Green Synthesis of Silver-Decorated Magnetic Particles for Efficient and Reusable Antimicrobial Activity

Metal and metal hybrid nanostructures have shown tremendous application in the biomedical and catalytic fields because of their plasmonic and catalytic properties. Here, a green and clean method was employed for the synthesis of silver nanoparticle (Ag NP)-SiO2-Fe2O3 hybrid microstructures, and biomolecules from green tea extracts were used for constructing the hybrid structures. The SiO2-Fe2O3 structures were synthesized using an ethanolic green tea leaf extract to form Bio-SiO2-Fe2O3 (BSiO2-Fe2O3) structures. Biochemical studies demonstrated the presence of green tea biomolecules in the BSiO2 layer. Reduction of the silver ions was performed by a BSiO2 layer to form Ag NPs of 5–10 nm in diameter in and on the BSiO2-Fe2O3 microstructure. The reduction process was observed within 600 s, which is faster than that reported elsewhere. The antimicrobial activity of the Ag-BSiO2-Fe2O3 hybrid structure was demonstrated against Staphylococcus aureus and Escherichia coli, and the nanostructures were further visualized using confocal laser scanning microscopy (CLSM). The magnetic properties of the Ag-BSiO2-Fe2O3 hybrid structure were used for studying reusable antimicrobial activity. Thus, in this study, we provide a novel green route for the construction of a biomolecule-entrapped SiO2-Fe2O3 structure and their use for the ultra-fast formation of Ag NPs to form antimicrobial active multifunctional hybrid structures.


Introduction
For the last seven decades since the discovery of antibiotics, their uncontrolled use has led to a generation of antibiotic-resistant strains that are unresponsive to currently available conventional antibiotics, creating a major concern for the health sector [1,2]. More newgeneration antibiotics have been introduced in the field of medicine; however, the toxicity and adverse effects of these antibiotics are worrying factors for implementation [3,4]. Therefore, alternate therapeutic strategies have been studied for the last two decades, and several types of nanomaterials have been tested for their antimicrobial activity that is nontoxic to human and nonhuman hosts [5][6][7]. Silver nanoparticles (Ag NPs) have been extensively studied because of their effective antimicrobial activity known since ancient times [8]. A detailed study of the antimicrobial activity of Ag NPs against a wide range of pathogenic microorganisms was performed; it was demonstrated that the Ag NPs act by releasing Ag+ ions, which act on bacteria through reactive oxygen species [8]. The antimicrobial activity of the Ag NPs decreases with aggregate formation, where Ag NPs form a bulky material that makes it difficult for them to act on small bacteria and viruses [9][10][11]. In addition, Ag NPs are not only toxic to the bacteria but also to mammalian cells, which is a major concern for the therapeutic use of Ag NPs [12]. Therefore, several functionalizations or immobilization procedures have been used to enhance their antimicrobial efficiency and avoid the aggregation and toxicity of Ag NPs before release into the environment [13]. Currently, extensive research is ongoing for the

Preparation of the Ethanolic Extract of Green Tea Leaves
Green tea leaves (1.2 g) were ground and refluxed for 4 h in anhydrous ethyl alcohol (99.9%) under magnetic stirring. The suspension was cooled at room temperature and centrifuged at 4000 rpm for 10 min to remove large particulates of green tea leaves. Next, the green tea leaf extract was filtered through Whatman filter paper to remove small leaf particulates. Before further experimental procedures, the purified ethanolic green tea extract was kept in the dark at 4 • C.

Synthesis of Fe 2 O 3 NPs
Fe 2 O 3 NPs were synthesized using a previously reported method [22]. Briefly, 1 M of iron nitrate Fe(NO 3 ) 3 was prepared in 50 mL of distilled water (DW) under N 2 gas bubbling for 30 min. The pH of this solution was maintained at more than 10 under vigorous stirring by using aqueous ammonium hydroxide (30%). Then, the solution was autoclaved at 150 • C for 10 h in a Teflon-lined stainless-steel reactor. The formed precipitate was centrifuged and washed several times with ethanol and water until a neutral pH was obtained. The particles were vacuum-dried at 50 • C for 12 h.

Synthesis of BSiO 2 -Fe 2 O 3 Particles
BSiO 2 -Fe 2 O 3 particles were synthesized using a previously reported method with modifications [23]. In the purified ethanolic green tea extract (40 mL), 50 mg of Fe 2 O 3 NPs were dispersed under sonication for 20 min. Two milliliters of TEOS was added dropwise to the Fe 2 O 3 NP suspension and further mixed with mechanical stirring for 1 h. The pH of the solution was adjusted to 11 by adding aqueous ammonium hydroxide dropwise. After 24 h of incubation, the formed particles were collected by centrifugation and repeatedly washed with ethanol and water. The BSiO 2 -Fe 2 O 3 particles were vacuum-dried at 50 • C for 12 h.

Synthesis of the Ag-BSiO 2 -Fe 2 O 3 Hybrid Microstructure
For the construction of the Ag-BSiO 2 -Fe 2 O 3 hybrid structure, the BSiO 2 -Fe 2 O 3 particles were dispersed in 10 mL of DW and sonicated for 20 min to disturb the agglomerates. Ten milliliters of aqueous AgNO 3 was poured into the solution to form a 10 mM AgNO 3 solution. The mixture was further incubated and stirred for 30 min and observed for color change. The formed hybrid structure was washed with water and ethanol several times, obtained with centrifugation, and vacuum-dried at 50 • C overnight.

Antimicrobial Activity
The antimicrobial activity of the Ag-BSiO 2 -Fe 2 O 3 hybrid microstructure was tested against Staphylococcus aureus and Escherichia coli. The growth media used for S. aureus and E. coli were trypticase soy broth and Luria-Bertani broth, respectively. The bacterial cultures were grown in the broths for 8 h to obtain the desired optical density (OD). Initially, the effect of the hybrid structure, streptomycin, AgNPs, and Fe 2 O 3 NPs was tested on the growth curve of the bacterial cells for 22 h. The growth of both microorganisms was monitored using UV-visible spectroscopy, that is, optical density at 610 nm. Various concentrations of the hybrid structure, from 50 to 90 µg mL −1 , were added to the broths (10 6 to 10 7 bacterial cells mL −1 ) and further incubated for 24 h at 37 • C. The antimicrobial activity of streptomycin, AgNPs, and Fe 2 O 3 NPs was also studied with varied concentrations. After incubation, 0.1 mL from the incubated broth was spread over the respective agar and incubated for 24 h and observed for growth, and the colony-forming units (CFUs) mL −1 were measured. The higher concentrations of the hybrid nanostructure inhibited the growth of the bacteria completely, and they were used for the antimicrobial reusability experiment, in which 200 µg mL −1 of the hybrid structure was inoculated in 10 6 to 10 7 bacterial cells mL −1 . To visualize the antimicrobial activity of the prepared particles, a live/dead staining assay was performed after the exposure of 50 and 200 µg mL −1 of the nanohybrid nanostructure to S. aureus and E. coli cultures, as specified in earlier reports. The cells were observed under CLSM (Carl Zeiss LSM 710, Jena, Germany) [24].

Estimation of Total Phenolic Content
The total phenolic content of the green tea extract prepared using water and ethanol was determined using the McDonald method [25]. Briefly, a standard solution of gallic acid monohydrate (100 µg mL −1 ) was prepared in methanol. Different concentrations of 0.1 to 1.0 mL of green tea leaf extract were mixed with a 1:1 mixture of Folin-Ciocalteu reagent and DW in 10 different tubes. To each tube, 7.5% sodium carbonate (2 mL) was added, and the mixture was allowed to react further for 30 min. The absorbance was measured at 765 nm, and the standard calibration curve was plotted. Then, 100 µL of the test samples was used to estimate the total phenolic content with a standard graph in terms of gallic acid equivalent (mg GAE g −1 extract).

DPPH Radical Scavenging Activity
The antioxidant properties of the ethanolic green tea extract and BSiO 2 NPs were measured based on the DPPH free radical scavenging activity [26]. In 100 mL of methanol, DPPH (6 mg) solution was prepared. The antioxidant activity was measured based on the discoloration of stable DPPH radicals in methanol, and at 517 nm, the absorbance was obtained. Different aliquots of the test samples were tested for the discoloration of DPPH solution. Using the given formula, the radical scavenging activity was calculated as follows: % Radical scavenging activity = absorbance (control) − absorbance (sample)/absorbance (control) × 100.

ABTS Radical Scavenging Assay
The ABTS radical scavenging assay was followed to demonstrate the antioxidant capability of the ethanolic green tea extract before and after TEOS hydrolysis by using a previously published method with some modifications [26]. Briefly, green tea extract and solution obtained from TEOS hydrolysis were diluted to different concentrations. They were added to 7 mM of ABTS standard solution prepared with 2.45 mM of potassium persulfate (K 2 S 2 O 8 ), and the mixture was further allowed to react for 6 min at 30 • C. After incubation, absorbance was measured at 734 nm. The radical scavenging activities of the extracts were calculated using the following equation and were compared with that of BHT: ABTS radical scavenging activity = absorbance (control) − absorbance (sample)/absorbance (control) × 100.

Analytical Instrumentation
UV-Visible spectroscopy was performed using a Schimadzu UV 1600 spectrophotometer with a 1 cm well-stoppered quartz cuvette. The morphology of the microstructures was evaluated by HR-TEM analysis using a JEM-3010 (JEOL) working at 300 kV and by energy-dispersive spectroscopy (EDS). Crystallography analysis of the nanostructures was performed using an X-ray diffractometer (Rigaku, Tokyo, Japan) with a Cu target. The magnetic properties of Fe 2 O 3 and Fe 2 O 3 hybrid structures were measured with a vibrating sample magnetometer (VSM; Lakeshore Cryotronics, Inc., Westerville, OH, USA). The antimicrobial activity was observed under CLSM. Dynamic light scattering (DLS) spectroscopy and zeta potential measurement of the particles in colloidal solution were performed using a NICOMP TM 380 ZIS (Santa Barbara, CA, USA) for the determination of the hydrodynamic diameter (HDD) and charge on the particles.

Synthesis of the Hybrid Microstructure
The plant extract-mediated synthesis of nanomaterials has been one of the green chemistry approaches followed for the preparation of various materials. Extracts from several plant parts have been studied for the synthesis of metal nanoparticles that contain various polysaccharides, proteins, vitamins, or alkaloids, functionalizing and capping the nanomaterials with nontoxic and biodegradable biomaterials [27]. The presence of biomolecules on the surface of the nanoparticles leads to an increase in the colloidal stability of the nanoparticles forming a biomolecular corona on the surface, which contributes to enhanced bioactivity of the nanoparticles [28,29]. Here, in the presented work, the magnetic, catalytic active hybrid structure was synthesized with plant leaf extract biomolecules entrapped in SiO 2 on the surface of the magnetic nanostructure and further dispersed in aqueous Ag + ions for the reduction process to form Ag NPs on the BSiO 2 -Fe 2 O 3 particle surface. Table S1 provides the total phenolic content of green tea in water and ethanol. Though the phenolic content in ethanol compared to water was low, ethanol is the most favorable for the extraction of total phenols because it prevents the oxidation of phenolic compounds. In addition, the antioxidant property of the ethanolic and water extract, obtained from ABTS and DPPH scavenging assays, revealed effective antioxidant activity in both solvents. In alkaline ethanolic green tea extract, TEOS was added for the formation the most favorable for the extraction of total phenols because it prevents the oxidation of phenolic compounds. In addition, the antioxidant property of the ethanolic and water extract, obtained from ABTS and DPPH scavenging assays, revealed effective antioxidant activity in both solvents. In alkaline ethanolic green tea extract, TEOS was added for the formation of the SiO2 layer, where the biomolecules present in the green tea extract become trapped, resulting in biomolecule-entrapped SiO2 (BSiO2) (Figure 1). The Fe2O3 NPs were synthesized using a previously reported hydrothermal method. Figure 2A,B show Fe2O3 NPs of an average diameter of 100 nm. To these magnetite NPs, the alkaline mixture of ethanolic green tea extract and TEOS was added and allowed to form the BSiO2 layer on the surface of the Fe2O3 NPs for 12 h at room temperature. In Figure 2C,D, the smooth layer of BSiO2 on the surface of the magnetite NPs can be observed to form a core-shell structure. The size of the magnetite NPs increased by approximately 30 nm, and a layer of 10-20 nm of BSiO2 was formed. The formed core-shell structure was exposed to aqueous AgNO3, and the active biomolecules of the BSiO2 layer reduced AgNO3 to form Ag NPs.
The synthesis of Ag NPs was confirmed using UV-visible spectroscopy ( Figure 3A). No absorbance for Fe2O3 and BSiO2-Fe2O3 NPs was observed in the range of 400-500 nm, whereas Ag-BSiO2-Fe2O3 NPs showed characteristic absorbance at 424 nm, confirming the formation of Ag NPs on the BSiO2-Fe2O3 hybrid nanostructure. There are several reports on the synthesis of Ag NPs using biomaterials originating from plants and bacteria such as carbohydrates, amino acids, enzymes, proteins, and polyphenols [30]. Moulton et al. proposed a mechanism for the synthesis of Ag NPs using epigallocatechin-3-gallate obtained from C. sinensis leaf extract [31]. In addition, tea polyphenols, flavonoids, and epicatechin present in C. sinensis leaf extract were found to function as reducing and capping agents in the Ag NP synthesis process [31]. DFT calculations showed that the catechol moiety of flavonoids has a low energy of dissociation for the O-H bond of -OH groups, which is less than that of the -OH groups present in other parts of flavonoids [32]. The -OH groups of the catechol moiety of flavonoids have been demonstrated to function as metal-ion-reducing molecules. Various solvents have been utilized for the extraction of polyphenols and epicatechins from various plant extracts to maintain their high antioxidant activity [33]. Green tea extracts prepared in ethanol have high polyphenol content and retain high antioxidant properties [33]. Here, microhybrid structures were fabricated using the concentrated catechin derivatives from ethanolic green tea extract with active reducing capacity. The Fe 2 O 3 NPs were synthesized using a previously reported hydrothermal method. Figure 2A,B show Fe 2 O 3 NPs of an average diameter of 100 nm. To these magnetite NPs, the alkaline mixture of ethanolic green tea extract and TEOS was added and allowed to form the BSiO 2 layer on the surface of the Fe 2 O 3 NPs for 12 h at room temperature. In Figure 2C,D, the smooth layer of BSiO 2 on the surface of the magnetite NPs can be observed to form a core-shell structure. The size of the magnetite NPs increased by approximately 30 nm, and a layer of 10-20 nm of BSiO 2 was formed. The formed core-shell structure was exposed to aqueous AgNO 3 , and the active biomolecules of the BSiO 2 layer reduced AgNO 3 to form Ag NPs.  formation of Ag NPs on the BSiO 2 -Fe 2 O 3 hybrid nanostructure. There are several reports on the synthesis of Ag NPs using biomaterials originating from plants and bacteria such as carbohydrates, amino acids, enzymes, proteins, and polyphenols [30]. Moulton et al. proposed a mechanism for the synthesis of Ag NPs using epigallocatechin-3-gallate obtained from C. sinensis leaf extract [31]. In addition, tea polyphenols, flavonoids, and epicatechin present in C. sinensis leaf extract were found to function as reducing and capping agents in the Ag NP synthesis process [31]. DFT calculations showed that the catechol moiety of flavonoids has a low energy of dissociation for the O-H bond of -OH groups, which is less than that of the -OH groups present in other parts of flavonoids [32]. The -OH groups of the catechol moiety of flavonoids have been demonstrated to function as metal-ion-reducing molecules. Various solvents have been utilized for the extraction of polyphenols and epicatechins from various plant extracts to maintain their high antioxidant activity [33]. Green tea extracts prepared in ethanol have high polyphenol content and retain high antioxidant properties [33]. Here, microhybrid structures were fabricated using the concentrated catechin derivatives from ethanolic green tea extract with active reducing capacity.  XRD spectra of the γ-Fe2O3 NPs (black), BSiO2-Fe2O3 particles (red), and Ag-BSiO2-Fe2O3 hybrid (blue) are shown in Figure 3B, demonstrating the crystallinity of the hybrid  (113), respectively (JCPDS card no. 969013048) [35]. The XRD pattern demonstrated the face-centered-cubic structure of metallic silver. A broad peak of 21-23 • at 2θ for SiO 2 was observed in the XRD of the Ag-BSiO 2 -Fe 2 O 3 hybrid structure. The VSM measurement with a peak field of 20 KOe at room temperature was used to calculate the magnetic hysteresis loops of γ-Fe 2 O 3 NPs (black), BSiO 2 -Fe 2 O 3 NPs (red), and Ag BSiO 2 -Fe 2 O 3 hybrid and to analyze changes in the magnetic behavior of the NPs with modifications ( Figure 3C, blue). The saturation magnetization (M s ) value of the γ-Fe 2 O 3 NPs was 61 emu g −1 at 300 K. After modification with BSiO 2 on the surface of the γ-Fe 2 O 3 NPs, the saturation magnetization value reduced to 39 emu g −1 , and it further declined to 23 emu/g because of the presence of Ag NPs in the BSiO 2 layer. The decrease in the saturation magnetization may be due to the mass replacement and shielding of the magnetic NPs with SiO 2 and Ag NPs. The remanent magnetization (Mr) and coercivity (Hc) of the bare Fe 2 O 3 were also affected by the modifications with SiO 2 and Ag NPs. The Mr decreased from 16.3 emu g −1 for γ-Fe 2 O 3 NPs to 11.5 emu g −1 for BSiO 2 -Fe 2 O 3 NPs and then to 6.4 emu g −1 for the Ag-BSiO 2 -Fe 2 O 3 hybrid structure. The Hc value of Fe 2 O 3 NP increased from 114 Oe to 121 Oe for the Ag-BSiO 2 -Fe 2 O 3 hybrid structure. The increase in Hc may be attributed to changes in the domain structure or surface coating anisotropy. Although the Ag-BSiO 2 -Fe 2 O 3 structure showed decreased saturation magnetization due to the presence of Ag NPs and SiO 2 , it displayed extreme magnetic behavior, which suggests its magnetic separation capability. Thus, the Ag-BSiO 2 -Fe 2 O 3 nanohybrid can be recovered and reused by applying an external magnetic field. Therefore, the proposed method for modifying the SiO 2 nanostructures can be used for the construction of various hybrid nanostructures. Figure 4A,B show HR-TEM images of the hybrid structure demonstrating the presence of small Ag NPs on the surface of the BSiO 2 -Fe 2 O 3 NPs, thus forming the Ag-BsiO 2 -Fe 2 O 3 hybrid structure. The Ag NPs were 5-10 nm in size and were distributed evenly in and on the BsiO 2 layer ( Figure 4C). EDS analysis confirmed the presence of elemental silver in the hybrid nanostructure ( Figure 4D). Figure 4E shows the elemental distribution of the Ag-BSiO 2 -Fe 2 O 3 structure displaying the elemental maps of C, Fe, O, Si, and Ag individually. The elemental mapping clearly shows that biomolecules from the green tea extracts were well distributed in the SiO 2 layer around the Fe 2 O 3 nanoparticles, which further led to the synthesis of Ag NPs to form the hybrid nanostructure. The FT-IR spectrum of the extract of green tea and Ag-BSiO 2 -Fe 2 O 3 NPs with the spectral range from 400 to 4000 cm -1 showed the presence of bands at~3327 cm −1 ,~2974 cm −1 , and 2880 cm −1 , corresponding to aliphatic OH groups, νCH 2 , and C-H stretching, confirming the presence of phenolic compounds in green tea and Ag-BSiO 2 -Fe 2 O 3 NPs ( Figure S1) [36].

Antimicrobial Activity
The effective antimicrobial activity of the NPs is dependent on the NPs aggregation and interaction of the NPs with microbial cells. Thus, the hydrodynamic diameter (HDD) and zeta potential studies of the NPs provide information regarding the size and charge on the NPs in the colloidal solution, demonstrating the stability of NPs. The average HDD of the AgNPs, Fe2O3 NPs, and Ag-BSiO2-Fe2O3 hybrid structure in the saline solution was 30 ( Figure S2A), 200 (Figure S2B), and 500 nm ( Figure S2C), respectively. The zeta potential of the AgNPs, Fe2O3 NPs, and Ag-BSiO2-Fe2O3 hybrid structure was −28.6 ( Figure  S3A), −5.36 ( Figure S3B), and −17.8 mV (Figure S3C), respectively. The antimicrobial activity of the streptomycin, AgNPs, Fe2O3 NPs, and Ag-BSiO2-Fe2O3 hybrid structure was analyzed using S. aureus (Gram-positive) and E. coli (Gram-negative). The effective concentration of the various structures was determined by analyzing the inhibitory effect on the growth curve of both microorganisms; the time-dependent growth of the microorganisms was monitored at 610 nm. Figure 5 shows that 50 µg mL −1 of the microhybrid affected the growth rates of S. aureus ( Figure 5A) and E. coli ( Figure 5B), but it did not completely inhibit both microorganisms. At 100 and 200 µg mL −1 , the growth of both microorganisms was hampered, and no changes in the absorbance were observed after 20 h of incubation

Antimicrobial Activity
The effective antimicrobial activity of the NPs is dependent on the NPs aggregation and interaction of the NPs with microbial cells. Thus, the hydrodynamic diameter (HDD) and zeta potential studies of the NPs provide information regarding the size and charge on the NPs in the colloidal solution, demonstrating the stability of NPs. The average HDD of the AgNPs, Fe 2 O 3 NPs, and Ag-BSiO 2 -Fe 2 O 3 hybrid structure in the saline solution was 30 ( Figure S2A), 200 (Figure S2B), and 500 nm ( Figure S2C), respectively. The zeta potential of the AgNPs, Fe 2 O 3 NPs, and Ag-BSiO 2 -Fe 2 O 3 hybrid structure was −28.6 ( Figure S3A), −5.36 ( Figure S3B), and −17.8 mV (Figure S3C), respectively. The antimicrobial activity of the streptomycin, AgNPs, Fe 2 O 3 NPs, and Ag-BSiO 2 -Fe 2 O 3 hybrid structure was analyzed using S. aureus (Gram-positive) and E. coli (Gram-negative). The effective concentration of the various structures was determined by analyzing the inhibitory effect on the growth curve of both microorganisms; the time-dependent growth of the microorganisms was monitored at 610 nm. Figure 5 shows that 50 µg mL −1 of the microhybrid affected the growth rates of S. aureus ( Figure 5A) and E. coli ( Figure 5B), but it did not completely inhibit both microorganisms. At 100 and 200 µg mL −1 , the growth of both microorganisms was hampered, and no changes in the absorbance were observed after 20 h of incubation with the Ag-BSiO 2 -Fe 2 O 3 structure. In similar experiments, a 10 µg mL −1 concentration of streptomycin and AgNPs showed an inhibitory effect on both microorganisms. The complete inhibition of S. aureus ( Figure S4A) and E. coli ( Figure S4B) was observed at 30 µg mL −1 of streptomycin, whereas 50 µg mL −1 of AgNPs showed complete inhibition of S. aureus ( Figure S5A) and E. coli ( Figure S5B). The Fe 2 O 3 and BSiO 2 -Fe 2 O 3 NPs showed no effect on the bacterial growth curve in 50-200 µg mL −1 for both microorganisms (Figures S6 and S7). It is clear that 50 µg mL −1 of the hybrid structure was most effective for S. aureus, whereas E. coli showed some inhibition at this concentration. However, both microorganisms were highly sensitive to 200 µg mL −1 of the Ag-BSiO 2 -Fe 2 O 3 hybrid structure. To determine the minimum bactericidal concentration, both bacterial strains were exposed to various concentrations of the Ag-BSiO 2 -Fe 2 O 3 structure. Although 50 µg mL −1 of the hybrid structure reduced the growth of the microorganisms, it did not inhibit growth completely. However, as the concentration of the microhybrid increased from 50 to 90 µg mL −1 , the growth of both microorganisms constantly decreased. At 90 µg mL −1 of the hybrid structure, complete inhibition was observed for S. aureus ( Figure 6A) and E. coli ( Figure 6B); therefore, it can be considered the minimum bactericidal concentration (MBC) for both bacterial strains. For streptomycin, 20 µg mL −1 and 30 µg mL −1 were inhibitory for S. aureus and E. coli, respectively, as no growth was observed on growth plates ( Figure S8). AgNPs inhibited both microorganisms at 30 µg mL −1 ( Figure S9). However, the Fe 2 O 3 and BSiO 2 -Fe 2 O 3 particles showed no effect on the microbial growth in 50 to 90 µg mL −1 (Figures S10 and S11). The AgNPs showed high stability and low HDD in the aqueous solution, which allowed the maximum interaction with microorganisms, leading to its effective antimicrobial activity, whereas the presence of plant extract and AgNPs on the surface Fe 2 O 3 NPs increased the stability of the hybrid nanostructure compared to Fe 2 O 3 NPs, allowing it to perform more effectively in antimicrobial activity. The CLSM study was performed to visualize the antimicrobial activity of the hybrid structure where a 200 µg mL −1 concentration of the hybrid structure is completely inhibitory to both species showing the red dead bacteria cells (Figures S12 and S13). with the Ag-BSiO2-Fe2O3 structure. In similar experiments, a 10 µg mL −1 concentration of streptomycin and AgNPs showed an inhibitory effect on both microorganisms. The complete inhibition of S. aureus ( Figure S4A) and E. coli ( Figure S4B) was observed at 30 µg mL −1 of streptomycin, whereas 50 µg mL −1 of AgNPs showed complete inhibition of S. aureus ( Figure S5A) and E. coli ( Figure S5B). The Fe2O3 and BSiO2-Fe2O3 NPs showed no effect on the bacterial growth curve in 50-200 µg mL −1 for both microorganisms (Figures S6 and  S7). It is clear that 50 µg mL −1 of the hybrid structure was most effective for S. aureus, whereas E. coli showed some inhibition at this concentration. However, both microorganisms were highly sensitive to 200 µg mL −1 of the Ag-BSiO2-Fe2O3 hybrid structure. To determine the minimum bactericidal concentration, both bacterial strains were exposed to various concentrations of the Ag-BSiO2-Fe2O3 structure. Although 50 µg mL −1 of the hybrid structure reduced the growth of the microorganisms, it did not inhibit growth completely. However, as the concentration of the microhybrid increased from 50 to 90 µg mL −1 , the growth of both microorganisms constantly decreased. At 90 µg mL −1 of the hybrid structure, complete inhibition was observed for S. aureus ( Figure 6A) and E. coli ( Figure  6B); therefore, it can be considered the minimum bactericidal concentration (MBC) for both bacterial strains. For streptomycin, 20 µg mL −1 and 30 µg mL −1 were inhibitory for S. aureus and E. coli, respectively, as no growth was observed on growth plates ( Figure S8). AgNPs inhibited both microorganisms at 30 µg mL −1 ( Figure S9). However, the Fe2O3 and BSiO2-Fe2O3 particles showed no effect on the microbial growth in 50 to 90 µg mL −1 (Figures S10 and S11). The AgNPs showed high stability and low HDD in the aqueous solution, which allowed the maximum interaction with microorganisms, leading to its effective antimicrobial activity, whereas the presence of plant extract and AgNPs on the surface Fe2O3 NPs increased the stability of the hybrid nanostructure compared to Fe2O3 NPs, allowing it to perform more effectively in antimicrobial activity. The CLSM study was performed to visualize the antimicrobial activity of the hybrid structure where a 200 µg mL −1 concentration of the hybrid structure is completely inhibitory to both species showing the red dead bacteria cells (Figures S12 and S13).  Because 200 µg mL −1 of the microhybrid caused complete inhibition of both pathogenic strains, the concentration of the hybrid structure was checked for repeated use of antimicrobial activity. The Ag-BSiO2-Fe2O3 hybrid was added to each bacterial suspension, and the mixture was incubated for 24 h. After incubation, the hybrid material was isolated with an external magnet, and the bacterial suspension was analyzed spectroscopically. The hybrid structure was washed with a buffer before each repeated use, and the new bacterial suspension was added to this washed buffer. This was repeated for the next 5 cycles. The Ag-BSiO2-Fe2O3 hybrid structure maintained 80% of its relative antimicrobial activity against S. aureus and 75% against E. coli (Figure 7). The loss of antimicrobial activity of the Ag-BSiO2-Fe2O3 hybrid structure may be due to the loss of the hybrid material during isolation from the bacterial suspension.

Conclusions
Here, we have developed a prominent, clean, robust method for the synthesis of an antimicrobial hybrid nanostructure using green tea active biomolecules. C. sinensis green tea ethanolic extract comprises polyphenols and other active ingredients and has a high antioxidant and reducing activity. The extract was allowed to become trapped in the SiO2 layer by hydrolysis. This process of entrapment was employed to synthesize the separable SiO2-coated Fe2O3 NPs magnetically. The reducing capacity of biomolecule-entrapped SiO2-Fe2O3 NPs was used to synthesize Ag NPs on the surface of the magnetic structure, Because 200 µg mL −1 of the microhybrid caused complete inhibition of both pathogenic strains, the concentration of the hybrid structure was checked for repeated use of antimicrobial activity. The Ag-BSiO 2 -Fe 2 O 3 hybrid was added to each bacterial suspension, and the mixture was incubated for 24 h. After incubation, the hybrid material was isolated with an external magnet, and the bacterial suspension was analyzed spectroscopically. The hybrid structure was washed with a buffer before each repeated use, and the new bacterial suspension was added to this washed buffer. This was repeated for the next 5 cycles. The Ag-BSiO 2 -Fe 2 O 3 hybrid structure maintained 80% of its relative antimicrobial activity against S. aureus and 75% against E. coli (Figure 7). The loss of antimicrobial activity of the Ag-BSiO 2 -Fe 2 O 3 hybrid structure may be due to the loss of the hybrid material during isolation from the bacterial suspension. Because 200 µg mL −1 of the microhybrid caused complete inhibition of both pathogenic strains, the concentration of the hybrid structure was checked for repeated use of antimicrobial activity. The Ag-BSiO2-Fe2O3 hybrid was added to each bacterial suspension, and the mixture was incubated for 24 h. After incubation, the hybrid material was isolated with an external magnet, and the bacterial suspension was analyzed spectroscopically. The hybrid structure was washed with a buffer before each repeated use, and the new bacterial suspension was added to this washed buffer. This was repeated for the next 5 cycles. The Ag-BSiO2-Fe2O3 hybrid structure maintained 80% of its relative antimicrobial activity against S. aureus and 75% against E. coli (Figure 7). The loss of antimicrobial activity of the Ag-BSiO2-Fe2O3 hybrid structure may be due to the loss of the hybrid material during isolation from the bacterial suspension.

Conclusions
Here, we have developed a prominent, clean, robust method for the synthesis of an antimicrobial hybrid nanostructure using green tea active biomolecules. C. sinensis green tea ethanolic extract comprises polyphenols and other active ingredients and has a high antioxidant and reducing activity. The extract was allowed to become trapped in the SiO2 layer by hydrolysis. This process of entrapment was employed to synthesize the separable SiO2-coated Fe2O3 NPs magnetically. The reducing capacity of biomolecule-entrapped SiO2-Fe2O3 NPs was used to synthesize Ag NPs on the surface of the magnetic structure,

Conclusions
Here, we have developed a prominent, clean, robust method for the synthesis of an antimicrobial hybrid nanostructure using green tea active biomolecules. C. sinensis green tea ethanolic extract comprises polyphenols and other active ingredients and has a high antioxidant and reducing activity. The extract was allowed to become trapped in the SiO 2 layer by hydrolysis. This process of entrapment was employed to synthesize the separable SiO 2 -coated Fe 2 O 3 NPs magnetically. The reducing capacity of biomolecule-entrapped SiO 2 -Fe 2 O 3 NPs was used to synthesize Ag NPs on the surface of the magnetic structure, thus forming the Ag-BSiO 2 -Fe 2 O 3 hybrid nanostructure. As the entrapped biomolecules have a high reducing capacity, the reduction of Ag + ions occurred quickly, which led to the formation of spherical Ag NPs with diameters in the range of 5-10 nm on the magnetic nanostructure. Elemental dot mapping revealed the uniform distribution of biomolecules and Ag NPs in the SiO 2 layer coated on Fe 2 O 3 NPs. The Ag-BSiO 2 -Fe 2 O 3 hybrid microstructure showed excellent antimicrobial activity against S. aureus and E. coli. As the hybrid nanostructure was magnetic in nature, it showed excellent reusable antimicrobial activity. Toxicology studies need to be performed to use the antioxidant hybrid structure for medicinal and environmental applications. The outcome of the presented work creates opportunities for the construction of various metal microhybrid structures using eco-friendly biomaterials.