**3. Results and Discussion**

### *3.1. Condition Optimization for SF@Cu-NFs Preparation*

In order to obtain the best formation morphology of SF@Cu-HNFs product, preparation conditions of SF concentration and reaction time were systematically investigated and optimized.

### 3.1.1. Effect of SF Protein on SF@Cu-NFs Formation

In order to investigate the effect of SF protein on the nanoflower formation, products with and without SF were firstly prepared and characterized. For the preparation of SF@Cu-NFs in this work, SF protein was simply added into the solution containing copper ions and phosphate [57]. Meanwhile, Cu3(PO4)2 particles were also prepared by the similar procedures without the addition of SF solution. With 24 h preparation time, the photography and SEM images were shown in Figure 1a for SF@Cu-NFs products prepared with 400 mg L−<sup>1</sup> SF and were shown Figure 1b for Cu3(PO4)2 particles without SF, respectively. As it can be seen, although the appearances were similar (insets in Figure 1a,c), the microscopic images showed significant differences between SF@Cu-NFs products and Cu3(PO4)2 particles. As shown in Figure 1a,b, SF@Cu-NFs products were successfully formed by self-assembly with uniform flower-like morphology similar to the Chinese national flowers peony (Inset in Figure 1b). However, Cu3(PO4)2 particles with irregular flakes and uneven particle size distribution were formed without SF addition (as shown in Figure 1c,d). The Cu3(PO4)2 particles were not flower-like but Chinese tremella like with loose structures (Inset in Figure 1d). The above results confirmed that SF protein was a key factor and had beneficial effects for the flower-like nanoflowers formation.

### 3.1.2. Effect of SF Concentration on SF@Cu-NFs Formation

Under the condition of 12 h reaction time, the effect of SF concentration on the product formation was investigated ranging from 50 to 400 mg L−1. As shown in Figure 2, it was notable to observe that there was a grea<sup>t</sup> variation in morphology of SF@Cu-NFs by regulating the protein concentrations. Interestingly, the SF@Cu-NFs became smaller with the increase of SF concentration (Figure 2a,b,e,f), which may be caused by the increasing number of nucleation sites on the SF molecular. However, some SF@Cu-NFs products would bind with each other with SF concentration at 200 and 400 mg L−<sup>1</sup> (Figure 2c,d,g,h), which was also disadvantageous for the adsorption with decreasing the SF@Cu-NFs number and surface. As a result, 100 mg L−<sup>1</sup> SF concentration was selected to prepare the SF@Cu-NFs with the proper product size and superior shape.

**Figure 1.** (**<sup>a</sup>**,**b**) SEM images of SF@Cu-NFs product with SF. Insets are photographies of SF@Cu-NFs and Chinese penoy flower. (**c**) and (**d**) SEM images of Cu3(PO4)2 particles without SF. Insets are photographies of Cu3(PO4)2 particles and Chinese tremella.

**Figure 2.** (**<sup>a</sup>**–**d**) SEM images of SF@Cu-NFs with different SF concentrations at 50 mg <sup>L</sup>−1, 100 mg <sup>L</sup>−1, 200 mg L−<sup>1</sup> and 400 mg <sup>L</sup>−1, respectively. (**<sup>e</sup>**–**h**) The orresponding SEM images enlarged with mangnification of individual products.

### 3.1.3. Effect of Reaction Time on SF@Cu-NFs Formation

In order to study the formation process of the three-dimensional hierarchical structures, the effect of preparation time on the product formation was investigated with the addition of 100 mg L−<sup>1</sup> SF concentration. Experiments were carried out by collecting samples from the reaction mixture and observing intermediates and products at different time intervals. As shown in Figure S1, the PBS solution containing SF changed into blue after the addition of CuSO4. Then the solution became turbid blue after 10 min, indicating the production of NFs products. The SEM images in Figure 3a–d and insets showed the appearance and solution changes of SF@Cu-NFs product ranging from 30 min to 24 h. The diameter distribution of products from 0 to 24 h was shown in Figure S2. The process of product formation can be divided into four corresponding stages.

The first stage is initial stage with reaction time from 10 to 30 min. At this stage, blue fine and visible particles began to appear in the solution (shown as inset in Figure 3a). As observing from corresponding SEM shown in Figure 3a, primary crystal of Cu3(PO4)2 was formed (encircled in red circle) and SF protein molecules complexed with Cu2+ on its surface (encircled in green circle). The product composited mainly through the coordination of amide groups in the protein backbone and was beneficial for the formation of larger nanosheet petals.

**Figure 3.** (**<sup>a</sup>**–**d**) SEM images of the nanostructures of SF@Cu-NFs products at different preparation times of 30 min, 3 h, 12 h, and 24 h. Insets are photographies of solution change. (**e**) Schematic illustration of the formation process of SF@Cu-NFs products at preparation time of 12 h.

The second stage is growth stage with reaction time from 30 min to 6 h. At this stage, the blue particles grew bigger and blue flocculent precipitation was observed in the solution (shown as inset in Figure 3b). As observing from corresponding SEM shown in Figure 3b, a series of SF@Cu-NFs products with complete flower-like shape have been formed (encircled in red circle). However, the morphology SF@Cu-NFs products were not uniformed and there were still some small petal products (encircled in green circle). This result indicated the nanoflowers needed to grow further.

The third stage is the formation stage with reaction time from 6 h to 12h. At this stage, small product particles furthermore grew up and deposited to the bottom ((shown as inset in Figure 3c)). As observing from corresponding SEM shown in Figure 3c, SF@Cu-NFs products with uniform flower-like shape and size were formed.

The fourth stage is the overgrowth stage with reaction time from 12 h to 24 h. At this stage, some SF@Cu-NFs grew up and deposited to the bottom (shown as inset in Figure 3d). This was because polar side chain of SF could promote the formation of large folding nanosheets through its hydroxyl, carboxyl, and amino groups. As observing from corresponding SEM shown in Figure 3d, the further growth made some SF@Cu-NFs bind with each other to form much bigger product (encircled in green circle). The overgrowth effect decreased the SF@Cu-NFs number and surface, which was disadvantageous for the adsorption. As a result, the incubation time of 12 h was considered to be the optimum preparation time for the SF@Cu-NFs products. The schematic illustration of the formation process of SF@Cu-NFs products at preparation time of 12 h was shown in Figure 3e.
