Graphene oxide and its derivatives as promising In-vitro bio-imaging platforms

Intrinsic fluorescence and versatile optical properties of Graphene Oxide (GO) in visible and near-infrared range introduce this nanomaterial as a promising candidate for numerous clinical applications for early-diagnose of diseases. Despite recent progresses in the impact of major features of GO on the photoluminescence properties of GO, their modifications have not yet systematically understood. Here, to study the modification effects on the fluorescence behavior, poly ethylene glycol (PEG) polymer, metal nanoparticles (Au and Fe3O4) and folic acid (FA) molecules were used to functionalize the GO surface. The fluorescence performances in different environments (water, DMEM cell media and phosphate buffer with two different pH values) were assessed through fluorescence spectroscopy and fluorescent microscopy, while Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) and Scanning electron microscopy (SEM) were utilized to evaluate the modifications of chemical structures. The modification of GO with desired molecules improved the photoluminescence property. The synthesized platforms of GO-PEG, GO-PEG-Au, GO-PEG-Fe3O4 and GO-PEG-FA illustrated emissions in three main fluorescence regions (blue, green and red), suitable for tracing and bio-imaging purposes. Considering MTT results, these platforms potentially positioned themselves as non-invasive optical sensors for the diagnosis alternatives of traditional imaging agents.

are further affected by the size, chemical functional groups, oxidation degree and other related factors 22,23 . GO can perform as an efficient quencher through either charge transfer or resonance energy 24 . In the customized dopamine biosensor utilizing GO fluorescence, the detection was based on the charge transfer between GO and dopamine quenching the fluorescence property of GO 25,26 . Based on another study, the GO sensor platform was used for the detection of metal cations 27,28 , where GO was applied as a fluorophore (electron donor) and the metal ions acted as the electron acceptor 29,30 .
The nature of fluorescence property of fluorophores, such as quantum dots and organic dyes, basically quenches by GO through the Fluorescence Resonance Energy Transfer (FRET) 31,32 . According to recent findings, quencher GO possesses a large number of bonding sites through oxygen-containing groups, which is ideal for targeting and delivery purposes 33,34 . The captured molecules on the surface of GO, including the fluorophorelabeled single stranded aptamers, double-stranded DNA molecules or antibodies/antigens [35][36][37] in the optimized distance, would turn on the fluorescence 38 . The dual role of GO, as a fluorophore and quencher, introduces that as a potential polymer for developing new sensors with multiplex detection capability, however, the broad fluorescence emission restricted its bio-imaging performances 39,40 . Proper modification of GO, using polymers, noble-metal nanoparticles and molecules, improves its fluorescence emission for definite detection/biosensing purposes 41,42 . For targeting delivery purposes, the modification with polymers increases the hydrophilicity and circulation of GO through the biological environment and reduces the steric hindrance between the targeting ligand and biomarker 43,44 . There are some reports on several polymers, including polyethyleneimine-polylactide (PEI-PLA) and polyethylene glycol (PEG), with bright and multi-color auto-fluorescence properties for theranostic systems 45,46 . Metal nanoparticles, on the other hand, are widely used to construct structures with unique electric, catalytic and photonic properties, such as local surface plasmon resonance (LSPR) 47 , surface-enhanced Raman scattering (SERS) 48,49 , and surface-enhanced fluorescence (SEF) 50 . Noble-metal nanoparticles-modified GO produce nano-platform with numerous applications in targeting, delivery, therapy, imaging and sensing properties 51,52 . Among various targeting ligands in delivery conjugates, the fluorescence spectroscopic behavior of folic acid (FA) was seen to be suitable for targeted imaging approaches 53 . These auto-fluorescence molecules had a large absorption cross-section bond overlapping with GO spectrum, where the emission peaks shifted from UV to NIR region, improving the detected fluorescence behavior 54 . These findings triggered our research interests towards investigating derivetized GO as a new theranostic agent for biomedical approaches. The fluorescent biosensors, used in the near-IR region by facilitating the effective interference-free signals, can avoid the interferences (e.g., auto-fluorescence and scattering light) in biological environments 55  Methods. Synthesis of nano-conjugates. GO-PEG. To prepare PEGylated graphene oxide (GO-PEG), GO was acylated using EDC and DMAP connecting PEG molecules via ester bonds [56][57][58][59] . In brief, 50 mg GO was dispersed in 100 ml deionized water (DI) with 50 mg EDC and DMAP under bath sonication followed by adding 100 mg PEG6000 at room temperature. The solution was kept stirring vigorously at 60 °C overnight. The final product, GO-PEG, was washed and purified in a dialysis tube (MW cut off: 12,000 KDa).
GO-PEG-Fe 3 O 4 . GO-PEG (400 mg) was added to 70 mL of 0.1 M NaOH solution and sonicated at room temperature for 45 min. The desired product was separated by centrifuging and washed with DI water for several times to adjust the pH to 6. The volume of 20 ml of degassed water was added to the isolated product and dispersed by an ultrasonic bath for 30 min. A solution of FeCl 3 ·6H 2 O (48 mg) and FeCl 2 ·4H 2 O (17.6 mg) in 5 ml degassed water was mixed dropwise with the suspension at 60 °C in an ultrasonic bath for 60 min. Then, ammonium hydroxide solution (23%) was added to the mixture through a funnel to adjust pH to 11-12. The final product, GO-PEG-Fe 3 O 4 , was separated by a magnet and washed several times 60 .
GO-PEG-Au. AuNPs were synthesized separately using Turkevich method based on reduction of the HAuCl 4 by citrate in water 61 . In brief, chloroauric acid solution (HAuCl 4 ) (200 ml of 0.01 wt. %) was heated for 20 min and refluxed in a 500 ml-round-bottom flask using a temperature-controlled hot plate under continuous stirring. A 4.5-ml aliquot of 1 wt. % sodium citrate solution was heated for 20 min and added to the boiling chloroauric acid solution, while heating under reflux for 15 min to reach the complete reaction. Then, the solution was allowed to cool down to room temperature with continuous stirring to yield citrate-capped AuNPs. In the following step, the synthesized gold nanoparticles were added to GO-PEG solution under sonication for 2 h; another stirring process was performed subsequently for further 5 h at room temperature. The final product, GO-PEG-Au, was obtained after the crude product was purified through dialysis (MW cut off 12,000 KDa).
GO-PEG-FA. GO-PEG (400 mg) was dispersed in 250 ml DMSO in a bath sonication followed by adding DCC (45 mg), DMAP (25 mg) and FA (67 mg) at room temperature. The solution was kept stirring vigorously at 60 °C under nitrogen gas for 36 h. The final product, GO-PEG-FA, was washed and purified in a dialysis tube (MW cut off 12,000 KDa) 62 .

Results and discussion
Characterization. GO The XRD patterns of GO, PEG and GO-PEG are displayed in Fig. 1b. GO has an influence on the arrangement of molecular chain of PEG in the crystal lattice, disturbing the order of its crystallization. This decreases the crystallinity of PEG and concludes in an effective conjugation of PEG to GO nano-sheets by ester bonding.
The XRD pattern of pure GO was analyzed based on a sharp diffraction peak at 2θ = 10.4°, corresponded to the (001) crystalline plane diffraction peak of GO 65 . The XRD diffraction pattern of PEG was confirmed by the characteristic diffraction peaks at 19.2°, 23.3° and 26.4°6 6 . The XRD diffraction pattern of GO-PEG represented the amorphic pattern of the nano-conjugate at 2θ = 15°. The diffraction peak at 2θ = 29° was expanded, indicating more amorphous structure of nano-conjugate. The morphology of GO and GO-PEG was further assessed using  (Fig. 4b).
EDX spectra further showed the corresponding peaks of carbon (C) oxygen (O) and iron (Fe) in the final conjugate (Fig. 4c).
GO-PEG-Au. The crystallographic structure of synthesized GO-PEG-Au was studied using XRD (Fig. 5) with distinguished peaks at 38.2°, 44.59° and 64.7° corresponded to (220), (200) and (111) planes, respectively, confirming the formation of AuNPs in the nano-conjugate 69 . Figure 6a shows   www.nature.com/scientificreports/ carbon, oxygen and gold elements throughout the surface of GO-PEG-Au (Fig. 6b). EDX spectra further showed the corresponding peaks of C, O and gold (Au) in the final conjugate (Fig. 6c).
GO-PEG-FA. The grafting GO-PEG with FA led to the introduction of new absorbance peaks in the FTIR pattern of GO-PEG-FA at 1640 cm −1 and 3400-3500 cm −1 , representing the N-H group, and the peak at 1400 cm −1 corresponded to the aromatic ring stretch of the pteridine ring and ρ-amino benzoic acid moieties of FA 70 . Another peak at ~ 1700 cm −1 was an evidence of the ester bonding between FA and GO-PEG surface (Fig. 7). FE-SEM images of the modified GO revealed a sheet like morphology with wrinkled structure for FA-functionalized GO-PEG (Fig. 8a) 71 . EDX analysis presented a meaningful picture for the element distribution on the surface of conjugate, verifying the presence of nitrogen in the folic acid structure (Fig. 8b). EDX spectra contained the related peaks of C, O and nitrogen (N) (Fig. 8c).  To study the solution effect on the intensity and pattern of detected fluorescence, GO derivatives were dissolved in three different solvents (water, PBS and DMEM cell media). Acidic pH particularly needs to be considered for the system design for imaging or tracking purposes, either inside (endosomes) or outside (tumor area) the cells. As discussed in the following sessions, apart from the type of functionalization, the solvent considerably affects the emitted fluorescence 72 .   (Fig. 9a). GO becomes the charge donor when modified with PEG (GO-PEG), its broad fluorescence spectra are quenched in water at the range of 450-550 and 650-750 nm. In return, GO acts as the energy acceptor quenching the emission peaks of PEG (energy donor) at 508 and 605 nm (Fig. 9b). Likewise, the behavior of PEGylated GO in DMEM cell media is similar to the detected emission in water, while the new peaks at ~ 300-450 and 650-800 nm are attributed to the cell media absorption. At the natural pH (7.4), the fluorescence emissions of both components (GO and PEG) are preserved and the emission peak at 400 nm is blue-shifted, representing GO-PEG as a blue fluorophore. Thus, GO-PEG can be introduced as a turn on/off biosensor in tumor studies, and a tracer for in vitro and in situ imaging. GO-PEG-Fe 3 O 4 acts similar to GO-PEG in both water and acidic pH, in which the fluorescence emission curve of GO is quenched in the acidic condition (Fig. 9c). Thereby, it could be applicable as a suitable switchoff biosensor in a tumor area. In cell media, however, the fluorescence of PEG and Fe 3 O 4 components are both preserved, while the fluorescence of GO is quenched. This fact shows that GO can behave as an energy acceptor and simultaneous energy donor in the cell media, introducing GO-PEG-Fe 3 O 4 conjugate as a potential tracer for in-vitro and in situ tumor imaging. Nevertheless, at natural pH (7.4), the fluorescence would be nearly quenched.
Au accelerates the blue emission (400 nm) with the new peak at red region (700 nm). This introduces GO-Au as a potential candidate for imaging and thermal therapy (Fig. 9d). GO-PEG-Au has a similar behavior in both water and DMEM cell media. As previously mentioned, the new detected peaks at 400 and 700 nm are contributed to the absorption of DMEM cell media. On the contrary, the fluorescence emission of GO would be quenched by Au NPs at both natural and acidic pH values, when GO acts as a charge donor (switch-off biosensor). Figure 9e indicates the interesting patterns of GO-PEG-FA in all various solvents (water, cell media, natural and acidic). Although the blue and red emissions are still detectable with the lower densities, green fluorescence (430-550 nm) is observed with strong peak at 460 nm.
Emission spectra at the excitation wavelength of 350 nm. The dissolved GO in water indicates a broad emission at 400-650 nm as well as a sharp peak at 700 nm (Fig. 10a). DMEM cell media and neutral pH affect the trend resulting in a blue shift to 400-550 nm, while acidic pH causes a red-shift with a maximum emission peak at 750 nm. According to Fig. 10b, PEG acts as an energy acceptor quenches the predicted fluorescence of GO. Likewise, the metal nanoparticles have a similar behavior in the wavelength of 350 nm (Fig. 10c, d). Interestingly,  www.nature.com/scientificreports/ FA accelerates the blue emission at 400-500 nm representing a strong fluoresce at the green zone (Fig. 10e), the range appropriated for bio-imaging applications 62 .
Emission spectra at excitation wavelength of 430 nm. At the excitation wavelength of 430 nm, GO has a broad emission (Fig. 11a) that is improved and reinforced with the PEG modification (Fig. 11b) at the red region (560-800 nm) in all solvents. GO-PEG-Fe 3 O 4 shows a single peak at 547 nm in water, while multiple emission peaks appear at 500, 570 and 650 nm when dissolved in DMEM cell media and PBS (Fig. 11c). Likewise, GO-PEG-Au illustrates the same behavior as GO-PEG-Fe 3 O 4 ; while the fluorescence intensity is lower when dissolved in water (Fig. 11d). In the excitation wavelength of 430 nm, GO-PEG-FA does not show any identified emission suitable for imaging purposes (Fig. 11e).
Emission spectra at the excitation wavelength of 480 nm. GO at the excitation wavelength of 480 nm has an inappropriate fluorescence behavior (Fig. 12a). Functionalization with PEG improves this fluorescence emission and results in a red shift maximized at 650 and 730 nm in natural and acidic conditions, respectively (Fig. 12b). Fe 3 O 4 , however, decreases the fluorescence intensity of the conjugate in acidic condition creating a blue shift in DMEM cell media (Fig. 12c). Due to the broad emission pattern of GO-PEG-Fe 3 O 4 in water, so, it is not suggested for the detection purposes. Au, on the other hand, increases the fluorescence intensity of the conjugate in both natural and acidic conditions (Fig. 12d). GO-PEG-Au indicates the multiple emission peaks with low intensity when dissolved in water. GO-PEG-FA decreases the fluorescence intensity of conjugate in all solvents, representing FA as an energy acceptor in the excitation wavelength of 480 nm (Fig. 12e).
According to Figs. 9-12, the GO derivatives are potentially traceable fluorophores for imaging purposes in biological environments. Figure 13 shows the fluorescence images of modified GOs dissolved in PBS solution in three different fluorescence regions. After surface modification 73 , the photoluminescence property of GO differs, either quenched or accelerated, disregarding the applied excitation wavelength. PEG polymer quenches the GO emission in blue and green regions, while the red emission is still detectable. In fact, modified conjugates with metal nanoparticles are stronger at all three fluorescence regions, leading to the detectable images. The red shift occurred with folate makes this derivative an ideal sensor for bio-imaging purposes.
Biocompatibility. MTT    When the excitement occurs at 430 nm, all four modified GO conjugates experienced interesting alterations. The observed red shift introduced GO-PEG ideal as the "switch-on biosensor". Metal NPs conjugates showed the multiple emission peaks, whereas folate quenched the predicated emissions at both blue and far red regions in GO-PEG-FA.
At the excitation of 480 nm, GO-PEG experienced a sharp peak and red shift in natural and acidic conditions, while metal NPs, in particular Au, as well as folate, accelerated the expected emission, representing GO-PEG-Au and GO-PEG-FA as the ideal theranostic agents.
GO and its derivatives in conclusion displayed an excited-state protonation of COOH groups in various pH conditions. The creation of localized sp 2 clusters and structural defects during GO reduction through the modification were more likely to be responsible for the enhancement of green fluorescence. Besides, it was found that the fluorescence of GO and its derivatives conjugates were tunable between ultraviolet, visible and NIR with a robust intensity. These features suggested that the fluorescence aspect of GO, specifically at the excitation of 480 nm could be readily incorporated in a variety of biomedical imaging applications; Likewise, GO may act not only as a fluorophore, it can also operate as a quencher, introducing the new chances for the next generation biosensors with multiplex detections.