Preparation of Graphene Oxide and Immobilization of biomolecules onto the large smooth surface for biosensor applications has attracted significant attention in recent years around the Globe. Besides, immobilization of biomolecules     onto surfaces of different material is essential in many fields of Chemical  , physical, biological sciences, including cell and molecular biology, medical diagnostics, analytical chemistry, tissue engineering, and bioprocess engineering. Since the discovery of the many advantageous properties of immobilized biomolecules, the challenges in this area have been to develop new support substrate materials with appropriate structures, surface morphology and functionality to widen the understanding of immobilized biomolecules. Recently, along with the development of nanostructured materials , a range of nanomaterials with different surface properties have been utilized as substrates for biomolecule immobilization . In particular, oxidized graphene  has been demonstrated as a well-defined, ordered, and atomically smooth surface . Graphene is comprised of a lattice of carbon atoms in a hexagonal array, creating an ordered structure against which morphology of immobilized biomolecules can be easily discerned. We have demonstrated here that the GO can be prepared from A-HDLC as large solid-state sheets to produce an ordered structure which can be used as a solid support substrate on which biomolecules can be immobilized, spontaneously adsorbed .
2. Abbreviations and Acronyms
Hydrogenated Diamond Like Carbon (HDLC), Graphene Oxide (GO), Oxidation of annealed HDLC (A-HDLC), Atomic force microscopy (AFM), Bovine Serum Albumin (BSA), BSA treated Graphene oxide sample (BGO), Transmission electron microscopy (TEM), Scanning electron microscopy (SEM).
3. Materials and Methods
Potassium Permanganate (extra pure AR, 99.5%, Sisco Research Laboratories Pvt. Ltd.), Potassium nitrate, (ACS reagent, ≥99.0%, Sigma Aldrich), Sodium Phosphate Dibasic Anhydrous, (99.5%, Sisco Research Laboratories Pvt. Ltd.), Bovine Serum Albumin (BSA) (96.96%, Sisco Research Laboratories Pvt. Ltd.). Ultra-pure water using Milli-Q system of Millipore Co. was used throughout the experiment.
3.2. Preparation Graphene Oxide from HDLC
We already have demonstrated earlier  a straightforward synthesis of HDLC film surfaces. The HDLC ﬁlm was heated to get Graphene-like and graphite-like structure  (A-HDLC) at a fixed temperature of 1000˚C for 30 min in high vacuum ~1 × 10−6 torr and cooled to room temperature after heating. Graphene oxide (GO) was prepared from A-HDLC according to the modified Hummers-Offeman method . In brief, about 10mm diameter A-HDLC sample was taken in 50 mL beaker and cold concentrated sulphuric acid added to it (1 mL, 98 wt%, dry ice bath) and potassium permanganate (KMnO4, 20 mg) gradually added with continuous vigorous stirring and cooling to prevent the temperature from exceeding 25˚C. The dry ice bath was removed and replaced by a water bath and the mixture heated to 35˚C for half an hour with gas release under continuous stirring, followed by slow addition of deionized water (2 mL), which produced a rapid increase in solution temperature up to a maximum of 90˚C. The reaction was maintained for 10 min to increase the oxidation degree of the graphene oxide product.
3.3. Immobilization of Protein onto the Surface of Graphene Oxide
0.2 M Phosphate buffers of pH 7.5 were prepared at room temperature. Graphene Oxide sample, cleaned by isopropyl alcohol and rinsed in ultra-pure water, dried with nitrogen and was immersed in BSA solution, prepared by dissolving 25 mg of BSA in 25 mL of 0.2 M Phosphate buffer, for 2 hours at room temperature under slow stirring was condition using a magnetic stirrer. The BGO was rinsed with ultra-pure water and dried with nitrogen.
3.4. Characterization of Samples
The cross sectional view and the morphology of the surface of the GO film surface have been analyzed by TEM, AFM and SEM respectively. Raman spectrum of the GO sample was obtained by the confocal Micro Raman spectrometer (LabRAM HR Vis, Horiba Jobin Yvon SAS France) which includes an 800 mm focal length Czerny-Turner type spectrograph equipped with mirrors (reflective optics) having high spectral resolution of 0.1 cm−1/pixel at 488 nm with an 1800 gr/mm grating. FTIR spectrum of the sample was obtained by Infrared Fourier Vacuum Spectrometer Vertex 70v (Bruker Optik GmbH Germany) with fixed 30˚ angle reflection with ATR crystal (Zn-Se) for solid GO sheet and sample. KBr beam splitter, DLaTGS detector, MIR source, Rock Solid Interferometer system, spectral resolution (>0.4 cm−1) and contamination free vacuum (<0.2 mbar) are the key technical features of Vertex 70 V. AFM topography image of the sample was obtained in non-contact mode by Multimode Scanning Probe Microscope (Agilent AFM 5500 series, USA) having multipurpose small scanner with low coherence laser (1 mW power, 670 nm wavelength, length of coherence (<50 mm), scan range: XY: 0 - 10 mm; Z: 0 - 2 mm, noise level: XY < 0.1 nm RMS, Z < 0.02 nm RMS.
4. Results and Discussion
Raman spectroscopy is a fingerprint and non-destructive technique to characterize graphitic materials, in particular to determine the defects and the ordered and disordered structures of graphene. Figure 1 shows the Raman spectra of the A-HDLC and GO sheets prepared from HDLC. The Raman spectra of GO sheet display two prominent peaks at 1358 and 1598 cm−1, corresponding to the well-documented D- and G-band. The G line represents the in-plane bond stretching motion of the pairs of C sp2 atoms (the E2g phonons); while the D line corresponds to breathing modes of rings or j-point phonons of A1g symmetry . Usually, the graphene-like sheets synthesized by annealing of HDLC approach show two clear D and G band in the Raman spectrum with the intensity ratio of ID/IG ≈ 1 due to the defects and partially disordered crystal structure of graphene-like sheets. It is noted here that for the GO, the intensity of the G band is significantly higher than that of the D band, suggesting that the prepared graphene Oxide has low defect content  . Figure 2 shows the thickness of the HDLC thin film.
The FT-IR analysis  of GO in Figure 3 shows the existence of H-bonded OH stretch at 3415 cm−1, C-H stretch at 2915 cm−1, C=O in carboxylic acid group at 1725 cm−1, C=C in conjugated ketones at 1628 cm−1 and primary alcohol C-O stretch at 1086 cm−1. The presence of oxygen-containing groups on GO is observed while Graphene-like/Graphite-like spectra have no significant peak for these groups  . FTIR analysis    in Figure 3 and
Figure 1. Raman spectra of the A-HDLC and GO sheets prepared from HDLC. (Black = GO, Blue = A-HDLC; Excitation wavelength 488 nm, laser power 7.5 mW, grating 1800 gr/mm, objective 100×, aperture 100 μm.
Figure 2. Typical TEM cross sectional view of the GO film measured with a Philips CM200 kV TEM machine operated at 200 kV with LaB6 filament; the sample of the cross section was prepared by the TEM sample preparation equipments of Gatan, USA.
Figure 3. Typical FTIR Spectra of GO sheet sample surface.
Figure 4 revealed that, after immobilization BSA onto GO, the peak intensity of GO at 1725 cm−1 (carboxylic acid), 1628 cm−1 (primary alcohol) and disappeared implying the reaction of such groups in GO. Characteristic peaks     of the functional groups present in the GO and BGO samples, as shown in Figure 3 and Figure 4, are assigned, and are given in Table 1(a) and Table 1(b), respectively. Figure 4 shows the immobilization of BSA, where the protein is immobilization on GO based on physical immobilization via π-π stacking and other molecular interactions. The physical immobilization onto GO occurs via different types of interactions, such as Van der Waals forces, electrostatic or hydrophobic π-π stacking interactions and hydrogen bonds between the oxygen functional groups of GO and nitrogen/oxygen groups of protein and chemical interaction i.e. covalent bond may among C=O of GO and -NH2 of Protein  .
Figure 5 and Figure 6 show the SEM images of Graphene-like A-HDLC and GO sheets composites, respectively. The sample shows plate-like forms without any amorphous or other kinds of crystallized phase particles. Morphology of GO   is observed as flaky texture reflecting its layered microstructure as shown in Figure 6. The larger interspaces of the layer and the thinner layer edges of GO can be clearly seen in Figure 6.
AFM was employed to characterize the degree of exfoliation of graphene-like sheets. As shown in Figure 7, a flat graphene-like sheet was selected for further investigation using the three-dimensional view.
From the profile of the atomic force microscopy (AFM) image, the roughness of the GO film in the GO device was estimated to be approximately 10 nm. We performed AFM to investigate the morphology of the GO sheets (Figure 8). The well-ordered surface improves resolution of immobilized protein by AFM. The root mean square roughness was 0.1 nm. It seemed that whole area was covered with GO sheets. We have shown that GO can serve as a substratum for immobilization of proteins. Since BSA protein is a complex molecule having a large variety of functional groups, at this stage it is very difficult to identify the exact route of reaction pathways for GO sheets with BSA protein. There were two reactive groups namely C=O and -OH groups in GO sheets. The more possibility for the reaction of BSA protein with GO sheets is electrostatic interaction/H-bonding/Vander waal’s force of attraction and covalent bonding. The
Figure 4. Typical FTIR Spectra of BGO sheet sample surface.
Figure 5. SEM image of Graphite-like sheet (A-HDLC) and Graphene-like sheet.
Figure 6. SEM image of GO sheets.
Figure 7. Non-contact mode AFM topography images of A-HDLC surface: scan size 2.25 µm × 2.25 µm.
Figure 8. Non-contact mode 2D and 3D AFM topography images of GO sheets: scan size 3 µm × 3 µm.
Table 1. (a) Tentative assignments of peaks in the FTIR spectra of GO sheet; (b) Tentative assignments of peaks in the FTIR spectra of BGO.
AFM image (Figure 9) shows the topography of the surface of BGO sample. Its roughness value (0.5 nm) is higher, by an order of magnitude, than that of the GO sheets surface. The immobilized proteins are globular in nature and they are aggregated as shown in the image Figure 9.
Figure 9. Non-contact mode 3D AFM topography images of BGO sheets: scan size 3 µm × 3 µm.
We have demonstrated that GO films comprised of an ordered structure of carbon atoms can be prepared on large-area-substrate surfaces. We further demonstrated that GO is an atomically smooth substratum useful for protein immobilization. Good AFM imaging of BSA protein onto GO Sheet and retention of the conformation of the immobilized protein are due to the atomically smooth surface with roughness < 0.10 nm. The ability to coat macroscopic surfaces presents GO Sheet as a potentially useful coating for biomedical devices, Spontaneous protein adsorption on Graphene Oxide nanosheets allowing efficient intracellular vaccine protein delivery . HDLC materials have been shown to exhibit biocompatible surface properties   . It can be anticipated that GO sheet will also be biocompatible   and may offer improved properties by harvesting  related to the highly ordered structure.
One (Hari Shankar Biswas) of the authors thank UGC Minor Project, Reference No.F.PSW-140/15-16 for funding. Authors also thank Mr. Biswarup Pal Choudhury, RKMVC College, Rahara, Dr. Susanta Debnath, SINP, Mr. U.S. Sil of PPD, SINP, Dr. Suchandra Chatterjee, Dr. Lalita Das, Department of Chemistry, Surendranath College, for characterization of sample and their technical help during the experiments in this work.
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