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MODIFICATION HYDROXYAPATITE WITH ZnO AS ANTIBACTERIALDwi Aprillia Wulandaria, Diana Vanda Welliaa , Novesar Jamaruna aMaterials Chemistry Laboratory Department of Chemistry, Science Faculty, University of Andalas

e-mail: [email protected]

Department of Chemistry, Faculty Unand, Limau Manis, 25163

Abstract

Modification with zinc oxide (ZnO) via sol-gel method has been successfully carried out. The analysis of X-Ray Diffraction (XRD) showed hexagonal crystal system and the addition of zinc can increase the intensity and crystallinity of the sample. The spectrum of Fourier Transform Infrared (FTIR) showed the presence of functional groups suitable for the identification of HA in wave numbers 3000-3600 cm-1, 1000-1045 cm-1 and 1300-1490 cm-1 indicating the presence of the O-H, P-O and C-O, respectively. Antibacterial test has been done using disc diffusion method and resulted that the capability of HA as antibacterial increasing with the increase of ZnO concentrations. HA/ZnO 1:1 with a concentration of 1000 ppm has inhibitory activity with a maximum diameter of 8 millimeters.keywords: Hydroxyapatite, ZnO, Sol gel, Antibacterial.I. IntroductionHydroxyapatite (HA), Ca 10 (PO 4) 6 (OH) 2, is the main mineral constituent of bone and tissue sources of calcium in living organisms. Synthetic HA is an inorganic material that shows the versatile nature of biocompatibility, bioactivity and osteoconductivity. HA also has the ability as an antibacterial that can inhibit biofilm formation and colonization of planktonic cells.1

HA is often modified with other metals to obtain superior biological properties again. Some form of modification in the form of the HA surface coating with another metal or metals deposited there as well with the the matrix HA.1 Several studies have been done conducted on several HA is coating with silver (HA/Ag) for the purpose of obtaining the optimum capability HA and Ag as antibacterial, against gram-positive bacteria and negatif2, as antifungal Candida albicans1, with Zirconia (HA/Zr) to improve the mechanical properties so more robust.3 In addition to Ag metal, other metals that can be used to modify HA to be able to enhance the biological capability as antibacterial, among others, can be used with heavy metals oligodinamic high power, one of which Zn. The use of Zn metal as a carrier of metal ions Zn2+ has high antibacterial properties, good chemical stability, and relatively low prices. Zn is known as an essential heavy metals, toxic metal though, it is needed by the body for growth in very small amounts. Toxicity owned Zn metal will effect when it enters the body in the sheer number of large organisms or exceeds the threshold toleransinya.4 Research on HA modified with Zn is expected to improve the ability of HA as new in the field of inorganic antibacterial.Tests conducted on the bacteria Escherichia coli (E.colli) by disc diffusion method to look at the diameter of the clear zone is not overgrown with bacteria. This bacterium is a reasonable amount of beneficial bacteria for digestion, but if excessive breeding can produce enterotoxin of E. coli as a cause of some cases of diarrhea and meningitis.5 In this study, the method used is the sol-gel method, because it has a better purity and can be used at low temperatures, one of which is in the process of synthesis HA.6II. Research Methodology 2.1. Chemicals, equipment and instrumentation

For the materials used in the study, among others, Synthetic hydroxyapatite obtained from the Materials Laboratory (Andalas University), Media Nutrient Agar (Merck), Nutrient Broth Media (Merck), cultures of E. coli, 65% HNO3 (Brataco), distilled water, (NH4)2HPO4 (Merck), NH4OH technical (Brataco), Zn (NO3) 2 (Merck).

While equipment used in the study, including glass cup, erlenmeyer, test tubes, measuring cup, ose, spatulas, hot plate stirrer, magnetic bars, analytical balance, buchner spiritus, sterile cotton, cotton buds, calipers, oven, incubator, autoclave, furnace, pulvurizer.Tools of instrumentation used X-Ray Diffraction (XRD; Shimadzu diffractometer 610 40 kV - 30 mA, Cu K), Fourier Transform Infrared (FTIR ; Jasco FTIR-410), Scanning Electron Microscopy-Energy Dispersive X-Ray (SEM - EDX; JEOL SEM).

2.2. Research procedures

2.2.1 Modification of HA/ZnO

Into a 500 mL HA gel was added Zn (NO3)2 with various concentrations of HA/ZnO 1: 0.25, 1: 0.5, 1: 0.75 and 1: 1. Stirring during the process of adding a constant speed. After the addition of Zn(NO3)2 is completed stirring process continued for 10 min and aging for 24 hours to form a gel. Obtained gel was heated in a 70 C oven for 24 hours, powder in furnace at a temperature of 600 C. Powder is obtained HA/ZnO with a variation of 1: 0.25, 1: 0.5, 1: 0.75 and 1: 1.2.2.2 Antibacterial Activity Test

The process begins with a media NB, then carried to the culture of E. coli, after 24 hours, the bacteria grow with marked turbidity in NB media. Testing is done by setting the inhibition of 2 pieces that have petridish NA containing solid media. Each sample is tested against HA and HA / ZnO with a variation of 1: 0.75 and 1: 1 with a concentration of 500 and 1000 ppm diluted with distilled water.

Petridis inserted into each each paper disc that has been soaked in antibacterial solution (HA and HA / ZnO). At the center of the petri paper disc placed as a negative control. After 24 hours of observations were made of the clear zone are not overgrown microbes. Measured against clear zone diameter and compared with a standard diameter of the inhibition of HA and Zn on the growth of bacteria.

III. Hasil dan Pembahasan3.1.Analysis of the results of characterizationThe addition of Zn (NO3)2 as a source of Zn2+ to form ZnO. Increasing the intensity of the color is directly proportional to the increase in the concentration of Zn(NO3)2 is added to the HA matrix, the composite ratio of HA/ZnO 1: 1 is the intensity of the green color is more concentrated. Powder HA/ZnO obtained at various concentrations is characterized by a wide range of instrumentation equipment under the same conditions. Characterization by XRD, conducted on 3 samples at calcination temperature of 600 oC with a ratio HA/ZnO 1: 1, 1: 0.75 and HA synthesis. XRD data of test results compared to the standards of the International Centre for Diffraction Data (ICDD) for ZnO with refference code 00-019-1463 and 01-075-9526 for refference code hydroxyapatite.

Figure 1. diffractogram XRD results of HA/ZnO concentration ratio 1:1 after calcination at 600 oC for 3 hours.The results of XRD analysis in Figure 1 in the form of HA/ZnO variation in concentration of 1:1 shows the best crystallinity with the highest intensity at 2 angles 31,74o, while the peak for ZnO is found at an angle 2 32,16o. HA crystals obtained system is hexagonal with space group P63/m. The difference of the three samples HA/ZnO 1:1, 1:0.75 and HA synthesis seen in crystallinity and increase the intensity of Zn(NO3)2. From the XRD data sample results, the more sources of Zn were added to the HA will increase the intensity of the peaks and minimize the width of the peak of HA. Thus modification of HA with ZnO will increase the peak intensity and narrow peak width thus increasing the crystallinity of HA.The analysis of Fourier Transform Infrared (FTIR) is very important to know the functional groups found in the sample, whether it is in accordance with the existing functional group on HA, Ca10(PO4)6 (OH)2. Some functional groups that can be identified by FTIR, among others, the hydroxyl group on the O-H bond P-O and C-O bond in the fingerprint region. Of the three FTIR spectra as found in figure 2 seen a wide valley that marks the OH stretching specific to HA over wave number 3000 cm-1 to 3600 cm-1. While the PO group stretching at 1000-1045 cm-1 wave number. CO group is found at 1300-1400 cm-1. At the peak of 1632 cm-1 seen the OH bending vibrations on the surface of the sample. Additions of Zn on the HA does not affect the results of its FTIR spectrum. So it can be concluded that, no new chemical bonds are formed between HA with ZnO.

Figure 2. FTIR spectra of the samples HA/ZnO concentration ratio 1: 1 after calcination at 600 C for 3 hours,3.2 Application Antibacterial Activity of HA and HA/ZnO

Antibacterial test activities as an antibacterial agent against E. coli is done by using the disc diffusion method. Where the inhibition of antibacterial can be seen in the diameter of the clear zone is not overgrown with bacteria and compared with the negative control.

Figure 5 showed antibacterial test results on samples of HA, HA/ZnO 1: 0.75 and HA/ZnO 1:1. The images show the diameter of the clear zone on the concentration of HA/ZnO at a concentration of 1000 ppm of 1:1 by 8 mm, for HA/ZnO 1:0.75 at a concentration of 1000 ppm obtained clear zone size of 6 mm, while for the HA concentration of 1000 ppm only obtained a diameter of 5.7 mm. The results obtained show HA/ZnO 1:1 coined the inhibitory effect on bacterial growth greater than HA/ ZnO 1: 0.75 and synthesis HA.

Figure 5. Clear zone around the growth of E. coli that has been given a antibacterial HA and HA/ZnO (at a concentration of 500 ppm and 1000 ppm).The ability of increasing inhibition, comparable with the addition of Zn to the HA. The positive charge of the Zn2+ ion and a negative charge on the bacterial cell membrane will cause attraction between Zn2+ ions and negatively charged areas on the bacterial cell wall to form an electrostatic bond. This will form an electrostatic bond stress leads to damage the cell wall permeability, reducing the normal intake of nutrients to sustain life, causes cells to become dead.4 Thus, a growing number of Zn2+ in the sample, the greater the ability to inhibit the growth of E. coli bacteria. IV. ConclusionModified hydroxyapatite with ZnO via sol gel method has been successfully carried out. Analysis X-Ray Difraction (XRD) showed that the addition of zinc can increase the intensity and crystallinity of the sample. Spectrum of Fourier Transform Infrared (FTIR) shows, the presence of functional groups suitable for the identification of HA which indicates the OH group, PO and CO. Antibacterial test by disc diffusion method showed the ability of HA as antibacterial increasing to an increase in the concentration of ZnO. HA/ZnO 1: 1 at a concentration of 1000 ppm maximum antibacterial activity against Escherichia coli with a diameter of 8 mm.V. AcknowledgementsAcknowledgements profusely, the two counselors, who have provided comments and suggestions in the smooth conduct of research and in the preparation of the journal.References1. Zamperini,C.A.,Andre,R.S.,Longo,V.M., 2013, Antifungal Applications of Ag-Decorated HA Nanoparticles, Hindawi Journal, No. ID 174398, Hal 9.

2. C.S.Ciobanu, 2012,Antibacterial activity of silver-doped HA nanoparticles against gram-positive and gram-negative bacteria, Nanoscale Research Letters, No.1, Vol.7, Hal 324.3. Vasconcelos,M.C.2011,Barreto, Tailoring the Microstructure of SolGel Derived Hydroxyapatite/Zirconia Nanocrystallin Composites, Nanoscale Research Letters, 2011, No.6, Hal. 20.4. Angraini, D.,2013,Sintesis Zn(II)/Silika dengan Metode Sol-Gel sebagai Material Antibakteri terhadap Escherichia coli dan Staphylococeus aureus. Thesis, FMIPA,program pasca sarjana, UGM, Yogyakarta.5. Kusuma,F.A., 2010, Escherichia Coli ,Fakultas Farmasi UNPAD,Bandung.6. Agrawal, K., Gurbhinder, S., Devendra, P., Satya, P., 2011, Synthesis and Characterization of Hydroxyapatite Powder by Sol-Gel Method for Biomedical Application, Metallurgical and Materials Engineering Journal, No. 8, Vol.10. c.

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