several distinct diffraction peaks reflected the lattice planes of (220), (311), (400), (511) and (422), which corresponds to the pure NiFe2O4 phase (JCPDS 10-0325). To invetigate the Brunauer–Emmett–Teller (BET) surface area of the NiFe2O4 NTs, the N2 adsorption–desorption isotherms was analyzed in Fig. 1C. The surface area of NiFe2O4 NTs is 104.4 m2 g−1, clarifying the large specific area of nanotubes structure of NiFe2O4 NTs.
In addition, the morphology and structure of h-BN was clarified in the transmission electron microscopy image. As expect, the almost transparent film-like structure with wrinkles and scrolls was distinctly observed in Fig. S2, reflecting the untrathin nature of h-BN.
3.2. EIS and ECL characterization of ratiometric ECL biosensor
As a reliable tool, the electrochemical impedance spectroscopy 
was conducted to track the stepwise assembly process of the electrode by utilizing [Fe(CN)6]3−/4− as redox probe. From the results in Fig. 2A,
compared to the bare GCE (curve a), a smaller impedance value was observed at the NiFe2O4 NTs modified GCE (curve b), resulting from the good conductivity of NiFe2O4 NTs. After the electrode was assembled with Envision complex (curve c), Ab1 (curve d), HE4 (curve e) and [email protected]@Ab2 bioconjugates (curve f) successively, the im-pedance values gradually increased because the Envision complex, Ab1, HE4 and [email protected]@Ab2 bioconjugates as the mass-transfer blocking layer which impeded the electron transport. Importantly, these results came to the conclusion that the biosensor was successful fabricated.