# FT-IR Characterization of Antimicrobial Hybrid Materials through Sol-Gel Synthesis
## 2 Materials and Methods  
Organic/inorganic hybrid materials MO\({}_{2}\)/PCL with M = Si or Ti were prepared, using the sol-gel process, from polycaprolactone of analytical reagent grade as the organic element, and tetramethyl orthosilicate (TMOS) as the source for SiO\({}_{2}\), or titanium butoxide as the source for TiO\({}_{2}\). The amount of the organic element was 6 wt.% in both PCL/SiO\({}_{2}\) and PCL/TiO\({}_{2}\) hybrid materials, as optimized in a previous work [15].  
In Figure 1, the flow chart of hybrid synthesis by the applied sol-gel route is depicted.  
The presence of hydrogen bonds between organic/inorganic elements of the hybrid materials was unraveled by acquiring their FT-IR spectra in the 400-4000 cm\({}^{-1}\) region using a Prestige 21 (Shimadzu, Kyoto, Japan) system with a resolution of 4 cm\({}^{-1}\) (45 scans). The spectrometer used a Deuterated Triglycine Sulfate (DTGS) detector with KBr windows. Disks with a diameter of 13 mm, a thickness of 2 mm, a weight of 200 mg and containing 1 wt.% of sample in KBr were obtained by pressing sample powders into a cylindrical holder using a Specac manual hydraulic press. The Prestige software (IR solution) was used to analyze the FT-IR spectra.  
The nature of MO\({}_{2}\) gel, PCL and PCL/MO\({}_{2}\) hybrid materials was confirmed by X-ray diffraction (XRD) analysis using a Philips diffractometer. Powder samples were scanned from 2\(\Theta\) = 5\({}^{\circ}\) to 60\({}^{\circ}\) using CuK\({}_{\alpha}\) radiation.  
The surface morphology and phase distribution of the organic/inorganic hybrid materials were investigated by a scanning electron microscope (Cambridge model S-240) and by an atomic force microscope (Digital Instruments Multimode) in contact mode in air. For SEM, the specimens were previously coated with a thin Au film to obtain a conductive surface.  
Bioactivity tests were performed on pieces of the hybrid materials soaked in a simulated body fluid (SBF) with the proper ion concentrations [16; 17] nearly equal to those of human blood plasma. During soaking, the temperature was fixed at 37 \({}^{\circ}\)C and the ratio between the exposed surface and the volume solution was kept constant (50 mm\({}^{2}\) mL\({}^{-1}\) of solution), due to its strong influence on the reactivity degree, as previously reported [17; 18; 19].  
The ability of the hybrid materials to form an apatite layer during the SBF soaking test was ascertained by FT-IR analysis on SiO\({}_{2}\)/PCL-reacted samples, where SEM and EDS analyses were performed on TiO\({}_{2}\)/PCL-reacted samples. An SEM outfitted with an energy-dispersive X-ray fluorescence system (EDS, Link AN10000, Oxford Microanalysis) was used to investigate the morphology of the coated sample and to complete it with qualitative elemental analysis.  
The antimicrobial activity of synthetized materials was investigated by a diffusion method. _Escherichia coli_ (ATCC 25,922) bacteria culture was diluted in distilled water to achieve a \(1.0\times 10^{6}\) CFU/mL suspension. This latter was inoculated in Tryptone Bile Glucuronide Agar (TBX) medium (Liofilchem, Roseto degli Abruzzi (TE), Italy). Sample hybrids and a control disk were placed at the center of the Petri dishes using a pair of sterile tweezers. Incubation was carried out for 24 h in an incubator at 44 \({}^{\circ}\)C. The microbial growth was evaluated by observing the diameter of the inhibition halo (ID). Antimicrobial tests were performed in quadruplicate on different days to ensure reproducibility, and values are expressed as the mean values \(\pm\) standard deviation (SD) of all the performed measurements.  
Figure 1: Flow chart of PCL/SiO\({}_{2}\) and PCL/TiO\({}_{2}\) gel synthesis.