ORIGINAL PAPER

Epigallocatechin-3-gallate-capped Ag nanoparticles:preparation and characterization

Shokit Hussain • Zaheer Khan

Received: 4 November 2013 / Accepted: 8 November 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract We used an aqueous leaf extract of Camellia

sinensis to synthesize Ag nanoparticles (AgNPs). A layer

of ca. 6 nm around a group of the AgNPs in which the

inner layer is bound to the AgNPs surface via the hydroxyl

groups of catechin has been observed. TEM analysis of

AgNPs showed the formation of truncated triangular

nanoplates and/or nanodisks and spherical with some

irregular-shaped polydispersed nanoparticles in absence

and presence of shape-directing cetyltrimethylammonium

bromide. The polyphenolic groups of epigallocatechin-3-

gallate (EGCG) are responsible for the rapid reduction of

Ag? ions into metallic Ag0. The free –OH groups are able

to stabilize AgNPs by the interaction between the surface

Ag atoms of AgNPs and oxygen atoms of EGCG

molecules.

Keywords Camellia sinensis � Epigallocatechin-

3-gallate � Oxidation � CTAB � Ag nanoparticles

Introduction

Electron-, proton- and ion-transfer reactions at the inter-

faces between two immiscible phases are fundamentally

important in understanding the phase transfer catalysis,

drug delivery and different phenomenon in membrane

chemistry [1]. The domain of surface science is perhaps

one of the most interdisciplinary areas of modern science,

nanotechnology, bionanotechnology and nanotoxicology.

There is a class of compounds called surface active

compounds or surfactant that decreases interfacial tension

or interfacial free energy of interfaces [2, 3]. Surfactant-

assisted synthesis has been considered to be an effective

methodology for the shape-controlled synthesis of

nanomaterials in the aqueous phase [4–6]. Generally, sur-

factants, ligands, polymers, or dendrimers have been used

to confine the growth in the nanometer regime [7, 8]. El-

Sayed et al. [9] discussed the mechanism of shape control

role of surfactants. Reetz et al. [10] reported the structures

other than spheres form as a result of specific interaction of

the capping agents with different growing faces of the

particles. Sau et al. [11] first time reported the synthesis

multi-pods gold nanoparticles (AuNPs) in the presence of

CTAB using the seeds growth method. Baruah et al. [12]

synthesized the stable AuNPs, bearing a bilayer of cetyl-

trimethylammonium bromide supported by N-nonylamine

as a cosurfactant. The synthesis and mechanism of AgNPs

and AuNPs bounded by interdigitated bilayers of CTAB

and fatty acids have been the subject of various investi-

gations in water [13–15]. Murphy et al. [14] also pointed

out that the uptake of organic molecules from the bulk

aqueous phase by nanomaterials is a little-explored phe-

nomenon that has useful implications for both biomedical

and environmental applications of advanced new nano

materials.

Jose-Yacaman and co-workers [16] first reported the

formation of Au and Ag NPs by living plants, acted as a

reducing and stabilizing agent. Sastry et al. used Aza-

dirachta indica leaf broth in the extracellular synthesis of

pure metallic Ag- and AuNPs and bimetallic Au/AgNPs

having flat, plate-like morphology. The constituents of

leaves extract, such as flavanone and terpenoid acted as the

surface active molecules stabilizing the nanoparticles [17].

The involvement of plants/parts of plants constituents

(proteins, polyphenols and carbohydrates) in the synthesis

S. Hussain � Z. Khan (&)

Nano-science Research Lab, Department of Chemistry, Jamia

Millia Islamia (Central University), New Delhi 110025, India

e-mail: drkhanchem@yahoo.co.in

123

Bioprocess Biosyst Eng

DOI 10.1007/s00449-013-1094-0

of metal nanoparticles has been discussed on several

occasions [18–21]. The use of plants, flowers, leaves

extracts is the safest option both to attain shape-controlled

morphologies and to preserve biofunctionalities [22]. Radu

et al. [23] reported a synthesis of well-dispersed AgNPs

with an approximate size of 4 nm using tea leaf extract

from Camellia sinensis without using any capping or dis-

persing agent. The chemistry of tea, C. sinensis is complex:

catechins, polyphenols, alkaloids, amino acids, glucosides,

proteins, water-soluble extracts, minerals. Catechins and

caffeine have been considered as the most biologically

active group of tea components that may affect the path-

ogenesis of various diseases (antioxidative, antimutagenic,

and anticarcinogenic). Out of catechins (Scheme 1), epi-

gallocatechin-3-gallate, a typical plant polyphenol and has

anti-cancer activity [24–26], is the major existing species

present in the C. sinensis leaves aqueous extract [27].

In the present work, we have proposed a method for

synthesizing AgNPs bearing a layer of biomolecules of C.

sinensis leaves extract in aqueous solutions. Various

parameters (agglomeration number, the average number of

silver atoms per nanoparticle, molar concentrations of

O

OH

OH

OH

OH

OH

O

O

OH

OH

OH

OH

O

OH

OH OH

Epicatechin Epicatechin-3-gallate(ECG)

OOH

OH OH

OH

OH

OH

O

OO

OH

OH

OHOH

OH

OH

OH OH

Epigallatocatechin (EGC) Epigallocatechin-3-gallate (EGCG)

O

O

OH

OH OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH OH

OH

OH

OH

O

OH

OOH

OOH

Theaflavin Thearubigin

N

NN

N

O

O

CH3

CH3CH3

Caffeine

Scheme 1 Molecular

structures of some constituents

of Camellia sinensis leaves

extract

Bioprocess Biosyst Eng

123

nanoparticle in solution, extinction coefficient and increase

in the Fermi energy) have been calculated with the help of

Mie theory for the first time to the AgNPs synthesized

using aqueous leaves extract [28]. In addition, an effect of

shape-directing CTAB has also been reported on the

morphology of biomolecules capped nanoparticles.

Experimental

Materials and instruments

Silver nitrate (AgNO3, 99 %) and cetyltrimethylammo-

nium bromide (CTAB, 99 %) were obtained from Merck

India and used as received. All solutions were prepared

with double-distilled deionized water. Glassware was

cleaned with aqua regia and rinsed thoroughly with extra

pure water. All other chemicals used were of analytical

grade. The morphology of AgNPs was observed by

Transmission Electron Microscopy (TEM, Hitachi,

H7100). The orange color silver sols were deposited on a

copper grid at room temperature. After drying, sample was

analyzed at 80 kV. The particle size distributions were

determined using UTHSCSA Image Tool Program (version

3.00; Dental Diagnostic Science, UTHSCSA, San Antonio,

TX, USA). The optical property of AgNPs was analyzed

via UV–Visible (UV–Vis, Perkin Elmer, Lambda 35)

absorption double-beam spectrophotometer with a deute-

rium and tungsten iodine lamp in the range from 300 to

600 nm at room temperature.

Synthesis of AgNPs

The 10 g fresh leaves of C. sinensis broth was throughly

washed with sterilized double-distilled water and finely cut

leaves in a 500 cm3 Erlenmeyer flask containing 250 cm3

water, and then heated the mixture at 60 �C for 20 min.

After heating, the solution was cooled, decanted, and fil-

tered through Whatman no. 1 filter paper, and the filtrate

was stored in amber-colored airtight bottle at 10 �C and

used within a week for the preparation of AgNPs. In a

typical experiment, aqueous AgNO3 solution (10.0 cm3 of

0.01 mol dm-3) was mixed in a solution containing leaves

extract (10 % v/v) and required amount of water for dilu-

tion. As the reaction proceeds, the colorless reaction mix-

ture containing leaves extract ? Ag? ions became brown

in color, indicating the formation of AgNPs [16, 17, 29].

The shape and size of the silver nanoparticles depend on

the reduction potentials of reactants, [CTAB], temperature

and time. Therefore, to establish the role of [Ag?],

[CTAB], [extract], and reaction time, a series of experi-

ments were performed under different experimental con-

ditions, i.e., [Ag?] = 5.0–40.0 9 mol dm-3, [CTAB] =

1.0–10.0 9 10-4 mol dm-3 and [extract] = 4–10 % v/v.

Among the various parameters the [Ag?], [CTAB] and

reaction time are particularly crucial for the control of

morphology and the size of AgNPs (vide infra).

Kinetic measurements

The kinetic measurements were carried out in a three-

necked reaction vessel fitted with a double surface con-

denser to check evaporation by adding the required con-

centrations of AgNO3, CTAB and water (for dilution

maintained). The progress of the reaction was followed

spectrophotometrically by adding the required concentra-

tions of leaves extract. The absorbance of the appearance of

yellowish-brown color was measured at 440 nm at definite

time intervals. At this wavelength maximum, reaction

mixture containing AgNO3 and aqueous CTAB has no

absorbance. Apparent rate constants were calculated from

the initial part of the slopes of the plots of ln (a/(1 - a))

versus time by a fixed time method (vide infra). The results

were reproducible to within ±5 % with average linear

regression coefficient, r C 0.998 for each kinetic run.

Results and discussion

Generally, the surface plasmon resonance (SPR) bands are

influenced by the size, shape, morphology, composition

and dielectric environment of the prepared nanoparticles

[30]. Previous studies have shown that the spherical AgNPs

contribute to the only one absorption bands at around

400 nm in the UV–Vis spectra [31]. The choice of C.

sinensis as reducing agent is based on its rich content of

polyphenolic compounds. Figure 1 shows the UV–Vis

spectra of the nanoparticles obtained on varying the reac-

tion time at constant [Ag?] = 10.0 9 10-4 mol dm-3,

[extract] = 4 % v/v, and temperature = 30 �C. In the

present observations, the anisotropic growth of AgNPs was

confirmed by the appearance of characteristic SPR band at

ca. 395 and 440 nm in the UV–Vis region. For a short

reaction time, the particles gave a very weak shoulder at

395 nm. On increasing the reaction time, a sharp peak

begins to develop at 440 nm in addition to the shoulder.

The shoulder at lower wavelength (395 nm) might be due

to the multiplasmon excitation of faceted and anisotropic

AgNPs [32, 33] and the peak at 440 nm depends on the

sharpness of corner of silver triangles (nanodisks; Fig. 1).

The nanoplates (nanodisks) could be formed by the dis-

solution of the corner atoms of truncated triangular nano-

plates. Figure 2 shows the TEM images of silver nanodisks

in this study (length 47 nm, width 10). On careful obser-

vation of TEM images, a thin shell (layer = 6 nm) of

EGCG moieties (major constituents of leaves extract)

Bioprocess Biosyst Eng

123

covered on the groups of various AgNPs is seen. The

capping is prominent on each particle and the same may

also be responsible for interparticle binding. Each silver

nanodisk is a group of several truncated triangular nano-

plates (indicated by arrow in Fig. 2a: large fraction of tri-

angles having round corners). The observed SPR bands at

390 and 440 nm are also congruent with the optical

extinction of Ag nanodisks perviously prepared by Chen

et al. [34] using algal where absorptions at 351, 420 and

475 nm were found for the Ag nanodisks. The single

crystallinity of these nanodisks was also confirmed by

electron diffraction patterns (Fig. 2b). The sixfold sym-

metry of the diffraction spots indicates that the surface of

nanodisks was bounded by {111}faces. The other sets of

spots could be indentified as {211}, {222} and {422}

planes according to the pure face-centered cubic (fcc) sil-

ver structure (JCPDS, File No. 4-0787). Bakshi prepared

the lipid-capped single AuNP having a 4 nm thickness of

the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol

(POPG) bilayer around the particle. Our results are in good

agreement and provides more confirmatory evidence that

AgNPs capped by the constituents of leaves extract which

takes up a surprisingly high number of organic molecules

[6].

UV–Visible spectroscopy is one of the widely used

techniques for characterization of AgNPs. The shape of the

spectra and position of the wavelength maximal give pre-

liminary information about the size and the size distribu-

tion of the AgNPs [35]. Therefore, the UV–Vis spectra of

the AgNPs formation were also recorded at different time

intervals (Fig. 3: one weak shoulder, sharp peak and broad

hump at 395 and 440 and 550 nm, respectively) for 6 %

300 400 500 600 700 8000

1

2

3

4A

bsor

banc

e

Wavelength (nm)

60 50 40 30 25 20 15 10 5 0

Time (nm)

Fig. 1 Time-resolved UV–Visible spectra of AgNPs prepared by

AgNO3 ? Camellia sinensis leaves extract. Reaction conditions:

[Ag?] = 10.0 9 10-4 mol dm-3, [extract] = 4 % v/v, temperature

= 30 �C

Fig. 2 TEM images (a) of AgNPs capped with a catechins bilayer of

6 nm thickness and selected electron diffraction ring patterns (b).

Reaction conditions: [Ag?] = 10.0 9 10-4 mol dm-3, [extract] =

4 % v/v, temperature = 30 �C

300 400 500 600 700 8000

1

2

3

4

Abs

orba

nce

Wavelength (nm)

60 50 40 30 25 20 15 10 5 0

Time (min)

Fig. 3 Time-resolved UV–Visible spectra of AgNPs prepared by

AgNO3 ? Camellia sinensis leaves extract. Reaction conditions:

[Ag?] = 10.0 9 10-4 mol dm-3, [extract] = 6 % v/v, temperature

= 30 �C

Bioprocess Biosyst Eng

123

v/v extract. The appearance of an additional broad hump at

550 nm with higher concentration might be due shape- and

size-controlled tendency of leaves extract. Since the peak

wavelength did not shift significantly during the reaction

with increasing [extract] and reaction time, indicating that

the morphology of the AgNPs was not affected by the

increase in [extract]. Figure 4 shows the growth of AgNPs

for different [Ag?] and it was observed that a minimum of

15.0 9 10-4 mol dm-3 [Ag?] was required for the

nucleation/growth of AgNPs. As can be seen in Fig. 4

(typical example), the absorbance of AgNPs remains the

same with increasing the [Ag?] from C20.0 mol dm-3,

indicating that nucleation and growth processes are not

directly proportional to the [Ag?] and new AgNPs were not

formed at higher [Ag?]. The reaction was very sensitive to

small concentrations of Ag? ions, a concentration of

C5.0 9 10-4 mol dm-3 being enough to the oxidation of

C. sinensis leaves extract. Apparent first-order rate con-

stants were calculated from the initial part of the slopes of

the plots of ln (a/(1 - a)) versus time, where a = At/Aa

(absorbance At at time t and Aa is the final absorbance) with

a fixed time method [36, 37]. Interestingly, the straight line

obtained by plotting ln[a/(1 - a)] versus time (Fig. 5). The

reaction follows fractional-order kinetics with respect to

[Ag?] (104 kobs = 0.5, 2.0, 6.2, 15.5, 24.8, 25.4, 25.5 and

25.4 s-1 for 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0 and

40.0 9 10-4 mol dm-3 [Ag?], respectively).

Before attempting to propose a mechanism to the

reduction of Ag? ions into Ag0 by EGCG, it is necessary to

know the chemical speciation of silver(I) in presence of

EGCG. The standard electrode potential of Ag?/Ag0 redox

couple is 0.799 V [38]. Generally, complexation decreases

the redox potential and hence the reducibility of Ag? ions

(redox potentials are 0.07, 0.24 and 0.37 V, respectively,

for AgBr, AgOH and [Ag(NH)2]?). EGCG consists of

multiple phenolic hydroxyls, and it has a strong driving

force to eject the phenolic proton. EGCG is the catechin

present in the largest amount, implicating it as the main

active ingredient. It is, therefore, reasonable to infer that

the decrease in superoxide scavenging of reactive oxygen

species radicals was due to reduction of EGCG content

[39]. Toschi et al. [40] have also observed that the anti-

oxidant activity of the green tea is higher in the teas that

contain higher levels of EGCG and EGC. Therefore, pre-

sence of poly –OH groups are responsible for the higher

reactivity of EGCG which easily transfers the proton to

Ag? leading to the formation of stable EGCG capped

AgNPs (Scheme 2). Scheme 2. Reduction of Ag? ions by

the –OH groups of EGCG.

In Scheme 2, reaction first represents the ejection of

proton (one-step one-electron oxidation–reduction mecha-

nism; rate-determining step), which leads to the formation

of Ag0 and EGCG radical. In the next reaction, EGCG

radical immediately converted into the stable product, i.e.,

corresponding quinone. The complexation of the formed

Ag0 atoms with Ag? ions yields Ag2? ions and then the

Ag2? ions dimerize to yield yellow color silver sol, i.e.,

(Ag42?-EGCG). We have also discussed the effect of phe-

nolic –OH group on the reactivity of acetanilide, paracet-

amol, and tyrosine on the nucleation and growth of MnO2

and AgNPs formation [32, 41–43].

Micelles are dynamic aggregates of amphiphilic mole-

cules that create highly anisotropic interfacial regions lin-

ing the boundary formed by the highly polar aqueous and

0 10 20 30 400

1

2

3

4

5

Abs

orba

nce

(at

440

nm)

104 [Ag+] (mol dm-3)

Fig. 4 Effects of [Ag?] on the maximum absorbance of SPR band as

a function of time. Reaction conditions: [extract] = 4 % v/v,

time = 20 (open circle) and 40 min (filled circle),

temperature = 30 �C

0 10 20 30 40 50 60 70 80 90

-2

0

2

4

6

0 10 20 30 40 50 60 70 80 90

-2

0

2

4

6

ln(a

/1-a

)

Time (min)

Fig. 5 Plots of ln [a/1-a] versus time. Reaction conditions:

[Ag?] = 30 (filled inverted triangle) and 20.0 9 10-4 mol dm-3

(filled diamond, filled triangle, filled circle, filled square),

[CTAB] = 0.0 (filled inverted triangle and filled diamond), 6.0 (filled

triangle), 4.0 (filled circle), 2.0 9 10-4 mol dm-3 (filled square),

[extract] = 4 % v/v, temperature = 30 �C

Bioprocess Biosyst Eng

123

nonpolar hydrocarbon regions, imparting new chemical

and physical properties to the system. Micelles, as well as

other association colloids, can alter nanoparticle’s shape,

size and other surface properties to different extent

depending up on their molecular structure, i.e., nature of

head group, length of hydrophobic tail and type of coun-

terions [44, 45]. Therefore, a series of experiments were

performed by varying [CTAB] (at lower concentrations

from 1.0 9 10-4 to 6.0 9 10-4 mol dm-3 and higher

concentrations C8.0 9 10-4 to 10.0 9 10-4 mol dm-3) at

fixed concentrations of other reagents. The observed results

(spectra at different time intervals) are depicted graphically

in Fig. 6 for the different [CTAB], which shows that the

shape of the spectra and position of the SPR bands strongly

depend on the [CTAB]. The common features of Fig. 6

spectra are the appearance of two SPR bands (one peak and

one shoulder at 395 and 470 nm, respectively) for a short

reaction time. Interestingly, it was observed that the AgNPs

show only a sharp peak at 410 nm instead of two bands in

presence of higher [CTAB] (Fig. 6d) on standing over-

night. The presence of various absorption bands indicates

the existence of AgNPs of various shapes, sizes with wide

distribution [46]. We did not observe any significant

change in the position of SPR band with an increase in

[CTAB]. The broadening and decreasing the absorbance of

the SPR band with [CTAB] indicate that initially reduced

AgNPs grow to form larger particles and finally CTAB acts

as a shape-directing agent. Comparison between the Figs. 1

and 6 suggests that reaction time and [CTAB] altered the

morphology of growing AgNPs and spherical nanoparticles

were formed at the end of the reaction in presence of higher

[CTAB].

At 465 nm, the absorbance increases with the [CTAB]

and until it reaches a maximum then decreases with

[CTAB]. These observations are depicted graphically in

Fig. 7 as an absorbance-[CTAB] profile. The increase–

O

O

OH

OHO

OH

OH

OH

OH

OH

OH

+

O

O

OH

OHO

OH

OH

OH

OH

O

OH

+

(EGCG radical)

O

O

OH

OHO

OH

OH

OH

O

O

OH

H

(EGCG)

O

O

OH

OHO

OH

OH

OH

O

O

OH

+

Ag0 + Ag+ fast Ag2+

Ag2+ + Ag2

+ fastAg4

2+

Ag0

(Ag42+)n + (EGCG)n [(Ag4

2+)n-(EGCG)n ] fast

+

(silver sol)

Ag0slowAg+

Ag+

(EGCG quinone)

fast

Scheme 2 Reduction of Ag? ions by the –OH groups of EGCG

Bioprocess Biosyst Eng

123

decrease behavior (hypo chromic shift) of absorbance

after a definite time interval (40 min) may be explained

in terms of the solubilisation and dilution effect [3]. In

the present case, incorporations and/or associations of

EGCG and other catechins into the micellar palisade and

Stern layer of CTAB micelles take place, which in turn,

decrease the surface area of the reactants. As a result,

morphology of the AgNPs might be changed [6, 32].

Bakshi et al. [47] and El-Sayed et al. [48] have observed

the same effects (similar blue and red shift) of phos-

pholipids-stabilized pearl necklace-type gold–silver

bimetallic nanoparticles and demonstrated this from

discrete dipole approximation simulation, respectively.

The typical change in the shape of the spectra might be

due to the dilution effect. Possibly the two effects

(partitioning and/or solubilization and reduction of Ag?

to Ag0) work simultaneously on [CTAB] addition and

the resultant effect is a function of shape-directing role

of [CTAB].

0

1

2

3

4Time (min)

Ab

sorb

ance

Wavelength (nm)

0 5 10 15 20 25 30 35 40 45 50 55 60 70 80 90 120 overnight baseline

0

1

2

3

4

Time (min)

Ab

sorb

ance

0 5 10 15 20 25 30 35 40 45 50 55 60 70 80 90 120 Overnight baseline

0

1

2

3

4

Time (min)

Ab

sorb

ance

0 10 20 30 40 50 60 70 80 90 120 Overnight baseline

300 400 500 600 700 800

Wavelength (nm)300 400 500 600 700 800

Wavelength (nm)300 400 500 600 700 800

Wavelength (nm)300 400 500 600 700 800

0

1

2

3

4Time (min)

Ab

sorb

ance

0 5 10 15 20 25 30 40 50 60 70 80 90 120 overnight

A B

C D

Fig. 6 Time-resolved UV–Visible spectra AgNPs prepared by AgNO3 ? Camellia sinensis leaves extract in CTAB. Reaction conditions:

[Ag?] = 10.0 9 10-4 mol dm-3, [extract] = 4 % v/v, [CTAB] = 2.0 (a), 4.0 (b), 6.0 (c) and 8.0 9 10-4 mol dm-3 (d)

0 2 4 6 8 100

1

2

3

Abs

orba

nce

(at

465

nm)

104 [CTAB] (mol dm-3)

Fig. 7 Effects of [CTAB] on the maximum absorbance of SPR band

as a function of time. Reaction conditions: [Ag?] = 10.0 9 10-4

mol dm-3, [extract] = 4 % v/v, time = 20 (open circle) and 40 min,

(filled circle) temperature = 30 �C

Bioprocess Biosyst Eng

123

Figure 8 shows the TEM of the AgNPs in presence of

CTAB. They show that the particles are spherical, poly-

dispersed with some small-sized nanodisks of diameter ca.

30 nm. Presence of CTAB, removed the bilayer thickness

from the surface of the AgNPs (Fig. 2). We observed a

dramatic difference in the capping ability of catechins, i.e.,

EGCG from that of CTAB. EGCG shows a compact

interfacial film formation, while this is not so in the case of

micelle-forming CTAB surfactant. The capping ability of

EGCG originates due to electrostatic interactions between

the polar hydroxy groups and charged AgNPs surface [49].

It is certainly possible that the positive surface of AgNPs

forms an ion pair with the lone pairs of –OH groups present

in the EGCG molecules. Interestingly, a faint thin layer of

other material was also visualized on the surface of parti-

cles in the TEM images of AgNPs (Fig. 8), which might be

due to the capping properties of organic materials of

extract. Thus, the capping- and/or shape-directing role may

be explained in terms of excess EGCG adsorption

(although highly schematic) onto the surface of AgNPs

(Scheme 3). The above explanations and proposed mech-

anisms are in good agreement with the hypothesis highly

developed by the Shi et al. [50] and Bakshi [6] to the

reducing-cum-stabilizing role of EGCG and shape-direct-

ing role of proteins to the size-controlled synthesis of

AgNPs, respectively. The EGCG and other catechins are a

good choice as reducing, capping, stabilizing, and shape-

directing agents particularly when nanomaterials are

planned for use for biological applications. Although the

Scheme 3 is in good agreement to explain the observed

results (capping ability of EGCG). However, the role of

other polyhydroxy constituents (epicatechin, ECG, EGC,

theaflavin and thearubigin) as reducing and capping agents

could not be ruled out completely.

Comparison of spectroscopic, kinetic and TEM data

clearly indicates that the shape, the size distribution,

aggregation, cross-linking, polydispersity and size of Ag-

NPs formation follow the different pattern in the absence

and presence of CTAB. Thus, we may safely conclude that

EGCG solubilized into the micellar pseudophase and acted

only as a source of electron transfer to the Ag? ions. In

presence of CTAB, it can now be stated confidentially that

the reduction of Ag? to Ag0 occurs in the stern layer of

CTAB micelles. As a result, AgNPs must be present in this

region.

The larger metal particles can also exhibit more bands

due to the excitation of quadrupole and higher multipode

plasmon excitations. According to the Mie theory, a dipole

approximation of the oscillating conduction electrons is

given by (Eq. 1).

a ¼ 9ðVemÞ3=2 � xe2ðxÞc � ðe1ðxÞ þ 2emÞ2 þ e2ðxÞ2

ð1Þ

Where a = absorption coefficient, V = spherical

particle volume, c = speed of light, x = light frequency

and xm = dielectric constant of the surrounding medium.

The functions e1 and e2 are the real and imaginary part of

the dielectric function of the particle material (x(k)). The

SPR is expected, when the denominator of Eq. 1 becomes

small (e2 (x) = -2 em). The absorption peak position is,

thus, size-dependent within the dipole approximation.

Theoretically, Mie theory has been used to explain the

SPR spectra, [49]. The following expression for the dipole

approximation and the extinction coefficient a (k) of small

metal nanoparticles has been proposed.

a ¼ 18pln10

� 105

k�Mn3

0

q� e2

ðe1 þ 2n20Þ

2 þ e22

; ð2Þ

where M and q are the molar mass and the density of the

metal, n0 is the refraction index of dispersive medium, k is

0 5 10 15 20 25 30 350

2

4

6

8

10

12

Fre

quen

ce %

Diameter (nm)

A

B

Fig. 8 a TEM images of AgNPs in presence of CTAB (4.0 9 10-4

mol dm-3). b Size distribution histogram of resulting AgNPs.

Reaction conditions: [Ag?] = 10.0 9 10-4 mol dm-3, [extract] =

4 % v/v, temperature = 30 �C

Bioprocess Biosyst Eng

123

the wavelength. The agglomeration number of Ag

nanoparticles (NAg) has been calculated using the Eq. (3)

[51]:

NAg ¼ ð4=3ÞY

R3qNAM�1; ð3Þ

where R = radius of particle, NA = Avogadro number,

q = density, and M = atomic weight of silver. The

agglomeration numbers obtained using the Eq. (3) is

plotted versus diameter of AgNPs (obtained from TEM

images) (Fig. 9). The agglomeration of AgNPs increases

exponentially with increasing the diameter. A sharp

increase in the agglomeration number after a certain

diameter was observed thus pointing to the increased

agglomeration tendency of the AgNPs, which could be due

to the adsorption of EGCG and others polyhydroxy phenols

onto the surface of metallic silver particles, which in turn,

increases the Fermi level of particles [49]. Additionally, the

dielectric functions are a function of the size for very small

particles, as e.g., the damping constants change with size.

The neutral nucleophiles and neutral stabilizing polymers

have strong effect on the plasmon absorption band of silver

and/or metal nanometer particles and donate the electron

density to the particles via lone pairs of electrons. These

results followed the Beer–Lambert law (Eq. 6). The results

are rationalized in terms of the theories, calculations and

their explanations proposed by the various investigators

[46, 52–54]. The average number of silver atoms per

nanoparticle (N) was calculated with Eq. (4), where

D = average core diameters of the particles (in nm). The

values of N were found to be 0.8, 1.2, 5.3, 11.6, and

53.8 9 10-23 for 9.3, 13.4, 21.8, 28.3, and 47.2 nm,

respectively.

N ¼ p6

qD3

M¼ 30:89602 D3 ð4Þ

On the other hand, the molar concentration of the nan-

osphere solutions was calculated by dividing the total

number of silver atoms (Ntotal = the initial amount of

AgNO3 added to the reaction solution) over the average

number of silver atoms per nanosphere [N: calculated from

Eq. (4)] according to Eq. (5).

C ¼ Ntotal

NVNA

ð5Þ

A ¼ ebC ð6Þ

where V is the volume of the reaction solution in litre. It is

assumed that the reduction from Ag? to Ag0 atoms was

100 % complete. Calculated concentrations of Ag nano-

particles (C) are plotted against the maximum absorbance

at the SPR band. A good linear fitting of the experimental

data was found (Fig. 10) with e395 = 24.7 9 103

mol-1 dm3 cm-1.

The adsorption and/or chemisorptions of a neutral

nucleophile onto the surface of metal nanoparticles would

be accompanied by a shift of the Fermi potential to a more

negative value. The changes in the Fermi potential have

been calculated using the following equations.

EF ¼2m

8pð3h3NeÞ2=3 ð7Þ

DEF ¼2

3EF

6r

R

� �d ð8Þ

DEF ¼7:0

d

� �d; ð9Þ

O

O

OH

OHO

OH

OH

OH

OH

OH

OHO

O

OH

OHO

OH

OH

OH

OH

OH

OH

Ag

+

+

+++

++

+

+

+

+++

++

+Ag

Scheme 3 Adsorption of

EGCG on the surface of AgNPs

0 10 20 30 400

200000

400000

600000

800000

1000000

1200000

Agg

lom

erat

ion

Num

ber

Diameter (nm)

Fig. 9 Plot of agglomerization number against diameter of AgNPs

Bioprocess Biosyst Eng

123

where EF = Fermi energy of bulk silver, Ne = electron

density of Ag nanoparticles, h = Planck’s constant,

m = the effective electron mass (taken to be 1.0 [46],

r = radius of the silver atom and d = diameter. The values

of DEF were calculated using the Eq.(9) (assuming

EF = 5.48 eV and r = 0.16 nm and d = 0.3) and found to

be 0.03, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.15 eV for 58.3,

47.3, 33.3, 25.0, 20.0, 16.6 and 13.3 nm, respectively. The

maximum increase in Fermi level in EF is ca. 0.24 eV.

These results are in good agreement with the observations

reported by Henglein [49].

Surfactants, polymers, oligonucleotides, carbohydrates,

plant extracts and organic solvents have been used to

obtain fine and stable noble metal particles by the chemical

and physical methods [55]. These chemicals acted as

reducing, stabilizing and/or capping agents. The use of

plants extract provides advancement over other stabilizers

as it is cost effective, environment friendly, easily scaled

up for large scale synthesis. In the present method, EGCG

acted as a reducing and stabilizing agent and there is no

need to use high pressure, temperature and toxic chemicals

[56]. Comparison between the TEM images of AgNPs

synthesized in the absence and presence of externally

added stabilizer, i.e., CTAB (Figs. 2 and 8) clearly dem-

onstrated that the nanodisks of truncated triangular nano-

plates converted into the spherical AgNPs. In addition, we

did not observe a layer of biomolecules (EGCG) around the

surface of AgNPs in presence of CTAB. Thus, we may

safely conclude that desired shape of advanced metal

nanoparticles would be achieved using a suitable greener

bioreductant.

Conclusions

Aqueous leaves extract of C. sinensis has been used to

synthesize silver nanodisks. The presence of several SPR

bands in the UV–Vis spectra is due to the highly aniso-

tropic growth of nanoparticles. Polyhydroxy groups of

catechins reduce Ag? into Ag0 and ultimately leads to the

formation of AgNPs (triangular nanoplates and/or nanod-

isks). Our results suggest that the catechin, i.e., EGCG as

the major constituent in the mixture, forms a bilayer of ca.

6 nm thickness structure around a group of silver nanod-

isks. The polydispersed mainly spherical nanoparticles

were formed in the presence of CTAB, only participated in

the solubilization process of EGCG and other biomolecules

present in the extract.

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