Graphene Based Material for Biomedical Applications

Graphene Based Materials for Biomedical
Applications
Sitansu Sekhar Nanda
Department of Chemistry
Myongji University
1

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

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 aTwo-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

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

 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

 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 are 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

Table
Some of the Major Research
on GO Related Biomedical
Application are Presented
Here in the Table.
7

Recent Trends in Graphene Research
8
 Illustrates Charge Transfer in Graphene Layers (For 1–10 Layers).
The Inset Shows the Raman Spectra of Graphite Before and After
Exposure to 60 Torr NO2. For Monolayer (1L) and Two Layers
(2L) Of Graphene, the So-called G Peaks Were Observed at 1614
Cm−1 and 1608 Cm−1 Respectively. In Case of the Three Layers
(3L) of Graphene, The G Peaks are Splitted into Two Peaks Such
As 1601.5 Cm−1 and 1584 Cm−1 Denoted by the G−, Lower
Energy Peak. As the number of Graphene Layers Exceeds Three,
The Signal Peak Occurred at 1582 Cm−1 and 1598 Cm−1 and also
Intensity of Lower Energy Peaks Increased Compared with Layer.
This Confirmed that NO2 Adsorbs on the Top and Bottom
Surfaces Which Does Not Intercalate. Due to the Heavy
Adsorption on Both Side Of 1L Graphene, It Produces a Single G
Peak.

 Illustrates Reduced Graphene Oxide-gold Nanostar
Nanocomposites Prepared by Seed-mediated Growth And
Their Use As Active SERS Materials for Anticancer Drug
(Doxorubicin; DOX) Loading and Release. By This Approach,
Both Optical Properties and Morphology of Nanohybrids can
be Controlled without Using Stabilizer Such As Polymer or
Surfactant. By Changing Growth Rate Parameter Optical
Properties and its SERS Sensitivity Towards Aromatic Ring
Molecules Can Be Increased.
9

 Illustrates the Use of Monolayer Graphene, Sliver, and
R6G Molecules to Enhance the Reproducibility and
Stability of SERS. In this Figure (A) Control Structure
(R6G/Ag) was Prepared by Using Rhodamine 6G (R6G)
Molecules Absorbed on Ag Surface.
 (B) (R6G/G/Ag) Structure was Prepared by R6G
Molecules Absorbed on Graphene/Ag Substrate.
 (C) (G/R6G/Ag) Structure was Prepared by
Incorporation Of R6G Molecules in Ag and Monolayer
Graphene.
 (D) Monolayer Graphene On Ag Substrate was Evaluated
with a Time Interval of 8 Minutes on Continuous Laser
Irradiation.
10

 Presents A Typical TERS Profile of Graphene. Strong CH
Bending and Stretching Modes are Confirmed by Hydrogen-
terminated Graphene. In this Figure All the Spectra Were
Dominated By SLG (Solid Blue Line). It Does Not Show Any
Defects, CH Contamination or Edge Problem. Weak Defects
or Contamination in a SLG Observed in Dashed Red Line.
Some G Band And CH Bending and Stretching Mode
Intensity Observed in Dotted Green Line. From this Figure it
Concluded that From a Constant Signal TERS can be
Measured.
11

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.
12

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.
13

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%.
14

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.
15

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.
16

Chapter.3. Study of Antibacterial Mechanism of
Graphene Oxide Using Raman Spectroscopy
 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.
 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, Amide and Proteins.
 Escherichia coli (E. coli) and Enterococcus faecalis (E.
faecalis) were Used as Model Micro-organisms for All
the Experiments Performed.
17

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.
18

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.
19

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.
20

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.
21

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.
22

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.
23

 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
Chapter.4. Oxidative Stress and Antibacterial Properties
of a Graphene Oxide-cystamine Nanohybrid
24

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. 25

Chapter-4
 Cytotoxicity and ROS Studies of Cystamine Conjugated GO. Uv–Vis Spectrum and FT-IR of Cystamine,
GO, and Cystamine-Conjugated GO. 26

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.
27

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.
28

Chapter.5. Future Work & Perspective
 Current Research Plan Aims to Put 1) Bacteria and 2) Cancer Cells on
Graphene Electronic Devices Containing Graphene Monolayer and Electrodes.
We Control the Electron Density on the Graphene Using a Power Station,
Then We Study the Interaction Between the Graphene and the Bio-analytes.
Using Techniques Called Raman Spectroscopy And Atomic Force Microscopy,
We can Study the Phenomena at the Interface of Graphene and Bio-analytes;
Graphene Donates Some of Its Electrons to Bacteria and Cancer Cells,
Changing its Charge State In a Predictable Way. This Insight Could Allow us to
Design Tiny Graphene-based Electronic Devices.
29

Chapter-5
30
 General Schematic of Tissue Engineering Strategy.
(1) Cells are Isolated from the Patient and
(2) Expanded in 2D Culture.
(3) Expanded Cells are then Combined with Various
Natural or Engineered Bioactive Molecules (e.g., Growth
Factors, Nanoparticles, or DNA) into Biocompatible
Scaffolds and
(4) Cultured in Vitro under Specific Culture Conditions to
Promote Tissue Formation.
(5) Finally, Functional Tissue-engineered Constructs are
Implanted into the Donor to Replace the Damaged Tissue.

Peer Reviewed Publications
32
 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: 5.5)
 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).
 Nanda Ss, Papaefthymiou Gc, Yi DK, (2015) Functionalization of Graphene Oxide and its Biomedical Applications, Critical
Reviews In Solid State and Materials Sciences (IF: 5.5).
 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).
 Nanda Ss, Wang T, Kim K, Yi DK, (2015) Study of Antibacterial Mechanism of Graphene Oxide using Raman Spectroscopy,
Scientific Reports (IF: 5.5)
 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).
 Nanda Ss, An Ssa, Yi DK, (2015) Raman Spectrum of Graphene With its Versatile Future Perspectives, Trends in Analytical
Chemistry (IF: 7.4)

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