Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Covalent and Non-covalent Conjugation of Few-Layered Graphene Oxide and Ruthenium(II) Complex Hybrids and Their Energy Transfer Modulation via Enzymatic Hydrolysis Frankie Chi-Ming Leung and Vivian Wing-Wah Yam* Institute of Molecular Functional Materials (Areas of Excellence Scheme, University Grants Committee (Hong Kong)) and Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, P. R. China S Supporting Information *
ABSTRACT: Pyrene-containing ruthenium(II) tris-bipyridine complexes have been prepared. These complexes have been noncovalently attached onto the few-layered graphene oxide (GO) sheets through their high binding affinity for flat π-surfaces. Alternatively, the reduced graphene oxide (rGO) sheets have also been covalently functionalized with the ruthenium(II) trisbipyridine complex. The prepared conjugates have been characterized by transmission electron microscopy (TEM), energydispersive X-ray analysis (EDX), X-ray diffraction (XRD), atomic force microscopy (AFM), Raman spectroscopy, thermogravimetric analysis (TGA), and UV−visible absorption spectroscopy. The energy transfer properties of the resulted conjugates between the graphene and transition metal complexes have been studied via esterase hydrolysis. The energy transfer efficiencies were found to vary with the separation between the donor and the acceptor units. KEYWORDS: graphene oxide, reduced graphene oxide, ruthenium(II), FRET, pyrene, esterase
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INTRODUCTION
applications in biological systems. Moreover, the functional groups on rGO, such as the carboxy groups (−COOH),11 are highly versatile platforms for further covalent functionalization, in terms of 1,3-dipolar cycloaddition by sarcosine and 4carboxybenzaldehyde, followed by esterification.12 Amongst the current developments of graphene-based Förster resonance energy transfer (FRET) sensors, graphene has commonly been used to act as a quencher.13 Because of the large surface area of the π-conjugated system, graphene can act as a widely-adaptable energy acceptor for most of the organic dyes or quantum dots (QDs) for fluorescence quenching. When compared with the conventional organic quenchers, graphene has a higher quenching efficiency, resulting in the salient features of low background noise and high signal-tonoise ratio.14 The broad absorption spectrum of graphene can effectively quench the emission of most fluorophores. Thus,
Graphene is an allotrope of carbon which is a single layer of graphite. Geim and Novoselov successfully isolated singlelayered graphene sheets from graphite flakes by mechanical methods.1,2 The peculiar properties of graphene, including large theoretical specific surface area and excellent quenching ability,3,4 have aroused much interest in the exploration of graphene oxide (GO) functional materials.5−7 It has largely been employed in the field of cell labeling and real-time monitoring.8,9 Graphene usually has a large binding affinity for aromatic compounds. The higher the number of six-membered rings of the aromatic compounds obtained, the stronger would the interaction with the graphene sheets be. Therefore, graphene could be functionalized through non-covalent interactions with compounds that contain the aromatic moiety such as pyrene.10 GO is an important derivative of graphene, which is the precursor for the preparation of reduced graphene oxide (rGO) by the reduction method. There are a variety of oxygen-containing groups present at the edge and surface of GO, resulting in a good water solubility which can assist © XXXX American Chemical Society
Received: December 7, 2017 Accepted: April 9, 2018
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DOI: 10.1021/acsami.7b18663 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
the respective EI and ESI mass spectroscopy. Table 1 summarizes the photophysical data of complexes 1−5. Complex 1 in buffer solution (10 mM tris-HCl, pH 8.0) shows intense high-energy absorption bands with molar extinction coefficients in the order of 104 dm3 mol−1 cm−1 at ca. 240−350 nm. According to the electronic absorption spectrum of the free ligand pyren-1-ylmethanol, the vibronicstructured absorptions at ca. 242 nm are assigned as intraligand (IL) [π → π*] transitions of the pyrene moieties. The relatively broad absorption at 288 nm is assigned to the IL [π → π*] transition of bipyridine. The lower energy absorption bands at ca. 400−500 nm with molar extinction coefficients of ∼103 dm3 mol−1 cm−1 are ascribed to the MLCT[dπ(Ru) → π*(bpy)] transitions, characteristic of the ruthenium(II) polypyridine complex systems.25−28 The electronic absorption spectra of complexes 2−5 are similar to that of complex 1 except for the differences in molar extinction coefficients. A control complex other than the ruthenium(II) bipyridine system that contains a different pyrene-containing bipyridine ligand, the rhenium(I) complex 6, was obtained according to the literature method.19,20 The synthesis and characterization of complex 6 are described in the Supporting Information. Preparation of the Non-covalent and Covalent Conjugates. The few-layered GO were obtained from graphite flakes by the top-down approach.29 The graphite flakes were initially pre-oxidized by the treatment with concentrated sulfuric acid, potassium persulfate, and phosphorus pentoxide. The subsequent oxidation by Hummers method with concentrated sulfuric acid,30 potassium permanganate and hydrogen peroxide gave the yellowish brown aqueous dispersion of GO. The aqueous dispersions of ruthenium(II) and rhenium(I) complexes non-covalently functionalized GO (1-GO, 2-GO, 3-GO, 4-GO, 5-GO, and 6-GO) were obtained by mixing the corresponding complexes 1−6 with GO overnight and purified by several centrifugation−redispersion cycles. For the covalent functionalization of the conjugates, the rGO was prepared from the mild reduction of GO by the treatment of hydrazine.31 To prepare a well-dispersed aqueous buffer solution of rGO, the surface of the graphene layers was treated with trimethyl-1-(pyren-1-yl)-2,5,8,11-tetraoxatridecan13-ammonium bromide (pyr-NMe3+), a positive chargecarrying pyrene to prevent the aggregation of graphene layers and to increase the water solubility.32,33 The obtained rGO were well-dispersed in N-methylpyrrolidone (NMP). The ruthenium(II) complex covalently functionalized few-layered graphene sheets (Ru-rGO) were obtained from 1,3-dipolar cycloaddition of rGO with sarcosine and 4-carboxybenzaldehyde in NMP at high temperature, followed by the esterification with [Ru(bpy)2(bpy-CH2OH)]Cl2 in the presence of EDC·HCl and hydroxybenzotriazole (HOBt) in DMF. Scheme 1 shows the preparation procedures for both noncovalent and covalent functionalization of few-layered graphene sheets with ruthenium(II) complexes. Characterization of the Non-covalent and Covalent Conjugates. 1-GO has been selected as the illustration for the characterization of the non-covalent conjugates of the Ru complexes. The dispersions of the graphene samples were deposited on a lacey carbon grid for performing the transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) analysis. Figures 2a−d shows the TEM images of GO, rGO, 1-GO, and Ru-rGO, respectively. GO shows a relatively transparent image among the samples, confirming the identity of the few-layered GO and its few-atom
graphene has been used in the development of optical probes. For instant, rGO has been used in the design of Hg2+ luminescence turn-on sensor by the recovery of the acridine orange dye emission.15 Besides metal cation sensing, watersoluble GOs are also compatible for use in the detection of biomolecules such as protein,16 adenosine triphosphate (ATP),14 and single-strand DNAs (ssDNAs) in aqueous medium.17 An advantage of these kinds of probes over the conventional molecular beacon is the absence of complicated synthetic steps. More importantly, the quenching brought about by graphene sheets is more efficient, resulting in higher sensitivity.18 Nevertheless, the use of graphene in the development of nanomaterials and sensors is rather limited when compared to gold nanoparticles (GNPs) and QDs and has attracted much attention. Recent works by us have demonstrated the determination of enzymatic hydrolysis kinetics of esterase and the application of HepG2 cell imaging by using the ruthenium(II) complex system and GNPs based on the FRET mechanism.19−21 However, despite ratiometric fluorescence sensors based on FRET being developed for sensing of protease,22−24 the corresponding application of graphene-based nano-materials for protease sensing has not been as well developed and studied as GNPs. Extension of the work to explore the utilization of the conjugates for the “proof-of-principle” development of potential biosensing probes for esterase using graphene as an energy acceptor as well as the conjugation of transition-metal complexes to graphene sheets would be made. In this work, both covalent and non-covalent functionalizations of graphene sheets with water-soluble luminescent ruthenium(II) complexes have been explored and studied. The effect of the donor and the acceptor separation along the spacers on the FRET efficiencies has also been investigated.
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RESULTS AND DISCUSSION Synthesis and Photophysical Studies of Complexes. The chemical structures of ruthenium(II) complexes are shown in Figure 1. The pyrene-containing bipyridine ligands with
Figure 1. Chemical structures of complexes 1−6.
different oligoether linkages were obtained from the esterification of the corresponding oligoether-containing pyrene with 4′-methyl-2,2′-bipyridine-4-carboxylic acid in the presence of ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 4-(dimethylamino)pyridine (DMAP) in dichloromethane. The ruthenium(II) complexes 1−5 were synthesized by the reactions of the respective ligands with cis[Ru(bpy)2Cl2] in ethanol. The identities of the ligands and the metal complexes were confirmed by satisfactory 1H NMR spectroscopy, infrared spectroscopy, elemental analyses, and B
DOI: 10.1021/acsami.7b18663 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces Table 1. Photophysical Data of Complexes 1−5 emission complex 1
2
3
4
5
absorptiona λabs/nm (ε/dm3 mol−1 cm−1)
medium (T/K)
242 (49500), 276 (38465), 288 (29275), 325 (19780), 340 (26830), 424 (4880), 455 (6030)
242 (50320), 276 (39120), 287 (30340), 325 (20140), 340 (27015), 425 (4950), 455 (6190)
242 (52035), 276 (40555), 288 (31435), 325 (20445), 341 (27115), 425 (5110), 456 (6240)
242 (53585), 276 (41225), 287 (32965), 325 (20960), 340 (27080), 426 (5480), 457 (6415)
242 (55060), 276 (42635), 288 (34285), 325 (21480), 341 (27230), 426 (5700), 458 (6580)
a
buffer (298) solid (298) solid (77) glass (77)b buffer (298)a solid (298) solid (77) glass (77)b buffer (298)a solid (298) solid (77) glass (77)b buffer (298)a solid (298) solid (77) glass (77)b buffer (298)a solid (298) solid (77) glass (77)b
λem/nm (τ0/μs) 642 645 616 609 644 648 618 608 644 650 619 608 645 655 623 608 645 653 624 610
(1.38) (
