Ag@AgI, Core@Shell Structure in Agarose Matrix as Hybrid: Synthesis

May 14, 2012 - Citation data is made available by participants in Crossref's Cited-by ... Raid A. Ismail , Ghassan M. Sulaiman , Mayyadah H. Mohsin , ...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/Langmuir

Ag@AgI, Core@Shell Structure in Agarose Matrix as Hybrid: Synthesis, Characterization, and Antimicrobial Activity Somnath 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 no toxicity. The synthesized core@shell structure is very well characterized by XRD, UV−visible, photoluminescence, and TEM. A detailed antibacterial studies including repetitive cycles are carried out on Gram-negative Escherichia coli (E. coli) and Grampositive Staphylococcus aureus (S. aureus) bacteria in saline water, both in dark and on exposure to visible light. The hybrid could be recycled for the antibacterial activity and is nontoxic toward human cervical cancer cells (HeLa cells). The water insoluble Ag@AgI in agarose matrix forms a good coating on quartz, having good mechanical strength. EPR and TEM studies are carried out on the Ag@AgI/agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria. both in the form of neutral silver (Ag0)26,30−32 and in the oxidation state of +1 (Ag+) in the form of a suitable silver compound,33−35 nanoparticles of zinc oxide of different sizes and shapes,36,37 titanium dioxide,38,39 silicon dioxide,40,41 and so on.42−46 The basic approach for the study of antibacterial activity is to fabricate these materials in the form of powder,31,36,38,45 film,30,39 or embedded in a matrix.32 As the environmental hazards of nanomaterials as such during production, handling, and while in use are still not being well understood,47 one should be very cautious while working with such materials. When a nanomaterial is in the form of powder, the chances of enhanced toxicity may be high due to the difficulty in handling and also because of high solubility in a given solvent. The other disadvantage of using antimicrobial nanomaterials in the powder form is the difficulty in recovering the same after use. On the other hand when the bactericide nanoparticles are in the form of a film or embedded in a suitable matrix, one may overcome these disadvantages. And also the added advantage is that matrix can stabilize these particles48,49 for long-term usage. This also helps to have an easy coating on the medical devices made of stainless steel30 or polyurethane50 and so on. Our interest is directed toward the

1. INTRODUCTION Perhaps the two major problems facing the microbiologist and practicing medical professionals are, in tackling the microbes to prevent biofilm formation on various implantable medical devices1−4 and diseases caused by microbes in drinking water.5,6 Biofilm related infections are a major cause for morbidity and mortality,7,8 and it has been estimated that ∼60% bacterial infections in hospitals are due to biofilm formation.2,7 On the other hand, at least one-sixth of world’s population lack access to safe drinking water9 and the main sufferers are the children of the third world countries.6,10,11 Over a period of time, the extensive use of bactericides and chemicals such as chlorine for water treatment has led to the growth of new bacterial strains, resistant to multiple drugs (MDR),12,13 and also resulting in the formation of secondary carcinogenic disinfection byproduct (DBP).14−16 This has led to the tremendous development of new antibiotics in the recent past.17,18 In order to have simple processing of material with good stability and having efficient antibacterial activity with low toxicity, many inorganic based materials are being tried.19−23 With the advantages of nanomaterials over the bulk, especially with regard to the surface property, such as increased surface area24 which internally facilitates better interaction between the particles and the microbes, the inorganic nanomaterials have been widely studied as bactericides in the recent past.25−29 The well studied inorganic nanomaterials as bactericides are silver, © 2012 American Chemical Society

Received: March 30, 2012 Revised: May 10, 2012 Published: May 14, 2012 8550

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

excess solvent and water were pumped out, and 5 mL of 0.25 mmol KI solution was added in steps of 1 mL, at an interval of 30 min to the above solution at 90 °C with constant mixing to obtain Ag@AgI in agarose matrix. At the end, 20 mL of this solution was poured into a plastic Petri dish (Φ = 90 mm) and dried at room temperature until the mass could be peeled off as a transparent free-standing film. By a similar procedure, AgI/agarose was also prepared by taking 0.5 mmol of AgNO3 in previously prepared agarose solution (1.0 g of chloride free agarose in 100 mL of Millipore water) containing 100 mg of PVP and adding at once 20 mL of 0.5 mmol KI solution. The solution is thoroughly mixed for 3 h at 90 °C in a rotary evaporator. In order to ensure good mechanical strength for the films, for some specific studies, which will be described later, 200 μL of as prepared Ag/agarose, AgI/agarose, and Ag@AgI/agarose solutions were taken and drop-casted separately over quartz slides to form a film of area 1.0 cm2. Before carrying out any experiments, the films are washed repeatedly in Millipore water to remove impurities such as potassium nitrate that is formed during the reaction. Zeta Potential Measurement. Zeta potentials were recorded by using a Malvern Zetasizer Nano Z instrument at various stages of preparation by suitably diluting the reaction mixture. X-ray Diffraction (XRD). XRD patterns of Ag, AgI, and Ag@AgI in agaorse films were recorded in a Phillips XRD ‘X’ PERT PRO diffractometer using Cu Kα radiation (λ = 1.5438 Å) as the X-ray source. UV−Vis Absorption. UV−vis absorption studies were carried at the end of the formation of Ag NPs in agarose and after each step addition of KI solution. Typically, 1 mL of solution was taken out at the end of every 30 min and further diluted to 5 mL in Millipore water and scanned over the wavelength range 300−800 nm in a PerkinElmer Lambda 35 UV−vis spectrophotometer using a quartz cuvette with Millipore water as background. UV−visible spectra of Ag/agarose, AgI/agarose, and Ag@AgI/agarose coated on quartz slides were also recorded against uncoated quartz slide as reference. Photoluminescence (PL) Study. Like in the UV−visible absorption study, PL spectra were taken at the end of Ag NPs formation and also after every step addition of KI to Ag/agarose solution in a Jobin Yvon FluoroLog 4 (Horiba) instrument, with an excitation wavelength of 470 nm. PL spectra of Ag/agarose, AgI/ agarose, and Ag@AgI/agarose coated on quartz slides were also recorded. Transmission Electron Microscopy (TEM). The size and morphology of Ag and Ag@AgI nanoparticles were analyzed by TEM using a TECNAI F 30 transmission electron microscope. Care was taken to prepare and mount the samples on the Cu grid each time to avoid any possible agglomeration of particles while taking the films in water. All samples were prepared by similar conditions, by placing a drop of well-sonicated 1 cm × 1 cm film dissolved in 10 mL of Millipore water on a carbon-coated copper grid and subsequently dried in air before transferring it to the electron microscope, which was operated at an accelerated voltage of 200 kV. The healthy and the dead bacteria were observed via TEM by preparing thin slices of bacteria embedded in a polymer by microtome. After contact with nanohybrid materials for 4 h, the corresponding bacterial suspension was washed twice and fixed with 2% glutaraldehyde for 1.5 h. Samples were then post fixed with 1% lead acetate and dehydrated in acetone series (35, 50, 70, 80, 95, and 100%) for 3 min each. After dehydration by 100% acetone, samples were then embedded in SPURR resin of suitable size. Ultrathin sections were cut using an ultra microtome (RMC MT-X) and stained with uranyl acetate and lead citrate. The thin sections were taken on to the Cu grid, and micrographs were obtained using the JEOL (100 CX II) transmission electron microscope at 100 kV. Surface Profilometry. In order to measure the thickness of drop casted films over quartz slides, surface profilometry using a Veeco Dektak 150 instrument was used. The tip of the profilometer was scanned from the uncoated quartz surface (reference) to the film coated quartz (sample) surface, and the profilogram was obtained and the estimated difference in height of two profiles gives the thickness of

fabrication of inorganic nanomaterials in a suitable biocompatible matrix for the study of antibacterial activity. In order to control the microbial contamination in drinking water, composite materials loaded with slow releasing biocides such as heavy metals, antibiotics, and so on are being used.51−53 Among these, silver-based materials are of special interest. The strong inhibitory and bactericidal effect as well as broad spectrum of antimicrobial activity of silver have been known for a long time.30−32 Silver nanoparticles (Ag NPs) have low toxicity toward mammalian cells and do not easily provoke microbial resistance.54,55 A large amount of literature may be cited 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 literature available in the study of antimicrobial activity with the combination of Ag NPs and Ag+ in the form of core@shell structure embedded in a matrix. The advantages of such a combination are as follows: (i) if the shell consists of a silver compound, such as silver halide having very low solubility in water, it would prevent leaching out of Ag or Ag+ from the matrix and in turn causes less toxic to mammalian cell; (ii) one can have synergic effect of both Ag and Ag+ toward the killing of microbes; (iii) by embedding such a system in a matrix, one can 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 matrix as inorganic−organic hybrid in the form of a free-standing film. Agarose is a polysaccharide, is biocompatible, and is a cheap material that is readily available. The prepared samples are characterized by X-ray diffraction (XRD), UV−visible spectroscopy (UV−vis), photoluminescence spectroscopy, and transmission electron microscopy (TEM). The water insoluble Ag@AgI in agarose matrix can be coated as thin film on quartz plates, having good mechanical strength. It exhibited good antibacterial 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 visible light. Based on the UV−visible, EPR, and TEM studies, a plausible mechanism has been proposed for the killing of the bacteria by the hybrid. The hybrid could be recycled for many times and was also found to be nontoxic toward HeLa cells, when kept in mineral water and also in saline solution for more than 2 months.

2. EXPERIMENTAL SECTION Materials. Silver nitrate (99.9%), potassium iodide (99.8%), absolute alcohol (99.9%) (SD Fine-Chem Pvt. Ltd., India), polyvinyl pyrrolidone (PVP), Mw ∼ 55 000 (Aldrich Chemical Co. Inc.), and agarose (electrophoresis grade, Hi-Media Laboratories Ltd., India) are procured. All chemicals were used as such without any further purification. Saline solution of 0.85% NaCl in MilliQ water and standard reference strains E. coli MTCC 1302 and S. aureus ATCC 25923 (supplied by M.S. Ramaiah Hospital, Bangalore) were used for the bacterial studies. Synthesis of Ag@AgI Nanoparticles in Agarose Matrix. In situ Ag NPs of size 15−25 nm were synthesized in an agarose matrix according to the procedure reported by us earlier.48 In a typical reaction, 1.0 g of chloride free agarose was dissolved in 100 mL of Millipore water under boiling conditions in a rotary evaporator with constant mixing. To this clear solution, 60 mL of ethanol and 100 mg of 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 Millipore water and added dropwise to the above solution and refluxed for 3 h at 90 °C in the rotary evaporator. At the end of 3 h, the solution turns to golden yellow, ensuring complete reduction of Ag+ ions to Ag0. The 8551

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

the film. This is repeated for three different samples and the average thickness 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 in a mechanical shaker. The cultured microbial concentration was diluted to 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 saline solution and incubated at 37 °C with gentle mechanical shaking. To one of them, only a sterilized glass slide (positive control) and to the other a Ag@AgI/agarose coated glass slide (test beaker) was kept. At an interval of 1 h, 100 μL of incubated solution was taken out for plating in nutrient agar plates. After overnight incubation at 37 °C, the viable colonies were visible to the naked eye and thus counted manually and compared with positive control. For reusability test (cycling), the slides were rinsed thoroughly with sufficient water; and after each bacterial study, the slides were dried in air, and further on a subsequent 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 glass slide was exposed to the light source of a metal halide lamp (OSRAM, 150W, power spectrum is provided in Figure S1 of the Supporting Information). The lamp was kept at an appropriate distance, such that the temperature was maintained between 37 and 39 °C near the sample, when the lamp was illuminated. The temperature maintenance was 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 water were detected by EPR-spin trapping technique using spin trapper 5,5dimethyl-1-pyrroline-N-oxide (DMPO, 0.02 M) (Sigma Aldrich). Ag/ agarose, AgI/agarose, and Ag@AgI/agarose coated glass slides were dipped in water containing DMPO for 2 h in the dark and similarly on exposure to visible light. A known volume of these solutions was drawn into quartz capillary tubes and placed in an EPR tube, and spectral studies were carried out on Bruker emx X-band EPR spectrometer. 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 ROS by incubating the samples in 30 mL of 0.05 m (M) DCFH-DA solution for 2 h, both in the dark and on exposure to visible light. The fluorescence intensity of the solution was measured in a Jobin Yvon Fluoro Log 4 (Horiba) instrument, with an excitation wavelength of 485 nm. Cytotoxicity Assay. In vitro cytotoxicity assay for the Ag@AgI nanoparticles embedded in agarose (Ag@AgI/agarose) was carried out by exposing human cervical cancer cell line (HeLa cells) in disposable plastic 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 and saline solution. Then after each hour, 100 μL of this water and saline solution was pipetted into 25 mL capacity wells, and then 0.1 mL freshly trypsinized HeLa cells cultured as a monolayer in Dulbecco’s modified Eagle's medium (DMEM) (containing 10% fetal calf serum and 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 and saline water were left as controls. After the incubation for different intervals, the cells were observed for healthy conditions and death, if any, under an inverted microscope. A similar experiment was repeated after keeping the Ag@AgI/agarose glass slides in mineral water and saline water separately for 2 months and observing for cytotoxocity.

Scheme 1. Schematic Illustration of Stepwise Formation of Core@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.

that a thin layer of AgI is formed over Ag core in the first step (this is further discussed in UV−visible absorption spectral studies). And on further addition of KI, the shell thickness increases as depicted in Scheme 1. The progress of the reaction in solution is monitored by the pH, UV−visible absorption spectra, and zeta potential (ζ) measurements. During the formation of the core@shell structure, the pH was found to be in the acidic range varying from 3.8 to 4.8. The formation of core@shell structure in the acidic medium may be explained by the following reaction as proposed by Mulvaney56 Ag n + mH+ + mI− → Ag(n − m)(AgI)m + (m /2)H 2

And ζ recorded, from the formation of Ag NPs to the core@ shell structure, increases gradually from very negative potential of −12.7 mV to −5.51 mV and to −3.51 mV on first addition to the fifth addition of KI solution. This change in surface charge may be presumed to the initial adsorption of negative charged species such as NO3− on Ag NPs and on addition of KI, the possible removal of NO3− ions from the surface as KNO3, along with the formation of the AgI shell. X-ray Diffraction (XRD). Figure 1 shows the XRD pattern of agarose, Ag/agarose, AgI/agarose, and Ag@AgI/agarose coated over quartz slides. The broad diffraction band around 20°−30° is seen for both amorphous agarose and Ag/agarose, and the absence of diffraction peaks corresponding to Ag in Ag/agarose is due to very low concentration of Ag present. The ICP-OES analysis of Ag present in Ag/agarose film coated on the slide is found to be 31.33 (±0.2371) μg cm−2. In the case of AgI/agarose and Ag@AgI/agarose, prominent peaks of low

3. RESULTS AND DISCUSSION Synthesis of Ag@AgI Nanoparticles in Agarose Matrix. In situ synthesis of Ag@AgI, core@shell structure in agarose matrix was achieved by the stepwise addition of KI solution of lower concentration (0.25 mmol) compared to the original silver concentration (0.5 mmol) taken. It is presumed 8552

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

Figure 2. UV−visible spectra showing (a) the stepwise addition of KI, (b) the subtracted spectra, (c) the deconvoluted spectra of the top three spectra 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.

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

temperature phases (β, γ) of AgI are observed. But it is difficult to assign the diffraction peaks exactly to any one of the known phases (β-AgI, γ- AgI), as their reflections are very close. However, on careful observation of the diffraction peaks of AgI/agarose (Figure 1c) and Ag@AgI/agarose (Figure 1d), one can see the absence of (102) and (112) and the decrease in 8553

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

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

(Figure 2a). The appearance of a surface plasmon resonance (SPR) band at 412 nm confirms the formation of Ag NPs in agarose. On addition of KI solution, the SPR seems to disappear, with the formation of a long absorption tail and the appearance of an exciton peak of AgI, when KI reaches a particular concentration. Further analysis of the spectra was carried out by subtracting the spectrum of Ag NPs, that is, without any addition of KI from each one of these spectra, and the resulting plots are shown in Figure 2b. The initial SPR band of AgNPs (412 nm) shows immediate dampening and broadening on addition of 10 μL of KI (Ag to I− mole ratio = 1:0.25 × 10−3). The dampening of the SPR band continues further 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) is added, along with the dampening of the spectra the evolution of Z1,2 exciton peak (421 nm) appears as a knee and is due to γAgI.58 The dampening of the SPR band is due to the initial adsorption 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− mole ratio = 1:6.25 × 10−3), the shape of the curve changes, with no visible turf due to dampening but a neat formation of exciton peak of AgI is seen; this perhaps indicates the formation of a complete shell of AgI on Ag NPs. On further addition of KI, the

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.

have AgI phase rich in Ag. Based on this reasoning, one may conclude that the AgI phase in the core@shell structure is predominantly γ-AgI, having a zinc blend structure. UV−Vis Absorption Study. In order to understand the formation of core@shell structures, the UV−visible spectral studies were carried out further on addition of very low aliquots from 10 to 1050 μL of KI solution (0.25 mmol in 20 mL) to Ag NPs in agarose and the spectra were recorded in solution 8554

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

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.

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

intensity of the exciton peak increases, with no shift in peak position. The top three plots of Figure 2b were further deconvoluted and are shown in Figure 2c. The Z1,2 exciton peak of AgI splits into two bands, centered at 420 nm and 415 nm (Figure 2c). This was also observed earlier by Mochizuki and Umezawa59 in their studies on optical properties on microcrystals of AgI, and they explain that the splitting may be due to 8555

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

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.

PL emission of Ag@AgI/agarose film is much higher than that of the AgI/agarose film, indicating the effect of core@shell structure but with no change in peak position. However, when compared with the same concentration of Ag@AgI/agarose in solution (curve 5 of Figure 4a), there is found to be a blue-shift. This is mainly due to change in dielectric constant of the medium. Transmission Electron Microscopy (TEM) Study. Figure 5a, b shows the TEM pictures of Ag/agarose and AgI/agarose, respectively. Both Ag NPs and AgI NPs are more or less spherical in shape, with the size in the range 18−27 nm. The TEM images of Ag@AgI/agarose (Figure 5c) shows an interior dark shade surrounded by a lighter shade for most of the particles, and this contrast indicates a core@shell structure. However, a small amount of Ag NPs, especially the very smaller ones forming only AgI particles, are not ruled out. Further, a HRTEM 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 estimated shell thickness is around 4.77 nm (Figure 5e). Surface Profilometry. Figure 6 shows the surface profilometry plot taken on the film Ag@AgI/agarose coated on a glass slide. The profilogram shows a thickness of ∼3.5 μm. The line obtained, all most parallel upon scanning ∼500 μm along the sample, indicates that the surface of the film is quite smooth. Antibacterial Activity. The antibacterial activity testing was done against both E. coli and S. aureus. Our interest in the study of a cheap and effective inorganic/organic hybrid based antimicrobial agent has earlier led to the development of Ag/ agar−agar films.48 Further attempts to improve on this system led to the study of the present work. Figure 7 shows the percentage survival versus exposure time in the dark and on exposure to light for films of all the samples synthesized on the glass slide. In dark, the percentage survival of E. coli remains above 80% at the end of contact time of 30 min for all the samples. But after this contact time, there is a steep decrease in survival 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 with Gram positive S. aureus, and with zero survival around 2.5 h for Ag@AgI/agarose film (Figure 7b). When the experiment was repeated on exposure of films to light, an interesting result was

effective diameter of AgI shell, calculated from Bruss equation,60 Enano = E bulk + [h2 /2m*d 2] − [1.8e 2 /2πCC0d]

where m* (reduced mass of exciton) = 0.185 me (reduced mass of electron), ε = 5.6, d = effective diameter of the particle, was plotted against the band gap energy obtained from UV−vis spectra (Figure 2b and c) and shown in Figure 2d and also a corresponding table including the amount of KI solution added in each case is shown as an inset. From the table, it may be inferred that the monolayer formation of AgI shell on AgNPs is taking place, when KI added is around 250 μL (Ag to I− mole ratio 1: 6.25 × 10−3), which fairly compares with the shape of UV−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 higher wavelengths (red-shift), that is, from 421 to 431 nm, an increase of 10 nm and also with increase in intensity (Figure 3a). This is due to the increase in thickness of the AgI shell.60−62 The UV−vis spectra of Ag NPs/agarose, AgI/ agarose, and Ag @AgI/agarose as film coated on quartz slide having the same area and thickness, containing equal concentrations of Ag, are shown in Figure 3b. The absorption intensity of Ag @AgI core@shell is much higher than that of the film containing only AgI; this is because of the increased electron population in the conduction band of AgI due to the transfer of electrons from excited Ag NPs to AgI.63 This further substantiates 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 most intense peak. The appearance of the 547 nm peak can be attributed to the exciton recombination.64 Upon stepwise addition of KI, the peak at 547 nm (Figure 4a) gets red-shifted to 566 nm, with an increase in intensity, accompanied by the decrease in full width at half maximum (FWHM) which indicates the subsequent growth of AgI shell over Ag core. As the thickness of the shell increases, the band gap decreases, causing a red-shift in the emission spectra. The PL emission spectra of agarose, Ag/agarose, AgI/agarose, and Ag@AgI/ agarose coated film over quartz slides are also shown in Figure 4b. Similar to the observed UV−vis spectra, the intensity of the 8556

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

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 after incubating with material under dark and light.

was seen on exposure to light (Figure 8b). These results were a bit surprising. What are the plausible reasons for the antibacterial activity of the samples in the dark and in the presence of light? What might be the possible mechanism for the killing of bacteria by the Ag@AgI/agarose hybrid film? We have made few attempts to answer these questions based on UV−vis, EPR, and TEM studies. The germicidal effect of nanoparticles is wellknown,30,36,38,40,42 but their mode of action is still inconclusive,65−69 though people have suggested several mechanisms including the killing due to production of reactive oxygen species (ROS),36,37 contact killing,69 that is, cell wall attachment and internalization,70,71 deactivation of enzymatic activity of bacterial respiratory chain,72,73 bacterial structural changes by the nanoparticles due to release killing,65,66 and so forth. But it

found. The rate of killing of the bacteria is still faster in the case of Ag@AgI/agarose, and it took only 45 min to kill both Gram negative and Gram positive bacteria in saline solution. In the case of Ag/agarose and AgI/agarose films, the antibacterial activity trend is almost the same as in the dark, indicating only a marginal 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 on exposure to light, the film was exposed to the bacteria in saline solution and is recycled up to five times by taking fresh bacterial solution each time. The percent survival was determined as before and plotted against the number of cycles as a bar chart (Figure 8). The performance of the film in the dark was good up to the fifth and fourth cycles in the case of E. coli and S. aureus, respectively (Figure 8a); however, no such performance 8557

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

Ag/agar−agar,48 where the decrease in the SPR band of Ag NPs was quite substantial during cycling, and we have accounted this for the loss of Ag NPs from the matrix to the saline solution (Supporting Information Figure S3). As the solubility product of AgI is very low [Ksp = 8.3 × 10−17],74 the chances 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 stable during cycling, and this accounts for the nature of the UV− visible spectra observed. Thus, the possibility of killing the microbes 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, and the results are shown in Figure 10. None of these samples showed any EPR signal (Figure 10a−c), indicating the nonproduction of ROS in dark. This is further proved in the fluorescence experiment carried out for the detection of ROS. Again, none of these materials kept in saline solution in the dark exhibited any fluorescence (Figure 10h). Thus, killing due to the production of ROS is also ruled out in the dark. The only other possibility left out is contact killing (bacteria coming in contact with the material). However, as the recycling of Ag@ AgI/agarose could not be carried out more than five times with zero survival in the dark, it is possible that the adherence of bacteria over the film might have prevented further action of the 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/agarose exhibits a quartet in EPR indicating the production of the hydroxyl radical (OH•)75 (Figure 10f). The quartet signal is due to the DMPO−OH adduct.76 But this disappears after the first cycle (Figure 10g). Also, Ag@AgI/agarose exposed to light in 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 contact killing and oxidative stress. This combinatorial action reduces the killing time to 45 min, when the bacteria were tested in the presence of light. Further, in UV−vis spectra (Figure 9b), in addition to the almost retention of intensity of the exciton peak (431 nm) on cycling in the presence of light, there appears a broad absorption band around 450−700 nm with a gradual increase in intensity up to the fifth cycle. This broad absorption band can be assigned to Ag obtained from photodecomposition of AgI and having a large size distribution. It is well-known that AgI in the presence of light photoreduces to Ag.77 The gradual dampening of the corresponding PL spectra after each cycle further confirms the photodecomposition of AgI with the increase in concentration of Ag (Supporting Information Figure S4). After photoreduction, the silver produced in the film quenches the production of hydroxyl radical (OH•) and thus could 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 diffused from the film and has have to the cell walls, causing a total change in morphology of the bacteria, indicating the loss of cellular integrity and the cytoplasm material flowing out,78,79 causing the death of the bacteria (Figure 11c, d). Whereas, in the 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 treated with water exposed to Ag@AgI/agarose and (b) cells treated with unexposed water (the numbers 5, 3.3 in panel (a) are from the lens and can be ignored).

is still not clear in many of the cases whether the killing is due to any one individual mechanism or due to the combination of more 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 is shown in Figure 9a. After each cycle, the intensity of the exciton peak of AgI slightly decreases, say about 5% decrease at the end of the sixth cycle. This is in contrast with our earlier study with 8558

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir of the bactericides seems to be different. It looks as if the end surfaces or peripheral membrane of the bacteria are being destroyed as shown by arrows (Figure 11e−h). But no Ag particles are found inside the cell, which indicates indirectly that no Ag particles have diffused from the agarose matrix. As AgI solubility 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 is ineffective to inhibit the bacterial growth; this may be due to the possibility of a considerable amount of bacteria attaching to the 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 that can kill or inhibit bacteria present in water with minimal toxicity. So it would be more appropriate to check the cytotoxicity effect of water after being exposed to Ag@AgI/ agarose coated glass with time. Upon conducting cytotoxicity experiments, the results obtained showed that the HeLa cells treated with exposed Ag@AgI/agarose were healthy with no granulations nor was there any death similar to controls (Figure 12). From these results, it is clear that Ag@AgI/agarose did not exhibit any toxicity to human cells exposed to either mineral water or saline water even when the core@shell was held in it for more than 2 months. Thus, the results show that the Ag@ AgI/agarose core@shell exhibited excellent bactericidal activity but almost no toxicity to human cells, indicating its potential use in the treatment of water to remove microorganisms for safe drinking water or for other uses.

■

REFERENCES

(1) Angelova, N.; Hunkeler, D. Rationalizing the design of polymeric Biomaterials. Trends Biotechnol. 1999, 17, 409−421. (2) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial Biofilms: A Common Cause of Persistent Infections. Science 1999, 284, 1318−1322. (3) Hall-Stoodley, L.; Costerton, J. W.; Stoodley., P. Bacterial Biofilms: From the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2, 95−108. (4) Langer, R.; Tirrell, D. A. Design materials for Biology and Medicine. Nature 2004, 428, 487−492. (5) Halabi, M.; Dorothea, O.; Katharina, G.; Wiesholzer-Pittl, M.; Kainz, M.; Schoberl, J.; Dierich, P. M.; Allerberger, F.; Wurzner, R. Prevalence of Shiga toxin-, intimin- and haemolysin genes in Escherichia coli isolates from drinking water supplies in a rural area of Austria. Int. J. Hyg. Environ. Health 2008, 211, 454−457. (6) Ashbolt, N. J. Microbial contamination of drinking water and disease outcomes in developing regions. Toxicology 2004, 198, 229. (7) Klevens, R. M.; Edwards, J. R.; Richards, C. L., Jr Estimating health care-associated infections and deaths in U.S. hospitals, 2002. Public Health Rep. 2007, 122, 160−166. (8) Lichter, J. A.; Van Vlietpa, K. J.; Rubner, M. F. Design of Antibacterial Surfaces and Interfaces: Polyelectrolyte Multilayers as a Multifunctional Platform. Macromolecules 2009, 42, 8573−8586. (9) Montgomery, M. A.; Elimelech, M. Water and Sanitation in Developing Countries: Including Health in the Equation: Millions suffer from preventable illnesses and die every year. Environ. Sci. Technol. 2007, 41, 17−24. (10) Bresee, S. J.; Hummelman, E.; Nelson, E. A. S.; Glass, I. R. Rotavirus in Asia: The Value of Surveillance for Informing Decisions about the Introduction of New Vaccines. J. Infect. Dis. 2005, 192, S1− S5. (11) Sadeghi, G. H.; Mohammadian, M.; Nourani, M.; Peyda, M.; Eslami, A. Microbiological Quality Assessment of Rural Drinking Water Supplies in Iran. J. Agric. Soc. Sci. 2007, 3, 31−33. (12) Ridgway, H. F.; Olson, B. H. Chlorine Resistance Patterns of Bacteria from Two Drinking Water Distribution Systems. Appl. Environ. Microbiol. 1982, 44, 972−987. (13) Alanis, J. A. Resistance to Antibiotics: Are We in the PostAntibiotic Era? Arch. Med. Res. 2005, 36, 697−705. (14) Krasner, S. W.; Weinberg, H. S.; Richardson, S. D.; Pastor, S. J. Occurrence of a New Generation of Disinfection Byproducts. Environ. Sci. Technol. 2006, 40, 7175−7185. (15) Richardson, S. D.; Thruston, A. D.; Collette, T. W. Multispectral Identification of Chlorine Dioxide Disinfection Byproducts in Drinking Water. Environ. Sci. Technol. 1994, 28, 592−599. (16) Li, Q.; Mahendra, S.; Lyon, D. Y.; Brunet, L.; Liga, M. V.; Li, D; Alvarez, P. J. Antimicrobial nanomaterials for water disinfection and microbial control: Potential applications and implications. Water Res. 2008, 42, 4591−4602. (17) Butler, M. S.; Cooper, A. M. Antibiotics in the clinical pipeline in 2011. J. Antibiot. 2011, 64, 413−425. (18) Clardy, J.; Fischbach, M. A.; Walsh, C. T. New antibiotics from bacterial natural products. Nat. Biotechnol. 2006, 24, 1541−1550. (19) Fang, M.; Chen, J.-H..; Xu, X.-L..; Yang, P.-H..; Hildebrand, H. F. Antibacterial activities of inorganic agents on six bacteria associated with oral infections by two susceptibility tests. Int. J. Antimicrob. Agents 2006, 27, 513−517. (20) Slenters, T. V.; Hauser-Gerspach, I.; Daniels, A. U.; Fromm, K. M. Silver coordination compounds as light-stable, nano-structured and anti-bacterial coatings for dental implant and restorative materials. J. Mater. Chem. 2008, 18, 5359−5362.

ASSOCIATED CONTENT

S Supporting Information *

Additional figures showing the power spectrum of the metal halide lamp, a schematic representation of the experimental setup, UV−vis spectra of SPR bands of Ag NPs on glass plates, and PL spectra of Ag@AgI/agarose film after each cycle in the presence of light. This material is available free of charge via the Internet at http://pubs.acs.org.

■

ACKNOWLEDGMENTS

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

4. CONCLUSION A novel synthetic method was adopted for the preparation of a hybrid containing a core@ shell structure of Ag@AgI in a biocompatible material such as agarose for the purpose of using it as an efficient antibacterial agent for safe drinking water. The concept of using a low solubility material (AgI) as the shell is to prevent the diffusion of Ag into water, thus preventing the toxicity of the material. The material is very well characterized and showed its efficacy as a good bactericidal agent for repeated use. The optical properties of the material have given some interesting results. EPR and TEM experiments were carried out to elucidate the possible mechanism for the killing action of the material, and it is concluded that the bactericide is mainly due to contact killing.

■

■

Article

AUTHOR INFORMATION

Corresponding Author

*Telephone: +91-80-22933310. Fax: +91-80-23601310. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 8559

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

(21) Gerasimchuk, N.; Gamian, A.; Glover, G.; Szponar, B. Light Insensitive Silver(I) Cyanoximates As Antimicrobial Agents for Indwelling Medical Devices. Inorg. Chem. 2010, 49, 9863−9874. (22) Bujdak, J.; Jurecekova, J.; Bujdakova, H.; Lang, K.; Sersen, F. Clay Mineral Particles As Efficient Carriers of Methylene Blue Used for Antimicrobial Treatment. Environ. Sci. Technol. 2009, 43, 6202− 6207. (23) Ahmad, S.; Isab, A. A.; Ali, S.; Al-Arfaj, A. R. Perspectives in bioinorganic chemistry of some metal based therapeutic agents. Polyhedron 2006, 25, 1633−1645. (24) Daniel, M. C.; Astruc, D. Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chem. Rev 2004, 104, 293−346. (25) Zhang, Y.; Peng, H.; Huang, W.; Zhou, Y.; Zhang, X.; Yan, D. Hyperbranched Poly(amidoamine) as the Stabilizer and Reductant To Prepare Colloid Silver Nanoparticles in Situ and Their Antibacterial Activity. J. Phys. Chem. C 2008, 112, 2330−2336. (26) Banerjee, M.; Mallick, S.; Paul, A.; Chattopadhyay, A.; Ghosh, S. S. Heightened Reactive Oxygen Species Generation in the Antimicrobial Activity of a Three Component Iodinated ChitosanSilver Nanoparticle Composite. Langmuir 2010, 26, 5901−5908. (27) Dutta, R. K.; Sharma, P. K.; Bhargava, R.; Kumar, N.; Pandey, A. C. Differential Susceptibility of Escherichia coli Cells toward Transition Metal-Doped and Matrix-Embedded ZnO Nanoparticles. J. Phys. Chem. B 2010, 114, 5594−5599. (28) Brayner, R.; Dahoumane, S. A.; Yepremian, C.; Djediat, C.; Meyer, M.; Coute, A.; Fievet, F. ZnO Nanoparticles: Synthesis, Characterization, and Ecotoxicological Studies. Langmuir 2010, 26, 6522−6528. (29) Ghosh, S.; Goudar, V. S.; Padmalekha, K. G.; Bhat, S. V.; Indi, S. S.; Vasan, H. N. ZnO/Ag nanohybrid: synthesis, characterization, synergistic antibacterial activity and its mechanism. RSC Adv. 2012, 2, 930−940. (30) Ghosh, S.; Ranebennur, T. K.; Vasan, H. N. Study of Antibacterial Efficacy of Hybrid Chitosan-Silver Nanoparticles for Prevention of Specific Biofilm and Water Purification. Int. J. Carbohydr. Chem. 2011, 2011, 693759. (31) Alonso, A.; Munoz-Berbel, X.; Vigues, N.; Macanas, J.; Munoz, M.; Mas, J.; Muraviev, D. N. Characterization of Fibrous Polymer Silver/Cobalt Nanocomposite with Enhanced Bactericide Activity. Langmuir 2012, 28, 783−790. (32) Gottesman, R.; Shukla, S.; Perkas, N.; Solovyov, L. A.; Nitzan, Y.; Gedanken, A. Sonochemical Coating of Paper by Microbiocidal Silver Nanoparticles. Langmuir 2011, 27, 720−726. (33) Wang, Q.; Yu, H.; Zhong, L.; Liu, J.; Sun, J.; Shen, J. Incorporation of Silver Ions into Ultrathin Titanium Phosphate Films: In Situ Reduction to Prepare Silver Nanoparticles and Their Antibacterial Activity. Chem. Mater. 2006, 18, 1988−1994. (34) Balogh, L.; Swanson, Douglas, D. R.; Tomalia, A.; Hagnauer, G. L.; McManus, A. T. Dendrimer-Silver Complexes and Nanocomposites as Antimicrobial Agents. Nano Lett. 2001, 1, 18−21. (35) Malcher, M.; Dmitry, V.; Heurtault, B.; Andre, P.; Schaaf, P.; Mohwald, H.; Voegel, J. C.; Sokolowski, A.; Ball, V.; Boulmedais, F.; Frisch., B. Embedded Silver Ions-Containing Liposomes in Polyelectrolyte Multilayers: Cargos Films for Antibacterial Agents. Langmuir 2008, 24, 10209−10215. (36) Raghupathi, K. R.; Koodali, R. T.; Manna, A. C. Size-Dependent Bacterial Growth Inhibition and Mechanism of Antibacterial Activity of Zinc Oxide Nanoparticles. Langmuir 2011, 27, 4020−4028. (37) Applerot, G.; Lipovsky, A.; Dror, R.; Perkas, N.; Nitzan, Y.; Lubart, R.; Gedanken, A. Enhanced Antibacterial Activity of Nanocrystalline ZnO Due to Increased ROS-Mediated Cell Injury. Adv. Funct. Mater 2009, 19, 842−852. (38) Kong, H.; Song, J.; Jang, J. Photocatalytic Antibacterial Capabilities of TiO2-Biocidal Polymer Nanocomposites Synthesized by a Surface-Initiated Photopolymerization. Environ. Sci. Technol. 2010, 44, 5672−5676.

(39) Fu, G.; Vary, P. S.; Lin, C. T. Anatase TiO2 Nanocomposites for Antimicrobial Coatings. J. Phys. Chem. B 2005, 109, 8889−8898. (40) Jang, J.; Kim, Y. Fabrication of monodisperse silica−polymer core−shell nanoparticles with excellent antimicrobial efficacy. Chem. Commun. 2008, 4016−4018. (41) Rosemary, M. J.; MacLaren, I.; Pradeep, T. Investigations of the Antibacterial Properties of Ciprofloxacin@SiO2. Langmuir 2006, 22, 10125−10129. (42) Pang, M.; Hu, J.; Zeng, H. C. Synthesis, Morphological Control, and Antibacterial Properties of Hollow/Solid Ag2S/Ag Heterodimers. J. Am. Chem. Soc. 2010, 132, 10771−10785. (43) Wang, X.; Wu, H. F.; Kuang, Q.; Huang, R. B.; Xie, Z. X.; Zheng, L. S. Shape-Dependent Antibacterial Activities of Ag2O Polyhedral Particles. Langmuir 2010, 26, 2774−2778. (44) Kim, Y. H.; Lee, D. K.; Cha, H. G.; Kim, C. W.; Kang, Y. C.; Kang, Y. S. Preparation and Characterization of the Antibacterial Cu Nanoparticle Formed on the Surface of SiO2 Nanoparticles. J. Phys. Chem. B 2006, 110, 24923−24928. (45) Cioffi, N.; Torsi, L.; Ditaranto, N.; Tantillo, G.; Ghibelli, L.; Sabbatini, L.; Bleve-Zacheo, T.; D’Alessio, M.; Zambonin, P. G.; Traversa, E. Copper Nanoparticle/Polymer Composites with Antifungal and Bacteriostatic Properties. Chem. Mater. 2005, 17, 5255− 5262. (46) Sileika, T. S.; Kim, H. D.; Maniak, p.; Messersmith, P. B. Antibacterial Performance of Polydopamine-Modified Polymer Surfaces Containing Passive and Active Components. ACS Appl. Mater. Interfaces 2011, 3, 4602−3410. (47) Musee, N.; Thwala, M.; Nota, N. The antibacterial effects of engineered nanomaterials: implications for wastewater treatment plants. J. Environ. Monit. 2011, 13, 1164−1183. (48) Ghosh, S.; Kaushik, R.; Nagalakshmi, K.; Hoti, S. L.; Menezes, G. A.; Harish, B. N.; Vasan, H. N. Antimicrobial activity of highly stable silver nanoparticles embedded in agar-agar matrix as a thin film. Carbohydr. Res. 2010, 345, 2220−2227. (49) Travan, A.; Pelillo, C.; Donati, I.; Marsich, E.; Benincasa, M.; Scarpa, T.; Semeraro, S.; Turco, G.; Gennaro, R.; Paoletti, S. Noncytotoxic Silver Nanoparticle-Polysaccharide Nanocomposites with Antimicrobial Activity. Biomacromolecules 2009, 10, 1429−1435. (50) Jain, P.; Pradeep, T. Potential of Silver Nanoparticle-Coated Polyurethane Foam As an Antibacterial Water Filter. Biotechnol. Bioeng. 2005, 90, 59−63. (51) Pavlukhina, S.; Lu, Y.; Patimetha, A.; Libera, M.; Sukhishvili, S. Polymer Multilayers with pH-Triggered Release of Antibacterial Agents. Biomacromolecules 2010, 11, 3448−3456. (52) Lalueza, P.; Monzon, M.; Arruebo, M.; Santamaria, J. Antibacterial action of Ag-containing MFI zeolite at low Ag loadings. Chem. Commun 2011, 47, 680−682. (53) Nguyen, P. M.; Zacharia, N. S.; Verploengen, E.; Hammond, P. T. Extended Release Antibacterial Layer-by-Layer Films Incorporating Linear-Dendritic Block Copolymer Micelles. Chem. Mater. 2007, 19, 5524−5530. (54) Gosheger, G.; Hardes, J.; Ahrens, H.; Streitburger, A.; Buerger, H.; Erren, M.; Gunsel, A.; Kemper, F. H.; Winkelmann, W.; von Eiff, C. Silver-coated megaendoprostheses in a rabbit model-an analysis of the infection rate and toxicological side effects. Biomaterials 2004, 25, 5547−5556. (55) Percival, S. L.; Bowler, P. G.; Russell, D. Bacterial resistance to silver in wound care. J. Hosp. Infect. 2005, 60, 1−7. (56) Mulvaney, P. Surface Plasmon Spectroscopy of Nanosized Metal Particles. Langmuir 1996, 12, 788−800. (57) Mochizuki, S.; Fujishiro, F. Structural, electrical and optical studies on AgI−anatase composites. J. Phys.: Condens. Matter 2003, 15, 5057−5072. (58) Cardona, M. Optical Properties of the Silver and Cuprous Halides. Phys. Rev. 1963, 129, 69−78. (59) Mochizuki, S.; Umezawa, K. Optical spectra of free and quasifree AgI microcrystals. Phys. Lett. A 1997, 228, 111−117. (60) Pedersen, D. V.; Wang, S. Iodination of Gas-Phase-Generated Ag Nanoparticles: Behavior of the Two Spin Orbit Components of the 8560

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561

Langmuir

Article

AgI Exciton in Ag@AgI Core−Shell Nanoparticles. J. Phys. Chem. C 2007, 111, 1261−1267. (61) Xu, Z.; Hou, Y.; Sun, S. Magnetic Core/Shell Fe3O4/Au and Fe3O4/Au/Ag Nanoparticles with Tuneable Plasmonic Properties. J. Am. Chem. Soc. 2007, 129, 8698−8699. (62) Ma, Y.; Li, W.; Cho, E. C.; Li., Z.; Yu, T.; Zeng, J.; Xie, Z.; Xia, Z. Au@Ag Core_Shell Nanocubes with Finely Tuned and WellControlled Sizes, Shell Thicknesses, and Optical Properties. ACS Nano 2010, 4, 6725−6734. (63) Hu, C.; Peng, T.; Hu, X.; Nie, Y.; Zhou, X.; Qu, J.; He, H. Plasmon-Induced Photodegradation of Toxic Pollutants with Ag-AgI/ Al2O3 under Visible-Light Irradiation. J. Am. Chem. Soc. 2010, 132, 857−862. (64) Zhao, L.; Wang, Y. X.; Chen, Z.; Zou, Y. M. Preparation, characterization, and optical properties of host−guest nanocomposite material SBA-15/AgI. Phys. B (Amsterdam, Neth.) 2008, 403, 1775− 1780. (65) Sondi, I.; Salopek-Sondi, B. Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J. Colloid Interface Sci. 2004, 275, 177−182. (66) Yang, W.; Shen, C.; Ji, Q.; An, H.; Wang, J.; Liu, Q.; Zhang, Z. Food storage material silver nanoparticles interfere with DNA replication fidelity and bind with DNA. Nanotechnology 2009, 20, 085102−085108. (67) Panacek, A.; Kolar, M.; Vecerova, R.; Prucek, R.; Soukupova, J.; Krystof, V.; Hamal, P.; Zboril, R.; Kvitek, L. Antifungal activity of silver nanoparticles against Candida spp. Biomaterials 2009, 30, 6333−6340. (68) Shrivastava, S.; Bera, T.; Roy, A.; Singh, G.; Ramachandrarao, P.; Dash, D. Characterization of enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology 2007, 18, 225103−225111. (69) Sanpui, P.; Chattopadhyay, A.; Ghosh, S. S. Induction of Apoptosis in Cancer Cells at Low Silver Nanoparticle Concentrations using Chitosan Nanocarrier. ACS Appl. Mater. Interfaces 2011, 3, 218− 228. (70) Feng, Q. L.; Wu, J.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. 2000, 52, 662−668. (71) Song, H. Y.; Ko, K. K.; Oh, L. H.; Lee, B. T. Fabrication of silver nanoparticles and their antimicrobial mechanisms. Eur. Cells Mater. 2006, 11, 58. (72) Klasen, H. J. A historical review of the use of silver in the treatment of burns. II. Renewed interest for silver. Burns 2000, 26, 131−138. (73) Kim, K. J.; Sung, W. S.; Suh, B. K.; Moon, S. K.; Choi, J. S.; Kim, J. G.; Lee, D. G. Antifungal activity and mode of action of silver nanoparticles on Candida albicans. BioMetals 2009, 22, 235−242. (74) CRC Handbook of Chemistry and Physics; Lide, D. R., Ed.; The Chemical Rubber Co.: Cleveland, OH, 1999. (75) Guo, J.-F..; Ma, B.; Yin, A.; Fan, K.; Dai, W.-L. Photodegradation of rhodamine B and 4-chlorophenol using plasmonic photocatalyst of Ag−AgI/Fe3O4@SiO2 magnetic nanoparticle under visible light irradiation. Appl. Catal. B, 2011, 101, 580−586. (76) Lipovsky, A.; Tzitrinovich, Z.; Friedmann, H.; Applerot, G.; Gedanken, A.; Lubart, R. EPR Study of Visible Light-Induced ROS Generation by Nanoparticles of ZnO. J. Phys. Chem. C 2009, 113, 15997−16001. (77) Rowland, S. C.; Layton, R. G.; Smith, D. R. photolytic activation of silver iodide in the nucleation of ice. J. Atmos. Sci. 1964, 21, 698− 700. (78) Yamanaka, M.; Hara, K.; Kudo, J. Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis. Appl. Environ. Microbiol. 2005, 71, 7589−7593. (79) Pal, S.; Tak, Y. K.; Song, J. M. Does the Antibacterial Activity of Silver Nanoparticles Depend on the Shape of the Nanoparticle? A Study of the Gram-Negative Bacterium Escherichia coli. Appl. Environ. Microbiol. 2007, 73, 1712−1720.

8561

dx.doi.org/10.1021/la301322j | Langmuir 2012, 28, 8550−8561