*2.4. Characterization*

#### 2.4.1. Size, Polydispersity Index, and Zeta Potential

The synthesized conjugated NPs were characterized by a particle size analyzer (PSA) using Zetasizer Nano ZS90 (Malvern Instrumentations, Malvern, UK). Moreover, 50 μL NPs were dispensed in a cuvette and disseminated in a 950 μL DDW to measure the percentage intensity at 25 ◦C.

## 2.4.2. Fourier Transform Infrared (FTIR) Spectroscopy

At 80 ◦C under 0.4 bar, the particles were freeze-dried using a freeze-dryer (Christ, Germany). As a cryoprotectant, mannitol (10%) was utilized. With a fine powder of 5.0 mg, Fourier transform infrared (FTIR) spectroscopy was accomplished. At an average temperature between 4000 and 400 cm<sup>−</sup>1, FTIR spectra were documented using potassium bromide (KBr) in a 1:10 ratio with an AVATAR 370 FTIR (Therma Nicolet, San Jose, CA, USA). A graph pattern was employed to determine the ionic interaction between samples to measure mancozeb loading. Spectroscopic Tools, 2019 (Thomas St. (http://www.scienceand-fun.de/tools/, accessed on 7 September 2022) was used to evaluate the data.

#### 2.4.3. Transmission Electron Microscopy (TEM)

The TecnaiTM (FEI, Hillsboro, OR, USA) TEM was used to confirm the mancozeb loading. Before analysis, samples were placed in a water bath sonicator. A drop of diluted conjugated nanoparticles was placed on a carbon-coated copper grid, followed by roomtemperature air-drying. The photos were acquired using a 200 kV operational voltage using a facility from SAIF, AIIMS, New Delhi.

#### 2.4.4. Differential Scanning Calorimetry (DSC)

At LPU, Jalandhar's CIF facility, a 3.0 mg sample was utilized to make thermographs using a DSC 4000 System (Perkin Elmer, Waltham, MA, USA) with a heating and cooling rate of 10 ◦C/min. Samples were heated from 30 to 445 ◦C. Pure nitrogen gas (99.99%) was pumped into the system at a rate of 20 mL/min.

#### *2.5. In Vitro Study*

#### 2.5.1. Encapsulation Efficiency (EE) and Loading Capacity (LC)

The supernatant was collected in a sterile tube after the nanocomposites were centrifuged at 15,000 rpm for 35 min. NanoDrop2000c (Thermo Scientific, Wilmington, DE, USA) was used to acquire mancozeb in the supernatant at 290 nm using the supernatant of their equal blank-attached nanoparticles as simple adjustments. The following equation was used to compute the encapsulation efficiency (EE%):

Total mancozeb added-mancozeb in supernatant/total mancozeb added ×100 = Encapsulation efficiency (percentage).

The following equation was used to compute the loading capacity (LC%):

(Mass of mancozeb in CSGA conjugated NPs)/(Weight of CSGA conjugated NPs recovered) × 100 (1)

#### 2.5.2. Slow-Release Profile of Conjugated CSGA NPs

Mancozeb's in vitro release from CSGA-conjugated nanoparticles was derived via dialysis tubing (Hi-Media Ltd., Mumbai). A total of 10 mg of the sonicated nanocomposite was added to 1.0 mL of sterile phosphate-buffered saline, PBS (pH 7.2). This was dished in a dialysis membrane with closed clips at one end and immersed in 10 mL of the same PBS in separate tiny beakers. At 37 ◦C, it was incubated in a shaker at 160 rpm; 1.0 mL of phosphate-buffered saline was pipetted out of each beaker after predetermined intervals of time (30 min), and the same volume of buffer was supplied to each beaker. The absorbance of the resultant solution was measured at 290 nm to estimate the quantity of mancozeb in the buffer using the standard curve.

#### 2.5.3. Antimicrobial Activity

The impact of NPs on the mycelial growth of a specific pathogen was established using the mycelium inhibition technique on potato dextrose agar (PDA, 2%) with certain modifications [23]. To make the final concentration of conjugated nanoparticles as 0.5, 1.0, and 1.5 ppm, autoclaved potato dextrose agar medium (Hi-Media, Mumbai, India; temp. 40 ◦C) was placed onto separate sterile Petri plates (90 mm × 15 mm) and allowed to harden. A 5.0 mm mycelial disc was removed from a seven-day-old test pathogen culture and placed in the center of the test Petri dish, where it was incubated at 28 ± 1 ◦C under constant monitoring. Growth was measured after four days for three replications, and the treated plates were compared to the control plates (without nanocomposites) to calculate the percent suppression of mycelia by NPs using an earlier approach [24].

$$\text{dc} - \text{dt/dc } 100 = \text{percent inhibition} \tag{2}$$

where dc is the control mycelial diameter and dt is the mycelial treatment diameter.
