**3. Characterization of DCF-SNP**

Various instruments are used to characterize the synthesized dichlorofluorescein silver nanoparticles. These are some of the techniques which we used in our investigations, such as FTIR analysis, that are used to classify a functional group that has a reducing and stabilizing properties. Finally, zeta potential and nanoparticles tracking analysis are used to track the surface charge and the size of the silver nanoparticles.

#### **3.1 Fourier transform infra red (FTIR)**

Dichlorofluorescein silver nanoparticles (DCF-SNPs) are used in the FTIR analysis to recognize the main functional groups involved in the reduction and capping of silver salt for the development of stable dichlorofluorescein silver nanoparticles (DCF-SNPs). In the FTIR instrument, the radiation falls on the sample and causes changes in the vibration and rotational motion of the molecules at a wavelength of 4000–440 cm − 1 consisting of near and far infrared frequencies and the FTIR spectrum of the formulation was recorded.

#### **3.2 Nanoparticles tracking analysis (NTA)**

The size of the synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) is calculated using Nanosight (LM-20, UK) at the Department of Biotechnology, Sant Gadge Baba Amravati University, India. Dichlorofluorescein silver nanoparticles (DCF-SNP) were diluted in 0.5 ml nuclease-free water and injected into the sample chamber. The instrument parameters are calibrated as specified and the device is analyzed nanoparticles samples to calculate the size of the nanoparticles. The data are summarized on the computer screen and are retrieved in PDF format for analysis.

#### **3.3 Zeta-potential analysis (ZP)**

The zeta potential characterization of dichlorofluorescein silver nanoparticles (DCF-SNPs) was tested by the Department of Biotechnology, Sant Gadge Baba Amravati University, Maharashtra, India. A zeta potential is used to assess the potential surface charge of Dichlorofluorescein Silver nanoparticles (DCF-SNP) using a zetasizer (nano ZS, malvern instrument Ltd., UK). In particular, the liquid samples of the dichlorofluorescein silver nanoparticles DCF-SNP (5 ml) were diluted with double distilled water (50 ml) using NaCl as an electrolyte suspension solution 2 M NaCl). In addition, the samples are injected into the sample slot of Zeta

**33**

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

values of the zeta potential ranged from +30 mV to-30 mV.

and root was calculated with the aid of a string and a scale [33].

shows typical and narrow absorption peak at approximately 400 nm.

Dichlorofluorescein silver nanoparticles (DCF-SNP) were successfully fabricated using AgNO3 (1 mM) and DCF (2.5 per cent) by boiling the reactants at 12 pH. The reaction is observed for color shift and is found to be dark brown in color

Dichlorofluorescein silver nanoparticles (DCF-SNPs) are used by different analytical methods to characterize them at the nanoscale level. The technique used for characterization were fourier transform infrared spectroscopy (FTIR), zeta potential (ZP) analysis and nanoparticles tracking analysis (NTA) using NanoSight LM-20. In addition, the synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) was used to successfully test its effect on the germination of Mung Bean (*V. radiata*) seeds. Essentially, the characterization of the synthesized dichlorofluorescein silver nanoparticles (DCF-SNP) was primarily carried out by observing the change in color of the reaction mixture from light green to dark brown. In addition different nanoparticles characterization techniques are used to chemically characterize nanoparticles. In the UV–visible spectrum analysis a single, strong, and broad surface plasmon resonance (SPR) peak was observed at 419 nm that confirmed the synthesis of Dichlorofluoresceine Silver nanoparticles. In same way, [34] UV–vis results displayed that the SPRs becomes sharper and shifts towards lower wavelength (*Escherichia hermannii* = 438 nm, *Citrobacter sedlakii* = 441 nm), shows that the particle size of AgNPs decreased. The work of [35] suggested that the green synthesis of gallic acid-coated silver nanoparticles UV–vis absorption spectrum

potential instrument and the good and important results data are reported in PDF formats. In each case, an average of three different measurements made while the

**3.4 Effect of dichlorofluorescein silver nanoparticles (DCF-SNPs) on seeds** 

Dichlorofluorescein silver nanoparticles (DCF-SNP) was used to investigate its effect on the germination of Mung Bean (*Vigna radiata*) seeds. Mung bean seeds were purchased from the market and stored in a dry place under room temperature in the dark. Here, four separate concentrations of 25 per cent, 50 per cent, 75 per cent and 100 per cent (v/v) of dichlorofluorescein silver nanoparticles (DCF-SNPs) dispersion were prepared in distilled water. The Whatman no.3 filter paper is layered on the sterile Petri dishes of 12 cm diameter and germination test analysis was carried out. In this test, the seeds were surface sterilized with 0.1% Hgcl2 solution and rinsed three times with distilled water In total, 10 seeds of Mung Bean (*V. radiata*) were placed in the respective Petri dishes. The solution of each concentration was transferred to each Petri dish and the treatment was administered daily at only enough doses to hydrate the seeds. The petri dish was then placed in a dark seed germinator and held at 25 °C. Seed with root tip 1 mm and above was considered germinated. Percent germination and root and shoot length (in mm) were recorded every 24 hours up to 72 hours. The root length was determined from the region under the hypocotylis to the end of the root cap. The length of the shoot was measured to the nearest millimeter of the root hypocotyl transission zone to the middle of the cotyledon. The length of the shoot

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

**germination of** *Vigna radiata*

**4. Results and discussions**

**4.1 Visualization of (DCF-SNPs)**

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

potential instrument and the good and important results data are reported in PDF formats. In each case, an average of three different measurements made while the values of the zeta potential ranged from +30 mV to-30 mV.

#### **3.4 Effect of dichlorofluorescein silver nanoparticles (DCF-SNPs) on seeds germination of** *Vigna radiata*

Dichlorofluorescein silver nanoparticles (DCF-SNP) was used to investigate its effect on the germination of Mung Bean (*Vigna radiata*) seeds. Mung bean seeds were purchased from the market and stored in a dry place under room temperature in the dark. Here, four separate concentrations of 25 per cent, 50 per cent, 75 per cent and 100 per cent (v/v) of dichlorofluorescein silver nanoparticles (DCF-SNPs) dispersion were prepared in distilled water. The Whatman no.3 filter paper is layered on the sterile Petri dishes of 12 cm diameter and germination test analysis was carried out. In this test, the seeds were surface sterilized with 0.1% Hgcl2 solution and rinsed three times with distilled water In total, 10 seeds of Mung Bean (*V. radiata*) were placed in the respective Petri dishes. The solution of each concentration was transferred to each Petri dish and the treatment was administered daily at only enough doses to hydrate the seeds. The petri dish was then placed in a dark seed germinator and held at 25 °C. Seed with root tip 1 mm and above was considered germinated. Percent germination and root and shoot length (in mm) were recorded every 24 hours up to 72 hours. The root length was determined from the region under the hypocotylis to the end of the root cap. The length of the shoot was measured to the nearest millimeter of the root hypocotyl transission zone to the middle of the cotyledon. The length of the shoot and root was calculated with the aid of a string and a scale [33].

#### **4. Results and discussions**

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

The dichlorofluorescein silver nanoparticles (DCF-SNPs) is prepared by mixing 97.5 ml of AgNO3 (1 mm) and 2.5 ml of dichlorofluorescein in conical flask at constant stirring. The complete reaction process is carried out under boiling condition for 1–2 min, the pH of the mixture is adjusted to 12 and the reaction completes after 24 hours. The chemical constituents or the functional group that are presents in the Dichlorofluoresceine (DCF) structure reduced the silver salt to form the silver nanoparticles called as dichlorofluorescein silver nanoparticles (DCF-SNPs). The complete process of synthesis of dichlorofluorescein silver nanoparticles (DCF-SNPs) using AgNO3 and dichlorofluorescein at 0 hr., 2 hr. and 24 hr. and observed changes in color from light green to dark brown, confirming the synthesis of silver

Various instruments are used to characterize the synthesized dichlorofluorescein silver nanoparticles. These are some of the techniques which we used in our investigations, such as FTIR analysis, that are used to classify a functional group that has a reducing and stabilizing properties. Finally, zeta potential and nanoparticles tracking analysis are used to track the surface charge and the size of the silver nanoparticles.

Dichlorofluorescein silver nanoparticles (DCF-SNPs) are used in the FTIR analysis to recognize the main functional groups involved in the reduction and capping of silver salt for the development of stable dichlorofluorescein silver nanoparticles (DCF-SNPs). In the FTIR instrument, the radiation falls on the sample and causes changes in the vibration and rotational motion of the molecules at a wavelength of 4000–440 cm − 1 consisting of near and far infrared frequencies and the FTIR

The size of the synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) is calculated using Nanosight (LM-20, UK) at the Department of Biotechnology, Sant Gadge Baba Amravati University, India. Dichlorofluorescein silver nanoparticles (DCF-SNP) were diluted in 0.5 ml nuclease-free water and injected into the sample chamber. The instrument parameters are calibrated as specified and the device is analyzed nanoparticles samples to calculate the size of the nanoparticles. The data are summarized on the computer screen and are retrieved in PDF format for analysis.

The zeta potential characterization of dichlorofluorescein silver nanoparticles (DCF-SNPs) was tested by the Department of Biotechnology, Sant Gadge Baba Amravati University, Maharashtra, India. A zeta potential is used to assess the potential surface charge of Dichlorofluorescein Silver nanoparticles (DCF-SNP) using a zetasizer (nano ZS, malvern instrument Ltd., UK). In particular, the liquid samples of the dichlorofluorescein silver nanoparticles DCF-SNP (5 ml) were diluted with double distilled water (50 ml) using NaCl as an electrolyte suspension solution 2 M NaCl). In addition, the samples are injected into the sample slot of Zeta

**2.3 Synthesis of dichlorofluorescein silver nanoparticles**

nanoparticles synthesis is given in **Figure 3**.

**3. Characterization of DCF-SNP**

**3.1 Fourier transform infra red (FTIR)**

spectrum of the formulation was recorded.

**3.2 Nanoparticles tracking analysis (NTA)**

**3.3 Zeta-potential analysis (ZP)**

**32**

Dichlorofluorescein silver nanoparticles (DCF-SNPs) are used by different analytical methods to characterize them at the nanoscale level. The technique used for characterization were fourier transform infrared spectroscopy (FTIR), zeta potential (ZP) analysis and nanoparticles tracking analysis (NTA) using NanoSight LM-20. In addition, the synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) was used to successfully test its effect on the germination of Mung Bean (*V. radiata*) seeds. Essentially, the characterization of the synthesized dichlorofluorescein silver nanoparticles (DCF-SNP) was primarily carried out by observing the change in color of the reaction mixture from light green to dark brown. In addition different nanoparticles characterization techniques are used to chemically characterize nanoparticles. In the UV–visible spectrum analysis a single, strong, and broad surface plasmon resonance (SPR) peak was observed at 419 nm that confirmed the synthesis of Dichlorofluoresceine Silver nanoparticles. In same way, [34] UV–vis results displayed that the SPRs becomes sharper and shifts towards lower wavelength (*Escherichia hermannii* = 438 nm, *Citrobacter sedlakii* = 441 nm), shows that the particle size of AgNPs decreased. The work of [35] suggested that the green synthesis of gallic acid-coated silver nanoparticles UV–vis absorption spectrum shows typical and narrow absorption peak at approximately 400 nm.

#### **4.1 Visualization of (DCF-SNPs)**

Dichlorofluorescein silver nanoparticles (DCF-SNP) were successfully fabricated using AgNO3 (1 mM) and DCF (2.5 per cent) by boiling the reactants at 12 pH. The reaction is observed for color shift and is found to be dark brown in color after 24 hours. The synthesized dichlorofluorescein silver nanoparticles DCF-SNP before and after the reaction and color shift are shown in **Figure 4**.

The silver nanoparticles have been synthesized successfully using DCF has been synthesized by mixing 97.5 ml of AgNO3 (1 mM) and 2.5 ml of dichlorofluoresceine in conical flask. It takes 75 sec to boil and the color changes from orangish to greenish color. However, [34] uses silver nitrate (AgNO3) of 10–3 M concentration to the reaction vessels containing the bacterial isolate supernatants and reaction completes after 24 hours.

#### **4.2 Fourier transforms infrared spectroscopy (FTIR) analysis**

The FTIR measurement were carried out in order to identify the involvement of different functional groups present in dichlorofluorescein compound solution responsible for the bioreduction of Ag + and the capping of dichlorofluorescein silver nanoparticles (DCF-SNPs). The observed FTIR intense bands for Dichlorofluorescein compound solution were compared with the standard IR band ranges and this enables to identify the functional group at 1042.809 cm−1, 1074.148 cm−1, 1153.629 cm−1, 1262.412 cm−1, 1373.712 cm−1, 1472.978 cm−1, 1634.676 cm−1, 1997.814 cm−1, 2113.782 cm−1, 2204.477 cm−1, 2261.689 cm−1, 2300.828 cm−1 and 3262.457 cm−1 for possibly capped the dichlorofluorescein silver nanoparticles. The FTIR spectrum of dichlorofluoresceine silver nanoparticles (DCF-SNPs) is shown in **Figure 5** and the details in terms of wave number, bond and intensity are given in (**Table 2**). **Table 3** illustrates a rather more specific FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) including that of the area covered, peak height from left to right edge and centre.

The fourier transform infrared spectroscopy results confirms that absorption bands at 1042.809 cm−1, 1074.148 cm−1, 1153.629 cm−1, 1262.412 cm−1, 1373.712 cm−1, 1472.978 cm−1, 1634.676 cm−1, 1997.814 cm−1, 2113.782 cm−1, 2204.477 cm−1, 2261.689 cm−1, 2300.828 cm−1 and 3262.457 cm−1 as the wave numbers for the functional groups amine, tertiary alcohol, aromatic ester, alkane, conjugated alkene, alkyne and isocynate that have taken part reducing the silver salt to form

**Figure 4.**

*Visual observation of color change during the dichlorofluorescein silver nanoparticles (DCF-SNPs) nucleation stage.*

**35**

**Table 2.**

**Figure 5.**

*dichlorofluorescein (DCF) solution.*

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

dichlorofluorescein silver nanoparticles (DCF-SNP). Similarly, [36] showed single aldehyde, OH stretching and aldehyde, amide, carbonyl, ethylene, methoxy compounds present in Cannonball Leaves extract and involved in the reduction of silver

*Shows the specifics of the FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) in terms of* 

*Shows the FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) which has been reduced via* 

**Sr. No. Wavenumber (cm−1) Bond Functional group Intensity** 1. 1042.809 C-N stretching Amine Medium 2. 1074.148 C-N stretching Amine Strong 3. 1153.629 C-O stretching Tertiary alcohol Strong 4. 1262.412 C-O stretching Aromatic easter Strong 5. 1373.712 O-H bonding Alcohol Medium 6. 1472.978 C-H bonding Alkane Variable 7. 1634.676 C-C stretching Conjugated alkene Medium 8. 1997.814 C=C=C stretching Allene Medium 9. 2113.782 C C stretching Alkyne Weak 10. 2204.477 C C stretching Alkyne Weak 11. 22161.689 N=C=O stretching Strong, broad Isocynate 12. 2300.828 — — — 13. 3262.457 O-H stretching Strong, broad Alcohol

Nanoparticles tracking and analysis (NTA) was carried out using NanoSight LM-20 to determine the dispersion characteristics, i. e. size and distribution of silver nanoparticles of dichlorofluoresceine (DCF-SNPs). The nanoparticles

salt for the creation of silver nanoparticles using the FTIR spectrum.

**4.3 Nanoparticles tracking (NTA) analysis**

*wave number (cm−1), bond, functional group and bonding strength.*

*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*

#### **Figure 5.**

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

before and after the reaction and color shift are shown in **Figure 4**.

**4.2 Fourier transforms infrared spectroscopy (FTIR) analysis**

The FTIR measurement were carried out in order to identify the involvement of different functional groups present in dichlorofluorescein compound solution responsible for the bioreduction of Ag + and the capping of dichlorofluorescein silver nanoparticles (DCF-SNPs). The observed FTIR intense bands for Dichlorofluorescein compound solution were compared with the standard IR band ranges and this enables to identify the functional group at 1042.809 cm−1, 1074.148 cm−1, 1153.629 cm−1, 1262.412 cm−1, 1373.712 cm−1, 1472.978 cm−1, 1634.676 cm−1, 1997.814 cm−1, 2113.782 cm−1, 2204.477 cm−1, 2261.689 cm−1, 2300.828 cm−1 and 3262.457 cm−1 for possibly capped the dichlorofluorescein silver nanoparticles. The FTIR spectrum of dichlorofluoresceine silver nanoparticles (DCF-SNPs) is shown in **Figure 5** and the details in terms of wave number, bond and intensity are given in (**Table 2**). **Table 3** illustrates a rather more specific FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) including that of the area covered, peak height

The fourier transform infrared spectroscopy results confirms that absorption bands at 1042.809 cm−1, 1074.148 cm−1, 1153.629 cm−1, 1262.412 cm−1, 1373.712 cm−1,

1472.978 cm−1, 1634.676 cm−1, 1997.814 cm−1, 2113.782 cm−1, 2204.477 cm−1, 2261.689 cm−1, 2300.828 cm−1 and 3262.457 cm−1 as the wave numbers for the functional groups amine, tertiary alcohol, aromatic ester, alkane, conjugated alkene, alkyne and isocynate that have taken part reducing the silver salt to form

*Visual observation of color change during the dichlorofluorescein silver nanoparticles (DCF-SNPs)* 

after 24 hours.

from left to right edge and centre.

after 24 hours. The synthesized dichlorofluorescein silver nanoparticles DCF-SNP

The silver nanoparticles have been synthesized successfully using DCF has been synthesized by mixing 97.5 ml of AgNO3 (1 mM) and 2.5 ml of dichlorofluoresceine in conical flask. It takes 75 sec to boil and the color changes from orangish to greenish color. However, [34] uses silver nitrate (AgNO3) of 10–3 M concentration to the reaction vessels containing the bacterial isolate supernatants and reaction completes

**34**

**Figure 4.**

*nucleation stage.*

*Shows the FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) which has been reduced via dichlorofluorescein (DCF) solution.*


#### **Table 2.**

*Shows the specifics of the FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) in terms of wave number (cm−1), bond, functional group and bonding strength.*

dichlorofluorescein silver nanoparticles (DCF-SNP). Similarly, [36] showed single aldehyde, OH stretching and aldehyde, amide, carbonyl, ethylene, methoxy compounds present in Cannonball Leaves extract and involved in the reduction of silver salt for the creation of silver nanoparticles using the FTIR spectrum.

#### **4.3 Nanoparticles tracking (NTA) analysis**

Nanoparticles tracking and analysis (NTA) was carried out using NanoSight LM-20 to determine the dispersion characteristics, i. e. size and distribution of silver nanoparticles of dichlorofluoresceine (DCF-SNPs). The nanoparticles


#### **Table 3.**

*Shows the more specific FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) including the area covered, peak height from left to right edge and Centre.*

tracking and analysis measure the size of individual nanoparticles in a suspension by their brownian motions from which the intensity of particle size distribution are obtained. The size of dichlorofluorescein silver nanoparticles (DCF-SNPs) from nanoparticles tracking and analysis was found to be less than 293 nm. The size distribution histogram of dichlorofluoresceine silver nanoparticles using nanoparticles tracking and analysis (NTA) is represented in **Figure 6** and the 3-D plot of dichlorofluoresceine silver nanoparticles size distribution intensity can be seen in **Figure 7**.

It could be seen extremely obviously from the histogram that the synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) ranged from 66 nm to 293 nm in size. The very few dichlorofluoresceine silver nanoparticles (DCF-SNPs) are

#### **Figure 6.**

*Shows the size distribution histogram of dichlorofluoresceine silver nanoparticles using nanoparticles tracking and analysis (NTA).*

**37**

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

112 nm, 159 nm, 215 nm and 293 nm in size. However the substantial majority of

*Shows the 3-D plot for size distribution intensity of dichlorofluorescein silver nanoparticles (DCF-SNPs).*

The synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) intensity of their size distribution in nanoscale clearly demonstrates that most dichlorofluorescein silver nanoparticles (DCF-SNPs) are similar in diameter (159 nm) and few are clustered in scales above 300 nm. The synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) are therefore stabled formulated which could be used

The synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) has a zeta potential value of-9.35 mV implying that the dichlorofluorescein silver nanoparticles (DCF-SNP) have a high negative surface load. The zeta variance efficiency was found to be 6.10 and 0.0196, reflecting that the zeta potential synthesized dichlorofluorescein silver nanoparticles are significant and could be used to associate other materials or molecules to achieve secondary effects. **Figure 8** displays the zeta potential graph for dichlorofluorescein silver nanoparticles (DCF-SNP). The dichlorofluoresceine silver nanoparticles (DCFs) are in ranged between 40–293 nm size in diameter confirmed by analyzing nanoparticles tracking and analysis (NTA). The size distribution and zeta potential of dichlorofluoresceine silver nanoparticles were determined by DLS and it is confirmed that the dichlorofluoresceine silver nanoparticles obtained are colloidal in nature, with average diameter approximately 159 nm and the corresponding average zeta potential for dichlorofluoresceine silver nanoparticles as −9.35 mV. In contrast, the work of Saeb et al. (2014) obtained silver nanoparticles using bacterial isolates gave the highest value of zeta potential of −30 mV, which indicates a good stability. However, unexpectedly, this zeta potential value was drastically decreased to −18.3 mV

dichlorofluorescein silver nanoparticles (DCF-SNP) are 159 nm wide.

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

for chemical analysis in agriculture.

and − 9.5 mV after 30 and 90 days respectively.

**4.5 Effect of DCF-SNPs on seeds germination of** *Vigna radiata*

The effect of dichlorofluorescein silver nanoparticles (DCF-SNPs) on seed germination of Mung bean (*V. radiata*) was conducted and the root and shoot

**4.4 Zeta potential (ZP) analysis**

**Figure 7.**

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

#### **Figure 7.**

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

**Peak Name Area Height Left Edge Right Edge Center Peakr1** −37.159 1.234 1052.004 1010.969 1042.809 **Peak2** −30.857 1.572 1104.232 1066.927 1074.148 **Peak3** −10.283 0.895 1160.189 1141.537 1153.629 **Peak4** −35.279 1.485 1279.566 1234.800 1262.412 **Peak5** −11.528 0.547 1384.020 1335.523 1373.712 **Peak6** −17.379 0.721 1481.013 1451.169 1472.978 **Peak7** −2365.076 25.540 1764.532 1563.085 1634.576 **Peak8** −8.886 0.502 2014.477 1984.633 1997.814 **Peak9** −151.214 2.514 2137.584 2070.434 2113.782 **Peak10** −6.076 0.452 2215.924 2197.272 2204.477 **Peak11** −1.151 0.104 2268.151 2245.768 2261.689 **Peak12** −8.086 0.027 2365.145 2297.996 2300.828 **Peak13** 1341.449 4.760 3290.312 2932.183 3262.457

*Shows the size distribution histogram of dichlorofluoresceine silver nanoparticles using nanoparticles tracking* 

tracking and analysis measure the size of individual nanoparticles in a suspension by their brownian motions from which the intensity of particle size distribution are obtained. The size of dichlorofluorescein silver nanoparticles (DCF-SNPs) from nanoparticles tracking and analysis was found to be less than 293 nm. The size distribution histogram of dichlorofluoresceine silver nanoparticles using nanoparticles tracking and analysis (NTA) is represented in **Figure 6** and the 3-D plot of dichlorofluoresceine silver nanoparticles size distribution intensity can be seen in **Figure 7**. It could be seen extremely obviously from the histogram that the synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) ranged from 66 nm to 293 nm in size. The very few dichlorofluoresceine silver nanoparticles (DCF-SNPs) are

*Shows the more specific FTIR spectrum of dichlorofluorescein silver nanoparticles (DCF-SNPs) including the* 

*area covered, peak height from left to right edge and Centre.*

**36**

**Figure 6.**

**Table 3.**

*and analysis (NTA).*

*Shows the 3-D plot for size distribution intensity of dichlorofluorescein silver nanoparticles (DCF-SNPs).*

112 nm, 159 nm, 215 nm and 293 nm in size. However the substantial majority of dichlorofluorescein silver nanoparticles (DCF-SNP) are 159 nm wide.

The synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) intensity of their size distribution in nanoscale clearly demonstrates that most dichlorofluorescein silver nanoparticles (DCF-SNPs) are similar in diameter (159 nm) and few are clustered in scales above 300 nm. The synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) are therefore stabled formulated which could be used for chemical analysis in agriculture.

#### **4.4 Zeta potential (ZP) analysis**

The synthesized dichlorofluorescein silver nanoparticles (DCF-SNPs) has a zeta potential value of-9.35 mV implying that the dichlorofluorescein silver nanoparticles (DCF-SNP) have a high negative surface load. The zeta variance efficiency was found to be 6.10 and 0.0196, reflecting that the zeta potential synthesized dichlorofluorescein silver nanoparticles are significant and could be used to associate other materials or molecules to achieve secondary effects. **Figure 8** displays the zeta potential graph for dichlorofluorescein silver nanoparticles (DCF-SNP).

The dichlorofluoresceine silver nanoparticles (DCFs) are in ranged between 40–293 nm size in diameter confirmed by analyzing nanoparticles tracking and analysis (NTA). The size distribution and zeta potential of dichlorofluoresceine silver nanoparticles were determined by DLS and it is confirmed that the dichlorofluoresceine silver nanoparticles obtained are colloidal in nature, with average diameter approximately 159 nm and the corresponding average zeta potential for dichlorofluoresceine silver nanoparticles as −9.35 mV. In contrast, the work of Saeb et al. (2014) obtained silver nanoparticles using bacterial isolates gave the highest value of zeta potential of −30 mV, which indicates a good stability. However, unexpectedly, this zeta potential value was drastically decreased to −18.3 mV and − 9.5 mV after 30 and 90 days respectively.

#### **4.5 Effect of DCF-SNPs on seeds germination of** *Vigna radiata*

The effect of dichlorofluorescein silver nanoparticles (DCF-SNPs) on seed germination of Mung bean (*V. radiata*) was conducted and the root and shoot

#### **Figure 8.**

*Shows the zeta potential graph for surface charge on the dichlorofluoresceine silver nanoparticles (DCF-SNPs).*

lengths were recorded every 24 hours. In the present experiment, the production of radicals exceeding 1 cm is interpreted to be positive growth otherwise indicated as negative. The effect of dichlorofluorescein silver nanoparticles (DCF-SNPs) on seed germination of Mung bean is shown in the **Figure 9**.

From the **Figure 9** it is noted that as the concentration of dichlorofluoresceine silver nanoparticles increases the root and shoot length decreases as compared to controls once. After 96 hrs, the Mung beans treated with 25% of dichlorofluoresceine silver nanoparticles (DCF-SNPs) shows growth in root and shoot length when compared with the positive control. However, the concentration of DCF-SNPs increases from 50% to 100%, there was inhibition of growth observed when compared to 25% of dichlorofluoresceine silver nanoparticles (**Figure 10**).

The effect of dichlorofluoresceine silver nanoparticles on seed germination of the Mung bean (*V. radiata*) was carried out and the size of root and shoot lengths were recorded after every 24 hour. Percentage of seed germination were substantially influenced by the addition of dichlorofluorescein silver nanoparticles (DCF-SNPs). The 25% concentration of DCF-SNPs treated Mung seeds indicates an excellent growth at 72 hours. In specific, the seed germination rate increases from 75% to 95%. While, as the concentration increases from 50% to 100%, the growth rate get varies or unpredicted. Our results are similar to the work of the [33] found that the as the concentration of ZnO NPs increased there was decrease in germination of seeds. Control showed statistically significant difference and could not improve shoot length. Besides, There was an increase in germination significantly with zinc oxide nano particles treated seeds at different concentrations viz., 20 mg shown 100%, 40 mg-95%, 60 mg-90%, 80 mg-90% and 100 mg of ZnO NPs shown 85% germination in Mung bean seeds [33].

The [37] study the effect of silver nanoparticles on the seed germination and plant growth and found that the highest germination rate for corn seeds, was 6.5 seeds/day, which was observed after exposure to 1.5 mg/ml of silver nanoparticles and the highest germination percentage (73.33%) and highest germination rate (1.59 seeds/day) for watermelon were recorded at 2 mg/ml silver nanoparticles.

**39**

**Figure 9.**

*(DCF-SNPs) on mung beans.*

shoot was more prominent.

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

Due to interactions of dichlorofluorescein silver nanoparticles, the percentage of germination and length of root and shoot has indeed been affected. The average length of root are measure after 24 hrs, 48 hrs and 72 hrs and found to be 6.6 mm, 22.2 mm and 41.7 mm respectively. Similarly, the average length of shoot are measure after 48 hrs and 72 hrs and found to be 2.4 mm and 9 mm respectively. The experiment showed the average length of root and shoot at 72 hours was highest

*Shows the time dependent toxicity effect of varying concentrations of dichlorofluoresceine silver nanoparticles* 

In the present study, the dichlorofluoresceine silver nanoparticles showed unpredicted effects on root and shoot length when treated with the various concentrations of dichlorofluoresceine silver nanoparticles (DCF-SNPs). The higher concentration of nanoparticles may be attributed to toxic level of nanoparticles which has been seen in present experimentation that above certain level of concentration the seedlings respond in different way and causes subsequent declines in growth. The work of [33] evidence the same results their study stating that at low concentrations the ZnO nanoparticles shows good effect on root and

which is 41.7 mm and 9 mm respectively (**Table 4**).

*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*

#### **Figure 9.**

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

lengths were recorded every 24 hours. In the present experiment, the production of radicals exceeding 1 cm is interpreted to be positive growth otherwise indicated as negative. The effect of dichlorofluorescein silver nanoparticles (DCF-SNPs) on seed

*Shows the zeta potential graph for surface charge on the dichlorofluoresceine silver nanoparticles (DCF-SNPs).*

From the **Figure 9** it is noted that as the concentration of dichlorofluoresceine silver nanoparticles increases the root and shoot length decreases as compared to controls once. After 96 hrs, the Mung beans treated with 25% of dichlorofluoresceine silver nanoparticles (DCF-SNPs) shows growth in root and shoot length when compared with the positive control. However, the concentration of DCF-SNPs increases from 50% to 100%, there was inhibition of growth observed when

compared to 25% of dichlorofluoresceine silver nanoparticles (**Figure 10**).

The effect of dichlorofluoresceine silver nanoparticles on seed germination of the Mung bean (*V. radiata*) was carried out and the size of root and shoot lengths were recorded after every 24 hour. Percentage of seed germination were substantially influenced by the addition of dichlorofluorescein silver nanoparticles (DCF-SNPs). The 25% concentration of DCF-SNPs treated Mung seeds indicates an excellent growth at 72 hours. In specific, the seed germination rate increases from 75% to 95%. While, as the concentration increases from 50% to 100%, the growth rate get varies or unpredicted. Our results are similar to the work of the [33] found that the as the concentration of ZnO NPs increased there was decrease in germination of seeds. Control showed statistically significant difference and could not improve shoot length. Besides, There was an increase in germination significantly with zinc oxide nano particles treated seeds at different concentrations viz., 20 mg shown 100%, 40 mg-95%, 60 mg-90%, 80 mg-90% and 100 mg of ZnO NPs shown

The [37] study the effect of silver nanoparticles on the seed germination and plant growth and found that the highest germination rate for corn seeds, was 6.5 seeds/day, which was observed after exposure to 1.5 mg/ml of silver nanoparticles and the highest germination percentage (73.33%) and highest germination rate (1.59 seeds/day) for watermelon were recorded at 2 mg/ml

germination of Mung bean is shown in the **Figure 9**.

85% germination in Mung bean seeds [33].

**38**

**Figure 8.**

silver nanoparticles.

*Shows the time dependent toxicity effect of varying concentrations of dichlorofluoresceine silver nanoparticles (DCF-SNPs) on mung beans.*

Due to interactions of dichlorofluorescein silver nanoparticles, the percentage of germination and length of root and shoot has indeed been affected. The average length of root are measure after 24 hrs, 48 hrs and 72 hrs and found to be 6.6 mm, 22.2 mm and 41.7 mm respectively. Similarly, the average length of shoot are measure after 48 hrs and 72 hrs and found to be 2.4 mm and 9 mm respectively. The experiment showed the average length of root and shoot at 72 hours was highest which is 41.7 mm and 9 mm respectively (**Table 4**).

In the present study, the dichlorofluoresceine silver nanoparticles showed unpredicted effects on root and shoot length when treated with the various concentrations of dichlorofluoresceine silver nanoparticles (DCF-SNPs). The higher concentration of nanoparticles may be attributed to toxic level of nanoparticles which has been seen in present experimentation that above certain level of concentration the seedlings respond in different way and causes subsequent declines in growth. The work of [33] evidence the same results their study stating that at low concentrations the ZnO nanoparticles shows good effect on root and shoot was more prominent.

#### **Figure 10.**

*Shows the dichlorofluorescein silver nanoparticles effect on a: Root length and B: Shoot length of mung beans (V. radiata) seed after 24 hr., 48 hr., 72 hr.*


#### **Table 4.**

*Indicates the average percentage of the observed root and shoot length of germinated mung bean after 24 hrs, 48 hrs, 72 hrs.*

Therefore, the 25% concentration of dichlorofluoresceine silver nanoparticles (DCF-SNPs) though show positive effects on the seed germinations of mung beans it could have an advantage of using as the tracking the bio active compound,

**41**

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

to visualize the fluorescent Zein nanoparticles translocation in sugar cane.

lation into the plant system using confocal laser scanning microscopy.

(M.S.), India, for providing research space and laboratory facility for

The authors declare no conflict of interest.

The authors would like to thank Rajiv Gandhi Biotechnology Centre, Rashtrasant Tukdoji Maharaj Nagpur University, L.I.T. Premises, Nagpur-440033

The FTIR analysis of dichlorofluorescein silver nanoparticles (DCF-SNPs) were done at the Narsamma Hirayya Arts Commerce & Science college, Amravati,

Author would like to thank the Principal and Dr. Khandekar, of Narsamma

Hirayya Arts Commerce & Science college, Kiran Nagar Near Farshi Stop,

In the current study, the synthesized dichlorofluoresceine silver nanoparticles (DCF-SNPs) were synthesized successfully by boiling method. In addition, dichlorofluoresceine silver nanoparticles (DCF-SNPs) were characterized by different techniques for measuring the particles size, morphology, functional group and surface charge. Moreover, the different concentrations of dichlorofluoresceine silver nanoparticles (DCF-SNPs) effects on germination of mung beans (*V. radiata*) and the length of root and shoot were studied. The synthesized dichlorofluoresceine silver nanoparticles (DCF-SNPs) are less than 159 nm size that interact and activates growth related gene. Therefore, 25% concentration of dichlorofluoresceine silver nanoparticles (DCF-SNP) is tested positive for shoot and root growth as compare to control. The 25% concentration of dichlorofluoresceine silver nanoparticles (DCF-SNPs) is good dye for conjugation with other bioactive compounds and useful for tracking the bio active compounds, bio uptake, biotransformation and bioaccumu-

fertilizers, pesticides, hormone, minerals transfer into the plant system. As the dichlorofluoresceine silver nanoparticles (DCF-SNPs) is a organic dye it could be coupled with the non toxic materials or polymer that could have avoid harmless to plants and at the same time assist to deliver bio active essential compound in plants. This bio uptake, biotransformation, and bioaccumulation of Fluorescent dichlorofluoresceine silver nanoparticles (DCF-SNP) could be studied using the Confocal Laser-Scanning Microscopy. A study done by [38] already used Confocal laser scanning microscopy (CLSM), Leica TCS SP2 microscope (Leica Inc., Buffalo Grove, IL)

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

**5. Conclusion**

**Acknowledgements**

experimentations.

**Footnotes**

**Thanks**

**Conflict of interest**

Maharashtra, India.

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

fertilizers, pesticides, hormone, minerals transfer into the plant system. As the dichlorofluoresceine silver nanoparticles (DCF-SNPs) is a organic dye it could be coupled with the non toxic materials or polymer that could have avoid harmless to plants and at the same time assist to deliver bio active essential compound in plants. This bio uptake, biotransformation, and bioaccumulation of Fluorescent dichlorofluoresceine silver nanoparticles (DCF-SNP) could be studied using the Confocal Laser-Scanning Microscopy. A study done by [38] already used Confocal laser scanning microscopy (CLSM), Leica TCS SP2 microscope (Leica Inc., Buffalo Grove, IL) to visualize the fluorescent Zein nanoparticles translocation in sugar cane.

## **5. Conclusion**

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

Therefore, the 25% concentration of dichlorofluoresceine silver nanoparticles

*Indicates the average percentage of the observed root and shoot length of germinated mung bean after 24 hrs,* 

*Shows the dichlorofluorescein silver nanoparticles effect on a: Root length and B: Shoot length of mung beans* 

1. 0% 7 mm 16.1 mm 49.8 mm — 4 mm 17.5 mm 2. 25% 7.1 mm 24.1 mm 50.2 mm — 2 mm 16.3 mm 3. 50% 6 mm 22.3 mm 34.5 mm — — 2.5 mm 4. 75% 7.1 mm 27.4 mm 42.2 mm — 1.3 mm 3.7 mm 5. 100% 6 mm 21.4 mm 32 mm — — 5.3 mm 6. Total Avg. 6.6 mm 22.2 mm 41.7 mm 2.4 mm 9 mm

**Average root length Average shoot length 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs**

(DCF-SNPs) though show positive effects on the seed germinations of mung beans it could have an advantage of using as the tracking the bio active compound,

**40**

**Table 4.**

*48 hrs, 72 hrs.*

**Figure 10.**

**Sr. no**

*(V. radiata) seed after 24 hr., 48 hr., 72 hr.*

**Conc. of DCF-SNPs**

In the current study, the synthesized dichlorofluoresceine silver nanoparticles (DCF-SNPs) were synthesized successfully by boiling method. In addition, dichlorofluoresceine silver nanoparticles (DCF-SNPs) were characterized by different techniques for measuring the particles size, morphology, functional group and surface charge. Moreover, the different concentrations of dichlorofluoresceine silver nanoparticles (DCF-SNPs) effects on germination of mung beans (*V. radiata*) and the length of root and shoot were studied. The synthesized dichlorofluoresceine silver nanoparticles (DCF-SNPs) are less than 159 nm size that interact and activates growth related gene. Therefore, 25% concentration of dichlorofluoresceine silver nanoparticles (DCF-SNP) is tested positive for shoot and root growth as compare to control. The 25% concentration of dichlorofluoresceine silver nanoparticles (DCF-SNPs) is good dye for conjugation with other bioactive compounds and useful for tracking the bio active compounds, bio uptake, biotransformation and bioaccumulation into the plant system using confocal laser scanning microscopy.

#### **Acknowledgements**

The authors would like to thank Rajiv Gandhi Biotechnology Centre, Rashtrasant Tukdoji Maharaj Nagpur University, L.I.T. Premises, Nagpur-440033 (M.S.), India, for providing research space and laboratory facility for experimentations.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Footnotes**

The FTIR analysis of dichlorofluorescein silver nanoparticles (DCF-SNPs) were done at the Narsamma Hirayya Arts Commerce & Science college, Amravati, Maharashtra, India.

#### **Thanks**

Author would like to thank the Principal and Dr. Khandekar, of Narsamma Hirayya Arts Commerce & Science college, Kiran Nagar Near Farshi Stop,

Amravati, Maharashtra, India, for FTIR analysis. In addition, authors thanks Dr. Aniket Gade, Sant Gadge Baba Amravati University, MS, India for the zeta potential and Nanoparticles tracking and analysis (NTA) characterization of formulated dichlorofluoresceine silver nanoparticles (DCF-SNP).
