Facile synthesis of Fe 3 O 4 /Sio 2 /Cu nanoparticles: investigation of physicochemical attributes of interaction in cationic micellar media and photo-antibacterial impact

Solubilization of core-shell magnetic nanoparticles has gained new popularity as a versatile partitioning method with the recent growth in nanotechnology and related biotechnology applications. In this sense, Fe 3 O 4 /SiO 2 /Cu nanoparticles (NPs) were designed and characterized by various techniques such as Ultra-violet visible (UV-vis), Fourier transforms infrared (FTIR), X-ray diffractometry (XRD), scanning and transmission electron microscopy (SEM & TEM), energy dispersive X-ray (EDX) and thermogravimetric (TGA) analyses. The size determined by TEM analysis of Fe 3 O 4 /SiO 2 /Cu NPs was found in the range of 24 nm. The diffractometer results verified the impurity-free synthesis of the desired nanoparticles. Micellar solubilization and thermodynamics of micellization were determined correspondingly using a UV-Vis spectrophotometer and conductometer. The thermodynamics of micellization in terms of enthalpy, entropy and standard Gibb’s free energy was determined using electrical conductometry. The solubilization parameters viz. partition constant (K c ), binding constant (K b ), partition coefficient (K x ), and the corresponding Gibb’s free energies were estimated from the absorption spectroscopic analysis. The experimental outcomes and their computation revealed the rise in the critical micelle concentration of CTAB due to the incorporation of the nanoparticles with the micelles. Moreover, the photo-antibacterial activity of the nanoparticles in the bacteria culture environment showed effectiveness in comparison to the control group. ______________________________ broth (5 ml) followed by overnight incubation at 37°C. After the centrifugation of the samples and these were washed with phosphate buffer saline (PBS) and their concentration was adjusted appropriately. The effect of Fe 3 O 4 /SiO 2 /Cu NPs on bacterial strains was evaluated by suspending the nanoparticles in Mueller-Hinton broth (2% w/w). The growth of the culture medium was also compared with the photocatalysts. Mueller-Hinton broth inoculated with bacteria served as the positive control. The sterile Microtubes were filled with 0.5 ml of stock solutions and the final concentration was made up to 1 mg/mL, followed by the addition of bacterial suspensions to each Microtube (approx. 105 colony-forming units/mL). The prepared systems were categorized into three sets, the first set was exposed to a UV light source (50W) for 16 min and the samples were drawn from the mixture at various time intervals viz. 0, 2, 4, 8, and 16 min. The second set was subjected to the visible light source (Xenon lamp) and the third set was left without any light source for 160 min the samples from these two groups were taken at intervals of 0, 20, 40, 80, and 160 min. The serial dilutions of the bacterial culture aliquots were prepared in PBS and the colony-forming number of viable cells (CFU/ml) was assessed by the pouring plate technique. All the measurements were observed in triplicate.


Introduction
To date, many methods of remediation of the industrial effluents have been in practice, but the methods based on biological, physical, and chemical treatments are very common [1]. The methods such as photocatalytic degradation, membrane process, reverse osmosis, ozonation, oxidation, photo-Fenton and electrochemical procedures have been extensively employed for remediation [2]. Most of these above-mentioned methods have some restrictions except photocatalytic degradation which acts as a very handy and smart tool due to being simple, efficient, and inexpensive [3]. Moreover, it could be operated under ambient conditions, and it has excellent biological and chemical sensing and minimal formation of byproducts. Among the many approaches to improving water and food quality against microbial agents, the use of nanoparticles has emerged on the horizon to contribute to this field [4]. Nowadays, the use of copper-containing semiconductor nanoparticles as photocatalysts for disinfection and purification of industrial and domestic effluents is due to its low cost, easy availability, chemical stability, anti-bacterial property and exceptional photocatalytic potential to degrade recalcitrant organic pollutants [5,6]. With the irradiation of photocatalysts with suitable light sources various reactive species such as superoxides, and hydroxyl radicals along with photo-generated electrons and holes are generated which can cause the deactivation of microorganisms [7]. Previously reported results by some researchers have shown that bare copper nanoparticles showed weak photocatalytic activity under visible light sources and had a narrow light-response range [8]. With the incorporation of copper in a suitable nanosystem, the photocatalytic performance, and separation efficiency of electron-hole pairs could be improved during the photocatalytic process [9]. The incorporation of the silicon oxide layer between the photocatalyst shell and the magnetic core prevents the recombination of electrons (e -) and holes (h + ) [10]. This could decline the negative effects of Fe 3 O 4 on the photocatalytic process of Cu, retain the magnetic properties, prevent oxidation of Fe 3 O 4 , and enrich the antibacterial efficiency [11].
Considering the biological applications of magnetic nanoparticles, the study of micellar interaction is of much importance [12]. Surfactants are amphiphilic moieties that form colloidal size micelles to facilitate the solubilization of insoluble or sparingly soluble substances in aqueous media [13,14]. The dynamic phenomenon of solubilization plays many key functions during biological and industrial processes [15]. Various industries, especially the textiles and pharmaceutical sector are expediting the research on the role of surfactant interactions with the help of models, procedures, and techniques [16,17].
In this study, our research group managed to synthesize Fe 3 O 4 /SiO 2 /Cu NPs and studied the interactions in the cationic micellar media of CTAB (cetyltrimethylammonium bromide) along with the photo antibacterial activity.

Experimental details
From Sigma Aldrich all reagents were purchased and were used without further purification. FTIR Bruker spectrometer (Bruker Tensor 27 FTIR) for the recording of IR spectra. The data were collected in the range of 400 to 4000 cm -1 . The crystallite size was measured by Bruker D8 Advance Powder x-ray Diffractometer with high-resolution LynxEye detector and Cu radiation source. Scanning Electron Microscope (SEM) Philips XL-30E was employed to observe the morphology of nanoparticles. The samples were carbon coated and their morphology was probed using SEM working at an operating voltage of 20 kV. The composition of the material was determined by energy-dispersive X-ray (EDX) spectroscopy. In the case of Transmission Electron Microscopy (TEM) images, the suspensions were dropped onto copper grids; the ethanol was eliminated via the freeze-drying technique; the specimens were subjected to morphological observations on a Transmission Electron Microscope (Philips EM208) with an accelerating voltage of 100 kV.

Synthesis of Fe 3 O 4 NPs
First, the solution was made by dissolving 1g of polyvinyl alcohol (PVA 15000) as a surfactant and 4.8 mmol FeCl 2 .4H 2 O in 15 mL of water followed by 4.8 mmol of FeCl 3 .6H 2 O. The stirring was done at 80°C for half an hour, followed by the drop-wise addition of hexamethylenetetramine (HMTA; 1 mol/L) with vigorous stirring to obtain a black solid at pH 10. The black product was immediately filtered, washed with ethyl alcohol, and then dried at 85 °C for 7 h.

Synthesis of Fe 3 O 4 /SiO 2 NPs
The Fe 3 O 4 /SiO 2 NPs have been synthesized by a modified Stober method [18]. In a 55 mL mixture of deionized water and ethanol (1:10), the 2.1 mmol of Fe 3 O 4 NPs and 0.2 mL of tetraethoxysilane (TEOS) were added followed by 5 mL of NaOH (10 % wt). The product (Fe 3 O 4 /SiO 2 NPs) was separated after 30 min, stirring at ambient temperature. The product was immediately washed with ethyl alcohol and then dried at 85 °C for 7 h.

Synthesis of Fe 3 O 4 /SiO 2 /Cu NPs
To synthesize Fe 3 O 4 /SiO 2 /Cu NPs, 50 mg of Fe 3 O 4 /SiO 2 NPs was dispersed in a 50 mL mixture of MeOH/ H 2 O (85:15). The pH was maintained at 4.5 by adding 1M HCl. The solution was placed in an ultrasonic bath for 2 h. After that 0.5 mmol of N-[ (3-trimethoxysilyl) propyl] ethylenediamine tri acetic acid was added along with a few drops of glacial acetic acid. The complete reaction mixture was again placed in the ultrasonic bath for 2h. The product was precipitated from the reaction mixture using a super magnet followed by acetone washing and then dried in a vacuum at room temperature for 30 min to obtain modified Fe 3 O 4 /SiO 2 NPs. Afterwards, the CuCl and modified Fe 3 O 4 /SiO 2 NPs in the equimolar ratio were added to deionized water and stirred for 3 h. The resulting product was separated using a magnet and washed successively with water (10 mL) and ethyl alcohol (10 mL) to get rid of any unreacted CuCl. The product was finally dried in a vacuum at room temperature to yield the Fe 3 O 4 /SiO 2 /Cu NPs.

Solubilization study 2.2.1. Electrical conductivity to estimate thermodynamic parameters
The thermodynamics of micellization (entropy, enthalpy, Gibb's free energy) were calculated from the conductivity data of CTAB in the presence and absence of nanoparticles under study. The conductivity of all the solutions was recorded on a Hanna conductivity meter (HI-99301; USA) at three different temperatures viz. 298K, 308 K, 318 K. The electrode coated with platinum black was used to minimize and avoid polarization within a range of 0.01 µS/cm to 199.9 mS/cm. The electrode was initially calibrated using standard potassium chloride solution within the required concentration range [19,20].

UV-Visible spectroscopy to estimate partition and binding parameters
The absorptions of aqueous and micellar solutions were measured with a double beam UV-Visible spectrophotometer (Perkin Elmer, USA). The absorption scanning and spectrum solutions in the absence and presence of Fe 3 O 4 /SiO 2 /Cu NPs were observed. After an initial screening of Fe 3 O 4 /SiO 2 /Cu NPs absorption, the absorption maxima were compared to foresee the extent of micellar interaction. Once the interaction was verified from the comparative absorption values (λ max ), the critical micelle concentration was monitored for varying CTAB concentrations. The simple and differential absorption spectra were recorded to estimate the extent of interaction. While measuring simple absorption, the reference was distilled water, whereas the stock solution of Fe 3 O 4 /SiO 2 /Cu NPs was the reference. In both cases, the micellar solution of Fe 3 O 4 /SiO 2 /Cu NPs was taken in the sample cells.

Photo antibacterial activity
The effectiveness of the synthesized nanoparticles was determined by their photocatalytic ability to decolorize bacterial strains. We selected the bacterial strains of Staphylococcus aureus (ATCC-6538; Gram-positive), and Escherichia coli (ATCC-25922; Gram-negative) to carry out pure culture investigation. The bacteria were inoculated by sterile aliquots of Mueller-Hinton broth (5 ml) followed by overnight incubation at 37°C. After the centrifugation of the samples and these were washed with phosphate buffer saline (PBS) and their concentration was adjusted appropriately. The effect of Fe 3 O 4 /SiO 2 /Cu NPs on bacterial strains was evaluated by suspending the nanoparticles in Mueller-Hinton broth (2% w/w). The growth of the culture medium was also compared with the photocatalysts. Mueller-Hinton broth inoculated with bacteria served as the positive control. The sterile Microtubes were filled with 0.5 ml of stock solutions and the final concentration was made up to 1 mg/mL, followed by the addition of bacterial suspensions to each Microtube (approx. 105 colony-forming units/mL). The prepared systems were categorized into three sets, the first set was exposed to a UV light source (50W) for 16 min and the samples were drawn from the mixture at various time intervals viz. 0, 2, 4, 8, and 16 min. The second set was subjected to the visible light source (Xenon lamp) and the third set was left without any light source for 160 min the samples from these two groups were taken at intervals of 0, 20, 40, 80, and 160 min. The serial dilutions of the bacterial culture aliquots were prepared in PBS and the colonyforming number of viable cells (CFU/ml) was assessed by the pouring plate technique. All the measurements were observed in triplicate.

Results and discussion
The path used to synthesize the Fe 3 O 4 /SiO 2 /Cu NPs is given in Fig. 1, where TEOS is a tetraethoxysilane molecule and TET is N-[ (3-trimethoxysilyl) propyl] ethylenediamine tri acetic acid molecule. Fig. 2 represent the UV-vis absorption spectra of Fe 3 O 4 NPs with and without a SiO 2 as well as Fe 3 O 4 /SiO 2 NPs decorated by Copper. No obvious peaks were observed in the spectrum of Fe 3 O 4 NPs. However, the presence of SiO 2 caused the appearance of a peak at 265 nm in the spectra of the Fe 3 O 4 /SiO 2 NPs. This peak may originate from the changes in the bandgap caused by the quantum size effect and surface effect of SiO 2 NPs [21], as well as from the Fe-O-Si bonds of the core-shell NPs [21]. Moreover, decorated Fe 3 O 4 /SiO 2 NPs with Cu (I) caused the appearance of a broad peak around 600 nm, which originates from the copper NPs [22]. Interestingly, the peak at 250 nm, which was visible in the UV-vis spectrum of Fe 3 O 4 /SiO 2 NPs, was also shifted in   Fig. 3 (Fig. 2b) shows two peaks at 1660 and 1385 cm -1 which are attributed to C=O and C-N groups of ligands, respectively and stretching vibration of Si-O appears at 1050, 1110 cm -1 . In the FTIR spectrum for Fe 3 O 4 /SiO 2 /Cu NPs (Fig. 2c), the appearance of a peak at 603.61 cm −1 was assigned to Cu-O stretching vibration.

SEM results
The field emission scanning electron microscopy (SEM) image in Fig. 4

HR-TEM results
As seen in TEM images (Fig. 5

), Fe 3 O 4 NPs (A) appear spherical while Fe 3 O 4 /SiO 2 NPs (B) it can be seen clearly that Fe 3 O 4 NPs are covered with silica coatings. Fe 3 O 4 /SiO 2 /Cu NPs (C)
appear roughly spherical having an average diameter of around 24 nm. The aggregation of nanoparticles may be due to strong interactions between the particles.

Thermogravimetric analysis
The TGA curves of Fe 3 O 4 /SiO 2 /Cu NPs are shown in Fig. 7. The TGA analysis was applied to investigate the thermal stability of Fe 3 O 4 /SiO 2 /Cu NPs. The decomposition was in several steps, which the first stage shows the evaporation of water that is physically absorbed on the surface of the nanoparticle and the second stage might be related to the removal of organic moiety involved in the synthesis process. The total weight loss calculated is 9.714 %.

Estimation of thermodynamics of micellization
Electrical conductivity was employed to estimate the thermodynamic and micellar parameters due to their reliability and sensitivity. On micelle formation, some noticeable change was observed in the electrical conductivity and critical micelle concentration was located at the intersectional point of pre-micellar and post-micellar straight lines in the conductivityconcentration plot, as shown in Figure 8, which represents conductivity-concentration plots of Fe 3 O 4 /SiO 2 /Cu NPs in the presence of CTAB at varying temperatures. The thermodynamic attributes were estimated from conductivity data using equations 1-4.
The thermodynamic parameters calculated at varying temperatures are summarized in Table 1   The relationship between temperature and CMC is not easy to understand. With the temperature rise, there occurs dehydration from the hydrophilic part of micelles facilitates micellization. On the contrary, the dehydration from hydrophobic parts of micelles increases the entropy which in turn disfavors micellization and it occurs at higher CMC. The thermodynamics attributes of micellization are presented in Table 1. The negative magnitudes of Gibb's free energy and entropy of micellization indicated the spontaneity of the Fe 3 O 4 /SiO 2 /Cu NPs/CTAB system. The involvement of hydrophobic and electrostatic interactions was responsible for the system's stability and spontaneity. The exothermicity of the micellar system was indicated by the negative magnitude of ∆H m . The continuously rising negative value of enthalpy revealed greater hydration of polar groups than the structure breaking effect of water molecules in the vicinity of hydrophobic moieties.

UV-Visible spectroscopic study
Absorption spectra of Fe 3 O 4 /SiO 2 /Cu NPs in the aqueous and micellar media were recorded at 298 K. The absorption maxima (λmax) of Fe 3 O 4 /SiO 2 /Cu NPs were spotted at 454 nm in aqueous media and this wavelength was used for subsequent absorption analysis. A shift of λmax was critical in deciding the extent of interaction of Fe 3 O 4 /SiO 2 /Cu NPs with CTAB. The hydrophobic force and electrostatic interactions pushed Fe 3 O 4 /SiO 2 /Cu NPs to re-adjust and relocate themselves within micelles. The gradual rise in the absorption with increasing CTAB concentration was observed, possibly due to the significant incorporation of Fe 3 O 4 /SiO 2 /Cu NPs in the micelles and its CMC value shifted from 0.9 mM to 0.917 mM (Fig. 9). The CMC obtained from UV-vis absorption data (0.917 mM) and conductivity data (9.14 mM) agreed. The slight difference in the values was mainly due to their different working principles of the techniques, and secondly, the values CMC is fixed rather it can be found within a certain concentration range between pre-and post-micellar regions. Differential spectroscopy was employed for quantitative and qualitative interpretation of micellar interactions. It facilitates estimating binding, partitioning constants, and their corresponding free energy changes. A similar trend was observed in differential absorbance based on gradual and continuous inclusion of Fe 3 O 4 /SiO 2 /Cu NPs within CTAB micelles, as depicted in Fig. 10b. Kawamura devised a model to estimate the partition constant (K c ) and partition coefficient (K x ) are given in equations 5 and 6 [23] and the other parameters were calculated using the following equations (5)(6)(7)(8)(9).

(a)
Partition constant (K c ): (c) Standard change in Gibb's free energy of partition: Binding constant (K b ): Standard change in Gibb's free energy of binding: In the above equations: ∆A = differential absorbance; K c = partition constant; K x = partition coefficient; C a = concentration of Fe 3 O 4 /SiO 2 /Cu NPs; ∆A ∞ = differential absorbance at infinite dilution; = C s -CMC o ; C s = concentration of CTAB, n w = number of moles of H 2 O per liter. Plots to calculate partition and binding constants are given in Figure 10 while their corresponding numerical values are given in Table 2.

Assembling of Fe 3 O 4 /SiO 2 /Cu NPs in the micelles of CTAB
The dynamic nature of solubilization caused the Fe 3 O 4 /SiO 2 /Cu NPs to stay for a different time in the exterior and interior of micelles and this affected UV-Vis absorbance, thus we witnessed random absorptions. The degree of solubilization and plausible location of Fe 3 O 4 /SiO 2 /Cu NPs in CTAB micelles was directly related to temperature, the concentration of the participating additives and surfactant, chemical and structural nature of additive molecules. The location and mechanism of the process rely on quantitative values of partition constant and partitioning coefficient within micellar and aqueous phases. Higher values of K x directed the Fe 3 O 4 /SiO 2 /Cu NPs to accommodate in the palisade layer of micelles.
The higher the value of Kx, the greater tendency of transference of Fe 3 O 4 /SiO 2 /Cu NPs towards the outer region of micellar media due to the interaction of carbonylic negative charge and cationic head of CTAB. The values of partition coefficient Kx (201 × 10 3 ) in the micelles were significantly higher as compared to Kb (4.33×10 3 dm 3 mol -1 ). This system was stable and partitioning of Fe 3 O 4 /SiO 2 /Cu NPs was spontaneous as indicated by negative values of ΔGp (-32.25 kJmol -1 ) and ΔGb (-20.75 kJmol -1 ) as summarized in Table 2. Based on portioning attributes, the possible locus of Fe 3 O 4 /SiO 2 /Cu NPs in the micelles of CTAB is presented in Figure 11.

Photo antibacterial properties
The results of the tests performed on S. aureus exposed to UV light sources are shown in Fig. 12A. The control group showed very small growth as compared to Fe 3 O 4 /SiO 2 /Cu NPs. The results of the sample exposed to a visible light source and under dark conditions are illustrated in Fig. 12B and 12C. As presented in Fig. 12B, the control group showed a significant increase in bacterial growth over time, but the presence of Fe 3 O 4 /SiO 2 /Cu NPs prevented bacterial growth. The bacterial growth in all samples under dark conditions demonstrated light dependence of antibacterial properties. The photo-antibacterial examination of E. coli in the UV light, visible light and dark conditions are shown in Fig. 13A, 13b and 13C correspondingly. In Fig. 13A it is very clear that the decline in colony count is attributed to Fe 3 O 4 /SiO 2 /Cu NPs and comparatively the control group showed no significant change. The control group experienced significant bacterial growth under visible light could be seen in Fig. 13B, whereas the presence of Fe 3 O 4 /SiO 2 /Cu NPs caused the highest decrease in growth. All the samples under dark conditions experienced growth in darkness with time (Fig. 13C). This trend highlighted the role and significance of the importance of light in reaction systems.

Conclusions
Fe 3 O 4 /SiO 2 /Cu NPs were successfully synthesized via the modified Stober method. Thermodynamic studies demonstrate the increases in the critical CMC of surfactant due to the incorporation of the Fe 3 O 4 /SiO 2 /Cu NPs in the micelles. From experimental findings, it was noticed that the value of critical micelle concentration in the Fe 3 O 4 /SiO 2 /Cu NPs/CTAB system was higher than pure CTAB. The CMC of pure CTAB shifted from 0.9 mM to 0.914 mM as revealed by conductivity data and to 0.917 mM as indicated by UV-Vis analysis. The photo antibacterial studies on E. coli and S. aureus by Fe 3 O 4 /SiO 2 /Cu NPs under dark conditions, UV, and visible light irradiation showed that the optimized nanoparticles have a good antibacterial impact.