*3.1. Materials*

*J. regia* leaves were picked from walnut trees in Tabriz, Iran. AgNO3, as silver salt, was bought from Dr. Mojallali (Dr. Mojallali Chemical Complex Co., Tehran, Iran). Standard solution of Ag NPs (with particle size of 10 nm and concentration of 1000 ppm) was purchased from Tecnan-Nanomat (Spain). *Escherichia coli* (PTCC 1270) and *Staphylococcus aureus* (PTCC 1112) were attained from microbial Persian-type culture collection (PTCC, Tehran, Iran). There is no ATCC (American type culture collection) number for *E. coli* and this bacteria is clinical isolate. However, the ATCC number of *S. aureus* is 6538. Nutrient agar was bought from Biolife (Biolife Co., Milan, Italy).

#### *3.2. Preparation of J. regia Leaf Extract*

The *J. regia* leaf was washed, dried (in dark room), powdered, and 1 g of the prepared powder was added into 100 mL of boiling distilled water for 5 min. After cooling the solution, it was filtered (Whatman No. 1 filter paper) using a Buchner funnel under vacuum pressure and the clear walnut leaf extract was stored at a cold temperature (4 ◦C).

#### *3.3. Ag NPs Synthesis Using J. regia Leaf Extract*

The Ag NPs solution was obtained by a domestic microwave-assisted synthetic approach. AgNO3 solution (1 mM) was made by dissolving 0.017 g of its powder in 100 mL of deionized double-distilled water. In a typical synthesis, different amounts of AgNO3 solution (15–25 mL) were mixed with different amounts of *J. regia* leaf extract (0.1–0.9 mL) and the mixture solutions were put into a microwave oven (MG-2312W, LG Co., Seoul, Korea) at a constant power of 800 W and microwave exposure time (180 s).

## *3.4. Physico-Chemical Assay*

#### 3.4.1. Fourier Transform-Infrared (FT-IR) Spectra Analysis

In order to identify the possible reducing and stabilizing biomolecules of *J. regia* leaf extract, FT-IR measurements were carried out. The FT-IR spectrum of the extract was recorded on a Bruker Tensor27 spectrometer (Bruker Co., Karlsruhe, Germany) using KBr pellets in the 4000–400 cm<sup>−</sup><sup>1</sup> region.

#### 3.4.2. Surface Plasmon Resonance

Ag NPs, due to their SPR, have a strong absorption of light, which is shown as broad emission peaks (λmax) in the wavelength ranging from 380 to 450 nm [8–10]. Therefore, formation of Ag NPs using *J. regia* leaf extract can be confirmed by the absorption spectrum of the mixture solutions containing fabricated Ag NPs by using a Jenway UV-Vis spectrophotometer 6705 (Cole-Parmer Co., Staffordshire, UK). Furthermore, by preparing several serial dilute Ag NPs solutions (10–1000 ppm) and establishing standard curves based on the defined concentrations of the Ag NPs solutions and their absorbance unit values, it is possible to determine the concentration of the formed Ag NPs using *J. regia* leaf extract [10].

#### 3.4.3. Particle Size, Particle Size Distribution, Polydispersity Index and Zeta Potential of the Synthesized Ag NPs

In order to measure values of the mean particle size (nm), PDI (ranging 0–1) and zeta potential (mV) of the fabricated Ag NPs and their PSD, a DLS particle size analyzer (Nanotrac Wave, Microtrac, Montgomeryville, PA, USA) was utilized. The DLS technique scatters a laser light beam at the surface of dispersed NPs, which results in the detection of the backscattered light. PDI is a dimensionless value which shows that the uniformity of the synthesized NPs and their surface electric charge are related to the PDI and zeta potential values of the synthesized NPs [14,15].

## *3.5. Antibacterial Assay*

Bactericidal activity of the formed Ag NPs was evaluated using the well diffusion method. For this reason, bacterial suspensions containing 1.5 × 10<sup>8</sup> colony-forming units of bacteria was prepared based on a 0.5 McFarland standard and 0.1 mL of that amount was spread on the surface of solid nutrient agar in the plates and a hole, 5 mm in diameter, was made in the solid agar. 10 μL of the produced Ag NPs solution was then poured into the created well and put in incubator at 37 ◦C for 24 h. The antibacterial activity of the synthesized Ag NPs correlated to the diameter of created clear zones around the holes. An Ampicillin disc 5mm in diameter (Oxoid, 10 μg/disc) was used as a positive control for both Gram-positive and Gram-negative bacteria strains and the antibacterial activity of the synthesized Ag NPs was compared to the Ampicillin.

#### *3.6. Experimental Design, Statistical Analysis and Optimization Procedure*

The experiment was planned using a central composite design (CCD) and response surface methodology (RSM) was used to evaluate the effects of two independent parameters, namely amount of leaf extract (X1) and amount of AgNO3 solution (X2), on the prepared Ag NPs. The studied response variables were broad emission peak (λmax) (Y1, nm) and concentration (Y2, ppm) of the synthesized Ag NPs. As clearly observed in Table 1, thirteen experimental treatments were assigned with five different levels for each independent parameter using Minitab software (v.16 statistical package, Minitab Inc., Pennsylvania State, PA, USA). In order to correlate the λmax (Y1) and concentration (Y2) of the synthesized Ag NPs to the studied synthesis variables, a second order polynomial equation (Equation (1)) was used. Where β0 is a constant, β1, β11, and β12 correspond to the linear, quadratic and interaction effects, respectively [16]. The suitability of the model was studied, accounting for the coefficient of determination (R2) and adjusted coefficient of determination (R2-adj) [17]. Analysis of variance (ANOVA) was also used to provide the significance determinations of the resulted models in terms of *p*-value. Small *p*-values (lower than 0.05) were considered as statistically significant [18].

$$\mathbf{Y} = \boldsymbol{\beta}\_0 + \boldsymbol{\beta}\_1 \mathbf{X}\_1 + \boldsymbol{\beta}\_2 \mathbf{X}\_2 + \boldsymbol{\beta}\_{11} \mathbf{X}\_1^2 + \boldsymbol{\beta}\_{22} \mathbf{X}\_2^2 + \boldsymbol{\beta}\_{12} \mathbf{X}\_1 \mathbf{X}\_2 \tag{1}$$

Numerical optimization was carried out to determine exact amounts of silver salt and *J. regia* leaf extract in the Ag NPs synthesis to produce NPs with minimum λmax (particle size) and maximum concentration. Graphical optimization was also used to better visualize the effects of the synthesis parameters on the response variables (λmax and concentration) [19]. Suitability and accuracy of the generated models in predicting the response variables in the defined range of the Ag NPs synthesis parameters were validated by synthesis of Ag NPs using obtained optimum synthesized conditions and comparison of the experimental values of the response variables and their predicted values [20].
