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 Page 1 of 54 Inorganic Chemistry Frontiers
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Biosynthesis of Amino Acid Functionalized Silver Nanoparticles for Potential Catalytic and

Oxygen Sensing Applications

Abhishek Chandra† and Man Singh†*

Inorganic Chemistry Frontiers Accepted Manuscript

†
School of Chemical Sciences, Central University of Gujarat, Gandhinagar-382030, India
Email: [email protected], [email protected]
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ABSTRACT

A green approach for biosynthesis of amino acid functionalized silver nanoparticles (AgNPs) using

Neem gum is reported. The AgNPs showed good colloidal stability with surfactants and dyes, which

to the best of our knowledge, is the first attempt with biosynthesized AgNPs. The reduction of

Methylene blue (MB) by SnCl2 in micellar mediums and Congo red/4-Nitrophenol by NaBH4

validated high catalytic efficiency of the AgNPs. To the best of our knowledge, it is the first report

on a detailed kinetic study of AgNPs catalyzed MB reduction in cationic, anionic and nonionic

micellar mediums. The highest MB reduction rate was observed in anionic micellar medium with

tryptophan functionalized AgNPs. The AgNPs were responsible for backward oxidation of reduced

MB in acidic nonionic micellar medium, which is the first ever demonstration of a one-cycle redox

MB clock reaction involving silver nanoparticles. The clock reaction elucidated potential use of the

AgNPs as low-cost sensors for oxygen leakage in vacuum-packed food packages, and for creating

oxygen deficient environment in acidic aqueous and micellar mediums, where catalyst poisoning by

O2 prevails. The deactivation of catalytic activity of tryptophan functionalized AgNPs observed

during Congo red and 4-Nitrophenol reduction may find application in such catalytic reduction,

where improved selectivity is required. With the possibility of large-scale production of the AgNPs

on economic scale possible, the AgNPs thus may hold great promise in effective and eco-friendly

wastewater treatment, with potential application in oxygen sensing.

KEYWORDS Green chemistry, Silver Nanoparticles, Methylene Blue, Plant gum, Surfactants,

Clock reaction, Congo red, 4-Nitrophenol

 Inorganic Chemistry Frontiers Page 2 of 54
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1. INTRODUCTION

Designing a new synthetic route for metal nanoparticles production, the key factors that

merit important consideration are utilization of nontoxic chemicals, renewable materials and

Inorganic Chemistry Frontiers Accepted Manuscript

environmentally benign solvents. As compared to physical and chemical processes, the biological

system based synthetic routes for the production of nanoparticles (NPs) are economic, and does
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not pose potential biological and environmental risks.1,2 Among the biological systems, the

production of NPs using plants as compared to biological resources is advantageous on account

of enhanced scalability and safety.3 Plant extract provides a more flexible control over size,

shape, crystallinity, and size distribution of the NPs, with faster synthesis and easy purification.4-6

Currently, many methods have been reported for the synthesis of copper, gold, silver, platinum

and iron NPs using plant extract.7-10 In these methods; the carbonyl, hydroxyl and carboxylic

functional groups present in the plant extract facilitates metal ion complexation and reduction.

Further, the proteins and polysaccharides present in plant extract increases the stability and

functionality of the NPs.11

Among the metallic NPs, silver NPs express optical sensing, catalytic and antimicrobial

properties. To exhibit the said properties efficiently, Silver NPs need to be stable and compatible

in different aqueous environments, avoiding aggregation and corrosion. For instance, their

synthesis methods using plant extract of leaf,12-16 fruit,17-21 gum,22-26 flower,27-28 seed,29-31 and

others,32-36 are well-established, but there are lacunae with regards to their colloidal stability in

aqueous mediums of high ionic strength, variable pH, surfactants, and dyes.

Since chemical routes for silver NPs synthesis are expensive and hazardous, usage of new

natural resources for their synthesis therefore becomes a natural need. Neem gum (NG) is a

water-soluble plant gum exudate from Azadirachta indica bark and contains proteins, complex

polysaccharides and many organic compounds which could facilitate the Ag+ ion reduction to

Ag(0).37-41 Such features of NG encouraged us to use this natural resource as a reductant for the
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biosynthesis and stabilization of silver nanoparticles (AgNPs). However, owing to the

complexity of the active functional groups in NG, good colloidal stability of the biosynthesized

AgNPs may be difficult to achieve. Amino acids are known to form coordinate complexes with

Inorganic Chemistry Frontiers Accepted Manuscript

Ag+ ion through various sites such as >C=O, -NH2, -COOH, thiol, imidazole and indole

groups.42-44 The formation of Ag+amino acid complexes may thus provide a compact domain for
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the controlled nucleation of Ag nuclei with defined shape and increased stability. Amino acids

like histidine (His), tryptophan (Trp), methionine (Met), asparagine (Asp), glutamic acid (Glu)

and alanine (Ala) have strong affinity for Ag+ ions.42 In NG, they are present in a very less

amount, not sufficient enough to provide controlled nucleation of the Ag nuclei.38 Thus, to

achieve controlled nucleation with defined shape and enhanced stability in various aqueous

environment, the AgNPs were functionalized with Ala, Trp, Met, His, Glu, and Asp, and are

noted as f-AgNPs.

Despite the structural potential of amino acids to interact with silver, and the presence of

electron rich active components in plant extract, no research work has been reported on their

combined impact on silver NPs formation, their colloidal stability and applicability. Our studies

on the f-AgNPs thus become a pioneer as it establishes an operational link between the AgNPs

and the structural activities of amino acids and electron rich active components in plant extract.

Furthermore, this study widens the applicability and database of the AgNPs. This study is a first

of its kind because our research methodology to investigate the effects of pH, ionic strength,

surfactant, and dye on the colloidal stability of the f-AgNPs fills up a wider gap vis-à-vis AgNPs

science and their correlation with natural resource for the advanced understanding of bio-

nanomaterials. Moreover, we have initiated studies based on the effect of cationic, anionic and

nonionic surfactants on the f-AgNPs colloidal stability. A similar model was extended to cationic

and anionic dyes, and such studies are yet to be reported.

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The good colloidal stability of the AgNPs prompted us to use them as catalyst for

reduction reactions. The catalytic efficiency of the AgNPs was studied on reduction of

Methylene blue (MB) by acidic SnCl2 in aqueous and micellar mediums, and on reduction of

Inorganic Chemistry Frontiers Accepted Manuscript

Congo red and 4-Nitrophenol by NaBH4 in aqueous. Very limited work is reported on silver NPs

catalyzed MB reduction in micellar medium, and their detailed kinetics. Ghosh et al

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demonstrated silver and gold NPs catalyzed reduction of MB by NaBH4 in the presence of

sodium arsenate, in sodium dodecyl sulfate (SDS) micellar system.45 However, the focus of their

work was on the quantitative estimation of arsenic and hence, no kinetic investigation was

offered on the NPs catalyzed MB reduction in anionic micellar system. Pal et al studied micellar

catalyzed MB reduction by ascorbate ion and observed that cationic micellar catalysis in

presence of ascorbate ion was much higher than the reduction rate in presence of silver NPs in

aqueous.46 Here also, no kinetic study was carried out on the silver NPs catalyzed MB reduction

in micellar systems. Jiang and co-workers in their study on silver NPs supported on silica spheres

catalyzed dye reduction showed that cationic, anionic and nonionic surfactant decreases the

reduction rate of the dyes by deactivating or poisoning the catalyst.47 Furthermore, the earlier

reports on redox MB clock reaction were fast and reversible upto many cycles,48-50 and hence

kinetic study on backward oxidation of reduced MB to oxidized MB was not possible. Earlier,

MB clock reaction did not have any practical application other than as an example of simple and

crowd-pleasing demonstration of a clock reaction. In this study, we for the first time, report the

kinetics of backward oxidation of reduced MB and illustrated a possible practical application of

the one cycle redox MB clock reaction in presence of the AgNPs as low cost sensors for oxygen

leakage in vacuum-sealed packages. The catalyzed MB reduction revealed that some of the f-

AgNPs could be successfully used to create O2 deficient environment in acidic aqueous and

micellar mediums, where catalyst poisoning by O2 prevails.

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In this study, the combinatorial science of AgNPs in association with NG and amino

acids on the catalytic reduction have revealed some new findings, such as pseudo-second order

kinetics for MB reduction, increased MB reduction rate in anionic micellar medium, and

Inorganic Chemistry Frontiers Accepted Manuscript

selective catalytic reduction, which to the best of our knowledge, are yet to be reported with

silver NPs. The present work thus has opened a new domain in the biosynthesis of AgNPs,
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wherein plant extract in combination with amino acid, flavonoid, proteins and many others may

act as valuable natural resources and could be used for manipulating AgNPs surface activities

with wider and potential applications.

2. EXPERIMENTAL SECTION

2.1. Materials and Methods

Neem gum (NG) was collected from neem trees located at Gandhinagar, India. L-alanine (Ala)

(A7469), D-tryptophan (Trp) (T9753), L-methionine (Met) (M9625), L-histidine (His) (H8000),

L-glutamic acid (Glu) (G1251), L-asparagine (Asp) (A0884), AgNO3 (209139), NaOH (S8045),

HCl (320331), NaCl (S7653), Dodecyltrimethylammonium bromide (DTAB) (D5047), Sodium

dodecyl sulfate (SDS) (L6026), Octyl β-D-glucopyranoside (OGP) (O8001), Dioctyl

sulfosuccinate sodium salt (AOT) (D1685), Tween-20 (T-20) (P1379), Sodium borohydride

(NaBH4) (71320), 4-Nitrophenol (4-NP) (1048), Stannous Chloride (SnCl2) (208256), Congo red

(CR) (C6277), and Methylene blue (MB) (28514), were procured from Sigma-Aldrich and used

as received.

2.2. Synthesis of Amino Acid Functionalized Silver Nanoparticles (f-AgNPs)

For the synthesis of f-AgNPs, briefly to 10 mL (2.75% w/v) aqueous NG, 100 μL (1.75

M) aqueous AgNO3 and 10 mL (0.1 M) aqueous Ala/Trp/Met/His/Glu/Asp was added. The

resulting reaction mixture was transferred to a 50 mL centrifuge tube (Tarson) and incubated for

20 min at 373.15 K. The f-AgNPs dispersion thus obtained was purified by centrifugation at

17500×g for 20 min followed by re-dispersion in 100 mL Milli-Q water. For the synthesis of
 Inorganic Chemistry Frontiers Page 6 of 54
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non-functionalized AgNPs, in place of 10 mL (0.1 M) aqueous amino acid, 10 mL water was

used. All the dispersions were stored in airtight amber colored bottle at 4C, to be used in later

experiments. The AgNPs thus synthesized are AgNP (non-functionalized AgNPs), and amino

Inorganic Chemistry Frontiers Accepted Manuscript

acid functionalized AgNPs (f-AgNPs): Ala-AgNP (Ala functionalized AgNPs), Trp-AgNP (Trp

functionalized AgNPs), Met-AgNP (Met functionalized AgNPs), His-AgNP (His functionalized

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AgNPs), Glu-AgNP (Glu functionalized AgNPs), and Asp-AgNP (Asp functionalized AgNPs).

UV-visible spectroscopy was used to optimize the reaction parameters such as NG and

AgNO3 concentration; and reaction time. To optimize the NG and AgNO3 concentration, 10 mL

(0.21 to 3% w/v) aqueous NG was added to 100 μL (1.75 M) aqueous AgNO3; and (2.5 to 100

μL) 1.75 M aqueous AgNO3 was added to 10 mL (2.75% w/v) aqueous NG, respectively,

followed by 20 min incubation at 373.15 K. For reaction time optimization, spectra after every 1

min was recorded for the reaction mixture containing 10 mL (2.75% w/v) aqueous NG and 100

μL (1.75 M) aqueous AgNO3, incubated at 373.15 K.

2.3 Surface Functionalization Efficiency of Amino Acids for f-AgNPs

UV-visible absorbance spectroscopy was used to investigate the surface functionalization

efficiency (S.F.E.) of the respective amino acids. The development of purple color (Ruhemann’s

purple) complex on reaction of ninhydrin with free α-amino group of amino acids was the

genesis of S.F.E.. For S.F.E. determination, the supernatants obtained from all the washing

cycles of the individual f-AgNPs was mixed together and aqueous ethanolic solution of

ninhydrin was added. The reaction mixture was maintained at 373.15 K for 15 min, for

completion of the reaction. The absorbance of the reaction mixtures at 570 nm (λmax of amino

acidninhydrin complex) was recorded and concentration of the respective amino acid in the

supernatant was calculated using the standard curve (absorbance vs concentration) of amino acid.

S.F.E. was calculated using the formula

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[Amino acid]i  [Amino acid]s

S .F .E.(%)  100
[Amino acid]i

where, [Amino acid]i and [Amino acid]s are the concentration of amino acid used for f-AgNPs

Inorganic Chemistry Frontiers Accepted Manuscript

synthesis and concentration of amino acid in the supernatant, respectively.

The absorbance spectra of the purple colored amino acidninhydrin complex may be
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sensitive to the other reactive moieties present in the medium. Since, the AgNPs supernatants

may contain some reactive moieties (due to NG), which may affect the spectra of the colored

complexes; the standard curve of the amino acidninhydrin complexes was obtained by

dissolving different concentrations of amino acids in the supernatant, obtained after washing of

AgNP (non-functionalized). Aqueous ethanolic solution of ninhydrin was added and the reaction

mixture was maintained at 373.15 K for 15 min. The absorbance of these amino acid containing

reaction solutions was recorded at 570 nm using UV-visible spectrophotometer. In all the S.F.E.

experiments, the absorbance measurements were performed in triplicates and the overall solution

volume, and quantity of ninhydrin used was kept constant. Figure S1 depicts the standard curve

for alanine-ninhydrin complex, illustrating unreacted Ala concentration during Ala-AgNP

synthesis.

For the f-AgNPs, the S.F.E. of Ala, Trp, Met, His, Glu, and Asp was found to be 64.59,

72.09, 78.07, 64.33, 54.13, and 59.58%, respectively. Thus, when 10-3 mole of amino acids was

used to synthesize 100 mL aqueous dispersion of f-AgNPs; 57.54 mg Ala, 147.23 mg Trp,

116.49 mg Met, 99.81 mg His, 79.63 mg Glu, and 78.72 mg Asp was involved in formation and

surface functionalization of Ala-AgNP, Trp-AgNP, Met-AgNP, His-AgNP, Glu-AgNP, and Asp-

AgNP, respectively. Interestingly, NG did not develop purple color on reaction with ninhydrin,

elucidating the absence of free amino group.

2.4. Structural Characterization of AgNPs

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The UV-visible absorbance spectra were recorded using a UV-visible spectrophotometer,

Spectro 2060 plus at 298.15 K. For AgNPs morphology, high-resolution transmission electron

microscopy (HR-TEM) was performed using a JEOL JEM-2100 HR-TEM, at 180 kV. The X-ray

Inorganic Chemistry Frontiers Accepted Manuscript

diffraction (XRD) was performed on Bruker, D8 Focus instrument of Cu-Kα wavelength (λ =

1.54 A), with scanning over a 2θ range of 20–90 with 0.017 step size. For high resolution
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UHV X-ray photoelectron spectroscopy (XPS), Omicron ESCA+ was used for the compositional

confirmation of the AgNPs. The Al K α radiations of 1486.7 eV energy were operated at 15 kV

and 20 mA, throughout the measurements. The high-resolution XPS spectra were collected at

pass energy 20. For the XPS analysis, dried powder samples of AgNPs were mounted on carbon-

coated tape. Malvern Zetasizer 3000 was used to determine the size distribution, polydispersity

index (PDI) and zeta potential of the AgNPs. The FTIR spectra were recorded using PerkinElmer

Spectrum 65 series FTIR Spectrophotometer. For FTIR sample preparation, the AgNPs

dispersions were dried in vacuum oven for 24 h at 313.15 K. The dried samples (2 mg) were

blended with KBr (200 mg) and pressed into pellet. The pellets were placed into the sample

holder and FTIR spectra were recorded in the range 4000–400 cm–1 in transmission mode. The

FTIR spectra were derived from the average of 64 scans with 1 cm−1 resolution. The 1H NMR

studies was performed on Bruker Advance 500 MHz instrument at 298.15 K. The second

derivative FTIR calculations and Gaussian curve fitting analysis were made using OriginPro 8

software. The total silver concentration of the AgNPs stock suspension was measured in

triplicate by inductively coupled plasma optical emission spectrometer (ICP-OES; model: 7300

DV, Perkin Elmer) against a standard AgNO3 solution.

2.5. Colloidal Stability of AgNPs

2.5.1. Influence of pH, Ionic Strength, Surfactant and Dyes on Colloidal Stability of AgNPs
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The influence of pH (2 to 14), ionic strength (0.1 to 2 M aqueous NaCl), surfactant

(0.0025 to 0.010 M) and dye (10 to 50 μM) on the AgNPs colloidal stability was investigated

using UV–visible spectroscopy, as reported in the Supporting Information.

Inorganic Chemistry Frontiers Accepted Manuscript

2.6. Catalytic Activity of AgNPs
The catalytic efficiency of AgNPs on reduction of MB by acidic SnCl2 in
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aqueous/surfactant micellar mediums, and reduction of CR and 4-NP by NaBH4 in aqueous was

monitored using UV–visible spectroscopy, as reported in the Supporting Information.

3. RESULTS AND DISCUSSION

3.1. Synthesis of AgNPs

The formation of AgNP by reduction of Ag+ ions with aqueous NG was illustrated by the

concomitant color change of the reaction mixture. As the biosynthesis proceeds, a change in

color occurs from colorless to yellowish within 2 min but after 20 min a brown color appeared

(Figure 1a). The spectra of the dispersion had a characteristic absorption maximum at 420 nm

(Figure 1), attributed to the surface plasmon resonance (SPR) band of silver NPs. For the

production of AgNP, the optimum condition of NG and AgNO3 concentration; and reaction time

were achieved by UV–visible spectroscopy. Figure 1 shows the UV–visible spectra recorded for

the optimized (a) NG concentration, (b) AgNO3 concentration and (c) reaction time. Figure 1b

depicts the UV–visible spectra of AgNP produced using 100 μL (1.75 M) AgNO3 with different

NG concentrations, after 20 min incubation at 373.15 K. The population of AgNP increased with

increasing NG concentration, depicted through increased absorbance (Figure 1b). The highest

absorbance at 420 nm with 2.75% (w/v) NG illustrates conversion of all Ag+ ions into Ag(0) and

the subsequent decrease in absorbance with > 2.75% (w/v) NG illustrates aggregation of the

AgNP. The production of AgNP with 2.75% (w/v) NG was also studied by varying AgNO3

concentration, as depicted in Figure 1c. The absorbance increased with increasing AgNO3

concentration due to enhancement in the oxidation of hydroxyl groups by Ag+ ions. No change in

the absorbance observed with > 100 μL (1.75 M) AgNO3 illustrates that at this AgNO3
 Inorganic Chemistry Frontiers Page 10 of 54
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concentration, all the reducing agents present in NG were consumed, with no further reduction of

Ag+ ion possible. The synthesis was also assessed by varying reaction time at fixed NG and

AgNO3 concentrations (Figure 1d). The reducing ability of NG increases with increasing

Inorganic Chemistry Frontiers Accepted Manuscript

incubation period, possibly because more and more hydroxyl groups were converted to carbonyl

groups through oxidation by dissolved oxygen present in water, which in turn reduced the Ag+
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ions. The initial blue shift observed in the SPR peak of AgNP within 3 min of incubation (Figure

1d) infers initiation of controlled nucleation rate, resulting in decreased particle size. No change

in the SPR peak position from 3 min onwards infers progress of nucleation at constant rate.

Meanwhile, no change in the absorbance after 20 min of incubation infers complete consumption

of the precursors. Thus, based on the optimal results from AgNP synthesis, 10 mL (2.75% w/v)

aqueous NG with 100 μL (1.75 M) aqueous AgNO3 and 20 min as reaction time, have been used

to synthesize the f-AgNPs with 10 mL (0.1 M) aqueous Ala/Trp/Met/His/Glu/Asp.

3.2. Characterization of AgNPs

The HR-TEM micrographs revealed that AgNP were irregular shaped, while the f-AgNPs

were primarily spherical shaped, with no agglomeration (Figure 2) and size ranging from in

between 5–50 nm (Table 1). The hydrodynamic diameter of the AgNPs, measured by DLS

showed an average diameter of 14-39 nm, with relatively narrow size distribution (PDI) (Table

1). It appears that amino acids with their high complexation ability toward Ag+ ion resulted in

ordered nucleation of the Ag nuclei, thereby providing a narrow size distribution for the f-

AgNPs. Furthermore, the amino acids could have associated around the NG constituents at the f-

AgNPs surface by H-bonding, thereby preventing further nucleation. In absence of amino acids,

disordered nucleation could have resulted in a wider size distribution for the AgNP colloids

(Table 1). Thus, amino acids play a monitoring role during the nucleation and growth process of

the f-AgNPs and based on their affinity towards Ag+ ion, vary the size distribution of the f-

AgNPs. Figure S2 depicts the HR-TEM micrographs of AgNPs revealing capping by proteins
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and polysaccharides in NG, wherein Trp-AgNP and His-AgNP exhibits thick layer of capping

over their surface

For investigating the crystalline structure of the AgNPs, XRD analysis was performed.

Inorganic Chemistry Frontiers Accepted Manuscript

The XRD patterns of the AgNPs are shown in Figure 3. The d-spacings obtained from XRD are

consistent with the standard cubic Ag2O and Ag phases. The sharp diffraction peaks indicated
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high crystallinity of the Ag2O and Ag crystallites, and were indexed to fcc cubic silver (JCPDS

PDF 04-0783)51 and cubic Ag2O (JCPDS 76-1393),52 reported in Table S1. The size of Ag and

Ag2O crystallites was calculated using Debye–Scherrer equation (Table S1). The cubic Ag

planes are in close agreement with the unit cell of fcc structure (JCPDS File No. 87-0720)53 with

a lattice parameter of a = 4.09 A°. The Ag2O planes are in close agreement with the unit cell of a

hexagonal structure (JCPDS File No. 76-1393) with a lattice parameter of a = 4.76 A°. The

100% intensity peak of Ag (111) planes illustrate the maximum concentration of Ag crystallites

in Trp-AgNP and His-AgNP. Figure S3 elucidates the percentage of Ag and Ag2O crystallite

planes in the AgNPs. Ala, Trp and His seems responsible for Ag (200) plane formation in Ala-

AgNP, Trp-AgNP and His-AgNP, respectively. Furthermore, Trp and His assist in Ag (111)

plane formation, while Ala, Glu, and Asp assist in Ag2O (111) plane formation. Moreover, Trp,

Met and His strongly suppresses the Ag2O (110) plane formation in the f-AgNPs. Hence, during

the nucleation and growth stages of the f-AgNPs, amino acids introduce peculiar features in their

crystalline structure, which may improve their functionality and stability. Figure S2 illustrates

the HR-TEM micrograph of AgNPs depicting the lattice spacing of Ag (111)/Ag2O (110) plane,

and are in good agreement with the XRD results. The biosynthesis of Ag/Ag2O nanocomposite

thus observed in this work, establishes a functional linkage between their formation and the

structural activities of amino acids and electron rich active components of plant extract and adds

to an otherwise limited literature.54,55

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The capping of f-AgNPs by proteins and polysaccharides in NG and their

functionalization with amino acids introduce electron rich sites at the f-AgNPs surface. With

these electron rich sites, a negatively charge electrostatic double-layer is formed around the f-

Inorganic Chemistry Frontiers Accepted Manuscript

AgNPs colloids in water. The overall negative charge these electrostatic double-layers carry,

governs the f-AgNPs colloidal stability. Hence, zeta potential measurement of the f-AgNPs
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colloids is inevitable, providing a clear view of their stability. NG contains complex

polysaccharides rich in uronic acid, which is majorly in the form of D-glucuronic acid and

aldobiuronic acid.37-41 The negative zeta potential of the AgNPs (Table 1), thus may be due to the

negatively charged carboxyl groups present in the complex polysaccharides of NG, which gets

adsorbed over the AgNPs surface. The higher negative zeta potential observed for the f-AgNPs

may be due to their functionalization with amino acids. Since amino acids contain carboxyl

group, the surface functionalization with amino acids thus may increase the concentration of

carboxyl groups adsorbed at the f-AgNPs surface, thereby making their surface more negatively

charged. A high value of zeta potential denotes a high electrical charge on the surface of AgNPs,

which can cause a strong electrostatic repulsive force among the nanoparticles to prevent

agglomeration, and with increased colloidal stability. Thus, functionalization with amino acids

may have resulted in higher colloidal stability of the f-AgNPs than AgNP (non-functionalized).

The AgNPs surface chemistry was further explored by FTIR spectroscopy. Figure 4

shows the FTIR spectra of NG, AgNP, and Trp-AgNP. The FTIR spectra of Trp-AgNP is also

the representative spectra of f-AgNPs, as the f-AgNPs spectra were similar. The IR spectrum of

NG shows specific peaks at 1076, 1307, 1554, 1644, 2090, 2934 and 3394 cm−1. The stretching

vibration band at 1307 cm−1 infer C-N aromatic functional group of proteins in NG.56 The peak

at 1644 cm-1 in the amide I band (1700-1600 cm-1) correspond to C=O stretching modes, while

the peak at 1554 cm−1 in the amide II band (1600-1500 cm-1) is due to N-H bending and C-N

stretching modes, of the peptide linkages of proteins in NG.57 The bands at 1076 and 3394 cm−1
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infer presence of amine and hydroxyl groups, respectively.58 The broad band at 2090 cm−1 could

be assigned to various carbonyl species, while band at 2934 cm-1 correspond to asymmetric

stretching, scissoring vibrations of various methylene groups present in NG.23 In comparison

Inorganic Chemistry Frontiers Accepted Manuscript

with the FTIR spectrum of NG, major shifts for the AgNPs were observed in the regions (1700-

1500 cm-1) and (3500-3300 cm-1); suggesting presence of carbonyl, carboxyl, hydroxyl and
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amino groups at their surface. The changes associated with the vibrational energies of these

groups present in NG and amino acids may provide detailed information on their association

with the AgNPs. In view of this, the FTIR spectra of NG, AgNP, and Trp-AgNP were

deconvoluted in the regions: (1700-1500 cm-1) and (3500-3300 cm-1) and a Gaussian multiple-

peak-fitting procedure was employed after baseline correction (Figure S4). The multiple peaks

resulting from the deconvolution infers changes associated with the vibrational energies of

carbonyl, carboxyl, hydroxyl and amino groups; while area under the deconvoluted peaks may

infer the qualitative changes associated with their composition. The relative changes (peak center

and peak area) perceived in the deconvoluted spectra of AgNP and Trp-AgNP, when compared

with of NG (Figure S4) confirmed that the carbonyl, carboxyl, hydroxyl, and amine groups

resulting from amino acid and NG proteins are strongly bound to the AgNPs surfaces. These

spectral changes originated mostly due to changes in N-H bending, C-N stretching and C=O

stretching vibrations of amide linkage of proteins, carboxylic stretching vibrations of uronic acid,

O–H stretching vibrations of polyphenols and stretching vibration of –COC linkages of

flavonoids present in NG along with N-H, carbonyl and carboxylic stretching vibrations in amino

acid. The variable area under the deconvoluted peaks (Figures S4) for AgNP and Trp-AgNP

infer their growth process being regulated by changing interaction dynamics induced by amino

acids. No specific band at ~877 cm-1 corresponding to Ag-O bonds is observed in the FTIR

spectra of AgNPs contrary to the XRD results which showed the presence of Ag2O. This may be
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due to strong binding of amino acids to the f-AgNPs surfaces and robust capping by the

polysaccharides and proteins of NG (see Figure S1).

For better understanding the surface chemistry of the AgNPs and their interaction with

Inorganic Chemistry Frontiers Accepted Manuscript

NG and amino acids, X-ray photoelectron spectroscopy (XPS) analysis was performed. Figure 5

shows the full scanned spectrum of the AgNPs in the range of 200 eV to 800 eV. In the XPS
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survey spectra, the atoms of C, N, O and Ag were detected, with no other peak, indicating the

high purity of the AgNPs. The high resolution Ag 3d spectrum of the AgNPs (Figure S5)

demonstrated two peaks at 368 eV (Ag 3d5/2) and 374 eV (Ag 3d3/2) with a peak splitting of

6.0 eV, which corresponds to metallic silver (Ag0).59 The peak shape of the Ag 3d5/2 and Ag

3d3/2 levels suggest that a single Gaussian peak function cannot be applied to fit each Ag 3d level

successfully. Therefore, each Ag 3d level were deconvoluted into two splitting peaks, and the

corresponding binding energies are tabulated in Table S2. The deconvoluted curve of the Ag

3d5/2 level were well fitted by two peaks centered 368.2 eV and 367.3 eV corresponding to

Ag0 and Ag2O, respectively,60,61 illustrating the presence of both forms of silver in the AgNPs

and supports the XRD results. The estimated percentage of Ag0 and Ag2O in the AgNPs was

calculated from the ratio of the area under the curve of the splitting peaks of Ag 3d 5/2 level and

are reported in Table S3. Table S3 infers that Trp-AgNP, Met-AgNP, and His-AgNP have >

70%, while AgNP, Ala-AgNP, and Asp-AgNP have < 40% Ag0 content.

Figure S5 shows the deconvoluted O 1s spectrum of the AgNPs, and the corresponding

binding energies are tabulated in Table S2. The peak at 532.9 eV is assigned to C–OH, C–O,

and C=O bonds available for silver ions to bind with,62 and is present only in the f-AgNPs

spectrum (Figure S5), inferring their functionalization by amino acids, which are rich in these

bonds. Functionalization is further supported by the presence of peak at 532-533 eV in the f-

AgNPs spectrum, attributed to the oxygen associated with the surface hydroxyl groups from the

carboxylic groups of the amino acids,63 which otherwise is absent in the AgNP spectrum (Figure
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S5). The additional peaks observed in the deconvoluted O 1s spectrum of the AgNPs in the range

528-532 eV suggest the presence of O2- in Ag2O and AgO bond formation.64

Figure S5 illustrates the deconvoluted C 1s spectrum of the AgNPs, and the

Inorganic Chemistry Frontiers Accepted Manuscript

corresponding binding energies are tabulated in Table S2. The C 1s spectrum were deconvoluted

into two distinct peaks for AgNP, His-AgNP, and Trp-AgNP; while into three distinct peaks for
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Ala-AgNP, Met-AgNP, Glu-AgNP, and Asp-AgNP. In the C 1s spectrum, the peak at 284.4

eV, 285, 286, 289, and 290 eV is attributed to C=C, CC, CO/CN, C=O, and OC=O,

respectively.65 The AgNP spectrum does not have peak at 286 and 290 eV, whereas for the f-

AgNPs, their spectrum either have atleast one of their peaks at 286 or 290 eV. This further

provides the strong evidence of the f-AgNPs being functionalized by the amino acids, as they

have the CO/CN and OC=O functional bonds.

Figure S5 shows the deconvoluted N 1s spectrum of the AgNPs, and the corresponding

binding energies are tabulated in Table S2. The deconvoluted peak at 399.2 eV for f-AgNPs

(except Ala-AgNP) is due to the C-N unit of the amino acids, signifying the interaction between

these N atoms and the f-AgNPs.66 The peak at 398.4 eV in AgNP and Ala-AgNP spectrum

suggest the presence of charged nitrogen atoms,67 inferring an electrostatic interaction with the

Ag0 surface. The peak at 402.4 eV for Trp-AgNP is attributed to N-Oxide, while peak at 400.1

eV for Met-AgNP is attributed to amide nitrogen.68 The deconvoluted N 1s spectrum of the

AgNPs further revealed that the area under the curve of the deconvoluted peaks is highest for

Asp-AgNP, while least for AgNP. Since, AgNP is capped by proteins and polysaccharides, while

the f-AgNPs are additionally functionalized with amino acids, the N content is thus least in

AgNP. Furthermore, among the amino acids used in this study, only Asp contains two –NH2

groups, hence the N content was highest in Asp-AgNP.

To validate the percentage of Ag0 and Ag2O content in each of the AgNPs as obtained

from the XPS analysis, UV-visible spectroscopic experiment was conducted. The genesis of this
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experiment is that: Ag2O (brown) dissolves in excess aqueous ammonia yielding a clear

transparent solution, represented by the equation:

Ag 2O( s )  4 NH 3  H 2O  2[ Ag ( NH 3 ) 2 ]OH(aq)

Inorganic Chemistry Frontiers Accepted Manuscript

For this, to a known volume of AgNPs aqueous dispersion (containing 1.68 mM Ag+ ions as

estimated from ICP-OES), an excess amount of aqueous ammonia (25% w/v) was added and
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stirred at 750 rpm for 30 min in dark. The AgNPs dispersion thus obtained, only contains Ag0 in

the dispersed phase, whereas all the Ag2O content of the AgNPs have been converted to

[Ag(NH3)3]OH (aq). The UV-visible absorbance spectra of the AgNPs dispersion before and

after addition of ammonia were recorded. In all the experiments, the overall volume of the

aqueous dispersion was kept constant. Since the absorbance of AgNPs dispersion (Ag0+Ag2O) at

their respective λmax is proportional to its concentration (known from ICP-OES analysis), AgNPs

having the highest Ag2O concentration will suffer the highest decrease in the absorbance on

ammonia addition. Thus, the absorbance recorded for the AgNPs aqueous dispersion after

addition of ammonia is only due to Ag0 left in the AgNPs dispersion. The change in absorbance

of the AgNPs dispersion before and after ammonia addition will be proportional to the Ag2O

concentration in the AgNPs dispersion. Figure S6 infers the absorbance spectra of the AgNPs

dispersion before and after addition of ammonia. Figure S6 elucidates that the λmax of the AgNPs

undergoes blue shift with decreased absorbance on ammonia addition, inferring decreased

particle size due to dissolution of Ag2O (s) crystallites into [Ag(NH3)3]OH (aq). The percentage

of Ag0 and Ag2O content present in the AgNPs as obtained from the absorbance spectra is

reported in Table S3. Table S3 infers that Trp-AgNP, Met-AgNP, and His-AgNP have > 78%,

while AgNP, Ala-AgNP, and Asp-AgNP have < 46% Ag0 content; which are in good agreement

with the XPS results.

The observations from FTIR inferred the participation of functional groups in NG in not

only the formation of Ag/Ag2O crystallites but also in retaining certain structure stabilizing
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groups, facilitated by amino acids. The binding of amino acids to the f-AgNPs surfaces were

further, confirmed using 1H NMR spectroscopy and are explained in the Supporting Information

(Figures S7-S14).

Inorganic Chemistry Frontiers Accepted Manuscript

3.3. Mechanism of AgNPs Formation

HR-TEM, XRD, FTIR and 1H NMR analysis depicted adequate information on the AgNPs
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formation mechanism. The hydroxyl and carboxylic groups in NG first facilitated the

complexation of Ag+ ions. Consequently, these Ag+ ions oxidize the hydroxyl groups to carbonyl

groups and were reduced to Ag(0). The dissolved oxygen also favored the oxidation of hydroxyl

groups to aldehydes and carboxylates, which in turn reduces more Ag+ ions to Ag(0). To begin

with, the Ag+ ions were trapped on the surface of proteins and polysaccharides present in the NG

through electrostatic interaction. The reducing agents present in NG such as flavonoids, reducing

sugars, and tannin then reduces the Ag+ ions (Scheme 1), resulting in nucleation of silver nuclei.

The oxidation product of these reducing agents were mainly acidic, confirmed from decreased

pH of the reaction mixture after AgNPs synthesis (see Scheme 1).69,70 In absence of amino acids,

the nucleation occurred over a longer period, resulting in uncontrolled growth of the AgNP,

confirmed from HRTEM (Figure 2). With amino acids, Ag+ ion forms dicoordinate (Ala) and

tricoordinate (Trp/Met/His/Glu/Asp) complexes. Shoeib et al optimized the structure of these

complexes and found the affinity of Ag+ ions for the amino acids as His > Met  Trp > Asp >

Glu > Ala.42 The optimized structure of amino acidAg+ complexes and Ag+ ion affinity are

illustrated in Scheme 2. The strong affinity of Ag+ ions for amino acids is the foundation for the

ordered nucleation of Ag nuclei, resulting in controlled growth of the f-AgNPs, confirmed from

HRTEM (Figures 2). The absence of amino acid functionalization in AgNP and the lower

affinity of Ag+ for Ala, Glu and Asp could have resulted in increased oxidation probability of Ag

nuclei undergoing Ostwald ripening, resulting high concentration of large sized Ag2O crystallites

in AgNP, Ala-AgNP, Glu-AgNP, and Asp-AgNP. The strong affinity of Ag+ ion for Trp, Met,
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and His resulted in decreased oxidation probability with low Ag2O crystallites concentration in

Trp-AgNP, Met-AgNP, and His-AgNP, well complimented with XRD results (Figure S3).

Furthermore, chances of Trp involving in Ag+-π interaction, additionally creates a compact and

Inorganic Chemistry Frontiers Accepted Manuscript

ordered atomic arrangement of Ag nuclei undergoing nucleation, resulting in smallest size Trp-

AgNP with highest Ag (111) and Ag (200) crystallites concentration (see Scheme 2, and Figure
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S2). Likewise, Ag+ ion having strongest affinity for His resulted in smaller sized NPs with high

Ag (111) and Ag (200) crystallites concentration (see Scheme 2, and Figure S2). Among the f-

AgNPs, the weakest Ag+ ion affinity for Ala resulted in highest Ag2O crystallite concentration in

Ala-AgNP, and in the formation of Ag (200) crystallites, which otherwise is absent in AgNP.

3.4. Influence of pH, Ionic Strength, Surfactants, and Dyes on AgNPs Colloidal Stability

FTIR and 1H NMR inferred that the amino acid supplemented NG matrices at the f-AgNPs

surfaces may significantly contribute to their colloidal stability in different environments. Since

catalytic activity, bioavailability, and toxicity of NPs depends on their colloidal stability, it is

therefore critical to study their stability in different aqueous environments. The stability of

AgNPs was therefore, investigated in different aqueous environment of variable pH, ionic

strength, surfactants, and dyes using UV-visible absorbance spectroscopy. The spectra were

analyzed for SPR peak shifting, SPR band broadening, and decrease in the absorption intensity.

Figure S15 shows the spectra of aqueous AgNPs at pH 2 to 14. The SPR band of

the AgNPs does not change in the pH 2–10 range, illustrating their good colloidal stability.

Figure S15 reveals that for Trp-AgNP, its SPR peak absorbance at pH 5 increases and for His-

AgNP, decreases at pH 2. Since the isoelectric point of His is 7.47, it seems that at pH 2, the -

COO group of His at His-AgNP surface is protonated, with a consequent reduction in

electrostatic repulsion. The formation of intermolecular H-bonding between protonated -COO

groups of surface-bound His molecules could also contribute to aggregation. The observed SPR

band broadening of AgNP and Ala-AgNP at pH > 10, and of Glu-AgNP and Asp-AgNP at pH >
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11 may be due to increased particle aggregation.71 For the AgNPs except Trp-AgNP, a

characteristic hyperchromic effect observed at pH 14 illustrates deprotonation of the surface-

bound -COOH group by OH ions, with increased repulsive electrostatic double-layer forces and

Inorganic Chemistry Frontiers Accepted Manuscript

stabilization.72

The colloidal stability of the AgNPs was further investigated by increasing ionic strength
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from 0.1 M to 2 M NaCl. Figure S16 illustrates the spectra of f-AgNPs at pH 7.0 with increasing

ionic strength. Except Met-AgNP, a blue shift in their respective SPR peak is observed at 0.1 M

NaCl. For AgNP and Ala-AgNP, the SPR peak shifts by 10 nm, while for Trp-AgNP, His-AgNP,

Glu-AgNP and Asp-AgNP; shifts by 5 nm. A further blue shift by 5 nm is observed for Trp-

AgNP at 1.5 M NaCl, and by 15 nm for Asp-AgNP at > 0.4 M NaCl. The decreased

agglomeration with increasing ionic strength may explain these blue shifts. The salt-induced

decreased agglomeration arises due to increased electrostatic repulsion between the negatively

charged nanoconjugates. Electrostatic repulsion is initiated by electrical double layer formation,

and for the AgNPs (except Met-AgNP), the Cl ions could have diffused into their electrical

double layer, with increased electrostatic repulsion. At 0.1 M NaCl, AgNP and Ala-AgNP

nanoconjugates exhibited the strongest electrostatic repulsion, depicted through 10 nm blue-shift.

For Trp-AgNP and Asp-AgNP, further diffusion of Cl- ions occurred at 1.5 M NaCl and > 0.4 M

NaCl, respectively. Asp-AgNP nanoconjugates with an overall 15 nm blue shift, inferred

strongest electrostatic repulsion with highest Cl- ion concentration in their electrical double layer.

For AgNP, Ala-AgNP, Glu-AgNP, and Asp-AgNP; the hyperchromic effect observed at [NaCl]

< 1 M, is due to their increased dispersibility, while the hypochromic effect observed at [NaCl] >

1 M infers formation of aggregates due to their surface charge neutralization by increasing Na+

ion population. For Met-AgNP, with upto 2 M NaCl, the observed hypochromic effect with no

SPR peak shifting infers that the negatively charged Met-AgNP electrical double layer is

neutralized with increasing electrolyte concentration, with Cl- ions having the least diffusion
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tendency. No considerable change in the absorbance of Trp-AgNP and His-AgNP dispersions

with increasing ionic strength confirms their high colloidal stability even at high ionic strength.

The AgNPs colloidal stability was further explored with cationic, anionic and

Inorganic Chemistry Frontiers Accepted Manuscript

nonionic surfactants. Figures S17 illustrate the UV-visible spectra of AgNPs with (0.0025 to

0.010) M aqueous surfactants. With increasing cationic surfactant (DTAB) concentration,

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significant changes in the AgNPs absorption maxima and peak broadening was observed. This

could be due to the surface charge neutralization by the cationic quaternary nitrogen of DTAB,

leading to aggregation with decreased colloidal stability. In aqueous nonionic surfactant (OGP),

SPR peak of the AgNPs showed no major peak broadening as compared to water, which may be

due to the absence of electrostatic interactions prevailing between the AgNPs surface and the

glucose head of OGP. Similarly, in aqueous anionic surfactants (SDS), the AgNPs illustrated

good colloidal stability, demonstrated through no major peak broadening. The existence of

repulsive Coulombic forces between the negatively charged AgNPs surface and the anionic

sulfate head may have resulted in their good colloidal stability. Figures S17 thus elucidates good

colloidal stability of Ala-AgNP in OGP; Trp-AgNP in OGP and SDS; Met-AgNP in OGP; His-

AgNP in OGP and SDS; Glu-AgNP in OGP; and Asp-AgNP in OGP. Figure S17 (inset)

illustrates the stability of AgNPs in aqueous surfactants, in terms of dispersion transparency.

With DTAB, the AgNPs dispersions are turbid, while transparent with SDS and OGP. With

increasing alkyl chain length and concentration of the cationic surfactant, the turbidity of AgNPs

dispersions increased, inferring the formation of larger aggregates with decreased colloidal

stability (image not reported).

The AgNPs colloidal stability was further investigated in aqueous cationic and

anionic dyes. The UV-visible spectra of each dye at 10 to 50 μM concentration and aqueous

AgNPs were taken separately. The two curves were combined mathematically, giving the

absorption spectra that would be expected if the AgNPs and dyes showed no interaction with
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each other. This combined spectrum is compared with the absorption observed for the AgNPs

with dyes. The comparison is shown in Figure S18. The observed spectra of Trp-AgNP, His-

AgNP, Glu-AgNP, and Asp-AgNP were similar to the mathematically combined absorption

Inorganic Chemistry Frontiers Accepted Manuscript

spectrum of solutions, illustrating their high stability in cationic dye (MB). Furthermore, AgNP,

Ala-AgNP, Trp-AgNP, Glu-AgNP, and Asp-AgNP were found to have good stability in anionic
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dye (CR). Possibly the dyes interact with the surface of the AgNPs via the –NH2, -COO and

OH groups. These groups may be engaged in H-bonding with the -COO and –NH2 groups

present over the AgNPs surface. There is an additional possibility of electrostatic interactions

between the -COO group present at the AgNPs surface and the delocalized positive charge

present over the aromatic rings in MB.73,74 These factors may have resulted in bringing the

AgNPs in the close vicinity of each other, leading to their agglomeration. This could have led to

enhanced retention of incident light by the larger agglomerates, with increased absorbance. The

extreme stability of Trp-AgNP with dyes may be due to the extreme hydrophobic surface

provided by the polymerization of Trp as predicted by 1H NMR (Figure S10), thereby restricting

the formation of hydrogen bonds.

3.5. Catalytic Activity of AgNPs on Methylene Blue (MB) Reduction

MB has the basic dye skeleton of the thiazine group, and in aqueous medium has an

absorption maxima at 665 nm (due to n–π* transition).75,76 Figure 6 illustrates the catalytic

activity of AgNPs on MB reduction by acidic SnCl2 in aqueous and AOT/SDS/DTAB micellar

mediums. Figure 6 infers that without AgNPs, MB reduction progressed, but with a very slow

rate. In aqueous and AOT/SDS/DTAB micellar mediums and in presence of 30 μM AgNPs, the

blue color of MB solution changes to green, and finally bleaches to colorless solution (except

with Trp-AgNP and His-AgNP), and the MB absorption maxima at 665 nm diminishes,

indicating reduction of MB to its reduced form Leuco MB (LMB). This confirmed the substantial

catalytic activity of the AgNPs on MB reduction in these mediums. With Trp-AgNP and His-
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AgNP, the green colored solution turns yellowish after complete MB reduction, depicting their

good stability in these reaction mediums. It is clear from Figure 6 that the reduction of MB

proceeds almost instantaneously, with more than 84% of MB reduced within the first 1 min in

Inorganic Chemistry Frontiers Accepted Manuscript

AOT and SDS micellar mediums. Comparatively, the MB reduction is slower in DTAB micellar

and aqueous mediums, with more than 63% and 57% of MB reduced, respectively, within the
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first 1 min. With the AgNPs, almost complete reduction of MB is achieved within 4 min in

micellar AOT and SDS, 5 min in micellar DTAB and 7 min in aqueous mediums. Trp-AgNP

exhibited the highest catalytic activity, with almost 100% MB being reduced within 40 sec in

AOT/SDS/DTAB micellar mediums.

In T-20 micellar medium and in absence of AgNPs, the intensity of MB absorption

maxima at 665 nm slowly increased with time (Figure 7), inferring incapability of acidic SnCl 2 to

reduce MB. Figure 7 shows the effect of AgNPs at different doses on the MB reduction by acidic

SnCl2. With AgNPs (except Trp-AgNP and His-AgNP), the MB reduction accelerates but

become reversible (i.e. re-appearance of blue color due to oxidation of LMB to MB) after 2-3

min at all AgNPs doses. Figure S19 illustrates the extent of MB reduction in T-20 micellar

medium after 3 min in presence of different AgNPs doses. After 3 min and at 30 μM AgNPs

concentration,  65% MB is reduced in presence of AgNP and Ala-AgNP, while 82, 87 and 80%

MB is reduced in presence of Met-AgNP, Glu-AgNP and Asp-AgNP, respectively. With Trp-

AgNP, a complete reduction is observed, while with His-AgNP, 92% reduction is observed. The

transition between blue and colorless solution and then from colorless to blue solution is a well-

known redox cycle called MB clock reaction. To check whether MB reduction was reversible in

presence of AgNPs in aqueous and AOT/SDS/DTAB micellar mediums, the MB reduction by

acidic SnCl2 was carried out with 20 μM AgNPs as catalyst and the progress of the reduction

reaction is illustrated in Figure S20. No backward oxidation of LMB to MB was thus observed,

depicted through decreased absorbance at 665 nm, and inferring irreversible MB reduction in
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these reaction mediums. Figure 8a illustrates the snapshots of MB + acidic SnCl2 in different

mediums, taken after 2 h of AgNPs addition. It strongly supports the fact that, except in micellar

T-20, no backward oxidation of LMB occurred in aqueous and AOT/SDS/DTAB miceller

Inorganic Chemistry Frontiers Accepted Manuscript

mediums, inferring good stability of LMB, which otherwise is unstable in aerated conditions.

Furthermore, if we shook these reaction mixtures, the solutions remained bleached. In all the MB
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solutions, AgNPs yellowish color diminished except for Trp-AgNP and His-AgNP, thus

exhibiting their good stability in such reaction mediums. There are only a few reports of a

reversible reaction involving MB in the acidic range.48,50 This work reports the first ever

demonstration of the MB clock reaction in acidic pH in the presence silver NPs and in nonionic

miceller medium. Since among the AgNPs in presence of which MB clock reaction was

observed, Glu-AgNP exhibited the highest MB reducing ability, it was chosen to represent the

MB clock reaction, showed in Figure 8b.

An interesting finding of this MB clock reaction is that, the reversible color alteration

goes on for one cycle irrespective to the catalyst concentration, which otherwise continues for

several cycles, as reported in the earlier studies.49,77 This phenomenon indicates the instability of

the AgNPs: AgNP, Ala-AgNP, Met-AgNP, Glu-AgNP and Asp-AgNP as catalyst in the reaction

medium as earlier depicted through their diminished yellowish color. This may be due to their

degradation due to exposure to a range of chemical species generated during the reduction

reaction. Such important phenomenon illustrating one redox cycle may find oxygen sensing

application. An example of this is illustrated in Figure 9, where two Whatman filter papers were

dipped in a 3 min old decolorized formulation of MB/SnCl2/T-20/Glu-AgNP (30 μM). The

Whatman filter paper kept inside a vacuum-sealed plastic package remained bleached, while the

one kept in open air regained its blue color within 90 min, confirming aerial oxidation.
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The AgNPs catalyzed MB reduction in aqueous and SDS/DTAB/AOT micellar mediums

followed a linear relationship observed for pseudo-second order kinetics (Figure 10), expressed

by the equation:

Inorganic Chemistry Frontiers Accepted Manuscript

1 1
  k2t
[C]t [C]0
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where [C]t is MB concentration at time t, [C]0 is initial concentration of MB. The k2 is slope of

the straight-line depicting second-order rate constant and are reported in Table 2. Moreover, Trp-

AgNP and His-AgNP catalyzed MB reduction in T-20 micellar medium followed pseudo-second

order kinetics and the k2 values are reported in Table 2. The k2 values established AOT micellar

medium as the most efficient medium for MB reduction, and Trp-AgNP with highest catalytic

efficiency. The order of efficient solvent medium for MB reduction revealed by k2 values is

AOT > SDS > DTAB > aqueous (Table 2). A possible explanation for pseudo-second order

kinetics is the formation of molecular associations of MB such as dimers or higher aggregates in

aqueous medium. AgNPs due to their negative surface charge must have resulted in strong

adsorption of cationic MB, thereby bringing them in close vicinity of each other. Thus, attractive,

noncovalent interactions between aromatic rings of the MB i.e. π–π stacking must have

developed, resulting in their increased extent of molecular association. Moreover, similar

behavior has been reported recently.75,78 Golz et al showed that MB does not follow the linear

relationship as predicted by Beer−Lambert law and concluded aggregation being the cause.79 This

characteristic behavior is common to many organic dyes of which aggregation was proposed to

be the reason.79

Until now, no study on the kinetics of the backward oxidation of LMB to MB is reported

because in the earlier studies, at the time of recording the backward reaction, the onset of

forward reaction simultaneously, created a problem. Figure 7 shows that in T-20 micellar

medium, from 3 minute onwards; the backward oxidation of LMB in presence of AgNPs (except
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Trp-AgNP and His-AgNP) followed a typical pattern, wherein the absorbance at 665 nm

increased linearly with time. The observation, thus elucidates that the AgNPs must have played a

crucial role in the backward oxidation of LMB to MB. For the backward oxidation of LMB to

Inorganic Chemistry Frontiers Accepted Manuscript

MB in presence of these AgNPs (20-30 μM), the plot of MB concentration vs time shows a

straight line with a positive slope (Figure 11), inferring zeroth-order reaction pathway, with the
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kinetics expressed by the equation:

[C]t  [C]0  k0t

where [C]t is MB concentration at time t, [C]0 is initial concentration of MB during onset of

LMB backward oxidation, and k0 denotes the zero-order rate constant. The k0 values reported

in Table 3 reveal that irrespective of the AgNPs concentration, k0 remains constant, and is

lowest for AgNP. Therefore, the rate of backwards oxidation LMB to MB is independent of the

AgNPs concentration, but depends on the nature of the AgNPs.

Figure S21 presents the absorbance spectra of the reaction mixture containing MB and

acidic SnCl2 in AOT and T-20 micellar mediums, recorded 20 min after addition of Trp-AgNP

and Glu-AgNP. The spectra observed with Trp-AgNP also represents that of His-AgNP, while

the spectra observed with Glu-AgNP also represents that of AgNP, Ala-AgNP, Met-AgNP and

Asp-AgNP because of similarity in Ag and Ag2O crystallite concentration. Figure S21 elucidates

that in AOT and T-20 micellar mediums; the SPR peak of Trp-AgNP suffers a blue shift with

decreased absorbance in presence of MB and acidic SnCl2, compared to its SPR peak in acidic

SnCl2. In AOT and T-20 micellar mediums, the SPR peak of Glu-AgNP disappears in presence

of MB and acidic SnCl2, compared to its SPR peak in acidic SnCl2. Furthermore, in AOT and T-

20 micellar mediums with Trp-AgNP, no peak is observed at 665 nm, which strongly infers the

complete reduction of MB to LMB with enhanced LMB stability. In AOT micellar medium with

Glu-AgNP, the absence of peak at 665 nm infers complete reduction of MB to LMB with
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enhanced LMB stability, while in T-20 micellar medium and with Glu-AgNP, the peak at 665

nm re-appears, indicating backward oxidation of LMB to MB.

3.5.1. Proposed Mechanism for MB redox reaction

Inorganic Chemistry Frontiers Accepted Manuscript

The pH of the aqueous and AOT/SDS/DTAB/T-20 micellar solutions containing MB, acidic

SnCl2 and AgNPs was  1.5, thus making the solutions acidic. In acidic medium, the standard
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reduction potential ( E 0 ) of Sn4+/Sn2+ is 0.15 V, whereas at pH ≈ 1.5, the E 0 value of MB/LMB

is 0.532 V.50 Thus in acidic medium, the redox reaction between Sn2+ and MB (depicted in eq. 1)

is thermodynamically favorable because the E 0 of the overall reaction is positive, thus making

the reaction spontaneous ( G0  nFE0  ve ).

Sn2  Sn4  2e E 0  0.15 V

MB  2e  LMB E 0  0.532 V
MB  Sn 2  LMB  Sn 4 E 0  0.382 V (1)

The reaction represented in eq. 1 does not happen on experimental time scale, may be due to the

low frequency of molecular collisions among the reactants. Micellar medium increases the

frequency of molecular collisions due to the close association of the reactants at the micellar

interface, while AgNPs helps in electron relay from donor to acceptor, thus catalyzing the MB

reduction by Sn2+. In acidic medium, oxidation of metallic Ag on the AgNPs surface by

dissolved O2 is thermodynamically favorable as E 0 of the reaction is positive, as depicted in eq.

2.

2  [2 Ag  2OH   Ag 2O  H 2O  2e  E 0  0.34 V
O2  4 H   4e  2 H 2O E 0  1.23 V
4 Ag  O2  2 Ag 2O E 0  0.55 V (2)

This elucidates the degradation of AgNPs in aqueous and AOT/SDS/DTAB micellar mediums,

thus explaining the disappearance of AgNPs yellowish color during MB reduction (Figure 8a).

Since among the AgNPs, Trp-AgNP and His-AgNP contains the highest Ag crystallites

concentration (Figure S2), high concentration of dissolved O2 is thus required to oxidize all the
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Ag crystallites, whereas low concentration of dissolved O2 is required in case of the other AgNPs

with much lower Ag crystallite concentration. Thus, with Trp-AgNP and His-AgNP in aqueous

and AOT/SDS/DTAB micellar mediums, dissolved O2 is completely utilized making the

Inorganic Chemistry Frontiers Accepted Manuscript

solutions O2 deficient, while with the other AgNPs, Ag crystallites are completely oxidized (eq.

2). This explains the persistence of yellowish color in solutions with Trp-AgNP and His-AgNP,
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while the disappearance of yellow color in the other AgNPs catalyzed MB reduction in aqueous

and AOT/SDS/DTAB/T-20 micellar mediums (Figure 8a). This important observation with Trp-

AgNP and His-AgNP may find major application in organic synthesis for generating O2 deficient

environment in acidic aqueous and micellar mediums, where catalyst poisoning by O2 prevails.

The same mechanism further explains the disappearance of the SPR peak of Glu-AgNP, while

persistence of SPR peak of Trp-AgNP as observed in Figure S21. The blue shift with decreased

absorbance observed for Trp-AgNP in Figure S21 is due to the decreased Ag crystallite

concentration due to their oxidation to Ag2O crystallites by dissolved O2 in acidic medium (eq.

2), followed by their dissolution.80 In acidic medium, the backward oxidation of LMB to MB,

depicted by eq. 3 is thermodynamically favorable due to the overall positive E 0 value.

2  [LMB  MB  2e  E 0  0.532 V
O2  4 H   4e   2 H 2O E 0  1.23 V
2 LMB  O2  4 H   2MB  2 H 2O E 0  0.166 V (3)

Among the solvent mediums studied in his work, T-20 micellar medium has strong affinity

towards aerobic O2.81-85 Thus, in comparison to aqueous and AOT/SDS/DTAB micellar media;

T-20 micellar medium contains higher concentration of dissolved O2. The reaction depicted in

eq. 3, thus becomes possible, confirmed by the generation of MB peak at 665 nm (Figure S21)

due to backward oxidation of LMB to MB. The reaction depicted in eq. 3 is not possible in the

other solvent mediums due to deficiency of dissolved O2 created by the reaction represented in

eq. 2, thus barring the backward oxidation of LMB to occur. The reason for not observing
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backward oxidation of LMB in presence of Trp-AgNP and His-AgNP in T-20 micellar medium

may be due to the high Ag crystallite concentration. The high Ag crystallite concentration in Trp-

AgNP and His-AgNP further engages the additional dissolved O2 incorporated in the reaction

Inorganic Chemistry Frontiers Accepted Manuscript

solution due to the affinity of T-20 for aerobic O2, as depicted by the oxidation reaction in eq. 2.

In T-20 micellar medium, in presence of the other AgNPs, their Ag crystallite concentration was
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not sufficient to utilize the overall dissolved O2. Therefore, dissolved O2 persisted in these

solutions, thus making the thermodynamically favorable backward oxidation of LMB possible,

as depicted in eq. 3. Since Ag2O is a widely used catalyst for aerobic oxidation reactions,86 it is

proposed here that with increased Ag2O concentration in the reaction solutions left after MB

reduction, they could have assisted the LMB oxidation to MB. The lowest k0 value observed for

AgNP may be due to the presence of large sized Ag2O crystallites in AgNP, among the AgNPs

(Table 3). The highest catalytic activity of Trp-AgNP followed by His-AgNP is therefore,

attributed to its highest Ag crystallite concentration. The highest negative zeta potential of Trp-

AgNP could have resulted in strong adsorption of MB, a cationic dye, over its surface. The

presence of π–π interaction between MB and indole group of Trp present over the Trp-AgNP

may have further facilitated MB adsorption. Furthermore, the highest content of Ag0 in Trp-

AgNP as shown by XPS and UV-visible spectroscopy results (Table S3) along with a bigger

active contact surface for MB due to its smaller size, must have contributed to its highest MB

reducing ability.

3.5.2. Proposed Mechanism for Micellar Medium Facilitated MB Reduction

To act as an efficient catalyst, the redox potential of the AgNPs should remain between the redox

potential of donor (Sn2+) and acceptor (MB). The AgNPs may then accept electrons from Sn2+

ions and transfer them to MB through a process called electron relay effect. 45 The MB reduction

by acidic SnCl2 in aqueous is not observed on the experimental time scale. It may be due to their

low encounter probability, wherein the reactants solvated nature generates a kinetic barrier.
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Surfactant micellar medium removes this barrier, thereby increasing the encounter probability. 45

MB has dye skeleton of the thiazine group, and exists as aggregates in water.79 Due to the

hydrophobic effect, MB has a tendency to form aggregates in aqueous solution so as to minimize

Inorganic Chemistry Frontiers Accepted Manuscript

the hydrophilic-hydrophobic interfacial area.79 MB is bound to anionic surfactant by both

electrostatic and hydrophobic interactions, and to cationic and nonionic surfactants, only by
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hydrophobic forces. The stronger force of attraction between anionic surfactants explains

qualitatively the faster MB reduction rate in AOT/SDS micelles. Anionic micelles ultimately

assist to increase the collision probability between MB and Sn2+ ions through their incorporation

in the anionic micellar Stern layer. Possibly, MB and Sn2+ ions align themselves around the

negatively charged SDS and AOT micellar surface, and forms a compact reaction zone. When

added to the anionic micellar medium, the negatively charged AgNPs align themselves around

the compact reaction zone in a MB-AgNPs-Sn2+ sequence. A compact electron relay is thus,

generated due to the large surface area of AgNPs, and electrons from Sn2+ could easily be

transferred to MB. The activation energy of reduction reaction is thus, considerably decreased,

making reduction fastest in AOT and SDS micellar mediums. In contrary, the positively charged

DTAB micellar surface disperse and scatter the MB and Sn2+ ions away from its surface due to

electrostatic repulsion, and thus not favoring a compact reaction zone. Since AgNPs aggregates

in aqueous cationic surfactant (Figures S17), once in DTAB micellar medium, they align around

the positively charged micellar surface, and aggregates with decreased surface area. Thus, a

compact electron relay is not generated, as in the case of AOT/SDS micellar mediums. The

AgNPs aggregation thus causes lower decrease in the activation energy of reduction reaction

with decreased reduction rate in DTAB micellar medium, compared to AOT/SDS micellar

mediums. In T-20 micellar media, the MB molecules may remain close to the micellar surface

due to hydrogen bonding and hydrophobic forces. When added to the T-20 micellar medium,

AgNPs align themselves around the uncharged micellar surface due to the electrostatic
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interaction between its negatively charged surface and cationic MB, and forms a weak MB-

AgNPs-Sn2+ sequence. A compact electron relay is thus not possible and hence, the reduction is

not as fast as in SDS/AOT/DTAB micellar mediums. The affinity of T-20 towards aerial

Inorganic Chemistry Frontiers Accepted Manuscript

oxidation may in-turn re-oxidize the reduced Leuco MB form, without completing the MB

reduction. A working model illustrating the sequence of MB, Sn2+ ions and AgNPs in aqueous
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and AOT, SDS, DTAB and T-20 micellar mediums is depicted in Scheme 3.

3.6. Catalytic Activity of AgNPs on Congo Red (CR) and 4-Nitrophenol (4-NP) Reduction

In aqueous, CR show absorption bands at 240 nm (nπ* transitions associated with di-

substituted benzene ring), 340 nm (nπ* transitions associated with naphthalene rings), and 500

nm (ππ* transitions associated with azo group).87 Figure 12 illustrates the catalytic activity of

AgNPs for reduction of CR by NaBH4. Without AgNPs, CR reduction progressed, but with a

very slow rate. In presence of AgNPs, the red color of CR bleaches to colorless solution (Figure

12), and absorption maxima at 500 nm diminished, indicating reduction of azo bonds, while

absorbance of 340 and 240 nm peaks decreased gradually, inferring reduction of naphthalene and

benzene derivatives. This confirmed the substantial catalytic activity of AgNP, Ala-AgNP, His-

AgNP, Glu-AgNP and Asp-AgNP on CR reduction. The AgNPs catalyzed CR reduction

followed a linear relationship observed for pseudo-first order kinetics (Figure 13). The

absorbance of CR is proportional to its concentration; the ratio of absorbance at time t (At) and

time t = 0 (A0) are equivalent to the ratio of CR concentration at time t (Ct) and time t = 0 (C0).

The rate constant ( k1 ) was determined from the linear plot of ln(Ct/C0) versus reduction time in

seconds, reported in Table 4. In order to compare the catalytic activity of the AgNPs with the

ones reported in the literature, the activity parameter   k1 / m , which is the ratio of pseudo-first

order rate constant ( k1 ) to the total mass of the catalyst addedwas used to compare the catalytic

efficiency of the AgNPs. The catalytic efficiency of AgNPs inferred by k1 values follows the
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trend AgNP > Asp-AgNP > Ala-AgNP > His-AgNP > Glu-AgNP > Trp-AgNP  Met-AgNP.

The highest Ag crystallite concentration in Trp-AgNP and aggregation behaviour observed for

Met-AgNP in aqueous CR may have played a crucial role on decreasing their catalytic activity.

Inorganic Chemistry Frontiers Accepted Manuscript

Since, amino acid functionalization decreases the Ag2O crystallite concentration in the f-AgNPs,

and AgNP, with highest Ag2O and lowest Ag crystallites content (Table S3) showing the highest
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catalytic efficiency; it seems that Ag2O crystallites are responsible for the catalytic activity on

CR reduction.

The catalytic activity of AgNPs for reduction of 4-NP by NaBH4 is shown in Figure 14.

Aqueous 4-NP shows an absorption maxima at 320 nm, but after NaBH4 addition, the peak

shifted to 400 nm with increased intensity due to the phenolate ion formation, which is fully

conjugated with benzene moiety.88 Without AgNPs, negligible change in the absorbance at 420

nm indicates ineffective reduction of 4-NP to 4-Aminophenol by NaBH4. In presence of the

AgNPs (except AgNP and Trp-AgNP), the yellow color of 4-nitrophenolate ion bleaches to

colorless solution, and absorption maxima at 400 nm diminished with appearance of a new peak

at 300 nm (Figure 14). This confirmed the reduction of 4-NP and formation of 4-Aminophenol.76

AgNP and Trp-AgNP were able to reduce 4-NP initially, but the reduction seized as the reaction

progressed (Figure 14). The AgNPs catalyzed 4-NP reduction followed a linear relationship

observed for pseudo-first order kinetics (Figure 15). The rate constant ( k1 ) was determined from

the linear plot of ln(Ct/C0) versus reduction time in seconds, reported in Table 4. In order to

compare the catalytic activity of the AgNPs with the ones reported in the literature, the  value

was used to compare the catalytic efficiency of the AgNPs. The catalytic efficiency of AgNPs

inferred by k1 values follows the trend Met-AgNP > Ala-AgNP > Asp-AgNP > Glu-AgNP >

His-AgNP. The lowest Ag crystallite concentration and unsymmetrical size of AgNP may have a

profound effect on limiting its catalytic activity, while possibility of Trp-AgNP surface poisoning

due to strong ππ interaction between Trp and 4-Aminophenol could have resulted in stalling its
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catalytic activity. The important observation on deactivation of catalytic activity of AgNP and

Trp-AgNP may be useful for such reduction reactions where improved selectivity (such as

Lindlar’s catalyst) is required.

Inorganic Chemistry Frontiers Accepted Manuscript

3.6.1. Proposed Mechanism for CR and 4-NP Reduction

In the catalytic reduction of CR and 4-NP, AgNPs due to their large surface area can act as an
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electron relay system. Since the redox potential of Ag cluster lies between that of BH4 (donor)

and CR/4-NP (acceptor), the electron relay effect involving AgNPs is thermodynamically

favorable. Just before the electron transfer reaction, CR/4-NP and BH4 ions are adsorbed on the

AgNPs surface. The catalytic reduction then proceeds by electron transfer from BH4, wherein

AgNPs accept electrons and conveys them to CR/4-NP, subsequently reducing them. NaBH4

ionizes in water to BH4−, and provides surface hydrogen for the reaction. The surface hydrogen

is first transferred to the AgNPs surface, which enables the reduction of CR/4-NP, as shown in

Scheme 4. Furthermore, as the reaction progresses, the H2 generated by NaBH4 can cause

convection of water, thus removing the reduction products away from the AgNPs surface,

consequently maintaining the high reactivity of the AgNPs.

3.8. Comparison with Literature

Owing to the uniqueness of the resources used in this work for the synthesis of the AgNPs, their

catalytic abilities may not be directly compared to those showed by other nanomaterials. An

example of this is the catalytic reduction of MB, which in this work followed a pseudo-second

order kinetics. Nevertheless, the AgNPs catalytic activity on reduction of MB, CR and 4-NP

presented in this work, when compared to the literature, are at par with those in the literature in

some cases and better in the other cases. For MB reduction, when compared to the literature,

either the amount of catalyst/reducing agent used is high or the reduction time or % MB

reduction is higher than with the AgNPs reported here. Furthermore, for CR and 4-NP reduction,

the  values with AgNPs reported here are much higher, when compared with literature. All the
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comparison parameters such as activity parameter, rate constant, amount of catalyst and reagents

used, % reduction and reduction time for the catalytic reduction of MB, CR and 4-NP are

summarized in Tables S4, S5, and S6; respectively. In this context, it is important to note that

Inorganic Chemistry Frontiers Accepted Manuscript

irrespective of the comparison, the results in this work are far better than those normally required

for the respective applications. This is because of the possibility for producing these cost-
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effective AgNPs on a large scale and their extreme colloidal stability in harsh aqueous

environment of variable pH, ionic strength, surfactants, and dyes.

The good catalytic activity of AgNPs is attributed to their high monodispersity and

stability in solution, their relatively smaller size and Ag/Ag2O nanocomposite polycrystallinity.

Moreover, the multiple functional groups present over the AgNPs surfaces due to amino acid

functionalization and various NG components provide a suitable environment for the

development of electrostatic interaction and H-bonding with the reagents, resulting in their

enhanced adsorption over their surface. Furthermore, AgNPs smaller size results in high surface-

to-volume ratio and more atoms on the surface as potential catalytic sites, thereby generating a

strong electron relay effect.

4. CONCLUSIONS

A green protocol is developed for the synthesis of amino acid functionalized AgNPs using Neem

gum as a multifunctional reagent. The XRD and XPS analysis of the AgNPs reveled them being

composed of Ag/Ag2O nanocrystallites. The f-AgNPs exhibited good colloidal stability against

high ionic strength, variable pH, surfactants and dyes solution. The catalytic efficiency of the

AgNPs was demonstrated for MB reduction by acidic SnCl2, and CR/4-NP reduction by NaBH4.

The order of most efficient solvent system for MB reduction was AOT > SDS > DTAB >

aqueous, and 100% MB reduction was achieved within 40 sec with Trp-AgNP in all solvent

systems. The catalytic efficiency of AgNPs on CR and 4-NP reduction by NaBH4 followed the

trend (AgNP > Asp-AgNP > Ala-AgNP > His-AgNP > Glu-AgNP > Trp-AgNP  Met-AgNP)
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and (Met-AgNP > Ala-AgNP > Asp-AgNP > Glu-AgNP > His-AgNP), respectively. Here, the

AgNPs-catalyzed clock reaction has been demonstrated using MB and acidic SnCl 2 in T-20

micellar medium. To the best of our knowledge, it is the first attempt to demonstrate a one-cycle

Inorganic Chemistry Frontiers Accepted Manuscript

redox MB clock reaction using silver NPs in acidic nonionic micellar medium. The clock

reaction suggest potential use of the AgNPs as low cost sensors for oxygen leakage in vacuum-
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packed food packages, and for creating oxygen deficient environment in acidic aqueous and

micellar mediums, where catalyst poisoning by O2 prevails. The deactivation of catalytic activity

observed for Trp-AgNP may find application in such catalytic reduction, where improved

selectivity is required.

ACKNOWLEDGMENTS

Man Singh gratefully acknowledges Defence Research and Development Organization (DRDO),

Government of India, (Research project No. ERIP/ER/1203062/M/01/1586), for financial

assistance. Abhishek Chandra thanks DRDO for providing financial assistance in the form of

Senior Research Fellowship (SRF) and Gajendra Inwati (SNS, CUG) for his timely assistance

during XPS analysis. The authors are thankful to the Central University of Gujarat, Gandhinagar

for providing infrastructure and instrumentation facilities.

ASSOCIATED CONTENT
Supporting Information

Notes: The authors declare no competing financial interest.

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REFERENCES

[1] P. Raveendran, J. Fu and S.L. Wallen, J. Am. Chem. Soc., 2003, 125, 13940-13941.

Inorganic Chemistry Frontiers Accepted Manuscript

[2] S. Narayanan, B.N. Sathy, U. Mony, M. Koyakutty, S.V. Nair and D. Menon, ACS Appl.

Mater. Interfaces, 2012, 4, 251−260.

[3] E.C. Njagi, H. Huang, L. Stafford, H. Genuino, H,M. Galindo, J.B. Collins, G.E. Hoag
Published on 28 November 2017. Downloaded by Fudan University on 29/11/2017 08:36:27.

and S.L. Suib, Langmuir, 2011, 27(1), 264–271.

[4] R. Varma, Green Chem., 2014, 16, 2027–2041.

[5] J. Virkutyte and R.S. Varma, ACS Symposium Series, 2013, 1124, 11–39.

[6] S.K. Das, E. Marsili and M. Rahman, Nanomaterials 2011, 253–278.

[7] A.T. Harris and R. Bali, J. Nanopart. Res., 2008, 10, 691–695.

[8] S. Ghosh, S. Patil, M. Ahire, R. Kitture, D.D. Gurav, A.M. Jabgunde, S. Kale, K. Pardesi,

V. Shinde, J. Bellare, D.D. Dhavale and B.A. Chopade, J. Nanobiotechnology, 2012,

10:17.

[9] M. Khan, S.F. Adil, M.N. Tahir, W. Tremel, H.Z. Alkhathlan, A. Al-Warthan and

M.R.H. Siddiqui, Int. J. Nanomedicine, 2013, 8(1), 1507–1516.

[10] M. Rai and A. Yadav, IET Nanobiotechnol., 2013, 7(3), 117–124.

[11] V.V. Makarov, A.J. Love, O.V. Sinitsyna, S.S. Makarova, I.V. Yaminsky, M.E.

Taliansky and N.O. Kalinina, Acta Naturae, 2014, 6(1), 35–44.

[12] S.S. Shankar, A. Rai, A. Ahmad and M. Sastry, J. Colloid Interface Sci., 2004, 275, 496–

502.

[13] Z. Khan, O. Bashir, J.I. Hussain, S. Kumar and E. Ahmad, Colloids Surf., B. 2012, 98,

85– 90.

[14] C. Dipankar and S. Murugan, Colloids Surf., B. 2012, 98, 112– 119.

[15] P. Kumar, M. Govindaraju, S. Senthamilselvi and K. Premkumar, Colloids Surf., B.,

2013, 103, 658– 661.

 Inorganic Chemistry Frontiers Page 36 of 54
View Article Online
DOI: 10.1039/C7QI00569E

[16] S.A. Kumar, S. Ravi, V. Kathiravan and S. Velmurugan, Spectrochim. Acta Mol. Biomol.

Spectrosc., 2014, 121, 88–93.

[17] S. Li, S.; Y. Shen, A. Xie, X. Yu, L. Qiu, L. Zhang and Q. Zhang, Green Chem., 2007, 9,

Inorganic Chemistry Frontiers Accepted Manuscript

852–858.

[18] G.A. Kahrilas, L.M. Wally, S.J. Fredrick, M. Hiskey, A.L. Prieto and J.E. Owens, ACS
Published on 28 November 2017. Downloaded by Fudan University on 29/11/2017 08:36:27.

Sustainable Chem. Eng., 2014, 2, 367−376.

[19] A.A. Kulkarni and B.M. Bhanage, ACS Sustainable Chem. Eng. 2014, 2, 1007−1013.

[20] M.N. Nadagouda, N. Iyanna, J. Lalley, C. Han, D.D. Dionysiou and R.S. Varma, ACS

Sustainable Chem. Eng., 2014, 2, 1717−1723.

[21] J. Puiso, D. Jonkuviene, I. Macioniene, J. Salomskiene, I. Jasutiene and R. Kondrotas,

Colloids Surf., B., 2014, 121, 214–221.

[22] P. Velusamy, J. Das, R. Pachaiappan, B. Vaseeharan and K. Pandian, Ind. Crops Prod.,

2015, 66, 103–109.

[23] A.J. Kora, S.R. Beedu and A. Jayaraman, Org. Med. Chem. Lett., 2012, 2:17.

[24] A.J. Kora, R.B. Sashidhar and J. Arunachalam, J. Carbohydr. Polym., 2010, 82, 670–679.

[25] A.J. Kora, R.B. Sashidhar and J. Arunachalam, Process Biochem., 2012, 47, 1516–1520.

[26] M. Thakur, S. Pandey, A. Mewada, R. Shah, G. Oza and M. Sharon, Spectrochim. Acta

Mol. Biomol. Spectrosc., 2013, 109, 344–347.

[27] I.M. Krishna, G.B. Reddy, G. Veerabhadram and A. Madhusudhan, Appl Nanosci., 2016,

6, 681–689.

[28] V.K. Vidhu and D. Philip, Spectrochim. Acta Mol. Biomol. Spectrosc., 2014, 117, 102–

108.

[29] V.K. Vidhu and D. Philip, Micron, 2014, 56, 54–62.

[30] H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S. Pyne and A. Misra, Colloids Surf., A, 2009,

348, 212–216.
 Page 37 of 54 Inorganic Chemistry Frontiers
View Article Online
DOI: 10.1039/C7QI00569E

[31] Y. He, F. Wei, Z. Ma, H. Zhang, Q. Yang, B. Yao, Z. Huang, J. Li, C. Zeng and Q.

Zhang, RSC Adv., 2017, 7, 39842-39851.

[32] G.A. Kahrilas, W. Haggren, R.L. Read, L.M. Wally, S.J. Fredrick, M. Hiskey, A.L.

Inorganic Chemistry Frontiers Accepted Manuscript

Prieto and J.E. Owens, ACS Sustainable Chem. Eng., 2014, 2, 590−598.

[33] X. Li, J.J. Lenhart and H.W. Walker, Langmuir, 2012, 28, 1095−1104.
Published on 28 November 2017. Downloaded by Fudan University on 29/11/2017 08:36:27.

[34] H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De and A. Misra, Colloids Surf., A, 2009,

339, 134–139.

[35] M.N. Nadagouda and R.S. Varma, Green Chem., 2008, 10, 859–862.

[36] A.A. AbdelHamid, M.A. Al-Ghobashy, M. Fawzy, M.B. Mohamed and M.M.S.A.

Abdel-Mottaleb, ACS Sustainable Chem. Eng., 2013, 1, 1520−1529.

[37] R.W. Soukup and K. Soukup, Switzerland; A.D. Kinghorn, H. Falk, J. Kobayashi (Eds.);

Springer-Verlag Wien Publishers, 1999; 453-588.

[38] S. Mukherjee and H. Srivastava, J. Am. Chem. Soc., 1955, 77(2), 422–423.

[39] M. Allman, M. Pena and D. Peng, Eur. J. Clin. Nutr., 1995, 49, 169-178.

[40] D. Anderson and A. Hendrie, Juss. Carbohyd. Res., 1971, 20, 259-268.

[41] T. Brindha and J. Mallika, Juss. Int. J. Pharm. Chem., 2015, 5(6), 227-231.

[42] T. Shoeib, K.W.M. Siu and A.C. Hopkinson, J. Phys. Chem. A, 2002, 106, 6121-6128.

[43] T. Shoeib and B.L. Sharp, Inorg. Chim. Acta, 2009, 362, 1925–1934.

[44] J.A. Jacob, S. Naumov, T. Mukherjee and S. Kapoor, Colloids Surf., B, 2011, 87, 498–

504.

[45] S.K. Ghosh, S. Kundu, M. Mandal and T. Pal, Langmuir, 2002, 18, 8756-8760.

[46] T. Pal, S. De, N.R. Jana, N. Pradhan, R. Mandal and A. Pal, Langmuir, 1998, 14, 4724-

4730.

[47] Z.J. Jiang, C.Y. Liu and L.W. Sun, J. Phys. Chem. B, 2005, 109, 1730-1735.

[48] T. Snehalatha, K.C. Rajanna and P.K. Saiprakash, J. Chem. Educ., 1997, 74(2), 228-233.
 Inorganic Chemistry Frontiers Page 38 of 54
View Article Online
DOI: 10.1039/C7QI00569E

[49] C. Ray, S. Dutta, S. Sarkar, R. Sahoo, A. Roy and T. Pal, RSC Adv., 2013, 3, 24313–

24320

[50] M. Basu, A.K. Sinha, M. Pradhan, S. Sarkar, A. Pal, C. Mondal and T. Pal, J. Phys.

Inorganic Chemistry Frontiers Accepted Manuscript

Chem. C, 2012, 116, 25741−25747.

[51] W. Wei, X. Mao, L. Ortiz and D. Sadoway, J. Mater. Chem., 2011, 21, 432–438.
Published on 28 November 2017. Downloaded by Fudan University on 29/11/2017 08:36:27.

[52] L. Xu, B. Wei, W. Liu, H. Zhang, C. Su and J. Che, Nanoscale Res. Lett., 2013, 8:536.

[53] S. Küünal, S. Kutti, P. Rauwel, M. Guha, D. Wragg and E. Rauwel, Nano Lett., 2016,

6(3), 191-197.

[54] E.E. Elemike, D.C. Onwudiwe, A.C. Ekennia, C.U. Sonde and R.C. Ehiri, Molecules,

2017, 22, 674.

[55] S. Ravichandran, V. Paluri, G. Kumar, K. Loganathan and B.R.K. Venkata, J. Exp.

Nanosci., 2016, 11(6), 445-458.

[56] V. Gopinath, D. MubarakAli, S. Priyadarshini, M. Priyadharshini, N. Thajuddin and P.

Velusamy, Colloids Surf., B 2012, 96, 69–74.

[57] F. Korkmaz, D.A. Erdogan and S.O. Yaman, New J. Chem., 2015, 39, 5676-5685.

[58] P. Shashi, L. Manu and S. Mika, Colloid Surf., A, 2010, 364, 34–41.

[59] S. Agnihotri, S. Mukherji and S. Mukherji, Nanoscale, 2013, 5, 7328–7340.

[60] Q. Chen, W. Shi, Y. Xu, D. Wu and Y. Sun, Mater. Chem.Phys., 2011, 125, 825-832.

[61] X. Yang, L. Xu, X. Yu and Y. Guo, Catal. Commun., 2008, 9, 1224.

[62] Y. Chi, L. Zhao, Q. Yuan, X. Yan, Y. Li, N. Li and X. Li, J. Mater. Chem., 2012, 22,

13571- 13577.

[63] S. Agnihotri, G. Bajaj, S. Mukherji and S. Mukherji, Nanoscale, 2015, 7, 7415–7429.

[64] T.C. Kaspar, T. Droubay, S.A. Chambers and P.S. Bagus, J. Phys. Chem. C, 2010, 114,

21562–21571.

[65] A.A. Taha, Adv. Nat. Sci.: Nanosci. Nanotechnol., 2015, 6, 045003.
 Page 39 of 54 Inorganic Chemistry Frontiers
View Article Online
DOI: 10.1039/C7QI00569E

[66] H. J. Kim, I.-S. Bae, S.-J. Cho, J.-H. Boo, B.-C. Lee, J. Heo, I. Chung and B. Hong,

Nanoscale Res. Lett., 2012, 7(1), 30.

[67] Z. Zhang, X. Zhang, Z. Xin, M. Deng, Y. Wen and Y. Song, Nanotechnology, 2011, 22,

Inorganic Chemistry Frontiers Accepted Manuscript

425601.

[68] E. Mazzotta, S. Rella, A. Turco and C. Malitesta, RSC Adv., 2015, 5, 83164-83186.
Published on 28 November 2017. Downloaded by Fudan University on 29/11/2017 08:36:27.

[69] M.A. Armani, A. Abu-Taleb, N. Remalli, M. Abdullah, V.V.S.S. Srikanth, RSC Adv.,

2016, 6, 44145-44153.

[70] N. Durán, P.D. Marcato, M. Durán, A. Yadav, A. Gade, and A. Rai, Appl Microbiol

Biotechnol., 2011, 90, 1609–1624.

[71] T.C. Prathna, N. Chandrasekaran and A. Mukherjee, Colloids Surf., A, 2011, 390, 216–

224.

[72] J. Hierrezuelo, A. Sadeghpour, I. Szilagyi, A. Vaccaro and M. Borkovec, Langmuir,

2010, 26(19), 15109–15111.

[73] D.V. Leff, L. Brandt and J.R. Heath, Langmuir, 1996, 12, 4723–4730.

[74] S.L. Westcott, S.J. Oldenburg, T.R. Lee and N.J. Halas, Langmuir, 1998, 14, 5396–5401.

[75] M.A. Rauf, M.A. Meetani, A. Khaleel and A. Ahmed, Chem. Eng. J., 2010, 157(2), 373–

378.

[76] R. Laoufi, J. Lazzari, O. Jupille, S. Robach, G. Garaud, P. Cabaih, H. Dolle, A.J. Crugel

and A. Baillly, J. Phys. Chem. C, 2011, 115(11), 4673–4679.

[77] S. Pande, S. Jana, S. Basu, A.K. Sinha, A. Datta and T. Pal, J. Phys. Chem. C, 2008, 112,

3619-3626.

[78] M. Hamity, R.H. Lema, C.A. Suchetti and H.E. Gsponer, J. Photochem. Photobiol. A:

Chem., 2008, 200, 445–450.

[79] E.K. Golz and D.A.V. Griend, Anal. Chem., 2013, 85, 1240−1246.
 Inorganic Chemistry Frontiers Page 40 of 54
View Article Online
DOI: 10.1039/C7QI00569E

[80] S. Eckhardt, P.S. Brunetto, J. Gagnon, M. Priebe, B. Giese and K.M. Fromm, Chem. Rev.,

2013, 113, 4708−4754.

[81] D. Leštan, M. Leštan and A. Perdih, Appl. Environ. Microbiol., 1994, 60(2), 606-612.

Inorganic Chemistry Frontiers Accepted Manuscript

[82] K. Miyashita, N. Inukai and T. Ota, Biosci. Biotech. Biochem., 1997, 61(4), 716-717.

[83] K.S. Rhee, J. Food Sci., 1978, 43, 1-9.

Published on 28 November 2017. Downloaded by Fudan University on 29/11/2017 08:36:27.

[84] Y. Galagan, W.F. Sua, J. Photochem. Photobiol., A, 2008, 195, 378–383.

[85] A. Mills, Chem. Soc. Rev., 2005, 34, 1003–1011.

[86] P. Das, S. Ray, A. Bhaumik, B. Banerjee and C. Mukhopadhyay, RSC Adv., 2015, 5,

6323-6331.

[87] L.G. Devi, S.G. Kumar and K.M. Reddy, Cent. Eur. J. Chem. 2009, 7(3), 468-477.

[88] W. Li, X. Ge, H. Zhang, Q. Ding, H. Ding, Y. Zhang, G. Wang, H, Zhang and H Zhao,

Inorg. Chem. Front., 2016, 3, 663-670.

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Table 1. Particle Size Range, Average Particle Diameter, Polydispersity Index (PDI), and Zeta
Potential Observed from HR-TEM and DLS Analysis for the AgNPs: AgNP, Ala-AgNP, Trp-
AgNP, Met-AgNP, His-AgNP, Glu-AgNP and Asp-AgNP

Size Range in Average PDI Zeta

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AgNPs nm Diameter in Potential
(HR-TEM) nm (mV)
(DLS)
AgNP 5-50 39±24 0.602±0.078 -20.68±1.01
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Ala-AgNP 5-40 40±7 0.176±0.025 -40.98±1.56

Trp-AgNP 5-20 14±3 0.215±0.029 -80.84±2.73
Met-AgNP 15-35 34±9 0.268±0.035 -53.93±2.15
His-AgNP 5-20 25±8 0.333±0.041 -72.22±2.12
Glu-AgNP 20-30 30±8 0.283±0.039 -63.85±2.29
Asp-AgNP 10-40 38±12 0.499±0.067 -47.50±1.89

Table 2. Pseudo-Second Order Rate Constant ( K 2 /103 M-1∙Sec-1) for Reduction of MB by Sncl2
in 0.01 M Aqueous SDS/DTAB/AOT Mediums Catalyzed by AgNPs: AgNP, Ala-AgNP, Trp-
AgNP, Met-AgNP, His-AgNP, Glu-AgNP and Asp-AgNP

AgNPs K 2 (AOT) K 2 (SDS) K 2 (DTAB) K 2 (T-20) K 2 (Aqueous)

AgNP 1.90 1.78 1.53 - 1.34
Ala-AgNP 1.60 1.57 1.27 - 0.91
Trp-AgNP 28.10 23.98 14.53 12.26 11.20
Met-AgNP 9.05 2.83 1.66 - 1.21
His-AgNP 12.03 8.30 4.38 1.93 1.56
Glu-AgNP 3.41 1.90 1.56 - 1.19
Asp-AgNP 3.56 3.08 1.77 - 0.49

Table 3. Zero-Order Rate Constant ( K 0 /10-9 Mol∙L-1∙Sec-1) for Reversible Oxidation of LMB in
0.01 M Aqueous T-20, Catalyzed by AgNPs: AgNP, Ala-AgNP, Met-AgNP, Glu-AgNP and
Asp-AgNP

AgNPs 20 μM 25 μM 30 μM
AgNP 2.7 2.8 3.0
Ala-AgNP 6.1 5.8 5.7
Met-AgNP 4.7 4.8 5.0
Glu-AgNP 5.3 4.9 5.0
Asp-AgNP 4.0 3.8 3.7
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Table 4. Pseudo-First Order Rate Constant ( K1 /Min-1) for CR and 4-NP Reduction by Nabh4 in
Aqueous Medium Catalyzed by AgNPs: AgNP, Ala-AgNP, Trp-AgNP, Met-AgNP, His-AgNP,
Glu-AgNP and Asp-AgNP

K1 (CR) K1 (4-NP)

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AgNPs
AgNP 0.2486 N/A
Ala-AgNP 0.1486 0.0729
Trp-AgNP 0.0152 N/A
Met-AgNP 0.0146 0.0741
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His-AgNP 0.1222 0.0169

Glu-AgNP 0.1162 0.0397
Asp-AgNP 0.2210 0.0541
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Figure 1. (a) Color change of reaction mixture during biosynthesis of AgNP, (b) optimization
plot for NG concentration, (c) optimization plot for AgNO3 concentration, and (d) optimization
plot for reaction time for the biosynthesis of AgNP

Figure 2. HR-TEM micrographs of (a) AgNP (b) Ala-AgNP (c) Trp-AgNP (d) Met-AgNP (e)
His-AgNP (f) Glu-AgNP (g) Asp-AgNP (In Fig.2 scale bar is not clear enough)
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Figure 3. XRD pattern of (a) AgNP (b) Ala-AgNP (c) Trp-AgNP (d) Met-AgNP (e) His-AgNP
(f) Glu-AgNP (g) Asp-AgNP
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Figure 4. FTIR spectra of NG and AgNPs: (a) AgNP (b) Ala-AgNP (c) Trp-AgNP (d) Met-
AgNP (e) His-AgNP (f) Glu-AgNP (g) Asp-AgNP

Figure 5. XPS spectral analysis of (a) AgNP (b) Ala-AgNP (c) Trp-AgNP (d) Met-AgNP (e)
His-AgNP (f) Glu-AgNP and (g) Asp-AgNP showing full scan
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Scheme 1. Proposed mechanism for chelation and reduction of silver ions by flavonoid, glucose,
gluconic acid, and tannin during AgNPs biosynthesis with NG. Inset: pH of reaction mixture
before and after biosynthesis of AgNPs
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Scheme 2. Schematic representation of formation of AgNP and f-AgNPs using NG, optimized
structures of amino acidAg+ complexes with NG, binding affinity of Ag+ ion with amino acids
and their co-relation with crystallite planes observed from XRD analysis of the AgNPs
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Figure 6. Plot of the absorbance at λmax (665 nm) of MB versus time with AgNPs in (a) in 0.01
M aqueous AOT (b) in 0.01 M aqueous SDS (c) in 0.01 M aqueous DTAB (d) in aqueous

Figure 7. Plot of absorbance at λmax (665 nm) of MB vs time with variable amount of AgNPs: (a)
AgNP (b) Ala-AgNP (c) Trp-AgNP (d) Met-AgNP (e) His-AgNP (f) Glu-AgNP (g) Asp-AgNP
in 0.01 M aqueous T-20
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Figure 8. (a) Snapshots of MB + acidic SnCl2 in aqueous and AOT/SDS/DTAB/T-20 micellar

mediums, taken after 2 h of 30 μM AgNPs addition; (b) Schematic representation of the MB
clock reaction involving MB, acidic SnCl2 and 30 μM Glu-AgNP in T-20 micellar medium

Figure 9. Photographs of Whatman filter paper dipped in a formulation: MB/SnCl 2/T-20/Glu-

AgNP (30 μM), (a) placed in open air (b) inside a vacuum sealed airtight plastic package
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Figure 10. The plot of [(1/Ct)-(1/C0)] versus time for the reduction of MB by acidic SnCl2 (a) in
0.01 M aqueous AOT (b) in 0.01 M aqueous SDS (c) in 0.01 M aqueous DTAB (d) aqueous (e)
in 0.01 M aqueous T-20; catalyzed by AgNPs: AgNP, Ala-AgNP, Trp-AgNP, Met-AgNP, His-
AgNP, Glu-AgNP, and Asp-AgNP

Figure 11. The plot of MB concentration versus time for the backward oxidation of LMB to MB
in AOT micellar medium; catalyzed by 30 μM AgNPs: AgNP, Ala-AgNP, Met-AgNP, Glu-
AgNP, and Asp-AgNP
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Scheme 3. Schematic arrangement of MB, Sn2+ ions and AgNPs during catalytic reduction of
MB by SnCl2 as reductant in aqueous and aqueous micellar mediums of SDS, AOT, DTAB and
T-20
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Figure 12. (A) Time dependent UV–visible absorption spectra for the catalytic reduction of CR
by NaBH4 (a) in absence of AgNPs and in presence of 30 μM AgNPs: (b) AgNP (c) Ala-AgNP
(d) Trp-AgNP (e) Met-AgNP (f) His-AgNP (g) Glu-AgNP and (h) Asp-AgNP

Figure 13. (B) The plot of ln(C/C0) versus time for the reduction of CR, catalyzed by (a) AgNP
(b) Ala-AgNP (c) Trp-AgNP (d) Met-AgNP (e) His-AgNPs (f) Glu-AgNPs (g) Asp-AgNPs
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Figure 14. (A) Time-dependent UV–visible spectra for the catalytic reduction of 4-Nitrophenol
by NaBH4 (a) in absence of AgNPs and in presence of 30 μM AgNPs: (b) AgNP (c) Ala-AgNP
(d) Trp-AgNP (e) Met-AgNP (f) His-AgNP (g) Glu-AgNP and (h) Asp-AgNP
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Figure 15. (B) The plot of ln(C/C0) versus time for the reduction of 4-NP catalyzed by (a) Ala-
AgNP (b) Met-AgNP (c) His-AgNP (d) Glu-AgNP (e) Asp-AgNPs

Scheme 4. Reaction mechanism for the AgNPs catalyzed reduction of 4-NP and CR by NaBH4