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Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology A:Chemistry

jo ur nal homep age: www.elsev ier .com/ locate / jphotochem

ynthesis of Ag nanoparticles in Span/Span-Tween mixed surfactantystem and its optical, kinetic and fluorimetric studies

ousumi Mukherjee, Ambikesh Mahapatra ∗

epartment of Chemistry, Jadavpur University, Kolkata 700 032, India

r t i c l e i n f o

rticle history:eceived 25 April 2014eceived in revised form 19 June 2014ccepted 23 July 2014vailable online 7 August 2014

eywords:pan-Tween surfactant

a b s t r a c t

Span and Span-Tween mixed surfactants have been used to stabilize the silver nanoparticles synthe-sized chemically and photochemically. This synthetic process of silver sol preparation has the advantageof producing homogeneous particles of very small size (∼1.65 nm) and finally aggregate brilliantly in asymmetric manner to form cubic clusters. Electromagnetic interaction in the close-packed assembly ofsilver aggregates generates its optical property that has been explained in the light of Maxwell-Garnetteffective medium theory. A small sized silver nanoparticle has quenching properties on the fluorophoreprobes of methylene blue and cresol red. The particle size dependent fluorescence quenching property has

urface plasmonggregationSAbinding constant

been rationalized considering its surface area. Steady state circular dichroism and time resolved spectro-scopic studies of Ag nanoparticles with bovine serum albumin (BSA) and human adult hemoglobin (Hb)proves the formation of a ground state BSA-Ag complex. The effect of different sizes of silver nanoparti-cle on the rate of electron transfer reaction between [CoIII(NH3)5Br](NO3)2 and Mohr’s salt (FeII) has alsobeen explained rationally.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Electrons move about freely in metal nanoclusters for theirlose-lying conduction and valence bands. The free movement oflectrons gives rise to a surface plasmon absorption band whichepends on both the cluster size and chemical environment ofurroundings. Thus the colors of the sols vary with the methodf preparation and state of aggregation. The net effect of devia-ion from the spherical shape is splitting of the dipole resonancento two absorption bands, in which the induced dipole oscillateslong and transverse to the spheroid axis. In the longitudinal res-nance, the band increases in cross-section and shifts to longeravelengths. In the transverse resonance, the band remains more

r less intact. The change-over from spherical to ellipsoidal shapeesults the shifting of absorption band in the UV–vis range. Theppearance of a new peak at a longer wavelength region is oftenscribed to the formation of loosely packed aggregates. The later

re formed due to dipole interaction between neighboring par-icles. The optical properties of Ag sols depend not only on theize and shape of the particles but also on the change in electron

∗ Corresponding author. Tel.: +91 33 2457 2770/33 2432 4586;ax: +91 33 2414 6223.

E-mail addresses: ambikeshju@gmail.com, amahapatra@chemistry.jdvu.ac.inA. Mahapatra).

ttp://dx.doi.org/10.1016/j.jphotochem.2014.07.020010-6030/© 2014 Elsevier B.V. All rights reserved.

density on their surface [1]. Chemical reduction methods wereused to synthesize silver nanoparticles, stabilized in polymers hasalready been reported [2–4]. The present study reports on the pho-tochemical reduction of Ag(I) to prepare silver sols. Syntheses ofsilver nanoparticles have also been carried out by chemical reduc-tion of Ag(I) where Span and Tween surfactant molecules act asstabilizer through template/capping agent.

The electron transfer step is important in many homogeneousand heterogeneous reactions [5,6]. Passage of an electron betweendonor and acceptor with large redox potential difference in theelectron transfer step may be restricted. An effective catalyst withdonor–acceptor partner acts as an electron relay system and helpselectron transfer. Metal or metal particles are well-known exam-ples of this type of redox catalyst. Recent studies suggest that whenmetal particles become smaller in size then their redox propertiesdiffer from the bulk metal. Their redox potential changes in thepresence of adsorbed foreign ions [7,8]. Thus the role of the metalparticle as an electron transfer catalyst is expected to vary withparticle size. We present here kinetic investigations to elucidatethe kinetics and mechanism of the reduction of pentamminebro-mocobalt(III) nitrate, [Co(NH3)5Br](NO3)2 by the reducing agentMohr’s salt, FeII with catalytic effect of silver nanoparticle. This

reaction is believed to proceed through an inner sphere mechanism.

In recent years, the size-selective synthesis of metallic nano-clusters has stimulated researchers because the size provides animportant control over many physical and chemical properties of

2 M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13

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cheme 1. (a) Structure of Bovine Serum Albumin with Tryptophan residues (greend green is iron-containing heme groups). (For interpretation of the references to

ano materials [9,10]. The unique physical and chemical proper-ies of the surfaces of solids are attracting attention in both basiccience and technology [11]. The technological uses of surfacerocesses are numerous; the most important ones are industrialatalytic uses and fabrication of the revolutionary new generationf microelectronic devices. Basic surface science traditionally haseen concerned with structural aspects of surfaces and adsorbatesnd mechanistic aspects of surface reactions. As the dimensions ofhe particles shrinks into the nanometer range, there are significanthanges in optical and electronic properties due to both quan-um size effects and increasingly important role of the surface inontrolling the overall energy of the particles [9]. Noble metal clus-ers show widely interesting size-dependent optical, electronic andhemical properties [12–20]. These interesting physicochemicalroperties have gained increasing scientific interest to the photo-hemists and photobiologists and to exploit their role in a numberf photophysical studies. Here we have studied the size effect ofilver nanoparticles on cresol red flurophore probe.

Nanoparticle probes act as biosensors in the chemical andiochemical fields, and their applications are increasing day byay. These probes have been applied to ultrasensitive detectionf proteins, DNA sequencing, clinical diagnostics, etc. Recentlye reported the interaction of colloidal Ag with bovine serum

lbumin and Human adult hemoglobin (Hb) (Scheme 1) usinguorescence spectroscopy [18]. Ag nanoparticle has attractedore attention because silver is an extremely noble metal in

he nano scale and has remarkable catalytic activity [19], sizend shape-dependent optical properties [20], and its antimicrobialctivity [21].

. Materials and methods

.1. Materials and instrument

All the reagents used were of either GR or AR grades. Silveritrate (AgNO3), sodium borohydride (NaBH4), ascorbic acid, gooduality tween surfactant of different chain lengths (Tween 20, 60,0 and 85), methylene blue and cresol red from Merck, India weresed as received without further purification. Span 20, 60, 80, 85rom Fluka; BSA from SRL (India) & Human adult Hb (Sigma Chemi-al Co.) were used as purchased. The [Co(NH3)5Br](NO3)2 complex

as been prepared by a standard procedure [22]. All aqueous solu-ions were prepared in deionised and double distilled water withecond distillation being carried out from alkaline permanganaten all pyrex still.

r), (b) Structure of human hemoglobin (red and blue are proteins’ � & � subunitsin this figure legend, the reader is referred to the web version of this article.)

The pH of the solutions was measured with a Systronics pH-meter. All kinetic and spectral measurements were recorded onAgilent 8453E UV-Visible Spectroscopy System using quartz cells(1.0 cm optical path length) from Hellma. Transmission ElectronMicroscopic (TEM) studies of the particles was carried out at a res-olution of 1.9 A unit of JEOL, JEM-2100 Electron Microscope fromJapan. TEM specimens were prepared by placing micro-drops ofnanoparticle solutions on a carbon film supported by a coppergrid. The Dynamic Light Scattering (DLS) results were obtainedwith a Nano ZX DLS instrument supplied by Malvern Instruments.Emission spectra were recorded on Perkin-Elmer LS55 fluorescencespectrophotometer. For Time-correlated single-photon-countingTCSPC measurement, the photoexcitation was made at 370 and450 nm using a ns diode laser (IBH, Nanoled-03) and (IBH, Nanoled-07) respectively in an IBH Fluorocube apparatus. The fluorescencedecay data were collected on a Hamamatsu MCP photomultiplier(R3809) and were analyzed by using IBH DAS6 software. For UVirradiation, a TUV 15W G15T8 lamp supplied by Philips (India) wasused.

To prepare phosphate buffer solution of pH 6.8, aqueous solu-tions of 51.0 mL of 0.2 mol L−1 monobasic sodium phosphate and49.0 mL of 0.2 mol L−1 dibasic sodium phosphate were mixed anddiluted to 200 mL. All experiments were performed in this buffersolution of pH 6.8.

2.2. Preparation of nanoparticles

AgNO3 as the precursor and NaBH4 as a reducing agentwere used to prepare silver nanoparticles. After complete mix-ing of 0.03 mL 1 × 10−4 mol L−1 AgNO3 solution with 1.50 mL1 × 10−4 mol L−1 of selected surfactant of Span 20, 60, 80 and 85and 1.50 mL 1 × 10−4 mol L−1 of selected surfactant of Tween 20,60, 80 and 85, the solution was purged with N2 gas for 2–3 minto remove all dissolved oxygen. Then 0.2 mL 1 × 10−2 mol L−1 of icecold NaBH4 solution was added drop by drop into it while shaking toget silver nanoparticle stabilized in the mixed surfactants. The solu-tion turned to yellow. This reduction reaction was also performedwith Span 60 solution of 1 × 10−3 mol L−1 to get blue colored silversol.

1.0 �L of an aqueous solution of AgNO3 (1 × 10−2 mol L−1) wereadded into the mixed micelle. The resulting mixture was shakenwell so as to get a clear solution. To this, 1.0 �L of an aqueous

solution of ascorbic acid (AA, 5.68 × 10−2 mol L−1) were added. Thesolution was taken in a 1 cm quartz cuvette and exposed to UV radi-ation at a distance of 8 cm from the lamp. The irradiation time forall the experiments was 10 min.

M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13 3

Table 1Size distribution of silver nanoparticles synthesized in different surfactants.

System (chemically) Size (nm) System (chemically) Size (nm) System (photochemically) Size (nm)

Sp 20-Tw 20 1.13 Span 20 3.00 Sp 20-Tw 20 7.004.00 Sp 60-Tw 60 8.004.50 Sp 80-Tw 80 8.605.00 Sp 85-Tw 85 10.00

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Table 2Dynamic light scattering (DLS) studies of various silver sols.a

Type of sol Diameter (nm) Polydispersity Diffusion coefficient(cm2 s−1) (D)

Blue Ag sol 95.01 0.90 0.52 × 10−7

Yellow Ag sol 12.85 0.22 3.99 × 10−7

Sp 60-Tw 60 1.85 Span 60Sp 80-Tw 80 2.00 Span 80

Sp 85-Tw 85 3.00 Span 85

.3. Circular dichroism

Circular dichroism (CD) spectra of BSA, its mixture withanoparticle, Hb and its mixture with nanoparticle were recordedith a JASCO J-815 CD Spectropolarimeter attached to a chiller to

ontrol the temperature of the electronic circuit (sample tempera-ure 308 K). For measurements in the far-UV region (200–250 nm),

quartz cell with a path length of 10 mm was used in nitrogentmosphere.

.4. Kinetic measurements

The kinetics of the reaction between Mohr’s salt (FeII) andCo(NH3)5Br](NO3)2 (CoIII complex) in the presence of nanometerized silver sol have been studied spectrophotometrically underseudo-first order condition with an excess amount of FeII at 298 K.he influence of the addition of FeII solution on the spectra of pureoIII complex solution was recorded. The change proceeds through

maximum at ca. 550 nm. The decrease in absorbance (At) of theeaction mixture occurs at 550 nm. The integrated rate equationsed to determine the observed pseudo-first order rate constant,obs values from the plot is: At = A∞ + a1exp(−kobst); which is firstrder exponential decay equation. The kobs values were repro-ucible within ±3% for all kinetic runs.

. Results and discussion

.1. Syntheses of nanoparticles chemically

Chemical reduction method is used to synthesize silveranoparticles in different micellar environments using a seriesf non-ionic Span and a mixture of Tween and Span surfactants.he silver ions are reduced to silver metal particles by sodiumorohydride and primarily the silver particles are homogeneouslyistributed through out the solution. This happens due to inherentydrophobic nature of surface of metal nanoparticle [24]. Subse-uently they tend to aggregate to form bigger particles and theemaining ions in the bulk get adsorbed on the surface of earlyormed particles where successive reduction takes place. Therebyilver sols have been prepared in aqueous solutions of a series ofpan and a mixture of Tween and Span surfactants which stabi-izes the sol particles for its hydrophobic nature. The uniquenessf this method is to prepare very small sized particles, lowesteing of the order of ∼1.6 nm. The nanoparticles prepared fromhe mixture of Tween and Span surfactants were of the small-st size, thus it proves the enhanced stabilizing agent of mixedicelles.In addition, the chain length of surfactants has a profound influ-

nce on the evolution of particle shape and also on their extentf aggregation. Surfactants with smaller chain lengths producemaller particles. Surfactants with smaller chain lengths give rise toigher concentrations of micelles with a lower aggregation numbernd hence smaller micelles encompass a smaller amount of silver

ons. Thus evolution of smaller Ag particles is logical.

The size distribution of particles has been obtained from TEMictures (Fig. 1A) and its homogeneous distribution has obtainedrom histograms of size distribution (Fig. 1B). This has been

aPrecursor salt used – AgNO3; scattering angle – 90◦; solvent viscosity – 0.89 cP;refractive index – 1.33 [D = kBT/(6��RH), kB = 1.3807 × 10−23 J K−1].

prepared by measuring individual particle size from TEM pictureswhich is reasonably non-agglomerated (Table 1). Most of the silversols are yellow in color except one which is blue, prepared usingdifferent concentration of Span 60, with distinct absorption peakat 650 nm and a hump at 450 nm. Nanoparticles were formed onlyabove the CMC (critical micelle concentration or CMC is defined asthe concentration of surfactants above which micelles form and alladditional surfactants added to the system go to micelles) of varioussurfactants used to stabilize the particles. The development of bluesol happens due to availability of macro aqueous environment ofnormal micelles. Its polymer like structure promotes the formationof aggregates. It is known that aggregation of small silver parti-cles leads to a broad plasmon band in the visible region [25]. Theblue colored silver nanoparticle solution is block shaped and formsa hollow nanostructure while the yellow colored one is spherical(Scheme 2). The apparent discrepancy between the diameter (d) ofsilver nanoparticles obtained by TEM and DLS of the sols (Table 2)can be attributed to the formation of micellar jacket surroundingthe nanoparticles. The larger diameter of the particle in blue solcompared to the yellow one is an indication of more aggregation ofparticles. The blue sol (Scheme 2(a)) has the highest polydispersity,p.d. 0.9. The larger diameter of the blue sol compared to the yellowone is an indication of the former being an aggregate. Thus the bluesol exhibits optical activity which has been explained later. The dis-persion stability of silver nanoparticle protected by these Span anda mixture of Tween and Span surfactants has been maintained atleast for a month.

The advantage of using mixture of Tween and Span surfactantis to prepare very small size nanoparticle while using only Tweensurfactant is to get large size nanoparticles [26]. Secondly, due toits variation in size from 2 to 70 nm, we have got a scope to study itsoptical activity. Thirdly, the small size nanoparticles increase sig-nificantly the rate of electron transfer reaction. Finally due to smallsize we have made a comparative effect of its quenching ability.

3.2. Syntheses of nanoparticles photochemically

The aim of this work was to prepare Ag sols in mixed micelles byUV irradiation. The interesting observation from this study is thefact that the surfactants mixture of Tween and Span promotes theformation of aggregated pink sol. Thus, the macro aqueous envi-ronment in normal micelles is conducive to the process of pink solformation. Another important observation is that the surfactantmixture with its polymer-like structure promotes the formation

of aggregates, whereas other surfactants like the cationic cetyltrimethyl ammonium bromide (CTAB) and the anionic sodiumdodecyl sulfate (SDS) fails to do so. This pink sol is [Fig. 1A I(d)]

4 M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13

Fig. 1. (A) TEM image of silver nanoparticles synthesized in different surfactants (I) photochemically synthesized in (a) Span 20-Tween 20; (b) Span 60-Tween 60 (i) SAEDimage (ii) TEM image; (c) Span 80-Tween 80; (d) Span 85-Tween 85 (i) SAED image (ii) TEM image and (II) chemically synthesized in (a) Span 20 (i) TEM image (ii) SAEDimage; (b) Span 60; (c) Span 80 (i) TEM image (ii) SAED image; (d) Span 85; and (III) chemically synthesized in (a) Span 20-Tween 20 (i) TEM image (ii) SAED image; (b) Span60-Tween 60; (c) Span 80-Tween 80 (i) TEM image (ii) SAED image; (d) Span 85-Tween 85. (B) Size distribution histogram of silver nanoparticles synthesized in differentsurfactants (I) photochemically synthesized in (a) Span 20-Tween 20; (b) Span 60-Tween 60 (i) SAED image (ii) TEM image; (c) Span 80-Tween 80; (d) Span 85-Tween 85 (i)SAED image (ii) TEM image and (II) chemically synthesized in (a) Span 20 (i) TEM image (ii) SAED image; (b) Span 60; (c) Span 80 (i) TEM image (ii) SAED image; (d) Span85; and (III) chemically synthesized in (a) Span 20-Tween 20 (i) TEM image (ii) SAED image; (b) Span 60-Tween 60; (c) Span 80-Tween 80 (i) TEM image (ii) SAED image; (d)Span 85-Tween 85.

M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13 5

Fig. 1. (Continued ).

Scheme 2. (a) A view of blue color Ag sol prepared chemically using Span 60 surfactant. (b) The effect of different particle shapes on the absorption spectrum. (c) A plot of��max as a function of particle size. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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M. Mukherjee, A. Mahapatra / Journal of Photoche

arger in size than yellow sol and the pink sol particles are closelyrranged while the yellow sol particles are widely spaced.

The concentration of the reductant, that is ascorbic acid, haseen found to be a deterministic factor in obtaining both the yellownd pink Ag sols photochemically. In this report, neutral ascorbiccid is the reducing species. The photochemical reduction processay proceed via a radical mechanism. The product formed from

he reaction is dehydroascorbic acid (MCD174) as evidenced fromhe GC–MS studies of the reaction mixture. This was followed byorrish type of cleavage leading to mass fragments at m/z 113, 95,6, 60 and 59. The photochemical transformation of ascorbic acido dehydroascorbic acid in aqueous Span and Tween medium in theresence of Ag(I) possibly follows a radical pathway. UV irradiationay stimulate the photolysis of organic compounds and produces

educing organic radicals [27].

.3. Evolution of silver nanoparticle aggregates: measuringurface plasmon oscillation

Two different sizes of silver nanoparticles have been employedo investigate the size effect on the aggregation behavior (i.e., size,hape and morphology of the aggregates) of metallic particles in theanometer range. The particle size varied within 3–70 nm wherehe concentrations of the silver are same in all cases. The silver par-icles have been synthesized with Span surfactant which offers thease of achieving mono dispersed silver colloids over a wide range.he color of the solution varies from yellow to blue depending onhe size of the particles. The surface plasmon resonance of the silverarticles is red shifted with increase in particle size in accordance toie theory [28]. Now, upon addition of surfactant of varying chain

ength to the particles, the color of the solution becomes blue indi-ating the formation of aggregates amongst the silver particles. Thehanges in the UV–vis spectra of the resultant colloids were mea-ured to study the size effect of metal nanoparticles on the surfacelasmon resonance due to aggregation. The salient feature is thatn extended plasmon band has been developed at longer wave-ength and a clear bathochromic shift in �max has been observed

ith increasing particle size from 3 to 70 nm (Scheme 2(b)). A lin-ar increase in peak displacement against particle size has alsoeen observed in the range of 3–70 nm, but deviating slightly for0–70 nm (Scheme 2(c)).

The optical absorption behavior of the silver particles with sizesn the nanometer range upon aggregation and its dependence onhe individual particle size in the closely packed assembly coulde accounted within the framework of Maxwell-Garnett theory29]. The optical properties of isolated colloidal particles and inarticular, their dependence on particle size have been extensively

nvestigated through Mie scattering theory. In particular, the Mieheory is a mathematical–physical description of the scatteringf electromagnetic radiation by spherical particles immersed in aontinuous medium. Since the inter-particle coupling in the nanocale assemblies is stronger than the coupling with the surround-ng medium, the Mie’s theory developed for very dilute solutionsnd isolated particles fails to describe the optical absorption spec-rum. The Maxwell-Garnet theory is an effective medium theory.t is now known theoretically and experimentally that when indi-idual spherical silver particles come into close proximity to onenother, electromagnetic coupling of clusters becomes effectiveor cluster–cluster distances smaller than five times the clusteradius (d ≤ 5R) [d = 10 nm, R = 35 nm. Here, 10 < 5R i.e., 10 < 175Scheme 2(a))] and may lead to complicated extinction spectraepending on the size and shape of the produced cluster aggre-

ate by splitting of the plasma resonance. The optical propertiesf the metallic nanoparticles are mainly determined by two con-ributions: (1) the properties of the particles acting as well-isolatedndividuals and (2) the collective properties of the whole ensemble.

and Photobiology A: Chemistry 294 (2014) 1–13

Thus, in an ensemble of large number of particles, if the particlescome close together, the oscillating electrons in one particle feelthe electric field due to oscillation of the electrons in the surround-ing particles and this leads to a collective plasmon oscillation ofthe aggregated systems. Under such situation the isolated-particlesapproximation breaks down and the electromagnetic interactionsbetween the particles play a determining role to offer a satisfactorydescription of the surface plasmon oscillations.

In the present experiment, the aggregation between the silvernanoparticles of different size was induced by the Span surfac-tant. Surfactant acts as a mediator to direct the self-assemblyof silver clusters into controlled ensembles with varied func-tional response. Thus, the interparticle distance correspondingto the molecular chain length of surfactant remains fixed for allsets of silver nanoparticles. However, the assembly process pro-vides modular collective optical behavior, as examined throughUV–vis spectroscopic measurements. As the particle size increasesfrom 3 to 70 nm, the extended plasmon band at longer wave-length shows a clear bathochromic shift in the absorption maxima(Scheme 2(b)). Thus, the magnitude of the spectral shift could beascertained to the proximity effects due to nearest neighbor inter-actions between the particles. When silver nanoparticles assembleinto aggregated structures, there is an increase in the dielectricconstant of the medium, shifting the plasmon peak to a lowerenergy. Both of these effects contribute to the red shift and broad-ening of the plasmon band to longer wavelength corresponding tothe aggregated particles. As the particle size (individual) increases,the particles come at a closer distances in making the nanoscaleaggregates.

3.3.1. Characterization of the silver aggregates and effect ofsurfactant concentration

The synthesized silver nanoparticle aggregates have a broaddistribution of sizes and shapes because of the random nature ofaggregate formation. This variety of sizes and shapes is appar-ent in the TEM images and DLS studies. Scheme 2(a) shows theTEM images of large aggregates of 70 nm silver nanoparticles. Theaggregate formation is understandable while we compare the TEMimages of non-aggregated Ag particles of two representative (3 and70 nm) [Fig. 1A III(d), Scheme 2(a)] sizes with their aggregates. Fromthe TEM images, it is evident that the variation in particle size areunable to produce samples containing aggregates with a controlledgeometry, rather the sizes and shapes of the aggregates varied overextended ranges.

Now, we have studied the nature of aggregation of the sil-ver nanoparticles with the variation in surfactant chain length.With variation in surfactant concentration, it is possible to shiftthe dipole plasmon substantially. It is now well established inthe literature that three main parameters affect the dipole plas-mon: aggregate size, shape, and individual particle size. Alterationin any of these parameters or their combination, thereof, willresult in drastic changes in peak intensity and position. Generally,as silver nanoparticle aggregates to larger size, the dipole plas-mon would be red shifted. However, aggregate shape also playsan important role in band position. Surfactant concentration andchain length undoubtedly affects not only shape but also size ofthe aggregates. The width of the dipole plasmon, however, is sim-ply dependent on the polydispersity of the size and shape of theaggregates. The blue sol exhibits high polydispersity (Table 2).Another evidence of this is the higher diffusion coefficient of theyellow sol compared to the blue one. The latter of aggregatedparticles will diffuse slower than the yellow sol which consists

of non-aggregated particles. This would be due to polydispersityof the medium or existence of non-spherical particles, which canresult from aggregation of individual spherical particles into blockstructure.

M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13 7

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ig. 2. Plots of kobs vs. [Ag]0 for the reaction. [CoIII]0 = 0.01 mol L−1, [FeII]0 = 0.11 mob) chemically prepared. (c) Chemically prepared in mixed micelle.

.4. Effect of nanoparticles on the reaction kinetics

In presence of nanoparticle the reaction is first order in CoIII

n view of the fact that the plot of absorbance (At) versus times) fits well with first order exponential decay curve on Microcalrigin version 6.1. The slope of the plot is overall second order

ate constant (k) of the reaction. This redox reaction between oxi-izing agent, CoIII complex salt and reducing agent, FeII salt inqueous medium proceeds through a bimolecular bromo-bridgednner sphere complex [30]. An electron can be transferred slowlyrom FeII to CoIII through the bromo bridge. The kobs value (inhe absence of silver nanoparticles) for the electron transfer reac-ion between [Co(NH3)5Br]2+ and Fe(ClO4)2 with ionic strength.7 mol L−1 [31] corroborates with data of this reaction with ionictrength 0.8 mol L−1. Presence of silver nanoparticle enhances thislectron transfer rate. Nanometer size silver particles can serve asfficient catalyst [26] in many redox reactions as they have largeurface area that acts as surface catalyst on which bound sub-trate readily transfer electron to the electron acceptor. Initiallyoth reactants are adsorbed on the surface of the silver metal parti-les. Subsequently the reductant transfers electron to CoIII complexhrough surface of the metal particle and CoIII complex is conse-uently get reduced. In the absence of the particles, the reactingpecies exhibit a rapid diffusion thus sinking the chances of fruitfulncounters. Both reactants have a tendency to come into contact

ith the entrapped metal particles and as a result an effective

lectron transfer takes place. This is another way of explaininghe nanoparticle catalyzed CoIII complex reduction with Mohr’salt.

ig. 3. (a) Effect of photochemically prepared silver sol on the fluorescence spectrum ofixture. 106[Ag]0, mol L−1 = (a) 0; (b) 3.72; (c) 11.16; (d) 16.74; (e) 24.18; (f) 29.76; [Inset

ffect of chemically prepared silver sol on the fluorescence spectrum of 1.83 × 10−5 mol Lol L−1 = (a) 0; (b) 3.72; (c) 11.16; (d) 16.74; (e) 24.18; (f) 29.76; [Inset] Stern–Volmer plo

onic strength = 0.8 mol L−1 and temperature = 298 K, (a) photochemically prepared

The rate of electron transfer reaction increases with nanopar-ticle concentration in each case of Tween surfactant. The plot ofkobs versus initial silver nanoparticle concentration, [Ag]0 (Fig. 2)is nonlinear with a saturation limit indicates the surface cat-alyzed phenomenon of silver nanoparticle. As the concentrationof nanoparticle increases, the surface area of heterogeneous cata-lyst increases and thus the electron transfer reaction becomes morefacile. The trend of the rate of electron transfer reaction graduallydecreases as the size of nanoparticle increases. This is because ofthe smaller the size of the nanoparticle the larger is the surface area.As a result by reason of its large surface area it acts as an efficientsurface catalyst on which the electron transfer reaction occurs withgood grace. It has been found that its rate increases with decrease ofsilver particle size and about ten times in presence of smallest sizesilver nanoparticle compared to in aqueous medium. Thus rate ofelectron transfer reaction has been observed highest in presence ofsilver nanoparticle stabilized chemically in Span 20 and Tween 20due to its being small in size and as a consequence of large surfacearea. This trend of gradual decrease of electron transfer rate withsize of nanoparticle is observed with the nanoparticle stabilized inother Tween and Span surfactants.

3.5. Fluorescence quenching property of silver nanoparticles

The effects of silver nanoparticles on fluorescence property ofcresol red and methylene blue have been studied (Figs. 3 and 4).On adding �L amount of silver sols to fluorophore, quenching offluorescence is observed (Table 3).

1.83 × 10−5 mol L−1 cresol red in 5 × 10−4 mol L−1 Span 85 & Tween 85 surfactant] Stern–Volmer plot for cresol red in presence of Ag nanoparticle (size ∼10 nm). (b)−1 cresol red in 5 × 10−4 mol L−1 Span 85 & Tween 85 surfactant mixture. 106[Ag]0,t for cresol red in presence of Ag nanoparticle (size ∼3 nm).

8 M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13

Table 3Studies of fluorescence quenching property of silver nanoparticles.

System studied Size of sol (nm) ˚f* KSV (mol−1 L) kq (mol−1 L s−1)

Cresol red in aqueous Sp 85 & Tw 85 – 0.6 ( ϕf0) – –

Yellow Ag sol (†C) 16.74 × 10−6 mol L−1 ∼3 0.33 2.7 × 104 2.48 × 1013

Pink Ag sol (†PC) 16.74 × 10−6 mol L−1 ∼10 0.44 7.4 × 102 6.8 × 1011

Methylene blue in aqueous Sp 20 & Tw 20 – 0.6 ( ϕf0) – –

Yellow Ag sol (†PC) 46.79 × 10−5 mol L−1 ∼8 0.46 4.3 × 102 1.6 × 1011

†PC – photochemically prepared; †C – chemically prepared; ϕf0 – quantum yield of 1.83

surfactant mixture; ˚f* – absolute quantum yields determined using quinine sulfate in 0.5

& 2.77 ns respectively.

Fig. 4. Effect of silver sol on the fluorescence spectrum of 1.83 × 10−5 mol L−1 methy-l −4 −1 5

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F(b

ene blue in 5 × 10 mol L Span 20 & Tween 20 surfactant mixture. 10 [Ag]0, mol−1 = (a) 0; (b) 12.10; (c) 30.25; (d) 46.79; (e) 64.53; [Inset] Stern–Volmer plot forethylene blue in presence of Ag nanoparticle.

Radiant fluorescence decay of the dyes shows different lifetimesn micellar environment and in presence of silver nanoparticles. Theuorescence decay curve of the dyes in aqueous solutions consistsf two resolvable exponential components (not shown). Fig. 5(a)hows the decay of methylene blue in mixed Span 20 and Tween0 solution (curve I) and in the presence of silver nanoparticlescurve II). The intensity of fluorescence as a function of time, I(t),or such a system follows the below equation

(t) = a1exp(−t/�1) + a2exp(−t/�2) (1)

here �1 and �2 are the short and long component of lifetimesespectively and t is the time in seconds. The decay parameters of

ig. 5. (a) Fluorescence decay of 1.83 × 10−5 mol L−1 methylene blue in the presenc30.25 × 10−5 mol L−1). The samples were excited at 675 nm to record the decay curve. (lue in 5 × 10−4 mol L−1 Span 20 & Tween 20 surfactant mixture. 105[Ag]0, mol L−1 = (a) 0

× 10−5 mol L−1 cresol red and methylene blue in 5 × 10−4 mol L−1 Span & Tween N H2SO4 as standard (�f = 0.55); lifetime (�f

0) of cresol red & methylene blue = 1.09

time correlation function are summarized in Table 4. The change inlifetime of the dye molecules in the presence of silver sol supportsthe interaction of the dye molecules with the metal particles.

The next issue to be addressed is the nature of quenching i.e.,whether it is a case of dynamic or static quenching. Ag sols absorbinsignificant amount of light at the exciting wavelength. Thus thequenching does not happen for absorption of light by Ag sols. Here,concentration quenching by dipolar energy transfer between thefluorophore molecules does not happen since the ratio of Ag solconcentration to probe concentration being ∼4. The probability offluorophore molecules crowding together is less. Moreover, if con-centration quenching happens to occur, then with increasing Ag solconcentrations efficiency of quenching would decrease, thus lead-ing to an increase in ϕf. However, this was not observed. Now, forboth static and dynamic quenching, the fluorophore and quenchermust be in contact. However, if dynamic quenching happens tooccur, then the ratio of ϕf of methylene blue in absence of quencherto that of its presence (�f

0/�f) would correspond to an equal changein its lifetime (�f

0/�f) [32]. However, such a correspondence is notobserved in the present case. Lifetime measurements reveal prac-tically no change in �f of 1.83 × 10−5 mol L−1 methylene blue in5 × 10−4 mol L−1 aqueous Span 20 and Tween 20, on addition ofAg sols. The lifetime in aqueous micelle, i.e., �f

0 (2.77 ns) decreasesonly very slightly to 2.12 ns in the presence of Ag sols (Fig. 5(a)).This small decrease is supposed to within an error limit of ±1.0 ns.Thus, quenching by Ag sols in this work happens for static quench-ing. Static quenching removes a fraction of the fluorophore fromobservation. The complexed fluorophores are non-fluorescent, thusfluorescence has been observed from the non-complexed fluo-rophore, i.e., remains unperturbed and gives unaffected lifetime.

Static fluorescence quenching usually results from the forma-

tion of a non-fluorescent complex between the fluorophore andthe quencher. The formation of a ground state complex altersthe absorption spectrum of the probe. In this work, the silverplasmon band (shape and height) has been affected slightly by

e of (I) Span 20 and Tween 20 (5 × 10−4 mol L−1), (II) (I) + silver nanoparticleb) Effect of silver sol on the excitation spectrum of 1.83 × 10−5 mol L−1 methylene; (b) 12.1; (c) 30.25; (d) 46.79.

M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13 9

Table 4Time-resolved fluorescence studies of dyes, BSA and Hb in various media.

System �1 (ns) �2 (ns) a1 a2 ‹�› (ns) 2

1.83 × 10−5 mol L−1 CR + 5 × 10−4 mol L−1 Sp 85 & Tw 85 0.20 1.09 0.09 0.02 52.16 1.161.83 × 10−5 mol L−1 CR + 5 × 10−4 mol L−1 Sp 85 & Tw 85 + 30.25 × 10−5 mol L−1 Ag np 0.01 0.7 4.95 0.01 39.90 0.951.83 × 10−5 mol L−1 MB + 5 × 10−4 mol L−1 Sp 20 & Tw 20 1.22 2.77 0.04 0.01 79.40 1.141.83 × 10−5 mol L−1 MB + 5 × 10−4 mol L−1 Sp 20 & Tw 20 + 30.25 × 10−5 mol L−1 Ag np 0.70 2.12 0.04 0.02 64.00 1.151.83 × 10−5 mol L−1 MB + 5 × 10−4 mol L−1 Sp 20 & Tw 20 + 46.79 × 10−5 mol L−1 Ag np 0.22 1.79 0.12 0.01 36.64 1.030.77 × 10−6 mol L−1 BSA + 5 × 10−4 mol L−1 Sp 20 & Tw 20 0.094 – 0.27 – – 1.080.77 × 10−6 mol L−1 BSA + 5 × 10−4 mol L−1 Sp 20 & Tw 20 + 30.25 × 10−5 mol L−1 Ag np 0.086 – 0.20 – – 1.020.77 × 10−6 mol L−1 Hb + 5 × 10−4 mol L−1 Sp 20 & Tw 20 0.060 0.39 910.20 0.009 54.62 1.060.77 × 10−6 mol L−1 Hb + 5 × 10−4 mol L−1 Sp 20 & Tw 20 + 30.25 × 10−5 mol L−1 Ag np 0.003 0.19 1768.97 0.090 5.33 0.94

�1 and �2 are the life time components arising out of bi-exponential fittings.a1 and a2are the corresponding amplitudes of these components.‹

aofaobs

qas(qcr[oe

Iitcft(fosicst(sftvocf

3p

tt

Table 5Aggregation number of dye molecules over different size of silver nanoparticles.

Set Particlediameter (nm)

Aggregationnumber

Surface-to-volumeatomic ratio (˛)

�› is the average lifetime given by (�1a1 + �2a2).2 is the fitting parameter Chi-squared, 2 close to 1 means a good fitting.

ddition of the probe implies the chemisorption of methylene bluento the sol surface. Thus, some sort of ground-state complex isormed between methylene blue and the Ag sol, which on excitationbsorbs light and immediately returns to the ground state with-ut emitting a photon. Excitation spectra of methylene blue haveeen obtained to check ground state of dye molecule (Fig. 5(b)). Thepectra clearly show it is a case of static quenching.

The dependence of the fluorescence intensity upon theuencher concentration is plotted and linear plots are obtained forll types of sols. This indicates that only one type of quenching i.e.,tatic quenching occurs. For static quenching, the dependence ofI0/I) on quencher concentration [Q] is similar to that for dynamicuenching except that the quenching constant is the associationonstant KSV [33]. Static quenching of methylene blue and cresoled by all types of Ag sols obeys a linear relation (Figs. 3 and 4Inset]), Eq. (2). This linearity (standard deviation ±0.89%, averagef five variants) is indicative of a single class of fluorophores, allqually accessible to the quencher

I0I

= 1 + KSV[Q ] (2)

0 = fluorescence intensity in absence of quencher; I = fluorescencentensity in presence of quencher. [Q] in Figs. 3 and 4 represents theotal quencher concentration. KSV is the Stern-Volmer/associationonstant for complex formation. Table 3 lists the KSV valuesor the different sols. The yellow sol prepared chemically formshe strongest complex with cresol red as KSV value is highest2.7 × 104 mol−1 L) for it. The higher value of KSV also observedor the sol prepared from small sized particle. A careful studyf Table 3 reveals that the strength of association of the Agol to methylene blue and cresol red can be directly linked tots efficiency as a quencher. The stronger fluorophore-Ag solomplex formation (i.e., high KSV) indicates the larger extent oftatic quenching i.e., high value of (I0/I). Considering the life-ime of methylene blue and cresol red in absence of quencher�f

0 = 2.77 ns, 1.09 ns), the ‘apparent bimolecular quenching con-tant’ kq, has been calculated from KSV (shown in Table 3) rangingrom 1.6 × 1011 to 24.8 × 1012 mol−1 L s−1. These values are largerhan those of possible for a diffusion-controlled reaction [32]. A kq

alue of 1 × 1010 mol−1 L s−1 is the largest possible value in aque-us solutions [32]. This fact further strengthens the contention thatomplex formation between methylene blue and Ag sol is the causeor the quenching rather than any dynamic diffusion process.

.5.1. Size effect of silver nanoparticles on fluorescence quenching

roperty using cresol red as fluorophore

We intend to study the size effect of silver nanoparticles onhe fluorescence quenching property of cresol red taking the spec-ral features of dye molecules into consideration (Fig. 3). For

Yellow color Ag sol ∼3 833.67 0.286Pink color silver sol ∼10 30,876.70 0.086

this purpose silver nanoparticles of two different sizes have beenemployed. It is found that the fluorescence of Span 85 and Tween85 incorporated cresol red has been quenched to the maximumextent by the small sized silver sol. The size dependence of the sil-ver nanoparticles can be accounted by considering the distributionof the probe molecules over the silver particles. We have calculatedthe aggregation number. The number is different from one set toanother as the size of the particles varies in different sets keepingthe amount of total silver constant. All these results are summarizedin Table 5. The size regime dependence of the silver nanoparticleson cresol red can be rationalized by correlating the structure ofthe nanoparticles in terms of their surface parameters. As the sil-ver concentration is same for all sets of particles, the larger is theparticle the lower is the concentration of the particles and the sur-face area is correspondingly lower. The number of dye moleculesassociated with each silver nanoparticle has been determined bycalculating the particle concentration in the silver sols [34]. At first,the aggregation number has been calculated using the formula:NAg = (59 nm−3)(�/6)(DMS)3, where DMS is the mean diameter ofthe particles. Thus, a silver particle of 3 nm diameter is composed of834 silver atoms and each particle corresponds to 13,000 cresol redmolecules The surface-to-volume atomic ratio (˛) of metal particlesis defined by, = 3d/R, where d and R denote the atomic diame-ter and particle size respectively. Assuming d = 2.86 A for atomicgold, the surface-to-volume atomic ratio, = 0.286 which indicatesamongst the 834 silver atoms in a 3 nm silver particle, 238 atomsare located at the surface whereas = 0.086 for 10 nm silver par-ticle. Thus, a 3 nm silver particle with a surface area of 28.26 nm2

and 238 atoms on the surface accommodate large number of cresolred molecules. In the present case, when the size of the particleschanges from 3 to 10 nm the density of cresol red molecules sur-rounding each silver particle becomes higher. For smaller size ofthe particle, the population of the cresol red molecules surround-ing each silver particle is such that the situation prevail the dyemolecules to form an ordered adlayer. As the size of the particleincreases, due to the increase in density of cresol red molecules sur-rounding the silver particles there is steric interactions among thedye molecules prevent to form an ordered arrangement on the par-

ticle surfaces. Therefore, no vast changes in the spectra have beenobserved in case of larger particle compared to smaller one. Thelow intense band [i.e., fluorescence quenching is less in presence

10 M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13

hylen

onefr

Nt

3b

aaaaap

isiabptotstto

Ft

Scheme 3. Representation of energy levels (S = Singlet; T = Triplet) of met

f 10 nm silver nanoparticle than that in presence of 3 nm silveranoparticle (Fig. 3)] observed in the presence of larger particlesssentially arises from the unbound dye molecules in the solutionrom which it can be concluded that some of the dye moleculesemain adsorbed on metal surface.

The aggregation number was calculated using the formula:Ag = (59 nm−3)(�/6)(DMS)3, where DMS is the mean diameter of

he particle.

.5.2. Aggregation effect of silver nanoparticles using methylenelue as fluorophore

The fluorescence spectra (Fig. 4) indicate that more quenchingt higher silver nanoparticle concentrations arises from the moreggregation number of methylene blue. Methylene blue exhibits

sharp monomeric band with a maximum at 675 nm. The dyebsorption renders the silver nanoparticles to coalesce. The red shiftnd broadening of the peak have been observed with increasingarticle size.

The red shift in the absorption maximum observed in Fig. 4ndicates that the methylene blue aggregates and forms colloidaluspensions are of J-type. In fact, the J-aggregates are character-zed by a red-shifted absorption band relative to the monomer bands a result of exciton delocalization over the molecular buildinglocks of the aggregate [35]. The molecular exciton theory wasroposed by McRae and Kasha [36] to afford the most satisfac-ory treatment of the electronic transitions in the dimeric formf the dye. This theory predicts that the excitonic singlet state ofhe dimer splits into two levels as a consequence of the two pos-

ible arrangements (in-phase and out-of-phase oscillation) of theransition dipoles of the chromphores in the dimer. Since the exci-on splitting depends on the oscillator strength of the transition,nly the singlet-excited state splits and the triplet-excited state

ig. 6. High resolution transmission electron micrographs of the silver colloids (preparedhe dye molecules.

e blue (MB) monomer and dimer that control the excited state dynamics.

remains nearly degenerate. As a result of splitting, one level is nowlower and the other is higher in energy than the correspondingmonomer singlet excited state. Transitions from ground state toeither excited state are possible and the number of bands actuallyobserved depends on the geometry of the dimer. For parallel dimers(H-type aggregates), the transition to the lower energy excited stateis forbidden and a single blue-shifted band is observed with respectto monomer. On the other hand, in case of head-to-tail dimers (J-type aggregates), the transition to the higher energy excited state isforbidden and the spectrum shows a single red-shifted band withrespect to monomer. A schematic presentation of the H- and J-type aggregates and representative energy levels that control theexcited state dynamics are shown in Scheme 3 [37]. The aggrega-tion effect is also reflected in the emission spectra of the dye andthe dye-nanoparticle interaction can be corroborated from lifetimemeasurements.

The observed dye aggregation in presence of smaller parti-cles can be interpreted by considering that dye molecules induceinter-cluster interactions within the smaller particles and suchaggregates which bring adsorbed dye molecules closer facili-tate dimer formation in the assembly of the dye molecules.Since, the molar surface area increases with decreasing parti-cle size, there results an increase in molar free energy of thesmall particles compared to larger ones and thus, the nanopar-ticle aggregation predominates in colloids with smaller particlesafter addition of the dye. So, aggregation of particles is the causeand dimerization of dye molecules is the effect. High resolutiontransmission electron micrographs (Fig. 6) of the silver colloids

(prepared photochemically with Tween 20 and Span 20) beforeand after addition of the dye molecules show that the silverparticles are aggregated in solution after addition of the dyemolecules.

photochemically with Tween 20 and Span 20) (a) before and (b) after addition of

M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13 11

+ n( )

dye

Ag nanopa rticle

So

piswitwfip

3

S

wflnq[icqco

Ff6

Fig. 8. Effect of silver sol on the fluorescence of 0.77 × 10−6 mol L−1 BSA in5 × 10−4 mol L−1 Span 20 & Tween 20 surfactant mixture, 105[Ag]0 (mol L−1) being;

cheme 4. A schematic diagrams illustrating the possible morphological changesf silver-dye cluster assembly.

During the formation of silver aggregates (Scheme 4), the closeacking of the dye molecules around the silver surface induces

nter-particle interactions. Fig. 7 shows an increase in the inten-ity of 658 nm peak, i.e., increases the concentration of the dimerith increase in the concentration of silver sols. Inset shows a linear

ncrease of the dimer concentration with increase in the concen-ration of silver sols indicating the extent of dimerization increasesith increased number of silver particles in the aggregate. The

ormation of dye aggregates in the presence of smaller particlesndicates that there is a dramatic increase in surface energy of thearticles as the diameter decreases below ∼10 nm.

.5.3. Quenching effect using BSA as fluorophoreThe fluorescence quenching phenomenon is described by the

tern–Volmer relation:

I0I

= 1 + KSV [Q ] = 1 + kq�0[Q ] (3)

here I0 and I are the fluorescence intensities of BSA (asuorophore) in the absence and presence of quencher (Aganoparticle), KSV is Stern–Volmer constant, kq is the bimolecularuenching rate constant, and �0 is the average lifetime of BSA [38],Q] is the molar concentration of quencher. Fig. 8 shows the effect ofncreasing colloidal Ag nanoparticle concentration on the fluores-

ence emission spectra of BSA. Addition of colloidal Ag nanoparticleuenches BSA fluorescence emission. The complex formed betweenolloidal Ag nanoparticle and BSA is responsible for the quenchingf BSA fluorescence emission. A linear plot [Inset, Fig. 7] of (I0/I)

ig. 7. Absorption spectra of 1.83 × 10−5 mol L−1 methylene blue in presence of dif-erent concentration of silver nanoparticles. [Inset] shows the plot of absorbance (at58 nm) as a function of silver concentration.

(a) 0; (b) 12.1; (c) 30.25; (d) 46.79; (e) 64.53. [Inset] Stern–Volmer plot for BSAbinding of Ag nanoparticle.

against [Ag nanoparticle]0 is obtained and from the slope we havefound the Stern–Volmer constant (KSV) as 2.05 × 103 mol−1 L. Thequenching is not dynamic in nature (Table 4); it depends on the for-mation of a complex between Ag nanoparticle and BSA (Scheme 5).

We also see a red shifted emission of BSA in presence of sil-ver sol. Therefore, it is advantageous that BSA helps us to studythe aggregation phenomena. Aggregation of nanoparticles forms anew species for which the shoulder peak is formed. These changesare due to changes in the size/shape of the silver nanoparticlesbrought about due to interaction with BSA. Thus, a BSA-nanoclusterassembly enables to study the aggregation phenomena.

3.5.3.1. Binding constant and number of binding sites. The relation-ship between the fluorescence intensity and quenching mediumcan be deduced from the following equilibrium:

nQ + BK�Qn − B (4)

where B is the biomolecule which is also fluorophore, Q is thequencher molecule, Qn − B is the quencher–biomolecule complexand the association/binding constant K is given by

K = [Qn − B][Q ]n[B]

(5)

If the overall amount of biomolecules (bound or unboundwith the quencher) is [B]0, then [B]0 = [Qn − B] + [B], here [B] isthe concentration of unbound biomolecules. Thus the relationshipbetween fluorescence intensity and the unbound biomolecule is[B]/[B]0 = I/I0, that is

log[

I0 − I

I

]= n log[Q ] + log K (6)

where K is the binding constant of Ag nanoparticle with BSA, whichcan be determined from the plot of log[(I0 − I)/I] versus log[Q]. Thuswe have found the binding constant “K” as 4.5 × 103 mol−1 L andbinding sites “n” as 1.2 for Ag nanoparticles with BSA from the

Scheme 5.

12 M. Mukherjee, A. Mahapatra / Journal of Photochemistry and Photobiology A: Chemistry 294 (2014) 1–13

F[t

ihb

optca

3d

ig(ftqIaKcsit

200 210 220 230 240 250-12 0

-10 0

-80

-60

-40

-20

0

20

CD

(mde

g)

wavel eng th (nm)

Hb Hb+Sp 20 &Tw 20 +Ag np

Fig. 11. Far-UV CD spectra of Hb in absence and presence of Ag nps.−5 −1 −4 −1

F1

ig. 9. Far-UV CD spectra of BSA in absence and presence of Ag nanoparticles.Ag]0 = 46.79 × 10−5 mol L−1 in 5 × 10−4 mol L−1 Span 20 & Tween 20 surfactant mix-ure, [BSA]0 = 0.77 × 10−6 mol L−1.

ntercept and slope. The good agreement between these values of Kighlights the validity of assumption proposed for the associationetween BSA and colloidal Ag nanoparticles.

CD spectroscopy is a sensitive technology to monitor the sec-ndary structure alteration of protein. CD spectra of BSA in theresence and absence of Ag nanoparticle was shown in Fig. 9, wherehe double peaked band with minima at 208 and 220 nm for eachurve has been observed which represents the transition of � → �*nd n → �* of ˛-helical structure.

.5.4. Quenching effect using Hb as fluorophore andetermination of binding constant

The potential interaction between Hb and Ag nanoparticle ismplied by the fluorescence peaks at 460 nm. The peak intensityradually decreases with increasing amounts of Ag nanoparticleFig. 10(a)). The observed fluorescence quenching probably arisesrom the energy transfer occurring between Hb and Ag nanopar-icle. The fluorescence quenching mechanism can be analyzeduantitatively with the Stern–Volmer equation [39], where I0 and

are the relative fluorescence intensities of Hb at 460 nm in thebsence and presence of quencher, Ag nanoparticle, respectively;SV is the Stern–Volmer quenching constant, and [Q] is the con-

entration of quencher (Ag nanoparticle). The Stern–Volmer plotshow that the fluorescence quenching at 460 nm by Ag nanoparticles in agreement with the Stern–Volmer equation when the concen-ration of Ag nanoparticle is low or equal to 64.53 × 10−5 mol L−1

ig. 10. (a) Effect of silver sol on the fluorescence of 0.77 × 10−6 mol L−1 Hb in 5 × 10−4 m2.1; (c) 30.25. (b) Stern–Volmer plot for Hb binding of Ag np. [Inset] is the Lineweaver–B

[Ag] = 30.25 × 10 mol L in 5 × 10 mol L Span 20 & Tween 20 surfactant mix-ture, [Hb] = 0.77 × 10−6 mol L−1.

(Fig. 10(b)). From the slope we have found the Stern–Volmer con-stant (KSV) as 1.03 × 103 mol−1 L. The quenching mechanism mainlyarises from static quenching (Table 4) [39]. As such, a ground statecomplex is probably formed between Hb and Ag nanoparticle thatleads to this fluorescence quenching.

The binding constant (K) between Hb and Ag nanoparticle havebeen obtained by employing the Lineweaver–Burk equation [39],

1I0 − I

= 1I0

+ 1KI0

1[Q ]

(7)

where I and I0 are fluorescence intensities of Hb in the presenceand absence of Ag nanoparticle, respectively; [Q] is the concen-tration of Ag nanoparticle. The values of binding constants K,determined from the Lineweaver–Burk equation at different tem-peratures, show that the order of magnitude of K is 1.94 × 103.The plot is shown in the inset of Fig. 10(b). CD spectroscopy, aquantitative technique to investigate the conformation of proteinsin aqueous solution, provides additional evidence for the possibleconformational changes of the adsorbed Hb. The CD spectrum ofHb exhibits an intensive positive peak at about 199 nm and pro-nounced negative bands at about 208 and 222 nm (Fig. 11), which

are characteristics of a high ˛-helical content [34]. The intensity ofthe negative bands at 208 and 222 nm is increased, whereas that ofthe positive peaks about 199 nm is decreased for the adsorbed Hb.

ol L−1 Span 20 & Tween 20 surfactant mixture, 105[Ag]0, (mol L−1) being; (a) 0; (b)urk plot for determination of binding constant.

mistry

4

sTshlifwlb

oscbt

[psp

uAat

mtai

A

oMeFiUatr

R

[

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M. Mukherjee, A. Mahapatra / Journal of Photoche

. Conclusions

Silver nanoparticles of small diameter (∼1.65 nm) have beenynthesized in binary mixtures of Span and Tween surfactant series.his surfactant mixture provides good control over nanoparticleize, shape and polydispersity. Spherical silver sols aggregate in aollow cubic fashion to produce blue sol. This novel class of hol-

ow nanostructures is expected to find application both in opticalmaging for early stage tumor detection and as a therapeutic agentor photo thermal cancer treatment. Another importance of thisork is the action of UV light in synthesizing nanoparticles. UV

ight induced cleavage of ascorbic acid to produce radical species iselieved to be responsible for the reduction of Ag(I).

Blue sol exhibits optical property. Inter particle coupling effectsn the surface plasmon resonance of silver particles with variableizes in the nanometer regime have been investigated and the opti-al absorbance behavior of the resulting nano scale aggregates haseen enlightened within the framework of Maxwell-Garnett effec-ive medium theory.

The electron transfer between oxidizing agent, CoII-complex saltCo(NH3)5Br](NO3)2 and reducing agent, FeII salt (Mohr’s salt) takeslace slowly from FeII to CoIII through the bromo bridge on theurface of silver nanoparticle. The rate is influenced significantly inresence of nanoparticles.

Fluorescence studies reveal that the dye such as methylene bluendergoes aggregation when bound to small silver nanoparticles.

better understanding of the aggregation of silver-dye clusterssembly is important for designing imaging systems, optoelec-ronic devices and light-energy conversion systems.

The interaction between colloidal Ag nanoparticles and BSA/Hbolecules clearly indicate that colloidal Ag nanoparticles quench

he fluorescence emission of BSA and Hb through a static mech-nism. The binding study of drugs with nanoparticles is of greatmportance in pharmacy, pharmacology and biochemistry.

cknowledgements

Financial help from Center for Advanced Studies, Departmentf Chemistry, Jadavpur University and Research Fellowship toousumi Mukherjee by UGC (New Delhi) are gratefully acknowl-

dged. The authors thank the Chairman of the Central Researchacility of Indian Institute of Technology, Kharagpur for TEM facil-ty. The authors thank Dr A Dasgupta, Professor of Biochemistry,niversity of Calcutta for DLS instrumental facility. Dr S Baitaliknd Dr S Das are acknowledged for their generosity in helpinghe authors to perform the lifetime and circular dichroism studiesespectively.

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