Humanin: A novel functional molecule for the green synthesis of graphene

doi:10.1016/j.colsurfb.2013.06.018

Colloids and Surfaces B: Biointerfaces

Volume 111, 1 November 2013, Pages 376-383

https://doi.org/10.1016/j.colsurfb.2013.06.018 Get rights and content

Highlights

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Graphene was synthesized via one-step reduction method from graphene oxide under mild condition in aqueous solution using mitochondrial peptide.
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The method is an environmentally friendly, simple and rapid.
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The prepared graphene is highly soluble in water.
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The peptide mediated synthesis of graphene could provide potential applications in various biological and biomedical fields.

Abstract

The synthesis of graphene nanosheets from graphene oxide is an interesting area of nanobiotechnology because graphene-based nanomaterials have potential applications in the biomedical field. In this study, we developed a green, rapid, and simple method for the synthesis of graphene from graphene oxide, which uses the mitochondrial polypeptide humanin as a reducing agent. Graphene was prepared via one-step reduction of graphene oxide under mild conditions in an aqueous solution, and the resulting substance was characterized using a range of analytical procedures. UV–vis absorption spectroscopy confirmed the reduction of graphene oxide to graphene. Fourier transform infrared spectroscopy was used to study the changes in the surface functionalities, and X-ray diffraction was used to investigate the crystal structure of graphene. High resolution scanning electron microscopy and atomic force microscopy were also employed to investigate the morphologies of the synthesized grapheme, and Raman spectroscopy was used to evaluate its single- and multi-layer properties. The results described here suggest that the potent reducing agent humanin may be used as a substitute for hydrazine during graphene synthesis, thereby providing a safe, biocompatible and green method for the efficient deoxygenation of graphene oxide that can be used for large-scale production and biomedical applications.

Graphical abstract

Introduction

Recently, carbon-based nanomaterials have opened up new avenues for the development of novel functional materials. Because of its significant high electrical conductivity, mechanical flexibility, and chemical stability, graphene is an emerging material of great interest [1], [2], [3], [4], [5]. The various unique nanostructures of graphene have promising potential applications for multiple technologies, including chemical sensors [6], [7], catalyst support [8], [9], Li ion batteries [10], [11], biosensor development [12], [13], imaging [14], drug delivery [15], [16], bacterial inhibition [17], [18], [19], and photothermal therapy [20], [21]. Several groups have proposed environmentally friendly approaches to the synthesis of graphene, including flash photo reduction [22], hydrothermal dehydration [23], solvo thermal reduction [24], catalytic [25], photocatalytic [26], [27], [28], and photodegradation [29] methods. The most promising method for the large-scale production of graphene is a multi-step process that involves the chemical oxidation of graphite (Gt) to Gt oxide (GtO), followed by conversion to graphene oxide (GO) and then subsequent reduction of GO to graphene using a chemical reducing agent [30], [31].

The chemical reduction of GO by a reducing agent is one of the most versatile, economical, easy, and scaleable methods for the production of graphene. However, the use of chemical methods leads to limited solubility or irreversible agglomeration during preparation in water and most organic solvents [32]. Currently, this obstacle can be overcome by chemical functionalization of GO with organic molecules or polymers, followed by their chemical reduction using hydrazine or its derivatives [33], [34]. Strong and toxic reducing agents and surfactants are essential to complete the reduction of GO in the aqueous phase [35]; however, hydrazine and its derivatives are highly toxic and explosive. To solve this problem, many studies have attempted to develop novel aqueous and environmentally friendly reduction procedures using bacterial respiration [36], polyallylamine [37], potassium hydroxide [38], polyvinylpyrrolidone [39], ascorbic acid [40], Baker's yeast [41], or proteins [13]. Fernandez-Merino et al. [42] synthesized graphene by using vitamin C as a reducing agent and the resulting products were highly stable in water and some common organic solvents, including dimethylformamide and N-methylpyrrolidinone. Several groups have attempted to use biological molecules, such as melatonin [43] or amino acids [44], as reducing agents. Shewanella [45], Escherichia coli [46], and Pseudomonas aeruginosa [47] have also been used to convert GO to graphene. Pham et al. [32] developed a simple approach to the production of graphene that involves the chemical reduction of GO by l-glutathione. However, the identification of additional novel biomolecules is essential to the development of a green method for the production of graphene.

Here, we developed a simple, quick, and environmentally friendly approach to graphene synthesis in an aqueous solution under mild conditions that uses humanin (HN) as a reducing agent. Humanin, a 24 amino acid mitochondrial polypeptide chain (Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala) with a molecular weight of 2656.3 Da, was first identified from a cDNA library of surviving neurons in the human Alzheimer's disease brain [48]. Since its initial discovery, several cDNAs with sequence homology to HN have been identified in plants, nematodes, and rodents, suggesting that the protein is evolutionarily conserved [49]. HN also plays a role in preventing cell death in tissues other than the nervous system [50].

Section snippets

Materials

HN was purchased from Peptide Institute Inc., Japan. Gt powder, NaOH, KMnO₄, anhydrous ethanol, 98% H₂SO₄, 36% HCl, and 30% hydrogen peroxide (H₂O₂) aqueous solution were purchased from Sigma–Aldrich (St Louis, MO, USA) and were used directly without further purification. All aqueous solutions were prepared using deionized water. Unless otherwise stated, all other chemicals were also purchased from Sigma–Aldrich.

Synthesis of GO

The aqueous dispersion of GO sheets was prepared as described previously [51].

Characterization of GO and HN-rGO

A distinctive color change from pale-yellow to black after reduction of GO indicates the successful synthesis of rGO [42]. GO was reduced by HN as described in the Materials and Methods section, and then UV–vis absorption spectroscopy was used to confirm the reduction of the oxygen-containing groups. The absorption peak of the GO dispersion was located at a wavelength of 227 nm and had a shoulder peak at approximately 300 nm (Fig. 1), which is consistent with a previous report [28]. After

Conclusion

This study describes the use of the mitochondrial protein HN as a green and simple reduction method for the large-scale synthesis of water-soluble graphene. The transition of GO to graphene was confirmed by various analytical techniques. Structural and morphological studies demonstrated that some of the oxygen functionalities in GO were removed by this method, and that HN can be functionalized on the resulting rGO sheets. Moreover, the highly soluble graphene sheets were obtained relatively

Acknowledgements

This paper was supported by the SMART-Research Professor Program of Konkuk University. Dr. Sangiliyandi Gurunathan was supported by Konkuk University SMART-Full time Professorship. This work was supported by BioGreen 21 program of the RDA (Grant No. PJ009625), ARPC (Grant No. 111047-5), Republic of Korea.

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The brown color of the GO dispersion changed to black as it went through the reduction procedure. Characterization was carried out using an X-ray diffractometer, the RigakuMiniflex II-C, which was purchased by the researchers (Gurunathan et al., 2013b, 2013c). Using scintillation counters (=1.5406 A), high-resolution XRD patterns from a 3kw source with a Cu target were obtained and analyzed in this study.
The larvicidalproperty of graphene oxide (GO) and thiourea-reduced graphene oxide (T-rGO)was assessed against Culexquinquefasciatuslarvae. A simple water-soluble material synthesis method was used. The transformation of graphene into graphene oxide was accomplished in a single step. Under mild conditions, grapheneoxidewasdissolved in water to form a solution. Structure, optical, and microstructural features of the synthesized samples wereevaluatedusing a variety of analytical tools to compare the samples. Both GO and RGO, as well as GO, showed strong larvicidal potential when used against the third instar larvae of the Culexquinquefasciatus mosquito, with LC ₅₀ and LC ₉₀ values of 1.71 and 5.17 ppm and 1.89 and 5.00 ppm, respectively.
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As per reports, plant and leaf extracts can be used as an alternative for reduced graphene oxide production [46]. Various biomolecules like ascorbic acid [49], amino acids [50], glucose [51], bovine serum albumin [52], melatonin [53], humanin [54], enhanced green fluorescent protein [55], and resveratrol, from grapes [26] are used for the synthesis of reduced graphene oxide. Functional groups of reduced graphene oxides interact with DNA, proteins, peptides, and enzymes and, therefore, can be easily chemically modified [56].
Nanomaterials have brought a paradigm shift in the field of science and technology leading to the development of futuristic materials with value addition. Carbon-based nanomaterials specifically graphene-integrated nanostructures have encouraged the field of bioengineering and medicine tangentially with greater insights. The applications are ramified, from disease detection, diagnosis, and treatment further leading to the repurposing of therapy which is increasing with time. Synthesis of graphene with newer strategies with fewer complications has added interest among the scientific fraternity. In this review, we attempt to explore and comprehend the recent advances in the synthesis and encapsulation of graphene-based nanomaterials. Further, the emphasis is laid on the implications of graphene-based nanomaterials for their applications in biomedicine with a prime focus on drug delivery systems. The major therapeutic application of graphene in various diseases like cancer, neuro-regeneration, anti-viral (Ebola and Zika) are described. Future directions, prospects, and limitations are highlighted to strengthen research for elucidating newer inputs and thrusts in the design and development of graphene-derived smart therapeutics which will lead to personalized therapy with better healthcare driving towards the pinnacle of human welfare.
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In the present study, the green approach has been adopted for the reduction of graphene oxide (GO) using cow urine and urea, which were further spray-coated to fabricate graphene nanosheets (GNs) based transparent conducting films (TCFs). Raman analysis reveals the I_D/I_G intensity ratio of ~1.25, 1.39 for synthesized GNs by cow urine and urea, respectively. It indicates the removal of oxygen spices from GO, which is further confirmed by elemental analysis and FTIR. XRD, Raman and Urbach energy confirm the enhanced defects and lattice disorders, which are attributed to nitrogen incorporation, vacancies, and other structural defects. The presence of pyridinic-N defects as revealed by XPS and other structural defects has changed the electrical and optical properties of GNs. A detailed comparative study has been done to compare the electrical and optical properties of spray-coated TCFs over the quartz substrate. The enhanced conductivity in CGF (coated film using GN synthesized by cow urine) and UGF (coated film using GN synthesized by urea) might be due to presence of pyridinic-N and oxygen species removal, while enhanced transmissivity is due to presence of less number of graphene layers. UGF, which is thermally annealed at 900 °C shows the best performance with sheet resistance ~1.78 kΩ/□ and transmittance ~71.16%.
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Humanin, a 24 amino acid mitochondrial polypeptide chain (Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala) with a molecular weight of 2656.3 Da, was first identified from a cDNA library of surviving neurons in the human Alzheimer's disease brain [48]. Gurunathan et al. [363] have used humanin as an agent for GO reduction. The good water dispersibility of the resulting rGO is reflected by the lower amount of protein dispersant residue found on its surfaces.
Graphene, reduced graphene oxide (rGO) and derived materials have emerged as promising solutions for applications in renewable energy storage/conversion devices. No alternatives are known to simultaneously exhibit large specific surface area, high electrical conductivity, good chemical stability, high mechanical strength and flexibility. This review article is a collection of some of the most relevant research efforts published in the last few years focusing on the synthesis and modification of graphene/rGO as well as doped and hybrid bi-dimensional carbon materials. For research on graphene growth, the choice of precursor and physical state (gas, solid or liquid) has been proved to be as important as the environment and synthesis approach. On the other hand, research focused on graphene oxide reduction has relied on the development of simple and efficient techniques for rGO conversion and device structuring. Modifications applied to graphene (during synthesis) or rGO (during reduction) have included doping, decoration with nanoparticles and the formation of composite microstructures. Fabrication of electrodes based on graphene/rGO for application in energy storage and conversion has been reported, including relevant performance data from real devices (supercapacitors, lithium ion batteries, fuel cells or solar cells). This review concludes with a brief discussion of some of the possible directions for promising research in the area of graphene/rGO fabrication for energy conversion and storage devices.
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A number of biomolecules, reducing sugars, phytoextracts, bacterial and fungal biomass have been used as reducing agents or stabilizers. However, the reduction efficiency of many of these bio-friendly reagents is not comparable with stronger reagents like hydrazine [42,46–57]. In addition, the insufficient reducing and stabilizing ability of many of these green reducers results in aggregation, diminishing the properties of graphene derivatives.
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Humanin: A novel functional molecule for the green synthesis of graphene

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Synthesis of GO

Characterization of GO and HN-rGO

Conclusion

Acknowledgements

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