## 3 Results and Discussion  
### Synthesis of AgNPs and Ag(II)PRO Nanoparticles  
We have previously reported the synthesis of high-valence silver nanoconjugates with higher antibacterial efficacy [20; 21]. The polydiguanide type compound chlorhexidine (CHX) possessed two ionizable guanidine moieties which were suitable for the synthesis of stable high-valence silver chlorhexidine (Ag-CHX) complex. The molar ratio 1:1 of chlorhexidine and AgNO\({}_{3}\) was used to make AgCHX complex as the ligand coordinated with four nitrogen donor atoms from two guanidine units. In this present work, propamidine has been used as a new ligand to make stable high-valence nanocrystalline silver complex. The diamidine compound propamidine is similar to guanidine type compound but not exactly the same as chlorhexidine structure. In this study, 2:1 molar ratio of propamidine and AgNO\({}_{3}\) is used to form high-valence silver complex as four nitrogen donor atoms are coming from two propamidine units. Particularly, propamidine has diamidine structure, which requires 2:1 molar ratio for the complexation between silver ion and propamidne. This approach may contribute to the generalization of synthesis of silver complex grown as silver nanoparticles. The synthetic scheme of high-valence Ag(II)PRO complex has been shown in Figure 1. The synthesis of Ag(II)PRO complex was achieved by different solvent conditions. Initially, equal and different ratios of DMSO, water, and methanol were used to optimize the reaction condition. Finally, the reaction was successfully performed in methanol:water at a 3:1 ratio. After confirming the formation of Ag(II)PRO complex by NMR, FT-IR, mass and XPS spectrum, we synthesized the high-valence Ag(II)PRO nanoparticles by microemulsion technique. Figure 2 shows the synthetic scheme for the synthesis of Ag(II)PRO nanoparticles by microemulsion method. Figure 1: Synthetic scheme for the preparation of silver(II) propamidine complex. Figure 2: Schematic representation of the synthesis of silver(II) propamidine nanoparticles by reverse microemulsion technique. Microemulsions are defined as a clear, thermodynamically stable, mixture of liquid phases (oil and water) and surfactant, frequently in combination with a co-surfactant. Surfactant is usually a chemical compound possessing both hydrophilic and lipophilic properties. The three key reaction steps were involved in the formation of Ag(II)PRO nanoparticles in microemulsion technique. Firstly, we have to make the ligand propamidine water soluble. The commercially available propamidine is not completely soluble in heptane-water system (oil-water). To overcome this solubility issue, we have added diluted sulfuric acid to dissolve the propamidine ligand (pH > 5) by forming the dihydrogensulfate salt of propamidine. The second step involves the oxidation of silver ion to high-valence silver complex by sodium peroxysulfate oxidation. Finally, trimethylamine was added for complexation reaction in a neutral homogenous microemulsion medium. This appropriate medium was maintained for 24 h at neutral condition (pH = 7.0) to achieve fine and uniform sized nanoparticles. The \({}^{1}\)H-NMR, FT-IR, and Q-TOF mass spectra were obtained to verify the formation of Ag(II)PRO complex. The disappearance of broad -NH band in IR and the absence of imine protons in NMR spectra of Ag(II)PRO complex clearly confirmed the coordination of the silver metal to propamidine ligand (Figure S1). In this study, XPS spectra was obtained to confirm the oxidation state of the silver atom in silver propamidine complex. XPS is one of the most widely used surface analysis technique to confirm the valence state of the atom in the synthesized products or compounds. Figure 3 shows XPS spectrum of high-valence silver propamidine complex. The Ag 3d\({}_{3/2}\) and Ag 3d\({}_{5/2}\) XPS peaks were observed at 373.40 eV and 367.90 eV, respectively, this was confirmed with literature values for Ag\({}^{2+}\).