Since the Nobel prize for Physics was awarded to Andre Geim and Konstantin Novoselov “for groundbreaking experiments regarding the two-dimensional material graphene”, the eyes of the scientific world have been focused on this so-called miracle material.

1. GRAPHENE OXIDE AND ITS BIOMEDICAL
APPLICATIONS
SITANSU SEKHAR NANDA
DEPARTMENT OF BIONANOTECHNOLOGY
GRADUATE SCHOOL, GACHON UNIVERSITY
UNDER SUPERVISION OF
PROF. DONG KEE YI
Presentation for Ph.D. Degree
1

2. TABLE OF CONTENTS
2
 Chapter.1. Introduction
 Chapter.2. Graphene Oxide Based Fluorometric
Detection of Hydrogen Peroxide in Milk
 Chapter.3. Study of antibacterial mechanism of
Graphene Oxide using Raman Spectroscopy
 Chapter.4. Oxidative stress and antibacterial properties
of a graphene oxide-cystamine nanohybrid
 Chapter.5. Future work & perspective

3. Chapter.1. Introduction
 Since the Nobel prize for Physics was awarded to Andre
Geim and Konstantin Novoselov “for groundbreaking
experiments regarding the two-dimensional material
graphene”, the eyes of the scientific world have been
focused on this so-called miracle material.
 GO is a two-dimensional material of exceptional
strength, unique optical, physical, mechanical, and
electronic properties. Ease of functionalization and high
antibacterial activity are two major properties identified with
GO.
 Due to its excellent aqueous process ability, surface
functionalization capability, surface enhanced Raman
scattering (SERS), and fluorescence quenching ability, GO
chemically exfoliated from oxidized graphite is considered
a promising material for biological applications.
3

4. CHAPTER-1
Graphene as exfoliated from graphite, is hydrophobic,
i.e., not dispersible in water, highly reactive and non-
biocompatible. However, upon oxidation to form Graphene
Oxide (GO), it becomes hydrophilic and therefore water
soluble and is amenable to a host of biomedical
applications.
Hybridization of GO with polymers, gold and magnetic
nanoparticles results in carbon-related Nano composites
used in a variety of biomedical and biotechnological
applications such as for phototherapy, bio-imaging, drug
and gene delivery, bio sensing, and antibacterial action.
4

5.  This data depicts the relative research activity on
different graphene-family of materials for biological
applications indicating that more than 50%of them
(62.6%) are biomedical in nature.
 Due to the small size, large surface area, and useful non
covalent interactions with aromatic ring molecules, GO
is a promising material for drug delivery.
 For this reason the subject of GO toxicity has attracted
special attention in order to increase the biosafety of
GO.
 The valid evaluation and actual confirmation of the
biocompatibility of GO is central to many scientific
investigations.
CHAPTER-1
5

6.  Marcano et al. prepared an improved method for the
synthesis of GO.
 They found that excluding NaNO3, increasing KMnO4 and a
mixture of H2SO4/H3PO4 can improve the oxidation process.
 A schematic presentation of their results is presented here.
 According to their reaction protocol, the reaction is not
exothermic and produces no toxic gas. This makes it useful
for biomedical applications.
CHAPTER-1
6

7. TABLE
Some of the major research
on GO related biomedical
application are presented
here in the table.
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8. Chapter.2. Graphene Oxide Based Fluorometric
Detection of Hydrogen Peroxide in Milk
 The analytical feature of our proposed method includes
low detection limit (10 mmol mL−1) and satisfactory
recovery values for samples.
 The presence of H2O2 in milk is a major concern
because it constitutes a public health hazard. Many milk
industries are using H2O2 as a preservative, but if the
concentration increases then it causes so many health
problems such as neurodegenerative disorders, cancer
and diabetes.
 Present methods show an easy way for detecting H2O2
generally require considerable time and laboratory
facilities. The chemical tests have sufficient sensitivity
to detect wide linear range of H2O2 concentration.
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8

9. CHAPTER-2
 In Fig. (a) typical field emission scanning electron microscopy
(FE-SEM) images of GO are shown.
 In Fig. (b) the main absorbance peak attributable to π-π*
transitions of C=C in synthesized GO occurs at around 232 nm.
 In Fig. (c) the XRD spectra of GO show a distinct peak at
15.10° corresponding to a d-spacing (in this case, the interlayer
distance between sheets) of approximately 7.15 Å that is due to
interlamellar water trapped between hydrophilic GO sheets.
 In Fig. (d) the Fourier transform infrared (FTIR) spectra of the
GO clearly show the presence of carboxyl (O-H deformation
1730-1700 cm-1), hydroxyl (O-H stretching vibration 3450 cm-1),
epoxy (750 cm-1), and carbonyl (C=O stretching 1050 cm-1)
groups.
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10. CHAPTER-2
 In Figure (a) the XPS spectra of GO are shown.
 In Figure (b) the deconvolution of the C1s peak in the
XPS spectrum shows the presence of four types of
carbon bonds: C–C (284.8 eV), C–O (hydroxyl and
epoxy, 288.2 eV), C=O (carbonyl, 292.7 eV), and O C–O
(carboxyl, 294.8 eV).
 By integrating the area of the deconvolution peaks, the
approximate percentage obtained for C–C is 45.36%.
Similarly, In Figure 2(c) the deconvolution of the O1s
peak in the XPS spectrum shows the presence of one
type of oxygen bond: O–H (530 eV).
 By integrating the areas of the deconvolution peaks, the
approximate percentage for O–H was obtained to be
38.72%.
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11. CHAPTER-2
 In This Figure the photoluminescence spectra (PL) of
milk containing GO and DCFH-DA are shown.
 The solution was excited at 350 nm and the emission
spectra were obtained at 420 nm and 480 nm,
respectively. Due to the absence of H2O2, the PL is
mostly similar to the spectra of GO.
 Initially, they show the distinctive band-edge absorption
peak at 420 nm. As the stacking deposition proceeds,
the absorption reaches to the shorter wavelengths down
to 480 nm and becomes more featureless.
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12. CHAPTER-2
 In this Figure the PL spectra of milk with GO, DCFH-DA
and H2O2 are shown. The solution was excited at 350
nm and the emission spectra were obtained at 530 nm.
 This newly proposed method has been used to
determine H2O2 in nine milk samples; A low limit of H2O2
(10 mmol mL−1) has been detected. When H2O2 was
added into the milk at nine different concentrations (10,
15, 20, 25, 30, 35, 40, 45 and 50 mmol mL−1 of H2O2 are
observed for all samples.
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13. Chapter.3. Study of antibacterial mechanism of
Graphene Oxide using Raman Spectroscopy
 Here we developed a new and sensitive fingerprint
approach to study the antibacterial activity of GO and
underlying mechanism, using Raman spectroscopy.
 Spectroscopic signatures obtained from biomolecules
such as Adenine and proteins from bacterial cultures
with different concentrations of GO, allowed us to probe
the antibacterial activity of GO with its mechanism at
the molecular level.
 Escherichia coli (E. coli) and Enterococcus faecalis (E.
faecalis) were used as model micro-organisms for all
the experiments performed. 13

14. CHAPTER-3
 In general, the Raman spectrum of graphite exhibits a
‘G band’ at 1580 cm-1 and a ‘D band’ at 1350 cm-1. The
G band is due to the first order scattering of the E2g
mode whereas the D band is related to the defect in the
graphite lattice.
 The Raman spectra of GO are shown in Figure, which
show the presence of a G band at 1660 cm-1 and a D
band at 1380 cm-1. The G band of GO is shifted towards
a higher wave number, an observation that co-relates
with the oxidation of graphite which results in the
formation of sp3 carbon atoms.
 Furthermore, the D band in the GO is broadened due to
the size reduction of the in-plane sp2 domains during
oxidation.
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15. CHAPTER-3
(a) SEM images of E. coli (control) (b) SEM images of E. coli when treated with 50µg/mL of GO (c) SEM images
of E. coli when treated with 100µg/mL of GO (d) SEM images of E. coli when treated with 150µg/mL of GO.
As the concentration of GO increases the shape of E.coli changes.
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16. CHAPTER-3
 Among the bands displaying obvious changes, the 729
cm−1 band was assigned to Adenine. As the
concentration of GO increased, the concentration of the
Adenine ring mode increased, as shown in Figure.
 The conformation about these bonds is related to the
structure of the protein. The S-S stretching vibration
occurred at 490 cm-1 as shown in Figure. The intensity of
the S-S stretching vibrations increased as the
concentration of GO increased.
 Here, the Amide VI band (CO-NH bending vibration)
occurred at 610 cm-1 which is shown in Figure. The
intensity of Amide VI band (CO-NH bending vibration)
increased as the concentration of GO increased.
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17. CHAPTER-3
 Bio- AFM images of E.coli cells, before and after
treatment with various concentrations of GO,
immobilized on a glass slide.
 (a) Images of untreated E.coli, showing a height profile
of 150.9 nm. (b) Images of E.coli after GO of 50 µg/mL
concentration was added showing an increased height
profile up to 207.8 nm. (c) Images of E.coli after GO of
100µg/mL concentration was added showing an
increased height profile up to 329.1 nm. (d) Images of
E.coli after GO of 150 µg/mL concentration was added
showing an increased height profile up to 551.9 nm. (e)
Plot of bacterial height profile (y-axis) vs. GO
concentration (x-axis); as the concentration of GO
increased the E.coli height profile increased.
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18. CHAPTER-3
 Bio- AFM images of E. faecalis cells, before and after
treatment with various concentrations of GO, immobilized on
a glass slide.
 (a) Images of untreated E. faecalis, showing a height profile
of 376.2 nm. (b) Images of E. faecalis after GO of 50 µg/mL
concentration was added showing an increased height profile
up to 571.2 nm. (c) Images of E. faecalis after GO of 100
µg/mL concentration was added showing an increased height
profile up to 711.2 nm. (d) A concentration of 150µg/mL of
GO was added to images E. faecalis after GO of 150 µg/mL
concentration was added showing an increased height profile
up to 727.7 nm. (e) Plot of bacterial height profile (y-axis) vs.
GO concentration (x-axis); as the concentration of GO
increased the E. faecalis, height profile increased.
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19. CHAPTER-3
 Herewith, by using morphological and spectroscopic
data we have confirmed experimentally and theoretically
that GO can induce the degradation of the outer and
inner cell membranes of E. coli and E. faecalis bacteria.
The work presented here demonstrates the great
antibacterial action of GO is due to the release of
Adenine and protein from Bacteria.
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20.  Cystamine has been successfully conjugated with
graphene oxide (GO) as a drug carrier.
 The current study used the microdilution method to
determine the minimum inhibitory concentrations of
cystamine-conjugated GO against four types of
pathogenic bacteria. Minimum inhibitory
concentrations values were 1 μg/mL against
Escherichia coli and salmonella typhimurium, 6 μg/mL
against Enterococcus faecalis, and 4 μg/mL against
Bacillus subtilis.
 Toxicity of the conjugate against squamous cell
carcinoma 7 cells was minimal at low concentrations,
but increased in a dose-dependent manner.
Chapter.4. Oxidative stress and antibacterial properties
of a graphene oxide-cystamine nanohybrid
20

21. CHAPTER-4
 Magnified AFM images of GO showed its height 0.8 nm
whereas cystamine-conjugated GO shows its height 1.2
nm.
 SEM image showed conjugation of cystamine with GO which is
confirmed by the reduction of the size of cystamine-conjugated
GO. 21

22. CHAPTER-4
 Cytotoxicity and ROS studies of cystamine conjugated GO UV–Vis spectrum and FT-IR of cystamine,
GO, and cystamine-conjugated GO. 22

23. CHAPTER-4
 XPS measurements provided additional information
about the nature of cystamine-conjugated GO. Both
conjugated and unconjugated GO exhibited C=C (sp2),
C-C (sp3), C=O (carbonyl), O-C=O (carboxyl), and C-
O/C-O-C (hydroxyl and epoxy) groups).
 Figure shows the C1S peaks of GO including C-O
(hydroxyl and epoxy, 288.1 eV), C=O (carbonyl, 291.4
eV), C=C/C-C (284.7 eV), and O=C-O (carboxyl, 294.8
eV) species. The C=C/C-C peak was 45.36% of the total
GO. C-O (hydroxyl and epoxy, 288.5 eV), C=O
(carbonyl, 291.9 eV), and O=C-O (carboxyl, 294.5 eV)
peaks are shown in Figure B and indicate cystamine
conjugation with GO. The major species, C=C/C-C
(284.7 eV), was reduced to 36.53% of the total.
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24. CHAPTER-4
 In our study, the differential toxicity of cystamine-
conjugated GO toward Gram-negative bacteria
compared to gram-positive bacteria may be related to
differences in the natures of their cell walls.
 A thin peptidoglycan layer (7−8 nm thickness) is present
in gram-negative bacteria whereas a thick peptidoglycan
layer (20−80 nm thickness) is present in gram-positive
bacteria. The thicker peptidoglycan layer in gram-
positive bacteria may explain why these bacteria are
more resistant to the antibacterial effects of cystamine-
conjugated GO.
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25. CHAPTER.5. FUTURE WORK & PERSPECTIVE
 Photothermal therapy employs photosensitizing
agents taken up by cells, which generate heat from
light absorption, leading to photo-ablation of the
cancer cell and subsequent death.
 Deep penetration and little non specific
photothermal heating in the NIR window are due to
the low absorption of light by tissues, which are
largely transparent in NIR wavelength range.

26. CHAPTER-5
 Recently, photothermal therapy employing graphene-
oxide as the photo-absorbing agent has emerged as an
alternative and promising noninvasive treatment for
cancer cells as well as not-cancer related diseases,
such as Alzheimer’s disease (AD). It can generate heat
from optical energy, leading to the “burning” of cancer
cells or AD amyloid- b peptides (A b).
 Our current ability to synthesize, manipulate, modify,
and functionalize GO in the form of nanohybrids opens a
wide vista in the area of biomedical applications. As the
living cell is of micrometer-size dimension, nanometer-
size entities can be made to address various membrane
cell receptors or enter the cell to deliver
pharmaceuticals, genes, or heat.
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27. CHAPTER-5
 In this figure, PLGA showed (C-O-C) bending vibration
band at 1250 cm -1 ,(CH3) stretching vibration band at
1385 cm-1 and carbonyl band 690 cm-1.
 Cystamine conjugated GO showed a temperature up to
650C at 633 nm laser irradiation.
 In future, we will make GO composite with PLGA and
check it for photothermal activity.
 Moreover, PLGA has different intensity in finger print
region. So, it can be used for surface enhanced Raman
scattering (SERS).
27

28.  AMARNATH CA, NANDA SS, PAPAEFTHYMIOU GC, YI DK & PAIK U, (2013) NANOHYBRIDIZATION OF LOW-
DIMENSIONAL NANOMATERIALS: SYNTHESIS, CLASSIFICATION, AND APPLICATION, CRITICAL REVIEWS IN SOLID
STATE AND MATERIALS SCIENCES (IF:2.714 - PUBLISHED)
 NANDA SS, AN SSA, YI DK, (2015) OXIDATIVE STRESS AND ANTIBACTERIAL PROPERTIES OF A GRAPHENE OXIDE-
CYSTAMINE NANOHYBRID, INTERNATIONAL JOURNAL OF NANOMEDICINE (IF:4. 195 - PUBLISHED).
 NANDA SS, PAPAEFTHYMIOU GC, YI DK, (2015) FUNCTIONALIZATION OF GRAPHENE OXIDE AND ITS BIOMEDICAL
APPLICATIONS, CRITICAL REVIEWS IN SOLID STATE AND MATERIALS SCIENCES (IF:2.714 - PUBLISHED).
 NANDA SS, KIM K, YI DK, (2015) GRAPHENE OXIDE BASED FLUOROMETRIC DETECTION OF HYDROGEN PEROXIDE IN
MILK, JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY (IF: 1.339 - ACCEPTED).
 NANDA SS, WANG T, KIM K, YI DK, (2015) STUDY OF ANTIBACTERIAL MECHANISM OF GRAPHENE OXIDE USING
RAMAN SPECTROSCOPY, CURRENT APPLIED PHYSICS (IF: 2.1 - SUBMITTED)
 NANDA SS, AN SSA, YI DK, (2015) MEASUREMENT OF CREATININE IN HUMAN PLASMA USING A FUNCTIONAL
POROUS POLYMER STRUCTURE SENSING MOTIF, INTERNATIONAL JOURNAL OF NANOMEDICINE (IF:4. 195 –UNDER
REVISION).
 NANDA SS, AN SSA, YI DK, (2015) RAMAN SPECTRUM OF GRAPHENE OXIDE, ITS DERIVATIVES AND GRAPHENE
OXIDE HYBRID MATERIALS, TRENDS IN ANALYTICAL CHEMISTRY (IF: 6. 6 – UNDER PREPARATION)
PEER REVIEWED PUBLICATIONS
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