Bioprocess Biosyst Eng DOI 10.1007/s00449-013-1094-0
ORIGINAL PAPER
Epigallocatechin-3-gallate-capped Ag nanoparticles: preparation and characterization Shokit Hussain • Zaheer Khan
Received: 4 November 2013 / Accepted: 8 November 2013 Ó 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-3gallate (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 Epigallocatechin3-gallate Oxidation CTAB Ag nanoparticles
Introduction Electron-, proton- and ion-transfer reactions at the interfaces 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 S. Hussain Z. Khan (&) Nano-science Research Lab, Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi 110025, India e-mail: [email protected]
compounds or surfactant that decreases interfacial tension or interfacial free energy of interfaces [2, 3]. Surfactantassisted synthesis has been considered to be an effective methodology for the shape-controlled synthesis of nanomaterials in the aqueous phase [4–6]. Generally, surfactants, ligands, polymers, or dendrimers have been used to confine the growth in the nanometer regime [7, 8]. ElSayed 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 cetyltrimethylammonium 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 investigations 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 phenomenon 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 Azadirachta 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
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active group of tea components that may affect the pathogenesis of various diseases (antioxidative, antimutagenic, and anticarcinogenic). Out of catechins (Scheme 1), epigallocatechin-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
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 dispersing 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 Scheme 1 Molecular structures of some constituents of Camellia sinensis leaves extract
OH OH HO
OH
O
OH
O HO
HO
O
O OH
OH
HO
OH
HO
Epicatechin
Epicatechin-3-gallate(ECG) OH OH
OH OH
HO
O OH O
HO
O
HO
O OH
OH
HO
OH
OH
Epigallatocatechin (EGC)
OH
Epigallocatechin-3-gallate (EGCG) OH
OH
OH
OH OH
OH
O
HO
O
O
HO
OH
HO
OH
O
O HO
O O
O OH
OH
HO
Theaflavin O CH3
H3C
N
N O
N CH3
Caffeine
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N
HO
OH
OH
Thearubigin
Bioprocess Biosyst Eng
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 cetyltrimethylammonium 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 deuterium 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 filtered 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 dilution. As the reaction proceeds, the colorless reaction mixture 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 experiments were performed under different experimental conditions, 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 threenecked reaction vessel fitted with a double surface condenser to check evaporation by adding the required concentrations of AgNO3, CTAB and water (for dilution maintained). The progress of the reaction was followed spectrophotometrically by adding the required concentrations 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 reaction 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 dissolution of the corner atoms of truncated triangular nanoplates. Figure 2 shows the TEM images of silver nanodisks in this study (length 47 nm, width 10). On careful observation of TEM images, a thin shell (layer = 6 nm) of EGCG moieties (major constituents of leaves extract)
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4
Time (nm)
Absorbance
3
60 50 40 30 25 20 15 10 5 0
2
1
0
300
400
500
600
700
800
Wavelength (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
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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
4 Time (min) 60 50 40 30 25 20 15 10 5 0
3
Absorbance
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 nanoplates (indicated by arrow in Fig. 2a: large fraction of triangles 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 symmetry 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) silver 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 preliminary information about the size and the size distribution 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 %
2
1
0
300
400
500
600
700
800
Wavelength (nm) 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
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
Absorbance (at 440 nm)
5
4
3
2
1
0 0
10
20
30
40
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
6
ln(a/1-a)
4
2
0
-2 0
10
20
30
40
50
60
70
80
90
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
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 constants 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 antioxidant activity of the green tea is higher in the teas that contain higher levels of EGCG and EGC. Therefore, presence 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 mechanism; 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., (Ag2? 4 -EGCG). We have also discussed the effect of phenolic –OH group on the reactivity of acetanilide, paracetamol, and tyrosine on the nucleation and growth of MnO2 and AgNPs formation [32, 41–43]. Micelles are dynamic aggregates of amphiphilic molecules that create highly anisotropic interfacial regions lining the boundary formed by the highly polar aqueous and
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Bioprocess Biosyst Eng OH
OH
O
OH HO
HO
O
O OH
OH
+
O OH
slow
Ag+
OH
OH
O
+
O OH
O
OH
OH OH
OH
(EGCG)
(EGCG radical) O
O
H
O
O HO
Ag0
HO
O OH
+
O OH
OH
fast
Ag+
+
O OH
OH
O
O
Ag0
OH
O
OH
OH OH
OH
(EGCG quinone) Ag0 + Ag+
fast
Ag2+
Ag2+ + Ag2+
fast
Ag42+
(Ag42+)n + (EGCG)n
fast
[(Ag42+)n-(EGCG)n ] (silver sol)
Scheme 2 Reduction of Ag? ions by the –OH groups of EGCG
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 counterions [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
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show only a sharp peak at 410 nm instead of two bands in presence of higher [CTAB] (Fig. 6d) on standing overnight. 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–
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Time (min)
3
Absorbance
B 4
0 5 10 15 20 25 30 35 40 45 50 55 60 70 80 90 120 overnight baseline
2
1
0 5 10 15 20 25 30 35 40 45 50 55 60 70 80 90 120 Overnight baseline
Time (min)
3
Absorbance
A 4
2
1
0
0 300
400
500
600
700
800
300
400
Wavelength (nm)
500
600
700
800
Wavelength (nm)
D 4
C 4
Time (min)
Time (min) 0 10 20 30 40 50 60 70 80 90 120 Overnight baseline
2
1
0 5 10 15 20 25 30 40 50 60 70 80 90 120 overnight
3
Absorbance
Absorbance
3
2
1
0
0 300
400
500
600
700
800
Wavelength (nm)
300
400
500
600
700
800
Wavelength (nm)
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)
Absorbance (at 465 nm)
3
2
1
0
0
2
4
6
8
10
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
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 phospholipids-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].
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A
B 12
Frequence %
10 8 6 4 2 0
0
5
10
15
20
25
30
35
Diameter (nm) 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
Figure 8 shows the TEM of the AgNPs in presence of CTAB. They show that the particles are spherical, polydispersed 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 particles 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
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(although highly schematic) onto the surface of AgNPs (Scheme 3). The above explanations and proposed mechanisms 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-directing 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 shapedirecting 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 AgNPs 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). a¼
9ðVem Þ3=2 xe2 ðxÞ c ðe1 ðxÞ þ 2em Þ2 þ e2 ðxÞ2
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. a¼
18p 105 Mn30 e2 ; ln10 k q ðe1 þ 2n20 Þ2 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
Bioprocess Biosyst Eng Scheme 3 Adsorption of EGCG on the surface of AgNPs
HO
+ + + + Ag + + OH + +
OH HO
O
OH HO O
HO
O
O OH + + + HO + Ag + OH + + + HO
OH O
HO
Agglomeration Number
OH O
OH
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.
1200000 1000000 800000 600000 400000
p qD3 ¼ 30:89602 D3 ð4Þ 6 M On the other hand, the molar concentration of the nanosphere 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).
N¼
200000 0
OH
0
10
20
30
40
Diameter (nm) Fig. 9 Plot of agglomerization number against diameter of AgNPs
the wavelength. The agglomeration number of Ag nanoparticles (NAg) has been calculated using the Eq. (3) [51]: Y NAg ¼ ð4=3Þ R3 qNA M 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
C¼
Ntotal NVNA
A ¼ ebC
ð5Þ 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 nanoparticles (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. 2m ð3h3 Ne Þ2=3 8p 2 6r DEF ¼ EF d 3 R 7:0 d; DEF ¼ d
EF ¼
ð7Þ ð8Þ ð9Þ
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Conclusions
8 Agglomeration Number
Absorbance (at 395 nm)
6000000
6
4000000
2000000
4
0
0
10
20
30
40
50
60
Diameter (nm)
2
0 0
5
10
15 -3
105 [Ag-nanoparticles] (mol dm ) Fig. 10 The Beer–Lambert plot to the formation of AgNPs [concentrations of particles were calculated using Eqs. (4) and (5)]. Reaction conditions: [extract] = 4 % v/v, [CTAB] = 4.0 9 10-4 mol dm-3, temperature = 30 °C. Inset plot of agglomeration number versus diameter plot in absence of CTAB
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 demonstrated that the nanodisks of truncated triangular nanoplates 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.
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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 anisotropic growth of nanoparticles. Polyhydroxy groups of catechins reduce Ag? into Ag0 and ultimately leads to the formation of AgNPs (triangular nanoplates and/or nanodisks). 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 nanodisks. 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.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Benjamin L (1996) Chem Rev 96:1449–1476 Tascioglu S (1996) Tetrahedron 52:11113–11152 Bunton CA (2006) Ad Colloid Interface Sci 123:333–343 Sun Y, Mayers B, Xia Y (2003) Nano Lett 3:675–679 Kim F, Sohn K, Wu J, Huang J (2008) J Am Chem Soc 130:14442–14443 Bakshi MS (2011) J Phys Chem C 115:13947–13960 Volokitin Y, Sinzig J, de Jongh LJ, Schmid G, Moiseev II (1996) Nature 384:621–623 Jana NR, Gearheart L, Murphy CJ (2001) Adv Mater 13:1389–1393 Ahmadi TS, Wang ZL, Green TC, Henglein A, El-Sayed MA (1996) Science 272:1924–1925 Bradley JS, Tesche B, Busser W, Maase M, Reetz MT (2000) J Am Chem Soc 122:4631–4636 Sau TK, Murphy CJ (2004) J Am Chem Soc 126:8648–8649 Baruah B, Craighead C, Abolarin C (2012) Langmuir 28:15168–15176 Nikoobakht B, El-Sayed MA (2001) Langmuir 17:6368–6374 Alkilany AM, Frey RL, Ferry JL, Murphy CJ (2008) Langmuir 24:10235–10239 Patil V, Mayya KS, Pradhan SD, Sastry M (1997) J Am Chem Soc 119:9281–9282 Gardea-Torresdey JL, Gomez E, Peralta-Videa J, Parsons JG, Troiani HE, Jose-Yacaman M (2003) Langmuir 19:1357–1361 Shankar SS, Rai A, Ahmad A, Sastry M (2004) J Colloid Interface Sci 275:496–502 Klaus T, Joerger R, Olsson E, Granqvist CG (1999) Proc Natl Acad Sci USA 96:13611–13614 Xie J, Lee JY, Wang DIC, Ting YP (2007) ACS Nano 1:429–439 Khan Z, Bashir O, Hussain JI, Kumar S, Ahmad R (2012) Colloid Surfs B 98:85–90 Hussain S, Akrema, Rahisuddin, Khan Z (2013) Bioprocess Biosyst Eng. doi:10.1007/s00449-013-1067-3 Li S, Shen Y, Xie A, Yu X, Qiu L, Zhang L (2007) Green Chem 9:852–858 Loo YY, Chieng BW, Nishibuchi M, Radu S (2012) Int J Nanomed 7:4263–4267 Higdon JV, Frei B (2003) Crit Rev Food Sci 43:89–143 Valcic S, Timmermann BN, Alberts DS, Wa¨chter GA, Krutzsch M, Wymer J, Guille´n JM (1996) Anticancer Drugs 7:461–468
Bioprocess Biosyst Eng 26. Gensler HL, Timmermann BN, Valcic S, Wa¨chter GA, Dorr R, Dvorakova K, Alberts DS (1996) Nutr Cancer 26:325–335 27. Cabrera C, Gimenez R, Lopez MC (2003) J Agric Food Chem 51:4427–4435 28. Kreibig U, Vollmer M (1995) Optical properties of metal clusters. Springer, Berlin. ISBN 978-3-540-57836-9 29. Khan Z, Singh T, Hussain JI, Al-Thabaiti SA, El-Mossalamy EH, Obaid AY (2013) Colloids Surfs B Biointerfaces 102:578–584 30. Kelly KL, Coronado E, Zhao LL, Schatz GC (2003) J Phys Chem B 107:667–668 31. Stamplecoskie KG, Scaiano JC (2010) J Am Chem Soc 132:1825–1827 32. Khan Z, Al-Thabaiti SA, Obaid AY, Khan ZA, Al-Youbi AO (2012) J Colloid Interface Sci 367:101–108 33. Jin RC, Cho YW, Markin CA, Kelly KL, Schatz GC, Zheng JG (2001) Science 294:1901–1903 34. Chen S, Fan Z, Carroll DL (2002) J Phys Chem B 106:10777–10781 35. Kim D-W, Shin S-I, Oh S-G (2003) In: Mittal KL, Shah DO (eds) Surfactant sciences series, vol 109. Marcel Dekker, New York, ISBN 0471746061 36. Huang Z-Y, Mills G, Hajek B (1993) J Phys Chem 97:11542–11550 37. Esumi K, Hosoyo T, Yamahira A, Torigoe K (2000) J Colloid Interface Sci 226:346–352 38. Goia DV, Matijevic E (1998) New J Chem 22:1203–1215 39. Toschi TG, Bordoni A, Hrelia S, Bendini A, Lercker G, Biagi PL (2000) J Agric Food Chem 48:3973–3980
40. Li C, Ni D (2009) Res Report 42:237–243 41. Kumar P, Khan Z (2006) Colloid Polym Sci 284:1155–1162 42. Malik MA, Basahel SN, Obaid AY, Khan Z (2010) Colloids Surf B Biointerfaces 76:346–353 43. Ahmad N, Malik MA, Al-Nowaiser FM, Khan Z (2010) Colloids Surf B Biointerfaces 78:109–114 44. Yu D, Yam VW-W (2004) J Am Chem Soc 126:13200–13201 45. Pal T, Sau TK, Jana NR (1997) Langmuir 13:1481–1485 46. Zhang J, Roll D, Geddes CD, Lakowicz JR (2004) J Phys Chem B 108:12210–12214 47. Bakshi MS, Possmayer F, Petersen NO (2007) J Phys Chem C 111:14113–14124 48. Jain PK, Eustis S, El-Sayed MA (2006) J Phys Chem B 110:18243–18253 49. Henglein A (1993) J Phys Chem 97:5457–5471 50. Wu H, He L, Gao M, Gao S, Liao X, Shi B (2011) New J Chem 35:2902–2909 51. Mucic RC, Storhoff JJ, Mirkin CA, Letsinger RL (1998) J Am Chem Soc 120:12674–12675 52. Pileni MP (1998) New J Chem 22:693–702 53. Shvalagin VV, Stroyuk AL, Kuchmii SY (2007) J Nanoparticle Res 9:427–440 54. Liu X, Atwater M, Wang J, Huo Q (2007) Colloids Surf B Biointerfaces 58:3–7 55. Bakshi MS (2009) Langmuir 25:12697–12705 56. Shervani Z, Yamamoto Y (2011) Carbohydr Res 346:651–658
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ORIGINAL PAPER
Epigallocatechin-3-gallate-capped Ag nanoparticles: preparation and characterization Shokit Hussain • Zaheer Khan
Received: 4 November 2013 / Accepted: 8 November 2013 Ó 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-3gallate (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 Epigallocatechin3-gallate Oxidation CTAB Ag nanoparticles
Introduction Electron-, proton- and ion-transfer reactions at the interfaces 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 S. Hussain Z. Khan (&) Nano-science Research Lab, Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi 110025, India e-mail: [email protected]
compounds or surfactant that decreases interfacial tension or interfacial free energy of interfaces [2, 3]. Surfactantassisted synthesis has been considered to be an effective methodology for the shape-controlled synthesis of nanomaterials in the aqueous phase [4–6]. Generally, surfactants, ligands, polymers, or dendrimers have been used to confine the growth in the nanometer regime [7, 8]. ElSayed 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 cetyltrimethylammonium 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 investigations 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 phenomenon 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 Azadirachta 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
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active group of tea components that may affect the pathogenesis of various diseases (antioxidative, antimutagenic, and anticarcinogenic). Out of catechins (Scheme 1), epigallocatechin-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
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 dispersing 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 Scheme 1 Molecular structures of some constituents of Camellia sinensis leaves extract
OH OH HO
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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 cetyltrimethylammonium 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 deuterium 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 filtered 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 dilution. As the reaction proceeds, the colorless reaction mixture 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 experiments were performed under different experimental conditions, 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 threenecked reaction vessel fitted with a double surface condenser to check evaporation by adding the required concentrations of AgNO3, CTAB and water (for dilution maintained). The progress of the reaction was followed spectrophotometrically by adding the required concentrations 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 reaction 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 dissolution of the corner atoms of truncated triangular nanoplates. Figure 2 shows the TEM images of silver nanodisks in this study (length 47 nm, width 10). On careful observation of TEM images, a thin shell (layer = 6 nm) of EGCG moieties (major constituents of leaves extract)
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Wavelength (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
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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
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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 nanoplates (indicated by arrow in Fig. 2a: large fraction of triangles 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 symmetry 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) silver 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 preliminary information about the size and the size distribution 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 %
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Wavelength (nm) 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
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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
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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
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 constants 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 antioxidant activity of the green tea is higher in the teas that contain higher levels of EGCG and EGC. Therefore, presence 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 mechanism; 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., (Ag2? 4 -EGCG). We have also discussed the effect of phenolic –OH group on the reactivity of acetanilide, paracetamol, and tyrosine on the nucleation and growth of MnO2 and AgNPs formation [32, 41–43]. Micelles are dynamic aggregates of amphiphilic molecules that create highly anisotropic interfacial regions lining the boundary formed by the highly polar aqueous and
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(Ag42+)n + (EGCG)n
fast
[(Ag42+)n-(EGCG)n ] (silver sol)
Scheme 2 Reduction of Ag? ions by the –OH groups of EGCG
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 counterions [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
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show only a sharp peak at 410 nm instead of two bands in presence of higher [CTAB] (Fig. 6d) on standing overnight. 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–
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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)
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10
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
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 phospholipids-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].
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Bioprocess Biosyst Eng
A
B 12
Frequence %
10 8 6 4 2 0
0
5
10
15
20
25
30
35
Diameter (nm) 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
Figure 8 shows the TEM of the AgNPs in presence of CTAB. They show that the particles are spherical, polydispersed 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 particles 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
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(although highly schematic) onto the surface of AgNPs (Scheme 3). The above explanations and proposed mechanisms 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-directing 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 shapedirecting 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 AgNPs 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). a¼
9ðVem Þ3=2 xe2 ðxÞ c ðe1 ðxÞ þ 2em Þ2 þ e2 ðxÞ2
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. a¼
18p 105 Mn30 e2 ; ln10 k q ðe1 þ 2n20 Þ2 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
Bioprocess Biosyst Eng Scheme 3 Adsorption of EGCG on the surface of AgNPs
HO
+ + + + Ag + + OH + +
OH HO
O
OH HO O
HO
O
O OH + + + HO + Ag + OH + + + HO
OH O
HO
Agglomeration Number
OH O
OH
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.
1200000 1000000 800000 600000 400000
p qD3 ¼ 30:89602 D3 ð4Þ 6 M On the other hand, the molar concentration of the nanosphere 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).
N¼
200000 0
OH
0
10
20
30
40
Diameter (nm) Fig. 9 Plot of agglomerization number against diameter of AgNPs
the wavelength. The agglomeration number of Ag nanoparticles (NAg) has been calculated using the Eq. (3) [51]: Y NAg ¼ ð4=3Þ R3 qNA M 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
C¼
Ntotal NVNA
A ¼ ebC
ð5Þ 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 nanoparticles (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. 2m ð3h3 Ne Þ2=3 8p 2 6r DEF ¼ EF d 3 R 7:0 d; DEF ¼ d
EF ¼
ð7Þ ð8Þ ð9Þ
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Bioprocess Biosyst Eng
Conclusions
8 Agglomeration Number
Absorbance (at 395 nm)
6000000
6
4000000
2000000
4
0
0
10
20
30
40
50
60
Diameter (nm)
2
0 0
5
10
15 -3
105 [Ag-nanoparticles] (mol dm ) Fig. 10 The Beer–Lambert plot to the formation of AgNPs [concentrations of particles were calculated using Eqs. (4) and (5)]. Reaction conditions: [extract] = 4 % v/v, [CTAB] = 4.0 9 10-4 mol dm-3, temperature = 30 °C. Inset plot of agglomeration number versus diameter plot in absence of CTAB
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 demonstrated that the nanodisks of truncated triangular nanoplates 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.
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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 anisotropic growth of nanoparticles. Polyhydroxy groups of catechins reduce Ag? into Ag0 and ultimately leads to the formation of AgNPs (triangular nanoplates and/or nanodisks). 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 nanodisks. 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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