Materials and Methods
3. Amine functionalized magnetic iron oxide nanoparticles: Synthesis, antibacterial activity and rapid removal of Congo red dye
3.4 Result and discussion
3.4.7 Antibacterial activity
Figure 3.11 (A) Antibacterial activity by disc diffusion method (a) shows the zone of inhibition when E.coli treated with the 160 µg/ml of FTT (b,c,d) Shows the zone of inhibition when E.coli treated with 80 µg/ml, 40 µg/ml, 20 µg/ml respectively where zone of inhibition is not clearly visible. (e) At middle shows the zone of inhibition, when E.coli treated with standard drug Gentamicin. (B) Antibacterial activity by disc diffusion method (a) shows the zone of inhibition when B.subtilis treated with the 160 µg/ml of FTT (b) Shows the zone of inhibition when B. subtilis treated with 80 µg/ml. (c) At middle shows the zone of inhibition, when B.subtilis treated with standard drug Gentamicin.
Disc diffusion method was performed to check the antimicrobial potential of the adsorbent material. Cell viability test of E. Coli and B. Subtilis with FTT were done by using the colony counting method.Bacterial growth kinetics was studied in presence of the FTT as well as in absence. FESEM analysis was done to check the effect of FTT on bacterial cell
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morphology.EDX analysis was further done to check the element deposited in the bacterial body. Concentration and Time-dependent antibacterial activity of nanomaterial was also carried out.Antibacterial activity of FTT was determined by the interaction of E.coli and B.
subtilis with the FTT by using a varying range of the concentration. To determine its propensity of killing bacteria, disc diffusion assay has been performed and antibacterial activity was observed at concentration 160 µg/ml and 80 µg/ml. We have observed the clear zone of inhibition at the concentration 160 µg/ml for FTT in E.coli and B.Subtilis (shown in Figure 3.11).
Figure 3.12 Graph representing the antibacterial activity of FTT with varying concentration against, E.coli and B. subtilis by showing the percentage loss in bacterial cell viability.
Figure 3.13 Growth kinetics of (a) E.coli and (b) B. subtilis in presence as well as the absence of FTT. The experiment performed in triplicate and the result showed as the Mean ± SEM.
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Zone of Inhibition also observed at 80 µg/ml but it was not clear. At low concentration zone of inhibition was not predominantly seen. Concentration-dependent activity of FTT was again examined by colony counting method, maximum inhibition observed at 160 µg/ml and it gradually decreases with decreasing concentration (shown in Figure 3.12).Bacterial growth kinetics were studied in presence as well as the absence of FTT (Figure3.13).We found that the stationary phase in bacterial growth can be observed at an early stage when bacteria treated with FTT at concentration 160 µg/ml.Bacterial growth inhibition is shown in the curve in comparison to control one.Production of reactive oxygen species due to oxidative stress induced by FTT is considered as the possible mechanism behind the antibacterial activity of FTT. To quantify the amount of ROS generated in the influence of FTT is measured by an assay employing the use of DCFDA dye is followed. DCFDA have the potential to penetrate the bacterial cell membrane and then gets hydrolyzed inside bacteria.
The hydrolyzed product gives the green fluorescence in presence of reactive oxygen species like singlet oxygen, superoxide radical, hydroxyl radical, peroxide and hydroperoxide radical.The fluorescence intensity is directly proportional to the presence of reactive oxygen species.More the amount of ROS, more the fluorescence intensity.The concentration- dependent effect of FTT on ROS generation in E.coli and B.subtilis has been depicted in
Figure 3.14 Fe3O4-TSPED-Tryptophyan induced ROS generation in (a) E.coli and (b) B.
subtilis. Fluorescence intensity shows the ROS generation at different concentration of Fe3O4-TSPED-Tryptophan. Higher fluorescence intensity indicates the excess generation of ROS.
Figure3.14 ROS production could also be observed in the absence of FTT treatment but it is less prominent in comparison to the treated one. Bacterial death was further characterized by alteration in normal morphology of bacteria which was done by FE-SEM shown in (Figure A4 and Figure A5), EDX analysis was also done, by which we detected the presence of silicon and iron in the bacterial body.We have chosen tryptophan as a coating material to
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modify the surface properties of iron oxide nanoparticle, which inflect the functional group and surface potential of the material.The amino group of tryptophan has a strong binding affinity toward the paramagnetic FTT by the help of hydrogen bonding or electrostatic force of interaction between the molecules.This tryptophan tagged FTT comprises of the carboxylic group which carries OH group which directly interact with the water molecule by hydrogen binding force of interaction to form the colloidal dispersion of FTT.
This colloidal dispersion facilitates the interaction with the bacteria. Substantial interaction also increases ROS production due to iron oxide[221]. It is well known that tryptophan is found in its zwitterions form which imparts the positive or negative surface potential which supports the Coulomb repulsion among the iron oxide nanoparticle which again enhances dispersivity of particles[222].It is already reported that the iron oxide is capable of inducing the reactive oxygen species (ROS) generation which increases the oxidative stress[223, 224]
and then bacterial death. The electrostatic force of interaction with bacteria also alters membrane integrity [225]. The above-shown result has shown that the current nanoparticles have the potential to kill bacteria in dose-dependent as well as concentration-dependent manner.