**4. Conclusions**

In this work, natural material of SF protein was used for the fabrication of protein–inorganic hybrid nanoflowers through self-assembly and the three-dimensional structure was applied to efficient adsorption of HMI Pb(II).

Through adsorption isotherms and kinetics, the adsorption performance of SF@Cu-HNFs for Pb(II) removal was systematically evaluated in detail. Langmuir and pseudo-second-order models indicated the monolayer adsorption and high capacity on the SF@Cu-NFs. Meanwhile, the adsorption thermodynamics showed that the spontaneous and exothermic process. As compared, SF@Cu-NFs indicated as an excellent adsorbent for Pb(II) treatment with the *Qmax* as high as 1908 mg g<sup>−</sup>1, which was about 3–20 folds greater than that of the other adsorbents.

By ascribing to its individual organic and inorganic component, the adsorption mechanism of SF@Cu-NFs for Pb(II) removal was discussed and revealed with two stages of fast adsorption and slow adsorption. On one hand, the flower 'stamen' of organic SF protein was designated as responsible adsorption site for fast adsorption of Pb(II). On the other hand, the flower 'petal' of inorganic Cu3(PO4)2 crystal was designated as responsible adsorption site for slow adsorption of Pb(II). This result clearly indicated that the silk fibroin protein-derived hybrid nanoflower could adsorb HMI Pb(II) well because of the adsorption site on the adsorbent surface.

In this work, we further understand the adsorption behavior and interaction process of HMI Pb(II) on the surface of silk fibroin derived hybrid nanoflowers. The present study has been successful in revealing the microscopic interaction process of Pb (II) adsorption that provides a new insight on understanding the adsorption mechanism. Also, based on interfacial adsorption, it is of grea<sup>t</sup> significance to comprehend the development of heavy metal ion removal applications. By fabricating SF@Cu-HNFs hybrid nanoflowers derived from SF protein, this work not only successfully provides insights on its adsorption performance and interaction mechanism for Pb(II) removal, but also significantly indicates its potential applications in contamination adsorption for environmental treatment.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1996-1944/13/5/1241/s1, Figure S1: Photography of solution change at reaction times of 0 min and 10 min., Figure S2: Diameter distribution of products from 0 to 24 h, Figure S3: (a) The surface zeta potential measurement of SF@Cu-NFs at pH=4.0–9.0; (b) Adsorption capacity of the prepared SF@Cu-NFs for Pb(II) at pH=4.0–9.0, Figure S4: The adsorption isotherms of Pb(II) fitting by (a) Langmuir model, (b) Freundlich model and (c)Temkin model, Figure S5: Evaluation of SF@Cu-NFs adsorption selectivity for (i) Pb(II), (ii) Cd(II) and (iii) Ni(II). Insets are HMIs solution after di fferent adsorption of 0, 5, 20, 40 and 90 min. The chromogenic reagen<sup>t</sup> for Pb(II) and Cd(II) are DTZ and for Ni(II) is DMG, Figure S6: XRD spectra of SF@Cu-NFs before (spectrum i) and after (spectrum ii) Pb(II) adsorption, Table S1: The related EDS data of SF@Cu-NFs, Table S2: Comparison maximum capacity of SF@Cu-NFs products in this work and other adsorbents for Pb(II) adsorption., Table S3: Thermodynamic data for Pb(II) adsorption by SF@Cu-NFs at di fferent temperatures.

**Author Contributions:** M.D., X.L. and Y.X. designed experiments; J.L., C.Z., S.Q. and X.L. carried out experiments; H.W., S.F., and R.Z. analyzed experimental results. X.L. and Y.X. analyzed sequencing data. X.L. and Y.X. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by [the National Natural Science Foundation of China] gran<sup>t</sup> number [51974266 and 51404203], [the China Postdoctoral Science Foundation Funded Project] gran<sup>t</sup> number [2017M612993] and [the Miaozi Project of Scientific and Technological Innovation of Sichuan Province] gran<sup>t</sup> number [2019091 and 2019093]. The APC was funded by [2019091 and 2019093].

**Conflicts of Interest:** The authors declare no competing financial interest.
