Supplementary Information Oxygenated graphene quantum dots (GQDs) synthesized using laser ablation for long term real time tracking and imaging

Fluorescence probes are essential for in vivo molecular imaging as well as cell tracking applications. Probes that emit in the far red wavelength penetrate better through biological tissue and the autofluorescence background is reduced at higher wavelengths, making such probes highly desirable. We report the application of Graphene Quantum Dots (GQDs) as efficient fluorescence probes for single cell tracking using high-resolution confocal microscopy as well as a potential in vivo fluorescent imaging agent. High-quality water soluble GQDs were synthesized by ablating highly oriented pyrolytic graphite (HOPG) in liquid using a nanosecond pulsed laser. Refluxing GQDs at 200 °C for 20 minutes and 1 hour produces excitation independent broad emission peaking at 600 nm and excitation dependent color-tunable emission, respectively. These emission properties can be attributed to the functional groups such as carboxyl and hydroxyl groups on the surface or edges. MTT assays with the breast cancer (MCF-7) cell line suggest that 20 min and 1 h GQDs had 80% viability post incubation with concentrations as high as 2 mg mL−1. The paper also explores the molecular tracking functionality of GQDs. Fluorescence microscopy showed that MCF-7 cells incubated for up to 48 hours with GQDs, internalized the GQDs, by caveolae-mediated endocytosis without any targeting moiety. In addition, high-resolution Z stacking and 3D confocal microscopy of a single live MCF-7 cell confirm successful uptake of GQDs into the cytoplasm by endocytosis. Fluorescence imaging of GQDs loaded in polyacrylamide gel and subcutaneously implanted near the thoracic region of an euthanized mice was done to explore the feasibility of in vivo imaging using GQDs. Deep red fluorescence (610 nm emission filter) was observed from the implanted region with relatively low autofluorescence background.

FT-Raman spectrum of pyrolytic graphite had a sharp peak of G band at 1590 cm -1 and D band at 1332cm -1 . 1 G-band is attributed to in-plane vibrations of sp 2 hybridized bonded carbon atoms whereas the D-band indicates sp 3 hybridized carbon atoms due to the presence of structural defects.Broad peak around 1800 cm -1 could bedue to the sp 3 hybridization of polyethylene glycol. 2 Broadening of D-band in GQDs was greatly enhanced by presence -OH and -COOH molecules compared to pure graphite. 3rthermore the I d /I g ratio of GQDs (0.6) was higher when compared to graphene (0.05) suggesting that GQDs had highly disordered structures due to the presence of functional groups at the edges.X-ray photon spectroscopy (XPS) was performed (Fig. S3b-d).The wide spectrum indicates that synthesized GQDs had major peaks corresponding only to Carbon and Oxygen.C 1s spectrum of GQDs was deconvolved into different bands suggesting that the 285, 286.9 and 288.8 eV binding energy values correspondto the sp 2 carbon chain (C-C), hydroxyl group (C-OH) and carboxyl groups (C=O) respectively.
O1s spectrum indicates that the presence of Oxygen in the GQDs was due to the carboxyl groups and hydroxyl groups.These groups were induced due to the refluxing and laser interaction of graphite material with PEG: water solution after ablation. 3,4 Stability of GQDs To understand the fluorescence emission properties of GQDs (Control GQDs, 20 min and 1 hr), particles were redispersed at a particular concentration in different pH ranging from 1-11 before obtaining fluorescence measurements.

Figure S1 .
Figure S1.Laser ablation setup of HOPG in beaker containing PEG:water solutionin a glass beaker connected to DC rotor (a).Zeta potential measurements show that refluxed GQDshad higher zeta potential compared to control GQDs (b).Dynamic Light scattering measurements of control, 20 min and 1 hr GQDs(c)

Figure S3 .
Figure S3.(a) FT-Raman spectra GQDs had a broad G and D band confirming that the particles possess sp2 and sp3 carbon atoms (b) Wide scan XPS spectrum of GQDs with major peaks of carbon and oxygen.(c) High resolution scanning of C1s (d) and O 1s XPS spectrum of GQDs.

Figure S5 .
Figure S5.Fluorescence microscopic images of MCF-7 cells incubated with GQDs for 24 hrsand excited at 365 and 400 nm.The fluorescence images for control , 20 min and 1 hr GQDs are shown.Blue (middle column)and green fluorescence emission(right column) were observed corresponding to excitation at 365nm and 400nm respectively.The corresponding bright field images are shown in the left column.

Figure S6 .
Figure S6.Confocal Z-stack images and 3D imaging of GQDs localization in single cell incubated for 48 hours with MCF-7, optically sectioned with a step size of 500 nm (Top).

Figure S7 .
Figure S7.White light image (left), Fluorescence image (middle), and blended fluorescence and white light image of euthanized mouse, subcutaneously implanted with a gel capsule containing GQDs.Imaging was done in trans-illumination mode with excitation wavelength of 530nm using commercial LEDs and the emission filtered at 590nm.