**2. Materials and methods**

*Silver Micro-Nanoparticles - Properties, Synthesis, Characterization, and Applications*

*Green synthesis of silver nanoparticles by different researchers using plant extracts [31].*

*Escherichia coli, and Pseudomonas jessinii* are some of the examples of micro organisms used for silver nanoparticles synthesis [26]. Besides, the production of silver nanoparticles form *Bacillus clausii* cultured form *Enterogermina* is explored [27]. The aqueous extract of cynobacterial *Oscillatoria limnetica* fresh biomass was used for the green synthesis of AgNPs and it takes about 30–60 hours for the reduction and stabilizing the synthesize of metallic nanoparticles that ranges

The plants part being organic and eco friendly are extensively used for synthesis of silver nanoparticles (AgNPs). The plant are the hot spots for the phytochemicals and secondary metabolites that are used constantly for various medicinal purposes including antimicrobial, antifungal, anti inflammatory, wound healing, antidiabatic etc. The plant sources such as leaves, stem, roots, flowers possessing the medicinal properties are used for the formulation of silver nanoparticles which carries the specific medicinal compound to reduce and capped the silver salt and present at the outer layer of silver to make them stable [29]. Therefore, the plant based silver nanoparticles possess the duel properties from silver and one from capped compounds from plants. The different plant leaf extracts for examples pine, ginkgo, magnolia, mango, neem, *oscimum scantum*, are used for their extracellular synthesis of silver nanoparticles. The biological silver nanoparticles production has the faster synthesis rates than the chemical methods and potentially be used in various foods, agriculture, chemical and medical application. The aqueous peel extract of *Annona squamosa* has been used successfully for synthesis of silver nanoparticles of irregular spherical in shape with the average particle size of 35 nm, at room temperature [30]. Green synthesis of silver nanoparticles by different plant extracts are described in **Table 1**. There are some of the synthesis methods that are constantly used for nanoparticles synthesis such as high temperature, pressure, sunlight condition, in autoclave [29]. In contrast, there are other publication that performed the synthesis process

Diversity of compounds, polymers, exopolysaccharides, proteins, lipids and other compounds such as natural dyes could be a good source for reducing metal salts to form stable stained nanoparticles which can have various applications such as pesticides, nutrients, hormones delivery for sustainable agriculture. In the light of the above addressed interesting information on dichlorofluorescein, silver nitrate, silver nanoparticles, it is evident that silver nanoparticles have an enormous application in various fields. It was noticed that none of the papers reported the synthesis of silver nanoparticles using any dye. The direct use of dichlorofluorescein (DCF) for the reduction of silver salt can also produce silver nanoparticles and could be used for the study of absorption and biotransformation in seeds and plants. The aim of this research is therefore to conduct the synthesis and

**Sr no Plants Size (nm) Plant's part Shape** 39. *Tribulus terrestris* 16–28 Fruit Spherical 40. *Vitex negundo* 5 & 10–30 Leaves Spherical 41. *Vitis vinifera* 30–40 Fruit circular, 42. Ziziphoratenuior 8–40 Leaves Spherical

**30**

at room temperature.

**Table 1.**

from 3.30–17.97 nm in size [28].

*1.5.2 Silver nanoparticles synthesis using plants*

The materials and the methodology adopted for the synthesis of dichlorofluorescein silver nanoparticles (DCF-SNP), characterization and application are described below. Material required: Requirement specification for the formulation of silver nanoparticles utilize dichlorofluorescein, NaOH, distilled water, AgNO3 (1 mm), conical flask, beaker, aluminum foil, volumetric flask, etc. Silver nitrate (AgNO3): silver nitrate was used for the synthesis of dichlorofluorescein silver nanoparticles (DCF-SNPs) and was then used with the seed germination assay. Instruments for study: The following instruments such as Fourier transform infrared spectroscopy (FTIR), Zeta Potential (ZP) and Nanoparticles tracking analysis (NTA) were used for characterization of synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) [32].

#### **2.1 Preparation of dichlorofluorescein (DCF) solution**

Dichlorofluorescein (DCF) solution is prepared using 10 mg concentrate in 100 ml of distilled water. The solution is entirely blended until it becomes a greenish liquid. A 2.5 mL of the prepared dichlorofluorescein (DCF) solution was used for the preparation of 100 mL silver nanoparticles.
