Facile Synthesis of Magnetic Iron-Based Nanoparticles from the Leach Solution of Hyperaccumulator Plant Pinus brutia for the Antibacterial Activity and Colorimetric Detection of Ascorbic Acid

It has been well known that metallic nanoparticles with striking properties possess wide application prospects in the processes of colorimetric detection, catalysis, disease diagnosis and treatment, energy, wastewater treatment, remediation, and antibacterial activity in recent years. Herein, iron-based nanoparticles (FeNPs), metallic nanoparticles, were synthesized via a facile chemical reduction method using a hyperaccumulator plant. Also, their use in antibacterial activity applications and colorimetric ascorbic acid (AA) detection was investigated. It was observed that FeNPs presented high antibacterial potency against Gram-positive bacteria of Listeria monocytogenes and Staphylococcus aureus and also Gram-negative bacteria of Escherichia coli(O157: H7), E. coli(ATCC 25922), Salmonella enteritidis, and Salmonella typhimurium. Moreover, it was found that FeNPs exhibited superior peroxidase-like activity to catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a blue color product, oxidized TMB (oxTMB), in the presence of H2O2. The colorimetric AA detection could be carried out by making the solution color lighter owing to the antioxidant property of AA. The quantitative detection of AA could be performed simply, selectively, and sensitively with FeNPs with a detection limit (LOD) of 0.5462 μM in a linear range of 30–200 μM.


INTRODUCTION
Nanoparticles can be synthesized by physical, chemical, and biological methods or hybrid methods, which are combinations of several of them. In this study, a new nanoparticle synthesis method was developed that combines the advantages of chemical and biological methods. According to this method, the iron-based nanoparticle (FeNP) synthesis can be carried out by the chemical reduction of the metal ions in the leach solution prepared from the iron hyperaccumulator plant of Pinus brutia. With this developed method, it can be possible to synthesize nanoparticles in a short time with high efficiency, which is one of the advantages of chemical methods, and also the process cost and the formation of toxic byproducts can be reduced using plants, which is one of the advantages of biological methods. In the literature, the metallic nanoparticles synthesized by various methods could be effectively used in various application areas such as bioremediation and decontamination applications, 1 biomedical applications, 2 drug delivery, 3 removal of various pollutants, 4 catalyst applications, 5 sensing applications, 6 antibacterial activity applications, 7 battery applications. 8 Among the metallic nanoparticles, ironbased nanoparticles have attracted much attention due to their outstanding properties such as high reactivity, adsorption capacity, biocompatibility, and mechanical, chemical, and thermal stability. 9 Additionally, they possess magnetic features, providing easy separation from the environment via an external magnet. 10 Because of the as-mentioned superior properties of iron-based nanoparticles, they have been widely utilized in the applications of catalysis, antimicrobial, anticancer, biosensor, energy, and wastewater treatment. 11 To the best of our knowledge, it is the first time to evaluate iron-based nanoparticles, synthesized using a hyperaccumulator plant as a metal ion source, in antibacterial activity applications and colorimetric AA detection in this study.
The researchers have focused on developing more effective antibacterial nanoparticles without side effects that are easy to enforce since the risk of biological and bacterial assaults, especially in the sectors of food, food packaging, and water, has increased gradually in recent years. The dimensions of metallic nanoparticles are between bulk materials and molecules/ atoms/ions, which interact with cells to make stable entities with less energy. 12 So, one of the most featured practical applications of iron-containing nanoparticles, metallic nanoparticles, is their utilization as antibacterial agents in the literature. 13−15 In this study, the antibacterial activity of FeNPs synthesized from a hyperaccumulator plant was investigated against various food-borne bacteria.
Ascorbic acid (AA) is a natural water-soluble vitamin, which is a puissant reducing and antioxidant agent that has important roles in battling bacterial infections, detoxifying reactions, and the formation of collagen in fibrous tissue, teeth, bones, connective tissue, skin, and capillaries. AA concentrations in the human bloodstream are generally between 0.6 and 2.0 mg/ dL; however, it varies from tissue to tissue. The deficiency of ascorbic acid (<0.4 mg/dL) in the body can cause the emergence of various diseases such as immunity decrease, anemia, and even scurvy. Taking more than the recommended amount of AA, which is more than 2.0 g per day, can result in side effects such as nausea and vomiting, diarrhea, abdominal pain and cramps, heartburn, insomnia, headache, fatigue, and kidney stones. Besides, AA has been gradually utilized in industries due to its strong reducing and antioxidant capacity. 16 Therefore, it is crucial to detect AA with a simple, convenient, inexpensive, and sensitive method. Various methods have been established and applied to detect AA, including titration, enzymatic methods, electrochemical techniques, fluorescence and chemiluminescence methods, capillary electrophoresis, and chromatography methods. 17,18 The detection of AA has been able to apply these methods with good sensitivities; however, these methods have some disadvantages such as the need for trained technicians, the requirement of expensive equipment or chemicals, and impractical and time-consuming operations. Among the traditional methods, the colorimetric method based on a chromogenic substrate producing color upon oxidation in the presence of the natural enzymes is more ideal and effective in detecting AA. However, natural enzymes are too sensitive to extreme experimental conditions, and so they can lose their catalytic activity easily at strong acidic/basic pHs and high-temperature values. In addition, there are some drawbacks of natural enzymes such as high cost, low stability, and difficulty in storage. 17,19 Nanozymes are inorganic nanomaterials with a more effective enzyme-like catalytic activity in comparison to natural enzymes because of their high catalytic activity, low cost, high stability, and wide range of applications. 20 Hence, to overcome these drawbacks, various nanozymes such as palygorskite@Co 3 O 4 nanocomposites, 21 polyacrylonitrile−CuO nanoflowers, 22 platinum nanoclusters, 23 gold nanoparticles, 24 cobalt-doped carbon quantum dots, 25 Cu−Ag bimetallic nanoparticles, 26 and Fe 3 O 4 /nitrogendoped carbon hybrid nanofibers 27 have been developed as peroxidase-like catalysts to catalyze the oxidation of TMB in the presence of H 2 O 2 for the colorimetric AA detection in the past decade. Since some difficulties have been faced with the sensitive detection of AA in complex biological media, the researchers are still focusing on improving the catalytic activity, sensitivity, selectivity, and stability of enzyme-mimetic nanomaterials. In this study, FeNPs synthesized from a hyperaccumulator plant were used instead of a natural enzyme in the oxidation reaction of TMB in the presence of H 2 O 2 , and the addition of the antioxidant agent AA to this reaction media enables simple, sensitive, and selective colorimetric detection of AA.

Synthesis and Characterization of Iron-Based Nanoparticles.
According to the iron-based nanoparticle (FeNP) synthesis method developed by our team, NaBH 4 , as a reductant agent, was added to the leach solution prepared from P. brutia instead of the synthetic iron salt solution under the required experimental conditions. The details of the synthesis method and the results of some characterization studies were presented in our previous work. 28 In this study, additional characterization studies using a Zeta-sizer via the dynamic light scattering technique (DLS), Fourier transform infrared (FTIR) spectra, and a vibrating sample magnetometer (VSM) were used to define the synthesized FeNPs.
2.2. Antibacterial Activity Test. Salmonella enteritidis, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (ATCC 25922), Staphylococcus aureus, andSalmonella typhimurium bacteria species, which are widely used and food-borne pathogens, were selected to determine the antibacterial activities of FeNPs. In the scope of the culture and inoculum preparation, the bacterial cultures were grown on tryptic soy agar (TSA) slants and kept at 4°C. Isolated colonies obtained from the TSA slants were inoculated into a tryptic soy broth (TSB) medium. The broth culture was incubated at 37°C for 24 h. The optical density of the culture was adjusted between 0.08 to 0.1 at 625 nm to obtain an inoculum size of 1 × 10 7 colony-forming unit (CFU)/mL. The antibacterial activity of FeNPs was investigated using the agar plate method. The surface of the agar plate (Mueller Hinton Agar) was inoculated by spreading the test microorganism over the entire surface. Then, a hole with a diameter of 6 mm was punched aseptically, and 0.0015 g of the test compound (FeNPs) and 30 μL of sterile distilled water were introduced into the well. The Petri dishes were incubated at 37°C for 24 h. The test compound diffused into the agar and inhibited the growth of the test microorganism. The diameters of inhibition growth zones were measured with a digital caliper. 29 2.3. Colorimetric Detection of Ascorbic Acid with FeNPs. The antioxidant property of AA was utilized for the colorimetric detection of AA. For this purpose, 500 μL of an acetate buffer solution (pH = 2.0), 500 μL of a 0.1 mM H 2 O 2 solution, and 100 μL of a 1.0 g/L FeNP solution were added sequentially onto 250 μL of a 0.5 mM 3,3′,5,5′-tetramethylbenzidine (TMB) solution in an UV−vis spectrophotometer cuvette. This solution was named "control" by us. After that, the spectrum scanning of the "control" containing the oxidation products formed as a result of the oxidation of TMB was performed with a UV−vis spectrophotometer at a 300−1100 nm wavelength range. Then, a series of control solutions were prepared, and 1000 μL of AA solutions at different concentrations (1.0−500 μM) were added separately to each prepared control solution. The reductions in the absorption peak intensities were determined with the addition of AA by performing repeat spectrum scans of the prepared solutions in the wavelength range of 300−1100 nm. For the determination of the minimum limit of detection (LOD) of FeNPs, the absorbance values of the control solutions containing the oxidation products (A i ) and AA solutions at different concentrations were added (A f ) were recorded with a UV−vis spectrophotometer at a 652 nm wavelength. In order to find the LOD value, a calibration line was formed by plotting the different concentrations of AA against the absorbance changes (ΔA = A i �A f ) at these concentrations. The limit of detection (LOD = 3σ/s) was calculated according to the signal, which is equivalent to 3 times the standard deviation of the blanks, where s is the slope of the calibration line and σ is the standard deviation of the control solution (the solution containing the oxidation products without adding AA). 21,30 2.4. Determination of Selectivity of FeNPs. The colorimetric detection of AA with FeNPs was carried out in the presence of different components that can be found in real samples such as NaCl, KCl, CuCl 2 , CaCl 2 , ZnCl 2 , MgCl 2 , Al(NO 3 ) 3 , (NH 4 ) 2 HPO 4 , glucose, lactose, maltose, fructose, sucrose, urea, uric acid, ascorbic acid, oxalic acid, lactic acid, L-cysteine, glutathione, dopamine, and melamine. To determine the selectivity of FeNPs, 500 μL of an acetate buffer solution (pH = 2.0), 500 μL of a 0.1 mM H 2 O 2 solution, and 100 μL of a 1.0 g/L FeNP solution were added sequentially into 250 μL of a 0.5 mM TMB solution, the solution containing all of the components was defined as "control", in a UV−vis spectrophotometer cuvette. The absorbance values of the control solutions containing the oxidation products were recorded with a UV−vis spectrophotometer at a 652 nm wavelength. After that, the solutions of NaCl, KCl, CuCl 2 , CaCl 2 , ZnCl 2 , MgCl 2 , Al(NO 3 ) 3 , (NH 4 ) 2 HPO 4 , glucose, lactose, maltose, fructose, sucrose, urea, uric acid, ascorbic acid, oxalic acid, lactic acid, L-cysteine, glutathione, dopamine, and melamine were added to each prepared control solution as a mixture with AA (500 μL of 1.0 mM AA + 500 μL of 1.0 mM interferents). The absorbance values of the final solutions containing different interferents were recorded with a UV−vis spectrophotometer at a 652 nm wavelength, and the absorbance changes (ΔA = A i − A f ) were calculated to determine the selectivity of FeNPs and the interactions of different components. 21,30 The recovery values were calculated with eq 1 given below where A i is the absorbance change in the presence of the interferent i and A r is the absorbance change in the presence of reference that is distilled water.

Method Validation.
In order to test the applicability of the colorimetric detection of AA with FeNPs in real samples, commercial AA-containing materials (vitamin C tablets and vitamin C water) and

Characterization of FeNPs.
The characterization results of XRD, EDX, and SEM of the synthesized FeNPs were presented in our previous work. 28 The XRD analysis results showed that FeNPs contained the crystal phases of Fe 3 O 4 , FeOOH, γ-FeOOH, and Fe 0 . The average particle size of FeNPs was obtained as 82.19 nm, and also the nearly spherical structures were observed in SEM images. It was determined  In this study, additional characterization studies such as FTIR, DLS, and VSM were carried out to determine the functional groups, the hydrodynamic diameter, and the magnetic properties of FeNPs. The hydrodynamic diameter of FeNPs was measured with a zeta-sizer via the DLS technique, and the average hydrodynamic diameter was found to be approximately 94 nm. The particle size distribution is also demonstrated in Figure 1. From Figure 1, it was observed that the particles with the sizes of 100−200 nm were obtained in the DLS analysis as a result of the agglomeration of the small particles. On the other hand, the structures larger than 200 nm are estimated to be the particles containing Ca, Na, Mg, and Cl elements originating from the hyperaccumulator plant used in the synthesis of FeNPs, which were also detected in the EDX analysis in our previous work. 28

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The functional groups of the synthesized FeNPs were determined by FTIR analysis, and the obtained FTIR spectrum is presented in Figure 2. It was obtained from Figure 2  Oe, respectively. The remanent magnetization value (M r < 1.125) obtained for FeNPs, less than 25% of the M s value, showed that FeNPs could be easily separated from the media with a permanent magnet and could be quickly dissolved in the solution without agglomeration. Since the H c value of FeNPs synthesized in this study was 125 Oe, it was determined that FeNPs were classified as semihard magnetic materials. It was concluded that FeNPs, which were classified as semihard magnetic materials, required more energy than soft materials to move in the loop and required a lower magnetic field than hard magnetic materials to reach saturation magnetization. Semihard magnetic materials have a wide range of uses such as magnetically coupled devices (brakes, clutches, tensioners), bias elements in product protection/safety systems, relay magnets, magnetic tool holders, sensor magnets, magnetic stirrers, and level sensors. 35−37 Accordingly, it can be said that FeNPs synthesized in this study could be used in the application of semihard magnetic materials.

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The saturation magnetization value (M s ) of FeNPs was compared with the iron-containing nanoparticles in the literature, and the results are summarized in Table 1. According to Table 1, it was determined that FeNPs synthesized in this study had a lower M s value than ironcontaining nanoparticles in the literature. It was thought that other components except for iron in FeNPs obtained in EDX analysis reduced the saturation magnetization (M s ) value of FeNPs.
3.2. Antibacterial Activity of FeNPs. The images showing the antibacterial activity of FeNPs on test microorganisms and the graph showing the inhibition zone diameters obtained for each microorganism are presented in Figures 4 and 5, respectively. The inhibition zone diameters of the sample compound for the bacterial species of S. enteritidis, L. monocytogenes, E. coliO157:H7, E. coli(ATCC 25922), S. aureus, and S. typhimurium were determined as 20.85 ± 2.98, 17.33 ± 2.62, 16.93 ± 1.80, 22.74 ± 1.09, 31.56 ± 1.50, and 20.35 ± 1.46 mm, respectively. Accordingly, it was proved that FeNPs were effective against all selected bacteria. As a result, it was concluded that the strong inhibitory effect of FeNPs synthesized in this study against the tested food-borne microorganisms will allow FeNPs to be used as a preservative agent in the food industry to reduce food contamination and provide longer-lasting storage. In this case, it can be recommended to use FeNPs synthesized from the hyperaccumulator plant of P. brutia for bacterial-resistant coating and antibacterial applications for biomedical devices.
The inhibition zone diameters of FeNPs were compared with the iron-containing nanoparticles in the literature, and the results are summarized in Table 2. It was concluded from Table 2 that the FeNPs synthesized in this study were at a competitive level with various iron-containing nanoparticles in the literature in terms of antibacterial activity.

Colorimetric Detection of Ascorbic Acid with FeNPs.
According to the predicted colorimetric AA detection steps given in Figure 6, FeNPs could catalyze the decomposition of H 2 O 2 via a Fenton-like reaction to generate  For the detection of AA with FeNPs, AA could discolor the blue color of oxTMB to colorless-TMB by means of the antioxidant property of AA. The UV spectrum scans of the solutions given in Section 2.3 containing the oxidation products of TMB were performed to verify the colorimetric AA detection steps. As shown in Figure 7, the absorbance intensities at 450 and 652 nm decreased gradually with the increase in AA concentrations from 1.0 to 500 μM, along with the solution color changing from blue to colorless. The color changes in 5 s with increasing AA concentration could also be visualized through the naked eye, discoloring from blue to colorless.
In order to determine the LOD value of FeNPs for the colorimetric detection of AA via TMB oxidation in the presence of H 2 O 2 , a calibration line given in Figure 8 (inset, the plot used to calculate the standard deviation value of the control solution) was formed by plotting the AA concentrations against the absorbance changes. The absorbance changes at 652 nm had a good linear regression equation ΔA = 0.001439 × C AA (μM) + 0.1973 (R 2 = 0.9894) with the AA concentration in a range of 30−200 μM. The limit of detection (LOD) of FeNPs for the colorimetric AA detection was calculated to be 0.5462 μM at an S/N (signal/noise) ratio of 3.0. Table 3 summarizes the comparison of FeNPs synthesized in this work with currently available peroxidase-like nanomaterials for colorimetric AA detection. As shown in Table 3, both the linear range and LOD of the synthesized FeNPs are comparable to or even better than some of the reported peroxidase-like nanomaterials synthesized from synthetic metal ion sources in the literature. These results demonstrated that the proposed FeNPs could be evaluated as effectively as peroxidase-like nanomaterials synthesized from synthetic metal ion sources in the literature.

Method Validation.
For the investigation of the reliability of the colorimetric AA detection with FeNPs, the method was applied to analyze AA content in real samples. As presented in Table 5, the recovery values were from 100.59 to 104.57%, and RSD % values were in the range of 1.14−3.28%. The results confirmed that the colorimetric AA detection method with FeNPs synthesized from the hyperaccumulator plant of P. brutia possessed the potential to be applied accurately and reliably to the detection of AA content in real samples.

CONCLUSIONS
In summary, we first developed a new facile method using the leach solution of hyperaccumulator plant Pinus brutia as a natural metal ion source to synthesize novel iron-based nanoparticles for the antibacterial activity and the colorimetric detection of ascorbic acid. The results showed that FeNPs exhibited a significant bactericidal effect toward Gram-positive (L. monocytogenes and S. aureus) and Gram-negative (E. coli(O157: H7), E. coli(ATCC 25922), S. enteritidis, and S. typhimurium) pathogenic bacteria. The findings suggested that the leach solution prepared from the iron hyperaccumulator plant of Pinus brutia could be used for developing antibacterial FeNPs against pathogenic bacteria. Furthermore, a sensitive and selective colorimetric AA detection system was successfully constructed using FeNPs as enzyme mimics. This colorimetric detection system using FeNPs could be applied to quantify AA concentration, with a linearity range of 30−200 μM and an LOD value of 0.5462 μM. It was observed that the colorimetric AA detection could be effectively carried out in the presence of various interfering substances except for uric acid, L-cysteine, L-glutathione, dopamine, and melamine. It was also tested to quantify AA in real samples, and their recovery values were 100.59−104.57% with RSD less than 4%, proving its feasibility in evaluating AA content in practical application. Considering all of these, the present work not only provides a novel antibacterial agent and a peroxidase-like catalyst but also inspires researchers to further explore various hyperaccumulator plants for the synthesis of metallic nanoparticles for a variety of applications.