The effect of different pHs, Surfactants and dialyses times on preparation of nano Rod Hydroxyapatite

Fateme Mirjalili ¹ (Department of material engineering,faculty of engineering,Islamic azad university Maybod beranch,Iran)

تاريخ الإرسال : 20 السبت , ذو القعدة, 1438 تاريخ التأكيد : 15 الأحد , رجب, 1439

الکلمات المفتاحية: Nano HA rod, precipitation method, XRD analysis,

ملخص المقالة :

Nano HA rod has been synthesized by precipitation method using Ca (NO3)24H2O and (NH4)2HPO4 as starting materials and ammonia solution as an agent for pH adjustment. The Ca/P molar ratio was maintained at 1.67. Then, the effect of different pH (4, 6, 8, 10 ,11), different surfactants and different times for dialyses on nano HA rod were studied. The samples were characterized by different techniques such as, X-ray diffraction, Fourier transform infrared spectra, and Transmission electron microscopies. The XRD analysis showed that the prepared HA nano rod was to be fully crystalline. The results showed that, the best pH for nano HA rod was 11and Span 20 as the surfactant had the better effect on dispersion and shape of nano rod. The HA nano-rods had an average diameter of 10 nm and length 70-80 nm after 12 hours of dialyses time.Selection and control of the precise dialyses time together with surface active agents proved to be important in controlling the rod size, degree of aggregation and the rod shape.

نص كامل:



The effect of different pHs, Surfactants and dialyses times on preparation of nano Rod Hydroxyapatite

 

Abstract

Nano HA rod has been synthesized by precipitation method using Ca(NO3)24H2O and (NH4)2HPO4 as starting materials and ammonia solution as an agent for pH adjustment. The Ca/P molar ratio was maintained at 1.67. Then, the effect of different pH (4, 6, 8, 10, 11), different surfactants and different times for dialyses on nano HA rod were studied. The samples were characterized by different techniques such as, X-ray diffraction, Fourier transform infrared spectra, and Transmission electron microscopies. The XRD analysis showed that the prepared HA nano rod was to be fully crystalline. The results showed that, the best pH for nano HA rod was 11and Span 20 as the surfactant had the better effect on dispersion and shape of nano rod. The HA nano-rods had an average diameter of 10 nm and length of 70-80 nm after 12 h of dialyses time.

Key word: Nano HA rod, precipitation method, XRD analysis

 Introduction:

Hydroxyapatite is calcium phosphate nearest to bone mineral and the most thermodynamically stable phase in the body [1-3].Calcium phosphates, such as hydroxyapatite for their biocompatibility and similarity to the mineral constituents of bones and teeth are good replacements for parts of the body which are damaged [4-6]. Hydroxyapatite raises the capability of bone growth and forms a strong chemical bond with bone tissue when the bone tissue grows in the surface layer of this ceramic [7,8].Nonetheless, it is a brittle material which leads to low mechanical failure and bounds its application in a position under the load.

Materials with nano scale features lead to an improved cellular response, hence they provide the current interest in all micro and nanotechnologies in biomedicine, principally in tissue engineering [9-11].The fibrous form of HA is particularly promising for use as a variety of implantable materials, e.g., directly as porous bone blocks and defect fillers or through formulation with degradable polymers in order to support cell functions and stimulate regeneration of new tissues [2,12,13]. 

Techniques that have been used for preparing nano HA rod include chemical precipitation, hydrothermal treatment, sol–gel, micro emulsion techniques, precipitation from complex solutions followed by microwave heating, wet chemical methods, mechanic chemical synthesis and electro deposition [13-20]. Bioactivity of Ca–P based materials is dependent on many factors such as the synthesis procedure, precursor reagents, impurity contents, crystal size and morphology, concentration and mixture order of reagents, pH and temperature [21-25]. Selection of the route of synthesis depends on the application. The present work reports a low temperature synthesis of hydroxyapatite nano rods by a modified and simple precipitation technique. To this end, the novelty of this report is the effect of different PH on preparation of HA nano rods and modify HA nano rods with different surfactants and then prepare HA nano rods via contorting of different dialyses times. 

2. Materials and methods

Nano crystalline hydroxyapatite rod was prepared by a solution-precipitation method via the reaction of calcium nitrate tetra-hydrate (Ca(NO3)2.4H2O, 98%,  Merck prolabo 0308821 142) with diammonium hydrogen phosphate ((NH4)2HPO4, 99%, Merck prolabo A0143307 037) with the molar ratio of Ca/P=1.67. The suspension of 1 M Ca (NO3)2.4H2O was vigorously stirred at 25°C with pH of 10.5-11. After that, 0.67 M (NH4)2HPO4 solution was slowly added drop wise to the solution; and the resulting solution was stirred for 1 h and aged for 12 h. Following the separation of the deposition of the reaction solution, the solution was washed with distilled water and dried in an air oven at 100 °C for 2 days to obtain a white powder.

In the next part, after preparing of the solution, different pH (4, 6, 8 and 10, 11) was adjusted to obtain the most percentage of HA phases. The suspensions of 1 M Ca (NO3)2.4H2O were vigorously stirred at 25°C with different pH. Then, 0.67 M (NH4)2HPO4 solution was slowly added drop wise to the solutions; and the resulting solutions were stirred for 1 h and aged for 12 h and dried in an air oven at 100 °C for 2 days as previously reported.

 In the next part, for improving the properties, different surfactants such as span 20 and Aliquid were added. After preparing the first solution of 1 M Ca (NO3)2 4H2O, the desired surfactants with weight percent of 2.wt% were added and then, the second solution of 0.67 M (NH4)2HPO4 was slowly added drop wise to the first solution. The resulting solutions were stirred for 1 h and aged for 12 h and then, washed with distilled water and dried for 2 days at 100 °C.

At last, for improving the properties, different times of dialyses (12, 15, 18 h) were used to provide the best condition for preparing the nano rods. At this stage, after providing the sol, it was dialyzed (dialysis bags-110, B'genei) against deionized water for different times, changing the water every 3 h. The gel obtained after dialysis was dried in an air oven at 100 °C for 2 days to obtain a white powder.

Phase identification was performed by X-ray diffraction (XRD, PW1800, Philips) using nickel filtered Cu Kα radiation in the range of 2θ = 10°-60° with a scanning speed of 5° min-1. Fourier transform infrared spectroscopy (FTIR, Perkin Elmer Spectrum 100) was carried out by the universal attenuated total reflection (UATR) method. Microstructures and morphology of powders were identified by transmission electron microscopy (Philips-Zeiss).

Results and Discussion

Synthesis of Hydroxyapatite nano rode:

In this report, HA nano rod was successfully provided by participation method. The reactions complicated in the formation of HA during the participation preparation and drying could be expressed as follows:

10Ca (NO3)2 · 4H2O + 6(NH4)2HPO4 20NH4OH Ca10 (PO4)6(OH)2 + 20NH4NO3 + 52H2O (1)

The formation of 20NH4NO3 (ammonium nitrate) by product was removed by repetitive washing with twice distilled water (15,16,26).

The XRD peaks of hydroxyapatite rod after drying at 100  for 1 h is illustrated in Fig. 1. The XRD pattern in Fig. 1 indicated that, the samples was mostly HA phase. On the other side, another crystalline phase (Tricalcium phosphate phase (TCP) ) found in the samples. These results were in close proximity with preparation of HA/Al2O3  nanocomposite  which reported previously in the literature (17). The reactions involved in the formation of β-TCP during the chemical precipitation could be expressed as follows:

 (Ca10 (PO4) 6 (OH) 2 → 3Ca3 (PO4) 2 + CaO + H2O)        [11, 12,17] (2)

 

Fig.1: XRD pattern of pure hydroxyapatite rod synthesized after drying at 100 ° C

The size of the crystalline was determined by the Scherrer method.

The equation was calculated as below:

t = 0.89 λ / β cos θ (3)

Where, t is grain size, λ is the wave length, β is peak width chosen at half height in radians and θ is the angle in degrees [15].

The crystallinity degree of hydroxyapatite phase by x-ray diffraction patterns of X , was calculated in equation 2:

Xc =1-(V112/300-I300) (14)

Where, Xc is the degree of crystallinity of the powder, V112 / 300 the intensity of the cavity between the diffraction peaks (112) and I300. [16, 17]

Accordingly, the degree of crystalline of pure hydroxyapatite was about 40% with the crystalline size about 20 nm.

FTIR analysis of pure HA is shown in Fig. 2. The FTIR shows the existence of phosphate (PO43-) bonds at 570, 600, 950, and 1100-1090 cm-1, hydroxyl (OH) bonds at 630 and 3567 cm-1and P-H band at 1989 and 2076 cm-1 ,which is consisted in previous researchers as mentioned in literature[8,17,18,27].

 

Fig. 2: FTIR curves of pure hydroxyapatite synthesized after drying at 100 ° C

Fig.3 illustrates the TEM morphology of nano HA short  rods. It was seen that separate rod-like HA crystals of uniform size and morphology had  diameter between 20 to 22 nm and length between 100 and 105 nm as shown in Fig. 3. This is agreement with  Kunjalukkal Padmanabhan result which prepared nano rod with 70–90 nm diameter and 400–500 nm length by using a simple sol–gel route and found the possible nanorod nucleation and growth could be related to the relative specific surface energies that connected with the different planes of HA crystal or nucleus (15). The short rods were slightly agglomeration as the size was too small to interweave into a network. This agglomeration between particles is attributed to the large surface area and energy associated with nanoparticles which is in according to other researchers (15,16,28).

 

Fig .3: TEM micrograph of the HA nano rod

The effect of pH on Hydroxyapatite nano rods

The XRD pattern of HA rods at pH=4 (H1), HA rod at pH=6(H2), HA rod at pH=8(H3), HA rod at pH=10(H4) and HA rod at pH=11(H5) after drying at 100 ℃ temperature for 1 h was shown in Fig.4. A comparison diffraction pattern of different pH indicated that, the formation of a HA phase was the dominant phase in pH=10-11, but with decreasing the pH , the calcium hydrogen phosphate phase was  appeared (pH=8). Moreover, at pH=4 and pH=6 the main phase was calcium hydrogen phosphate and some mirror HA phase. A comparison diffraction patterns indicated that with the decreasing of pH, decomposition of HA to calcium hydrogen phosphate phase increased, and the intensity peaks of hydroxyapatite phase decreased and according to Z. Evis, it was responsible for increasing the degradation rate of hydroxyapatite phase (20). When lower pH increased, the reaction between CaO derived from hydroxyapatite analysis and phosphate phase and formation of calcium hydrogen phosphate phase was increased and caused  the degradation rate of hydroxyapatite phase which is consisted in precious researcher that examined the effect of pH on nano HA particles and found the best pH for HA phase is 11(17,29). 

 

Fig.4: XRD pattern of HA rod at different PH after drying at 100 ℃

The FTIR curves(Fig.5) show the existence of phosphate (PO43-) bonds at 570, 600, 950, and 1100-1090 cm-1 and hydroxyl (OH) bonds at 630 and 3567 cm-1 and the existence of phosphate (P2O7) bonds at 800,850 cm-1[2,9,19,20,30]. However, with increasing the pH to 10 and 11 the intensity of phosphate of (PO43-) bonds at 570, 600, 950, and 1100-1090 cm-1 was increased which identified the highest percentages of HA phase at these PHs. This is consisted mainly of Tayyebi results who found, with increasing deposition of alumina at lower pH, more calcium aluminate phases were formed in the crystal lattice of HA/Al2O3 nanocomposite(17). Taken together, the results showed that the obtained material after sintering was mostly crystalline HA.

 

Fig.5: FTIR curves of HA rod at different PH after drying at 100 ℃

Regarding to Figs. 4 and 5, the best pH for preparing nano HA rod was 10- 11 that had the most HA phase comparison with other phosphate phase. This result consisted in TEM result (Figure 7) which shows the rods had the finest diameters about 15-20 nm and 100-110 nm length with good dispersion.

 As previous mentioned ,the probable nano rod nucleation and growth could be credited to the relative specific surface energies related to the different planes of HA crystal or nucleus, which had unlike surface energies  and the plans could define the amount of OH− absorbance from the solution [1,5,7,21-23,30].In our case, since the solution prepared was preserved at a high pH= 11, the different planes would have different OH− concentration, which would  control the growth rate and morphology of the crystal. The surface with high energy would display increase in OH− concentration, and this perhaps restricted the movement of Ca2+ and PO43− in the formed nucleus in one particular direction. In this situation, OH− patterned the nucleation process where, free Ca2+ and PO43− responded with OH in uniaxial direction (planes with less OH− concentration), nucleating into HA nano rods were in agreement with other published data [5]. This is schematically represented in Fig.6.

 

Fig.6. Schematic representation of nucleation of HA crystals [5]

Fig.7 shows the TEM image of nano HA rod at pH of 11. Certain agglomeration of rods was observed which was related to the large surface area and energy associated with nano rods. The average length of the HA rod seemed to vary between 120 and 150nm, while the average diameter was found to be between 20 and 25 nm.

 

Fig.7: TEM micrograph of the HA nano rod at pH of 11

 

Fig.8 shows the XRD patterns of HA nano rods with different surfactants. As Fig. 8 shows, in both patterns the dominant phase was HA, and there was not the difference between the patterns. The average crystallite size obtained for the nano rods, using Scherrer's equation, was 15 nm for the span 20 and 25 nm for the Aliquad samples which is in agreement with previous researcher results. Triethanolamine [11] and TEA [23] were used as capping agents for synthesis of nano particles. Additionally, various primary, secondary and tertiary amines are suggested to have shape controlling properties in synthesis of nano particles [22]. Gold nanowires were developed from HAuCl4 and TEA where TEA acted as a precursor for the formation of the gold threads [24,30]. Rod like nano HA powder is believed to be the most favorable building block to construct dental materials [14,31]. It has been shown that dense bioceramics with high strength and fracture toughness can be prepared using 1D HA nanostructures such as nano rods, nanowires, and nano fibers [24,32].

Fig.9 shows the FTIR curves of HA rod with different surfactants.

 

Fig.8: The XRD pattern of HA rod with Aliquad as a surfactant (H6) and Span 20 as a surfactant (H7) after drying at 100 ℃ 

According to Figure 9, there was not the difference between the patterns. The characteristic signature HA peak near 600 cm−1was related to the bending modes of P–O bonds in phosphate groups with contribution from the –OH of the apatite group at about 630 cm−1[1,12,23,30]. The small peaks in the region 3300 cm−1 were attributed to –OH bonds and those observed near 3472 cm−1were  associated to OH−1stretching vibration of HA [6]. The peaks noted around 1100–1000 cm−1were recognized to the stretching modes of the PO4 3−bonds in HA [7,24]. The peaks in the 2500–2400 cm−1region were associated with asymmetric C–H stretching, symmetric and asymmetric CH3 vibrations, C stretch etc [5,7,25,31]. This confirmed the presence of HA phase which is in agreement with XRD result.

 

Fig. 9: FTIR curves of HA nano rod with Aliquad as a surfactant (H6) and Span 20 as a surfactant (H7) after drying at 100 ℃ 

TEM image of nano HA rod with span 20 is shown in Fig.10.It was shown that, the rods had the good dispersion with diameter of 15 nm and 80 nm at length. The TEM micrograph of nano HA rod with Aliquad as a surfactant is illustrated in Fig.11.

 

Fig.10: TEM micrograph of the HA rod in presence of span 20

Based on Fig.11, with adding the Aliquad as a surfactant, the rods became more agglomerated and there was not a good dispersion and they had the diameter of 20 nm and 100 nm at length.

 

Fig.11: TEM micrograph of the HA rod in presence of Aliquad

The addition of the surfactants resulted in the formation of an amorphous phase coupled with some hydrates of surfactant. This might be the result of the adsorption of surfactant layer on the surface of the initial HA nuclei, which prevented the aggregation and grain growth of HA, in the absence of surfactants that would allow the formation of crystalline phase [10,26,27,32]. However, addition of Aliquad did not play a significant role in the formation of an amorphous HA phase coupled with some hydrates of surfactant, and the most difference observed is in the presence of Span 20 surfactant which is displayed in Fig. 10.

The data suggests that presence of Span 20 was responsible for constrained growth in the directions perpendicular to c axis. Shape control of particles in wet systems was performed by shape controllers adsorbed onto specific surfaces of the particle which reduced the growth rate normal to the adsorbed planes which is in agreement with Gao research that worked on different surfactants such as CTAB and SDS and found CTAB as a surfactant had better results [16].

The HA nano rods , dried at 100 °C, obtained from both the dialyzed sols were white in color with the powders obtained after dialysis being more free-flowing. Actual removal of adsorbed ions was achieved through repeated change (every 3 h) of the de-ionized water during dialysis. Solution route synthesis of HA nano rods have been reported where nano rods have been obtained at low temperature.

Fig.12 shows the XRD pattern of HA nano rod with different time of dialyses. As Fig.12 shows in all of patterns the main phase was HA, and there was not the difference between them.

 

Fig. 12: The XRD pattern of HA nano rod for 12 h of dialyses (H8) , 15 h of dialyses (H9) and 18 h of dialyses (H10) after drying at 100 ℃

 Comparing Fig. 1(H5) and Fig 12 (H8) shows the XRD of the HA powders obtained from

dialyzed and un-dialyzed sols. Well-developed crystalline peaks were noticed in H8 sample with very little background absorption indicating almost complete crystallization rather than H5sample that may be attributed to the efficient removal of adsorbed ions during dialysis.

The average crystallite size obtained for the nano rods, using Scherrer's equation, was 10 nm for the dialyzed after 12 h and 30 nm for the dialyzed after 15nm h and 40 nm for the dialyzed after 18 h samples. The lower size noticed for the dialyzed samples after 12 h might be attributed to the efficient removal of adsorbed ions during dialysis but, with increasing the dialyzed time the diameter sizes of nano-rods were increased which was donated to more water evaporated from sol and caused the supersaturated solution and provided material necessary to bond colliding particles and formed agglomerates. 

Fig.13 shows the FTIR curves of HA rod with different dialyses time. The FTIR spectra demonstrated that the main phase of all three nano rods was mainly HA however, no significant difference was observed between them. Nonetheless, comparing with Fig.10, there were some small bonds which were only observed in the un-dialyzed sample showing that, the effective removal of the organics was achieved through dialysis. Regarding to Fig.13, there was not apparent difference between the patterns. As previous patterns, the characteristic signature HA peak near 471,566 and 603 cm−1 were attributed to the bending modes of PO43- bonds in phosphate groups. [1,28,29]. Moreover, the peaks recognized to phosphate bonds at H9 and H10 were sharper than H8 which was related to dialysis times [Fig.13].

 

 

Fig.13: FTIR curves of HA rod with different dialyses time

Figs.14(a,b) shows TEM image of HA rods after 12 h dialyzed(a) and after 18 h dialyzed(b). The TEM micrograph of the dialyzed sample after 12 h (Fig.14 a ), shows discrete rod-like HA rods of uniform size and morphology, having diameter between 10 and 15 nm and length between 50 and 60 nm , indicating elongation along the HA c axis. systems is performed by shape controllers adsorbed onto specific plan, but with increasing the dialyzed time the diameters sizes of nano rods were increased and provided some agglomeration.

 The data suggests that presence of Span 20 and dialyzed time about 12 h were responsible for constrained growth in the directions perpendicular to c axis. This is in agreement with other researcher’s results which explored that presence of triethylamine was responsible for constrained growth in the directions perpendicular to c axis (18).

                         

(a)                                                                                   (b)

Fig 14: TEM micrograph of the HA rod a) after 12 h dialyzed, b) after 18 h dialyzed

According to Mirjalili [19] agglomeration of solid products from two liquid ionic solutions A and B is as follows: An+ +Bn- C

This reaction involves instantaneous (mixing controlled) chemical reaction, subsequent crystallization of the product (i.e. nucleation and growth of crystals) and its agglomeration.

At low super saturation there were usually negligible effects of agglomeration, and the crystals size distribution was mainly affected by competition between nucleation and growth of crystal. At high super saturation the process was dominated by agglomeration. As it preceded more than 12 h, more water evaporated from sol, the supersaturated solution thus supplied material necessary to bond colliding particles and formed agglomerates. Local concentrations also determined electrical interaction between small colloidal particles, because most particles in aqueous media were charged, and resulting repulsion force depended on solution composition [11,12, 13,33]. Hence, it would further enhance the agglomeration process for these reasons after 12 h of analyses time which we could see the strongly agglomeration in samples.

Conclusion

This study reports synthesis of crystalline HA nanorods of 10 -20 nm diameter and 60-100 nm length using precipitation method. The synthesis was done at pH= 10.5-11 condition for 12 h followed by drying at 100 °C. The precipitation method was used for the synthesis of nano HA rod which were coupled with the presence of surface active agents, like Span 20 and Aliquad. The results showed that, the nano HA rod was obtained at pH of 11 with  Span 20 as the surfactant  and it  had the better effect on dispersion and shape of nano rod than aliquad as a surfactant. The HA nano-rods had an average diameter of 10 nm and length 70-80 nm after 12 h of dialyses time. Selection and control of the precise dialyses time together with surface active agents proved to be important in controlling the rod size, degree of aggregation and the rod shape. It was seen that the separate rod-like HA crystals of uniform size with good dispersion without agglomeration.

References:

1. A.I .Misions, T.C.Vaimakis, C.C. Trapalis, Ceram.Inter., 36, 623(2010).

2.  C.Ergun, Z. Evis, T.J.Webster, F.C.Sahin , Ceram.Inter., 37, 971(2011).

3. F.N.Okatar, Matter. Let., 60, 2207 (2006).

4.F. Deravi , R. Sinatra, O.Christophe, P. Nesmith, H. Yuan, K. Deravi, A. Goss, A. MacQueen, R. Badrossamy, M. Gonzalez, D. Phillips, K. Parker, Mac. Mats. Eng.,1(2017).

5.  H.Ji, P. M. Marquis, Bon.Min. Res.,13,744(1992).

6. A.Haider, S. Haider, S.Han ,I.Kang, R. Soc. Chem.,7,7442 (2017).

7. Z.E.Erkmen, Y. Gens , F.N.Oktar, J.A. Ceram. Soc., 90, 2885 (2007).

8.T.Stephanie ,C. Tang, A. Kennedy, S. Talwar,S. A. Khan, J.F.Hydo.,35,36(2014).

 

9. N.Bahlawane, T.Watanabe, J.A. Ceram. Soc., 9, 2324(2000).

10.Y.Zhao, Y.Qiu, H. Wang, Y.Chen, S. Jin, S. Chen, Inter.J. Poly. Sci.,2016,1(2016).

11. R.T.Franceschi ,B.S. Lyer , Bon. Min. Res.,7, 235(1992).

12. S.Assafi, N.Bruijn, M. Jumaily, W. J. N. Sci. Eng., 6, 58(2016).

13. R.Vaben , D. Stover , J.Mat. Pro. Tech.,92, 84(1999).

14.Y. Lee,S. Fu,C. Lin, 1st Global Conference on Biomedical Engineering & 9th Asian-Pacific Conference on Medical and Biological Engineering, Thailand , pp.9-12(2014).

15.S. Kunjalukkal Padmanabhan , A.Balakrishnan, J.Particulogy,7,466(2009).

16. S. Gao  ,K. Sun , A.Li , H.Wang  , Maters. Res. Bul, 48, 1003 (2013).

17.S.Tayyebi,F.Mirjalili,H.Samadi,J.Ceram.Pross.17,1034(2016).

18. S. Jadalannagari , S.More, M. Kowshik , S.Ramanan ,Maters .Sci. Eng., 31, 1534 (2011).

 19. F. Mirjalili, M. Hasmaliza  ,L. Chuah Abdullah, Ceram. Inter. , 36 ,1253(2010).

20. Z. Evis  and R. Doremus , Maters. Res. Bul., 43, 2643 (2008).

21. P.Q. Franco, C.F.C. João, B.J.C.Silva , J.P.Borges , Maters.Let., 67, 233(2012).

22. A.Z.Zinchenko , R.H. Davis ,J.Phys. Fluids., 7, 2310(1995).

23. M. Mirjalili,S. Zohoori, J.Nanostruct.Chemis.,6,207(2016).

24. S. Ifuku  H. Maeta ,  H. Izawa,  M. Morimoto,  H. Saimoto, RSC. Adv. ,44, 267(2015).

25. M. Maas, P. Guo, M. Keeney, F. Yang, T. M. Hsu,  G. Fuller,  R. Martin, R. N. Zare, Nano. Lett., 11, 1383(2011).

26. Y. Zhao, Y.Qiu, H.Wang, Y.Chen, S.Jin, S.Chen, Inter. J.Pol.Sci., 2016,17(2016).

27. J. Li, A.He, C. Han, D.Fang, S. Hsiao,B. Chu ,Macro. Molecul. Rap.Com., 27, 114(2006).

28. R.Anamarija, App.Sur. Sci., 296 , 221(2014).

29. W.Cui, Y.Zhou, J Chang, Sci. Technol. Adv. Mater.,11,1(2010).

30. J. Venugopal,S. Low, A.Choon, A. Bharath Kumar, S.Ramakrishna, Art.Org.,32,388(2008).

 

31. F. Ejaz Ahmed, B. Lalia, R.Hashaikeh, J.Desal., 356 ,15(2015).

32.H.Won,H.Lee,J.Knowless, Bio.M. Res.,79,643(2006).

33.N.Nega,T.Hoai,P.viet, Colls. Sur. B: Bio.Inter.,128,506(2015).

 

 

 

 

 

 

 

---