*2.4. Characterization*

#### 2.4.1. Scanning Electron Microscopy

The microstructure of the nanofiber mats and cotton fabric was studied by scanning electron microscope (JSM-IT100, JEOL Ltd., Tokyo, Japan) with acceleration of voltage 20 KV. Before the imaging process, all the samples were sputter-coated with gold.

#### 2.4.2. Fourier Transform Infrared Spectroscopy

Fourier transform infrared spectroscopy (FT-IR, Tensor-37, Bruker, Berlin, Germany) was used to determine the characteristics of the microstructure of NIPAM, sericin, PNI-PAM, sericin/PNIPAM/PEO nanofibers and the functionalized cotton fabric with the KBr technique at wavelengths ranging between 400 cm<sup>−</sup><sup>1</sup> and 4000 cm<sup>−</sup>1. The samples were obtained in a vacuum dryer for 4 h before acquiring the spectra.

#### 2.4.3. Differential Scanning Calorimetry

Differential scanning calorimeter (DSC 7, Perkin Elmer, Waltham, MA, USA) was applied to evaluate thermal behaviors of untreated cotton fabric and functionalized cotton fabric under nitrogen with the flow rate at about 20 mL/min. The swollen sample weight was about 10 mg. The above samples were put in the aluminum sample holder and frozen below −20 ◦C. The samples were operated at 0–50 ◦C, 2 ◦C min−1. The samples performed heating/cooling cycles three times.

#### 2.4.4. Contact Angle Measurements

Static water contact angle tests were accomplished by using a drop shape analyzer (PT-705, Shanghai, China) with the needle method at flow rate of 1.0 μLs−<sup>1</sup> in 16% of relative humidity and temperature ranging from 15 ◦C to 45 ◦C. The Young Laplace drop profile fitting method was applied to evaluate the static contact angle results. The needle specification is shown as follows: diameter 0.5 mm and the water droplet volume ~5 μL. The standard deviation of the measurement series might create the error bars.

#### 2.4.5. pH-Responsive Swelling

Sericin is an inherently weak amphipathic polyelectrolyte with the acidic side and alkaline side base, which makes it sensitive to pH [32,33]. The swelling behaviors of sericin/PMIPAM/PEO hydrogels were conducted within a variety of pH values (1.0~11.0), in view of the crucial aspect of the parameters in responsiveness.

In order to determine the swelling ratio, the dried samples were immersed in various pH buffers for 24 h in the temperature 15 ◦C and 37 ◦C that completes with the hydrogels' low critical solution temperature range. Before swollen sample mass testing, it was necessary to remove the surface water of specimens. The average of five samples was used to obtain the final results. The swelling ratio (SR) was calculated as follows [34]:

$$\rm{SR(\%)} = \rm{[(Wt\_t - W\_d)/W\_d]} \times 100\tag{1}$$

where Wt and Wd are the masses of the swollen sample and dried sample, respectively.

#### 2.4.6. Antimicrobial Activity Measurements

The antibacterial activity of samples was investigated against Gram-positive bacteria and Gram-negative bacteria such as *Staphylococcus aureus* and *Escherichia coli* on the basis of the AATCC 100 test method and GB/T 31713-2015. Before the assay, all samples were made into small pieces at about 0.5 × 0.5 cm2. Additionally, UV exposure was applied to sterilize the above samples at about 30 min. The above samples were put, respectively, in the bacterial suspension by using sterile forceps and then immersed in a flask including phosphate-buffered saline at 0.3 mM/70 mL. The phosphate-buffered saline consisted of monopotassium phosphate and the cell culture solution. The cell concentration was about 1 × 105–4 × 10<sup>5</sup> (CFU)/mL. We shook the above flask on a rotary shaker at 150 rpm, 24 ◦C, 1 h. The 0.5 mL of culture solution was obtained, respectively, from incubated samples before and after shaking for 1 h, and then diluted and placed on the agar plates that were cultivated at 37 ◦C, 1 h. The inhibitory rate (%) was decided as follows [35]:

$$\mathbf{R(\%)} = \mathbf{[(B - A)/B]} \times 100\tag{2}$$

where R, B and A were the percentage of bacterial reduction and the bacterial colonies before and after shaking for 24 h, separately.

#### **3. Results and Discussion**

## *3.1. SEM*

The surface morphology of the sericin/PNIPAM/PEO nanofibers and functionalized cotton fabric under optimal conditions was decided by SEM (see Figure 3). The final images of the sericin/PNIPAM/PEO nanofibers indicated that the fibers were uniform and continuous without any bead formation (Figure 3a). As shown in Figure 3a, there was breakage in nanofibers when the spinning solution had more NIPAM. The cause might be that the increasing NIPAM caused the bad mechanical properties of nanofibers. This indicates that sericin, NIPAM and PEO were well blended. Based on the morphological analysis of the functionalized cotton fabrics (Figure 3b,c), the sericin/PNIPAM/PEO nanofibers were overlapped and fixed on the surface of the cotton fabric formation bilayer structure. The cross-linked reaction mechanism between sericin and cotton fabric is shown in Figure 3d. The FTIR spectra analysis also confirms the chemical cross-linking reaction of the cotton fabrics and sericin in the presence of glutaraldehyde [36]. The results of SEM showed that the functionalized cotton fabric was also successfully prepared by a combination of electrospinning technology and an interpenetrating polymer network technology.

**Figure 3.** The morphology of (**a**) Sericin/PNIPAM/PEO nanofibers: ((**a1**) 25/45/30; (**a2**) 40/30/30; (**a3**) 55/15/30); (**b**,**<sup>c</sup>**) Functionalized cotton fabric; (**d**) Reaction mechanism between sericin and cotton.
