Graphene / Graphene Oxide Based Pyrimidine Hybrid Materials for Metal Ion Sensing and DFT Study

Chemical and biological sensors are gaining wide popularity in day-to-day life and significantly help to increase the survivability by providing early warning for explosives, metal pollutant, and chemical warfare. GR analog based sensor devices have several advantages for chemical and biological sensing. The structural or chemical modifications of GR remarkably improve the properties of such device applications. Keeping this in mind, we have designed and synthesized pyrimidinedione-functionalized graphene oxide (FGO) and functionalized graphene (FG) sequentially. Synthesis of the hybrid materials was done using the simple hydrothermal method. The materials were characterized by various spectroscopic and analytical techniques. XRD study showed formation of well exfoliated GO sheets in the composite. FTIR data indicates the formation of GO-NO-Ur composites. Density functional theory (DFT) calculation was also investigated to understand the various non-covalent interactions of the NO-Ur and FGO. For the detection of metal ions, synthesized nanocomposite was analyzed to sense many metal ions (Ag, Cd, Cu, Fe, Hg, Mo, Ni, and Zn) and we observed strong binding mood against Fe ions having LOD and LOQ value of 0.0032 μM and 0.01 μM respectively.


Introduction
Metals play an important role in the biological system as well as in ecosystems [1].However, they have an adverse effect when concentration increases more than the permissible limit.Longterm exposure to heavy metals affects plants, as it may accumulate in plants and causes potential health risk towards living organism and food safety issues by the bio-magnification process [2].Metals also accumulate in the tissues of Epinephelus microdon, Cyprinus carpio fishes [3,4].Cereals, vegetables, and certain seafood can intake heavy metals.Its consumption can be harmful to human and other living beings.In marine animals concentration of heavy metals can be double or triple compared with terrestrial foods [5].Heavy metals have become a major issue for living being on earth, causing many of the diseases for example; 'Hg' causes Minamata, 'As' is carcinogenic in nature, 'Cd' damages kidney, etc [6,7].Heavy metals such as arsenic, mercury, cadmium, chromium, and lead; originated by various human activities such as use of pesticides, automobile emission, burning of coal and microelectronics, etc. causes water and soil pollution [8,9].
Graphene/Graphene oxide (GR/GO) is two dimensional (2D) crystals and nanomaterial systems, where a thick sheet of sp 2 -bonded carbon atoms are arranged in honeycomb hexagonal lattice [10,11,12].It is gaining popularity due to its unique physicochemical properties, such as a high surface area (theoretically, 2630 m 2 g -1 for single-layer graphene) [13], excellent electrical and thermal conductivity [14] (in-plane thermal conductivity 5300 Wm -1 K -1 at room temperature), a high mechanical strength [15], superior biocompatibility [16], and optical properties (e.g.high opacity, able to quench fluorescence).GR-based materials have shown a wide range of applications including biomedical imaging, video imaging, pH monitoring, gas sensing, chemical sensing and energy storage devices, etc. [17,18].
Recent developments in nanotechnology have further increased the interest of researchers to develop materials which adsorb and provide innovative systems to improve environmental remediation as well as metals ion sensing.[26].GR-silver nanocomposite (GR-Ag NPs), [27] and 1-Butyl-3-methylimidazolium tetrafluoroborate (BmimBF4, ionic liquid) functionalized GO (GO-IL) [28] have developed for the detection of Hg 2+ .Amino pyrene (AP) was grafted onto gold nanoparticles (AuNP) to form AP-AuNPs composite.The synthesized AP-AuNP composites were mixed with GO and quenched the fluorescent properties by GO.In the presence of Pb 2+ and sodium thiosulphate, AP-AuNP started leaching, and fluorescence switched on.This method or sensor applied to detect the Pb 2+ in aqueous solution as well as environmental water samples [29].
GR/GO-based heterocyclic molecule composites materials also gained wide popularity as a sensor for the detection of metal ions.Mao et al. have reported GR/GO-based bipyrene composites for detection of Mn 2+ by using the fluorescent technique.GO-based bipyrene composite was synthesized and then reduced the composites by a chemical method using hydrazine.The RGO then applied to sense various metal ions (Mn 2+ , Zn 2+ , Cr 2+ , Ca 2+ , Ni 2+ ) and Mn 2+ accelerated the fluorescence of composite or switched on the fluorescence of composite materials.All the experiments were done in an aqueous medium, and Mn 2+ was detected not only in aqueous medium but also in a cell.This sensor may be widely used for detection of Mn 2+ in the biological system as well as for environmental sensor [30].Crown[n]ether has been used with GO to sense alkali metal ions.For e.g.aza-9-crown-3 ether functionalized GO was capable to bind Li + , Na + and K + cations [31,32], 1-aza-15-crown [5]ether functionalized GO was used to determine Li + [33] , and 18 crown [6]ether-based GO hybrid material was found to detect alkali metal ion (K + ) [34].
Pyrimidine is a heterocyclic compound, a class of nitrogenous base, including uracil, cytosine and thymine.Pyrimidine has gained wide popularity due to its versatile action mechanism like anticancer agent [35], antiviral, antimicrobial [36] and medicinal properties [37].Many research articles are available based on pyrimidine derivatives as a metal ions sensing agents [38,39,40].
Recently we have reported that different functional groups in pyrimidines or its derivatives also provide a large variety of bonding behavior, while the presence of several hydrogen bond donor and acceptor sites together with a pyrimidine ring may favor diverse non-covalent interactions, thus ensuring a rich metallosupramolecular/coordination chemistry of such compounds [41,42,43,44].So pyrimidine derivatives also act as very good ligating properties and bind metal ions easily.It is found that organic compound functionalized GR/GO nanocomposite materials have capacity to sense the metal ions (Hg 2+ , Cd 2+ , Cu 2+ etc.) by quenching or enhancing the fluorescence activity [45].Only few literatures are found on GR/GO-based pyrimidine composites and which are limited to detect only Hg 2+ [46,47,48].Therefore functionalization of pyrimidine derivatives with GR/GO can be excellent materials for various metal ion detection due to their combination of properties.In the present work we have focused on the synthesis of pyrimidine functionalized GR/GO nanocomposite materials using hydrothermal method and its application toward metal ions sensing.

Materials and methods
The GO and nitrosouracil (NO-Ur) were synthesized in our lab as described in 2.1 and 2.2 and samples were characterized using FTIR (Shimadzu, IR-Affinity-1S), by making the KBr pellet of sample, within the range 4000-400 cm -1 .The compounds were characterized also by using UVvis spectrophotometry (Thermo, Evolution 300) in the wavelength range of 200-600 nm and seen the metal sensing properties of compound.The composite material was dispersed in distilled water (Millipore) 0.04 mg/ml.After preparing the solution of GO, the metal ion of 10 -3 M concentration were added separately, according to required concentration of solution.Synthesized composites materials was examined by X-ray diffraction (XRD) with Cu K radiation (= 1.54 °A).Characterization was done in control condition (temperature maintained 25 C) so that error can be minimized due to environmental factors.

Synthesis of GO
GO was synthesized by modified Hummer's method from naturally occurring graphite powder (purchased) using KMnO4 and H2SO4 as oxidizing agents [49].

Theoretical method
The energies of pyrimidine derivatives and its corresponding composites included in this study were computed at the BP86-D3/def2-TZVP level of theory.The geometries have been fully optimized unless otherwise noted.For instance to evaluate the non-covalent interactions observed in the solid state.The calculations have been performed by using the program TURBOMOLE version 6.5 [50].For the calculations we have used the BP86 functional with the latest available correction for dispersion (D3) [51].For NO-Ur, only the major component of the disordered isonitroso group was considered for DFT calculations.

Results and discussion
In this work, GO-nitrosouracil (GO-NO-Ur) composite material was synthesized for the detection of metal ions such as Cd 2+ , Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Mo 2+ , Ni 2+ , and Zn 2+ .First GO was prepared by modified Hummer's method and then functionalization of GO with NO-Ur was done by a simple stirring process as shown below (Scheme 1).The reaction between GO, and 1,3-dimethyl-6amino-5-nitrosouracil (NO-Ur) was carried out in alkali medium in the presence of NaOH.The reaction was completed in 24 hrs and monitored by the change in color of GO from yellowish brown to dark brown due to partial deoxygenation of GO.The bonding between GO and NO-Ur due to amidation reaction between free COOH of GO and NH2 of NO-Ur [49].After completion of the reaction, the precipitate was centrifuged, followed by washed in water/ethanol mixture and dried at 60±5ºC in a hot air oven.In the XRD spectra of GO, a characteristic peak appeared at two theta = 11 o .This peak disappears in the XRD spectra of GO-NO-Ur composite which suggests exfoliation of GO as well as a good interaction between the GO and NO-Ur (Figure 1).Thus the XRD study reveals well exfoliated GO sheets in the composite.In the FTIR spectra of NO-Ur, a band appeared at 3024 cm -1 for -OH group.Peak for -NH2 and C=O group is observed at 3545.16 cm -1 and 1715 cm -1 respectively.Aromatic C=C band peak shows at 1600 and 1475 cm -1 .Peak for N=O stretch and CH3 band appeared at 1577 cm -1 and 1450 cm -1 respectively.In case of GO the spectral reflect the confirmation of synthesis, the spectral band at 3205.69 cm -1 for -OH peak of carboxylic group and free -OH peak overlap each other.The peaks for C=O, (C-O-C) epoxy group and C=C groups were observed at 1716.65 cm -1 , 1037.70 cm -1 , and 1618.28 cm -1 respectively.Whereas in case of GO-NO-Ur, some spectra of respective functional group disappeared.The peak at 1650 cm -1 is found for C=O due to amide formation.The spectral band shifted from 1716.65 to 1650 cm -1 and band of -NH2 group was disappeared (shown in NO-Ur), due to amide formation.The functional groups of GO were found

Theoretical point of view:
The theoretical study is focused to the analysis of the various non-covalent interactions observed in the solid-state architecture of NO-Ur with preferably to the lone pair(lp)-π interactions.In the previous report, we have found from the computational study that uracil derivatives have wonderful ligating properties i.e. binding sites and coordination sites of uracil derivatives easily interact with metal ions [52].For better understand, we also studied the influence of the conformation adopted by the NO-Ur on the π-binding ability of the ring.Therefore we have computed the molecular electrostatic potential (MEP) surface of the compound, exploring several conformations and complexes.The energetic value at the π-hole is more positive when the intra-molecular H-bond is formed.Interestingly, when a water molecule is included in the calculation of the MEP in the same position of that in the crystal structure, the energetic value at the π-hole is identical to the conformer with the intramolecular H-bond.Therefore the ability of the ring to establish lp-π interactions is enhanced by the presence of the water molecule.From literature survey employing DFT methods, it is found that the oxidation reactions mainly proceeds via transformation of hydrogen atoms from the organic starting materials to GO surface, which means that the hydrogen atoms reacts with the epoxide moieties on the functionalized GO surface; whereas the epoxides converted into diols that were able to undergo further dehydration [53].From experimental investigation it is clear that amidation reaction helps to the overall stability of composites materials.

Conclusion
Heavy metal pollution is a key issue in these days for human welfare, because it causes many diseases and through food cycle/ food chain/ food wave it comes to a higher organism.We have successfully synthesized GO-NO-Ur composite and studied for the detection of the Fe 3+ metal ion.XRD, FTIR and UV-vis spectrophotometry confirmed the successful formation of the composites.XRD data reveal the well exfoliated GO sheets during the formation of the composite.
From FTIR data it is also confirmed that GO-NO-Ur composites formed via the amidation reaction.The various non-covalent interactions of the NO-Ur and its corresponding composites were studied by density functional theory (DFT) calculation.It is clear that NO-Ur has very good binding and coordination sites for the formation of stable composites with GO.However, detail noncovalent interactions of GO-NO-Ur and GO-NO-Ur-Fe using DFT calculation is going on in our laboratory.We observed a significant result for the detection of Fe 3+ ions in water sample having LOD and LOQ value 0.0032 μM and 0.01 μM respectively.Thus the composite has good potential for future application in the environmental study as well as in the quantitative and qualitative measurements for sensor and biosensor applications.Fe is an essential component of blood in our body and its concentration is very crucial as both higher and lower amount of Fe in body can cause disease.

Scheme 1 :
Scheme 1: Different stages (including synthesis and metal sensing application) of development of a sensor for the detection of Fe 3+ .

Figure 1 .
Figure 1.XRD spectra of GO and GO-NO-Ur composite.

2 .
at their own position, shown in Figure The shifting of peaks in the FTIR spectra indicates the successful development of the composite.

Figure 2 .
Figure 2. FTIR spectra of GO, NO-Ur, and GO-NO-Ur.For metal sensing application of the composite material, GO-NO-Ur (0.04mg/ ml) was dispersed into distilled water using ultra-sonication bath.Solution of metal salts (Cd, Ag, Cu, Fe, Hg, Mo, Ni, and Zn) were prepared of 10 -3 M concentration and amount of salt solution was added into the solution of dispersed GO-NO-Ur accordingly like, for 10 -5 -50 μl added, and for 10 -6 -5 μl added to make up the volume (5 ml) of a solution.UV-vis spectrophotometric technique was done for the application of metal sensing.UV-vis spectrophotometry determination was made in the range of 200-600 nm and found that GO has an absorption band at 237 nm as shown in figure3A.NO-Ur has two absorption bands at 221 and 318 nm, whereas, GO-NO-Ur composite material has only one absorption at 266 nm, as shown in figure 3B.Absorption band shifted from 221nm to 266nm, because of n-π transition of NO-Ur.The changes in absorption band, confirms the formation of GO-NO-Ur.The absorption band at 266 nm of GO-NO-Ur disappeared with Fe solution.This change in spectra confirms the metal was sensing properties of the composite material, due to binding of Fe 3+ to GO-NO-Ur composite as shown in scheme 1.The comparison of GO-NO-Ur and GO-NO-Ur-Fe confirms that changes occurred in the absorption band due to the interaction of Fe 3+ ion with GO-NO-Ur composite shown in figure 3B.The value of the limit of detection (LOD) and limit of quantification (LOQ) was determined with the help of Stern-Volmer plot for the absorbance of GO-NO-Ur in water at a various concentration of Fe solution at 266 nm as shown in figure 4. LOD and LOQ values were calculated by the equation of 3.3 × 0.001/ 0.931 and 10 × 0.001/ 0.931, and the values were obtained as 0.0032 μM and 0.01 μM, respectively.

Figure 3 .Figure 4 .
Figure 3. Representation of UV-vis spectra of A) GO at low concentration B) GO, NO-Ur, GO-NO-Ur and GO-NO-Ur-Fe [23]ee et al.have used thiacalix[4]arene based fluorescent sensor for the detection of Zn +[19].Song et al. have reported 2-[(1-hydroxynaphthalene-2yl)methyleneamino]acetic acid for the detection of Zn 2+ and F − on the basis of fluorescent sensor technique[20].The derivative of the 2, 5-diphenyl-1,3,4-oxadiazole-based fluorescent sensor for the detection of Zn 2+ was reported by Tang et al[21].Pandey et al. reported xanthene-based fluorescent sensor probe for Mg 2+[22].Zhu et al. showed functionalized nucleic acid-GR based sensors for the detection of Pb 2+ , Hg 2+ , and Ag + .The author also used two categories method for the detection of metal ions viz.direct assay and the amplification-based assay[23].