Eggshell Membrane-Templated MnO2 Nanoparticles: Facile Synthesis and Tetracycline Hydrochloride Decontamination

Taking advantages of reticular proteins and reductive groups on the surface, eggshell membrane (ESM) was selected to synthesize MnO2 nanoparticles from potassium permanganate through a super simple way in which ESM acted as both template and reductant. This process avoided harsh reaction conditions or complicated aftertreatments and thus owned the merits of green synthesis, handy operation, low cost, and easy purification. The ESM-templated MnO2 nanoparticles (MnO2 NPs/ESM) were characterized, and the content of nanomaterials on the template was tested. MnO2 NPs/ESM showed a good capacity for decontamination of tetracycline hydrochloride (TCH). The macroscopical materials can be separated easily by taking the membrane out to stop the degradation instead of centrifugation or filtration. It was studied that 72.27% of TCH (50 mg/L) was decontaminated in 20 min by 0.1920 g/L MnO2 nanoparticles, and removal efficiency could reach 83.10% after 60 min under buffered condition. The kinetics was studied with or without buffer, and it was concluded that the degradation process followed a pseudo-second-order model. The facile synthesis of materials and effective degradation would facilitate the nano-MnO2-based decontamination applications. Electronic supplementary material The online version of this article (10.1186/s11671-018-2679-y) contains supplementary material, which is available to authorized users.


Background
Pharmaceuticals and personal care products (PPCPs) are a kind of emerging water pollutions and are concerned closely by researchers in consideration of ecology and human health [1][2][3][4][5]. Antibiotics as a medicine to treat and prevent bacterial infections are used worldwide, accompanying with which upsetting risks to the environment have gradually appeared [6]. As a representative, tetracycline (TC) medicines have been used in veterinary science and aquaculture for years [7]. However, TCs can hardly be degraded in the environment and thus persist for a long time [8,9], which lead to a various negative influence on the ecosystem or human health [10][11][12][13]. Therefore, screening a facile and effective way to decontaminate TC-contaminated water has become a hotspot of research. One promising technique may be the assistance of manganese dioxide nanomaterials.
Manganese dioxide nanomaterials have been extensively studied owing to their unique merits of high surface area, tunable structure, catalytic oxidation activity, and eco-harmless [14,15]. Therefore, nano-MnO 2 -based applications have covered various fields ranging from catalysis [16,17], sensors [18,19], and capacitors [20,21] to drug delivery [22,23] and cancer therapy [24,25]. By the same token, MnO 2 nanomaterials with adsorption and oxidation properties have applied to wastewater treatment. Water pollutants including heavy ions [26], organic dyes [27], and phenols [28] treated by MnO 2 nanomaterials were reported. Meanwhile, antibiotics such as levofloxacin [29], ciprofloxacin [30], norfloxacin [31], sulfamethoxazole [32], sulfadiazine [33], cefazolin [34], lincosamide [35], and TCs [36,37] have been successfully decontaminated through MnO 2 treatment. Specific to TC antibiotics, highly porous MnO 2 nanosheets were utilized to degrade tetracycline, and pH, temperature, and dose-based kinetics were investigated [38]. A MnO 2 -based scheme was applied to remove tetracycline hydrochloride (TCH) and As(III) simultaneously, and the interactive effect on arsenic and antibiotics during MnO 2 treatment was studied [39]. Degradation of tetracycline antibiotics by MnO 2 was performed, and transformation kinetics and pathways were reported [40]. Though high removal efficiency of TCs was obtained in the abovementioned works, however, the degradation operation usually involved in the centrifugation or filtration in order to separate the material from antibiotics solutions, which took much of the treatment time and made the process complicated.
Eggshell membrane (ESM) as a unique biomaterial with extraordinary properties has been utilized in materials science extensively [41]. The main composition of fiber in ESM is a protein which endows ESM the ability to bind metal. Noble metal nanomaterials like Ag NPs and Au NPs were successfully synthesized using ESM as a template [42][43][44]. In addition, metal oxide nanomaterials such as ZnO [45], Co 3 O 4 [45], PbO [45], Mn 3 O 4 [46], and TiO 2 [47] were also prepared through ESM templating, which made the synthesis facile and under control and therefore provided a novel path for the synthesis of metal or metal oxide nanoparticles.
In this work, eggshell membrane-templated MnO 2 nanoparticles (MnO 2 NPs/ESM) were synthesized simply and quickly by a bio-templating method. Eggshell membrane played dual roles as a template and a reductant making nanoparticles dispersed uniformly on the macroscopical membranes. Combining the oxidizing MnO 2 nanoparticles with the easy-to-manipulate membrane, MnO 2 NPs/ESM were further applied to tetracycline hydrochloride decontamination, in which nanomaterials could be separated easily by simply taking out of solutions.

Materials and Apparatus
Deionized water with a conductivity of 18.2 MΩ cm −1 was used in this experiment from a water purification system (ULUPURE, Chengdu, China). Potassium permanganate (KMnO 4 , M w = 158.03), MnO 2 powder, and other reagents were at least of analytical grade and purchased from Kemiou Chemical Co. Ltd. (Tianjin, China). Tetracycline hydrochloride (TCH, USP grade) and glutathione (GSH, 98%) were purchased from Aladdin Reagents Company (Shanghai, China). Eggshell membrane (ESM) was peeled off carefully from a fresh eggshell which is obtained from Hongye student mess hall of Taiyuan Institute of Technology. PBS buffer solutions (0.2 M, pH = 7.0) were prepared by mixing 39 mL NaH 2 PO 4 solution (0.2 M) and 61 mL Na 2 HPO 4 solution (0.2 M), and PBS solutions with different pH values were prepared by titrating the abovementioned solution with sodium hydroxide or hydrochloric acid solution (both concentrations were 0.2 M) to the required pH values.
Scanning electron microscopy (SEM) of MnO 2 NPs/ ESM was carried out on a Quanta 200 FEG scanning electron microscope for the morphology observation. Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) of MnO 2 NPs were performed on a Tecnai-G20 transmission electron microscope. The size distribution of as-prepared MnO 2 NPs was obtained at a laser particle sizer (Malvern Nano-ZS90). X-ray photoelectron spectroscopy (XPS) was collected on an AXIS ULTRA DLD electron spectrometer (Kratos) with monochromatic Al Kα radiation for the surface composition and chemical state test of the product. Thermogravimetry (TG) analysis of ESM and MnO 2 NPs/ ESM was measured in air at a heating rate of 10°C/min on a Rigaku TG thermal analyzer (Rigaku Co. Japan). Fourier transform infrared spectroscopy (FTIR) from 4000 to 400 cm −1 of ESM and MnO 2 NPs/ESM was recorded in KBr discs on a Tensor II FTIR spectrometer (Bruker, Germany), and the spectra were processed through deconvolution. The ultraviolet-visible (UV-vis) absorption spectra of TCH were recorded on a TU-1901 UV-vis spectrophotometer (Puxi, China).

Synthesis of ESM-Templated MnO 2 Nanoparticles
The eggshell membrane-templated MnO 2 nanoparticles (MnO 2 NPs/ESM) were synthesized through a straightforward and facile method. In a typical process, the eggshell membrane was firstly peeled off from a fresh eggshell manually and washed ten times with deionized water to remove the needless egg white. After drying under room temperature, the clean ESM was then cut into slices with 5 mg weight each. Upon synthesis, ten slices of ESM were soaked into 20 mL KMnO 4 solution (1 mmol/L) and the open system kept stirring under room temperature. Thirty-five minutes later, the ESM slices were taken out and washed ten times with deionized water to remove the redundant solution. At last, the obtained membranes were dried and stocked at room temperature for further characterization and use.

Decontamination of TCH
The decontamination of TCH was performed by adding MnO 2 NPs/ESM into the TCH solutions simply and stirred under room temperature. Twenty slices of MnO 2 NPs/ESM were placed into 15 mL TCH solutions (50 mg/L) which were diluted by PBS buffer solutions and kept stirring for 60 min. The UV-vis spectra of TCH solutions after treatment were recorded immediately at room temperature. All of the absorption intensities of TCH measurement were set at a wavelength of 358 nm.
The removal efficiency (R, %) was calculated using the equation below: where C 0 and C (mg/L) stand for the initial and final concentrations of TCH in the treatment solutions, respectively.

Mechanism and Monitoring of MnO 2 NPs/ESM Synthesis
The synthesis of MnO 2 NPs/ESM was performed in an open system with ESM as biotemplate. The eggshell membrane was composed of many fibrous proteins on which lots of reductive groups like -OH, -NH 2 , -SH, etc. were interspersed. An in situ redox reaction was triggered once the KMnO 4 was introduced. While MnO 2 was generated, it grew gradually around these active groups. As a consequence, it was dispersed uniformly on the surface of fibrous proteins to form ESM-templated MnO 2 NPs. Figure 1a displayed the photos of synthesis system at different times, in which purple KMnO 4 solution turned into light brown gradually, and meanwhile, the white ESM slices became brown (Fig. 1b, c). To monitor the synthetic process, the absorption intensity of KMnO 4 at 525 nm and pH of this system were investigated in Fig. 1d, e. As shown, the absorption intensity of KMnO 4 decreased with time prolonged, and pH, conversely, improved gradually. Two sets of data both showed a platform after 35 min, and therefore, the synthesis time was selected. The increasing of pH was attributed to the formation of -OH during the reaction and a reaction course was speculated below:

Characterization of MnO 2 NPs/ESM
The morphology of the obtained MnO 2 NPs/ESM was investigated by scanning electron microscopy (SEM) in Fig. 2. Multilayered and intersecting fibrin network was observed in Fig. 2a, b. After further amplification, lots of particles were found uniformly coated on the surface of fibrous proteins. Therefore, it was concluded that ESM acted not only as a reductant but also a template during the synthesis. To further investigate the size of MnO 2 particles, a laser particle sizer test was carried out. In order to rule out the possibility that the particles with 4.8 nm were decomposed proteins, MnO 2 NPs/ESM and  Figure S1 that the average size of MnO 2 NPs was 4.8 nm. The photos of MnO 2 NPs/ESM before and after NaOH treatment were displayed in Additional file 2: Figure S2A. It was obvious that the brown color of the membrane faded evidently while the membrane kept unchanged after NaOH treatment, indicating that the MnO 2 NPs were released from the template. In thinking about the problem that the size of the protein from the eggshell may interfere with the results, the filtrated solutions after NaOH treatment were obtained from both blank ESM and MnO 2 (Additional file 2: Figure S2B) to be colorless and brown, respectively. In addition, the size distribution data of ESM after NaOH treatment displayed an average size of 1.7 nm in Additional file 2: Figure S2C. Therefore, the possibility that the particles with 4.8 nm were decomposed proteins from ESM itself was ruled out. Based on this, TEM was captured after the aforementioned filtrate was dialyzed. As shown in Fig. 2c, spherical nanoparticles were observed and the size was consistent with the one in Additional file 1: Figure S1. The HRTEM image in Fig. 2d indicated a lattice spacing of 2.5 Å that was well coincident with the (400) lattice plane of α-MnO 2 [48].
Besides, X-ray photoelectron spectroscopy (XPS) technique was carried out for the surface composition and elemental analysis of the obtained MnO 2 NPs/ESM. The full-scan spectrum (Fig. 3a) indicated that the synthesized material was composed of elements Mn 2p, O 1s, N 1s, and C 1s. Element C 1s, N 1s, and partial O 1s came from the template ESM. The partial XPS spectra of Mn 2p and O 1s were measured to study the details. As shown in Fig. 3b, two peaks at 653.8 and 642.0 eV can be assigned to Mn 2p 1/2 and Mn 2p 3/2 , respectively. The O 1s spectrum (Fig. 3c) can be divided into three component peaks with binding energy at 532.6, 531.4, and 530.5 eV, which were attributed to H-O-H, Mn-O-H, and Mn-O-Mn, respectively. The above data demonstrated that the as-prepared material was ESM-templated MnO 2 NPs.
To further verify this result, GSH solution was applied to the test thus obtained material inspired by a special reaction between GSH and MnO 2 [49,50]. As shown in Additional file 3: Figure S3, the brown color of MnO 2 disappeared after soaking into GSH solution for 1 min, indicating that the materials coated on ESM were MnO 2 . Furthermore, thermogravimetry (TG) analysis was carried out to measure the mass content of MnO 2 on ESM. The black and red curves in Additional file 4: Figure S4 stood for the mass changes of ESM only and MnO 2 NPs/ESM, respectively. The relative quality of ESM was almost zero at 600°C indicating that ESM was totally burnt out. However, the relative quality of ESM-templated MnO 2 NPs remained at 2.61% after ESM was burnt out. It was reported that MnO 2 was thermally decomposed at 500°C and conversed into  [51]. Moreover, further thermal decomposition of Mn 2 O 3 to Mn 3 O 4 occurred above 1000°C [52]. Therefore, the mass content of 2.61% at 800°C obtained in this experiment reflected the content of Mn 2 O 3 . According to the mass conservation of Mn, the original MnO 2 content loading on the ESM was calculated to be 2.88%.
FTIR spectra (Additional file 5: Figure S5) of ESM and MnO 2 NPs/ESM were collected after grinding the materials into powder. The interactions between proteins and nanoparticles mainly involve secondary structure changes, which are reflected on the band of amide I~1650 cm −1 (which may shift a bit) or amide II~1550 cm −1 . However, there were no obvious changes of peak position around 1650 or 1550 cm −1 of ESM before and after MnO 2 was involved, which was different from the previously reported results that could demonstrate the structural change of protein [53]. In order to get into the details and avoid missing any minor changes, deconvolution was applied to these spectra. Even though no observable peaks were shown up around 1650 or 1550 cm −1 , a new peak at 506 cm −1 appeared after MnO 2 NPs loading, and it was associated with the characteristic vibrational mode of Mn-O [54].
Mn has various oxidation states, so there are a few types of oxides, such as Mn 2 O 3 , MnO, and MnO 2 . The binding energy of Mn 2 O 3 is close to that of MnO 2 . In order to examine the oxidation state of Mn in this work, the HRTEM of as-prepared materials was imaged and displayed in Fig. 2d. The lattice spacing of 2.5 Å detected is well coincident with the (400) lattice plane of α-MnO 2 [48]. Moreover, our Mn materials were obtained based on the redox reaction between KMnO 4 and ESM under the neutral condition that favored the formation of MnO 2 instead of other oxidation states [55]. Importantly, as-prepared materials possess the reaction activity with GSH (Additional file 3: Figure S3), which is also a testimonial that the nanoparticle is MnO 2 [49,50]. It was also reported that MnO 2 could thermally be decomposed at 500°C and conversed into Mn 2 O 3 [51]. The TG curve of the as-prepared materials in Additional file 4: Figure S4 shows an obvious weight loss around 500°C, indicating the transformation from MnO 2 to Mn 2 O 3 , which is another testimony that the oxidation state of Mn is MnO 2 .

TCH Decontamination by MnO 2 NPs/ESM
Taking advantages of oxidative MnO 2 NPs and macroscopical template, MnO 2 NPs/ESM were applied to tetracycline hydrochloride (TCH) decontamination owing to the effective removal and easy operation. Figure 4a displayed the time-dependent absorption intensity of TCH at 358 nm treated by ESM only (black) and MnO 2 NPs/ESM (red). It was shown that absorption intensity kept unchanged in the presence of ESM only. However, it dropped sharply first and flattened out gradually under MnO 2 NPs/ESM treatment. This evident contrast demonstrated the capacity of MnO 2 NPs/ESM for TCH decontamination. Similarly, the UV-vis absorption spectra of TCH after ESM treatment hardly changed, but the absorption peak at 358 nm decreased obviously after MnO 2 NPs/ESM decontamination (Fig. 4b). Figure 4c investigated the absorption spectra variation of TCH, in which the absorption peak at 270 nm lowered in the first 10 min, but another peak at 358 nm decreased along with time was observed. The time-dependent removal efficiency by MnO 2 NPs/ESM decontamination was calculated in Fig. 4d, and it was found that removal efficiency was 72.27% at 20 min and it can reach 83.10% in 60 min.

Effect of pH and Buffer on TCH Decontamination
The pH played an important role in MnO 2 -based oxidative degradation, and the effect of pH on TCH decontamination in this work was investigated. Figure 5a displayed the absorption intensity of TCH before and after MnO 2 NPs/ESM treatment for 60 min under different pH, and the corresponding removal efficiency was calculated in Fig. 5b. It was demonstrated that the optimal decontamination of TCH by MnO 2 NPs/ESM was achieved under PBS buffer with a pH of 3.0. Moreover, TCH decontamination by MnO 2 NPs/ESM without buffer solution was investigated in Fig. 5c, in which the absorption intensity of TCH decreased gradually and pH of the degradation system was steadily improved. The same phenomenon of pH increase during the decontamination process was also reported in a previous work [38]. It was worth noting that the removal efficiency without buffer increased more rapidly than that under a buffered condition at the beginning (first 20 min). Then as the time passed, however, the removal efficiency with buffer exceeded the one without buffer after 30 min (83.10% for buffered and 78.37% for the unbuffered condition at 60 min). Removal efficiencies were monitored through concentration variations of TCH which were calculated from linear calibration plot (Additional file 6: Figure S6 and Additional file 7: Figure S7). Under the buffered condition, saline ions from PBS hindered the diffusion of TCH molecules onto the surface of MnO 2 NPs for further reaction, and hence, the reaction rate was lower than that of without a buffer. However, pH increase of reaction system along with time under unbuffered condition limited the oxidative capacity of MnO 2 NPs, and thus, the removal efficiency cannot reach the one obtained under controlled optimal pH.

Kinetic Study of TCH Decontamination
To further understand the TCH degradation by MnO 2 NPs/ESM, the kinetic study was carried out by changing the TCH initial concentration or dose of MnO 2 . We investigated the kinetics of degradation by different amounts of MnO 2 under buffered conditions. Figure 6a displayed the time-dependent absorption intensity of TCH degraded by different doses of MnO 2 (0.0960, 0.1440, and 0.1920 g/L), and the corresponding removal efficiencies were calculated in Fig. 6b. And linear kinetic plots by pseudo-first-order and pseudo-second-order were fitted in Fig. 6c, d, respectively. Moreover, degradation at different initial concentrations of TCH (30, 50, and 70 mg/L) with buffer was studied through monitoring the absorption intensity (Fig. 7a) and removal efficiency (Fig. 7b) at different times. Figure 7c, d fitted the linear first/second-order kinetic plots to investigate the kinetics. Similarly, degradation kinetics at different amounts of MnO 2 NPs and different initial TCH concentrations under unbuffered conditions were studied in Additional file 8: Figure S8 and Additional file 9: Figure S9, respectively. Table 1 exhibited the kinetic data obtained from different conditions. The correlation coefficients were linear-fitted and calculated to demonstrate the kinetic of TCH degradation by MnO 2 NPs/ESM. Generally, the correlation coefficients calculated through the pseudo-second-order model were higher than that through pseudo-first-order, indicating this process was more consistent with the pseudo-second-order model. In detail, this pseudo-second-order model had higher correlation coefficients at small doses of MnO 2 or high initial concentrations of TCH. And either way, correlation coefficients were closer to 1 in the buffered conditions compared with the degradation without a buffer.

Comparison of Commercial MnO 2 Powder and Other Reported Materials
To illustrate the advanced property of as-prepared MnO 2 NPs/ESM, the equal amount of commercial MnO 2 powder was contrastively tested for TCH decontamination under the same conditions. Figure 8 showed the removal efficiency from as-prepared MnO 2 NPs/ESM and commercial MnO 2 powder with or without a buffer. It was indicated that MnO 2 NPs/ESM showed a prominent advantage over commercial MnO 2 powder under both conditions. Though removal efficiency of around 80% through MnO 2 decontamination was obtained in previous work [38,39], it could reach up to 98% under pH = 6.5 through a MnO 2 and zero-valent iron (ZVI)-based permeable reactive barrier (PRB) system [56], which was attributed to the multiple effects from ZVI coupling with MnO 2 . Besides, other materials were also applied to TC decontamination. Immobilized TiO 2 nanobelts modified by Au and CuS nanoparticles (Au-CuS-TiO 2 NBs) displayed a removal efficiency of 96% towards oxytetracycline (OTC) owing to their superior photocatalytic activity [57]. Graphene oxide (GO) as an efficient adsorbent showed a good removal for TC after 24 h (R = 96%) [58]. Powder activated carbon/Fe 3 O 4 magnetic nanoparticles (PAC/Fe 3 O 4 MNPs) were applied as a catalyst to H 2 O 2 -assisted TC degradation, and removal efficiency of 94.5% was obtained [59]. It was noticed that the removal efficiency could be enhanced by prolonging the treatment time or increasing the material doses [39]. Nevertheless, all the work needs complicated degradation measurement and subsequent processing which increase the labor and test time. The handy operation of our method such as