Synthesis of double-clickable functionalised graphene oxide for biological applications

Azide- and alkyne-double functionalised graphene oxide (Click2 GO) was synthesised and characterised with ATR-FTIR, TGA, and Raman spectroscopy.


Preparation of GO by modified Hummer's method
The modified Hummer's method reported by Ali-Boucetta et al. 1 was used initially to prepare GO for comparison only. In brief, graphite powder (300 mg) and sodium nitrate powder (NaNO 3 , 150 mg) were dry-mixed in a 250 mL round bottom flask.
Sulfuric acid (H 2 SO 4 (96 -98 %, ~ 7 mL) was added and the mixture was kept stirring at 0 °C on an ice bath. Potassium permanganate powder (KMnO 4 , 900 mg) was added slowly (portion-wise) to the suspension. The addition rate was carefully controlled so that the temperature of the suspension was kept below 20 °C then stirred on an ice bath (0 -10 °C) for 10 minutes. The ice bath was then removed and replaced by a water bath to allow the temperature to rise to 35 ± 3 °C, which was maintained for at least 30 minutes. Deionised water (DI H 2 O) (14 mL) was then added slowly 4 into the pasty mixture. The temperature started to increase to ~ 98 °C with the appearance of a violet vapour. The mixture was kept stirring for 30 minutes then

Preparation of GO by modified Kovtyukhova-Hummer's method
Modified Hummer's method reported by Kovtyukhova et al. was adapted with some modification. 2 Steps involved in preparation of GO are described in details below and schematically summarised in Scheme S1.
Sulfuric acid (H 2 SO 4 , 96 -98%, ~ 100 mL) was added and the mixture was kept stirring at 0 °C on an ice bath. When the powders were fully dispersed, potassium 5 permanganate powder (KMnO 4 , 12 g) was added slowly to the suspension. The addition rate was carefully controlled so that the temperature of the suspension was kept below 20 °C. After the addition of KMnO 4 was complete, the suspension was stirred on an ice bath (0 -10°C) for an additional 10 minutes.
(iii) Medium temperature stage: The ice bath was removed and replaced with a water bath to allow the temperature to rise gradually to 35 ± 3 °C. The temperature was maintained for at least 2 hr until the dispersion became a brown pasty mixture.
The mixture was then heated to 60 °C.

Synthesis of GO-N 3 with epoxidised GO (epo-GO)
Direct epoxidation of GO with meta-chloroperoxybenzoic acid (mCPBA) has not been reported in the literature. In this work, mCPBA was used to introduce epoxides on GO surface to form epo-GO. Methods of epo-GO preparation is described below and summarised in Scheme S4.
The aqueous layer was then transferred into a 50 mL centrifuge tube loaded with 20 mL CH 2 Cl 2 and centrifuged at 3,214 × g (3900 rpm) for 2 minutes. The upper aqueous layer was discarded and refilled with DI H 2 O. The process was repeated for 10 times. CH 2 Cl 2 was then removed; GO-N 3 was resuspended in 50 mL DI H 2 O. The final product (GO-N 3 ) was obtained by freeze-drying and stored for subsequent characterisation using TGA and ATR-FTIR.

Synthesis of Click 2 GO (azide-, alkyne-double clickable GO)
In order to introduce azide and alkyne functional groups, a one-pot sequential synthesis strategy for azide and alkyne double functionalised GO (Click 2 GO) was attempted in this study. Reaction steps are summarised in Scheme 1 and described as below: (i) GO epoxidation: epo-GO was firstly prepared from GO (50 mL, 3mg/mL) using the mCPBA pre-treated method described in the previous section. However, after the reaction was complete, the entire suspension was then used directly for the next step without purification.
(ii) Azide introduction: NaN 3 (300 mg/each condition) was then mixed with epo-GO suspension. pH was adjusted to 4.5 using 1N NaOH and 1N HCl solutions. The mixture was stirred overnight at room temperature to afford mono-functionalised GO-N 3 . The suspension was used directly for the next step without purification. suspension from previous step and stirred overnight at room temperature. This step was carried out to introduce the second functional group, the alkyne, onto GO-N 3 through Steglich esterification. 3 EDC was used for the coupling instead of N,N'dicyclohexylcarbodiimide (DCC). The isourea by-product generated from EDC was water-soluble compared to water-insoluble dicyclohexylurea (DCU) from DCC so the former can be easily removed by water in the washing step.
(iv) Washing step: Reagents were removed by filtration. Reaction crude was washed with water (4 × 200 mL) followed by methanol (3 × 100 mL) and filtered using a   freeze-dried and characterised by ATR-FTIR and TGA (Figure 3 and S10).

Thermogravimetric analysis (TGA)
Liquid samples e.g. GO aqueous dispersions (75 µl) were firstly loaded on platinum sampling pans, dried at 120 °C for 2-minute. The concentration of the aqueous GO dispersions were calculated based on the residual weight after drying and the volume of loaded samples was expressed by the following equation: Eq. S1 To monitor the degree of chemical modification on the sample, dried aqueous or powder samples were then equilibrated at 100 °C for 10 minutes before heating up with a ramp of 10 °C /min from 100 to 1000°C under nitrogen with a flow rate of 90 mL/min using TGA model TGA-Q500 (TA instrument, USA). The TGA thermal curve is displayed as the weight (mg) or residual weight percentage (%) as a function of the temperature (°C) within the defined range.

Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR)
ATR-FTIR was performed using PerkinElmer ® Frontier™ FT-IR equipped with ATR accessory (diamond ATR polarization accessory with 1 reflection top-plate and pressure arm). The pressure arm was used for all solid samples at a force gauge setting between 100 ~ 120 units; no compression was used for liquid/oil samples. The number of scans was set at 15. Samples were loaded on the reflection top-plate at a quantity sufficient enough to cover the entire diamond surface. GO derivatives were dispersed in a suitable solvent (usually water for hydrophilic samples) and freeze dried into a loose sponge-like structure. The container was sealed immediately after freeze-drying. Moisture absorption was minimised when warming to room temperature in order to obtain a good spectrum resolution with minimum interferences.

Raman spectroscopy
Raman spectroscopy was performed using Renishaw ® inVia-Reflex spectrometer (UK) with an excitation wavelength set to 785 nm and 0.1 to 1 % laser power.
Aqueous dispersion of GO derivatives were deposited and dried on a calcium fluoride (CaF 2 ) slide (Crystran Ltd, UK) on a hot plate before the measurement.
Data were acquired and analysed using Renishaw's WiRE 4.0 (Windows-based Raman Environment) software.

Atomic force microscopy (AFM)
The surface topography of GO samples deposited on a dry mica surface was studied by AFM using tapping mode. GO samples were prepared at a concentration of 0.

Transmission electron microscopy (TEM)
TEM was performed on Philips CM 12 (FEI Electron Optics, The Netherlands) equipped with Tungsten filament and a Veleta -2k × 2k side-mounted TEM CCD Camera (Olympus, Japan). The accelerating voltage is 80 KV. The spot size was set at 3. Objective aperture was used with all samples. GO aqueous dispersions at a concentration of 0.2 mg/mL were deposited on carbon-film on 300 mesh copper grids or lacey carbon films on 300 mesh copper grids for the measurement.

GO flake surface area analysis
The surface area of the GO flake was analysed using Image J 1.49i software from National Institute of Health (USA). Images from AFM were used for analysis with a 13 total measurement number no less than 100 flakes for each sample. Histogram of surface area distribution and statistical calculation was generated and performed using DataGraph 3.2 (Visual Data Tools, Inc., USA).

Statistical analysis
The data were expressed as mean ± standard deviation (mean ± S.D.). All the statistical analyses were implemented using Minitab ® v16 (Minitab Inc., UK). The comparison between the control and experiment groups was analysed using student t-14 test of one-way ANOVA followed Tukey's HSD (honest significant difference) tests.
Mean differences with p <0.05 were considered significant.

GO characterization
Thermogravimetric analysis (TGA) was performed to analyse the thermaldecomposition profile of graphite, pre-oxidised graphite and GO (100-978 °C). As shown in Fig. S2A, the thermal deoxygenation of graphene derivatives was reflected by the reduced residual weight upon heating (101°C min -1 in nitrogen). The final residual weight at 978 1C was 99.36%, 94.15% and 38.16% for graphite, pre-oxidised graphite and GO, respectively. Raman spectroscopy confirmed the generation of GO (Fig. S2B). D, G and 2D peaks were identified for both graphite and GO. The spectra were normalised to G peak (intensity equals 1). G peak at 1580 cm -1 (graphite) or