# Biosynthesized Zinc Oxide Nanoparticles Disrupt Established Biofilms of Pathogenic Bacteria
## 3 Results and Discussion  
### Green Synthesis of ZnO-NPs and Its Characterization  
The aqueous extract of _P. zeylanica_ was used for the synthesis of zinc oxide nanoparticles. Zinc acetate solution was used as precursor for the synthesis. The addition of plant extract to zinc acetate solution resulted in precipitate formation. The ZnO-NPs synthesized using _P. zeylanica_ extract were obtained by centrifugation followed by drying in hot air oven. The initial characterization of ZnO-NPs was performed using UV-Vis spectroscopy. The absorption spectrum is presented in Figure 1A. ZnO-NPs exhibited a sharp absorption at 373 nm which is due to the surface plasmon resonance (SPR) and it is assigned to the intrinsic band-gap absorption of ZnO owing to the electron transitions from valance band to conduction band [24]. Our finding agrees with an earlier report in which ZnO-NPs synthesized using flower extract of _N. arbor-tristis_ exhibited a sharp band at 365 nm [25]. Moreover, a study optimized the synthesis of ZnO-NPs, where the particles were prepared in three different conditions. The synthesized nanoparticles dried in vacuum at 30 \({}^{\circ}\)C did not exhibit any absorption peak from 300 to 600 nm. However, samples prepared by heating the zinc oxide nanoparticles at 60 \({}^{\circ}\)C and 100 \({}^{\circ}\)C showed the absorption maxima at 377 nm [26]. UV-Vis data suggested the formation of phases of ZnO NPs. The band-gap energy (\(E\)) of ZnO-NPs was computed using [PERSON]'s equation:  
\[E=\frac{hc}{\lambda} \tag{1}\]  
where \(h\) is [PERSON]'s constant, \(c\) is light velocity, and \(l\) is light wavelength. The \(E\) (band-gap energy) for ZnO-NPs was found to be 3.326 eV, which is coherent with previous reports [27]. The crystal nature of ZnO-NPs was confirmed by analyzing X-ray diffraction. The ZRD pattern of ZnO-NPs is shown in Figure 1B. ZnO-NPs exhibited seven diffraction intense peaks at 31.09, 33.71, 35.56, 46.88, 55.96, 62.19, and 67.34 degrees. The detail of each peak is listed in Table 1. The most prominent peak was recorded at 35.56\({}^{\circ}\). The narrow and sharp peaks denote that ZnO-NPs were well crystalline in nature. These peaks match with the JCPDS file number 361451 [26]. It is reported that ZnO-NPs synthesized using _N. arbor-tristis_ extract exhibited XRD peaks at 31.80, 34.44, 36.24, 47.48, 56.62, 62.88, 66.42, and 68.0\({}^{\circ}\), which were attributed to the hexagonal phase of zinc oxide [25]. Using the Debye-Scherrer equation (Equation (2)), the crystalline size of ZnO-NOPs was calculated [28]:  
\[D=\frac{K\lambda}{\beta\ *\ cos\theta} \tag{2}\]where \(D\) is size of the ZnO-NPs; \(\beta\) is full width of XRD peak at the half maximum; \(\lambda\) is wavelength (1.5406 A) of radiation, and K is the Debye-Scherrer constant. The average size of ZNO-NPs was found to be 24.62 \(\pm\) 2.74 nm. Moreover, the particle size calculated using the most intense peak (35.56\({}^{\circ}\)) was found to be 24.38 nm. The finding corroborates previous results in which the size of ZNO-NPs synthesized using \(A\). _indica_ extract was found to be 18 nm [24]. Transmission electron microscopy was performed to decipher the size and shape of ZnO-NPs. A TEM image of ZnO-NPs at 50,000X magnification is shown in Figure 2A. Most of the ZnO-NPs were irregular in shape with variation in sizes. The size of nanoparticles ranged from 8 to 40 nm (Figure 2B). Moreover, the morphological analysis of ZnO-NPs was performed by SEM analysis, and the micrograph is shown in Supplementary Figure S1. The nanoparticles were observed as spherical to oval or spheroidal in shape, further validating the TEM results. SEM images can be seen as huge clusters of particles as they were taken from the powdered form of ZnO-NPs. Variation in shape and size of green synthesized nanoparticles have been documented previously [25]. Such variation in the shape of green synthesized nanoparticles is due to the fact that some of the nanoparticles are capped and stabilized at lower size while other particles are stabilized at higher size.