Ecofriendly fruit-switches based on graphene oxide for programed fruit

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Biological and Medical Applications of Materials and Interfaces

Ecofriendly fruit-switches based on graphene oxide for programed fruit preservatives delivery to extend shelf life Sandeep Sharma, Badal Kumar Biswal, Divya Kumari, Pulkit Bindra, Satish Kumar, Tsering Stobdan, and Vijayakumar Shanmugam ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02048 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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ACS Applied Materials & Interfaces

Ecofriendly fruit-switches: Graphene oxide based wrapper for programed fruit preservatives delivery to extend shelf life Sandeep Sharma,1‡Badal Kumar Biswal,1‡Divya kumari,1‡Pulkit Bindra,1Satish Kumar,1Tsering Stobdan,2and Vijayakumar Shanmugam*1 1

Institute of Nano Science and Technology, Habitat Centre, Phase- 10, Sector- 64, Mohali,

Punjab – 160062, India. 2

Defence Institute of High Altitude Research, Leh, India.

KEYWORDS: Nano-agri, smart fruit pack, non-toxic nano-preservative, graphene oxide ABSTRACT: According to FAO 2015 report, post-harvest agricultural loss accounts for 20-50% annually, on the other hand reports about preservatives toxicity are also increasing. Hence, preservative release with response to fruit requirement is desired. In this study, acid synthesized in the over ripen fruits were envisaged to cleave acid labile hydrazone to release preservative salicylaldehyde from graphene oxide (GO). To maximize loading and to overcome the challenge of GO reduction by hydrazine, 2 step activation with ethylenediamine and 4-nitrophenyl chloroformate respectively, are followed. The final composite show efficient preservative release with the stimuli of the over ripen fruit juice and improves the fruit shelf life. The composite shows less toxicity as compared to the free preservative along with the additional scope to reuse.

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The composite was vacuum filtered through 0.4 µm filter paper, to prepare a robust wrapper for the fruit storage.

INTRODUCTION Globally 40–50% of the fruits and vegetables produced in the field are lost before consumption.1 Fruit preservatives control the ripening rate, microbial infection and cold injury, thus gifts the luxury to have desired fruits round the year, irrespective of geography. Common preservatives include weak organic acids and its derivative viz., acetic, lactic, benzoic, sorbic acids, aldehydes and esters. The preservatives have been prepared as wax formulation and applied to form a thin coating,2 which may enter human while consumption. The cells fragment the preservatives into ions to maintain the physiological balance,3 which results in toxic ion accumulation, membrane disruption, essential metabolite inhibition and energy drain to restore the homeostasis.4-6 Consumption of preservatives like benzoic acid was observed to cause acidosis, convulsions hyperpnoea and idiosyncratic.7,8 Preservatives like sorbic acid were identified to be a mutagen and cause genotoxicity.9-11Usual practice of excess preservative application may evolve pathogen with tolerance to preservative by enzymatic degradation and preservative efflux.12-15 Recently, to avoid preservative toxicity, natural extracts were explored,16-18for instance, pomegranate peel extract was tested for the preservation of guava.19Volatile plant secondary metabolites viz., phenolic esters were also studied.20 Salicylates (Parabens) are natural phenols that delay ripening, trigger phyto-immune factors to extend shelf life,21 with an efficient antimicrobial property.22,23These phenolics have been approved for food preservation,24in spite of toxicity like chronic allergy, physiological complication, etc.25-27 Contradicting to the above toxicity, these phenolics also have therapeutic claims,28 this difference in opinion have been due


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to the concentration. This phenomenon have been person dependent, for instance, as less as 2.6 mg salicylate were found to develop broncho constriction in some individuals.29Even the digestible preservatives like nisin were frequently reported to form elementary glycated product.30 The food preservatives standards, specify the limits for the preservative consumption, 31,32

which is not being followed strictly, especially in developing countries. Further, the food

consumption volume differs from individuals and hence the preservative intake also differs. Hence, a packing material that can deliver the preservative based on the demand from the fruit, can customize. Thus, the tradeoff between the fruit preservation and non-target effect on the humans can be narrowed down. In health science, highly precise stimuli controlled targeted delivery through size,33 charge34, pH,35light triggered36 change and their combinations were explored. But in agriculture science, such triggered systems are not explored often. Recently, we identified a combination of external and internal stimuli for controlled and targeted pesticide delivery in plant ecosystem, however these stimuli were pertinent to pest (Animalia).37In plants, the fruits are the most expressive domain, hence in this study, the benefit of climacteric fruits to synthesize acid at ripening stage,38,39 is taken as an advantage for the switchable preservative release. Banana fruits have been one such fruit, rich in antioxidants and other healthy phytochemicals.40 In this study, fruit preservative delivery from the nano-packing in response to the signal from fruit stimuli is demonstrated. An inert platform like graphene oxide,41 with a very high surface area to accommodate maximum preservative has been chosen. This platform has been loaded with the model preservative salicylaldehyde. In addition, the surface area of GO can


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complement in ethylene sequestration,42,43which have been the primary signal from the ripening fruits44and in cold stress,45-50to induce oxidation.51Further, the ability of the composite to serve as fruit preservative carrier for multiple times is also demonstrated. To avoid the environmental risk of the GO based composite through direct fruit application, a stable membrane of the composite have been prepared through vacuum filtration with the porous filter paper support. EXPERIMENTAL SECTION Materials.Graphite powder, ethylenediamine (ReagentPlus), hydrazine (98%), 4-nitrophenyl chloroformate (97%), sodium carbonate (BioReagent), salicylaldehyde (≥99%), folin and ciocalteu reagent, gallic acid, thiobarbituric acid (≥98%), sodium citrate monobasic (BioXtra), citric acid, ninhydrin, sulfosalicylic acid, pyridine and trichloroacetic acid (ACS reagent) was purchased from Sigma Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), dimethyl sulphoxide (DMSO), phosphate buffered saline (PBS), fetal bovine serum (FBS) and 3-(4,5dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Himedia. Dialysis bags (cut off molecular weight: 7,000) was purchased from Thermo Fischer Scientific. Orthophosphoric acid (BP grade), hydrogen peroxide (ISO grade), Sulfuric acid, potassium permanganate, ethanol, sodium dihydrogen phosphate, methanol, acetone, glacial acetic acid, dichloromethane (DCM), dimethylformamide (DMF), di-sodium hydrogen phosphate, toluene and diethyl ether (ACS grade) was purchased from Merck. Transwell plate was purchased from Corning. Synthesis of Graphene Oxide (GO).Improved Hummer’s method has been adopted for the synthesis of graphene oxide.52 Briefly, concentrated H2SO4/H3PO4mixture in the ratio of 9:1 was added to graphite flakes and KMnO4 mixture. Then, the mixture was heated to 50 ˚C temperature


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for 12 h under stirring. Then, the reactant was poured into ice after cooling it to room temperature. After that, 30% H2O2was added to the above aliquot followed by the centrifuge and decanting of the supernatant. Then, the pellet was washed sequentially with water, HCl and ethanol followed by the coagulation with ether. After that, the pellet was dialyzed against distilled water in order to neutralize the pH. Finally, at room temperature, the material was vacuum dried overnight. Functionalization of GO. In typical synthesis, 10 mg of GO was dispersed in 95% ethanol by sonication. Then, 65 µL of ethylenediamine was added to the GO dispersion followed by the stirring for 24 h at room temperature. After that, the product was washed several times with ethanol, methanol and acetone followed by the drying under reduced pressure. This results in the formation of GOA. To activate the above synthesized amine functionalized GO (GOA) for further conjugation, as synthesized GOA was dispersed in 25 mL DCM and activated by adding 60 mg of 4-nitrophenyl chloroformate followed by dropwise addition of 41 mg of pyridine at 0 ˚C. After that, the reaction was carried out at room temperature for 12 h under nitrogen environment (for brevity this will be represented as acti-GOA). The activated acti-GOA was precipitated by adding chilled diethyl ether followed by drying overnight under reduced pressure. To functionalize the above synthesized acti-GOA, acti-GOA was dispersed in 2 mL of DMF followed by the addition of 300 µL of hydrazine and incubated at room temperature for 12 h under nitrogen environment. The hydrazine functionalized GO (hyd-GOA) was precipitated by adding chilled diethyl ether followed by drying overnight under reduced pressure. Salicylaldehyde (SA) loading in hyd-GOA composite. Briefly, 2 mg of hyd-GOA was dispersed in 2 mL of methanol followed by the addition of 8 mg of SA. After that, the reaction was allowed to proceed at room temperature for 24 h under nitrogen environment. Then, the product was


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centrifuged and washed several times. This results in the formation of SAhyd-GOA. The supernatant was collected and concentrated to 1 mL by using rotary vacuum evaporator and unloaded SA in the supernatant was quantified with HPLC with the help of standard SA curve. The drug loading efficiency (DLE) and encapsulation efficiency (EE) were calculated as follows: DLE (w/w %) = (weight of loaded SA/weight of Nanocomposite) ×100% EE (w/w %) = (weight of loaded SA/ theoretical weight of SA) ×100% Characterization. Cary series UV-Visible spectrophotometer (Agilent Technologies) was used to measure absorption spectra. Powder X-ray diffraction (PXRD) patterns analysis was carried out by using a Cu Kα1 radiation source (λ = 1.5406 Å) at 25 mA and 40 kV with a Bruker D8 Advance diffractometer. Fourier transform infrared (FT-IR) spectrum analysis was performed by using the Cary 600 series FT-IR spectrophotometer (Agilent technologies). Transmission electron microscope (TEM) was carried out with JEOL JEM-2100 (200 kV) microscopy. Raman spectra were recorded with the Confocal Raman system (WITec focus innovations) by using the 532 nm laser. The zeta potential analysis and dynamic light scattering were carried out with a Zetasizer Nano ZSP (Malvern) instrument. Salicylaldehyde was quantified by using the high pressure liquid chromatography (HPLC). HPLC system consisted of a control unit, binary pump (Waters 1525), photodiode array detector (Waters 2998) and an autosampler (Waters 2707). The chromatographic separation was achieved by using a chromatographic column (4.6×250 mm) with Zorbax eclipse XDB-C18, 5 µm column (Agilent). A high-pressure gradient was used with the organic solvent (96% methanol/4% glacial acetic acid (v/v) and HPLC grade water as mobile phases with the following percentages of organic solvent: 0 min-5%, 15 min-47%, 30 min-95%, 32 min-5%, 35 min-5% was used. The flow rate was used 1mL/minute and salicylaldehyde was


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detected by measuring the absorbance at 253 and 325 nm wavelength. Atomic force microscope images were recorded in tapping mode by using the Atomic force microscopy (AFM) (NanoScope 9.1, Bruker Multimode 8). pH triggered release of SA. The release behavior of SA from the SAhyd-GOA was studied in different pH buffer solution at room temperature. Briefly, 1 mL sample of SAhyd-GOA having 0.5 mg of SA was placed into the dialysis bags (cut off molecular weight: 7,000), which were dialyzed separately in pH 4 and pH 7 buffer solutions. Samples were withdrawn at definite time intervals and filled by the fresh buffer. The withdrawn samples were injected into the HPLC for quantification. Further, release behavior of SA from the SAhyd-GOA was also studied in fermented (pH 3.6) and fresh banana juice (pH 5.2) at room temperature. In brief, fresh and fermented banana was separately mess up with pestle and mortar. After that, filter the banana juice followed by centrifugation at 7000 rpm for 10 minutes in order to completely pellet down. Then, the supernatant was separated and used to study the release behavior of SA from SAhydGOA. For this study, 1 mL sample of SAhyd-GOA having 1.5 mg of SA was placed into the dialysis bags, which were dialyzed separately in fermented (pH 3.6) and fresh banana juice (pH 5.2). Samples were withdrawn at definite time intervals and replaced by the fresh banana juice. The withdrawn samples were injected into the HPLC for quantification. Fruit Biochemical assay. Malondialdehyde (MDA) content was quantified by using thiobarbituric acid (TBA) reaction method.53In brief, 5 mg of SAhyd-GOA was sprayed on the banana peel. After that, 1.07 gram of banana peel was homogenized in 4 mL of 5% trichloroacetic acid followed by the centrifuge at 12,000 rpm for 20 min. Then, the supernatant was mixed with 2 mL of 0.67% of TBA followed by heating at 100 ˚C for 30 min and then immediately cooled on ice. After centrifugation at 3000 rpm for 10 min, supernatant absorbance


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was measured at 450, 532 and 600 nm by using the spectrophotometer. The concentration of MDA was quantified by using the following formula: 6.45 × (A532-A600)-0.56 × A450.The study was performed up to 25 days. The total phenolic content (TPC) was quantified by using modified Singleton and Rossi protocol.53 In brief, 5 mg of SAhyd-GOA was sprayed on the banana peel. After that, 1.07 gram of banana peel was homogenized in 4 mL of 80% ethanol for 1 minute followed by extract filtration and centrifugation at 10,000 rpm for 15 min. Then, 1 mL of supernatant and 1 mL of folin– ciocalteu reagent was added into 10 mL of 7% sodium carbonate, followed by adding distilled water to make up the volume to 25 mL. Then, the mixture was allowed to settle for 1 h followed by measuring the absorbance at 750 nm by using the spectrophotometer. TPC content was quantified by prepared the standard curve of gallic acid. The study was performed up to 25 days. The proline content was quantified by using the reported method.54For the proline content, 5 mg of SAhyd-GOA was sprayed on the banana peel. Then, 0.507 g of banana flesh was homogenized in 10 mL of 3% aqueous sulfosalicylic acid followed by extracts filtration and centrifugation for 30 min. Then, 2 mL of glacial acetic acid and 2 mL of acid ninhydrin reagent was added in 2 mL of filtrate followed by heating at 90 ˚C in a water bath for 1 h. After 1 h, the reaction was terminated by putting the mixture on ice bath. Then, the reaction mixture was extracted by adding 4 mL of toluene followed by vigorously mixing and warming to room temperature. Finally, the absorbance was measured at 520 nm by using the spectrophotometer. The concentration of proline was quantified by using the standard curve and estimated on the basis of fresh weight as given: [µmoles proline/g of fresh weight material: (µg proline/mL × mL toluene)/115.5 µg/µmole]/[(g sample)/5]. The study was performed up to 25 days.


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Antibacterial property. E. coli and Pseudomonas syringae bacteria were maintained on LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per litre). For antibacterial activity, E. coli and P. syringae were cultured on LB medium at 37 ˚C and 30 ˚C respectively for 12 h before they were diluted in fresh LB medium to reach OD600= 0.2. The antibacterial effect of SA and SAhyd-GOA was tested by determining its minimum inhibitory concentration. Different concentration of SA and SAhyd-GOA was added to the culture medium followed by incubation at 37 ˚C for E. coli and 30 ˚C for P. syringae respectively. SA and SAhyd-GOA free bacterial culture were used as a control. After that, 100 µL of the sample was drawn out and placed on LB agar plates followed by the colony counting after overnight incubation. Composite biocompatibility and recyclability. The in vitro toxicity of the free SA and SAhyd-GOA was tested in NIH3T3 fibroblast cell lines and compared. 1×104 cells/well were seeded in transwell insert multiple well plate. Firstly, 600 µL of DMEM medium with cells was added to multiple well plates followed by adding the transwell inserts and incubated 37 ˚C in 5% CO2 incubator for 24 h. After that, the culture medium was replaced with the fresh medium. Then, 100 µL of medium with SA and SAhyd-GOA at the concentration of 10 mM, 1 mM and 0.1 mM was added in transwell inserts and incubated for 24 h. After 24 h, cell viability was measured by MTT assay. Fresh medium with MTT dye was added to each well and incubated for 4 h in 5% CO2 incubator at 37 ˚C. After that, the medium was removed and DMSO was added to each well and incubated. After 10 minutes, the plate was shaken gently to solubilize formazan crystal and resultant absorbance was measured at 570 nm and cell mortality was calculated. Finally, to show the advantage of the composite over the free SA application, the recyclability of the composite was tested. For this study, initially 5 mg solution of SAhyd-GOA


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was sprayed on the banana. After incubation for 25 days at room temperature, the material was recovered from the banana peel by soft brushing and weighed. Statistical analysis. GraphPad Prism 5 software (GraphPad software Inc. CA, USA) was used for all the calculations and Student t-test (two tailed) was used to calculate significant difference at various levels. Preparation of SAhyd-GOA composite wrapper and testing its potential for the application. For the preparation of wrapper, 9mg of SAhyd-GOA composite dispersed in 10mL of DD water was vacuum filtered with 0.4 µm filter paper support. Thus prepared composite wrapper was air dried and characterized with the SEM. The physical stability were tested by bending the wrapper. To ensure that there is no physical damage, the wrappers were folded and the resultant dust was quantified with the UV-VIS spectroscopy. Taking advantage of the reduced graphene oxide to conduct electrons, the stability of the wrapper after folding was also quantified by measuring the current at different applied voltage potential. The ability of the wrapper to release the preservative in response to the pH stimuli and the antibacterial property was also tested. Finally the wrapper was also tested for its ability to preserve the fruit by monitoring the MDA, proline and total phenol content.

RESULTS AND DISCUSSION Synthesis and characterization. In this study, to reduce the preservative toxicity in fruits, graphene oxide (GO) is used as the platform to demonstrate the stimuli triggered fruit


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preservative release. The GO was synthesized by Hummer’s exfoliation method with slight modification and characterized through TEM and spectroscopy techniques viz., UV-Visible, FTIR and Raman (Figure 1).52 Further, the X-ray diffraction pattern of 002 plane of GO was also confirmed at 2θ=11.4˚ (Figure S1).

Scheme 1.Synthetic scheme of graphene oxide preservative composite (SAhyd-GOA) tethered through acid labile hydrazone link.

The GO was tethered with salicylaldehyde (SA) a plant immune modulator through hydrazone linkage after 2 step activation as given in scheme 1 (step 1st and 2nd). Hydroxyl and epoxide are the abundant functional groups in GO, hence it has been targeted rather than other groups, which are very few in number.55In the first step, the GO was allowed to react with nucleophilic ethylenediamine, so that the electrophilic epoxide ring was opened into hydroxyl and amine (for brevity this material will be represented as GOA).56In this step, fewer nontargeted carboxyl groups would get amine functionalized. Thus, ethylenediamine conjugation has not only prepared the material for the next step but also significantly increase the functional groups. In the second step, both the hydroxyl (from epoxide opening and in native GO) and


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amine groups on the GOA were activated with multipurpose coupler viz.,4-nitrophenyl chloroformate to carbonates and carbamates respectively (acti-GOA).57,58These groups were compatible to form hydrazine derivative on a reaction with hydrazine (hyd-GOA). In addition, the C=O may also react to form hydrazone,59 however since C=O distribution have been less, this will not be discussed further. Finally, hyd-GOA was reacted with SA to form final hydrazone assisted SA tethered GO (SAhyd-GOA).60The whole process was followed with UV-Vis, FTIR and Raman spectroscopy (Figure 1B-D). Here, the hydrazone linkage is selected, because it has fastest hydrolysis rate and is more sensitive to the stimuli as compared to other acid cleavable linkages.61,62 The loading percent was quantified to be ~180% with the HPLC. Considering 25% oxygen in GO, with 60% of this distributed as hydroxyl and epoxy groups at 2/3 ratio will have 0.9 X 1023 sites in 1 gram GO63. The loading percentage shows slightly >5% of the groups were unoccupied, which may be due to the steric hindrance. As prepared aqueous GO dispersion showed the typical absorbance at 230 nm wavelength.64 This peak is red shifted in GOA, with the amine binding in GO, which has been observed before.65 In the final composite, the red shift is accompanied with a prominent SA peak at 325 nm. As synthesized GO confirmed the functional groups with a broad peak at 3000-3500 and a narrow peak at 1400 cm-1corresponding to –OH stretching vibrations, 1725 cm-1 corresponding to C=O (carbonyl and carboxyl) stretching vibrations and 1225 cm-1 corresponding to epoxy C-O stretching vibrations.62,66-74 With the addition of ethylenediamine, the signals from above GO functional groups disappeared or the intensity decreased, especially for –OH and C-O groups and an intense new peak at 1580 cm-1 appeared corresponding to N-H in-plane stretching vibration.61While following the steps, especially the activation with the 4nitrophenyl chloroformate, there is a significant enhancement in the carbonyl stretching vibration


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at 1730 cm-1,75 and appearance of the aromatic-NO2 band at 1530 cm-1 (Figure S2).76 This conjugation also stabilize the GO sheet from undesired reduction by the addition of hydrazine in the next step. Finally, with the addition of SA, a strong peak at 1620 cm-1appeared,65 which confirms the hydrazone (-C=N-) link between the functional groups in the GO, which is not observed in any of the previous synthesis stages.66,77-79 The characteristic 1580 cm-1peak of the SA is also found prominent in the composite.80 Raman spectra of the GO and the composite shows the typical D and G band at 1365 and 1577 cm-1 wavenumber respectively.81-86 The G band of GO shows a shift in the band maxima towards higher wavenumber, from 1577 cm-1 to 1581 cm-1 and 1585 cm-1 with the amine and final conjugation respectively.87 Further, the ratio of D/G intensity increased after the functionalization, which confirms the association with nitrogen by forming C-N bond, which causes distortion.88-91 This increase in the intensity of D band could also be due to the prevention of agglomeration by the functional groups and by undesired fragmentation of the GO sheet through reduction, which was known to increases the edges.89,92,93In the Raman spectra beautiful 2D peaks are reported based on the number of layers, 94

but in our material this is not so pronounced. The thickness of the GO sheet before and after functionalization [GO (SAhyd-GOA)], viz.,

was measured with the AMF (Figure 2 A,B). The height profile of the naked GO with the average thickness of 2 nm confirmed 5-6 layer.95 In case of SAhyd-GOA, the thickness is found to increase by more than 2-3 folds,96-98 which confirms the huge amount of SA loading on the GO. Similar trend in increase of the lateral height of the GO with the organic loading was reported before.99


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Figure 1. Characterization of GO, GOA, acti-GOA, hyd-GOA and SAhyd-GOA composite. (A) TEM image of GO showing thin layers of sheet. (B) UV absorbance spectra of GO, GOA, SA and SAhyd-GOA. (C) FTIR spectra of GO, GOA, SA and SAhyd-GOA. (D) Raman spectrum of GO, GOA, SA and SAhyd-GOA showing the D and G band at 1343 and 1600 cm-1, respectively.


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Figure 2. Characterization of GO tethered with SA through hydrazone link (SAhydGOA composite) at different stages of synthesis. (A and B) AFM image with the height profile of GO and SAhyd-GOA composite. (C) Hydrodynamic size of GO, GOA, acti-GOA, hyd-GOA, SA and SAhyd-GOA composite. (D) Zeta potential of GO, GOA, acti-GOA, hyd-GOA and SAhydGOA composite.

The hydrodynamic radius at each stage of synthesis from GO to the final composite SAhyd-GOA were measured with dynamic light scattering (Figure 2C). The size of the naked GO was found to be 300 nm, which supports the AMF observation. With the subsequent conjugation of GO with ethylenediamine (GOA), 4-nitrophenyl chloroformate (acti-GOA), hydrazine (hyd-GOA) and final SA (SAhyd-GOA), the size increased sequentially to 380, 469, 561 and 1150 nm respectively. This could be due to the organic layer crowding. Simultaneously, the surface charge


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of the particles at every stage of SAhyd-GOA composite synthesis was measured (Figure 2D). The GO synthesized by the hummer method was holding -31 mV100-102that turned into +15.5 mV with the ethylenediamine conjugation (GOA), which may be due to the free amine groups. In the next step of activation with 4-nitrophenyl chloroformate (acti-GOA), the surface charge again returned to -15.9 mV, which may be due to the non-availability of the free amine. In the final two reactions with hydrazine followed by SA, there were no more significant changes in the surface charge, probably due to the neutral behavior. pH triggered release of SA. Fruits especially the starchy fruits like the banana have been known to increase the acid contents 3 to 4 times in the transition from the pre-climacteric to post climacteric period.38Hence, the release of SA from the composite with response to pH change was quantified with HPLC (Figure 3A). The composite SAhyd-GOA incubated for 50 h in pH 4 buffer showed ~50% SA release, whereas in the neutral pH only 15% of the SA was released. Further, the release behavior of the SA from SAhyd-GOA was tested in fermented banana juice collected from post climacteric fruit exudation having pH 3.2. In 5 h of incubation, the composite showed 11.5% SA release (the quality of the fermented juice goes vary bad, hence the release after 5 h was not followed), whereas in the same incubation time with fresh juice

Ecofriendly fruit-switches based on graphene oxide for programed fruit

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