Polymer Nano-fibers synthesis using electrospinning incorporated with Graphene Oxide nanoparticles and followed by Vapour Phase Crosslinking

1. 1
“Fabrication and Characterization of Graphene
Oxide Nanoparticles Incorporated in Poly (Vinyl
Alcohol) Electro-spun Nanofibers and its Vapour-
phase Crosslinking”
■ Research fellow: Engr.Vinod Kumar
Supervisor
Prof. Dr. M. Ishaque Abro
Dept. of Metallurgy and Materials
Engineering, MUET Jamshoro

2. Contents
Introduction
Aims and Objectives
Literature Review
Experimental Work
Results and
Discussions
Conclusion
References
2

3. Introduction
 Now a days Nanotechnology is becoming a vast and
emerging field for the researchers of materials science,
atomic physics, medical chemistry and many other
fields
 Materials with strong antibacterial characteristics are
become basic need of daily life to protect human
health from various pollutants, including organic,
inorganic and biological substances.
 The incorporation of nanomaterials in polymer solutions
have become area of focus by of materials scientists, it
yields great improvement in overall properties of
resulting nanocomposite material. (Ghobadi et al.,
2015)
 Polymer solutions are further changed into polymer
membranes using various methods. The size of fibers in
membranes may vary from mm (millimeters) to nm
(nanometers).
3
Electrospinning setup

4. Introduction (Contd.)
 Electrospinning, is most common, simple
technique for production of nanofibers of desired
diameter using various nanofillers (Ceretti et al.,
2017).
 In this process, Jetting polymer solution through
electrostatic forces generates the fibres. (Li et al.,
2016)
 Polymers used so for the synthesis of nanofibers via
electrospinning technique are PVA, PLA, PMMA
etc
 The use of nanofillers improves the electrical,
mechanical, thermal and chemical properties of
electrospun matrix and make it applicable for
antibacterial activity and drug delivery.
 Graphene and its derivatives have very
constructive effect due to its superior
characteristics along with extremely high specific
surface area which makes it as best candidate as
nanofillers resulting its applications in many areas.
4

5. Introduction (Contd.)
 Recent advances in materials science have
allowed the use of Graphene in fields of biological
and medical sciences.
 The most reliable bio-degradable precursors for the
preparation of bio-compatible materials is Poly
(vinyl alcohol) (PVA). It has interesting performance
in various applications when electrospun with
nanofillers. (Sawada et al., 2012)
 The hydrophilic nature of PVA, make nanofibrous
membrane gets dissolved instantly in water when
immersed, which can be water insoluble
(hydrophobic) by cross-linking(Ahmad et al., 2012).
 Glutaraldehyde (GA) is considered as most
effective agent used for cross-linking purpose.
(Destaye et al., 2013)
 Figure shows the applications of Nanofibrous
membrane.
5

6. Aims and Objectives
The aim of this research project was to develop an efficient polymer
fibres loaded with graphene. In this regard, following objectives were set
for the proposed project.
 To synthesize and characterize the Graphene Oxide (GO).
 To synthesize and characterize Polymer Nanofiber Mat loaded with
GO.
 Crosslinking of Polymer nanofiber mat using Vapour-phase method.
 To evaluate the properties for the Biomedical applications i.e.
Antibacterial activity against gram negative bacteria. ( E.Coli,
Proteus and S. Aureus).
6

7. Literature Review
 The treatment and prevention of bacterial infections is being done by using antibacterial
drugs for last 80 years. Hence, among all other medicines the antibacterial drugs are most
misused due to easy availability and low cost (Demain and Sanchez, 2009).
 It has been confirmed through many reports that the spread of antimicrobial resistance
(AMR) and toxicity is due the widespread use of antibacterial drugs (Berendonk et al.,
2015).
 AMR occurs, when the bacteria become resistant and proliferate continuously in the
presence of antibacterial agent; in other words, the bacterium evolves and become
completely resistant to antimicrobial agents (Bos et al., 2015).
 The big challenge for pharmaceutical industry is to increase the antibacterial drugs
dissolution for poorly water-soluble agents (Marano et al., 2016).
 For this purpose, researchers have been working on design of nanotechnological
antibacterial delivery system such as Nano-capsules (Kobeissi et al., 2018) and
nanoparticles (Helmlinger et al., 2016).
 Polymer-based composites come in different forms, among which nanofibers are of
interests due to a big variety of important applications.
7

8. Literature Review (Contd..)
 Nanofibers’ minimum systemic toxicity, high loading capacity and high encapsulation
efficiency of antibacterial agents make it superior to other nanotechnological drug delivery
systems. (Chung et al., 2017)
 The general fabricating methods of polymer-based composite nanofibers are:
 Electrospinning,
 Self-assembly,
 Phase separation,
 Nano-porous template.(Beachley and Wen, 2010)
 THE ELECTROSPINNING PROCESS, patented by Formhals, is a very important technique for
making nanofibers, because of its simplicity and flexibility in producing homogeneous fibers
with adjustable diameter and microstructure with various nanofillers. . (Li et al., 2016)
 Moreover, the high electrical field present during the spinning process may improve
interactions between the nanofiller and the polymer matrix, thus improving the filler/matrix
coupling at the interface in molecular scale.
8

9. Literature Review (Contd..)
 The morphology and other characteristics of synthesized nanofibers largely
depends on some parameters includes solution concentration, conductivity,
viscosity, flow rate, volatility, Voltage applied and the distance between the
collector and injector.
 Nanofibers can be easily incorporated with antibacterial agents to inhibit
microbial colonization(Rieger et al., 2013)
 The continuous work by researchers to identify materials with comparable or
better antibacterial activity than current technologies that are also
environmentally friendly and should be economic to produce. The materials
of graphene family appear to fit this description.
 The incorporation of Graphene and its derivatives, such as graphene oxide,
doped and functionalized graphene materials, is the major focus in materials
science research, large surface area and high antibacterial characteristics
of graphene are the underlying reasons for its extensive usage in medical
applications. (Ghobadi et al., 2015, p.)
9

10.  GO effects the survival of common: Gram-positive bacteria (Enterococcus faecalis and
Bacillus subtilis),
Gram-negative bacteria (E.Coli and Salmonella typhimurium ) and Fungal pathogens (
Fusarium graminearum and Fusarium oxysporum ), (Chen et al., 2014)
 The antibacterial effects of GO result due to:
1) Basal Plane: The lateral dimensions of GO sheets in E.coli suspensions has effect on
antibacterial activity. When the GO has larger nanosheets, that can cover the cells and
prevent proliferation resulting strong antibacterial effects (Liu et al., 2012).
2) Entrapment: It is not possible for bacteria to proliferate due to blocked gas exchange and
bacteria are separated from their environment when they are trapped in aggregated GO
nanosheets. (Jayanthi et al., 2016)
3) Membrane Stress: There is large number of oxygen groups in GO as compared to rGO,
which can be well dispersed in suspension of bacteria and provide more chances to
generate cell membranes damage with sharp edges (X. Wang et al., 2013)
4) Photothermal Effect: The antibacterial activity of GO can be enhanced largely by its
synergistic effects and laser energy. (Shahnawaz Khan et al., 2015)
5) Oxidative Stress: The cellular components (lipids and proteins) are damaged by oxidative
stress produced by GO suspension. The DNA damage and mitochondrial dysfunction is
damaged by OS.(Romero-Vargas Castrillón et al., 2015)
10
Literature Review (Contd..)
ANTIBACTRAIL MECHANISIM OF GO

11. Literature Review (Contd..)
11
GRAPHENE SYNTHEIS TECHNIQUE
Top Down Method
Mechanical Exfoliation
Adhesive
Tape
AFM Tips
Chemical
Exfoliation
Chemical Synthesis
Sonication
Reduced
Graphene
Oxide
Bottom Up Methods
Pyrolysis
Epitaxial
Growth
CVD
Thermal Plasma
Other

12. Literature Review (Contd..)
12
CHEMICAL METHODS FOR
SYNTHESIS OF GO
BrodiesMethod
(1859)
Graphite,
Fuming HNO3,
KCLO3
Staudenmaier
method
(1898)
Graphite,
Fuming HNO3,
Conc. H2SO4
KClO3
Hoffman
Method
(1937)
Graphite,
Conc. HNO3
Conc. H2SO4
KClO3
HummersMethod
(1958)
Graphite,
Conc. H2SO4
KMnO4,
Na2NO3
Hummers
Method
Modified
Hummers
Method
Improved
Hummers
Method
FURTHER CLASSIFIED AS

13. Experimental Work
Selection of Materials 13
S.no Name of Chemical Company
1 Graphite Flakes Merck-Schuchardt, Germany
2 Sodium Nitrate (NaNO3) Merck-Schuchardt, Germany
3 PVA (average molecular weight 72,000 98% purity) Merck-Schuchardt, Germany
4 Potassium Permagnete (KMnO4) Rankem
5 Hydrochloric Acid (HCL,37%) Rankem
6 Hydrogen per oxide ( H2O2) Scharlas
7 Sulphuric Acid (H2SO4) ACS
8 Glutaraldehyde (GA, 25% aqueous solution DAEJUNG

14. Experimental Work
14
Description available: experimental details.pptx

15. Results and Discussions
Characterization of Graphene Oxide
SEM Analysis: (Comment No.4:
Further image Resolution &
Magnification needed in SEM)
➢ It could be observed that GO
has layered structure, with
ultrathin homogeneous films.
➢ These films are continuously
folded and the edges of
individual sheets are
distinguished in the image.
Exfoliation of sheets could also
be observed from images.
➢ This can be compared from
reported work (Arthi G and Bd,
2015)
15
a) b)
c) d)

16. EDS Analysis: (Comment No. 2:
Counter-check the synthesis of GO
through other techniques &
Comment 5: Benchmark the results)
 The synthesis of GO was also
confirmed using EDS that
showed the composition of
GO as 67 wt. % Carbon and
33 wt. % oxygen, as shown in
Fig a).
XRD ANALYSIS:
a) The peak of pure graphite 2θ=
26.7o, can be observed and
the disappearance of
graphite peak in Fig. b)
b) Sharp peak at 2θ= 12.1o
confirms the complete
oxidation of graphite.
c) Similar peak were previously
reported by (Wang et al.,
2008) & (Monteagudo et al.,
2019) that confirming the
oxidation of graphite.
16
b) c)
Results and Discussions
Characterization of Graphene Oxide
a)

17. Results and Discussions
Characterization of Graphene Oxide
FT-IR Analysis: ( Fig. a ) (Comment No. 3:
Reattempt the FTIR)
 The presence of two absorption peaks
observed, at 1570 cm-1 and 1060 cm−1 are
the attribute of stretching vibration of C=C
and C=O that represent the carboxylic acid
and carbonyl groups presence on the
edges of graphene oxide.
 The peak at 1710 cm-1 corresponding to the
stretching and bending vibration of OH
groups of water molecules adsorbed on
graphene oxide
 However the absorption peaks of 2340 cm-1
to 2670 cm -1 are representing the symmetric
and anti-symmetric stretching vibrations of
C-H2 Meanwhile the oxidation of graphite is
confirmed by the presence of oxygen-
containing groups. (Guo et al., 2009).
17
Fig. ATR-FTIR of Graphene Oxide
a)

18. Results and Discussions
Characterization of PVA Nanofibrous Membrane 18
a) c)
Fig a) shows the surface morphology of nanofibrous membrane.
Fig b) Histogram shows the average diameter of PVA nanofibers
422±133nm.
Fig c) ATR-FT-IR curve of PVA,
b)

19. Results and Discussions
Characterization of PVA/GO Nanofibrous membrane 19
SEM Analysis:
► Fig a) shows NFs of bare polymer PVA.
► Fig. b) shows PVA/GO NFs having 0.25
wt. % GO incorporated.
► Fig. c) shows PVA/GO NFs having 0.5
wt. % GO incorporated.
► Fig. d) shows PVA/GO NFs having 0.25
wt. % GO incorporated.
► The surface morphology of
electrospun PVA nanofibers that
confirms the incorporation of GO
particles in nanofibers. The filler
content can be further observed from
Figure Next Slide.
► The improved surfaces morphological
conditions can be obtained by well-
dispersing GO within PVA solution
media or by employing the organic
solvent.

20. 20
GO-
Particles
Results and Discussions
Characterization of PVA/GO Nanofibrous membrane
SEM Analysis: (Comment No. 7)
► In 1 wt. % GO incorporated NFs membrane, the large
amount of GO NPs were observed.
► Solution preparation for electrospinning involves the,
dispersion of GO NPs in DI- water for 2 hours under
sonication and then mixing with PVA solution through
stirring for half hour.
► Both processes: Ultra-sonication and stirring have
resulted the breaking GO particles much smaller sizes
and its incorporation can be confirmed in NFs.
► Similar conditions have also observed and reported
by (Harito et al., 2017).
► Few other factors that affect the morphology and
diameter of nanofibers are the filler quantity and
the fluctuations of parameters during
electrospinning process. Fig. b) represents the
average diameter of nanofiber is 274.16±93.23nm
b)
a)

21. Results and Discussions
Characterization of PVA/GO Nanofibrous
membrane
 FT-IR Analysis:
 The incorporation of graphene oxide
nanoparticles in electrospun nanofibers
membrane were confirmed using FT-IR analysis,
which have brought little shuffles in peaks of
pure polymer (PVA) membranes, the difference
can be observed in a) and b) spectra of Figure
a.
 The peaks between 900-1150 cm-1 mentioned
to be determining peak for C-O or -OH group.
(see in Region I, II, III and IV in Fig 6) shows
prominent peaks of both membranes.
 Peaks of GO NPs could be matched from GO
FT-IR as studied earlier.
21
a)

22. Results and Discussions
Characterization of Vapour- Phase Crosslinked
PVA/GO Nanofibrous membrane
SEM/ (Surface Morphology): The difference between a) and b) shows the effect of
Vapour-Phase Crosslinking on NFs membranes.
22
b)a)

23. Results and Discussions
Characterization of Vapour- Phase Crosslinked
PVA/GO Nanofibrous membrane
 EFFECT OF CROSSLINKING TIME ON DIAMETER
(Figures on Next slides)
 The change in morphology of nanofibers
membranes (Bare PVA polymer, 0.25 wt. % , 0.5
wt. % GO and 1 wt. % GO loaded in polymer)
by exposure to GA vapours of at 12h, 24h and
48h can be observed from (c, e, g ) of Fig 1, Fig
2, Fig 3, Fig 4 on next slides respectively.
 The significant change in diameters of as-spun
nanofibers can be observed from (f, g and h) of
Fig 1, Fig 2, Fig 3, Fig 4 at mentioned hour.
 Whereas a) and b) part of following figures shows
without crosslinked samples.
 Column Bar, shows average diameter of all as-
spun crosslinked samples.
 It can be interpreted that the exposure time
has direct effect on the diameter of
nanofibers. Other factors may also vary the dia
of nanofibers.
23

24. 24
2)1)

25. 25
4)3)

26. Results and Discussions
Characterization of Vapour- Phase Crosslinked
PVA/GO Nanofibrous membrane
FT-IR Analysis
 As shown in Figure, the crosslinking of PVA nanofibrous
membranes were also confirmed using FTIR.
 The three major bands observed in FTIR spectra that has
changed upon crosslinking by exposure to GA (2M
conc.) vapor, as reported (Jiang et al., 2018)
 The first large band shows the stretching of hydroxyl (-
OH)group resulting from intramolecular hydrogen
bonds (region I in Fig 9) between 3000-3650 cm-1.
 The decreasing intensity indicates the involvement of
more -OH group in acetal bridge formation.(Mansur et
al., 2008)
 The C=O stretching observed at between 1500-1750
cm-1 shows the unreacted end of aldehyde group and
due to presence of GO NPs in nanofibrous membranes
(region II of Fig 9).
 The gradual board peak at1000-1150 cm-1, resulted
due to increase in crosslinking time associated with O-
C-O vibration of acetal group (region III of Fig 9)
26

27. Results and Discussions
Antimicrobial Test
 Zone inhibition method was employed as shown in
Figure.
 PVA nanofibrous loaded with 1 wt % has shown
significant antibacterial activity against E.coli, gram
negative bacteria and most common pathogen,
involved in water born disease.
 It was observed that PVA/GO crosslinked membrane
have shown higher resistance (14mm) as compared to
PVA/GO sample (12mm) as shown in Fig 10 after 24
hours of incubation.
 This might be due to change in average diameter size
because crosslink membrane posses larger diameter as
compared to without crosslinked membrane of
PVA/GO.
 Similarly Bingan Lu, et al have reported the
antibacterial activity of chitosan–PVA nanofibers
containing graphene against E.coli (Lu et al., 2012).
27

28. 28
1
2
2.5
3
6
4
8
9
6.5
8
5.5
11.5
12
11
9.5
14
0
2
4
6
8
10
12
14
16
Without Crosslinked 12hrs CL 24hrs CL 48hrs CL
Inhibition Zone against E.Coli (mm)
Bare PVA PVA/GO_0.25% PVA/GP_0.5% PVA/GO_1%
Results and Discussions
Antimicrobial Test
 Other samples that have been
incorporated with GO have
also shown some activity, their
inhibition zone can be observed
from the histogram in Figure. It
can also be evaluated that the
vapour phase cross-linking has
significant effect on the
antibacterial activity of as-spun
membranes.
 Procedure for Antibacterial test:
experimental details.pptx

29. Conclusion
 Poly (Vinyl Alcohol) Electro-spun nanofibers mate loaded with graphene oxide
nanoparticles have been successfully fabricated and characterized the and further
crosslinked with Vapours of GA.
 It is concluded that the vapour exposure time has direct effect on the diameter of
nanofibers.
 In addition, crosslinked membrane shows significant antibacterial activity as compare
to PVA/GO nanofibrous membrane.
 The 1 wt % incorporation of GO is found much effective against bacteria E.coli as
compared to other wt % of 0.25 and 0.5. (results are available at )
 GO is found much active against E.coli of gram negative bacteria than others like
S.Aauers and Protues.
 The present work provides a basis for further studies of this novel nanofibrous material for
further medical applications.
29

30. Recommendations
 This work provides a base to work on nanofibrous membrane incorporated with
GO nanoparticles and following are the suggestions.
 Use of organic solvent can improve the morphology and dispersion of GO
nanoparticles in suspensions.
 Study of Thermal stability of membrane,
 Drug release profile of loaded GO nanoparticles and work on optimization is
suggested.
30

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34. Thank you…
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