CPEG-40 and PVP-40 silver nanoparticles were found to be the most stable based on characterization of size, zeta potential, and UV-Vis absorption over 8 months of aging. 2CPEG-5 silver nanoparticles also showed good stability. These stabilized nanoparticles exhibited over 90% viability when tested on MCF-7 and HEP-2 cell lines. Zinc oxide nanoparticles were assembled using Heliotropium crispum plant extract and characterized as near spherical particles with organic capping. These particles showed good biocompatibility on Huh7 cells. The zinc oxide nanoparticles were used to fabricate an electrochemical sensor with carbon dots for hydrogen peroxide detection, exhibiting a low detection limit of 2.4 nM

1. Improving the Stability of Biocompatible Nanoparticles and
their Applications
By: Faria Khan
Research Supervisor: Dr. Hussnain Janjua
Department of Industrial Biotechnology
Atta-ur-Rahman School of Applied Biosciences,
National University of Sciences and Technology,
Islamabad, Pakistan
MS Thesis Defense
7th November 2016
NUST201463581MASAB92514F
1

2. TABLEOFCONTENTS 01 - INTRODUCTION
• Hypothesis
• Objectives of the study
• Scientific issues addressed in study
• Novelty of our study design
02 –BACKGROUND
• AgNPs: Need to control surface-capping
agents in biocompatible way
• Biogenic Ag NPs: size and capping control
• ZnO NM: Need to explore green synthesis
methodology
• ZnO/C-dots: A biocompatible H2O2 sensor
03 - METHODOLOGY
• Synthesis, characterization and screening of
biogenic AgNPs
• Stability screening: heat resistant, ageing
and bio-stable AgNPs
• ZnO nanomaterial assembly in HC-extract
• Fabrication of H2O2 sensor system using
ZnO/C-dots
04 – RESULTS-PART I
• Size and SPR of 32 AgNPs (derivative and
primary AgNPs) for screening
• Functional group and size determination
of screened AgNPs
• Application as a stable and biocompatible
colloid
05 – RESULTS PART II
• Assembly of ZnO Nano-bulk in HC-extract
• Characterization & Biocompatibility check
on Huh7 cell lines
• Fabrication & electrochemical assessment
of ZnO/ C-dots based H2O2 sensor
08 – FUTURE PROSPECTS
• AgNPs: a biocompatible drug carrier tool
or a possible targeted delivery agent
• Commercial: a stable colloid for long-term
research applications
• ZnO: Further invitro and invivo monitoring
• Commercial: A green, cost effective
sensing device
06 –IMPORTANT HIGHLIGHTS
• The novel findings of our study
• Scientific justification of our findings
• Review of all the application results
07 - CONCLUSION
• The extend to which scientific issue was
addressed in our study
• Ag-NPs: three different application routes
• ZnO: Real time H2O2 electrochemical
sensor
2

3. Biogenic
Source
INTRODUCTION Ag
NPs
ZnO
NM
Green Synthesis Methodology:
• Less Toxic Way to Assemble Nanomaterials
• Biocompatibility for Biological Applications
Cost of Production for Commercialized Application:
• Simple Production Method
• Less Biomaterials and Starting Materials
• Controlled Reaction Conditions
Biological Applications:
• Stability Check in Environment/ Working Platform
• Efficacy Check in Biological System
• Modulation to Enhance/Shift Biological Properties
Stability Check for Research Application:
• Governed by Complex Colloidal Properties
• Modulation of Surface Chemistry
• Control of Size & Electrochemical Applications
Heliotropium
crispum
Identified
Biomaterial
2
1
3
4
Surface Chemistry:
Modulation
Enhancement
Control
Applications
NOVELTY
3

4. INTRODUCTION 1
2
3
4
Tested Synthesis Method
&
Optimization Details
Reduction Potential
&
NMs Assembly
Organic Groups &
Surface Capping
Biological Activity Tested
&
Modulation to Achieve More
4

5. EXAMPLE WITH 6 PARTS AND SILHOUETTE
5
01
02
03
04
05
• Aggregation & Dispersion
• SSA and Quantum Effect
• Stability & toxicity control
Colloidal AgNPs
• Electrostatic repulsion
• Steric Repulsion
• Electrosteric Repulsion .
Surface Capping
• Plant extract: electrosteric:
• Citrate-capped: electrostatic
• PEG and PVP: adsorption/
steric
Biocompatible Surfactants
• Chemical: Oxidation, reduction,
dissolution, sulfidation, photo-
reactivity etc.
• Biological: Chemical bio-nano
interaction & Corona formation
• Physical: Homo/ hetero-
aggregation & agglomeration
Transformation 1
.
Improving Stability
• Biological: Self stabilized but no
surface capping modulation
option available
• Chemical: Surfactants can be
charged materials or polymers
Silver Nanoparticles: Stability & ApplicationsBACKGROUND
1: Lowry et al. 2012 2: Baun et al., 2008 3: Christian et al., 2008

6. 02
Current
Techniques
01
H202 : Toxic
By-product
Some title here
03Our Solution
BACKGROUND ZnO Nanomaterial: H2O2 Sensing System
1:Marinho, Real, Cyrne, Soares, & Antunes, 2014
Limitations
Short
life
time Low
access
Difficult to
handle
High
cost
Easy Fabrication
route
Material
selection
Precise
monitoring
Cost Effect
Selection of
Simple
Technique
Applicability &
Stability6

7. AgNPs: Stability & Size Control
7

8. METHODOLOGY:
SILVER
2-Polymer
recapping
3-Ligand
exchange
Hit &
Trial:
NaBH4
1-Direct
method
3-Romer et al, 2011
1- Hoppe et al, 2006
2- Mulfinger et al, 2007
8

9. METHODOLOGY:
SILVER
2nd
screening
3rd
screening
4th
screening
1st
screening

10. RESULTS
0.5 HC Ag
5.0 HC Ag
Citrate-Ag
Primary AgNPs: Characterization
Peak SPR: Specific to adsorbate type & concentration
dH: 0.5HC-AgNPs< 5.0HC-AgNPs< citrate-AgNPs
430: 13nm 425: 12nm 406: 14nm
Fingerprint region
10

11. RESULTS
6 Derivative AgNPs Groups: UV-Vis Characterization
• Shape: Near-Spherical
• SPR: Dielectric constant and capping
• Polydispersity: FWHM *
• Shifts in λmax**: local NPs environment
*Full Width at Half Maximum
**Peak Surface Plasmon Resonance
11

12. RESULTS 6 Derivative AgNPs Groups: Zeta Potential
• Zeta range: -13mV to -32mV
• General trend
 PVP capping gave more close to 0 zeta value
 Varied with the concentration of polymer added to each group
 Steric repulsion and surface oxidation/ reduction of functional groups
 More negative zeta meant more electrostatic repulsion dominance
 Degree of stability not judged by Zeta Value in our case
Type of capping
 Electrostatic
 Steric
 Electrosteric
12

13. RESULTS 6 Derivative AgNPs Groups: DLS Size-Number Distribution
• Too much peaks……Too much confusion
• Variable no. of NPs in various size ranges: 10-100 d.nm
• From each group we selected less polydispersed and less size variability AgNPs sample
• Single or near single peaked sample selected for further studies only
13

14. RESULTS CPEG -40 & CPVP-80 AgNPs
CPEG-40 CPVP-80
CPEG-40 : 13.47nm
CPVP-80 : 14.84nm
Same citrate-group: Functional group orientation
14

15. RESULTS CPEG -40 & CPVP-80 AgNPs
dH: Z-size distribution & Zeta Potential
CPEG-40: 137nm (PDI= 0.413) (-26mV)
CPVP-80: 84nm (PDI= 0.419) (-13mV)
No FTIR peak change: No polymer addition: Enhanced/
modulated capping of citrate-plant compounds
15

16. RESULTS 2CPEG -05 & 2CPVP-20 AgNPs
2CPVP-202CPEG-05
Same citrate-group (400-410): Functional group orientation
2CPEG-5 : 13.98nm
2CPVP-20 : 16.02nm
16

17. RESULTS 2CPEG -05 & 2CPVP-20 AgNPs
dH: Z-size distribution & Zeta Potential
2CPEG-05: 57.69nm (PDI= 0.53) (-22mV)
2CPVP-20: 104.1nm (PDI= 0.45) (-21mV)
FTIR peak change: Ligand Exchange
Orientation of attachment important for size
17

18. RESULTS
PVP-40
PEG-20
PVP-40
PEG -20 & PVP-40 AgNPs
PEG-20 : 15.07nm
PVP-40 : 14.10nm
HC-group (420-430): Functional group orientation
2 morphologies: preferential adsorption of ligands
:specific crystallographic facets

19. RESULTS PEG -20 & PVP-40 AgNPs
dH: Z-size distribution & Zeta Potential
PEG-20: 137nm (PDI= 0.39) (-17mV)
PVP-40: 176nm (PDI= 0.57) (-19mV)
FTIR peak change: No polymer addition: polymer induced
partial capping on surface….increased steric effect
HC AgNPs: amine, carboxylate and ester (COC, SOR)
SOR: least stable and was removed in every derivative form
Amine: protein derived from PE keeping configuration compact
as removal always contributed towards increased size
19

20. RESULTS Ageing & Heat Resistant AgNPs
Ageing Stability
Two trends noted: Decrease in FWHM and shifts in λmax with respect to time
FWHM: narrower range with time hence time-dependent decrease in Polydispersity and Skewing to one specific
size
FWHM: Citrate-AgNPs, HC-AgNPs and PEG-20 AgNPs: 8th month increased PDI & size: 4 month stability
λmax: CPEG-40, CPVP-80, 2 CPEG-5 and PVP-40 AgNPs : no significant dH morphology change at 4-8 months
Most stable: PVP-40, CPVP-80, and 2CPEG-5 AgNPs as skewed most towards monodispersion & stable λmax
Heat Resistant
λmax: Blue shift means all AgNPs were not de-stabilized but decreased in size
CPEG-40 and 2CPEG-5 showed a red absorbance shift
FWHM: Aggregation level low as small absorbance range
>50nm capping diameter hence confer high resistance
CPEG-40 AgNPs and PVP-40 AgNPs :no significant FWHM and λmax change.
20

21. RESULTS Bio-stability of 0.5HC-AgNPs
Only concentration and dosage of AgNPs showing more than 90% viability were categorized as “biocompatible” carrier
entities in our study
70%
MCF-7 cell lines
• 82% viable at 0.5mg/mL
• 93% viable at 1.5mg/mL
• 79% viable at 2.0mg/mL
HEP-2 cell and HCEC cell lines
• 0.5mg/mL more than 90% cellular viability
• 1.0mg/mL: 100% viability: high recovery
• 1.5mg/mL: 87% viability
Important Trends:
Uptake of 0.5HC-AgNPs was high in MCF-7 cell lines
1.5mg/mL was significant concentration
Biocompatibility
• the uptake rate of AgNPs
• the decrease in viability at concentrations of AgNPs.
21

22. RESULTS Biocompatibility of Stability Tested AgNPs
70%
AgNPs MCF-7 HEP-2
5HC-AgNPs 88% 89.5%
Citrate-AgNPs 82% 78%
CPEG-40 AgNPs 83% 81%
CPVP-80 AgNPs 83% 74%
2CPEG-5 AgNPs 92% 82%
2CPVP-20 AgNPs 77% 92%
PEG-20 AgNPs 82% 88%
PVP-40 AgNPs 91% 87%
Fixed 1.5mg/mL
difference in cellular viability was observed
the response differed significantly in both cell lines
Cellular uptake and Cytotoxic response
 surface-capping layer composition
ˣ diameter of surface-capping exhibit *CPEG-40: 144nm
2CPEG-5 AgNPs : 92% viability in MCF-7 , 82% in HEP-2
2CPVP-20 AgNPs: 77% viability in MCF-7, 92% in HEP-2
cell membrane mediated endocytosis: receptor-ligand response22

23. ZnO NMs: Electrochemical Sensor
23

24. METHODOLOGY
24

25. RESULTS UV-Vis Absorbance Bands of ZnO NMs
• Exciton A: 250-280nm….corresponds to amine functional group
• Exciton B: 300-320nm….corresponds to ZnO annealed at low temperature
• UV-range: green emissions and dangling ZnO bonds can easily trap UV
• High plant concentration: High ZnO concentration present
• ZnO annealing: Dependent upon temperature and pH
25

26. RESULTS
Compositional Analysis of ZnO NMs
• OH bonded
• Carbohydrate chains
• Amine/amides: Plant Protein
Zn O
C N
26

27. RESULTS
Size and Morphology Growth of ZnO NMs
dH: 814nm
Zeta Vaue: -4mV
HR-TEM
Near spherical core of ZnO
Organic capping
• 1 day: nucleation> seed growth
• 2 week: bulk formation through growth
Agglomeration :
• hydrophilic environment
• functional groups Van der Waal’s forces
27

28. RESULTS Viability of ZnO NMs in Huh7 cells: Temporal Response
Hepatocyte Derived Cellular Carcinoma: High H2O2 activity
Cell Viability: Concentration- and Time Dependent Response
• 1-10ug/ml
day 1 viability was reduced on an average of 92%
day 2 the cells achieved 99% viability again (exocytosis)
• 15-100ug/ml
baseline control viability could not be rescued even after 2 days
higher concentrations (15-100ug/ml) once flooded into the
cellular system cannot be removed as efficiently
Mann-Whitney Rank test & ANOVA
P = 0.186 & P = 0.174
Difference due to random sampling
28

29. RESULTS Surface Chemistry of ZnO & ZnO/C-dots: XPS
High resolution binding energy spectra of bulk ZnO/C-dots and
confirm ZnO, functional groups & C-dots adsorption
Why add C-dots
Electro-catalytic activity
Enhance O-R potential
• Zn2+
1028 and 1050 eV in Zn2p: orbital states Zn2p3/2 and Zn2p1/2
• O2-
Single peak at O1s region at 532eV higher: loosely bound in
ZnO wurtzite structure
• Adventitious Carbon on surface: Polydopamine & PE
• N1s: PE nitrogen containing amines
Photo-electronic orbital states
29

30. RESULTS Working Electrode of H2O2 Detection: Electro-Catalytic Activity
A- Electro catalytic activity: 0.1 M PBS solution containing 5 mM (Fe(CN)6)3 /4 at a scan rate of 100 mVs−1
B- CV measurements of the ZnO in (a) buffer (b) H2O2
C- ZnO/C-dot composite electrodes in 0.1 M PBS solution (a) buffer (b) H2O2
 Both ZnO and ZnO/ C-dots show well defined redox peaks
 ZnO: electron transport activity mobility enhanced by C-dots
 Catalytic working efficiency of electrode at a very low applied
potential
30

31. RESULTS Amperometric Response of ZnO/C-dot: H2O2 Sensing & Reliability
A- The current-time responses of the ZnO/C-dot composite at a very low applied potential toward
successive doses of H2O2 .
B- The detection limit of our system 2.4 nM and the calculated linear range was ̴ 800 nM
 Peak Stabilization & Sensitivity Range
 Reliability of sensing: 20nM….50nM
 2.4nM: low detection limit & 800nM: saturation point31

32. RESULTS
Amperometric Response of ZnO/C-dot: H2O2 Selectivity in Biomolecule Presence
Amperometric Measurements:
The effect of interfering components on the
selectivity of H2O2 was investigated through
by initially adding constant concentrations
(50 nM) of various interferences such as UA,
AA, DA, Glu, KCl and NaCl to PBS solution
containing H2O2
 Low applied potential: energy efficiency
 Structure and surface capping provide
good specificity
 Sensor: significant response only toward
H2O2
 H2O2 is a small molecule so diffusion rate
high & so is detection
 Cost effective material selection
 Selective & Sensitive to H2O2
 Energy efficient sensing system
 H2O2 real time monitoring
 Reliable & reproducible results
32

33. Stability of Ag-NPs
Surface capping
modulation
Orientation of
surfactants
Ageing: Related to
capping type
Heat: Related to dH
Biostability:
endocytosis
HC-AgNPs:
Application Related
Drug carrier
Heat Resistant
Ageing Stable
Electrochemical
Sensor
Cost effective
Biocompatible
Reliable
Non-enzymatic
Longetivity
ZnO Nano Material
Unique Amines
Organic Coating
Simple materials
Modulation of yield
33
 
Important Highlights & Conclusion

34. In-vitro study of ZnO/ C-dots
Further electrochemical characterization
Physiological monitoring of H2O2 levels
Development of real-time monitoring
system
Range of detection callibration
Development of a real-time cost
effective device for research and
diagnostic
FUTURE
PROSPECTS
Further surface capping studies: pH
response
Stability ageing: Monitoring of SPR
absorbance
Cytotoxic evaluation: Temporal
Response
Dose dependent response
Drug trafficking at receptor-drug
interface
Drug loading and release study: pH, Heat
etc
AgNPs
ZnONMs

35. ACKNOWLEDGEMENTS
• First of all, I would like to offer my sincere gratitude to my supervisor Dr Hussnain
Janjua for his support, encouragement & guidance. In a subtle way he brings out the
true researcher in his students.
• Dr Irshad Hussain for expert advise and insightful discussions throughout my research
phase and providing me with the sample analysis facilities at LUMS
• I would also like to extend my utmost gratitude to my GEC members Dr Fahd Ehsan
and Dr Shahrukh Abbas for their precious input in my research
• I want to appreciate the HOD, Dr Sadaf Zaidi, administrative body at ASAB, especially
Dr Peter John (Principle ASAB) and all the faculty members.
• Special thanks to Dr Naeem Akhtar for introducing me into the nano-sensor research
world & Dr Nauman Khalid for his detailed discussions upon colloids and AgNPs
properties. Mr Zajif (LUMS) for his guidance in operation of lab instruments & analysis
• Lastly, all of my research fellows & lab mates for providing me with the best research
atmosphere.
35

36. Q&A SESSION
THANK YOU !
36