Sustainable Graphene Aerogel as an Ecofriendly Cell Growth

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Biological and Medical Applications of Materials and Interfaces

Sustainable Graphene Aerogel as an Ecofriendly Cell Growth Promoter and Highly Efficient Adsorbent for Histamine from Red Wine Shruti Shukla, Imran Khan, Vivek K. Bajpai, Hoomin Lee, TaeYoung Kim, Ashutosh Upadhyay, Yun Suk Huh, Young-Kyu Han, and Kumud Malika Tripathi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02857 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

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ACS Applied Materials & Interfaces

Sustainable Graphene Aerogel as an Ecofriendly Cell Growth Promoter and Highly Efficient Adsorbent for Histamine from Red Wine

Shruti Shuklaa, Imran Khanb, Vivek K. Bajpaia, Hoomin Leeb, Tae Young Kimc, Ashutosh Upadhyayd, Yun Suk Huhb,*, Young-Kyu Hana,* and Kumud Malika Tripathic,*

a Department

of Energy and Materials Engineering, Dongguk University-Seoul, 30 Pildong-ro

1-gil, Seoul 04620, Republic of Korea b Department

of Chemical Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon 22212,

Republic of Korea c

Department of Bionanotechnology, Gachon University, 1342 Seongnam-daero, Sujeong-gu,

Seongnam-si, Gyeonggi-do 461-701, Republic of Korea d

Department of Food Science and Technology, National Institute of Food Technology

Entrepreneurship and Management (NIFTEM), Sonipat, Haryana 131028, India

Running head: 3D graphene aerogel from Pyrus pyrifolia for histamine removal

*Corresponding

authors: Yun Suk Huh ([email protected]); Young-Kyu Han

([email protected]); Kumud Malika Tripathi ([email protected])

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ABSTRACT: The utilization of sustainable and lightweight graphene aerogel (GA), synthesized from crude biomass, as a cell growth promoter and an adsorbent for the efficient removal of histamine (HIS), a food toxicant, from the real food matrix have been explored. Due to the self-supported three-dimensional nanoporous honeycomb-like structure of the graphene framework and the high surface area, the synthesized GA achieved an 80.69% ± 0.89% removal of HIS from red wine (spiked with HIS) after just 60 min under both acidic (3.0) and neutral (7.4) pH conditions. Furthermore, simple cleaning with 50% ethanol and deionized water, without any change in weight, allowed them to be reused more than 10 times with a still significant HIS removal ability (over 71.6% ± 2.57%). In vitro cell culture experiments demonstrated that the synthesized GA had non-toxic effects on the cell viability (up to 80.35%) even at higher concentrations (10 mg mL-1), as determined via the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay by using human lung bronchial epithelial cells. Interestingly, GA promotes the wound healing ability on the scratched epithelial cell surfaces via enhancing the cell migrations as also validated by the western blot analysis via expression levels of epithelial β-catenin and E-cadherin proteins. The distinct structural advantage along with non-toxicity of the green synthesized GA will not only facilitate the economic feasibility of the synthesized GA for its practical real-life applications in liquid toxins and pollutants removal from the food and environment; but also broaden their applicability as promising biomaterials of choice for biomedical applications.

KEYWORDS: green synthesis, graphene aerogel, histamine toxin, food safety, wound healing

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INTRODUCTION

The increasing demand for contamination-free food products to nourish the rapidly growing worldwide population is raising serious ecological and environmental issues associated with the detection and removal of food toxins. Histamine (HIS), as the most important biogenic amine, is a major cause for allergies and food poisonings when taken in high concentration.1,2 In particular, its poisoning can cause skin rash, diarrhea, nausea, urticaria, vomiting, flushing, hypo- or hypertension, tingling, itching, and anaphylactic shock.1,3 The severity of these effects considerably depends on the HIS amount ingested and its susceptibility to the individual4 and is worsened in patients already taking monoamine oxidase inhibitor drugs.5 Nevertheless, the HIS level is considerably high in rotten food and some fishes, inducing serious foodborne diseases.1 This can be accompanied with foodborne bacteria producing high HIS concentrations (>500 mg kg–1) in a very short time, as confirmed by the US-FDA.3,6 Therefore, the detection, monitoring, and removal of HIS are crucial but still challenging to ensure food quality and safety.7,8 Research efforts are currently focused on the development of low-cost, environmentally friendly, and renewable materials as alternatives to the traditional natural preservative-based ones for the removal of toxins from food and beverage.1,9 We recently reported microbial cultures and plant-based formulations for an efficient reduction of HIS to overcome the aforementioned issues.6 However, there are still several disadvantages associated with the sensory quality of food in some cases. Recent research concerns have been moved toward the development of green non-toxic nanocarbon-based adsorbents for the removal of diverse pollutants and toxins due to the highly versatile morphology, high aspect ratio, and physiochemical properties that could be applied to individual household systems.10-12 Despite the advances, recovery and recycling difficulties along with the low loading capacity of certain 3



toxins including HIS are major obstacles that must still be overcome. Hydrophobic and porous nanocarbons with a large specific surface area and delocalized π electrons can serve this purpose owing to their distinct functional properties and exceptional physicochemical robustness.13 In the foreseeable future, low-cost and green nanocarbons with excellent recovery and recycling capabilities will most likely be the dominant adsorbent materials.14 Among the nano-carbons exploited, three-dimensional (3D) carbon nanostructures have been widely investigated because of their versatile characteristics and inherent fascinating properties, such as light weight, high internal surface area, low density, versatile porosity, chemical inertness, low thermal conductivity, and excellent electrical conductivity.15-18 The open and interconnected 3D networks, with continuous nanopores, of the aerogels are expected to provide excellent adsorption performance for different applications ranging from pollutant removal, oil–water separation and electromagnetic wave absorption, ink writing to osteoblast growth.19-22 In the past decades, the application of biomass-based graphene aerogels (GA) for environmental remediation has received much attention due to the easy processing of their natural sources, the low cost, and the reduced environmental footprint.16,23-25 The implementation of GAs is predominantly benefited from their inherent merits of structural interconnectivities, well-formed porosity, and excellent property in solid-liquid interphase for HIS-wine separation. Despite their high adsorption efficiency, GAs still present many obstacles such as a low-yield and complex fabrication process. Nevertheless, the intrinsic limitations of the texture and porous architecture of the bulk biomass have limited the nanoarchitecture and porosity of aerogels, which is a major challenge for the targeted synthesis. The cellular structure and consistency of the carbonaceous constituents of the precursors have dictated their effective properties.26 Hence, appropriate, low-cost, and green carbon precursors have considerable impacts on the aerogel synthesis and the achievement of their promising properties.26,27 From this perspective, we chose Pyrus pyrifolia, a pear species, as a promising 4


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sustainable natural precursor material for the synthesis of lightweight, porous, and honeycomblike nanostructured GA.28 Wine is an important beverage in world trade. HIS contamination in wine largely depends upon its type and origin. The European Union (EU) has adopted legal threshold limit of HIS as 10 mg L-1, excluding previously investigated ~34% wine from international market. The HIS contamination of grapes can even occur in the vineyard considering that red wine intolerance might be a natural marker symptom and, thus, it has been recommended as a HIS intolerance model.29 Therefore, the HIS quantification and removal are key to define the critical control points for precautionary measures against wine product contamination. Although many studies on GA as adsorbents have revealed their different performances, such as water pollutants adsorption,30 environmental hazards removal,31 oil–water separation,32,33 and radioactive hazards removal,34 their applicability as non-toxic green adsorbents for food safety and hygiene in real food matrix toward the removal of hazardous toxins, including HIS, has not been explored, yet. Therefore, the main motive of the presented research work was to develop a non-toxic adsorbent material which can absorb HIS contaminants from liquid-based fermented food products without any adversary effect on taste and sensory characteristics, as an alternative to conventional charcoal additives. To address bottleneck requirements such as non-toxicity along with negligible use of organic solvent without any adversary effect on the food sensory quality, we propose the sustainable synthesis of GA with 3D interconnected honeycomb-like graphene interfaces by using pear (Pyrus pyrifolia) as a raw material. The hierarchical 3D interconnected structure provides high surface area of ~480 m2 g–1 and appropriate pore size distribution, which allowed the substantially excellent adsorption (82.07% ± 0.89%) of HIS from red wine samples in just 60 min. More significantly, the reduction in the cell counts of HIS producing bacterial contaminants (Cronobacter sakazakii, Staphylococcus aureus, and Aeromonas sp.) was also 5



noticed in red wine samples, after few hours of treatment with GA. This study first time explores the use of low-cost GA as an effective tool to remove HIS from liquid food products. Moreover, we observed excellent recyclability of the synthesized GA that could be reused more than 10 times showing the same adsorption capability, after simple washing with ethanol; GA tended to retain its shape even after various cycles without any weight loss, which is quite beneficial for easy recycling and recyclability. The fabricated GA provides novel opportunities to effectively manage biomass for its extended application in toxins removal from beverage and food without any change in sensory quality and color. Moreover, the non-toxic behavior and efficient wound healing ability of the sustainable GA were determined via cell growth promotion assay and determination of protein expression levels of human lung bronchial epithelial cells by western blot analysis, thus facilitating the non-invasive wound healing and monitoring.

RESULTS AND DISCUSSION

Hierarchical 3D interconnected GA was synthesized through a combined hydrothermal and post-pyrolysis process directly from the raw pear, as illustrated in Scheme 1. A series of dehydration, polymerization and aromatization reactions followed by carbonization via intramolecular dehydration resulted in carbonaceous aerogel with retention in shape. GA was collected by freeze-drying a frozen carbonaceous aerogel, which was ultimately carbonized in flowing argon at 1000 °C to afford graphitization. At 1100 °C, the generation of syngas such as CO, CO2 and CH4 in argon atmosphere and removal of volatile impurities slowly develop a 3D network.35 While the generation of syngas causes the 3D network of graphene to expand or swell up to form porous cylindrical structures. The ejection of gases leads to collapse of few graphene layers and volume contractions resulting in porous structures of diverse shapes and 6


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sizes.35 The as-synthesized GA piece could stand steadily on a dandelion head, revealing its lightweight nature. The GA volume could be easily tuned just by cutting the pear into appropriate sizes. Then, the resulting lightweight sponge-like 3D GA was tested as adsorbents for the removal of biogenic HIS from red wine as a real food matrix and wound healing.

Scheme 1. A schematic representation of the synthesis of 3D interconnected GA (using pear as raw material) and corresponding optical image of synthesized GA stabilized on a dandelion head denotes the fabrication of the ultralight three dimensional aerogels. The application of GA was explored as histamine adsorbent in contaminated red wine samples and as potential wound healer.

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Morphological characterization. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and high-resolution TEM (HRTEM) were performed to investigate the morphology and microstructure of the synthesized GA. The low-resolution SEM micrograph shown in Fig. 1a reveals its honeycomb-like 3D macroporous interconnected structure. The high-resolution SEM (HRSEM) micrograph in Fig. 1b reveal that GA is composed of graphene sheets without any spherical or other morphological impurities. HRSEM of a honeycomb type cylindrical pore in Fig. 1c shows that the pore walls consisted of folded, crumpled, and disordered graphene layers with a thickness of 15.9 nm (Inset of Fig. 1c).

Figure 1. (a) Low-resolution SEM image of GA; (b) high resolution SEM images of GA, (c) HRSEM image of macro-porous walls. (d) Low-magnification TEM image of GA; (e) high magnification TEM image of GA and; (f) HRTEM image of GA showing number of graphitic layers and high density of surface defects as missing graphitic planes, highlighted by white circles.

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The internal GA microstructure was more precisely analyzed via the TEM micrographs; the low-resolution TEM image shown in Fig. 1d displays its elegantly interconnected continuous nanoarchitecture and the pores formed by this network. The high-magnification TEM image shown in Fig. 1e indicates that the synthesized GA basically consisted of wrinkled nanosheets, which contributed to the enhancement of the active surface area. The HRTEM image shown in Fig. 1f reveals the graphitic structure of GA, consisting of 6-10 individual layers of graphene with a lattice spacing of 0.30 nm corresponding to the (002) graphite plane36 and also highlights the missing graphitic layers and surface defects of the graphene sheets by white circles.

Structural characterization. The GA was further characterized with X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, thermogravimetric analysis (TGA), and X-ray diffractometry (XRD). Structural insights about the GA composition were acquired with the XPS analysis; the XPS spectrum shown in Fig. S2 reveals peaks at 285 eV and 532 eV corresponding to the presence of C (84.43%) and O (15.57%), respectively. The deconvoluted C 1s spectrum (Fig. 2a) shows four peaks (284.5 eV, 285.5 eV, 286.2 eV, and 287.0 eV) indicating four different bondings for the carbon atoms (respectively, C‒C, C=C, C‒O, and C=O) in the GA,37,38 which had a graphene-based structure. Fig. 2b shows the deconvoluted O 1s spectra indicating the two bonding states of oxygen; the carboxyl (C=O) and hydroxyl (C‒O) groups can be observed at 531.8 eV and 533.2 eV, respectively.39 The Raman spectrum (Fig. 2c) shows typical D and G bands at 1351 cm–1 and 1547 cm–1, respectively. The former was attributed to the disorder-induced mode of the defected graphitic structures and the later was assigned to the in-plane bond-stretching motion of the sp2 carbon atoms.40 The integrated intensity ratio between the D and G bands (ID/IG) reflected the disorder extent in the GA (Fig. 2c); its high value (1.3) suggested the presence of high-density surface defects in the form of sp3 carbon atoms.41,42 9



Figure 2. Structural analysis of GA. XPS spectra of GA; (a) C 1s spectrum of GA; (b) O 1s spectrum of GA. (c) Raman spectrum, showing the characteristic D and G bands of carbon nanostructures; (d) XRD pattern with sharp peak at ~ 21.7 º, showing the presence of graphitic carbon; (e) TGA thermogram; and (f) Nitrogen adsorption/desorption isotherms for surface area measurement by BET analysis. The XRD pattern in Fig. 2d shows peaks at 2θ = 21.7º and 44.05º corresponding, respectively, to the (002) and (101) reflections of the disordered graphene and the formation of interlayer condensation.43 The thermal stability of the synthesized GA was studied via TGA analysis from room temperature to 1000 oC under inert atmosphere. The weight loss 10


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corresponding to the temperature change, as shown in Fig. 2e, indicates a noticeable and gradual decrease in weight loss. The GA exhibited excellent thermal stability, with a total weight loss of only 16.5 % at 1000 oC resulting from the removal of the oxygenated functional groups.40 The surface area and pore size of the synthesized GA was examined via N2 adsorption–desorption measurements, which revealed a high Brunauer–Emmett–Teller (BET) surface area of ~480 m2 g–1 with a pore volume of 0.27 cm3 g–1. GA exhibited a type Ib isotherm along with a slight hysteresis (Fig. 2f), indicating diversity in the pore size distribution, from micro- to mesoporous.43

GA cytotoxicity. The non-toxic nature of the synthesized GA was evaluated through a cell viability assay by using healthy human lung bronchial epithelial cells, COS-7 and HaCaT. Regardless of the concentration (1–20 mg mL–1), GA had no effect on the viability of the cells tested in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay (Fig. 3a and 3b). A microscopic examination was also performed to determine the morphological changes in the cells (Fig. 3c–g). Cell viability was reported as 80.35% for 10 mg of GA. A very small change in the cell viability (77.25%) was observed in human lung bronchial epithelial cells even at higher concentrations of GA (20 mg mL–1) (Fig. 3a-b). The cell viability experiments were also performed with other normal cells, including COS-7 and HaCaT cells via MTT assay and morphological observations. As expected, the GA was found to be non-toxic up to its higher concentration of 20 mg mL–1 (Fig. S3) for both the cells. All the results further confirmed that the synthesized GA is 100% safe for use in both food media and oral administration.

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Figure 3. Cytotoxicity evaluation of GA by using human lung bronchial epithelial cells (BEAS-2B, ATCC® CRL-9609) (a) MTT assay; and (b) LDH assay. Morphological evaluation after treatment with different doses of GAs; (c) control; (d) 1 mg mL-1; (e) 5 mg mL-1; (f) 10 mg mL-1 and (g) 20 mg mL-1. NS: non-significant. Scale bar denoting 100 µM in each image. In addition, the ability of GA to retain the beneficial microflora (Bacillus and Lactobacillus strains) inoculation present in red wine was also analyzed via the agar plate count method (with the malt yeast peptone (MYP) and deMan–Rogosa–Sharpe (MRS) media, respectively) during the testing of toxin removal. Table S1 confirms that there was no significant reduction or elimination in the beneficial bacterial counts of the red wine samples treated with GA, when compared with their initial microflora before GA inoculation. 12


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Wound healing capability/Cell migration assay. The biomedical applicability of the synthesized GA was further explored as significant (p60% of HIS was initially adsorbed under both acidic and neutral pH and reached ~75% after 60 min. Such a high HIS adsorption was attributed to the well-developed meso- and micro-pores, including high surface area of the synthesized GA. The GA is basically composed of graphene nanosheets in 3D interconnected network, which consisted delocalized π electrons resulting in weak basicity.46 The highly hydrophobic surface of GA with oxygenated functional groups is attributed to predominant adsorption of HIS from wine. As revealed by Raman and TEM analysis, the high surface area of the GA with surface active sites in the form of surface defects results in high density of active sites for adsorption. In addition, the π-π interaction between aromatic ring of HIS and graphene nanosheets of GA significantly contribute to increase adsorption efficiency.46 Moreover, the electrostatic interaction between amine moieties of HIS and oxygenated functional groups of GA also significantly contribute in adsorption, depends upon the pH of the medium. A slight decrease in HIS adsorption was observed from pH 9 to pH 11, while its maximum was observed at pH 3.0 and pH 7.4, which is the preferable 16


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physiological condition in body fluids. The probable consideration of increasing HIS adsorption in this pH range could be owing to electrostatic and hydrogen bonding attractions among the amine moieties and the surficial functional groups of the GA, while, above pH 9.0, the electrostatic repulsions among negatively charged surfaces could reduce the HIS adsorption.30,47-49 The adsorption of HIS by GA attributed to both electrostatic and π-π interactions, along with hydrogen bonding interactions.19 At acidic and neutral pH hydrogen bonding interactions between surficial oxygenated groups of GA and HIS amine moieties are dominated, which in consequence lead to the higher adsorption efficiency.

Figure 6. HPLC Chromatograms of standard HIS solution in DI water at pH 3.0. Concentration of HIS; (a) prior to GA addition. (b) After 10 min; (c) after 40 min; and (d) after 60 min. of GA addition. The adsorption efficiency of GA at different concentrations (1, 5, and 10 mg 100 mL–1) was also analyzed under neutral pH (7.4). Fig. 7c shows that, at 5 mg 100 mL–1, GA exhibited higher HIS adsorption after 60 min compared to both lower (1 mg 100 mL–1) and higher (10 mg 100 mL–1) GA concentrations. Thus, this concentration was used in all the successive 17



adsorption studies. Furthermore, the adsorption efficiency of GA was also tested by varying the HIS concentration since, in real samples, this amine can be found in different amounts. The HIS adsorption ranged between 51.6% – 75.4% (Fig. 7c) after 60 min of reaction with variable HIS concentrations (50, 30, 10, and 5 mg 100 mL–1) under both acidic (3.0) and neutral (7.4) pH conditions, confirming the applicability of the synthesized GA for the removal of both high and low concentrated contaminants from food samples or environmental matrixes under physiological pH conditions.

Figure 7. Optimization studies for the efficient adsorption of HIS adsorption with GA; (a) standard calibration curve for HIS with different concentration (prior to GAs addition); (b) the influence of pH on the adsorption of HIS; (c) effect of GA concentration with time for HIS adsorption; and (d) HIS adsorption at constant reaction time (60 min) on varied HIS concentration at acidic and neutral pH conditions. 18


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Practical case study: A food matrix. The applicability of GA was further investigated on a real food matrix by using a South Korean red wine available in supermarkets. The adsorption experiments were performed on an artificially HIS-spiked red wine (50 mg 100 mL–1), as shown in Fig. 8a. Significant adsorption of HIS (80.69%, 84.43%, and 88.18%) was achieved after 60 min, 90 min, and 120 min, respectively, in red wine samples containing 5 and 10 mg 100 mL–1 of GA (Figs. 8a, b). A slight decrease in HIS adsorption compared to buffer systems (Fig. 7c) was observed that might be because of food matrix effect, where various background components compete and interfere with it,6 the adsorption percentage was still significantly high.

Figure 8. Application of GA for HIS adsorption in realistic food sample as HIS spiked red wine; (a) effect of different concentration of GA on His adsorption; (b) chromaticity (redness) 19



change of red wine after reacting with GA for different times; (c) visual color change in red wine before and after 60 min of GA addition; and (d) spectrophotometric analysis of red wine before and after GA addition, which confirms no significant difference in peak intensity and position. The consumer acceptability as a color preferable recommendation without any change was also tested with different red wine samples. The redness (a*) value is considered as the most important parameter for the consumer acceptability of red wine. As shown in Fig. 8b, when treated with GA, no significant difference was observed in the wine color after filtration. A visual color (Fig. 8c) and spectrophotometric absorbance analysis at wide wavelength confirmed the absence of significant changes in the characteristic peak intensity and position at ~320 nm and ~520 nm after the GA treatment (Fig. 8d). Hence, the synthesized GA exhibited a considerable potential for the effective removal of histamine from beverage and food samples.

Reduction of HIS producing bacterial contaminants in wine. Several microbial contaminants are responsible for the increase in HIS content in wine during its production and storage. Therefore, it is worth to evaluate the effect of GA on the reduction of HIS producing bacterial contaminants in wine; especially the microbes with amino acid-decarboxylating activity such as Staphylococcus aureus,50 Aeromonas species, and Cronobacter sakazakii.51

Not only HIS adsorption but a significant reduction in microbial cell count was also observed in each bacterial strain spiked wine samples treated with GA at the time intervals of 2, 4, 6, 8 and 10 h as compared to untreated red wine samples. Fig. S4 shows that the colony count was dropped significantly from 5 log CFU mL–1 to 1 log CFU mL–1 after 8 h of interaction with GA in red wine samples. These results clearly indicate the practical applicability of GA for the removal of HIS and reduction in HIS producing bacterial contaminants from red wine samples in wine processing industry from the early stages of its fermentation to end storage point. 20


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Regenerative properties and reusability. The adsorbent recyclability is crucial for long term applicability in contaminants removal and, along with the cost efficiency, determines the overall sustainability and industrial applicability of any material. Our synthesized GA exhibited extreme recyclability and recovery efficiency for HIS removal in artificially HIS-spiked red wine samples. The GA was reactivated by simply washing with 50% ethanol and DI water, followed by subsequent drying at 40 ºC. The recyclability results are shown in Fig. 9a; there was no significant decrease in HIS adsorption capacity even after 10 adsorbing-washing cycles. Moreover, the GA maintained its shape integrity and weight even after 10 cycles, which is quite significant for easy recycling and recovery without any centrifugation, confirming their excellent mechanical properties (Fig. S5 and Table S2).

Figure 9. Demonstration of the reusability of GA towards HIS adsorption; (a) in different lots of HIS spiked wine samples; and (b) in same lot of wine samples. When using the same lot of HIS-spiked red wine samples, ~ 75.06 % of HIS removal was observed after first 60 min of cycle and increased to 85.22% and 93.66% after second and a third run (after washing), respectively (Fig. 9b). On the other hand, the decrease in adsorption performance of GA after the first cycle in the same wine lot was due to lesser remained HIS amounts. These findings are in accordance with those of a previous study on the sustainable 21



renewability of agarose-derived aerogel as an oil adsorbent.42 These results confirmed that the remaining amount of HIS from the first cycle was completely removed at the third one, which approaches industrially applicable levels of HIS removal, suggesting the possibility of multiple time use of the activated GA for the complete elimination of toxins in the wine industry during the production and storage process.

Industrial model design. In the viewpoint of industrial applicability, both large- and smallscale model design for the lot-to-lot decontamination of wine during its processing in the food industry required to be investigated; this would reduce the lot-to-lot variations for the removal of unwanted toxins including HIS. In this complex design, GA filter pockets could be easily installed in the pipeline by using edible wool interval settings (Fig. 10). The designed setup can be installed in both long and small length glass columns containing liquid food samples including contaminated wine, which will pass through with optimized incubation conditions, and the flow rate can be managed by using the N2 pressure management system. A practical concept as shown in Fig. 10 for the real quality testing of wine was performed by designing a manual GA packed column with regular intervals of edible wool for fine separation and/or to avoid the release of GA fine materials into wine samples (Fig. S6). Flow rate of wine samples passing via column was also optimized considering the proper interaction of GA with HIS moieties. Further, the quality testing of GA column passed HIS spiked wine samples was analyzed for reduced HIS level through HPLC quantitative analysis. Similar HIS removal ability of about 70% was observed to approve the overall studies. However, a column designed with digitalized quality testing devices is still needed to be installed in future.

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Figure 10. A schematic representation for the possible design of industrial model for both small and large scale HIS removal with efficient recovery of reusable GA.

CONCLUSIONS In summary, 3D interconnected hierarchical graphene aerogels has been synthesized from a green approach using crude biomass Pyrus pyrifolia for the utilization in toxin removal from food and wound healing. The as-synthesized GAs possessed a 3D macroscopic honeycomblike network with high internal surface area and porosity. Importantly, GAs showed excellent biocompatibility and had negligible cytotoxicity effects on the cell viability (up to 80.35%) even at a higher dose (10 mg mL-1), when tested in both LDH and MTT assay by using human 23



lung bronchial epithelial cells, which made it more attractive for environmental and biological applications. Furthermore, GAs accelerated wound healing on the scratched epithelial cell surfaces via enhancing the cell migrations. Moreover, GAs exhibited high adsorption capacity when added to HIS-spiked red wine, and attained the highest HIS removal (82.07% ± 0.89%) just after 60 min under both acidic (3.0) and neutral (7.4) pH conditions. Distinct structural advantage of GA offers excellent recyclability without any loss in removal efficiency. Moreover, GA is also effective for reduction of HIS producing bacterial contaminants in red wine samples suggesting its beneficial use in wine processing industry from the process of fermentation to end storage point of wine. Non-toxicity of GAs along with high performance not only facilitate the feasibility for practical real-life applications for food and environment safety toward toxin/pollutant removal but also broadened the applicability in biomedical application.

EXPERIMENTAL SECTION

Chemicals and reagents. Fresh and high grade quality pear fruits were procured from the local super market in Korea, and were used as raw materials for the synthesis of GA, without any further pretreatment. Histamine dihydrochloride, acetone, and dansyl chloride, were obtained from Sigma-Aldrich. Sodium hydroxide, ammonium hydroxide, perchloric acid, and sodium hydrogen carbonate were provided by Junsei Chemicals. HPLC grade solvent such as methanol, ammonium acetate (0.1 M), and acetonitrile were purchased from Merck. Other reagents and chemicals used were of high analytical grade.

Synthesis of 3D interconnected GA. Naturally, highly ordered structures, high water content (~84 %), unique inherent grainy, meso/macroporous texture and micro-tubular channels endow 24


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pear (Pyrus pyrifolia) reported that structural specificity of the biomass can be inherited in nano-carbons by choosing appropriate synthetic processes. Peeled pear slices of appropriate shapes were washed with hot water, and heated in a Teflon-lined stainless steel autoclave at 250 oC for 36 h under self-generated pressure, and then naturally cooled to room. The resulting carbonaceous hydrogels were immersed in hot water to remove the soluble impurities and then dried at -48 oC at 40 Pa by using a freeze dryer (FD-1A-50, BiLon; China). The as-obtained aerogels were carbonized at 1100 °C for 1 h at a heating rate of 5 °C min–1 under argon atmosphere and then the furnace was naturally cooled to room temperature, yielding black colored ultralight GA.

Instrumentation. The surface morphology of the synthesized GA was observed by using a JEOL JSM-7500F (Tokyo, Japan) scanning electron microscope operating at 20 kV. TEM and HRTEM were performed on an FEI Tecnai G2 F30 instrument at 300 kV. XPS spectra were acquired to quantitatively analyze the chemical composition of the GA by using a ULVACPHI X electron system with an Al Kα X-ray source. The thermal stability of the synthesized GA was evaluated with a Mettler thermogravimetric analyzer (SDT Q600 V20.9, Build 20) at a heating rate of 10 °C min–1, between room temperature and 1000 °C under nitrogen atmosphere. Raman spectra were recorded on a WITec Raman spectrometer with a 405 nm excitation wavelength. A powder XRD pattern for 2θ values from 10° to 80° was recorded by using a Rigaku RINT-2000 X-ray diffractometer with Cu Kα radiation. The BET surface area of the synthesized GAs was determined via the BET nitrogen adsorption–desorption technique by using a TriStar 3000 analyzer (Quantachrome Instruments; USA) at liquid nitrogen temperature.

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HIS adsorption and removal. The HIS adsorption/removal capacity of 3D GA was analyzed by batch adsorption experiments, individually for HIS (spiked with different HIS concentrations) both in water and various buffer media. The GA applicability for real food matrixes was also tested by using red wine samples. The GA (5 mg) was added in solutions (100 mL) spiked with HIS (50 mg 100 mL–1), which simulated a highly HIS contaminated wine. Then, the solutions were incubated at room temperature with shaking; after the specified mixing time at the storing conditions, the samples were filtered through a 0.45 μm filter and the HIS concentration was analyzed via HPLC. The experiments were performed in triplicate and the data were expressed as the mean values. The GA recyclability was tested after washing with DI water and 50% ethanol, followed by a second rinse with DI water only for their reactivation and the removal of surface contaminants. Kinetic experiments were performed for different contact times (10 min, 20 min, 40 min, 60 min, 80 min, and 120 min) with a 50 mg 100 mL–1 HIS solution at physiological pH (7.4). After the optimization of the reaction time, the solutions were separated by filtration with a 0.45 μm filter paper and the HIS concentration was measured via standardized HPLC. The pH effect on the HIS solution was also tested in a pH range (3.0–9.5). The initial pH of the 50 mg 100 mL–1 HIS solution was adjusted by using a 0.1M HCl or NaOH solution. The GA addition was followed by incubation for 60 min; then, the solutions were filtered and the reduction of the HIS content was verified via HPLC. Additional studies about the HIS levels were carried out under neutral (7.4) and acidic (3.0) pH conditions to test the use of GA in grape juice or wine samples. The experiments were performed by mixing different concentrations of HIS with a constant dose of adsorbent (5 mg 100 mL–1) for 60 min; the spiked HIS concentrations were in the 1–50 mg-100 mL–1 range.

Sample derivatization for HPLC analysis of HIS. The adsorbed amounts of HIS were 26


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analyzed by HPLC. Prior to it, all the samples were derivatized according to a previously reported technique;6 briefly, 1 mL of each reacted sample or standard HIS solution was mixed with 200 mL of 2M NaOH and 300 mL of a saturated solution of NaHCO3. Then, 2 mL of the dansyl chloride solution (10 mg mL–1 in acetone) was added to the mixture, which was successively incubated in a water bath at 40 C for 45 min; next, 100 µL of a 25% NH4OH solution was added to stop the reaction and remove the residual dansyl chloride. After 30 min of incubation at room temperature, the volume of the reaction mixture was adjusted to 5 mL with acetonitrile and then centrifuged at 2500 rpm for 5 min. The supernatant was filtered through a 0.2 µm syringe filter and assayed by HPLC. In each experiment set, standard HIS solutions were tested for method standardization. The HPLC system consisted of two pumps and an ultraviolet (UV)–visible light (VIS) detector. The separation was achieved by using a C-18 column (4.6 mm width ×150 mm length) with a pore size of 5 mm at 30 ºC. The mobile phase consisted of 0.1 M ammonium acetate (solvent A) and acetonitrile (solvent B) and the flow rate was 0.8 mL min–1, with a gradient elution program for 35 min. The sample volume was 10 mL and the samples were monitored at 254 nm.

Method validation. The analytical HPLC procedure was internally validated by the calibration and evaluation of the linearity, precision, and recovery ranges. The linear response of the detector was determined in a range of HIS concentrations (12.5–150 µg L–1), giving a correlation factor r2 > 0.99. All the experiments were carried out in triplicate and the statistical analysis of the data was performed at a significance level p < 0.05 by using the SPSS software (IBM SPSS Statistics 22, IBM Corp; New York, US).

Effect of GA on HIS producing microbial contaminants. Selected HIS producing bacterial 27



contaminants such as Staphylococcus, Aeromonas sp., and Cronobacter sakazakii were cultured in Luria-Bertani (LB) broth at 37 ºC for 16-18 h, after which cultures were serially diluted with 0.1% peptone water (w/v) to prepare the optimum concentration of bacterial cells. In brief, red wine samples (10 mL) were spiked with 100 μL of bacterial inoculum of each HIS contaminant to obtain the final inoculum level in wine as ~106 CFU mL–1 of each culture and then GA (5 mg) was inoculated. After that reduction in cell counts of each HIS producing bacterium was then measured at every 4 h of interval using a conventional standard spread plate count method on nutrient agar (NA) plates. After incubation, typical colonies were counted and results were reported as log CFU mL–1.

Cytotoxicity assessment

Cell culture. The human lung bronchial epithelial cells (BEAS-2B, CRL-9609), COS-7 fibroblast cells (ATCC CRL-1651) and HaCaT human epidermal keratinocyte cells (ATCC PCS-200-011) were obtained from the American Type Culture Collection (ATCC). For culturing the cells, RPMI-1640 and DMEM medium supplemented with fetal calf serum (10% (v/v) and the cocktail of penicillin–streptomycin (1%) was used under 5% CO2 at 37oC. The culture medium from the cells was changed after every two days.

MTT and LDH release assay. The cytotoxic effect of GA was first determined via the MTT assay. The epithelial cells, COS-7, HaCaT were seeded in 24-well culture plates at a density of 5 × 104 cells/well in a 500 μL culture medium and incubated for 24 h to reach a 90% confluency. Then, the confluent cells were exposed to GA at various concentrations (1, 5, 10 and 20 mg mL–1) and successively incubated at 37 °C for 24 h. After that, the cells were treated with an MTT solution (5 mg mL–1) to produce dark blue colored formazan crystals, afterwards 28


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dissolved in dimethyl sulfoxide (50 μl). Finally, the absorbance was measured at 540 nm on a Varioskan LUX multimode microplate reader spectrophotometer. The cytotoxicity was also determined via the LDH assay. To quantify the cell plasma membrane damage, the cytosolic enzyme LDH was measured by using the LDH assay kit (Sigma-Aldrich; St. Louis, USA). After the GA treatment followed by incubation for 24 h, the cell-free supernatants were collected and centrifuged at 12000 rpm for 10 min; 50 μL of each supernatant was reacted with 50 μL of the reaction mixture (LDH substrate mix and assay buffer) for 30 min, protected from light. The initial and final absorbance was measured at 490 nm and 680 nm, respectively, with the microplate reader and, then, used to evaluate the cytotoxicity. Furthermore, the morphological changes in all tested cells after GA exposure (1, 5, 10 and 20 mg mL–1) were determined via microscopic examination.

In vitro wound healing capability/ Cell growth migration assay. Lung epithelial cells (5×104 cells/well) were seeded in a 12 well plate till the dense single layered with full confluence growth of cells was observed. After that the surface of plate was scratched using a sterile microtip in order to generate a cell-free zone. Free cells were then washed off with PBS, and further treated with GA at various concentrations (1, 5, and 10 mg mL–1) followed by incubation at 37 °C for 24 h. The area of wound closer and the number of cells migrated from one side to other were observed under inverted microscope and photographed after 12 h.

Western blot analysis of cell growth promoting proteins. After confirming the cell growth promotion or wound healing abilities of GA on the morphology of human lung bronchial epithelial cells, these results were further reconfirmed by visualizing the role of GA on up-and down-regulating the expression levels of wound healing protein markers, such as β-catenin and E-cadherin proteins by western blot analysis. In brief, lung epithelial cells (5×104 cells/well) 29



were scratched and treated with different concentrations of GA (1, 5, and 10 mg mL–1) by incubating them at 37 °C for 24 h. Total proteins were separated by using RIPA lysis buffer with protease & phosphatase inhibitors and an equal amount of protein lysate was separated in reducing polyacrylamide gel and transferred into a PVDF membrane (BioRad) by electroblotting. The membrane was subsequently probed with appropriate primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibody, and visualized by enhanced chemiluminescence (ECL) according to the recommended procedure.

ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT and Future Planning of Korea (2017M2A2A6A01020938 and 2017R1D1A1B03035373). This work was also supported by the Gachon University research fund of 2018. (GCU-2018-0370)

Supporting Information Effect of GA inoculation on beneficial microflora present in red wine, effect on weight of GA after regeneration process using in red wine, SEM image of honeycomb architecture, wide scan XPS spectrum of GA, cytotoxicity evaluation of GA using COS-7 and HaCaT cells, efficiency of GA on the reduction of histamine producing bacterial contaminants, structural integrity of GA, practical HIS removal applicability as supporting information.

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Graphic for Manuscript 310x164mm (96 x 96 DPI)


Sustainable Graphene Aerogel as an Ecofriendly Cell Growth

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