Biocompatible nano hydroxyapatite – curcumin bi-coated antibacterial activated carbon for water purification

Abstract

Activated carbon has been used for water purification since ancient times due to its well-known sorption properties. However it is not capable of disinfecting water borne pathogens such as bacteria. The main objective of this study was to incorporate antibacterial properties while maintaining the existing properties of Granular Activated Carbon (GAC). This was achieved by a biocompatible double coating on to GAC which consists of hydroxyapatite (HAP) nanoparticles and on top of those curcumin molecules. Coating of GAC with HAP was carried out using in situ precipitation of HAP under basic conditions. A layer of curcumin molecules was then attached on top of the HAP coating in order to obtain HAP-curcumin bi-coated GAC (HAP/C/GAC). Synthesized HAP/GAC and HAP/C/GAC were characterized using FT-IR spectroscopy, scanning electron microscopy (SEM), powder X-ray diffractometry (PXRD) and thermogravimetry (TGA). Characterization revealed that needle shaped HAP nanoparticles (50–100 nm in width and approximately 200–500 nm in length) can be anchored and immobilized successfully on GAC which in turn enhances the adhesion of curcumin on it. Antibacterial properties of pure GAC, HAP/GAC and HAP/C/GAC were then investigated using both Gram negative (Escherichia coli) and Gram positive (Staphylococcus aureus) bacteria. The results show that the antibacterial properties of HAP/C/GAC are remarkably higher than that of HAP/GAC and the antibacterial activity of pure GAC is negligible.

---

Introduction

Planet Earth is often called the “blue planet”¹ due to its vast surface covering of water. But in spite of this, the amount of water that is available for drinking is only less than 2.5%,¹ the remainder being either inaccessible or inconsumable.¹ Furthermore, a considerably large portion of potable water gets polluted as a result of human activities² while the usage of fresh water in agriculture and industries further limits its availability for consumption³ exacerbating the situation. Therefore the scarcity of clean and safe drinking water has become a crisis which needs attention.⁴ Moreover it has been identified that about 10% of the diseases that jeopardize the global population can be prevented if drinking water, hygiene and sanitation are improved.⁴ The basic idea of purifying water is to remove turbidity caused by other materials and microbial contaminants in potable water sources.⁵ Activated carbon is a widely used purification agent due to its well-known role as a sorbent, low cost abundance and environmental benignness.⁶ Also it is capable of improving the quality of drinking water by removing dust, sand, rust, color, objectionable tastes and odors.⁷ However early studies have shown that there's no significant difference between water filtered through activated carbon and unfiltered tap water with respect to the microbial contaminants.⁸ Therefore, often a separate disinfector like chlorine,⁹ ozone¹⁰ or UV is used in the filtration apparatus where activated carbon is present as the matrix.¹¹ On the contrary, these disinfector agents are more likely to form harmful DBP's (disinfection by-products)¹² such as trihalomethanes, haloaceticacids and aldehydes¹³ whenever it's used in water treatment due to high oxidative capacity.^12,13 Consequently in continuing their use, we often have to compromise between the effectiveness of disinfection and the formation of harmful DBP's.¹² Even though nanotechnology promises to give solutions for this dilemma, it has also been recorded that for a nanomaterial to be an effective and reliable purifier, these nano particles should be anchored thoroughly onto a base material such as GAC.¹² This dilemma pulls the attention towards inventing disinfectants which are not strong oxidants in this case, nanotechnology appears to be a promising relief.¹²

As for nanomaterials, silver nanoparticles,^12,14 metal oxide nanoparticles such as TiO₂ ¹² and ZnO,¹² fullerenes¹² and carbon nanotubes¹² are reported in water purification as non-DBP producers.¹² However silver nanoparticles are recognized to cause intoxication in humans leading to various health issues.¹⁵ High water solubility of ZnO brings down the opportunities for it in water treatment.¹² Furthermore concentrations >5 mM of ZnO nanoparticles have reported to reduce human T cell viability.¹⁶ The susceptibility of coagulation of fullerenes by the salts present in water, and the toxicity to mammalian cells¹² limits its audience in water treatment grounds.¹⁷

HAP is a calcium phosphate ceramic material with the molecular formula Ca₁₀(PO₄)₆(OH)₂ and nano HAP is a well-known material for removing heavy metal ions from water. HAP nanoparticles have become imperative tools in the medical industry due to its high biocompatibility with human physiology and has made it one of the best choices in drug and gene delivery.^18–20 Also it has been reported in antibacterial applications, as a surface for adhesion of silver.²¹ Apart from that, it's evident that HAP/GAC nanocomposite has high heavy metal sorption capacity.²²

On the other hand curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is a hydrophobic polyphenol, a natural compound extracted from the rhizome of plant Curcuma longa (turmeric). It has been in use for more than decades in ayurvedic medicine and Asian cuisine²³ due to its antioxidant, anti-inflammatory, antimicrobial and anti-carcinogenic properties.²⁴ Curcumin has well proven antibacterial properties as it inhibits the bacterial endotoxin induced cytokines secretion and related processes thereby directly suppressing the bacterial cell growth.^25,26

The main objective of this study is to develop a multifunctional biocompatible filtering matrix with enhanced anti-bacterial properties. In our investigation, GAC was selected as the matrix material. By keeping the inherent properties of GAC intact, well known antibacterial materials such as nano HAP and curcumin were introduced to the GAC to improve the lacking antibacterial property.

Coating of HAP nanoparticles on to GAC was carried out by modifying our previously reported procedure.²² This HAP layer was further functionalized with curcumin molecules to develop HAP-curcumin bi-coated GAC. These two compounds were then tested for their antibacterial activity against E. coli and S. aureus as Gram negative and Gram positive bacteria respectively.

Experimental section

Materials and methods

Granular activated carbon (coconut shell) and curcumin (80%) were purchased from Sigma-Aldrich Ltd. Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O), minimum assay 98% from Techno Pharmchem, India was used in the synthesis along with sodium dihydrogenphosphate dihydrate (NaH₂PO₄·2H₂O) of BDH Chemicals and ammonia solutions were prepared from 25% pure NH₃ from MERCK, Germany. All these chemicals were of reagent grade, and double distilled water was used throughout. All the solid reactants and synthesized nanoparticles were weighed in the analytical balance (CAS-CAY 120). Drying was done using the electric oven (National, Japan) and pH of the solutions were measured using pH meter (EUTECH – pH 510).

Synthesis of HAP coated GAC (HAP/GAC)

The synthesis was done by mixing (Ca(NO₃)₂·4H₂O) with NaH₂PO₄·2H₂O in 1.67 Ca/P molar ratio in 300.0 ml of double distilled water and heated with vigorous stirring. When the solution reached 85 °C, 300.0 ml of 25% NH₄OH and GAC (12.0 g) was added at once and the mixture was stirred for another 2 hours maintaining the temperature at 85 °C. Stirring was discontinued after two hours and the mixture was aged at 85 °C for 24 hours. The mixture was then allowed to cool under the room temperature and solid HAP/GAC was allowed to settle. After decanting the solution, HAP/GAC was washed with double distilled water until the pH of the washings become 7. The resultant product was filtered and oven dried at 80–100 °C for 3 hours. This was characterized using FT-IR, SEM, PXRD and TGA.

Synthesis of HAP-curcumin bi-coated GAC (HAP/C/GAC)

HAP/GAC nano composite synthesized as described above, was further coated with curcumin following the same procedure. Curcumin was added in 1 : 1 molar ratio with HAP prior to the addition of NH₄OH and GAC. Photographs showing the color changes during synthesis of (HAP/C/GAC) are given in ESI.†

Characterization techniques

The presence of functional groups of curcumin on HAP layer was confirmed using Fourier Transform Infrared Spectrometer AVATAR-320 (Thermo Nicolet). The FT-IR spectra were obtained over the region 400–4000 cm⁻¹ using KBr pellet technique. Pellet was prepared by mixing 2.0 mg of the sample with 200.0 mg of oven dried spectroscopy grade KBr (Sigma-Aldrich). The presence of curcumin was further confirmed by carrying out TGA analysis. The TGA analysis was performed using nearly 10.0 mg in Thermo-gravimetric Analyser (SDT Q600). The temperature range used for the analysis was 25–1000 °C. The surface morphology and microstructural features of synthesized nano composites were studied using Scanning Electron Microscope (HITACHI SU6600). X-Ray diffractometry was carried out on a Bruker D8 Focus X-ray powder Diffractrometer using CuKα radiation (λ = 0.154 nm) over a 2θ range of 3–60°, with a step size of 0.02° and a step time of 1 s.

Microbiological techniques

Lysogenic broth (LB) Agar media were purchased from the manufacturer Hardy Diagnostic and used as the medium for bacterial growth. Medium preparation was done by dissolving 10.0 g of LB and 2.5 g of agar in 500.0 ml of double distilled water. Inhibition zone study was done taking gentamicin (Mycin®, NEON laboratories Ltd) as the positive control and double distilled water as the negative control.

All the microbiological work was performed at the Laminar hood (BioBase). Bacterial growth media were autoclaved in Autoclave machine (ALP Co. Ltd. Model KT – 305D −230 V, 50/60 Hz) before use and all the glassware including Petri plates were incubated at 180 °C for 2 h in oven (Memmert Beschickung loading model 100-800) before use. Bacteria inoculated plates were incubated at 37 °C in incubator (Memmert Beschickung loading model 100-800) overnight for growth. For the sonication purposes Sonorox super RK 1028 CH, BANDELIN Electronics, Berlin was used. For the bacterial cell counting, haemocytometer (Neubauer, Germany) was used together with the optical microscope (OLYMPUS CK X41).

Disc diffusion assay

Sterilized paper discs were loaded with 10 μl of sonicated nanoparticle suspensions (HAP, HAP coated with curcumin) and mounted on E. coli spread plates. Nanoparticle suspensions were prepared by dissolving 0.1 g of nanoparticles in 10.0 ml of autoclaved distilled water. Prepared suspensions were sonicated for 15 min before use. 10 μl aliquot of 1000 ppm gentamicin was used as the positive control. The plates were then incubated at 37 °C for 24 h and then observed.

Column technique

Suspensions of E. coli and S. aureus were prepared in autoclaved double distilled water. All the filter materials were autoclaved before use. Autoclaved pure GAC, HAP nanoparticles and the two nano composites: HAP/GAC and HAP/C/GAC were loaded into separate syringes to form mini-water filters with 5 mm diameter and 2.5 cm length. Double distilled water was passed through each filter for 2 minutes prior to the suspension at a rate of 0.03 ml min⁻¹. Then the bacterial suspension was allowed to drain through the filter in the same rate under the laminar hood.

Filtered water was collected into autoclaved Pyrex tubes and was kept in an ice bath to inhibit bacterial multiplication. Bacterial contents of these water samples were analysed qualitatively using impregnation methods: by seeding 50.0 ml of autoclaved LB Agar media with 200.0 μl of the filtered water samples. The capacity of prolonged antibacterial efficacy of nano composites were analysed by draining bacterial suspension over 5 minutes and the resultant filtered water samples were analysed for the bacterial content using the same procedure. Quantitative analysis was done using the haemocytometer and spread plate technique.

Results and discussion

Characterization of HAP, HAP/GAC using FT-IR spectrophotometry

HAP nanoparticles were synthesized on GAC as given in the Experimental section. Pure nano HAP was synthesized according to previously published method.²² The presence of HAP coating on GAC was characterized using FT-IR spectroscopy. The FT-IR spectra obtained for pure GAC, pure HAP and HAP/GAC are given in the Fig. 1.

Fig. 1 FT-IR spectra of pure (a) GAC (b) HAP/GAC (c) pure nano HAP.

As seen in the Fig. 1, FT-IR spectrum of HAP/GAC and FT-IR spectrum of pure HAP are very much similar to each other. In both HAP/GAC and pure HAP, the broad peak that appears at 3000–3500 cm⁻¹ region accounts for the stretching vibration mode of H bonded OH groups present in HAP.²⁷ Peak at 1092 cm⁻¹ corresponds to the stretching mode of PO₄³⁻ groups and the peaks at 603, 831 cm⁻¹ are the bending modes of PO₄³⁻ groups.²⁷ The absence of broad band, at 2600–3000 cm⁻¹ region and, peaks at 1600 cm⁻¹ and 875–880 cm⁻¹ which are characteristics of amorphous calcium phosphate, carbonate apatites and Ca deficient HAP respectively, indicates that these contaminant compounds are not formed during the synthesis of HAP nanoparticles.²⁸ Therefore this confirms the presence of pure HAP nanoparticles on GAC. With compared to pure GAC spectrum, the HAP/GAC spectrum shows a broad band at 3000–3500 cm⁻¹ indicating the increased amount of OH groups on GAC as a result of coating it with HAP.

Morphology and nature of coating of HAP, HAP/GAC using SEM

The morphology and the size of HAP nanoparticles synthesized on GAC was determined using scanning electron microscopy. The SEM micrographs obtained for pure HAP and HAP/GAC are given in the Fig. 2.

Fig. 2 SEM images of (a) pure nano HAP (b) HAP/GAC low magnification (c) HAP/GAC high magnification.

Fig. 2(a) shows the needle shaped HAP nanoparticles with the aspect ratio of approximately 50–100 nm in width and around 200–500 nm in length.

The SEM images obtained for HAP/GAC (Fig. 2(b) and (c)) show that the coating of HAP on GAC is not even as previously observed²² and instead, the needle shaped particles are scattered on the GAC surface permitting most of the GAC surface to be exposed. This observed change in coating may be attributed to the addition of ammonia solution at once, during the synthesis of HAP on GAC thereby allowing fast nucleation and quick separation between nucleation and nanoparticle growth leading to a monodispersed nanoparticle formation as suggested by LaMer model.²⁹ According to Fig. 2(b) and (c), almost all the micropores of GAC are uncovered with HAP and this can be considered as an advantage since most of the adsorption occurs mainly at the pore sites.³⁰ The image also reveals that HAP nanoparticles are held well in the porous matrix of GAC allowing a successful coat. It can also be suggested that the HAP nanoparticles are held strongly by the carbon matrix as calcium leaching was not observed for the leaching test carried out.

Characterization of HAP, HAP/GAC using PXRD

Further confirmation on this coating is revealed in the PXRD pattern of the HAP coated GAC which is shown in Fig. 3. The comparison given with standard HAP peaks shows that synthesized nanocomposite comprises HAP nanoparticles that are in compliance with the standard hexagonal crystal system of HAP and the absence of other contaminants. The Peaks at 2θ positions of 26°, 29°, 32°–34°, 40°, 46°–54° are in good agreement with the previously reported crystalline HAP.^31,32 This shows that our synthesized HAP was nano sized with crystalline structure. The broad nature observed specially at the 2θ regions 26 and 40 are in good agreement with the presence of GAC matrix on which crystalline HAP has been coated. Also sharp XRD peaks for HAP present on GAC matrix account for its high crystallinity in contrast to the previously established data.²²

Fig. 3 Comparison of PXRD pattern of HAP/GAC with standard HAP.

Characterization of HAP/C/GAC using FT-IR spectrophotometry

HAP coated GAC nanocomposite was further functionalized by coating it with curcumin molecules as given in the Experimental section. The physical appearance of GAC, HAP/GAC and HAP/C/GAC is given in the Fig. 4. HAP/GAC has a whitish touch in its color than uncoated GAC. The presence of curcumin on the HAP layer is very clear due to the yellow color appeared on HAP/C/GAC. This was obtained after washing the HAP/C/GAC with plenty of water until the absence of peak at 1628 cm⁻¹ in FT-IR^33,34 and 435 nm peak in UV-Vis³⁴ which corresponds to the curcumin.

Fig. 4 Comparison of physical appearances of synthesized nano composites with GAC.

HAP-curcumin bi-coated GAC was analyzed using FT-IR spectroscopy and the spectra obtained for three samples namely, HAP/C/GAC, pure curcumin and HAP/GAC are compared in the Fig. 5.

Fig. 5 Comparison of FT-IR spectra of (a) HAP coated GAC (b) HAP-curcumin bi-coated GAC (c) curcumin.

The spectrum of curcumin in Fig. 5 contains the broad band around 3500 cm⁻¹ which is an indication of its H bonded phenolic OH group. The peak at 1628 cm⁻¹ is due to the aryl substituted C=C bond.³⁴ The shoulder appearing on to its right at 1599 cm⁻¹ accounts for the conjugated C=C bonds of the aromatic ring. The peak at 1150 cm⁻¹ stands for the C–O bond of the ether group (OCH₃).³⁵ These values are in accordance with the FT-IR peaks of curcumin reported previously.³⁶ When comparing the three spectra the spectrum of HAP/C/GAC has similarities to both HAP/GAC as well as to pure curcumin. The broad peak presents in the region of 3000 cm⁻¹ to 3500 cm⁻¹ indicates the presence of both HAP coating as well as curcumin. However, the characteristic band of curcumin's carbonyl group seen at 1628 cm⁻¹ present in HAP/C/GAC is absent in HAP/GAC. This confirms the successful coating of curcumin on top of the HAP coating. It was also confirmed that curcumin coats specifically onto HAP layer and not onto the carbon matrix. For this we carried out an experiment to coat curcumin onto pure GAC and pure HAP using the same coating procedure under HAP/C/GAC. The FT-IR spectra obtained for these experiments, (given in the ESI†) support the absence of curcumin on pure GAC. This selective adhesion of curcumin onto HAP can be attributed to the metal ion chelating ability of curcumin which ensures its binding to HAP via HAP's calcium ions.

Characterization using thermo gravimetric analysis

Synthesized nano-composites and their precursors were subjected to TGA analysis and the resulted thermograms are shown in Fig. 6.

Fig. 6 Comparison of TGA curves obtained for (a) curcumin (b) GAC (c) nano HAP (d) HAP/curcumin/GAC (e) HAP/GAC.

As seen in the Fig. 6, it is evident that two of the main precursors present in synthesized nano-composites are not thermo-stable up to 1000 °C. GAC and curcumin has burnt under air leaving no residue during 600–800 °C. On contrary, HAP has shown a significant stability towards temperature. The first endothermic region of HAP's TGA curve corresponds to the removal of physically adsorbed water from the crystal lattice.³² Furthermore, absence of any other endothermic regions assure the absence of other contaminant compounds like CaHPO₄, Ca(OH)₂ thereby confirming the purity of HAP.³⁷ Also it's visible that both the nanocomposites maintain a non-zero mass when 1000 °C is achieved. This is a clear evidence that these two nano composites contain HAP nanoparticles in them which withstand the higher temperatures.

Determination of antibacterial activity of synthesized nano-composites

Nano composites containing GAC, HAP and HAP/C were subjected to antibacterial activity using disc diffusion assay. Gentamycin (10 μg ml⁻¹) was used as the positive control and water as the negative control and the results obtained are shown in Fig. 7. The activity of each material is expressed as the diameter of growth inhibition area in cm.

Fig. 7 Inhibition zones. (A) Positive control (1.2 cm); (B) negative control (0.0 cm); (C and D) nano HAP (1.2 cm, 1.1 cm); (E and F) HAP coated with curcumin (1.5 cm, 1.3 cm).

Compared to the positive control gentamycin (10 μg ml⁻¹), HAP and HAP/C (100 μg ml⁻¹) showed similar level of antibacterial activity against E. coli. These results clearly show that nano composites containing HAP and HAP/C have an effective antibacterial activity with the highest in HAP/C.

The ability of synthesized HAP/GAC and HAP/C/GAC nano-composites in removing bacteria in water, has been experimented using a known amount (5.2 × 10⁹ CFU ml⁻¹) of E. coli and S. aureus bacterial suspensions. This bacterial suspension was passed through columns prepared with HAP/GAC and HAP/C/GAC. The filtrates were analyzed using impregnation method. The results obtained are given in the Fig. 8.

Fig. 8 (a) Bacterial suspension used for filtration (b) filtrate of GAC filter (c) filtrate of HAP filter (d) filtrate of HAP/GAC filter.

According to the results highest number of bacterial colonies is present in the filtrate of uncoated GAC filter. This is in accordance with the reported data where it mentions the formation of a biofilm on GAC which can facilitate the multiplication of coliforms rather than reducing their number.¹ In general both the nanoparticle containing filters seemed to have reduced the bacterial contamination in water. However no bacterial colonies can be found in water samples that were passed through the columns containing pure HAP. Antibacterial activity observed with HAP/GAC nanocomposite is in between pure GAC and pure HAP. And it is evident that the antibacterial properties of synthesized HAP are retained even if they are immobilized by coating on to GAC.

The extended experimental studies on antibacterial activity of synthesized filter materials namely GAC, HAP/GAC and HAP/C/GAC were carried out in order to compare the antibacterial properties of HAP/C/GAC to pure GAC. The results obtained are shown in the Fig. 9.

Fig. 9 Antibacterial activity against E. coli and S. aureus.

In this experiment antibacterial activity was demonstrated against number of colony forming units (CFU). Compared to the number of colonies present in the original sample, the number of colonies found in HAP/GAC filtrate and HAP/C/GAC filtrate are extremely low for the case of S. aureus. A close observation shows that absolutely no colonies are found in the 1^st ml for HAP/C/GAC as well as in HAP/GAC column. In HAP/GAC, a similar number of colonies are observed in the 2^nd and 3^rd ml followed by a slight increment in the 4^th and 5^th ml. In HAP/C/GAC, similar number of colonies are there in 2^nd and 3^rd ml and slight increment is observed up to 5^th ml. However, compared to HAP/GAC the number of colonies are significantly low and this can be attributed to the presence of curcumin.

A spread plate technique was carried out for filter systems containing pure GAC, HAP/GAC and HAP/C/GAC in order to compare their activity against S. aureus. The results are given in the Fig. 10. As seen in the figure, the amount of growth of bacteria in the filtrate of pure GAC has given rise to a lawn of bacteria in the spread plate and the thickness of the lawn has increased when going from the 3^rd towards 5^th. The reason can be the same that was mentioned before, facilitation of the growth of bacteria by the filter bed rather than a reduction.

Fig. 10 Comparison of the effects of HAP/GAC and HAP/C/GAC filters with the GAC filter.

Nevertheless in both nanocomposite containing filters, the viable cell count in the filtrate has tremendously decreased compared to the pure GAC filter. The filtrate obtained from HAP/C/GAC shows absolutely no growth for the first fraction. Further, it is clear that the filtrate obtained from HAP/C/GAC for 2^nd and 3^rd fractions are having only a very few and similar number of colonies with the slight increment going from 4^th to 5^th. However with compared to the HAP/C/GAC the amount of colonies at the HAP/GAC has a significant increment in number. On the other hand, the gradual reduction of the antibacterial activity observed in both filters may be due to the saturation of the activity sites with bacteria due to the repeated use of filter. Therefore further studies should be carried out to determine the capacity of the filters towards bacterial disinfection.

Conclusion

A novel biocompatible granular activated carbon nanocomposite was synthesized by immobilizing HAP nanoparticles and curcumin on GAC to develop a filter material in water purification applications. This will successfully overcome the health issues related to the release of metal and metal oxide nanoparticles to water during purification process using current metal and metal oxide based anti-bacterial water filters. Furthermore, HAP-curcumin bi-coated GAC carries a significant capacity to purify contaminated water with the antibacterial properties experienced by the presence of both curcumin and HAP. The novel material will be developed as a point-of-use filter material to be used in rural areas in Sri Lanka. Experiments to explore the possibilities of regeneration of the filter material are also in progress.

Acknowledgements

We are thankful to Department of Chemistry, University of Colombo and Sri Lanka Institute of Nanotechnology (SLINTEC) for providing us with the facilities.

References

1. T. Oki and S. Kanae, Science, 2006, 313, 1068–1072.
2. M. A. Shannon, P. W. Bohn, M. Elimelech, J. G. Georgiadis, B. J. Marinas and A. M. Mayes, Nature, 2008, 452, 301–310.
3. R. P. Schwarzenbach, B. I. Escher, K. Fenner, T. B. Hofstetter, C. A. Johnson, U. V. Gunten and B. Wehrli, Science, 2006, 313, 1072–1077.
4. M. A. Hanjra and M. E. Qureshi, Food Pol., 2010, 35, 365–377.
5. World Health Organization, Safer water – Better health, Geneva, 2008.
6. C. Ray and R. Jain, Drinking Water Treatment: Focusing on Appropriate Technology and Sustainability, Springer, 2011, p. 10.
7. B. I. Dvorak and S. O. Skipton, Drinking Water Treatment: Active Carbon Filtration, NebGuide, University of Nebraska-Lincoln Extension, Institute of Agriculture and Natural Resources, 2013, p. G1489.
8. J. V. Fiore and R. A. Babineau, Appl. Environ. Microbiol., 1977, 34, 541–546.
9. G. J. Jarocki, US Pat., 5 611 937, 1997.
10. K. Xiandong and D. Ping, CN Pat., 200910175831, 2011.
11. D. I. Collias, F. J. Derbyshire, S. G. Fishter, M. Jagtoyen and M. E. Trembly, WO Pat., 2000004977 A2, 2000.
12. Q. Li, S. Mahendra, D. Y. Lyon, L. Brunet, M. V. Liga, D. Li and P. J. J. Alvarez, Water Res., 2008, 42, 4591–4602.
13. N. Savage and M. S. Diallo, J. Nanopart. Res., 2005, 7, 331–342.
14. World Health Organization, Silver: water disinfection and toxicity, 2014, p. 1.
15. C. M. Jones and M. V. Hoek, J. Nanopart. Res., 2010, 12, 1531–1551.
16. K. M. Reddy, K. Feris, J. Bell, D. G. Wingett, C. Hanley and A. Punnoose, Appl. Phys. Lett., 2007, 90, 1–6.
17. C. M. Sayes, J. D. Fortner, W. Guo, D. Lyon, A. M. Boyd, K. D. Ausman, Y. J. Tao, B. Seetharaman, L. J. Wilson, J. B. Hughes, J. L. West and V. L. Colvin, Nano Lett., 2004, 4(10), 1881–1887.
18. K. Agrawal, G. Singh, D. Puri and S. Prakash, J. Miner. Mater. Charact. Eng., 2011, 10, 727–734.
19. L. Chen, J. M. Mccrate, J. C. M. Lee and H. Li, Nanotechnology, 2011, 22, 1–10.
20. N. F. Mohammad, R. Othman and F. Yee-yeoh, Rev. Adv. Mater. Sci., 2014, 38, 138–147.
21. J. J. Buckley, A. F. Lee, L. Olivi and K. Wilson, J. Mater. Chem., 2010, 20, 8056–8063.
22. S. Fernando, W. R. M. De Silva and K. M. N. De Silva, Appl. Surf. Sci., 2015, 351, 95–103.
23. G. Grynkiewicz and P. Slifirski, Acta Biochim. Pol., 2012, 59, 201–212.
24. K. Bairwa, J. Grover, M. Kania and S. M. Jachak, RSC Adv., 2014, 4, 13946–13978.
25. G. Liang, S. Yang, L. Jiang, L. Zhao, J. Xiao, F. Ye, Y. Li and X. Li, Chem. Pharm. Bull., 2007, 56(2), 162–167.
26. D. Rai, J. K. Singh, N. Roy and D. Panda, Biochem. J., 2008, 410, 147–155.
27. L. Chen, J. M. Mccrate, J. C. M. Lee and H. Li, Nanotechnology, 2011, 22, 1–10.
28. L. B. Cimdina and N. Borodajenko, Research of Calcium phosphates using Fourier Transform Infrared Spectroscopy, in Infrared Spectroscopy–Materials Science, Engineering and Technology, ed. T. Theophile, Intech, Shanghai, 2012, pp. 123–148.
29. V. K. LaMer and R. H. Dinegar, J. Am. Chem. Soc., 1950, 72, 4847–4849.
30. National Research Council (US) Safe Drinking Water Committee, Drinking Water and Health, National Academies Press (US), Washington (DC), 1980, ch. 4, vol. 2.
31. M. P. Mahabole, R. C. Aiyer, C. V. Rramakrishna, B. Sreedhar and R. S. Khairnar, Bull. Mater. Sci., 2005, 28, 535–545.
32. H. Eslami, M. Solati-Hashjin and M. Tahriri, Iran. J. Pharm. Sci., 2008, 4(2), 127–134.
33. M. A. Rodrigues, J. N. Fernandes, R. Ruggiero and W. Guerra, Am. J. Chem., 2012, 2(3), 157–159.
34. M. A. Subhan, K. Alam, M. S. Rahaman, M. A. Rahman and M. R. Awal, J. Sci. Res., 2014, 6(1), 97–109.
35. J. Coates, in Encyclopaedia of Analytical Chemistry, ed. R. A. Meyers, John Wiley & Sons Ltd, Chichester, 2000, pp. 10815–10837.
36. P. Anand, S. G. Thomas, A. B. Kunnumakkara, C. Sundaram, K. B. Harikumar, B. Sung, S. T. Tharakan, K. Misra, I. K. Priyadarsini, K. N. Rajasekharan and B. B. Aggrawal, Biochem. Pharmacol., 2008, 76(11), 1590–1611.
37. K. C. B. Yeong, J. Wang and S. C. Ng, Biomaterials, 2001, 22, 2705–2712.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra11518c

---