Ag@AgI, Core@Shell Structure in Agarose Matrix as Hybrid:Synthesis, Characterization, and Antimicrobial ActivitySomnath Ghosh,† A. Saraswathi,† S. S. Indi,‡ S. L. Hoti,§ and H. N. Vasan*,†

†Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore-560 012, India‡Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560 012, India§Vector Control Research Centre, Medical Complex, Indranagar, Puducherry-605006, India

*S Supporting Information

ABSTRACT: A novel in situ core@shell structure consisting of nanoparticles of Ag (Ag Nps) and AgI in agarose matrix (Ag@AgI/agarose) has been synthesized as a hybrid, in order to have an efficient antibacterial agent for repetitive usage with notoxicity. The synthesized core@shell structure is very well characterized by XRD, UV−visible, photoluminescence, and TEM. Adetailed antibacterial studies including repetitive cycles are carried out on Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria in saline water, both in dark and on exposure to visible light. The hybrid couldbe recycled for the antibacterial activity and is nontoxic toward human cervical cancer cells (HeLa cells). The water insolubleAg@AgI in agarose matrix forms a good coating on quartz, having good mechanical strength. EPR and TEM studies are carriedout on the Ag@AgI/agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria.

1. INTRODUCTIONPerhaps the two major problems facing the microbiologist andpracticing medical professionals are, in tackling the microbes toprevent biofilm formation on various implantable medicaldevices1−4 and diseases caused by microbes in drinkingwater.5,6 Biofilm related infections are a major cause formorbidity and mortality,7,8 and it has been estimated that∼60% bacterial infections in hospitals are due to biofilmformation.2,7 On the other hand, at least one-sixth of world’spopulation lack access to safe drinking water9 and the mainsufferers are the children of the third world countries.6,10,11

Over a period of time, the extensive use of bactericides andchemicals such as chlorine for water treatment has led to thegrowth of new bacterial strains, resistant to multiple drugs(MDR),12,13 and also resulting in the formation of secondarycarcinogenic disinfection byproduct (DBP).14−16 This has ledto the tremendous development of new antibiotics in the recentpast.17,18 In order to have simple processing of material withgood stability and having efficient antibacterial activity with lowtoxicity, many inorganic based materials are being tried.19−23

With the advantages of nanomaterials over the bulk, especiallywith regard to the surface property, such as increased surfacearea24 which internally facilitates better interaction between theparticles and the microbes, the inorganic nanomaterials havebeen widely studied as bactericides in the recent past.25−29 Thewell studied inorganic nanomaterials as bactericides are silver,

both in the form of neutral silver (Ag0)26,30−32 and in theoxidation state of +1 (Ag+) in the form of a suitable silvercompound,33−35 nanoparticles of zinc oxide of different sizesand shapes,36,37 titanium dioxide,38,39 silicon dioxide,40,41 andso on.42−46 The basic approach for the study of antibacterialactivity is to fabricate these materials in the form ofpowder,31,36,38,45 film,30,39 or embedded in a matrix.32 As theenvironmental hazards of nanomaterials as such duringproduction, handling, and while in use are still not being wellunderstood,47 one should be very cautious while working withsuch materials. When a nanomaterial is in the form of powder,the chances of enhanced toxicity may be high due to thedifficulty in handling and also because of high solubility in agiven solvent. The other disadvantage of using antimicrobialnanomaterials in the powder form is the difficulty in recoveringthe same after use. On the other hand when the bactericidenanoparticles are in the form of a film or embedded in asuitable matrix, one may overcome these disadvantages. Andalso the added advantage is that matrix can stabilize theseparticles48,49 for long-term usage. This also helps to have aneasy coating on the medical devices made of stainless steel30 orpolyurethane50 and so on. Our interest is directed toward the

Received: March 30, 2012Revised: May 10, 2012Published: May 14, 2012

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fabrication of inorganic nanomaterials in a suitable biocompat-ible matrix for the study of antibacterial activity.In order to control the microbial contamination in drinking

water, composite materials loaded with slow releasing biocidessuch as heavy metals, antibiotics, and so on are being used.51−53

Among these, silver-based materials are of special interest. Thestrong inhibitory and bactericidal effect as well as broadspectrum of antimicrobial activity of silver have been known fora long time.30−32 Silver nanoparticles (Ag NPs) have lowtoxicity toward mammalian cells and do not easily provokemicrobial resistance.54,55 A large amount of literature may becited for the use of silver ions33−35 or silver nanoparticles30−32

alone in a composite in the study of antimicrobial activity.However, to our knowledge, there is no reported literatureavailable in the study of antimicrobial activity with thecombination of Ag NPs and Ag+ in the form of core@shellstructure embedded in a matrix. The advantages of such acombination are as follows: (i) if the shell consists of a silvercompound, such as silver halide having very low solubility inwater, it would prevent leaching out of Ag or Ag+ from thematrix and in turn causes less toxic to mammalian cell; (ii) onecan have synergic effect of both Ag and Ag+ toward the killingof microbes; (iii) by embedding such a system in a matrix, onecan recover the bactericide and reuse.With these advantages in mind, we report a novel method of

in situ preparation of Ag@AgI (core@shell) in agarose matrixas inorganic−organic hybrid in the form of a free-standing film.Agarose is a polysaccharide, is biocompatible, and is a cheapmaterial that is readily available. The prepared samples arecharacterized by X-ray diffraction (XRD), UV−visible spec-troscopy (UV−vis), photoluminescence spectroscopy, andtransmission electron microscopy (TEM). The water insolubleAg@AgI in agarose matrix can be coated as thin film on quartzplates, having good mechanical strength. It exhibited goodantibacterial activity against Gram-negative Escherichia coli (E.coli) and Gram-positive Staphylococcus aureus (S. aureus)bacteria in saline water both in dark and on exposure to visiblelight. Based on the UV−visible, EPR, and TEM studies, aplausible mechanism has been proposed for the killing of thebacteria by the hybrid. The hybrid could be recycled for manytimes and was also found to be nontoxic toward HeLa cells,when kept in mineral water and also in saline solution for morethan 2 months.

2. EXPERIMENTAL SECTIONMaterials. Silver nitrate (99.9%), potassium iodide (99.8%),

absolute alcohol (99.9%) (SD Fine-Chem Pvt. Ltd., India), polyvinylpyrrolidone (PVP), Mw ∼ 55 000 (Aldrich Chemical Co. Inc.), andagarose (electrophoresis grade, Hi-Media Laboratories Ltd., India) areprocured. All chemicals were used as such without any furtherpurification. Saline solution of 0.85% NaCl in MilliQ water andstandard reference strains E. coli MTCC 1302 and S. aureus ATCC25923 (supplied by M.S. Ramaiah Hospital, Bangalore) were used forthe bacterial studies.Synthesis of Ag@AgI Nanoparticles in Agarose Matrix. In

situ Ag NPs of size 15−25 nm were synthesized in an agarose matrixaccording to the procedure reported by us earlier.48 In a typicalreaction, 1.0 g of chloride free agarose was dissolved in 100 mL ofMillipore water under boiling conditions in a rotary evaporator withconstant mixing. To this clear solution, 60 mL of ethanol and 100 mgof PVP were added after lowering the temperature to 50 °C. Further,0.5 mmol (0.085 g) of AgNO3 was dissolved in 20 mL of Milliporewater and added dropwise to the above solution and refluxed for 3 h at90 °C in the rotary evaporator. At the end of 3 h, the solution turns togolden yellow, ensuring complete reduction of Ag+ ions to Ag0. The

excess solvent and water were pumped out, and 5 mL of 0.25 mmol KIsolution was added in steps of 1 mL, at an interval of 30 min to theabove solution at 90 °C with constant mixing to obtain Ag@AgI inagarose matrix. At the end, 20 mL of this solution was poured into aplastic Petri dish (Φ = 90 mm) and dried at room temperature untilthe mass could be peeled off as a transparent free-standing film.

By a similar procedure, AgI/agarose was also prepared by taking 0.5mmol of AgNO3 in previously prepared agarose solution (1.0 g ofchloride free agarose in 100 mL of Millipore water) containing 100 mgof PVP and adding at once 20 mL of 0.5 mmol KI solution. Thesolution is thoroughly mixed for 3 h at 90 °C in a rotary evaporator.

In order to ensure good mechanical strength for the films, for somespecific studies, which will be described later, 200 μL of as preparedAg/agarose, AgI/agarose, and Ag@AgI/agarose solutions were takenand drop-casted separately over quartz slides to form a film of area 1.0cm2. Before carrying out any experiments, the films are washedrepeatedly in Millipore water to remove impurities such as potassiumnitrate that is formed during the reaction.

Zeta Potential Measurement. Zeta potentials were recorded byusing a Malvern Zetasizer Nano Z instrument at various stages ofpreparation by suitably diluting the reaction mixture.

X-ray Diffraction (XRD). XRD patterns of Ag, AgI, and Ag@AgI inagaorse films were recorded in a Phillips XRD ‘X’ PERT PROdiffractometer using Cu Kα radiation (λ = 1.5438 Å) as the X-raysource.

UV−Vis Absorption. UV−vis absorption studies were carried atthe end of the formation of Ag NPs in agarose and after each stepaddition of KI solution. Typically, 1 mL of solution was taken out atthe end of every 30 min and further diluted to 5 mL in Millipore waterand scanned over the wavelength range 300−800 nm in a Perkin-Elmer Lambda 35 UV−vis spectrophotometer using a quartz cuvettewith Millipore water as background. UV−visible spectra of Ag/agarose,AgI/agarose, and Ag@AgI/agarose coated on quartz slides were alsorecorded against uncoated quartz slide as reference.

Photoluminescence (PL) Study. Like in the UV−visibleabsorption study, PL spectra were taken at the end of Ag NPsformation and also after every step addition of KI to Ag/agarosesolution in a Jobin Yvon FluoroLog 4 (Horiba) instrument, with anexcitation wavelength of 470 nm. PL spectra of Ag/agarose, AgI/agarose, and Ag@AgI/agarose coated on quartz slides were alsorecorded.

Transmission Electron Microscopy (TEM). The size andmorphology of Ag and Ag@AgI nanoparticles were analyzed byTEM using a TECNAI F 30 transmission electron microscope. Carewas taken to prepare and mount the samples on the Cu grid each timeto avoid any possible agglomeration of particles while taking the filmsin water. All samples were prepared by similar conditions, by placing adrop of well-sonicated 1 cm × 1 cm film dissolved in 10 mL ofMillipore water on a carbon-coated copper grid and subsequentlydried in air before transferring it to the electron microscope, which wasoperated at an accelerated voltage of 200 kV.

The healthy and the dead bacteria were observed via TEM bypreparing thin slices of bacteria embedded in a polymer by microtome.After contact with nanohybrid materials for 4 h, the correspondingbacterial suspension was washed twice and fixed with 2%glutaraldehyde for 1.5 h. Samples were then post fixed with 1% leadacetate and dehydrated in acetone series (35, 50, 70, 80, 95, and100%) for 3 min each. After dehydration by 100% acetone, sampleswere then embedded in SPURR resin of suitable size. Ultrathinsections were cut using an ultra microtome (RMC MT-X) and stainedwith uranyl acetate and lead citrate. The thin sections were taken on tothe Cu grid, and micrographs were obtained using the JEOL (100 CXII) transmission electron microscope at 100 kV.

Surface Profilometry. In order to measure the thickness of dropcasted films over quartz slides, surface profilometry using a VeecoDektak 150 instrument was used. The tip of the profilometer wasscanned from the uncoated quartz surface (reference) to the filmcoated quartz (sample) surface, and the profilogram was obtained andthe estimated difference in height of two profiles gives the thickness of

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the film. This is repeated for three different samples and the averagethickness obtained.Antibacterial Activity. Cell viability was assessed by counting

colony-forming unit (CFU) according to the following procedure. E.coli was cultured in a nutrient broth solution at 37 °C for overnight ina mechanical shaker. The cultured microbial concentration was dilutedto 104 CFU/mL by serial dilution. From the diluted bacterial sample,0.1 mL was taken into two beakers containing 9.9 mL of sterile salinesolution and incubated at 37 °C with gentle mechanical shaking. Toone of them, only a sterilized glass slide (positive control) and to theother a Ag@AgI/agarose coated glass slide (test beaker) was kept. Atan interval of 1 h, 100 μL of incubated solution was taken out forplating in nutrient agar plates. After overnight incubation at 37 °C, theviable colonies were visible to the naked eye and thus countedmanually and compared with positive control. For reusability test(cycling), the slides were rinsed thoroughly with sufficient water; andafter each bacterial study, the slides were dried in air, and further on asubsequent day the experiment was repeated as before.As AgI is photosensitive and in order to find the influence of visible

light over the antibacterial activity, the Ag@AgI/agarose coated glassslide was exposed to the light source of a metal halide lamp (OSRAM,150W, power spectrum is provided in Figure S1 of the SupportingInformation). The lamp was kept at an appropriate distance, such thatthe temperature was maintained between 37 and 39 °C near thesample, when the lamp was illuminated. The temperature maintenancewas further ensured by keeping packs of ice surrounding the sample(schematically shown in Supporting Information Figure S2).Electron Paramagnetic Resonance (EPR) Spectroscopy.

Hydroxyl radicals produced by the synthesized compounds in waterwere detected by EPR-spin trapping technique using spin trapper 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 0.02 M) (Sigma Aldrich). Ag/agarose, AgI/agarose, and Ag@AgI/agarose coated glass slides weredipped in water containing DMPO for 2 h in the dark and similarly onexposure to visible light. A known volume of these solutions wasdrawn into quartz capillary tubes and placed in an EPR tube, andspectral studies were carried out on Bruker emx X-band EPRspectrometer.Fluorescence Study for ROS Detection. Further, a fluorescence

experiment was also carried out using a sensitive probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for the detection of ROSby incubating the samples in 30 mL of 0.05 m (M) DCFH-DAsolution for 2 h, both in the dark and on exposure to visible light. Thefluorescence intensity of the solution was measured in a Jobin YvonFluoro Log 4 (Horiba) instrument, with an excitation wavelength of485 nm.Cytotoxicity Assay. In vitro cytotoxicity assay for the Ag@AgI

nanoparticles embedded in agarose (Ag@AgI/agarose) was carried outby exposing human cervical cancer cell line (HeLa cells) in disposableplastic plates as described in the literature.48 Each one of the Ag@AgI/agarose coated glass slides was dipped in 10 mL of mineral water andsaline solution. Then after each hour, 100 μL of this water and salinesolution was pipetted into 25 mL capacity wells, and then 0.1 mLfreshly trypsinized HeLa cells cultured as a monolayer in Dulbecco’smodified Eagle's medium (DMEM) (containing 10% fetal calf serumand antibiotic solution) to active growth phase (18 000 cell per well)were added and incubated for 20 h at 35 °C in 5% CO2 atmosphere.Wells containing an equal number of cells of bare mineral water andsaline water were left as controls. After the incubation for differentintervals, the cells were observed for healthy conditions and death, ifany, under an inverted microscope. A similar experiment was repeatedafter keeping the Ag@AgI/agarose glass slides in mineral water andsaline water separately for 2 months and observing for cytotoxocity.

3. RESULTS AND DISCUSSION

Synthesis of Ag@AgI Nanoparticles in AgaroseMatrix. In situ synthesis of Ag@AgI, core@shell structure inagarose matrix was achieved by the stepwise addition of KIsolution of lower concentration (0.25 mmol) compared to theoriginal silver concentration (0.5 mmol) taken. It is presumed

that a thin layer of AgI is formed over Ag core in the first step(this is further discussed in UV−visible absorption spectralstudies). And on further addition of KI, the shell thicknessincreases as depicted in Scheme 1. The progress of the reactionin solution is monitored by the pH, UV−visible absorptionspectra, and zeta potential (ζ) measurements. During theformation of the core@shell structure, the pH was found to bein the acidic range varying from 3.8 to 4.8. The formation ofcore@shell structure in the acidic medium may be explained bythe following reaction as proposed by Mulvaney56

+ + → ++ −−m m mAg H I Ag (AgI) ( /2)Hn n m m( ) 2

And ζ recorded, from the formation of Ag NPs to the core@shell structure, increases gradually from very negative potentialof −12.7 mV to −5.51 mV and to −3.51 mV on first addition tothe fifth addition of KI solution. This change in surface chargemay be presumed to the initial adsorption of negative chargedspecies such as NO3

− on Ag NPs and on addition of KI, thepossible removal of NO3

− ions from the surface as KNO3, alongwith the formation of the AgI shell.

X-ray Diffraction (XRD). Figure 1 shows the XRD patternof agarose, Ag/agarose, AgI/agarose, and Ag@AgI/agarosecoated over quartz slides. The broad diffraction band around20°−30° is seen for both amorphous agarose and Ag/agarose,and the absence of diffraction peaks corresponding to Ag inAg/agarose is due to very low concentration of Ag present. TheICP-OES analysis of Ag present in Ag/agarose film coated onthe slide is found to be 31.33 (±0.2371) μg cm−2. In the case ofAgI/agarose and Ag@AgI/agarose, prominent peaks of low

Scheme 1. Schematic Illustration of Stepwise Formation ofCore@Shell Structure of Ag@AgI in Agarose Matrix

Figure 1. XRD pattern of (a) agarose, (b) Ag/agarose, (c) AgI/agarose, and (d) Ag@AgI/agarose coated over glass slides.

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temperature phases (β, γ) of AgI are observed. But it is difficultto assign the diffraction peaks exactly to any one of the knownphases (β-AgI, γ- AgI), as their reflections are very close.However, on careful observation of the diffraction peaks ofAgI/agarose (Figure 1c) and Ag@AgI/agarose (Figure 1d), onecan see the absence of (102) and (112) and the decrease in

intensity of (100) and (110) reflections of β-AgI and increasein intensity of β(002)/γ(111) in the case of Ag@AgI/agarose.Such an observation was also made earlier in AgI-anatasecomposites,57 and also it is known that the Ag rich phase of AgItends to crystallize in γ-AgI. In Ag@AgI/agarose, the AgI shellis in close contact with Ag at the atomic scale and will naturally

Figure 2. UV−visible spectra showing (a) the stepwise addition of KI, (b) the subtracted spectra, (c) the deconvoluted spectra of the top threespectra of (b), and (d) plot of the effective diameter of AgI shell versus band gap.

Figure 3. UV−visible spectra of (a) Ag/agarose after synthesis and after stepwise addition of KI, (b) of agarose, Ag/agarose, AgI/agarose, and Ag@AgI/agarose coated film over glass slides.

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have AgI phase rich in Ag. Based on this reasoning, one mayconclude that the AgI phase in the core@shell structure ispredominantly γ-AgI, having a zinc blend structure.UV−Vis Absorption Study. In order to understand the

formation of core@shell structures, the UV−visible spectralstudies were carried out further on addition of very low aliquotsfrom 10 to 1050 μL of KI solution (0.25 mmol in 20 mL) to AgNPs in agarose and the spectra were recorded in solution

(Figure 2a). The appearance of a surface plasmon resonance(SPR) band at 412 nm confirms the formation of Ag NPs inagarose. On addition of KI solution, the SPR seems todisappear, with the formation of a long absorption tail and theappearance of an exciton peak of AgI, when KI reaches aparticular concentration. Further analysis of the spectra wascarried out by subtracting the spectrum of Ag NPs, that is,without any addition of KI from each one of these spectra, andthe resulting plots are shown in Figure 2b. The initial SPR bandof AgNPs (412 nm) shows immediate dampening andbroadening on addition of 10 μL of KI (Ag to I− mole ratio= 1:0.25 × 10−3). The dampening of the SPR band continuesfurther at 20 μL of KI (Ag to I− mole ratio = 1:0.50 × 10−3).When 30 μL of KI (Ag to I− mole ratio = 1:0.75 × 10−3) isadded, along with the dampening of the spectra the evolutionof Z1,2 exciton peak (421 nm) appears as a knee and is due to γ-AgI.58 The dampening of the SPR band is due to the initialadsorption of I− on to the Ag NPs as predicted by Mulvaney.56

When the KI of the aliquot is around 250 μL (Ag to I− moleratio = 1:6.25 × 10−3), the shape of the curve changes, with novisible turf due to dampening but a neat formation of excitonpeak of AgI is seen; this perhaps indicates the formation of acomplete shell of AgI on Ag NPs. On further addition of KI, the

Figure 4. Normalized PL emission (λext = 470 nm) spectra (a) of Ag/agarose after synthesis and after stepwise addition of KI, (b) of agarose, Ag/agarose, AgI/agarose, and Ag@AgI/agarose coated film over quartz slides.

Figure 5. TEM images of (a) Ag/agarose, (b) AgI/agarose, (c) Ag@AgI/agarose, (d) HRTEM Ag@AgI/agarose, and (e) one of the Ag@AgI NP showing shell thickness.

Figure 6. Surface profilometry plot of Ag@AgI/agarose.

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intensity of the exciton peak increases, with no shift in peakposition. The top three plots of Figure 2b were furtherdeconvoluted and are shown in Figure 2c. The Z1,2 exciton peakof AgI splits into two bands, centered at 420 nm and 415 nm(Figure 2c). This was also observed earlier by Mochizuki andUmezawa59 in their studies on optical properties on micro-crystals of AgI, and they explain that the splitting may be due to

various reasons such as splitting of the doubly degenerate state

and so on. However, we are unable to explain the exact reason

for this splitting of the band in our system. The ratio of the area

of these two peaks along with the volume of KI solution added

is shown in the inset table and is found to increase slightly on

addition of KI. A good linear plot is obtained, when the

Figure 7. Viable cell count test by nutrient agar plating: percentage survival bacteria versus contact time for (a) E. coli and (b) S. aureus in the dark,and (c) E. coli and (d) S. aureus in the presence of light.

Figure 8. Plot of percent bacterial survival versus number of cycles: (a) in the dark and (b) in the presence of light.

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effective diameter of AgI shell, calculated from Brussequation,60

* π= + −E E h m d e CC d[ /2 ] [1.8 /2 ]nano bulk2 2 2

0

where m* (reduced mass of exciton) = 0.185 me (reduced massof electron), ε = 5.6, d = effective diameter of the particle, wasplotted against the band gap energy obtained from UV−visspectra (Figure 2b and c) and shown in Figure 2d and also acorresponding table including the amount of KI solution addedin each case is shown as an inset. From the table, it may beinferred that the monolayer formation of AgI shell on AgNPs istaking place, when KI added is around 250 μL (Ag to I− moleratio 1: 6.25 × 10−3), which fairly compares with the shape ofUV−visible spectra (Figure.2b) as discussed earlier.At higher dosages of KI, that is, from 1 mL (Ag to I− mole

ratio = 1:25.0 × 10−3) to 5 mL (Ag to I− mole ratio = 1:125.0 ×10−3), there is a marked shift of the exciton peak to higherwavelengths (red-shift), that is, from 421 to 431 nm, anincrease of 10 nm and also with increase in intensity (Figure3a). This is due to the increase in thickness of the AgIshell.60−62 The UV−vis spectra of Ag NPs/agarose, AgI/agarose, and Ag @AgI/agarose as film coated on quartz slidehaving the same area and thickness, containing equalconcentrations of Ag, are shown in Figure 3b. The absorptionintensity of Ag @AgI core@shell is much higher than that ofthe film containing only AgI; this is because of the increasedelectron population in the conduction band of AgI due to thetransfer of electrons from excited Ag NPs to AgI.63 This furthersubstantiates the formation of core@shell structure.Photoluminescence (PL) Study. Room temperature PL

emission spectra (excited at 470 nm) are presented in Figure 4.Emission intensities are normalized with respect to the mostintense peak. The appearance of the 547 nm peak can beattributed to the exciton recombination.64 Upon stepwiseaddition of KI, the peak at 547 nm (Figure 4a) gets red-shiftedto 566 nm, with an increase in intensity, accompanied by thedecrease in full width at half maximum (FWHM) whichindicates the subsequent growth of AgI shell over Ag core. Asthe thickness of the shell increases, the band gap decreases,causing a red-shift in the emission spectra. The PL emissionspectra of agarose, Ag/agarose, AgI/agarose, and Ag@AgI/agarose coated film over quartz slides are also shown in Figure4b. Similar to the observed UV−vis spectra, the intensity of the

PL emission of Ag@AgI/agarose film is much higher than thatof the AgI/agarose film, indicating the effect of core@shellstructure but with no change in peak position. However, whencompared with the same concentration of Ag@AgI/agarose insolution (curve 5 of Figure 4a), there is found to be a blue-shift.This is mainly due to change in dielectric constant of themedium.

Transmission Electron Microscopy (TEM) Study. Figure5a, b shows the TEM pictures of Ag/agarose and AgI/agarose,respectively. Both Ag NPs and AgI NPs are more or lessspherical in shape, with the size in the range 18−27 nm. TheTEM images of Ag@AgI/agarose (Figure 5c) shows an interiordark shade surrounded by a lighter shade for most of theparticles, and this contrast indicates a core@shell structure.However, a small amount of Ag NPs, especially the very smallerones forming only AgI particles, are not ruled out. Further, aHRTEM image of a Ag@AgI particle is shown in Figure 5d,clearly showing the lattice fringes with a spacing of 0.37 nm,corresponding to AgI (002) of the AgI shell. The estimatedshell thickness is around 4.77 nm (Figure 5e).

Surface Profilometry. Figure 6 shows the surfaceprofilometry plot taken on the film Ag@AgI/agarose coatedon a glass slide. The profilogram shows a thickness of ∼3.5 μm.The line obtained, all most parallel upon scanning ∼500 μmalong the sample, indicates that the surface of the film is quitesmooth.

Antibacterial Activity. The antibacterial activity testingwas done against both E. coli and S. aureus. Our interest in thestudy of a cheap and effective inorganic/organic hybrid basedantimicrobial agent has earlier led to the development of Ag/agar−agar films.48 Further attempts to improve on this systemled to the study of the present work. Figure 7 shows thepercentage survival versus exposure time in the dark and onexposure to light for films of all the samples synthesized on theglass slide. In dark, the percentage survival of E. coli remainsabove 80% at the end of contact time of 30 min for all thesamples. But after this contact time, there is a steep decrease insurvival and zero is reached at the end of 2 h in the case of Ag@AgI/agarose film compared to the other samples (Figure 7a).More or less, a similar trend is observed for all the samples withGram positive S. aureus, and with zero survival around 2.5 h forAg@AgI/agarose film (Figure 7b). When the experiment wasrepeated on exposure of films to light, an interesting result was

Figure 9. UV−visible absorption spectra of Ag@AgI/agarose film after each cycle (a) in the dark and (b) in the presence of light.

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found. The rate of killing of the bacteria is still faster in the caseof Ag@AgI/agarose, and it took only 45 min to kill both Gramnegative and Gram positive bacteria in saline solution. In thecase of Ag/agarose and AgI/agarose films, the antibacterialactivity trend is almost the same as in the dark, indicating only amarginal influence of light (Figure 7c, d).In order to check the stability of the film Ag@AgI/agarose

on repeated cycling for the bacterial activity in the dark and onexposure to light, the film was exposed to the bacteria in salinesolution and is recycled up to five times by taking fresh bacterialsolution each time. The percent survival was determined asbefore and plotted against the number of cycles as a bar chart(Figure 8). The performance of the film in the dark was goodup to the fifth and fourth cycles in the case of E. coli and S.aureus, respectively (Figure 8a); however, no such performance

was seen on exposure to light (Figure 8b). These results were abit surprising. What are the plausible reasons for theantibacterial activity of the samples in the dark and in thepresence of light? What might be the possible mechanism forthe killing of bacteria by the Ag@AgI/agarose hybrid film? Wehave made few attempts to answer these questions based onUV−vis, EPR, and TEM studies.The germicidal effect of nanoparticles is well-

known,30,36,38,40,42 but their mode of action is still incon-clusive,65−69 though people have suggested several mechanismsincluding the killing due to production of reactive oxygenspecies (ROS),36,37 contact killing,69 that is, cell wall attach-ment and internalization,70,71 deactivation of enzymatic activityof bacterial respiratory chain,72,73 bacterial structural changes bythe nanoparticles due to release killing,65,66 and so forth. But it

Figure 10. EPR spectra of DMPO + (a) Ag/agarose, (b) AgI/agarose, and (c) Ag@AgI/agarose (all in dark) and (d) Ag/agarose, (e) AgI/agarose,(f) Ag@AgI/agarose, and (g) Ag@AgI/agarose after 1st cycle (all in the presence of light). (h) Fluorescence spectra of DCFH-DA solution afterincubating with material under dark and light.

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is still not clear in many of the cases whether the killing is dueto any one individual mechanism or due to the combination ofmore than one mechanism.The UV−visible spectrum of Ag@AgI/agarose film was

taken after each cycle of exposure to bacteria in the dark and isshown in Figure 9a. After each cycle, the intensity of the excitonpeak of AgI slightly decreases, say about 5% decrease at the endof the sixth cycle. This is in contrast with our earlier study with

Ag/agar−agar,48 where the decrease in the SPR band of AgNPs was quite substantial during cycling, and we haveaccounted this for the loss of Ag NPs from the matrix to thesaline solution (Supporting Information Figure S3). As thesolubility product of AgI is very low [Ksp = 8.3 × 10−17],74 thechances of silver diffusing into the solution from Ag@AgI/agarose film is very little. There is no loss of either Ag or Ag+

from the matrix, and thus, the matrix remains almost stableduring cycling, and this accounts for the nature of the UV−visible spectra observed. Thus, the possibility of killing themicrobes by the mechanism of release killing may be ruled out.The other possibility is the production of ROS by Ag@AgI/agarose in contact with water in the dark.To test for the production of ROS by the samples in dark,

EPR experiments were carried out for Ag/agarose, AgI/agarose,and Ag@AgI/agarose along with DMPO in saline solution, andthe results are shown in Figure 10. None of these samplesshowed any EPR signal (Figure 10a−c), indicating thenonproduction of ROS in dark. This is further proved in thefluorescence experiment carried out for the detection of ROS.Again, none of these materials kept in saline solution in thedark exhibited any fluorescence (Figure 10h). Thus, killing dueto the production of ROS is also ruled out in the dark. The onlyother possibility left out is contact killing (bacteria coming incontact with the material). However, as the recycling of Ag@AgI/agarose could not be carried out more than five times withzero survival in the dark, it is possible that the adherence ofbacteria over the film might have prevented further action ofthe material after the fifth cycle.In the presence of light, Ag/agarose and AgI/agarose do not

show any EPR signal (Figure 10d,e), while Ag@AgI/agaroseexhibits a quartet in EPR indicating the production of thehydroxyl radical (OH•)75 (Figure 10f). The quartet signal isdue to the DMPO−OH adduct.76 But this disappears after thefirst cycle (Figure 10g). Also, Ag@AgI/agarose exposed to lightin saline solution exhibited fluorescence (Figure 10h),indicating the production of ROS. In the presence of light,the killing of bacteria is due to the combined action of contactkilling and oxidative stress. This combinatorial action reducesthe killing time to 45 min, when the bacteria were tested in thepresence of light. Further, in UV−vis spectra (Figure 9b), inaddition to the almost retention of intensity of the exciton peak(431 nm) on cycling in the presence of light, there appears abroad absorption band around 450−700 nm with a gradualincrease in intensity up to the fifth cycle. This broad absorptionband can be assigned to Ag obtained from photodecompositionof AgI and having a large size distribution. It is well-known thatAgI in the presence of light photoreduces to Ag.77 The gradualdampening of the corresponding PL spectra after each cyclefurther confirms the photodecomposition of AgI with theincrease in concentration of Ag (Supporting Information FigureS4). After photoreduction, the silver produced in the filmquenches the production of hydroxyl radical (OH•) and thuscould not be recycled efficiently.This is further confirmed by TEM studies conducted on the

bacteria (Figure 11). Figure 11a, b shows healthy E. coli and S.aureus bacteria, respectively, before contact. In the case of Ag/agarose, one can see the fine Ag particles which are diffusedfrom the film and has have to the cell walls, causing a totalchange in morphology of the bacteria, indicating the loss ofcellular integrity and the cytoplasm material flowing out,78,79

causing the death of the bacteria (Figure 11c, d). Whereas, inthe case of AgI/agarose and Ag@AgI/agarose films, the attack

Figure 11. TEM images of bacteria: healthy (a) E. coli and (b) S.aureus. Thin-section TEM images demonstrating the effect of (c, d)Ag/agarose, (e, f) Ag@AgI/agarose, and (g, h) AgI/agarose on (c, e,g) E. coli and (d, f, h) S. aureus.

Figure 12. Effect of Ag@AgI/agarose on HeLa cells: (a) cells treatedwith water exposed to Ag@AgI/agarose and (b) cells treated withunexposed water (the numbers 5, 3.3 in panel (a) are from the lensand can be ignored).

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of the bactericides seems to be different. It looks as if the endsurfaces or peripheral membrane of the bacteria are beingdestroyed as shown by arrows (Figure 11e−h). But no Agparticles are found inside the cell, which indicates indirectly thatno Ag particles have diffused from the agarose matrix. As AgIsolubility is very low in water, the Ag+ ions or the whole core@shell particle diffusing into the solution is also ruled out.However, after the fifth cycle, the Ag@AgI/agarose film isineffective to inhibit the bacterial growth; this may be due tothe possibility of a considerable amount of bacteria attaching tothe film, as the experiments are carried out in the static mode.In Vitro Cytotoxicity Assay. Our main objective is to

develop an organic−inorganic antimicrobial recyclable film thatcan kill or inhibit bacteria present in water with minimaltoxicity. So it would be more appropriate to check thecytotoxicity effect of water after being exposed to Ag@AgI/agarose coated glass with time. Upon conducting cytotoxicityexperiments, the results obtained showed that the HeLa cellstreated with exposed Ag@AgI/agarose were healthy with nogranulations nor was there any death similar to controls (Figure12). From these results, it is clear that Ag@AgI/agarose did notexhibit any toxicity to human cells exposed to either mineralwater or saline water even when the core@shell was held in itfor more than 2 months. Thus, the results show that the Ag@AgI/agarose core@shell exhibited excellent bactericidal activitybut almost no toxicity to human cells, indicating its potentialuse in the treatment of water to remove microorganisms forsafe drinking water or for other uses.

4. CONCLUSION

A novel synthetic method was adopted for the preparation of ahybrid containing a core@ shell structure of Ag@AgI in abiocompatible material such as agarose for the purpose of usingit as an efficient antibacterial agent for safe drinking water. Theconcept of using a low solubility material (AgI) as the shell is toprevent the diffusion of Ag into water, thus preventing thetoxicity of the material. The material is very well characterizedand showed its efficacy as a good bactericidal agent for repeateduse. The optical properties of the material have given someinteresting results. EPR and TEM experiments were carried outto elucidate the possible mechanism for the killing action of thematerial, and it is concluded that the bactericide is mainly dueto contact killing.

■ ASSOCIATED CONTENT

*S Supporting InformationAdditional figures showing the power spectrum of the metalhalide lamp, a schematic representation of the experimentalsetup, UV−vis spectra of SPR bands of Ag NPs on glass plates,and PL spectra of Ag@AgI/agarose film after each cycle in thepresence of light. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Telephone: +91-80-22933310. Fax: +91-80-23601310. E-mail:vasan@sscu.iisc.ernet.in.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors thank Ms. Bhagyashree (Physics Department,IISc) for recording EPR spectra and the Institute for theinternal funding for carrying out this work.

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