**Abstract**

The main objective of this study was to investigate whether dichlorofluorescein (DCF) is adequate for the formulation of stable dichlorofluorescein-induced silver nanoparticles under the boiling method to analyze their effects on the seed germination of Mung seeds *(Vigna radiata)*. Preliminary dichlorofluoresceine nanoparticles (DCF-SNPs) synthesis evidence by noticing the solution color transformed from a light green color to a dark brown color. The 2.5 ml of dichlorofluoresceine (DCF) solution was found sufficient for the formulation of dichlorofluoresceine induced silver nanoparticles at boiling conditions. Purified dichlorofluoresceine nanoparticles (DCF-SNPs) measure an average diameter of 293 nm where the majority of nanoparticles were around 159 nm in size with the surface load of-9.35 mV zeta potential value. The impact of dichlorofluorescein silver nanoparticles (DCF-SNPs) on the germination percentage of *V. radiata* has shown that, the 25% concentration of DCF-SNPs is excellent for the growth of Mung seeds (*V. radiata*). Overall, the dichlorofluorescein silver nanoparticles may be constructive for improving the percentage of seed germination at 25% of its concentration and may also be useful for fluorescent measurement using the confocal microscopy technique. Hence, dichlorofluorescein silver nanoparticles (DCF-SNPs) are proposed as an efficient detection system for nanoparticles in agrochemicals for plants.

**Keywords:** dichlorofluoresceine (DCF), *Vigna radiata*, dichlorofluoresceine silver nanoparticles (DCF-SNP), Zeta potential, confocal microscopy studies

#### **1. Introduction**

Dyes are colored entities that are chemically attached to the matrix fiber and accumulate during the drying period, providing color through the systematic absorption of light and increasing the speed of the fiber dyeing process [1]. There are two types of dyes which are natural or organic dyes and chemically synthesized dyes. In this chapter we are going to deal with organic dyes.

#### **1.1 Organic dye**

Natural dyes are derived from natural resources and, are typically categorized as plant, animal, mineral and microbial dyes, though plants are the key sources of natural dyes [2]. Examples of natural and organic coloring dyes are blue dye which derived from plant leaves, while red dye, Madder and Morinda from roots. Brazil Wood an old-world dye comes from wood of plant. Safflower and saffron dye made from their flowers, however, rhizomes of turmeric use to make dye. On the other hand yellow dye derived from roots, leaves, stems and flowers [2]. The organic dyes are colored as they appear in the visible light spectrum (400–700 nm) comprising of one chromosphere, a conjugate framework with a dual bond alternating arrangement with a single bond and an electron resonance that stabilizes in organic compounds [3]. Based on the chemical structure and characteristics properties, dyes are classified as *Azo* dyes, *Anthraquinone* dyes, Nitro dyes*, Diphenlmethane* dyes*, Triphenylmethane* dyes*, Xanthene* dyes, *Phthaleins* dyes, *Indigoid* and *Thionidigoid* dyes [4]. These organic coloring powders are smaller, thicker, finely fragmented crystalline solids, which are water soluble, whereas they are insoluble in application media such as ink or paint for the use of optical media, image sensors [1], photosensitizer and coloration [5]. Improved color functionality has been studied in recent times by adding functional properties such as antimicrobial, UV protection, insect repellent, etc. [6]. In addition, organic coloring is a major supplier for floral, dried flowers, pesticides, ice-melt, deicing, reservoir, lake, water tracing, leak detection, greenhouse, livestock, seed treatment, crop protection, fertilizer staining, food, cosmetic and environmental issues [7]. The multitude application of organic dyes has enable to use them for coloring, detection, biosensor, tracking in the test samples. One of the best examples of tracking dye is dichlorofluorescein (DCF).

#### **1.2 Dichlorofluoresceine (DCF)**

Fluorescein based titrations shows effective results at 0.005 N concentrations of chloride and if the formulation has neutral alkaline conditions [8]. 2′,7'-Dichloroand 2′,7′-difluorofluoresceins are superior alternatives to underivatized fluorescein. Quadrupole ion trap mass spectrometer for the analysis of the gas phase properties of three charge states of fluorescein, indicating that dianions and cations do not emit detectable fluorescence in the gas phase. Monoanions, on the other hand, do fluorescence and are useful for experiments [9]. Dichlorofluorescein (DFC) is a natural, crystalline organic, coloring agent that substitutes for chloride at 2 and 7 positions and originated in the fluorescein family. **Figure 1** illustrates the molecular

**Figure 1.**

*Demonstrates the molecular structure of Dichlorofluorescein with empirical formula as C20H10cl205 [10].*

**27**

**Figure 2.**

*Shows the molecular structure of silver nitrate [16].*

*Synthesis, Characterization of Dichlorofluorescein Silver Nanoparticles (DCF-SNPs)…*

chloride in the sample as specified in the Fajans method [11].

structure of dichlorofluorescein. The molecular weight of dichlorofluorescein (PubChem SID: 24894041) is 401.20 g/mol, a melting point of 280 °C and it is used as an indicator which is not prone to soluble, absorbs, disintegrates or infuses silver or halide ion, but changes color at the end of precipitate due to absorption phenomena [8]. It was therefore used as a quantitative agentometry titrant indicator, consisting of a known concentration of silver nitrate, to estimate the molarity of

Organic Dichlorofluorescein (DCF) applications are even less documented, however it has demonstrated application as an indicator for halide titration argentometry, gaseous dianion studies, targeting it as a probe for imaging, heamotherapy and histological applications [8, 10, 12]. Generally, non fluorescent 2′ 7' Dichlorodihydrofluorscein diacetate is used in human hepatocellular carcinoma cells to monitor the oxidation of 2′,7′-dichlorofluorescin-diacetate (DCF-DA) to an extremely fluorescent 2′,7′ dichlorofluorescein (DCF) compound due to the presence of reactive oxygen species using a fluorometric microplate assay [13]. And thereby, 2′,7′ dichlorofluorescein (DCF) may be very significant in the assessment of the human organ culture model. In the same way, organic dyes can be used to research the tracing of in the host body of insects, plants and animals [12]. DCF can be the reducing agent for metallic salt (iron, copper, zinc, silver, gold, etc.) for the

formation of metallic nanoparticles for various agriculture applications.

state at a temperature is 210 °C corresponds to 3.97 g/cm3

ment, dormancy and by spraying it on the growth tip of plants [17].

Silver nitrate (AgNO3) is an inorganic compound that appears to have a colorless, white crystalline composition with molecular weight of 169.873 g/mol. In its solid state, it has a density of 4.35 grams per cubic centimeter and its density in the liquid

points of silver nitrate are 414 °F and 824 °F respectively. In 1800s, silver nitrate was used for the treatment of ulcerations and infected wounds and stomach ulcers [14–15]. Silver nitrate is a chemical with a wide range of applications including antiseptic, suturing, eye disease disinfectant, burned wounds, wart and granulation tissue reduction, ulceration, dental cavity retention, etc. [16]. Other noteworthy medical applications of silver include wire or coated suture topical therapy for osteocutaneous fistulae, and foil coverings for burn wounds [14]. Silver nitrate compound is a productive source of creation for many other silver compounds used for the medical, biotechnological, nanotechnological, pharmaceutical as well as several other industries. **Figure 2** shows the silver nitrate molecular structure. Silver nitrate is frequently used chemical in several areas in agriculture including control growth, flowering develop-

. The melting and boiling

*DOI: http://dx.doi.org/10.5772/intechopen.96756*

*1.2.1 Dichlorofluorescein (DCF) applications*

**1.3 Silver nitrate (AgNO3)**

*Synthesis, Characterization of Dichlorofluorescein Silver Nanoparticles (DCF-SNPs)… DOI: http://dx.doi.org/10.5772/intechopen.96756*

structure of dichlorofluorescein. The molecular weight of dichlorofluorescein (PubChem SID: 24894041) is 401.20 g/mol, a melting point of 280 °C and it is used as an indicator which is not prone to soluble, absorbs, disintegrates or infuses silver or halide ion, but changes color at the end of precipitate due to absorption phenomena [8]. It was therefore used as a quantitative agentometry titrant indicator, consisting of a known concentration of silver nitrate, to estimate the molarity of chloride in the sample as specified in the Fajans method [11].

#### *1.2.1 Dichlorofluorescein (DCF) applications*

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

Natural dyes are derived from natural resources and, are typically categorized as plant, animal, mineral and microbial dyes, though plants are the key sources of natural dyes [2]. Examples of natural and organic coloring dyes are blue dye which derived from plant leaves, while red dye, Madder and Morinda from roots. Brazil Wood an old-world dye comes from wood of plant. Safflower and saffron dye made from their flowers, however, rhizomes of turmeric use to make dye. On the other hand yellow dye derived from roots, leaves, stems and flowers [2]. The organic dyes are colored as they appear in the visible light spectrum (400–700 nm) comprising of one chromosphere, a conjugate framework with a dual bond alternating arrangement with a single bond and an electron resonance that stabilizes in organic compounds [3]. Based on the chemical structure and characteristics properties, dyes are classified as *Azo* dyes, *Anthraquinone* dyes, Nitro dyes*, Diphenlmethane* dyes*, Triphenylmethane* dyes*, Xanthene* dyes, *Phthaleins* dyes, *Indigoid* and *Thionidigoid* dyes [4]. These organic coloring powders are smaller, thicker, finely fragmented crystalline solids, which are water soluble, whereas they are insoluble in application media such as ink or paint for the use of optical media, image sensors [1], photosensitizer and coloration [5]. Improved color functionality has been studied in recent times by adding functional properties such as antimicrobial, UV protection, insect repellent, etc. [6]. In addition, organic coloring is a major supplier for floral, dried flowers, pesticides, ice-melt, deicing, reservoir, lake, water tracing, leak detection, greenhouse, livestock, seed treatment, crop protection, fertilizer staining, food, cosmetic and environmental issues [7]. The multitude application of organic dyes has enable to use them for coloring, detection, biosensor, tracking in the test samples. One of the best examples of tracking dye is dichlorofluorescein (DCF).

Fluorescein based titrations shows effective results at 0.005 N concentrations of chloride and if the formulation has neutral alkaline conditions [8]. 2′,7'-Dichloroand 2′,7′-difluorofluoresceins are superior alternatives to underivatized fluorescein. Quadrupole ion trap mass spectrometer for the analysis of the gas phase properties of three charge states of fluorescein, indicating that dianions and cations do not emit detectable fluorescence in the gas phase. Monoanions, on the other hand, do fluorescence and are useful for experiments [9]. Dichlorofluorescein (DFC) is a natural, crystalline organic, coloring agent that substitutes for chloride at 2 and 7 positions and originated in the fluorescein family. **Figure 1** illustrates the molecular

*Demonstrates the molecular structure of Dichlorofluorescein with empirical formula as C20H10cl205 [10].*

**1.1 Organic dye**

**1.2 Dichlorofluoresceine (DCF)**

**26**

**Figure 1.**

Organic Dichlorofluorescein (DCF) applications are even less documented, however it has demonstrated application as an indicator for halide titration argentometry, gaseous dianion studies, targeting it as a probe for imaging, heamotherapy and histological applications [8, 10, 12]. Generally, non fluorescent 2′ 7' Dichlorodihydrofluorscein diacetate is used in human hepatocellular carcinoma cells to monitor the oxidation of 2′,7′-dichlorofluorescin-diacetate (DCF-DA) to an extremely fluorescent 2′,7′ dichlorofluorescein (DCF) compound due to the presence of reactive oxygen species using a fluorometric microplate assay [13]. And thereby, 2′,7′ dichlorofluorescein (DCF) may be very significant in the assessment of the human organ culture model. In the same way, organic dyes can be used to research the tracing of in the host body of insects, plants and animals [12]. DCF can be the reducing agent for metallic salt (iron, copper, zinc, silver, gold, etc.) for the formation of metallic nanoparticles for various agriculture applications.

#### **1.3 Silver nitrate (AgNO3)**

Silver nitrate (AgNO3) is an inorganic compound that appears to have a colorless, white crystalline composition with molecular weight of 169.873 g/mol. In its solid state, it has a density of 4.35 grams per cubic centimeter and its density in the liquid state at a temperature is 210 °C corresponds to 3.97 g/cm3 . The melting and boiling points of silver nitrate are 414 °F and 824 °F respectively. In 1800s, silver nitrate was used for the treatment of ulcerations and infected wounds and stomach ulcers [14–15]. Silver nitrate is a chemical with a wide range of applications including antiseptic, suturing, eye disease disinfectant, burned wounds, wart and granulation tissue reduction, ulceration, dental cavity retention, etc. [16]. Other noteworthy medical applications of silver include wire or coated suture topical therapy for osteocutaneous fistulae, and foil coverings for burn wounds [14]. Silver nitrate compound is a productive source of creation for many other silver compounds used for the medical, biotechnological, nanotechnological, pharmaceutical as well as several other industries. **Figure 2** shows the silver nitrate molecular structure. Silver nitrate is frequently used chemical in several areas in agriculture including control growth, flowering development, dormancy and by spraying it on the growth tip of plants [17].

**Figure 2.** *Shows the molecular structure of silver nitrate [16].*

Silver nitrate has a long use in nanotechnology for acting as a protective layer in stabilizing nanoparticles from further agglomeration. During the reduction of metal salt formation narrow size of nanoparticles are obtained. It absorbs on the surface of particle provide stabilization & diffusion barrier in the growth of particle [18]. Therefore, this silver salt are the best source for silver nanoparticles synthesis.

#### **1.4 Silver nanoparticles (AgNPs)**

Silver nanoparticles (AgNPs) range from 1 to 100 nm in size, which are fundamentally synthesized by physical, chemical and biological approaches. Silver nanoparticles were being used as antimicrobial agents in a wide variety of applications, which include disinfecting medical instruments and home appliances to water treatment [19]. Even though the other biological properties of silver nanoparticles such as antimicrobial, antifungal, anti-inflammatory, anti-cancer and anti-angiogenesis [20] already have enabled them to be extensively used in the fields of medicine and dentistry, diagnostics, therapeutic, medical care, health and food applications [21]. The other medical applications, including wound repair, bone healing, dental applications, vaccine adjuvant, antidiabetic agent, and biosensing [22]. The techniques of synthesis of metal silver nanoparticles have certain benefits as well as drawbacks. Therefore, depending on the application, the selection of the procedure is presumed and therefore, depending upon the application the selection of the methodology is considered. A well recorded manuscript available in the literature on the physical, chemical and biological preparation of silver nanoparticles. Nanoparticles have proved to be efficient agrochemical agents in order to improve the crop productivity, reducing the pests, increasing the nutrient uptake, inhibiting the pathogens and act as 'magic bullets' serving as herbicides, pesticides and fertilizers etc. [23–24]. The biological activity of AgNPs depends on factors including surface chemistry, size, size distribution, shape, particle morphology, particle composition, coating/capping, agglomeration, and dissolution rate, particle reactivity in solution, efficiency of ion release, and cell type, and the type of reducing agents used for the synthesis of AgNPs are a crucial factor for the determination of cytotoxicity [20]. The physicochemical properties of nanoparticles enhance the bioavailability of therapeutic agents after both systemic and local administration and other hand it can affect cellular uptake, biological distribution, penetration into biological barriers, and resultant therapeutic effects [20]. There are several methods for creating nanoparticles, including co precipitation, hydrothermal synthesis, inert gas condensation, ion sputtering scattering, micro emulsion, microwave, pulse laser ablation, sol–gel, sono chemical, spark discharge, template synthesis, and biological synthesis. We shall now briefly look into the methods for the synthesis of nanoparticles [25].

#### **1.5 Synthesis of silver nanoparticles**

There are large number of mrthods for bthe synthesis of silver nanoparticles. While in the present study we will discuss using microorganisms, plant extract and we proposed to use DCF for silver nanoparticles synthesis.

#### *1.5.1 Silver nanoparticles synthesis using microorganism*

There are various microorganism that have been explored for the synthesis of silver nanoparticles due to their advantage of reliable and ecofriendly process. The microorganisms used for reducing and capping the silver salts produces the various size, shape and morphology of silver nanoparticles. Novel *Nocardiopsis species, Brevibac teriumfrigoritolerans* strain, *Klebsiella pneumoniae,* 

**29**

*Synthesis, Characterization of Dichlorofluorescein Silver Nanoparticles (DCF-SNPs)…*

**Sr no Plants Size (nm) Plant's part Shape** 1. *Abutilon indicum* 7–17 Leaves Spherical 2. Acalyphaindica 0.5 Leaves — 3. Acalyphaindica 20–30 Leaves Spherical 4. *Acorus calamus* 31.83 Rhizome Spherical 5. *Allium sativum* 4–22 Leaves Spherical 6. *Aloe vera* 50–350 Leaves Spherical, 7. Alternanthera dentate 50–100 Leaves Spherical 8. *Argyreia nervosa* 20–50 Seeds — 9. Boerhaaviadiusa 25 Whole plant Spherical 10. *Brassica rapa* 16.4 Leaves — 11. *Calotropis procera* 19–45 Plant Spherical 12. *Carica papaya* 25–50 Leaves circular, 13. Centellaasiatica 30–50 Leaves Spherical 14. *Citrus sinensis* 10–35 Peel Spherical 15. Cocciniaindica 10–20 Leaves — 16. Cocous nucifera 22 Inflorescence Spherical 17. Cymbopogancitratus 32 Leaves — 18. *Datura metel* 16–40 Leaves Quasilinear 19. Eclipta prostrate 35–60 Leaves pentagons, 20. Eucalyptus hybrid 50–150 Peel spherical 21. Ficuscarica 13 Leaves — 22. *Garcinia mangostana* 35 Leaves — 23. Melia dubia 35 Leaves Spherical 24. Memecylonedule 20–50 Leaves hexagonal 25. *Moringa oleifera* 57 Leaves — 26. Musa paradisiacal 20 Peel — 27. *Nelumbo nucifera* 25–80 Leaves triangular 28. Nelumbo nucifera 25–80 Leaves triangular 29. *Passiflora foetida* — Leaves Coral 30. Pistaciaatlantica 10–50 Seeds Spherical 31. Pogostemonbenghalensis >80 Leaves — 32. *Portulaca oleracea* <60 Leaves — 33. Premnaherbacea 10–30 Leaves Spherical 34. Psoraleacorylifolia 100–110 Seeds — 35. Swieteniamahogani 50 Leaves — 36. Tea extract 20–90 Leaves Spherical 37. *Thevetia peruviana* 10–30 Latex Spherical 38. Trachyspermumammi 87, 99.8 Seeds —

*DOI: http://dx.doi.org/10.5772/intechopen.96756*


*Synthesis, Characterization of Dichlorofluorescein Silver Nanoparticles (DCF-SNPs)… DOI: http://dx.doi.org/10.5772/intechopen.96756*

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

**1.4 Silver nanoparticles (AgNPs)**

Silver nitrate has a long use in nanotechnology for acting as a protective layer in stabilizing nanoparticles from further agglomeration. During the reduction of metal salt formation narrow size of nanoparticles are obtained. It absorbs on the surface of particle provide stabilization & diffusion barrier in the growth of particle [18]. Therefore, this silver salt are the best source for silver nanoparticles synthesis.

Silver nanoparticles (AgNPs) range from 1 to 100 nm in size, which are fundamentally synthesized by physical, chemical and biological approaches. Silver nanoparticles were being used as antimicrobial agents in a wide variety of applications, which include disinfecting medical instruments and home appliances to water treatment [19]. Even though the other biological properties of silver nanoparticles such as antimicrobial, antifungal, anti-inflammatory, anti-cancer and anti-angiogenesis [20] already have enabled them to be extensively used in the fields of medicine and dentistry, diagnostics, therapeutic, medical care, health and food applications [21]. The other medical applications, including wound repair, bone healing, dental applications, vaccine adjuvant, antidiabetic agent, and biosensing [22]. The techniques of synthesis of metal silver nanoparticles have certain benefits as well as drawbacks. Therefore, depending on the application, the selection of the procedure is presumed and therefore, depending upon the application the selection of the methodology is considered. A well recorded manuscript available in the literature on the physical, chemical and biological preparation of silver nanoparticles. Nanoparticles have proved to be efficient agrochemical agents in order to improve the crop productivity, reducing the pests, increasing the nutrient uptake, inhibiting the pathogens and act as 'magic bullets' serving as herbicides, pesticides and fertilizers etc. [23–24]. The biological activity of AgNPs depends on factors including surface chemistry, size, size distribution, shape, particle morphology, particle composition, coating/capping, agglomeration, and dissolution rate, particle reactivity in solution, efficiency of ion release, and cell type, and the type of reducing agents used for the synthesis of AgNPs are a crucial factor for the determination of cytotoxicity [20]. The physicochemical properties of nanoparticles enhance the bioavailability of therapeutic agents after both systemic and local administration and other hand it can affect cellular uptake, biological distribution, penetration into biological barriers, and resultant therapeutic effects [20]. There are several methods for creating nanoparticles, including co precipitation, hydrothermal synthesis, inert gas condensation, ion sputtering scattering, micro emulsion, microwave, pulse laser ablation, sol–gel, sono chemical, spark discharge, template synthesis, and biological synthesis. We shall now briefly look into the methods for the synthesis

There are large number of mrthods for bthe synthesis of silver nanoparticles. While in the present study we will discuss using microorganisms, plant extract and

There are various microorganism that have been explored for the synthesis of silver nanoparticles due to their advantage of reliable and ecofriendly process. The microorganisms used for reducing and capping the silver salts produces the various size, shape and morphology of silver nanoparticles. Novel

*Nocardiopsis species, Brevibac teriumfrigoritolerans* strain, *Klebsiella pneumoniae,* 

**28**

of nanoparticles [25].

**1.5 Synthesis of silver nanoparticles**

we proposed to use DCF for silver nanoparticles synthesis.

*1.5.1 Silver nanoparticles synthesis using microorganism*


#### **Table 1.**

*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 from 3.30–17.97 nm in size [28].

#### *1.5.2 Silver nanoparticles synthesis using plants*

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 at room temperature.

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

**31**

**Figure 3.**

*Synthesis, Characterization of Dichlorofluorescein Silver Nanoparticles (DCF-SNPs)…*

transformation in the living plant using confocal microscopy.

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

for the preparation of 100 mL silver nanoparticles.

**2.2 Synthesis of silver nitrate (AgNO3) solution**

characterization of dichlorofluorescein induced dichlorofluorescein silver nanoparticles (DCF-SNPs) under boiling method to evaluate their effects on the seed germination of *Vigna radiata* and to propose that they would be used for real-time

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

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

The silver nitrate solution is prepared by using 1.698 g silver nitrate powder in 100 ml of distilled water to form 100 mM concentration. From his prepared 100 ml of silver nitrate solution; 1 ml is used for preparation of 1 mM silver nitrate solution for silver nanoparticles synthesis. The complete process is performed

*Displays the synthesis of dichlorofluorescein silver nanoparticles (DCF-SNP) employing AgNO3 and dichlorofluorescein under heating conditions. The change in color of the reaction mixture was noticed from light* 

*green to dark brown, demonstrating the formation of silver nanoparticles.*

*DOI: http://dx.doi.org/10.5772/intechopen.96756*

**2. Materials and methods**

nanoparticles (DCF-SNPs) [32].

under absence of light.

*Synthesis, Characterization of Dichlorofluorescein Silver Nanoparticles (DCF-SNPs)… DOI: http://dx.doi.org/10.5772/intechopen.96756*

characterization of dichlorofluorescein induced dichlorofluorescein silver nanoparticles (DCF-SNPs) under boiling method to evaluate their effects on the seed germination of *Vigna radiata* and to propose that they would be used for real-time transformation in the living plant using confocal microscopy.
